Journal of Ethnopharmacology 103 (2006) 439–447 Cocaine distribution in wild Erythroxylum species Stefan Bieri a , Anne Brachet b , Jean-Luc Veuthey a , Philippe Christen a,∗ a Laboratory of Pharmaceutical Analytical Chemistry, School of Pharmaceutical Sciences EPGL, University of Geneva, 20 Bd d’Yvoy, 1211 Geneva 4, Switzerland b Battelle, Agrochemical Product Development, 7 Route de Drize, 1227 Carouge-Geneva, Switzerland Received 30 May 2005; received in revised form 10 August 2005; accepted 16 August 2005 Available online 30 September 2005 Abstract Cocaine distribution was studied in leaves of wild Erythroxylum species originating from Bolivia, Brazil, Ecuador, Paraguay, Peru, Mexico, USA, Venezuela and Mauritius. Among 51 species, 28 had never been phytochemically investigated before. Cocaine was efficiently and rapidly extracted with methanol, using focused microwaves at atmospheric pressure, and analysed without any further purification by capillary gas chromatography coupled to mass spectrometry. Cocaine was reported for the first time in 14 species. Erythroxylum laetevirens was the wild species with the highest cocaine content. Its qualitative chromatographic profile also revealed other characteristic tropane alkaloids. Finally, its cocaine content was compared to those of two cultivated coca plants as well as with a coca tea bag sample. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Erythroxylaceae; Erythroxylum; Erythroxylum laetevirens; Cocaine; Tropane alkaloids; Gas chromatography; Mass spectrometry; Focused microwaveassisted extraction 1. Introduction The last 30 years have seen an increasing interest in cocaine analysis resulting from its expanding illicit use in Western Europe and North America. Regardless of the importance of cultivated coca plants from an economical point of view, these species always played a key role for South American natives (Grinspoon and Bakalar, 1981; Naranjo, 1981; Schultes, 1981; Plowman, 1984a). Coca chewing in South America has persisted from ancient times, but is still poorly understood from many points of view. This traditional habit is largely considered noxious by many regulatory authorities. The family Erythroxylaceae is composed of four genera: Aneulophus, Erythroxylum, Nectaropetalum and Pinacopodium (Hegnauer, 1981). The genus Erythroxylum, by far the most wellknown genus of the family comprises roughly 230 species of tropical trees and shrubs, which are widely distributed in South America, Africa and Madagascar (Plowman and Hensold, 2004). In 1907, Schulz divided this genus into 19 sections, providing a useful scheme for comparative phytochemical considerations. ∗ Corresponding author. Fax: +41 22 379 33 99. E-mail address: philippe.christen@pharm.unige.ch (P. Christen). 0378-8741/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2005.08.021 Erythroxylum and more particularly Erythroxylum coca and Erythroxylum novogranatense, as well as their varieties, is the only natural source of cocaine (Plowman, 1984b). Even if some attention has been focused on non-cultivated Erythroxylum species for the possible presence of cocaine, systematic investigation of the genus is still incomplete and several species used in traditional medicine remain unknown (Evans, 1981). Aynilian et al. (1974) reported the concentration of cocaine in herbarium specimens of seven Erythroxylum species. Holmstedt et al. (1977) analysed 62 samples of 13 tropical South American species by capillary gas chromatography coupled to mass spectrometry (GC-MS). Cocaine was found only in the leaves of two species, Erythroxylum coca and Erythroxylum novogranatense, but no measurable amount of cocaine was detected in any of the other 11 species. Subsequently, Plowman and Rivier (1983), using more sensitive assays, detected trace amounts of cocaine in 13 neotropical wild Erythroxylum species representing five sections of the