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Isolation and identification of unique marker compounds from the Tasmanian poppy Papaver somniferum N.
Forensic Science International 175 (2008) 202–208 www.elsevier.com/locate/forsciint
Isolation and identification of unique marker compounds from the Tasmanian poppy Papaver somniferum N. Implications for the identification of illicit heroin of Tasmanian origin Luke R. Odell a, Jana Skopec b, Adam McCluskey a,* a
Chemistry Building, School of Environmental and Life Sciences, The University of Newcastle, University Drive, Callaghan, NSW 2308, Australia b Australian Government Analytical Laboratories, 1 Suakin Street, Pymble, NSW 2073, Australia Received 20 October 2006; received in revised form 13 June 2007; accepted 9 July 2007 Available online 31 August 2007
Abstract Tasmanian opium accounts for 25% of the world’s legal supply of opium straw, and in 1998–99 sufficient numbers of flower pods (66,013) to manufacture ca 500 kg of heroin were stolen. Whilst the heroin signature program has been developed to determine the origin of heroin from other key producers, no such signature currently exists for Tasmanian derived heroin. Tasmanian poppies contain a unique alkaloid, oripavine, which is the source of ‘marker’ impurities in illicit heroin produced from Tasmanian poppy straw. Treatment of oripavine (500 mg) under Thiboumery and Mohr heroin processing conditions, followed by simple evaporative workup afforded 613 mg of a dark orange residue, which upon extensive chromatographic purification yielded oripavine 3-acetate (2) 22 mg; 3-acetyl-N-acetyldesthebaine (3) 35 mg; 3-acetyl-6-methoxy-4,5-epoxyphenanthrene (4) 5.8 mg; 3,4-diacetyl-6-methoxyphenanthrene (5) 27 mg; and 3,4,6-methoxy-5-[2(N-methylacetamido)]ethylphenanthrene (6) 52 mg. Compounds (2–6) are derived from oripavine and are unique to heroin derived from the Tasmanian poppy Papaver somniferum N. Analysis of illicit heroin samples seized from Turkey, Pakistan, Columbia and Myanmar did not reveal any of the aforementioned marker compounds. We have, however, identified four of these marker compounds (3–6) in seized heroin samples from Australia suggesting that they are of Tasmanian origin. Complete details of the isolation and identification of these compounds are provided. Crown Copyright # 2007 Published by Elsevier Ireland Ltd. All rights reserved. Keywords: Forensic science; Illicit drugs; Heroin; Heroin signature program; Oripavine; Tasmanian poppies; Papaver somniferum N
1. Introduction The continual growth in the manufacture, trafficking and abuse of illicit heroin are major areas of concern for most governments today, with Australia being no exception. Globally, the amount of drug seized rises with the most recent world drug report indicating a 9% increase in opium/heroin seizures in 2004 [1]. Within Australian there has been a noticeable downturn in the quantity of opium/heroin, but an increase in the actual number of seizures in recent years [2]. The recent Australia National Drug Household survey showed that demand for the drug is also on the decrease, with the 12,000
* Corresponding author. E-mail address: [email protected] (A. McCluskey).
