Liquid chromatographic analysis of cocaine and benzoylecgonine in plasma of traditional coca chewers from Bolivia during exercise

Journal of Ethnopharmacology 56 (1997) 173 – 178

Liquid chromatographic analysis of cocaine and benzoylecgonine in plasma of traditional coca chewers from Bolivia during exercise C. Rerat a, M. Sauvain b,*, P.P. Rop c, E. Ruiz a, M. Bresson c, A. Viala c b

a Instituto Boli6iano de Biologı´a de Altura, Uni6ersidad Mayor de San Andres, CP 717 La Paz, Boli6ia Institut Franc¸ais de Recherche Scientifique pour le De6eloppement en Coope´ration (ORSTOM), Departement Sante, 213 rue La Fayette, F-75480 Paris Cedex 10, France c Laboratoire de Police Scientifique, 97 boule6ard Camille Flammarion, 13248 Marseille cedex 04, France

Received 3 July 1996; accepted 28 August 1996

Abstract The purpose of the present study was to determine the amount of cocaine and benzoylecgonine in the plasma of Aymara Indians from the Bolivian Andes after traditional chewing of coca leaves during exercise performance. The determination was carried out by high performance liquid chromatography after solid-liquid extraction. The results showed that such use of coca leaves is well correlated with pharmacologically active concentration of cocaine in plasma. © 1997 Elsevier Science Ireland Ltd. Keywords: Coca chewing; Cocaine; Benzoylecgonine; Plasma concentrations; HPLC; Aymara Indians

1. Introduction Erythroxylum coca Lamarck var. coca (Erythroxylaceae) is a shrub found in Bolivia and Peru. It represents one of the four varieties of coca widely grown in the Andes (Plowman, 1984). In Bolivia, the production is partly used (10 000 metric tons per year) on site, especially by means of chewing (Carter and Mamani, 1986). The * Corresponding author.

purpose of the present study was to determine the plasma levels of cocaine and benzoylecgonine in Aymara Indians of the Bolivian Andes after this traditional use of coca and link this with the physiological parameters of the natives (blood glucose, free fatty acid, norepinephrine and insulin concentrations). Those parameters were obtained in previous studies performed to assess the effects of coca chewing on oxygen uptake and metabolic responses during graded exercise (Spielvogel et al., 1996, Favier et al., 1996).

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2. Materials and methods (Rop et al., 1993)

2.1. Reagents and glassware All reagents were of analytical grade. Sodium carbonate, sodium bicarbonate and isopropanol (Normapur) were purchased from Prolabo (Paris, France). Chloroforme (RPE, reagente puro erba), diethylether (RPE), acetonitrile (RS, reagente speciale, for HPLC), methanol (RS, for HPLC) and hydrochloric acid (RPE) were purchased from Carlo Erba (Milano, Italy). Ammonium acetate and 3 ml Extrelut® pre-packed columns were from Merck (Nogent sur Marne, France). All glassware was washed with a 3% ‘RBS 25’ biodegradable alkaline solution obtained from Serflam (Marseille, France) which contains a mixture of anionic and non-ionic detergents, and then rinsed successively with distilled water, acetone and extraction solvent.

2.2. Drugs and internal standard Cocaine hydrochloride (Cooper, France), benzoylecgonine tetrahydrate (Sigma) and bupivicaine hydrochloride (Roger Bellon, France) as internal standard were kindly supplied by the respective manufacturers. Stock solutions were made in methanol at a concentration of 1 mg/ml and stored at − 20°C. They were stable for at least 1 month. They were diluted to 500, 200, 100 and 50 ng/ml in the same solvent before use.

2.3. Apparatus and chromatographic parameters The chromatographic analysis was performed on a component ‘Waters’ system consisting of two M510 pumps, a WISP 712 automatic sample injection module, a micro Bondapack C18 column (30 cm× 3.9 mm I.D.; particle size 10 mm, ambient temperature) connected to a micro Bondapak C18 T.M. guard pak column (5 mm ×6 mm I.D.; two filters, one at each end, held the packing in place and provided a 2 mm filtering capability), and a UV-VIS M990 photodiode-array detector (which permits scanning of chromatographic and spectral data) coupled with an 840 control station.

The mobile phase was a mixture of two liquids distributed by the (A) pump: 0.1 M ammonium acetate, and the (B) pump: acetonitrile + methanol (50:50, v/v) with an ultimate phase of 0.1 M ammonium acetate/acetonitrile-methanol mixture (40:60, v/v) at a flow rate of 1 ml/min. The UV detection was monitored at a wavelength of 230 nm. The UV spectrum of each compound corresponding to a peak was established between 200 and 350 nm.