genus. Besides, they found that two species from Venezuela, namely Erythroxylum recurrens and Erythroxylum steyermarkii, contained cocaine amounts comparable to those found in cultivated species. This study is part of a large investigation of the genus Erythroxylum for tropane and related alkaloids (Brachet et al., 1997, 2002; Christen et al., 1993, 1995; Brock et al., 2005). We report 440 S. Bieri et al. / Journal of Ethnopharmacology 103 (2006) 439–447 here on the specific investigation of cocaine in 51 wild species. Due to the presence of an appreciable amount of cocaine, Erythroxylum laetevirens was qualitatively investigated for the presence of other tropane alkaloids and its chromatographic profile was compared to those of two cultivated species, together with a coca tea bag sample. 2. Materials and methods 2.1. Plant material and chemicals Most species were collected in South America between 1979 and 1984 by the late T. Plowman and kindly provided by Dr. Laurent Rivier (Lausanne, Switzerland). Collection details are given in Table 1. Two species originating from sites other than South America were also included in this study, namely Erythroxylum areolatum from the Bahamas (USA) and Erythroxylum macrocarpum from Mauritius. A voucher specimen of all plants is deposited at our Institute. For each species, only the leaves were analysed. Dry plant material was ground to a fine homogenous powder by a ballmill (MM 200 RETSCH, Switzerland) and finally sieved to an average particle size of less than 125 ␮m. Cocaine hydrochloride (COC) and Methadone hydrochloride (MET) were obtained from Siegfried Handel (Zofingen, Switzerland) and Hänsler (Herisau, Switzerland), respectively. 2.2. Extraction procedure Extractions were performed using focused microwaves at atmospheric pressure at a frequency of 2450 MHz using a Soxwave 3.6 apparatus (Prolabo, France) with a programmable heating power. Typically, 100 mg of powdered plant material was placed into a 20 mL quartz extraction vessel and hydrated with 10 ␮L of water prior to the addition of 5 mL methanol. The extraction was carried out at 125 W for 30 s. Each extract solution was filtered on a 0.45 ␮m PTFE filter (Brachet et al., 2002). Solutions obtained from wild species were evaporated to dryness and taken up in 1 mL methanol containing 10 ppm internal standard (methadone), while solutions from cultivated species were diluted four times with methanol. All samples were analysed by GC-MS without any further purification. 2.3. Gas chromatography GC-MS analyses were carried out using a Hewlett-Packard 5890 series II chromatograph coupled to a HP 5972 mass selective detector (Agilent Technologies, Palo Alto, CA, USA). The mass detector operated in the electron impact ionisation mode at 70 eV. Injections were performed in the splitless mode at 250 ◦ C with a splitless period of 60 s and with purge and septum purge flow rates of 30 and 3 mL/min, respectively. Injections of 1 ␮L were carried out with a HP 6890 series fast automatic liquid sampler (Agilent Technologies). A laminar liner (Restek, Bellefonte, PA, USA) was used as well as a standard syringe with a 42 mm long needle and a cone tip. Helium was used as carrier gas and operated in the constant flow mode (1 mL/min). For qualitative analysis, a HP5-MS column, 30 m × 0.25 mm i.d. × 0.25 ␮m film thickness was used with an initial oven temperature of 70 ◦ C (1 min hold) and a linear temperature program from 70 to 285 ◦ C at 5 ◦ C/min and hold at the final temperature for 15 min. Spectra were recorded in the mass range 30–500 Th with 1.3 scan/s and the MS transfer line was set at 280 ◦ C. For quantitative cocaine analysis, the oven was initially set at 70 ◦ C (1 min hold) and linearly increased to 285 ◦ C (5 min hold) at 30 ◦ C/min. GCMS (SIM mode) was performed using the selective ion 303 Th (molecular ion of cocaine), the qualifier ion 272 Th and the target ion 182 Th (base peak of cocaine). Methadone (MET) was used as internal standard with target ion 294 Th (molecular ion) and qualifier ion 72 Th (base peak of methadone). In order to enhance sensitivity, the potential of the electron multiplier was increased by a 400 V increment for a period of time of 2 min which included elution of the internal standard and cocaine. 