fewer Australian heroin users in 2004 than in 2001, a change in the previous growth in user numbers since 1988 [3]. In an attempt to combat the significant social and economic costs associated with heroin abuse, a number of countries have implemented ‘Heroin Signature Programs’ (HSP’s). In an HSP, seized samples are analyzed by a number of different analytical procedures with the aim of providing crucial drug trafficking and distribution intelligence information. HSP’s typically analyze and evaluate major synthetic impurities and adulterants, and these approaches have been extensively researched and previously reported [1,4–10]. Almost 25% of the World’s legal poppy straw is produced in Tasmania. The Tasmanian poppy industry is largely based on Papaver somniferum L. (PSL), which typically produces between 40 and 80% morphine and related alkaloids. Indeed, Tasmania is currently the worlds largest producer of opium
0379-0738/$ – see front matter. Crown Copyright # 2007 Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.forsciint.2007.07.002
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alkaloids [11]. However, since 1994 there has been a concerted effort by Tasmanian Alkaloids, the biggest grower of opium poppies, to develop a high thebaine poppy variety, and to simplify the processing of the alkaloids, thereby increasing production efficiency [12]. Thebaine is a more useful and lucrative product versus morphine. There have been previous reports of spontaneous Papaver somniferum mutants containing high thebaine concentrations; however these mutants only possessed a chromosomal abnormality, eventually resulting in revision back to a morphine type [13]. Ensuing screening and mutagenesis programs by Tasmanian Alkaloids ultimately led to the development of Papaver somniferum Norman. This cultivar produces approximately the same quantities of alkaloids per hectare, but with thebaine and oripavine (not morphine) as the major constituents. Routine screening by Tasmanian Alkaloids estimates the major alkaloid content of the Papaver somniferum N. (PSN) cultivar as a function of dry weight to be; thebaine (2.0%), oripavine (0.8%), codeine (0.01%) and morphine (0.05%) compared with the traditional Papaver somniferum L. (PSL) cultivar with morphine (2.4%), codeine (0.1%), oripavine (0.03%) and thebaine (0.1%) by dry weight. Morphine is present as a by-product from PSL infestation as a weed in the PSN crop and there is significant season-to-season variation in these minor alkaloids (morphine and codeine) [14]. Since initial plantings in 1996/7 (500 ha), PSN now accounts for 60% of the crop under cultivation [15]. Isolation of morphine from PSN is via harvesting and extraction of the poppy straw (PSN does not ‘bleed’ opium sap). In 1998–99, sufficient flower pods (66, 013) were stolen with the potential to produce ca 500 kg of heroin. The distribution of PSL versus PSN in these stolen flower pods was not reported. Accordingly, there is a need to identify HSP markers that will specifically identify heroin of PSN origin. In this regard, researchers have identified the alkaloid, oripavine (1) (Fig. 1) as being Tasmanian PSN specific [12,13,15–20]. Consequently, the presence of oripavine and its reaction by-products derived from refluxing in acetic anhydride in seized heroin samples identifies Tasmanian origin heroin derived from PSN. Herein we wish to report the isolation and identification of oripavine derived byproducts from the heroin from Tasmanian PSN opium.
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Unless otherwise stated all other reagents were used as supplied.
2.2. Fourier transform infrared and nuclear magnetic resonance spectroscopy Fourier Transform Infrared Spectra (FTIR) were recorded at 4 cm 1 resolution as NaCl films or casts using a Perkin-Elmer Paragon 1000 FT-IR Spectrophotometer and chloroform as the solvent. 1 H and 13C NMR spectra were recorded on a Bruker Avance-300DPX 300 MHz NMR spectrometer, using either deuterated chloroform (CDCl3) or methanol (CD3OD) as the solvent. For spectra recorded in CDCl3 the residual signals at d7.24 ppm for 1H and the triplet at d77.0 ppm for 13C were used as internal standards. When recorded in CD3OD the residual quintet at d3.35 ppm for 1H and the septet at d49.0 ppm for 13C were used as internal standards.
2.3. Thin layer and preparative chromatography Thin layer chromatography (TLC) was performed on Merck aluminum backed sheets pre-coated with silica gel 60 F254 (0.2 mm layer thickness). Visualization was achieved through exposure to UV light, followed by dipping in a phosphomolybdic and ceric sulfate mixture (2.5% H2SO4, 2% H3PO3MoO4, 1% Ce(SO4)2), and subsequent charring. Short column (‘speedy’) chromatography was performed using a sintered filter funnel (70 mm 55 mm) packed with approximately 30 g of silica gel HF254 (Merck). A gradient solvent system was employed commencing with 3 20 ml portions of dichloromethane:hexane (60:40). The polarity of the system was then increased maintaining a 20 ml portion size throughout: dichloromethane:hexane, 80:20; 100:0, dichloromethane:ethyl acetate; 60:40; 40:60; and finally the addition of three portions of ethyl acetate:methanol: 80:20; 60:40; and 40:60. The gel was then washed with 4 50 ml portions of acetone. Centrifugal chromatography (Chromatotron) separations were carried out on a Harrison Research Chromatotron using glass rotors coated with 2 mm of gypsum binder containing silica gel 60 PF254.
