Forensic Science Znternationul, 57 (1992) 99 - 107 Elsevier Scientific Publishers Ireland Ltd.
GAS CHROMATOGRAPHlC DETECTION OF COCAINE AND COCAETHYLENE IN HAIR OF MICE CHRONICALLY INJECTED WITH COCAINEORCOCAETHYLENEANDFEDETHANOL
SERGEI V. PIROZHKOVa~C, RONALD R. WATSONb*C and CLEAMOND D. ESKELSONC aResearch Institute for Medico-Biological Problems of Addictions, Moscow 121921 Russia, bDe-partments of Family and Community Medicine and %wgical Biology, NZAAA Alcohol Research Center, University of Arizona Health Sciences Center, Tucson, AZ 85724 (USA) (Received February 12th, 1992) (Accepted August 3Oth, 1992)
Summary GC and GClMS analysis was used to detect cocaine and cocaethylene in hair extracts of mice injected with 20 mgikg cocaine hydrochloride or an equivalent dose of cocaethylene fumarate twice daily for 3 weeks. Some mice were fed liquid Lieber-DeCarli diets containing ethanol (26% of total calories) and injected twice daily with the same doses of cocaine or cocaethylene or combination of cocaine and morphine (5 mg/kg). The average concentrations of cocaine in different experimental groups were in the range of 0.9-2.4 ng/mg of hair and for cocaethylene, 2.4-2.8 ng/mg of hair. There were no significant differences in hair concentrations of cocaine among groups receiving cocaine treatment, nor were there significant difference in cocaethylene concentration in hair in the two groups administered cocaethylene. In hair extracts of mice treated with cocaine and ethanol, levels of cocaethylene were below the limit of detection. Key words: Hair analysis; Cocaine; Cocaethylene;
Gas chromatography
Introduction Hair analysis for cocaine has been stated to be a useful tool for the identification of cocaine users, for the estimation of the amount of drug taken and to indicate the temporal pattern of consumption [l]. Cocaine was reliably identified and quantitated in hair samples from ten heavy cocaine users in a recent study using gas chromatography/mass spectrometry (GUMS) [2]. Another study reported a high quantity of cocaine (up to 98 ng/mg) in hair of a person who died 24 days after accidental cocaine overdose [3]. Not only cocaine but a number of its metabolites including benzoylecgonine, ecgonine methyl ester, norcocaine and cocaethylene were identified using GUMS. Correspondace to: Ronald R. Watson, Department of Family and Community Medicine, NIAAA Alcohol Research Center, University of Arizona Health Sciences Center, Tucson, AZ 85724, USA. 0379-0738/92/$05.00
0 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
100
Cocaethylene is synthesized in the body by transesterification of cocaine with ethanol. This compound was found in significant quantities in the postmortem blood, liver and brain of motor vehicle accident victims who used cocaine concurrently with alcohol shortly before the accident [4]. Cocaethylene possesses high pharmacological activity being equipotent to cocaine in binding to the dopamine transporter in human striatal membranes. It is much less potent in binding to the norepinephrine binding site of the occipital cortex membranes, or to serotonin transporter in the frontal cortex [4]. The LDsOfor cocaethylene is 1.5 times lower than for cocaine [5]. Concentrations of cocaethylene found in blood and tissues of drug addicts are comparable to or higher than those of cocaine [4,61.