2.4. Quantification Standard calibration curve was obtained with cocaine solutions at seven concentration levels between 0.1 and 100 ppm (0.1, 0.5, 1, 5, 25, 50 and 100 ppm) containing a fixed concentration (10 ppm) of methadone. Quantitative determination was based on the peak area ratio of the target ions of cocaine over methadone. A correlation coefficient of 0.9992 was obtained. The relative standard deviations (R.S.D.) for six consecutive injections with a cocaine standard solution at 5 ppm was inferior to 5%, and inferior to 10% at 0.1 ppm, corresponding to the limit of quantification (LOQ). For any concentration level, the three cocaine ions were detected at the corresponding elution time (between 9.55 and 9.57 min). Cocaine was considered to be present in a species, but not quantified (NQ) when ions 182 and 303 Th were detected with a signal to noise ratio of at least 2. When the target ion 182 Th was not detected, the symbol ND was used, meaning that no cocaine was present. 3. Results and discussion Before any discussion of the results, it is important to emphasize that the time elapsed between plant harvesting and analysis is between 20 and 25 years. Since it is believed that a cocaine leaf content may vary with time, the quantitative results reported should be viewed from that perspective despite the fact that the preservation of cocaine in Erythroxylum coca leaves has been shown in 44 year-old herbarium samples (Aynilian et al., 1974). A straightforward sample preparation method involving focused microwave-assisted extraction (FMAE) was used as already described by Brachet et al. (2002). This procedure was particularly well suited for mass limited samples, as it required no more than 100 mg of fine powdered plant material. Indeed, sample amounts of the various examined species at our disposal varied between a few hundred milligrams and a hundred and fifty grams. Furthermore, this method was extremely rapid (30 s), required low amount of organic solvent (5 mL) and thus allowed the extraction of numerous samples in a short period of time. In addition, it is environmentally friendly and does not necessi- Table 1 Cocaine content in wild Erythroxylum species Sections and species (Schulz, 1907) Macrocalyx (Sect. II) Erythroxylum macrophyllum Cav. (syn. Erythroxylum lucidum H.B.K.) Erythroxylum macrophyllum Cav. Erythroxylum suberosum A. St. Hil. Erythroxylum campestre A. St. Hil. Erythroxylum citrifolium A. St. Hil. Erythroxylum deciduum A. St. Hil. Erythroxylum fimbriatum Peyr. Erythroxylum laetevirens O.E. Schulz Erythroxylum mikanii Peyr. Erythroxylum mucronatum Benth. Erythroxylum myrsinites Mart. Erythroxylum passerinum Mart. Erythroxylum polygonoides Mart. Erythroxylum rufum Cav. Leptogramme (Sect. IV) Erythroxylum ovalifolium Peyr. Erythroxylum pulchrum A. St. Hil. Erythroxylum ulei O.E. Schulz Heterogyne (Sect. V) Erythroxylum areolatum L. Field museum accession number Cocaine content Literature (% m/m) Mexico. Selva alta Perennifolia. 18◦ 18′ N, 94◦ 47′ W. Alt. 300 m Coll. M. Nee, G. Diggs, F. Ramirez R.; Jul. 1982, No 25112 Coll. T. Plowman, H. Kennedy, No 5804 Coll. Hahn; Jan. 1983, No 1787 Brazil. State of Goiás Coll. M.J. Balick et al.; Nov. 1981, No 1308 F1921436 + 0.0003 (Holmstedt et al., 1977) F1763768 F1948034 F1945882 ND ND ND 0 (Holmstedt et al., 1977, Plowman and Rivier, 1983) 0 (Hegnauer, 1966) 0 (Hegnauer, 1966) F1931666 NQ 0 (Holmstedt et al., 1977) F1948025 + 0.00014 (Aynilian et al., 1974) F1931672 NQ 0 (Holmstedt et al., 1977) F1954563 + 0.0008 (Aynilian et al., 1974) F1899111 ++ ++++ 0–0.0011 (Holmstedt et al., 1977) – F1944044 ND – F1898255 ND 0 (Holmstedt et al., 1977) F1954578 ND – F1947948 ND – F1916635 ND – F1934034 ND 0 (Holmstedt et al., 1977) F1916623 ND – Venezuela. Edo. Miranda. Baruta. Alt. 1200 m Coll. T. Plowman; Feb. 1979, No 13413 Paraguay Coll. Hahn, No 1768 Venezuela. Estado Falcón. 