Identification and Quantitation of Cocaine and Its Metabolites ...

Journal of Analytical Toxicology, Vol. 16, September/October 1992

Identification and Quantitation of Cocaine and Its Metabolites, Benzoylecgonineand Ecgonine Methyl Ester, in Hair of Bolivian Coca Chewers by Gas Chr0matography/Mass Spectrometry'* M.R. M611er** and P. F e y

Institute of Legal Medicine, State University of Saarland, D-6650 Homburg, Germany S. R i m b a c h

Department of Gynaecology, University Heidelberg, D-6900 Heidelberg, Germany Abstract

I

Twenty hair samples obtained from Bolivian mine workers who chewed 3-8 g of coca leaves daily for several years were analyzed for cocaine and Its main metabolites, benzoylecgonine (BZE) and ecgonine methyl ester (EME). A new method was developed for the detection and quantltation of cocaine and its metabolites, BZE and EME, from hair in a single procedure. The hair samples were washed, cut Into 56 segments (2-cm length), pulverized, and incubated with phosphate buffer and the enzyme ~-glucuronidasearylsulfatase. After solid phase extraction and derlvatization with pentafluoropropionic anhydride/pentafluoropropanol, the drugs were identified and measured by gas chromatography/ mass spectrometry (GC/MS) using deuterated cocaine, BZE, and EME as Internal standards. The method is reproducible (cocaine, CV = 8%; BZE, CV = 14%) and the detection limit for cocaine and BZE was 0.1 ng/mg, for EME 1 ng/mg. In the different hair segments, cocaine was found to be present in concentrations between 1.4 to 50.6 ng/mg, benzoylecgonine from 0.4 to 17.6 ng/mg, and ecgonine methyl ester traces below the calibration curve of approximately 12.9 ng/mg. In 95% of the cases cocaine exceeded BZE and EME in concentration 9

Introduction

Because of its sensitivity and specificity, gas chromatography/ mass spectrometry (GC/MS) is the method of choice for detecting cocaine and its metabolites. Several GC/MS methods (1-9), summarized in Reference 11, are described for testing cocaine and its metabolites from hair. Martz (3) found cocaine and BZE, and Kidwell identified (4) cocaine, BZE, and EME in hair of cocaine abusers. Both used MS/MS, which is available only in a few forensic laboratories. Cocaine and its metabolites were analyzed in hair by EIGC/MS (7,8). Only traces of the drug and metabolites could be found in authentic samples (6). Cone (8) detected cocaine, norcocaine, and cocaethylene in hair of cocaine users. The purpose of this study is to describe a new, reproducible method for the simultaneous analysis of cocaine, BZE, and EME 9 Presented in part at the Third Winter Meeting of the German Society tot Pharmacology and Toxicology, Hannover, Germany, December 4-6, 1991 (t0). "" Author to whom correspondence should be addressed.

in human hair and to report on the measurement of hair samples of cocaine chewers. This method also allows for the simultaneous detection and quantitation of morphine, codeine, and amphetamine ( 11 )