As cocaethylene is only formed in the body from cocaine and ethanol and its presence in hair cannot be a result of external contamination, it has been proposed as a more reliable marker of cocaine abuse than cocaine itself [2]. Detection of cocaethylene in hair, however, requires at least concurrent consumption of cocaine and ethanol. Pharmacokinetic models of cocaethylene formation and elimination and specifically its retention in hair as related to cocaine and ethanol intake levels have not yet been proposed. Previously, a linear correlation between dose of cocaine and its concentration in hair of C57BL mice was demonstrated using a radioimmunoassay (RIA) [l]. Although RIA is sufficiently sensitive it lacks specificity since it does not distinguish cocaine metabolites like cocaethylene. We report here a quantitative GC detection of cocaine and cocaethylene in hair of mice chronically treated with cocaine or cocaethylene, or their combination with ethanol and morphine. These experiments were part of a study designed to compare immunotoxicity and hepatotoxicity of chronic injection of both drugs alone or in combination with ethanol and morphine. Materials and Methods
Cocaine hydrochloride and cocaethylene fumarate were obtained from National Institute of Drug Abuse (NIDA), morphine sulfate was obtained from NIDA via Mallinckrodt (St. Louis, MO). Hexane, isoamyl alcohol, methanol, ammonium hydroxide and potassium phosphate dibasic were purchased from EM Science (Gibbstown, NJ). Hexane and methanol were redistilled before use. The detergent Nonoxynol 9 was obtained from GAF Corporation (New York, NY). Animals and treatment procedure Male C57BL mice, 60 days old, were obtained from the National Cancer Institute. Animals were housed at 23”C, 50% relative humidity, with a 12-h light/dark cycle. Cocaine hydrochloride, 8.2 mg/ml, or cocaethylene-1.5 fumarate, 11.2 mglml of saline was given by intraperitoneal (i.p.) injections twice daily at 09:OO h and 16:00 h, 6 days per week. The doses of cocaine-HCl were gradually increased: 10 mglkg for the first 2 days, 15 mglkg for the next 5 consecutive days and then 20 mg/kg until the end of the experiment. Cocaethylene fumarate was injected similarly but all the doses were increased 1.37-fold as its molecular weight
101
is greater than that of cocaine HCl. Morphine was mixed with cocaine solution immediately before injecting the mixture into mice at doses of 2.5 mg/kg twice daily for the first week and then 5 mg/kg of morphine sulfate thereafter. Controls were similarly treated with saline. Ethanol (26% by total calories) was administered in a liquid Lieber-DeCarli diet (Dyets, Bethlehem, PA). Ethanol was substituted for an isocaloric amount of maltose-dextrin in the control diet. All diets were given ad libitum. In Experiment I mice were randomly distributed into 5 experimental groups: Injection Saline Saline Cocaine Cocaine Cocaine + morphine
Diet supplement None E than01 None E than01 E than01
During the first week of the study drug injections were administered to mice fed regular pelleted food. Consumption of liquid diets began on day 8. Hair was collected from the back area on day 21 and 22 of the experiment. In Experiment II the following groups were studied: Injection Saline Cocaine Cocaine Cocaethylene Cocaethylene
Diet supplement None None E than01 None E than01
The mode and duration of treatment
were the same as in Experiment
I.
Hair preparation and wash procedure Hair samples were placed on a funnel with Whatman no. 1 filter paper and washed with 10 ml of 5% solution of detergent Nonoxyno19. Then hair was rinsed under vacuum with 0.5 1 of distilled water and dried overnight at room temperature. Extraction procedure A sample of washed hair (50 - 100 mg) was weighed and placed in a glass silicon tube with a screw cap. Methanol (3 ml) was added and samples were incubated 2 h at 60°C. Methanol extracts were transferred to another set of tubes by a glass Pasteur pipette and evaporated under N2 at 40°C. The dry residue was redissolved in 2 ml 0.1 N HCl and extracted with 4 ml hexane containing 1% isoamyl alcohol (ISA-hexane). The hexane layer was discarded after centrifugation for 5 min at 2500 rev./min. The acid phase was made alkaline to pH 9.2 by adding 0.03 ml ammonium hydroxide and 0.5 ml 10% K2HP04 and extracted with 4 ml of ISA-hexane. The hexane layer was transferred to another set of
102
tubes after centrifugation and re-extracted with 2 ml 0.1 N HCl. The upper hexane layer was discarded and the acid phase was made alkaline as described and re-extracted with 4 ml ISA-hexane. The hexane phase was transferred into conical glass tubes and evaporated under Nz at 30°C. The residue was finally dissolved in 0.1 ml methanol and 3 ~1was injected into the gas chromatograph. Cocaine hydrochloride or cocaethylene fumarate (0.5; 1.0 and 1.75 pg in 0.05 ml methanol) was added to the hair sample (after methanol extraction) from control mice which had received saline only. These samples were treated in a manner similar to that used for the authentic samples. Calibration curves relating the amount of cocaine or cocaethylene to areas were developed and used in calculations. GC analysis
A Hewlett Packard 5890 gas chromatograph with flame ionization detector, equipped with HP 3396A integrator was used for analysis of drugs in hair extracts. The column was Drug One NON PAKD capillary column (Alltech Assoc.), 10 m x 0.53 mm i.d. The carrier gas was helium at a flow rate of 12 mllmin. The injection port temperature was 250°C. The oven temperature was maintained at 145°C for 7 min and then programmed to 220°C at 3 deg/min. GCIMS analysis
The presence of cocaine or cocaethylene in hair extracts was confirmed by GUMS analysis of two randomly selected samples from each of the experimental groups of Experiment II. GUMS analysis was performed with a Hewlett Packard 5890 gas chromatograph equipped with a HP 5970 electron ionization mass spectrometer. The column was a HP-5 cross-linked 5% phenylmethylsilicone fused-silica capillary column (25 m x 0.2 mm i.d., 0.33 pm film thickness). Helium was used as the carrier gas. The injection port temperature was 250°C and the GC interface temperature was 300°C. The oven temperature was programmed from 150°C to 280°C at 20 deg/min, with an initial hold of 1 min and a final hold time of 4 min. The following ions were monitored for each compound at their respective retention times (R3: cocaine, mlz 82, 182, 303; cocaethylene rnlz 82,196,317. Analytes were identified based on comparison of retention times and relative abundance of three confirming ions to the corresponding values of authentic standards run on a daily basis. Statistical analysis
All results are expressed as the mean * SE. Differences between groups were analyzed by Duncan 5% level multiple range test [7]. Results
As shown on Fig. 1, gas chromatography of the final extract provided peaks of cocaine and cocaethylene which were clearly separated from any contaminating peaks. The limit of detection for cocaine or cocaethylene was 0.2 nglmg of hair. In hair extracts from mice injected with saline no peaks with a retention time close to cocaine or cocaethylene were observed.