2.4. HPLC and GCMS instrumentation and conditions
2. Methods and materials
Gas chromatography/mass spectrometry was carried out using a Shimadzu QP–5050A GCMS. Chromatographic separation was achieved on a HP-5MS (5% phenyl methyl siloxane) capillary column (length 30 m, I.D 0.25 mm, film thickness 0.25 mm) with helium carrier gas at a flow of 1.2 ml/min (pressure 88.9 kPa). The injection temperature was maintained at 250 8C, with 1 ml splitless injection. The column was heated at 100 8C for 1 min, then increased at a rate 28 8C/min to 240 8C, then 2 8C/min to 280 8C, followed by 6 8C/min to 320 8C. High performance liquid chromatography (HPLC) was performed on a Waters 600 series system equipped with a Waters 996 diode array detector measuring at 290 nm. Chromatographic separation was achieved using a semipreparative steel column (10 mm silica gel, 250 mm 10 mm, Phenomenex) at a flow rate of 5 ml/min with a mixture of hexane and ethyl acetate as the mobile phase.
2.1. Sample purity and resourcing
2.5. Heroin signature analysis
All solvents used were HPLC grade or bulk solvents re-distilled from glass before use. Oripavine was obtained as a gift from the National Analytical Reference Laboratory (NARL), Pymble NSW.
Heroin hydrochloride (70%, 50 mg) was taken up in 5% H2SO4 (4 ml) and a 60:40 mixture of CH2Cl2 and diethyl ether (4 ml) added. The mixture was vortexed for 10 min and centrifuged for 10 min. The upper solvent layer (4 ml) was carefully removed and evaporated to dryness under a stream of nitrogen at 60 8C. The residue was derivatised prior to analysis with the addition of 250 mM of a derivatising solution containing 50% CH2Cl2 and 50% BSTFA (containing 1% trimethylchlorosilane (TMCS). Finally 50 ml of the internal standard (500 ppm benzopinacolone) was added and the mixture was heated at 60 8C for 30 min, and subjected to GC–MS analysis (as described in Section 2) [5].
2.6. Acetylation of Tasmanian poppy straw
Fig. 1. Oripavine (1).
Acetic anhydride (600 ml) was added slowly to Tasmanian PSN poppy straw concentrate (200 mg) and the resulting solution was stirred and heated to 80 8C. After 2.25 h the reaction mixture was allowed to cool to room temperature, and
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then to 0 8C. The pH was adjusted with cold 10% sodium carbonate and the mixture was then extracted with CHCl3 (3 10 ml), dried over MgSO4, and evaporated to afford 177.2 mg (87%) of crude heroin base.
2.7. Purification and identification of oripavine acetylation product 2.7.1. Oripavine 3-acetate (2) Crude (2) was obtained from the MeOH:EtOAc ‘speedy’ column fractions and purified by flash chromatography (silica gel, 4 g) using CHCl3:MeOH (9:1) as the mobile phase to yield (2) (22%) as a brown oil. 1 H NMR (CDCl3): dH 1.8–1.85 (1H, m), 2.2–2.26 (1H, m), 2.26 (3H, s), 2.57 (3H, s), 2.76–2.84 (1H, m), 2.89 (1H, dd, J = 6.9, 18.9 Hz), 3.02 (1H, dt, J = 3.5, 13.2 Hz), 3.45 (1H, d, J = 18.6 Hz), 3.62 (3H, s), 3.82 (1H, d, J = 6.9 Hz), 5.06 (1H, d, J = 6.5 Hz), 5.31 (1H, s), 5.66 (1H, d, J = 6.5 Hz), 6.64 (1H, d, J = 8.2 Hz), 6.78 (1H, d, J = 8.2 Hz). 