Methods

Reagents and standards. All reagents used were of analytical grade. Ecgonine methyl ester hydrochloride and ecgonine-d3methyl ester hydrochloride were purchased from Sigma Chemical (Steinheim, Germany). Cocaine, benzoylecgonine, cocained 3, and benzoylecgonine d 3 were purchased from Radian Corporation. ~-Glucuronidase (12 U/mL)-arylsulfatase (60 U/mL) was obtained from Merck (Darmstadt, Germany). The solid phase extraction was carried out with Chromabond | Ca8 (200 mg, 3 mL) extraction columns (Macherey-Nagel, Dtiren, Germany and VacElut | SPS 24 system (Analytichem International, Frankfurt, Germany). The ball mill (type MM2) was purchased from Retsch (Haan, Germany). Pentafluoropropionic anhydride (PFPA) and pentafluoropropanol (PFPOH) were purchased from Aldrich Chemical (Steinheim, Germany). Instrumentation. The following Hewlett-Packard instruments were used: 7673A automatic sampler, 5890 series II gas chromatograph, 5971A mass selective detector, Vectra QS/16S Workstation, and GI034B software. The gas chromatograph was equipped with an HP-Ultra 2 capillary column (crosslinked 5% phenyl methyl silicone, 12 m x 0.2 mm x 0.33-~m film thickness, temperature-programmed from 70~ (3 min hold) to 180~ at 15~ to 240~ at 5~ and to 300~ at 30~ (5 min hold)), directly inserted into the ion source. The injector temperature was 260~ and the GC/MS interface temperature was 280~ The helium carrier gas flow rate was 2 mL/min. The mass spectrometer was operated in the El mode with electron energy of 70 eV. Sample collection. The hair samples were obtained from 20 Bolivian mine workers, ages 13 to 60 years (mean age 31), who mined silver, zinc, tin, and lead. To bear their adverse conditions (hard physical work, dust, high temperatures, little food) they chewed 3 to 8 g of coca leaves daily over years. A continuous cocaine intake was thus expected. However, an accurate amount of daily intake for each individual was not known. The hair samples had an average length of 6 cm (4--11 cm) and were cut near the

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291


scalp and secured with adhesive tape. The root and tip were marked. A band of hair about the thickness of a pencil was taken from each worker. The samples were cut into 2-cm segments. The 56 segments obtained were examined for concentrations of cocaine, BZE, and EME. Blank control hair samples were obtained from five co-workers of the authors' institute. Procedure. The strands of hair were fixed with strings so that the hair could not shift. They were washed with warm water (5 min) and acetone (1 min) to eliminate external contamination. The samples were then dried in a stream of warm air. The strands of hair were cut into 2-cm segments which were separately pulverized in a ball mill. To the pulverized hair (10-30 mg), 2 mL phosphate buffer (pH 7.6), and 100 ng of the deuterated standards (cocaine, benzoylecgonine, and ecgonine methyl ester) were added. The sample was hydrolyzed with 75 ~tL [3-glucuronidase-arylsulfatase for 2 h at 40~ After centrifugation, the supemant was removed to a clean vessel and 2 mL phosphaste buffer was again added to the residue, shaken, and centrifuged. The two buffer fractions were combined. 1 mL of a 0.1M K2HPO+ solution was added to adjust the pH to 8. The sample was transferred to a Chromabond Ct8 extraction column that had been conditioned with 6 mL methanol and 3 mL H20 in a Vac-Elut | system and m

drawn through. The column was washed with 3 mL of H20, 3 mL of 0.25N acetic acid, and 3 mL of H20. Finally, it was dried by passing air through the column (10 rain) and centrifuging at 4000 rpm (15 min). The adsorbed drugs were eluted with 3 x 500 ILL acetone-dichlolromethane ( 3:1). Derivatization. The eluent was evaporated to dryness under a stream of nitrogen at 60~ The residue obtained was derivatized with 100/.tL pentafluoropropionic anhydride (PFPA) and 70 pL pentafluoropropanol (PFPOH) for 30 min at 60~ The mixture was again taken to dryness using a stream of nitrogen at 60~ GC/MS analysis. The derivatized residue was reconstituted in

Table I. Selected Ions and Retention Times Used for the Identification of Cocaine, BZE, and EME* Substance EME BZE C0C

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30 IJL of ethyl acetate and 2 IJL was injected into the GC/MS system. The mass spectrometer was operated in the selected ion mode (SIM). Table I shows the ions monitored for cocaine (COC), benzoylecgonine-PFP (BZE), ecogonine methyl esterPFP (EME), and the deuterated internal standards (IS), their percent abundance (%), and the retention times (RT). The substances were identified by their retention times and the relative abundance of the two ions monitored in comparison with the deuterated internal standards. Standard calibration curves were obtained by adding 10, 50, 100, 250, 500, and 1000 ng of cocaine, 10, 50, 100, 250, and 500 ng BZE and EME, and 100 ng of deuterated standards to 20 mg of pulverized blank control hair (corresponding to 0.5, 2.5, 5.0, 12.5, 25.0, and 50.0 ng/mg hair). The recovery rates were determined by adding 1130 ng of un-

deuterated cocaine, BZE, and EME to 20 mg of pulverized blank control hair. After extraction, 100 ng of the deuterated analogues were added. The solution was derivatized and quantitatively analyzed by GC/MS.