103
L Fig. 1. GC recordings of standards and hair extracts from mice injected with cocaine or cocaethylene. Panel A: hair extract from a control mouse spiked with 1.75 pg of cocaine hydrochloride. Arrow indicates the peak of cocaine. Panel B: hair extract from a mouse injected twice daily with 20 mglkg of cocaine_HCl for 3 weeks. Panel C: hair extract from a control mouse spiked with 1.75 kg of cocaethylene-fumarate. Arrow indicates the peak of cocaethylene. Panel D: hair extract from a mouse injected twice daily with 27 mg/kg of cocaethylene-fumarate (equivalent to 20 mglkg of cocaine-HCl) for 3 weeks.
Fig. 2. Mass spectra of standards and extracts from mice injected with cocaine or cocaethylene. Designation of panels as on Fig.1. Records were made at Rt 8.92min for cocaine and 9.29 min for cocaethylene.
105
TABLE I CONCENTRATIONS OF COCAINE AND COCAETHYLENE IN HAIR OF MICE CHRONICALLY INJECTED WITH COCAINE OR COCAETHYLENE OR THEIR COMBINATION WITH ETHANOL AND MORPHINE Data are means * SE. The number of animals in each group is indicated in parentheses. Concentration of cocaine or cocaethylene is expressed in ng of free-base form per mg of hair. Mice were injected with 20 mg/kg of cocaine-HCl, or 27 mg/kg cocaethylene fumarate (equivalent to 20 mg/kg of cocaine-HCl) twice daily for 3 weeks and received liquid modified Lieber-DeCarliu diet containing ethanol (26% of total calories) or isocaloric amount of maltose-dextrin as described in Materials and Methods. Some mice received combined injection of cocaine and morphine (5 mg/kg twice daily). Injection
Diet supplement
Experiment I
Experiment II
Cocaine (ng/mg of hair) Cocaine Cocaine Cocaine
None Ethanol Ethanol + morphine
1.2 f 0.2 (19) 2.1 zt 0.7 (25) 0.9 ?? 0.2 (19)
1.4 f 0.1 (7) 1.6 + 0.4 (9)
Cocaethylene (nglmg of hair) Cocaethylene Cocaethylene
None Ethanol
2.8 f 0.8 (12) 2.4 f 0.3 (11)
Examples of GUMS verification of cocaine and cocaethylene in hair extracts from mice chronically treated with these drugs are presented in Fig.2. Mass spectra of the hair samples were compared with the spectra of the authentic drugs and the presence of cocaine or cocaethylene was confirmed. Mean concentrations of cocaine in hair of mice receiving different diet treatment and cocaine injections were in the range of 0.8 - 2.4 ng/mg (Table 1). No statistically significant effects of ethanol or morphine were observed. Concentrations of cocaethylene were slightly higher than those of cocaine suggesting a longer half-life in the body, more efficient transport into hair or more effective binding of cocaethylene to hair matrix. Discussion In recently published studies using GUMS analysis it has been demonstrated that not only cocaine but its metabolites are retained in the hair matrix of drug users [2,3,8]. Thus hair analysis can be used as a complementary method for identification of cocaine abuse especially when blood or urine are not available or when it is necessary to confirm cocaine use several weeks or months prior to testing. Hair analysis can help verify quality of treatment for drug dependence, or in pre-employment screening. The relationship between the severity of abuse and concentration of cocaine in hair is not clear. While the levels of cocaine and metabolites detected by RIA correlated well with the injected dose of cocaine [l] in mouse hair, human studies of six cocaine abusers did not show this relationship [8]. The absolute values of cocaine in hair of drug users vary widely with ranges
106