11◦ 11′ N, 69◦ 41′ W. Alt. 1100–1200 m Coll. T. Plowman, P.E. Berry, R. Wingfield; March 1984, No 13424 Brazilia. Municı́pio de Curitiba Coll. T. Plowman, P.E. Berry, F. Juarez; Jan. 1985, No 4457 Coll. T. Plowman, No 11400 Venezuela. Edo. Sucre. Mochima. Alt. 50 m Coll. T. Plowman; March 1979, No 7800 Brazil. Municı́pio de Ilhéus Coll. T. Plowman, L.A.M. Silva, T.S. dos Santos; Feb. 1985, No 13959 Brazil. Municı́pio de Ilhéus Coll. T. Plowman, No 11375 Brazil. Municı́pio de Balsa. Alt. 885 m Coll. T. Plowman, P.E. Berry; Jan. 1985, No 4490 Brazil. Municı́pio de Cabo Frio. Sea level Coll. T. Plowman, D. Araujo; Feb. 1985, No 13936 Brazil. Estado da Bahia. Municı́pio de Maracás. 13◦ 28′ S, 40◦ 29′ W. Alt. 850–900 m Coll. T. Plowman, A.M. de Carvalho; Feb. 1983, No 12820 Venezuela. Ter. Fed. Amazonas. Depto Átures Coll. T. Plowman, F. Guánchez; April 1984, No 13765 Brazil. Municı́pio de Maricá. 22◦ 58′ S, 42◦ 54′ W. Sea level Coll. T. Plowman; Feb. 1983, No 12840 Brazil. Municı́pio de Cabo Frio. Sea level Coll. T. Plowman, D. Araujo; Feb. 1985, No 13940 Coll. T. Plowman, M. Ramirez, P.M. Rury, No 11419 F1947953 + 0.00008–0.0004 (Holmstedt et al., 1977, Aynilian et al., 1974) F1896991 ND 0 (Holmstedt et al., 1977, Plowman and Rivier, 1983) USA. Chicago. Field Museum of Natural History F1979181 ++ 0.0014 (Holmstedt et al., 1977) S. Bieri et al. / Journal of Ethnopharmacology 103 (2006) 439–447 Rhabdophyllum (Sect. III) Erythroxylum amazonicum Peyr. Collection site and collector number 441 Sections and species (Schulz, 1907) Collection site and collector number Field museum accession number Cocaine content Literature (% m/m) F1929656 + 0 (Zuanazzi et al., 2001) NQ 0 (Holmstedt et al., 1977) F1930165 NQ 0 (Holmstedt et al., 1977, Plowman and Rivier, 1983) F1916625 + – F1947952 ND – F1932110 + 0.0015 (Holmstedt et al., 1977, Plowman and Rivier, 1983) F1859518 ND 0 (Plowman and Rivier, 1983) F1973368 ++ 0.007 (Plowman and Rivier, 1983) F1944049 ND – F1934045 ND 0 (Plowman and Rivier, 1983) F1916659 ND – F1921739 ND – F1962321 ND – F1824664 F1947945 + + 0.0004–0.0005 (Holmstedt et al., 1977) – F1852976 ND 0 (Holmstedt et al., 1977) Paraguay, Coll. Hahn, No 1782 F1948031 NQ – Brazil. Estado da Bahia. 13◦ 40′ S, 39◦ 07′ W Coll. T. Plowman, A.M. de Carvalho; Feb. 1983, No 12810 Venezuela. Ter. Fed. Amazonas. Puerto Ayacucho. Alt. 85 m Coll. T. Plowman; Feb. 1979, No 7725 Brazil. Municı́pio de Macaé. Alt. 1150–1250 m Coll. T. Plowman, C. Farney, G. Martinelli, S. Pessoa, A. Costa; Nov. 1985, No 585 F1916662 ND – NQ – NQ – 442 Table 1 (Continued ) Coll. T. Plowman; Dec. 1986, No 14431 Archerythroxylum (Sect. VI) Erythroxylum argentinum O.E. Schulz Erythroxylum cumanense H.B.K. Erythroxylum densum Rusby Erythroxylum frangulifolium A. St. Hil. Erythroxylum gracilipes Peyr. Erythroxylum havanense Jacq. Erythroxylum aff. impressum O.E. Schulz Erythroxylum ochranthum Mart. Erythroxylum orinocense Kunth Erythroxylum martii Peyr. Erythroxylum mexicanum H.B.K. Erythroxylum roraimae Klotsch ex O.E. Schulz Erythroxylum shatona Macbride Erythroxylum subrotundum A. St. Hil. Erythroxylum undulatum Plowman Microphyllum (Sect. IX) Erythroxylum cuneifolium (Mart) O.E. Schulz Erythroxylum cuspidifolium Mart. Erythroxylum divaricatum Peyr. Erythroxylum gonocladum (Mart) O.E. Schulz Coll. S. Mori, B. Rabelo, R. Cardoso; Dec. 1984, No 17485 Coll. T. Plowman, No 6046 Brazil. Municı́pio de Cabo Frio. Armação de Búzios Coll. T. Plowman, D. Araujo; Feb. 1985, No 13942 Venezuela. Dist. Fed. Caracas. Alt. 900–950 m Coll. T. Plowman; Feb. 1979, No 7685 