Results

Identification. The EI mass spectra of cocaine and the PFP derivatives of benzoylecgonine and ecgonine-methyl ester and their deuterated analogues are shown in Figure 1. Figure 2 shows

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Table II. Mean Amounts of Cocaine, BZE, and EME in the 56 Segments of the Hair of 20 Coca Chewers No.

Age

Coca use since

Length(cm)

No. of Seg.

COC (ng/mg)

BZE (ng/mg)

EME (ng/mg)

COC/BZE

COC/EME

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

13 16 18 19 22 23 24 24 24 27 27 31 35 35 36 38 45 46 60 60

1 3 6 7 8 8 10 11 13 13 13 17 20 18 23 24 31 26 45 47

4 11 7 4 7 6 4 5 5 10 6 5 7 6 7 6 6 7 4 4

2 5 3 2 3 3 2 2 3 4 3 3 3 3 3 3 2 3 2 2

6.9 9.2 6.3 41.5 16.8 7.8 5.6 38.8 19.6 15.2 15.6 1.7 31.7 33.1 24.4 45.2 5.8 12.8 9.6 15.9

9.9 3.0 1.7 8.9 6.4 3.1 1.8 13.4 8.7 3.8 4.2 0.6 14.3 9.3 9.3 13.9 4.3 4.8 2.8 5.1

5.8 1.8 T* 5.6 10.4 3.6 1.8 8.0 2.4 1.9 1.7 T* 3.9 6.8 4.6 7.5 1.0 5.4 1.9 3.1

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1.2 5.2 10.3 7.4 1.8 2.2 3.2 4.8 8.2 8.0 9.0 2.5 8.1 4.9 5.4 6.1 2.8 2.9 5.3 5.3

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the SIM chromatograms of cocaine, benzoylecgonine-PFP, and ecgonine methyl ester-PFP extracted from an authentic hair sample (sample no. 8, segment 1, cocaine: 40.1 ng/mg, BZE: 12.1 ng/mg, EME: 8.2 ng/mg). Quantitation. Figure 3 shows the calibration curves of the three substances. The correlation coefficients were r = 0.9995 (cocaine), 0.9998 (BZE), and 0.9915 (EME). The detection limit for cocaine and BZE was 0.1 ng/mg, for EME 1 ng/mg hair. Recove O' and analyze stability. The recovery for cocaine was 90%, tbr BZE 75%, and for EME 50%. The possible conversion of cocaine to BZE or BZE to cocaine during the extraction procedurc was checked in two different experiments. 100 ng of cocaine and BZE was addled to 20 mg of pulverized blank control hair and extracted. 100 ng of deuterated internal standards was added before derivatization. The derivatization mixture was subjected to GC/MS analysis. It was found that 5% of cocaine was converted to BZE, but no BZE was converted to cocaine. The enzymatic pretreatment had no influence on the yield of cocaine, BZE, and EME. However, the method used is our routine procedure, and is also used for the detection of opiates and amphetamine. Repmducibili~.,. A mixture of hair samples of the subjects was analyzed (n = 7) for cocaine and BZE. The mean value for cocaine was 12.2 + 1 ng/mg (CV = 8%) and for BZE 2.8 + 0.4 (CV = 14%). Washing procedure. To investigate the part of cocaine and its metabolites that can be washed out by the described procedure, three different unpulverized hair samples (nos. 5,14,15) were divided in two parts. One part was extracted unwashed. The other one was washed three times alternately with warm water and acetone. The three aqueous solutions, the unwashed, and the washed hair samples from the three subjects were extracted and analyzed according to described procedure. The acetone phases were evaporated to dryness. After addition of the internal standards the residues were derivatized and analyzed as described. The three wash procedures extracted about 7.5% (5.7-9%) of cocaine and 7.9% (5.6-11.1%) of BZE. 70-95% of the extracted material was