of 8.2- 1’78ng/mg [2] and 0.12-5.7 ng/mg hair [8]. The use of acid extraction in the first case and enzymatic digestion of hair in the second may be partially responsible for the discrepancies in results. We used a 2-h extraction with methanol at 60°C since acid extraction for 18 h had no significant advantage in the recovery of cocaine and its metabolites [9]. The procedure for purification of hair extracts was a modification of a published method for extraction of cocaine from plasma [lo]. We introduced one additional extraction step with 0.1 N HCl and re-extraction into hexane to achieve better purification. Extention of the incubation period to 4 h did not yield higher concentrations of cocaine in the methanolic extracts of mice hair. We did not use a wash procedure with methanol as was proposed for human hair [2] in order to avoid loss of cocaine and cocaethylene from the hair matrix. In spite of this, concentrations of cocaine in mice hair usually did not exceed 4 ng/mg, while in human hair the mean value in ten cocaine users was 10.8 ng/mg [2]. Based upon the loss of cocaethylene and norcocaine during the methanol wash procedure this level could have been at least twice as high. We failed to detect cocaethylene in hair of mice receiving cocaine in combination with ethanol. In human hair the levels of cocaethylene were on average 15 times lower than that of cocaine [2] thus the expected concentration of cocaethylene in mouse hair is below the limit of detection. Relatively low levels of cocaine in hair of mice compared to humans, despite a more favorable hair/body mass index, may be explained by a lower rate of hair growth. When we shaved the hair on the back in a group of mice there was no significant growth in the bald area even after 1 month. Combined treatment with cocaine (or cocaethylene) plus ethanol, or cocaine and morphine plus ethanol produced serious liver injury in mice (unpublished data). This may have led to a decrease of cocaine or cocaethylene elimination due to loss of liver parenchymal function. There was no association between the extent of liver damage and concentration of cocaine or cocaethylene in hair. In conclusion GC analysis confirmed by GUMS demonstrated retention of cocaine and cocaethylene in mouse hair. Thus, mice may be a useful model in studies of the mechanism of cocaine and its metabolites incorporation into the hair matrix and its dependence on dose and rate of hair growth. References 1
W.A. Baumgartner, V.A. Hill and W.H. Bland, Hair analysis for drugs of abuse. J. Forensic sci., 34 (1990) 1433 - 1453. 2 E.J. Cone, D. Yousefnejad, W.D. Darwin and T. Maguire, Testing human hair for drugs of abuse. II. Identification of unique cocaine metabolites in hair of drug abusers and evolution of decontamination procedure. J. And. Toxicol., 15 (1991) 250-255. 3 R. Martz, B. Donelly, D. Fetterolf, L. Lasswell, G.W. Hime and W.L. Hearn, The use of hair analysis to document a cocaine overdose following a sustained survival period before death. J. Anal. Toxicol., 15 (1991) 279-281. 4 W.L. Hearn, D.D. Flynn, G.W. Hime, S. Rose, J.C. Cofino, E. Mantero-Atienza, C.V. Welti and D.C. Mash, Cocaethylene: a unique cocaine metabolite displays high affinity for the dopamine transporter. J. Neurochem., 56 (1991) 698-701.
107 5
W.L. Hearn, S. Rose, J. Wagner, A. Ciarlegio and D.C. Mash, Cocaethylene is more potent than cocaine in mediating lethality. Phurmecol. Biochem. Behav., 39 (1991) 531- 533. 6 P. Jatlow, J.D. Elsworth, C.W. Bradberry, G. Winger, J.R. Taylor, R. Russel and R.H. Roth, Cocaethylene: a neuropharmacologically active metabolite associated with concurrent cocaineethanol ingestion. Lifi Sci., 48 (1991) 1787 - 1794. 7 D.B. Duncan, Multiple range and multiple F tests. Biomettis, 11 (1955) l-42. 8 M.R. Harkey, G.L. Henderson and C. Zhou, Simultaneous quantitation of cocaine and its major metabolites in human hair by gas chromatography/chemical ionization mass spectrometry. J. Anal. To&ol., 15 (1991) 260-265. 9 D. Vale&e, M. Cassini, Pigliapochi and M. Vansetti, Hair as the sample in assessing morphine and cocaine addiction. Cl&. Cha., 27 (1981) 1952 - 1953. 10 P. Jatlow, Cocaine by GLC and a Nitrogen Detector. In I. Sunshine (ed.), Methodology of Analytical Z’oticology, Vol. 2, CRC Press, Cleveland, 1975, pp. 133- 137.