13 C NMR (CDCl3): dC 168.2 (s), 152.5 (s), 147.1 (s), 133.1 (s), 131.8 (s), 131.5 (s), 128.4 (s), 121.8 (d), 119.0 (d), 113.7 (d), 95.5 (d), 88.8 (d), 60.5 (d), 54.6 (q), 44.7 (t), 45.2 (s), 41.7 (q), 34.9 (t), 30.7 (t), 20.9 (q). FTIR (cm 1): 3018 (m), 1758 (s), 1604 (m), 1445 (m), 1214 (s). MS: m/z 339 (M+, 99), 297 (M–CH2CO, 100), 282 (20), 240 (20), 165 (8), 43 (65). 2.7.2. 3-Acetyl-N-acetyldesthebaine (3) A mixture of (1) and (6) was obtained from the DCM:EtOAc ‘speedy’ column fractions. Separation from (6) was achieved using the Chromatotron (centrifugal chromatography) (2 mm plate thickness) and a five-step gradient elution system (EtOAc:hexane (a) 6:4; (b) 8:2; (c) 1:0; (d) EtOAc:MeOH 9.5:0.5; (e) 9:1). (3) (147 mg, 30%) degraded upon storage in EtOAc and was repurified by flash chromatography (Redisep, Silica Gel, 4 g) using ether as the mobile phase to yield (3) (35 mg, 7% of oripavine mass) as a yellow oil. The 1H and 13C NMR spectra of (3) indicated the presence of two conformers. 1 H NMR (CD3OD): dH 1.5–1.87 (2H, m), 1.87 and 1.95 (3H, s), 2.29 and 2.30 (3H, s), 2.75 and 2.84 (3H, s), 3.19–3.36 (2H, m), 3.36–3.62 (2H, m), 3.9
(3H, s), 5.8 and 5.83 (1H, d, J = 10.4 Hz), 6.1–6.15 (1H, m), 6.15 and 6.18 (1H, d, J = 10.3 Hz), 6.91 (1H, d, J = 8.9 Hz), 6.92 (1H, d, J = 9 Hz). 13 C NMR (CD3OD): dC 171.0 and 170.9 (s), 167.9 (s), 148.1 and 148.0 (s), 139.8 and 139.6 (s), 138.9 and 138.8 (s), 131.5 and 131.0 (s), 130.75 and 130.7 (s), 130.62 and 130.6 (s), 130.5 and 130.48 (s), 127.0 and 126.6 (d), 123.2 and 123.1 (d), 121.3 and 121.1 (d), 120.8 and 120.65 (d), 120.6 and 120.1 (d), 57.7 (q), 46.2 (s), 45.3 and 42.5 (t), 35.5 and 34.1 (q), 36.2 and 34.9 (t), 29.3 and 29.2 (t), 19.7 and 18.9 (q), 18.5 and 18.4 (q). FTIR (cm 1): 2979 (m), 1766 (s), 1651 (s), 1634 (s), 1490 (m), 1214 (s), 751 (s). MS: m/z 381 (M+, 36), 339 (M–CH2CO, 10), 266 (18), 238 (16), 101 (82), 86 (58), 44 (100). 2.7.3. 3-Acetyl-6-methoxy-4,5-epoxyphenanthrene (4) A mixture of (4) and (5) (83 mg) was obtained from the DCM:hexane ‘speedy’ column fractions. Separation from (5) was achieved using the Chromatotron (1 mm plate thickness) and EtOAc:hexane (6:4) as the mobile phase. (4) was obtained as a yellow solid (27 mg, 5.4% of oripavine mass). M.p. 110– 112 8C. 1 H NMR (CD3OD): dH 2.47 (3H, s), 4.34 (3H, s), 7.47 (1H, d, J = 8.3 Hz), 7.53 (1H, d, J = 8.2 Hz), 7.78 (1H, d, J = 8.2 Hz), 7.79 (1H, d, J = 8.3 Hz), 7.82 (1H, d, J = 8.9 Hz), 7.91 (1H, d, J = 8.9 Hz). 13 C NMR (CD3OD): dC 167.0 (s), 144.3 (s), 141.1 (s), 140.8 (s), 129.5 (s), 125.6 (s), 125.1 (s), 124.7 (s), 121.9 (s), 125.0 (d), 123.3 (d), 122.9 (d), 121.5 (d), 120.7 (d), 116.7 (d), 58.5 (q), 20.0 (q). FTIR (cm 1): 1766 (s), 1626 (m), 1500 (m), 1435 (m), 1199 (s), 1149 (m), 1011 (m), 887 (w), 756 (m). MS: m/z 280 (M+, 25), 238 (M–CH2CO, 100), 223 (70), 139 (15), 75 (28). 2.7.4. 3,4-Diacetyl-6-methoxyphenathrene (5) Was obtained as a dark orange solid (52 mg, 10.5% of oripavine mass) with m.p. 156–160 8C, as outlined above.