294

in the first aqueous solution. In the acetone phases only traces of cocaine BZE could be found. Mine workers results. Table II shows the amounts of cocaine, BZE, and EME in the 56 segments of hair from the Bolivan mine workers. Cocaine, BZE, and EME could be identified and quantitated in all samples. The mean value for cocaine was 18.2 ng/mg, for benzoylecgonine 6.6 ng/mg, and for ecgonine-methyl ester 4.0 ng/mg. Figure 4 shows the values in the corresponding segments of the hair samples. The ratio of cocaine to BZE was greater than 1, with one exception.

Discussion The hair specimens were from American Indians working in the "Mina Candelaria" in Potosi, Bolivia. The mine was opened about 300 years ago was famous for its silver ore, which was mined until recently. Today the miners are looking for zinc, lead, and tin with little yield. The working conditions in the mine started out bad and haven't changed much in the last 300 years. The mining is done by hand, without machines. The Indians carry the ore to the surface in bags, using primitive ladders and makeshift arrangements. The mine workers are convinced that their ability to work under these harsh conditions--10-12 h work without eating or drinking at 4000 m above sea level at temperatures of 40~ and above--would not be possible without chewing coca leaves. They use about 3-8 g coca leaves daily by mixing it with the ash of the quinua plant and chewing it during their shift. Although their life expectancy is only 35--40 yr, some of the subjects, according to their own reports, had already worked and chewed coca leaves for more than 40 years. In the population studied, no dependence of the concentration of cocaine or its metabolites in hair to the age of the subjects or duration of cocaine use was observed. Our results indicate that


the mine workers consumed a constant amount of coca leaves, at least during the time frame of growth of these samples (approximately 4-11 months). However, the concentrations of cocaine and metabolites varied from subject to subject. All 56 hair segments analyzed were positive for cocaine, BZE, and EME (Table II). The mean value for cocaine was 18.2 ng/mg (range = 1.4-50.6), for benzoylecgonine 6.6 ng/mg (range = 0.4-14.9) and for ecgonine methyl ester 4.0 ng/mg (traces to 12.9). In two thirds of the mine workers the concentration of cocaine in the different segments was nearly identical. In most cases the BZE and EME in each subject were almost the same, differing only slightly between the segments (Figure 4). Subject no. 18 showed the highest intraindividual variation for cocaine, BZE, and EME in difSO bil;b SO

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ferent segments. The mean quotient of cocaine to its main metabolite BZE was 3 (range = 1.3-4.7), with one exception (subject no. 1). This was the youngest subject, who also reported the shortest time of coca chewing. In his hair sample the ratio of cocaine to BZE was 0.7. He also had the lowest quotient of cocaine/EME. These variations may be caused by genetic differences (12). All the subjects were identical in race, sex, and hair color. We also assume that their way of life was rather similar, including food intake, exposure of hair to light, dust, and environmental contaminations, as well as their hair washing procedures. Unfortunately, we do not have detailed information about these subjects, so we do not know whether these variations contrary to the assumptions above, are the result of different living habits, environmental conditions, irregular intake of coca leaves, different body metabolism (12), disease, or hair treatment. However, in most subjects tested, there was a constant individual rate of metabolism of cocaine to BZE and EME (Figure 5). The GC/MS detection of cocaine in hair of a drug user was first described in 1987 by Balabanova and Homoki (1). However, quantitation was made by RIA, measuring the amount of cocaine and its metabolites. Brunner et al. (2) reported the detection of BZE in the hair of cocaine abusers. The semiquantitative determination after derivatization gave a value of 9 ng/mg. Reuschel and Smith (5) tested the hair of 48 jail detainees for cocaine by RIA and confirmed the positive samples by GC/MS. They found between 0.3 and 18 ng/mg of cocaine in the hair samples. Kidwell (4) used pyrolysis CI/MS/MS performed on single hairs to detect cocaine and its metabolites, BZE and EME. Regardless of the route of application, in all cases cocaine exceeded its metabo-