GAS CHROMATOGRAPHlC DETECTION OF COCAINE AND COCAETHYLENE IN HAIR OF MICE CHRONICALLY INJECTED WITH COCAINEORCOCAETHYLENEANDFEDETHANOL
SERGEI V. PIROZHKOVa~C, RONALD R. WATSONb*C and CLEAMOND D. ESKELSONC aResearch Institute for Medico-Biological Problems of Addictions, Moscow 121921 Russia, bDe-partments of Family and Community Medicine and %wgical Biology, NZAAA Alcohol Research Center, University of Arizona Health Sciences Center, Tucson, AZ 85724 (USA) (Received February 12th, 1992) (Accepted August 3Oth, 1992)
Summary GC and GClMS analysis was used to detect cocaine and cocaethylene in hair extracts of mice injected with 20 mgikg cocaine hydrochloride or an equivalent dose of cocaethylene fumarate twice daily for 3 weeks. Some mice were fed liquid Lieber-DeCarli diets containing ethanol (26% of total calories) and injected twice daily with the same doses of cocaine or cocaethylene or combination of cocaine and morphine (5 mg/kg). The average concentrations of cocaine in different experimental groups were in the range of 0.9-2.4 ng/mg of hair and for cocaethylene, 2.4-2.8 ng/mg of hair. There were no significant differences in hair concentrations of cocaine among groups receiving cocaine treatment, nor were there significant difference in cocaethylene concentration in hair in the two groups administered cocaethylene. In hair extracts of mice treated with cocaine and ethanol, levels of cocaethylene were below the limit of detection. Key words: Hair analysis; Cocaine; Cocaethylene;
Gas chromatography
Introduction Hair analysis for cocaine has been stated to be a useful tool for the identification of cocaine users, for the estimation of the amount of drug taken and to indicate the temporal pattern of consumption [l]. Cocaine was reliably identified and quantitated in hair samples from ten heavy cocaine users in a recent study using gas chromatography/mass spectrometry (GUMS) [2]. Another study reported a high quantity of cocaine (up to 98 ng/mg) in hair of a person who died 24 days after accidental cocaine overdose [3]. Not only cocaine but a number of its metabolites including benzoylecgonine, ecgonine methyl ester, norcocaine and cocaethylene were identified using GUMS. Correspondace to: Ronald R. Watson, Department of Family and Community Medicine, NIAAA Alcohol Research Center, University of Arizona Health Sciences Center, Tucson, AZ 85724, USA. 0379-0738/92/$05.00
0 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
100
Cocaethylene is synthesized in the body by transesterification of cocaine with ethanol. This compound was found in significant quantities in the postmortem blood, liver and brain of motor vehicle accident victims who used cocaine concurrently with alcohol shortly before the accident [4]. Cocaethylene possesses high pharmacological activity being equipotent to cocaine in binding to the dopamine transporter in human striatal membranes. It is much less potent in binding to the norepinephrine binding site of the occipital cortex membranes, or to serotonin transporter in the frontal cortex [4]. The LDsOfor cocaethylene is 1.5 times lower than for cocaine [5]. Concentrations of cocaethylene found in blood and tissues of drug addicts are comparable to or higher than those of cocaine [4,61.