Fig. 2. Synthesis and purification of acetylated oripavine products.
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Fig. 3. Chemical structures of compounds 2–6. 1
H NMR (CDCl3): dH 2.38 (3H, s), 2.54 (3H, s), 3.97 (3H, s), 7.28 (1H, dd, J = 2.2, 8.8 Hz), 7.47 (1H, d, J = 8.7 Hz), 7.57 (1H, d, J = 8.9 Hz), 7.66 (1H, d, J = 8.9 Hz), 7.8 (1H, d, J = 8.7 Hz), 7.81 (1H, d, J = 8.8 Hz), 8.57 (1H, d, J = 2.2 Hz). 13 C NMR (CDCl3): dC 168.5 (s), 167.8 (s), 158.2 (s), 158.2 (s), 140.7 (s), 139.7 (s), 132.5 (s), 130.0 (s), 129.9 (d), 127.7 (s), 127.5 (d), 127.4 (s), 127.0 (d), 124.5 (d), 121.6 (d), 116.6 (d), 108.5 (d), 55.3 (q), 21.0 (q). FTIR (cm 1): 1772 (s), 1617 (m), 1512 (m), 1452 (m), 1206 (s). MS: m/z 324 (M+, 16), 282 (M–CH2CO, 13), 240 M – 2 CH2CO, 100), 139 (11), 43 (37). 2.7.5. 3,4-Diacetyl-6-methoxy-5-[2-(N-methylacetamido)]ethylphenanthrene (6) Separation of (6) from (3) was achieved as outlined above. Final purification of (6) was carried out via HPLC (normal phase) with EtOAc:hexane (Gradient, Start 9:1, End 1:0) as the mobile phase to obtain a yellow oil (5.8 mg, 1.2% of oripavine mass). 1H and 13C NMR spectra of (6) showed the presence of two conformers. 1 H NMR (CDCl3): dH 1.42 and 1.64 (3H, s), 2.1 and 2.12 (3H, s), 2.35 (3H, s), 2.17 and 2.54 (3H, s), 3.2–3.38 (2H, br m), 3.45–3.55 (2H, br m), 3.99 and 4.02 (3H, s), 7.26 (1H, d, J = 8.7 Hz), 7.36 (1H, d, J = 8.5 Hz), 7.41 (1H, d, J = 8.8 Hz), 7.47 (1H, d, J = 8.8 Hz), 7.68 (1H, d, J = 8.7 Hz), 7.73 (1H, d, J = 8.5 Hz). 13 C NMR (CDCl3): dC 169.9 (s), 169.9 (s), 168.0 (s), 156.5 (s), 140.2 (s), 137.8 (s), 131.8 (s), 127.9 (s), 127.4 (s), 124.1 (s), 122.0 (s), 126.85 and 126.8 (d), 126.6 and 126.4 (d), 125.3 and 124.9 (d), 122.3 and 122.2 (d), 121.0 and 120.7 (d), 111.9 and 111.4 (d), 55.8 and 55.4 (q), 50.5 and 46.5 (t), 34.6 and 32.5 (t), 28.9 and 27.8 (t), 20.1 (q), 20.05 and 20.0 (q), 19.9 and 19.6 (q). FTIR (cm 1): 2936 (m), 1770 (s), 1633 (s), 1591 (m), 1500 (m), 1434 (m), 1201 (s). MS: m/z 423 (M+, 12), 381 (M–CH2CO, 13), 339 (M – 2 CH2CO, 16), 266 (39), 251 (43), 248 (36), 100 (12), 44 (100).