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295

Journalof AnalyticalToxicology,Vol.16, September/October1992

lites in concentration. Martz compared the GC/MS and the CI/MS/MS techniques for the detection of cocaine and BZE in hair samples. He demonstrated the superiority of CI/MS/MS in sensitivity. However, he concluded that GC/MS using deuterated internal standards is the preferred method of analysis for identification and quantitation of cocaine. Cone et al. (8) described the simultaneous detection of cocaine, BZE, and EME in hair of cocaine users in an outpatient treatment facility. They found higher values and also an average of 5-10 for the quotient of cocaine/ BZE, compared to 1.3-4.7 in this study, although no differences in bioavailability were reported between intranasal and oral administration (13). Perhaps the higher ratio found by Cone et al. may be attributed more to the use of purified cocaine than to the route of administration. In conclusion, enteral or parenteral cocaine consumption leads to the excretion of cocaine, BZE, and EME into hair. Oral consumption of coca leaves leads to cocaine concentrations that exceed the amounts of the metabolites by 1-5 times. Further investigations are necessary to examine the interindividual differences in concentration of cocaine and its metabolites due to race, sex, amount and method of consumption, metabolism, and hair treatment.

References 1. S. Balabanova and J. Homoki. Determination of cocaine in human hair by gas chromatography/mass spectrometry. Z. Rechtsmed. 98:235-40 (1987). 2. H. Brunner, S. Balabanova, J. Homoki, and H.U. Wolf. Bestimmung von Benzoylecgonin im menschlichen Kopfhaar nach Kokain-Abusus durch Gaschromatographie-Massenspektrometrie. Beitr. GerichtL Med. 46:127-34 (1988). 3. R.M. Martz. The identification of cocaine in hair by GC/MS and MS/MS. Crime Lab Digest 15:67-73 (1988)

296

4. D.A. Kidwell. Analysis of drugs of abuse in hair by tandem mass spectrometry, 36th American Society of Mass Spectrometry and Allied Topics, San Francisco (1989). 5. S.A. Reuschel and F.M. Smith. Benzoylecgonine (cocaine metabolite) detection in hair samples of jail detainees using radioimmunoassay (RIA) and gas chromatography/mass spectrometry (GC/MS). J. Forens. ScL 36:1179-85 (1991). 6. 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. ToxicoL 15:260-265 (1991) 7. E.J. Cone. Testing human hair for drugs of abuse. I. Individual dose and time profiles of morphine and codeine in plasma, saliva, urine, and beard compared to drug-induced effects on pupils and behavior. J. Anal ToxicoL 14:1-7 (1990). 8. 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 evaluation of decontamination procedures. J. Anal ToxicoL 15:250-55 (1991). 9, R. Martz, B. Donnelley, D. Fetterolf, L. Lasswell, G. 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 15:279-81 (1991). 10. M.R. MSIler and P. Fey. Detection of cocaine, benzoylecgonine and methlecgonine in human hair by gas chromatography/mass spectrometry. Naunyn-Schmiedeberg's Arch. Pharmacol. Supplement 2 to Vol. 344, R 120 (1991). 11. MR. MSIler.Drug detection in hair by chromatographicprocedures. Review. J. Chromatogr., 580:(1 + 2): 125-34 (1992) 12. H.K. Watanabe, B. Hoskins, and I.K. Ho. Effects of subacute treatment with cocaine on activities of N-demethylase, UDP-glucuronyltransferase and sulfotransferase in WKY and SHR rat liver-sex and strain differences. Life-Sci. 42:79-86 (1988). 13. P. Wilkinson, C. Van Dyke, P. Jatlow, P. Barash, and R. Byck. Intranasal and oral cocaine kinetics. C/in. PharmacoL Ther. 27: 386-94 (1980). Manuscript received February 15, 1992; revision received June 28, 1992.