As cocaethylene is only formed in the body from cocaine and ethanol and its presence in hair cannot be a result of external contamination, it has been proposed as a more reliable marker of cocaine abuse than cocaine itself [2]. Detection of cocaethylene in hair, however, requires at least concurrent consumption of cocaine and ethanol. Pharmacokinetic models of cocaethylene formation and elimination and specifically its retention in hair as related to cocaine and ethanol intake levels have not yet been proposed. Previously, a linear correlation between dose of cocaine and its concentration in hair of C57BL mice was demonstrated using a radioimmunoassay (RIA) [l]. Although RIA is sufficiently sensitive it lacks specificity since it does not distinguish cocaine metabolites like cocaethylene. We report here a quantitative GC detection of cocaine and cocaethylene in hair of mice chronically treated with cocaine or cocaethylene, or their combination with ethanol and morphine. These experiments were part of a study designed to compare immunotoxicity and hepatotoxicity of chronic injection of both drugs alone or in combination with ethanol and morphine. Materials and Methods
Cocaine hydrochloride and cocaethylene fumarate were obtained from National Institute of Drug Abuse (NIDA), morphine sulfate was obtained from NIDA via Mallinckrodt (St. Louis, MO). Hexane, isoamyl alcohol, methanol, ammonium hydroxide and potassium phosphate dibasic were purchased from EM Science (Gibbstown, NJ). Hexane and methanol were redistilled before use. The detergent Nonoxynol 9 was obtained from GAF Corporation (New York, NY). Animals and treatment procedure Male C57BL mice, 60 days old, were obtained from the National Cancer Institute. Animals were housed at 23”C, 50% relative humidity, with a 12-h light/dark cycle. Cocaine hydrochloride, 8.2 mg/ml, or cocaethylene-1.5 fumarate, 11.2 mglml of saline was given by intraperitoneal (i.p.) injections twice daily at 09:OO h and 16:00 h, 6 days per week. The doses of cocaine-HCl were gradually increased: 10 mglkg for the first 2 days, 15 mglkg for the next 5 consecutive days and then 20 mg/kg until the end of the experiment. Cocaethylene fumarate was injected similarly but all the doses were increased 1.37-fold as its molecular weight
101
is greater than that of cocaine HCl. Morphine was mixed with cocaine solution immediately before injecting the mixture into mice at doses of 2.5 mg/kg twice daily for the first week and then 5 mg/kg of morphine sulfate thereafter. Controls were similarly treated with saline. Ethanol (26% by total calories) was administered in a liquid Lieber-DeCarli diet (Dyets, Bethlehem, PA). Ethanol was substituted for an isocaloric amount of maltose-dextrin in the control diet. All diets were given ad libitum. In Experiment I mice were randomly distributed into 5 experimental groups: Injection Saline Saline Cocaine Cocaine Cocaine + morphine
Diet supplement None E than01 None E than01 E than01
During the first week of the study drug injections were administered to mice fed regular pelleted food. Consumption of liquid diets began on day 8. Hair was collected from the back area on day 21 and 22 of the experiment. In Experiment II the following groups were studied: Injection Saline Cocaine Cocaine Cocaethylene Cocaethylene
Diet supplement None None E than01 None E than01
The mode and duration of treatment
were the same as in Experiment
I.
Hair preparation and wash procedure Hair samples were placed on a funnel with Whatman no. 1 filter paper and washed with 10 ml of 5% solution of detergent Nonoxyno19. Then hair was rinsed under vacuum with 0.5 1 of distilled water and dried overnight at room temperature. Extraction procedure A sample of washed hair (50 - 100 mg) was weighed and placed in a glass silicon tube with a screw cap. Methanol (3 ml) was added and samples were incubated 2 h at 60°C. Methanol extracts were transferred to another set of tubes by a glass Pasteur pipette and evaporated under N2 at 40°C. The dry residue was redissolved in 2 ml 0.1 N HCl and extracted with 4 ml hexane containing 1% isoamyl alcohol (ISA-hexane). The hexane layer was discarded after centrifugation for 5 min at 2500 rev./min. The acid phase was made alkaline to pH 9.2 by adding 0.03 ml ammonium hydroxide and 0.5 ml 10% K2HP04 and extracted with 4 ml of ISA-hexane. The hexane layer was transferred to another set of
102
tubes after centrifugation and re-extracted with 2 ml 0.1 N HCl. The upper hexane layer was discarded and the acid phase was made alkaline as described and re-extracted with 4 ml ISA-hexane. The hexane phase was transferred into conical glass tubes and evaporated under Nz at 30°C. The residue was finally dissolved in 0.1 ml methanol and 3 ~1was injected into the gas chromatograph. Cocaine hydrochloride or cocaethylene fumarate (0.5; 1.0 and 1.75 pg in 0.05 ml methanol) was added to the hair sample (after methanol extraction) from control mice which had received saline only. These samples were treated in a manner similar to that used for the authentic samples. Calibration curves relating the amount of cocaine or cocaethylene to areas were developed and used in calculations. GC analysis
A Hewlett Packard 5890 gas chromatograph with flame ionization detector, equipped with HP 3396A integrator was used for analysis of drugs in hair extracts. The column was Drug One NON PAKD capillary column (Alltech Assoc.), 10 m x 0.53 mm i.d. The carrier gas was helium at a flow rate of 12 mllmin. The injection port temperature was 250°C. The oven temperature was maintained at 145°C for 7 min and then programmed to 220°C at 3 deg/min. GCIMS analysis
The presence of cocaine or cocaethylene in hair extracts was confirmed by GUMS analysis of two randomly selected samples from each of the experimental groups of Experiment II. GUMS analysis was performed with a Hewlett Packard 5890 gas chromatograph equipped with a HP 5970 electron ionization mass spectrometer. The column was a HP-5 cross-linked 5% phenylmethylsilicone fused-silica capillary column (25 m x 0.2 mm i.d., 0.33 pm film thickness). Helium was used as the carrier gas. The injection port temperature was 250°C and the GC interface temperature was 300°C. The oven temperature was programmed from 150°C to 280°C at 20 deg/min, with an initial hold of 1 min and a final hold time of 4 min. The following ions were monitored for each compound at their respective retention times (R3: cocaine, mlz 82, 182, 303; cocaethylene rnlz 82,196,317. Analytes were identified based on comparison of retention times and relative abundance of three confirming ions to the corresponding values of authentic standards run on a daily basis. Statistical analysis
All results are expressed as the mean * SE. Differences between groups were analyzed by Duncan 5% level multiple range test [7]. Results
As shown on Fig. 1, gas chromatography of the final extract provided peaks of cocaine and cocaethylene which were clearly separated from any contaminating peaks. The limit of detection for cocaine or cocaethylene was 0.2 nglmg of hair. In hair extracts from mice injected with saline no peaks with a retention time close to cocaine or cocaethylene were observed.