(500 mg) was treated with acetic anhydride (2 ml) at 80 8C for 2 h, and then the reaction mixture was evaporated in vacuo to leave a dark orange residue (613 mg). Subsequent chromatographic fractionation techniques yielded five pure compounds 2–6 (Figs. 2 and 3). Of these 3–6 have not previously been reported and 2 has only been reported, without spectral data, as an oxidation product of heterocodeine 3-acetate [16]. Separately, we utilized the same experimental condition, and identified compounds 3–6 in the resulting heroin product (Fig. 4). 4. Discussion The isolation and structural elucidation of 2–6 provides a simple and effective method for the detection of Tasmanian PSN opium derived heroin. These compounds are well resolved and present in sufficient quantities to allow detection using the current Australian National Heroin Signature Program GC–MS procedure for trace organic analysis (see Section 2) [5]. Fig. 4 shows the appearance of 3–6 in a typical ‘Tasmanian PSN’ heroin sample, i.e. a sample of heroin prepared by the
3. Results In order to simplify the identification and isolation of oripavine-derived species, we commenced our studies with pure oripavine (supplied by the National Analytical Reference Laboratory) and subjected it to the standard Thiboumery and Mohr methodology for heroin synthesis [21]. Thus, oripavine
Fig. 4. Identification of oripavine derived products 3–6 in a typical Tasmanian heroin sample. Note: the PSN does not yield high quantities of morphine, hence low heroin yields.
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Fig. 5. GC–MS traces of illicit heroin seized with various geographical origins. Key marker compounds have been highlighted: heroin; acetyl codeine (AC); 3monoacetylmorphine (3-MAM); and 6-monoacetylmorphine (6-MAM). (a) GC–MS trace of heroin identified as being of SWA (Turkey) origin. (b) GC–MS trace of heroin identified as being of SWA (Pakistan) origin. (c) GC–MS trace of heroin identified as being of SA (Colombia) origin. (d) GC–MS trace of heroin identified as being of SWA (Myanmar) origin. (e) GC–MS trace of heroin identified as being of Tas PSL origin.
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Fig. 5. (Continued ).
acetylation of opium extracted from Tasmanian PSN poppies. The advantage of this technique is that, unlike other profiling methods, which rely on differences in production processes that result in quantitative differences in the ratios of commonly encountered compounds, it is based on ‘marker’ impurities that are unique to Tasmanian grown PSN poppies [15,22]. Thus, a heroin sample containing 3–6 can only be explained by the presence of oripavine in the original opium extract, which occurs only in Tasmanian grown PSN poppies. These findings were consistent across a number of Tasmanian PSN heroin samples examined (n = 5). In all HSP except the U.S., distinction between SEA, SWA, South American, or Mexican heroin samples is based solely on the morphine extraction and acetylation procedures employed in its production (see Section 2), referred to as ‘processing’ origin. Although these methods may be accurate, to a degree, in determining processing origin, they do not guarantee that a sample made using a typical procedure actually originated from that geographical origin. Consequently, as long as oripavine remains unique to Tasmanian PSN poppies, the detection of 3–6 in seized samples will provide unequivocal evidence of its origin. The
U.S. HSP procedures use a combination of impurity profiling, trace solvent analysis, and isotopic ratio mass spectrometry, and are typically 95% accurate in assigning geographical origin. The GC–MS trace for heroin based on Tasmanian PSN poppies (Fig. 4) clearly shows 3–6. Our initial assignments of these compounds were tentative via mass spectral analysis prior to analysis and complete characterization of these analogues in this work. The acetylation of oripavine afforded sufficient quantities of compounds 3–6 for identification purposes. The presence of 3–6 in typical ‘clan-lab’ preparations (Fig. 4) clearly shows that our efforts to maximize retrieval for identification did not contribute to the formation of these impurities. It is noteworthy that Tasmanian PSN derived heroin samples actually have low levels of heroin. This is a consequence of the low morphine concentration in the PSN poppy, typically 0.05%, however this is often contaminated with the more traditional PSL with a morphine content of 2.4%. Given that the thieves are not (normally) aware which cultivar they steal, PSN derived heroin is often contaminated with PSL derived heroin, and this is further exacerbated by inappropriate collection of
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both PSN and PSL cultivar by seasonal harvesters. Fig. 5 shows the corresponding GC–MS traces for heroin seized from a variety of geographical origins; Turkey, Pakistan, Columbia, Myanmar, and Tasmania (PSL cultivar). In all of these instances the GC–MS traces display significant differences from those observed with that of Tasmanian PSN origin. Most notably, Fig. 5 shows no evidence of 3–6, whilst these compounds are readily identified in the Tasmanian PSN sample. Acknowledgements LO wishes to acknowledge generous financial support from the Australian Forensic Drug Laboratory (Pymble, NSW, Australia). We also acknowledge the generous donation of oripavine from the National Analytical Reference Laboratory (Pymble, NSW, Australia). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.forsciint.2007.07.002. References [1] United Nations Drug Control Program, World Illicit Drugs Report 2006, New York, 2001. http://www.unodc.org/unodc/world_drug_report.html. [2] Australian Crime Commission, Illicit Drug Report 2005-6; http:// www.crimecommission.gov.au/html/pg_iddr2005-06.html. [3] Australian Institute of Health and Welfare, 2004 National Drugs Strategy Household Survey 2004: First Results; http://www.aihw.gov.au/publications/index.cfm/title/10122. [4] R. Dams, T. Benijts, W.E. Lambert, D.L. Massart, A.P. De Leenheer, Heroin impurity profiling: trends throughout a decade of experimenting, Forensic Sci. Int. 123 (2001) 81–88. [5] R.-B. Myors, P.-T. Crisp, S.-V. Skopec, R.-J. Wells, Investigation of heroin profiling using trace organic impurities, Analyst 126 (2001) 679– 689.