F1950280 S. Bieri et al. / Journal of Ethnopharmacology 103 (2006) 439–447 Erythroxylum glazioui O.E. Schulz Bolivia. Depto. Tarija. Prov. 21◦ 25′ S, 64◦ 16′ W. Alt. 1700 m Coll. J.C. Solomon; May 1983, No 10404 Venezuela. Dtto Federal. Alt. 900–950 m Coll. P.E. Berry; Feb. 1984, No 4297 Venezuela. Dtto. Federal. Alt. 900–950 m Coll. P.E. Berry; Feb. 1984, No 4298 Brazil. Municı́pio de Maricá 22◦ 58′ S, 42◦ 54′ W. Sea level Coll. T. Plowman; Feb. 1983, No 12860 Brazil. Municı́pio de Cabo Frio. Sea level Coll. T. Plowman, D. Araujo; Feb. 1985, No 13939 Venezuela. Dist. Fed. Los Caracas Coll. T. Plowman, P.E. Berry; March 1984, No 13355 Venezuela. Dtto Fed. Alt. 900–950 m Coll. P.E. Berry; 1979, No 3494 Peru. Depto Cajamarca. Prov. Jaén. Alt. 520 m Coll. T. Plowman; July 1986, No 14253 Brazilia. Municı́pio de Ilhéus Coll. T. Plowman, L.A.M. Silva, T.S. dos Santos; Feb. 1985, No 13968 Venezuela. Ter. Fed. Amazonas. Depto Átures. Savanna Coll. T. Plowman, F. Guánchez; April 1984, No 13755 Brazil. Estado da Bahia. Municı́pio de Ituberá. 13◦ 40′ S, 39◦ 07′ W Coll. T. Plowman, A.M. de Carvalho; Feb. 1983, No 12805 Mexico. Municı́pio San Andrés Tuxtla. 18◦ 27′ 30′′ N, 95◦ 11′ 15′′ W. Alt. 300 m Coll. M. Nee, G. Diggs, G. Schatz; July 1982, No 24756 Brazil. Amapà. Municı́pio de Mazagão. 0◦ 10′ N, 51◦ 37′ W Pachylobus (Sect. XVII) Erythroxylum macrocarpum O.E. Schulz Non classified species Erythroxylum andrei Plowman Erythroxylum ligustrinum var. ligustrinum D.C. (Erythroxylum aturense Plowman sp. nov.ined.) Erythroxylum confusum Britton Erythroxylum hypoleucum Plowman Erythroxylum leal-costae Plowman Erythroxylum mattossilvae Plowman Erythroxylum foetidum Plowman (Erythroxylum nervosum Plowman sp. nov. ined.) Erythroxylum ruizii Peyr. Erythroxylum macrophyllum Cav. var. savannarum Plowman (Erythroxylum savannarum Plowman) Erythroxylum splendidum Plowman Brazil. Estado da Bahia. 14◦ 04′ S, 38◦ 58′ W. Sea level Coll. T. Plowman, A.M. de Carvalho; Feb. 1983, No 12785 Venezuela. Ter. Fed. Amazonas. Depto Átures. 5◦ 22′ N, 67◦ 33′ W Coll. T. Plowman; April 1984, No 13773 Brazil. Municı́pio de Santa Maria Madalena 21◦ 56′ S, 41◦ 52′ W. Alt. 450 m Coll. T. Plowman, H.C. de Lima; Feb. 1983, No 12950 Mexico. Quintana Roo Coll. T. Plowman; May 1982, No 20642 Venezuela. Ter. Fed. Amazonas. Depto Rio Negro Coll. T. Plowman; April 1984, No 13700 Brazil. Estado da Bahia. Salvador. 12◦ 55′ S, 38◦ 21′ W. Sea level Coll. T. Plowman; Jan. 1983, No 12770 Brazil. Municı́pio de Ilhéus Coll. T. Plowman, L.A.M. Silva, T.S. dos Santos; Feb. 1985, No 13970 Venezuela. Ter. Fed. Amazonas. Depto Átures. Puerto Ayacucho Coll. T. Plowman; April 1984, No 13731 Ecuador. Prov. Manabı́. Cerro Montecristi. Alt. 300–400 m Coll. T. Plowman, P. Alcorn; July 1986, No 14340 Venezuela. Ter. Fed. Amazonas. Depto Átures Coll. T. Plowman, F. Guánchez; April 1984, No 13757 Brazil. Municı́pio de Valença. Guaibim. 13◦ 18′ S, 39◦ 00′ W. Sea level Coll. T. Plowman, A.M. de Carvalho; Feb. 1983, No 12818 ND 0 (Al-Said et al., 1986) F1916654 +++ – F1934043 + – F1916492 ND – F1910848 NQ – F1932963 ND – F1916645 ND – F1944101 ND – F1933543 ND – F1973408 ND – F1934047 ND – F1916633 ND – S. Bieri et al. / Journal of Ethnopharmacology 103 (2006) 439–447 Erythroxylum bradeanum O.E. Schulz Mauritius, Coll. V. Ridges, No 5 (–) species not previously investigated; (++++) >0.005% cocaine per gram leaf; (+++) 0.001–0.005%; (++) 0.0005–0.001%; (+) 0.0001–0.0005%. NQ: not quantified (presence of target ion 182 Th and selective ion 303 Th without confirmation ion 272 Th. ND: Total absence of molecular ion 303 Th. 443 444 S. Bieri et al. / Journal of Ethnopharmacology 103 (2006) 439–447 tate additional sophisticated sample treatment before analysis. After extraction, all of the investigated samples were qualitatively and quantitatively analysed by GC-MS in scan and SIM modes, respectively. The leaves of 51 Erythroxylum species