103
L Fig. 1. GC recordings of standards and hair extracts from mice injected with cocaine or cocaethylene. Panel A: hair extract from a control mouse spiked with 1.75 pg of cocaine hydrochloride. Arrow indicates the peak of cocaine. Panel B: hair extract from a mouse injected twice daily with 20 mglkg of cocaine_HCl for 3 weeks. Panel C: hair extract from a control mouse spiked with 1.75 kg of cocaethylene-fumarate. Arrow indicates the peak of cocaethylene. Panel D: hair extract from a mouse injected twice daily with 27 mg/kg of cocaethylene-fumarate (equivalent to 20 mglkg of cocaine-HCl) for 3 weeks.
Fig. 2. Mass spectra of standards and extracts from mice injected with cocaine or cocaethylene. Designation of panels as on Fig.1. Records were made at Rt 8.92min for cocaine and 9.29 min for cocaethylene.
105
TABLE I CONCENTRATIONS OF COCAINE AND COCAETHYLENE IN HAIR OF MICE CHRONICALLY INJECTED WITH COCAINE OR COCAETHYLENE OR THEIR COMBINATION WITH ETHANOL AND MORPHINE Data are means * SE. The number of animals in each group is indicated in parentheses. Concentration of cocaine or cocaethylene is expressed in ng of free-base form per mg of hair. Mice were injected with 20 mg/kg of cocaine-HCl, or 27 mg/kg cocaethylene fumarate (equivalent to 20 mg/kg of cocaine-HCl) twice daily for 3 weeks and received liquid modified Lieber-DeCarliu diet containing ethanol (26% of total calories) or isocaloric amount of maltose-dextrin as described in Materials and Methods. Some mice received combined injection of cocaine and morphine (5 mg/kg twice daily). Injection
Diet supplement
Experiment I
Experiment II
Cocaine (ng/mg of hair) Cocaine Cocaine Cocaine
None Ethanol Ethanol + morphine
1.2 f 0.2 (19) 2.1 zt 0.7 (25) 0.9 ?? 0.2 (19)
1.4 f 0.1 (7) 1.6 + 0.4 (9)
Cocaethylene (nglmg of hair) Cocaethylene Cocaethylene
None Ethanol
2.8 f 0.8 (12) 2.4 f 0.3 (11)
Examples of GUMS verification of cocaine and cocaethylene in hair extracts from mice chronically treated with these drugs are presented in Fig.2. Mass spectra of the hair samples were compared with the spectra of the authentic drugs and the presence of cocaine or cocaethylene was confirmed. Mean concentrations of cocaine in hair of mice receiving different diet treatment and cocaine injections were in the range of 0.8 - 2.4 ng/mg (Table 1). No statistically significant effects of ethanol or morphine were observed. Concentrations of cocaethylene were slightly higher than those of cocaine suggesting a longer half-life in the body, more efficient transport into hair or more effective binding of cocaethylene to hair matrix. Discussion In recently published studies using GUMS analysis it has been demonstrated that not only cocaine but its metabolites are retained in the hair matrix of drug users [2,3,8]. Thus hair analysis can be used as a complementary method for identification of cocaine abuse especially when blood or urine are not available or when it is necessary to confirm cocaine use several weeks or months prior to testing. Hair analysis can help verify quality of treatment for drug dependence, or in pre-employment screening. The relationship between the severity of abuse and concentration of cocaine in hair is not clear. While the levels of cocaine and metabolites detected by RIA correlated well with the injected dose of cocaine [l] in mouse hair, human studies of six cocaine abusers did not show this relationship [8]. The absolute values of cocaine in hair of drug users vary widely with ranges
106