2.4. Plant material The description of the specimens used in this study was given in a previous study (Sauvain et al., 1996). The coca leaves used for the traditional chewing were grown on the eastern slopes of the Andes (Yungas) and were bought to the La Paz market at the dry season. The mean concentration of cocaine, was 0.60/100 g of dry leaves.

2.5. Extraction procedure The plasma sample (2 ml) was added to 2 ml of bidistilled water, 1 ml of a sodium carbonate/bicarbonate saturated solution and a known amount (1 mg) of the internal standard. After vortex-mixing for 1 min, the mixture was passed through a 3 ml Extrelut® cartridge. Elution was carried out with 15 ml of a mixture of chloroform/isopropanol (90:10, v/v). The eluate was collected in a 20 ml evaporation glass tube and evaporated to dryness under a stream of nitrogen in a 40°C water bath. The residue was dissolved (by vortex-mixing) in 100 ml of 0.01 M hydrochloric acid. The acid extract obtained was washed with 3 ml of diethylether for 20 s on a whirlmixer and centrifuged for 5 min at 2800×g. The ether layer was discarded. Finally 25 ml of the acid solution was injected into the chromatograph.

2.6. Calculation The ratios between the peak areas of the analyzed drugs and the internal standard were calculated and plotted against the concentrations of the tested drugs added to blank samples at increasing concentrations of each drug (50, 100, 200 and 500

C. Rerat et al. / Journal of Ethnopharmacology 56 (1997) 173–178

ng/ml) with a constant amount of the internal standard (Fig. 1). Within these concentrations ranges the relations were linear for the two compounds. The recovery was ]70% and the linear regression curves for cocaine were y= 0.0020x+ 0.049 and for benzoylecgonine y = 0.0038x+ 0.045.

2.7. Clinical protocol Twenty-five male volunteers, high altitude natives from the Altiplano (Bolivia; 3800 m altitude) were studied. They had documented histories of frequent coca chewing during the previous years. Each subject has chewed the quantity habitually. Blood samples (10 ml) were first drawn at the end of 45 min chewing, and secondly after 20 min exercise (without chewing). The samples were collected into tubes containing 80 ml of 125 mg/ml NaF solution to prevent hydrolysis (Barnett et al., 1981). In the exercise, each subject had to perform an incremental energetic test on a bicycle ergometer (Spielvogel et al., 1996). After immediate centrifugation, the plasma samples were transferred into separate tubes and then frozen at − 80°C until analysis. The subjects included in the study has chewed leaves for 2 to 29 years (with an average of 8.6 years). The mode of chewing of Bolivian peasants is similar to that of the other Andean natives from Peru or Ecuador: coca leaves are placed together, with an alkaline material called llipta, in the mouth and are ‘chewed’ (rolled around with the tongue and wetted) into a quid which is held in the cheek and periodically chewed again (Holmstedt et al., 1979).

3. Results Two experiments were carried out (Table 1). Firstly, the quantity of chewed leaves was very distinct for every subject, from 3 to 77 g with an average of 319 21 g for the 17 subjects (age: 389 7 years). Plasma concentrations of cocaine after the chewing and before the exercise were between 28 and 289 ng/ml with an average of 989 75 ng/ml; after the exercise the the levels were from 30 to 211 ng/ml with an average of

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869 54 ng/ml. Plasma concentrations of benzoylecgonine were higher than those of cocaine: 2199 197 ng/ml after chewing and before the exercise and 4149 234 ng/ml after the exercise. In both cases cocaine and benzoylecgonine plasma levels were not correlated with the quantity of coca leaves chewed (P\ 0.05). The increase in benzoylecgonine concentration between the two measurement times was significant (P= 0.0002). In the second experiment which included eight subjects (age: 339 10 years), the quantities of coca leaves chewed were more regular: 129 3 g. More regular plasma concentrations of cocaine were therefore obtained: 89 9 25 ng/ml after chewing and before bioenergetic exercise; 80 9 22 ng/ml after exercise. The concentrations of benzoylecgonine were 2789 151 ng/ml after chewing and 3489 117 ng/ml after exercise. In addition, in this case plasma concentrations of cocaine and benzoylecgonine were not correlated with the quantity of coca leaves chewed (P\ 0.05): the increase in benzoylecgonine concentration between the two measurement times was also significant (0.010B PB 0.005).