belonging to seven different sections as described by Schulz (1907) were examined for their cocaine content. Among them, 28 species had not been investigated previously. According to the age of the investigated plant material and due to the low cocaine content, concentration ranges rather than exact concentrations are reported. Four domains, expressed in percentage of cocaine per gram dry mass, have been defined, namely: (++++) >0.005%; (+++) 0.001–0.005%; (++) 0.0005–0.001%; (+) 0.0001–0.0005%. The LOQ of the method turned out to be 0.0001% of cocaine per gram dry leaf. Retention time repeatability on the target ion (182 Th) was excellent (R.S.D. = 0.01%, n = 6) considering the high oven temperature program rate used for quantitative analyses. Fig. 1 shows the extracted ion profiles in the case of Erythroxylum argentinum, which was the wild species with the lowest quantified cocaine content. It demonstrates the specificity of the method, which requires the simultaneous presence of the three cocaine ions, together with the precise retention time. Cocaine was detected in 23 of the 51 species examined (Table 1). All the investigated sections except one (Pachy- lobus) contained at least one cocaine-producing species. This suggests, as indicated by previous studies (Aynilian et al., 1974; Plowman and Rivier, 1983), that cocaine is widely distributed among the genus Erythroxylum, irrespective of the sections. Fourteen species are reported to contain cocaine for the first time (Erythroxylum amazonicum, Erythroxylum citrifolium, Erythroxylum laetevirens, Erythroxylum argentinum, Erythroxylum cumanense, Erythroxylum densum, Erythroxylum frangulifolium, Erythroxylum subrotundum, Erythroxylum cuneifolium, Erythroxylum divaricatum, Erythroxylum gonocladum, Erythroxylum andreii, Erythroxylum aturense, and Erythroxylum confusum). Among them, Erythroxylum laetevirens, a shrub with pale-greenish flowers and green fruits, was the wild species with by far the highest cocaine content (0.011% dry weight). Thus, its alkaloid profile was accurately determined and compared with those of two cultivated Erythroxylum coca species, as well as with a “Mate de coca ” commercially available on the market in La Paz in Bolivia (Table 2). Quantitative results showed that even if the cocaine content in Erythroxylum laetevirens was markedly higher than in all the other investigated wild species, it was nonetheless much lower than in the “Mate de coca”, as well as in the cultivated species. In the literature, it has been reported that in these species, the lowest cocaine content was found in the ipadu variety (0.11–0.41%) and the Fig. 1. Quantitative GC-MS analysis with an oven program from 70 (1 min hold) to 285 ◦ C (5 min hold) at 30 ◦ C/min. Single ion monitoring traces of cocaine in Erythroxylum argentinum: (A) pseudo-molecular ion trace 303 Th of cocaine (selective target ion); (B) ion trace 272 Th – less abundant ion – (qualifier ion); (C) ion trace of cocaine base peak 182 Th (target ion); (D) pseudo-molecular ion trace 294 Th of internal standard (methadone). S. Bieri et al. / Journal of Ethnopharmacology 103 (2006) 439–447 Table 2 Cocaine content in Erythroxylum laetevirens and cultivated Erythroxylum coca species Species Cocaine content (% m/m) Erythroxylum laetevirens Trujillo cocaa Erythroxylum coca var. ipadu “Mate de coca” tea bag 0.0107 0.71 0.215 0.60 ± ± ± ± 0.0005 0.02 0.005 0.02 Plowman, 1984b. a E. novogranatense var. truxillense. highest content in the truxillense variety (0.42–1.02%), while the cocaine content in the novogranatense variety ranges from 0.17 to 0.76% (Holmstedt et al., 1977; Plowman and Rivier, 1983). Our results (Table 2) are in good agreement with these values and suggest that the “Mate de coca” probably consists in coca leaves, but not from the ipadu variety (Plowman, 1981; Schultes, 1981). According to Engelke and Gentner, 1991, herbal tea bags