of 8.2- 1’78ng/mg [2] and 0.12-5.7 ng/mg hair [8]. The use of acid extraction in the first case and enzymatic digestion of hair in the second may be partially responsible for the discrepancies in results. We used a 2-h extraction with methanol at 60°C since acid extraction for 18 h had no significant advantage in the recovery of cocaine and its metabolites [9]. The procedure for purification of hair extracts was a modification of a published method for extraction of cocaine from plasma [lo]. We introduced one additional extraction step with 0.1 N HCl and re-extraction into hexane to achieve better purification. Extention of the incubation period to 4 h did not yield higher concentrations of cocaine in the methanolic extracts of mice hair. We did not use a wash procedure with methanol as was proposed for human hair [2] in order to avoid loss of cocaine and cocaethylene from the hair matrix. In spite of this, concentrations of cocaine in mice hair usually did not exceed 4 ng/mg, while in human hair the mean value in ten cocaine users was 10.8 ng/mg [2]. Based upon the loss of cocaethylene and norcocaine during the methanol wash procedure this level could have been at least twice as high. We failed to detect cocaethylene in hair of mice receiving cocaine in combination with ethanol. In human hair the levels of cocaethylene were on average 15 times lower than that of cocaine [2] thus the expected concentration of cocaethylene in mouse hair is below the limit of detection. Relatively low levels of cocaine in hair of mice compared to humans, despite a more favorable hair/body mass index, may be explained by a lower rate of hair growth. When we shaved the hair on the back in a group of mice there was no significant growth in the bald area even after 1 month. Combined treatment with cocaine (or cocaethylene) plus ethanol, or cocaine and morphine plus ethanol produced serious liver injury in mice (unpublished data). This may have led to a decrease of cocaine or cocaethylene elimination due to loss of liver parenchymal function. There was no association between the extent of liver damage and concentration of cocaine or cocaethylene in hair. In conclusion GC analysis confirmed by GUMS demonstrated retention of cocaine and cocaethylene in mouse hair. Thus, mice may be a useful model in studies of the mechanism of cocaine and its metabolites incorporation into the hair matrix and its dependence on dose and rate of hair growth. References 1
W.A. Baumgartner, V.A. Hill and W.H. Bland, Hair analysis for drugs of abuse. J. Forensic sci., 34 (1990) 1433 - 1453. 2 E.J. Cone, D. Yousefnejad, W.D. Darwin and T. Maguire, Testing human hair for drugs of abuse. II. Identification of unique cocaine metabolites in hair of drug abusers and evolution of decontamination procedure. J. And. Toxicol., 15 (1991) 250-255. 3 R. Martz, B. Donelly, D. Fetterolf, L. Lasswell, G.W. Hime and W.L. Hearn, The use of hair analysis to document a cocaine overdose following a sustained survival period before death. J. Anal. Toxicol., 15 (1991) 279-281. 4 W.L. Hearn, D.D. Flynn, G.W. Hime, S. Rose, J.C. Cofino, E. Mantero-Atienza, C.V. Welti and D.C. Mash, Cocaethylene: a unique cocaine metabolite displays high affinity for the dopamine transporter. J. Neurochem., 56 (1991) 698-701.
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W.L. Hearn, S. Rose, J. Wagner, A. Ciarlegio and D.C. Mash, Cocaethylene is more potent than cocaine in mediating lethality. Phurmecol. Biochem. Behav., 39 (1991) 531- 533. 6 P. Jatlow, J.D. Elsworth, C.W. Bradberry, G. Winger, J.R. Taylor, R. Russel and R.H. Roth, Cocaethylene: a neuropharmacologically active metabolite associated with concurrent cocaineethanol ingestion. Lifi Sci., 48 (1991) 1787 - 1794. 7 D.B. Duncan, Multiple range and multiple F tests. Biomettis, 11 (1955) l-42. 8 M.R. Harkey, G.L. Henderson and C. Zhou, Simultaneous quantitation of cocaine and its major metabolites in human hair by gas chromatography/chemical ionization mass spectrometry. J. Anal. To&ol., 15 (1991) 260-265. 9 D. Vale&e, M. Cassini, Pigliapochi and M. Vansetti, Hair as the sample in assessing morphine and cocaine addiction. Cl&. Cha., 27 (1981) 1952 - 1953. 10 P. Jatlow, Cocaine by GLC and a Nitrogen Detector. In I. Sunshine (ed.), Methodology of Analytical Z’oticology, Vol. 2, CRC Press, Cleveland, 1975, pp. 133- 137.