[6] Office on drugs and crime, Methods for Impurity Profiling of Heroin and Cocaine, United Nations, New York, 2005. [7] T. Bora, M. Merdivan, C. Hamamci, Levels of trace and major elements in illicit heroin, J. Forensic Sci. 47 (2002) 959–963. [8] A.C. Allen, D.A. Cooper, J.M. Moore, M. Gloger, H. Neumann, Illicit heroin manufacturing by products: capillary gas chromatography, determination and structural elucidation of narcotine and norlaudanosine related compounds, Anal. Chem. 56 (1984) 2940–2947. [9] P. Esseiva, L. Dujourdy, F. Anglada, F. Taroni, P. Margot, A methodology for illicit heroin seizures comparison in a drug intelligence perspective using large databases, Forensic Sci. Int. 132 (2003) 139–152. [10] V. Dufey, L. Dujourdy, F. Besacier, H. Chaudron, A quick and automated method for profiling heroin samples for tactical intelligence purposes, Forensic Sci. Int. 169 (2007) 108–117. [11] International Narcotics Control Board, Narcotic Drugs, estimated world requirements for 2007, United Nations, New York 2007; http://www.incb.org/incb/en/narcotic_drugs.html. [12] J.C. Laughlin, B. Chung, B.M. Beattie, in: J. Bernath (Ed.), Poppy – The Genus Papaver, Harwood Academic Publishers, Amsterdam, 1998, pp. 249–277. [13] U. Nymann, O. Hall, Some varieties of Papaver somniferum L. with changed morphine alkaloid content, Hereditas 84 (1976) 69–76. [14] A.J. Fist, Tasmanian Alkaloids, Westbury, Tasmania, 7303, Australia, Personal communication. [15] A.J. Fist, The Tasmania poppy industry: a case study of the application of science and technology, in: Proceedings 10th Agronomy Conference, Hobart, Australia, 2001. [16] R.B. Barber, H. Rapport, Synthesis of thebaine and oripavine from codeine and morphine, J. Med. Chem. 18 (1975) 1074–1077. [17] A. Allen, D. Cooper, J. Moore, C. Teer, Thebaine rearrangements: nonclassical ring migrations, J. Org. Chem. 49 (1984) 3462–3465. [18] G.A. Cordell, Introduction to Alkaloids, 1st ed., John Wiley and Sons, Brisbane Australia, 1981. [19] Merck and Co., The Merck Index, 12th ed., Merck Research Laboratories, Whitehouse Station, 1996. [20] C.V. Trenerry, R.J. Wells, Analysis of drugs of abuse by micellar electrokinetic capillary chromatography, Progress in HPLC 5 (1996) 1–55. [21] D.A. Cooper, in: M. Klein, F. Sapienza, H. McClain, I. Khan (Eds.), Clandestinely Produced Drugs and Precursors, United States Department of Justice, Drug Enforcement Administration, Washington, DC, 1989. [22] B. Neilsen, J. Roe, Oripavine – a new opium alkaloid, Planta Med. 48 (1983) 205–207.