sold under the name “Health Inca Tea” or “Mate de Coca” 445 are commercially available since 1981 in Peru. The authors mentioned that the investigated tea bags were produced and packed in Peru from the leaves of Erythroxylum novogranatense var. truxillense by a national enterprise. Even if the product was claimed to be decocainized, the percentage of cocaine present in the plant tissue raised up to 0.37%, corresponding to about 3.7 mg of cocaine per tea bag. Similarly, Jenkins et al. (1996), analysed coca tea bags from Peru and Bolivia and indicated that cocaine, benzoylecgonine, ecgonine methyl ester and trans-cinnamoylcocaine were present in variable quantities. After exhaustive extraction, they found an average cocaine content of roughly 5 mg per tea bag consisting of 1 g plant material. When they prepared tea according to the labelling instructions, an average of about 4 mg cocaine was found per cup. These results were in good agreement with other investigations (Rivier, 1981; ElSohly et al., 1986; Siegel et al., 1986; Jackson et al., 1991). The cocaine content in the “Mate de coca” measured in the present study (0.60%), together with the qualitative chromatographic profile, indicate that the investigated tea bags consisted of basically pure coca leaves (Fig. 2A). Fig. 2. Qualitative GC-MS analysis with an oven program from 70 (1 min hold) to 285 ◦ C (15 min hold) at 5 ◦ C/min. Chromatographic profiles: (A) “Mate de coca” tea bag; (B) Trujillo coca; (C) Erythroxylum coca var. ipadu; (D) Erythroxylum laetevirens. For alkaloid structures and their abbreviations see Fig. 3. The compounds were identified according to their mass spectra and by injecting authentic references. MET: methadone. 446 S. Bieri et al. / Journal of Ethnopharmacology 103 (2006) 439–447 Fig. 3. Structure of cocaine and detected tropane alkaloids together with the biosynthetic precursor hygrine present in Erythroxylum laetevirens and in the cultivated Erythroxylum species. Finally, as Erythroxylum laetevirens had not been investigated previously, a qualitative chromatographic profile of its alkaloid content was carried out and compared with those of cultivated coca species (Fig. 2). All chromatographic profiles displayed a similar tropane alkaloid pattern. Indeed, hygrine, anhydro-ecgonine methyl ester, ecgonine methyl ester, cocaine, and two characteristic cinnamoylcocaines (Fig. 3) were unambiguously identified in all samples. The material that appeared between 20 and 27 min in all chromatographic profiles consisted mainly of fatty acids. 4. Conclusions In the present study, the leaf samples of 51 different Erythroxylum species were investigated for their cocaine content. Twenty-eight species had not been examined previously and cocaine was detected in 23 wild Erythroxylum species. Cocaine content was less than 0.001% for all wild species, except for Erythroxylum laetevirens in which a 10 times higher amount was determined. The qualitative chromatographic profile of the latter species was very similar to that of cultivated coca species. In particular, the characteristic cinnamoylcocaines were present. Comparison of GC profiles and quantitative results showed that the so-called “Mate de coca”, also known as “Health Inca tea”, was mainly composed of pure coca leaves. Consequently, the consumption of coca tea will result in ingestion of varying amounts of cocaine, together with other related tropane alkaloids. Before any overall chemotaxonomic conclusions are drawn regarding the occurrence of cocaine throughout the genus, further phytochemical investigations on more species are required. It appears from the present study that cocaine, even in trace amounts is not specifically produced by species belonging to a single section of the genus. Rather, it is widely distributed and thus cannot be used as a specific marker for the genus. 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