4. Discussion Paly et al. (1980) found significant amounts of cocaine in the plasma samples of native chewers from Peru: between 45 and 859 ng/ml depending on the chewing mode. The authors concluded that chewing coca seems to be as effective as oral absorption of medium doses of cocaine (2 mg/kg). According to another study, including six subjects who were not habitual chewers (Holmstedt et al., 1979), the maximum concentration reached was 149 ng/ml after 1 h chewing. Our work showed that the traditional use of coca leaves, especially chewing, was well correlated with pharmacologically active concentrations of cocaine in plasma. However, levels were found to be lower than those (120–474 ng/ml) obtained by intranasal cocaine introduction (1.5 mg/kg) (Fleming et al., 1990). In fact, the corresponding doses of cocaine for chewing must be considered to be low, when they are compared with the quantities used in intravenous injection

Fig. 1. Chromatograms and UV Spectra of blank plasma (2 ml) after solid-liquid extraction of Cocaine, Benzoylecgonine and internal standard (Bupivacaine) at 500 ng/ml.

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Age

43 23 24 46 35 33 37 48 37 44 39 33 46 37 35 37 45 389 7 41 20 43 25 22 37 45 31 339 10

Subject no.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Average 18 19 20 21 22 23 24 25 Average

59 62 59 62 56 65 71 57 52 62 60 55 51 51 52 51 44 57 97 70 55 71 59 54 57 51 64 609 7

Weight (kg)

27 9 15 35 61 14 21 20 77 42 33 41 57 3 9 20 51 31 9 21 11 8 14 10 17 11 10 12 12 93

ND 28 49 218 119 20 109 60 72 84 49 77 91 32 76 289 196 98 975 90 56 96 89 131 93 101 53 89 925

Cocaine (ng/ml) 210 173 157 667 431 165 65 69 35 85 69 205 520 35 98 193 546 219 9 197 206 184 186 277 606 154 387 223 278 9151

Benzoylecgonine (ng/ml)

Dose (g) of chewed coca Plasma concentration before exercise

Table 1 Amounts of cocaine and benzoylecgonine in the plasma of 25 coca chewers

41 43 129 84 79 ND 91 36 77 40 211 106 51 42 30 129 180 869 54 87 47 59 82 110 94 97 62 809 22

Cocaine (ng/ml)

649 288 615 626 758 121 313 136 223 162 311 679 797 216 193 447 510 4149 234 402 249 160 439 471 272 478 309 348 9 117

Benzoylecgonine (ng/ml)

Plasma concentration after exercise

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(Barnett et al., 1981). Indeed, a 100 mg dose taken intravenously resulted in a maximum plasma concentration at of about 1000 ng/ml of cocaine 5 min after the injection. The 200 mg dose gave a maximum value of 4000 ng ml. Those doses in i.v. use are 10–40 times higher than those absorbed by the traditional chewing. The significant amount of cocaine in the blood of chewers can be correlated with the results of one of our previous studies (Favier et al., 1996). This provides experimental evidence of the physiological effects of coca chewing (higher levels of free fatty acid, plasma epinephrine and blood glucose and a reduction in insulin plasma level), that could explain the better ability of coca users to sustain strenuous work for an extended period of time. The large concentration of benzoylecgonine in the plasma of chewers must be also emphasised. It appears to be related to the hydrolysis of a part of the cocaine in the mouth because of the high pH ( \10) produced by the use of alkaline substances in the practice of chewing. A previous study (Rivier, 1981) showed that 10% of cocaine was hydrolyzed to benzoylecgonine at this pH. Another explanation may be that the principal metabolite of cocaine in the human body is benzoylecgonine and that the half-life time of cocaine is short (1 h). The elimination of benzoylecgonine goes on for some hours, this compound may therefore be considered as a metabolite of accumulation (Tebbet and McCartney, 1988, Apple and Roe, 1990). This would explain the increase in its plasma concentration after exercise. Novak et al. (1984) have discussed the possible pharmacological activity of benzoylecgonine. The results of our study may suggest that benzoylecgonine plays a role in the use of the coca chewing (Cabieses, 1992).

Acknowledgements We express our profound gratitude to the subjects without whose dedication, cooperation, and spirit this work could not have been completed. This study was supported by a grant from Le Ministe`re des Affaires Etrangeres (France). .

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