Isolation and identification of unique marker compounds from the Tasmanian poppy Papaver somniferum N. Implications for the identification of illicit heroin of Tasmanian origin Luke R. Odell a, Jana Skopec b, Adam McCluskey a,* a
Chemistry Building, School of Environmental and Life Sciences, The University of Newcastle, University Drive, Callaghan, NSW 2308, Australia b Australian Government Analytical Laboratories, 1 Suakin Street, Pymble, NSW 2073, Australia Received 20 October 2006; received in revised form 13 June 2007; accepted 9 July 2007 Available online 31 August 2007
Abstract Tasmanian opium accounts for 25% of the world’s legal supply of opium straw, and in 1998–99 sufficient numbers of flower pods (66,013) to manufacture ca 500 kg of heroin were stolen. Whilst the heroin signature program has been developed to determine the origin of heroin from other key producers, no such signature currently exists for Tasmanian derived heroin. Tasmanian poppies contain a unique alkaloid, oripavine, which is the source of ‘marker’ impurities in illicit heroin produced from Tasmanian poppy straw. Treatment of oripavine (500 mg) under Thiboumery and Mohr heroin processing conditions, followed by simple evaporative workup afforded 613 mg of a dark orange residue, which upon extensive chromatographic purification yielded oripavine 3-acetate (2) 22 mg; 3-acetyl-N-acetyldesthebaine (3) 35 mg; 3-acetyl-6-methoxy-4,5-epoxyphenanthrene (4) 5.8 mg; 3,4-diacetyl-6-methoxyphenanthrene (5) 27 mg; and 3,4,6-methoxy-5-[2(N-methylacetamido)]ethylphenanthrene (6) 52 mg. Compounds (2–6) are derived from oripavine and are unique to heroin derived from the Tasmanian poppy Papaver somniferum N. Analysis of illicit heroin samples seized from Turkey, Pakistan, Columbia and Myanmar did not reveal any of the aforementioned marker compounds. We have, however, identified four of these marker compounds (3–6) in seized heroin samples from Australia suggesting that they are of Tasmanian origin. Complete details of the isolation and identification of these compounds are provided. Crown Copyright # 2007 Published by Elsevier Ireland Ltd. All rights reserved. Keywords: Forensic science; Illicit drugs; Heroin; Heroin signature program; Oripavine; Tasmanian poppies; Papaver somniferum N
1. Introduction The continual growth in the manufacture, trafficking and abuse of illicit heroin are major areas of concern for most governments today, with Australia being no exception. Globally, the amount of drug seized rises with the most recent world drug report indicating a 9% increase in opium/heroin seizures in 2004 [1]. Within Australian there has been a noticeable downturn in the quantity of opium/heroin, but an increase in the actual number of seizures in recent years [2]. The recent Australia National Drug Household survey showed that demand for the drug is also on the decrease, with the 12,000
* Corresponding author. E-mail address: [email protected] (A. McCluskey).
fewer Australian heroin users in 2004 than in 2001, a change in the previous growth in user numbers since 1988 [3]. In an attempt to combat the significant social and economic costs associated with heroin abuse, a number of countries have implemented ‘Heroin Signature Programs’ (HSP’s). In an HSP, seized samples are analyzed by a number of different analytical procedures with the aim of providing crucial drug trafficking and distribution intelligence information. HSP’s typically analyze and evaluate major synthetic impurities and adulterants, and these approaches have been extensively researched and previously reported [1,4–10]. Almost 25% of the World’s legal poppy straw is produced in Tasmania. The Tasmanian poppy industry is largely based on Papaver somniferum L. (PSL), which typically produces between 40 and 80% morphine and related alkaloids. Indeed, Tasmania is currently the worlds largest producer of opium
0379-0738/$ – see front matter. Crown Copyright # 2007 Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.forsciint.2007.07.002
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alkaloids [11]. However, since 1994 there has been a concerted effort by Tasmanian Alkaloids, the biggest grower of opium poppies, to develop a high thebaine poppy variety, and to simplify the processing of the alkaloids, thereby increasing production efficiency [12]. Thebaine is a more useful and lucrative product versus morphine. There have been previous reports of spontaneous Papaver somniferum mutants containing high thebaine concentrations; however these mutants only possessed a chromosomal abnormality, eventually resulting in revision back to a morphine type [13]. Ensuing screening and mutagenesis programs by Tasmanian Alkaloids ultimately led to the development of Papaver somniferum Norman. This cultivar produces approximately the same quantities of alkaloids per hectare, but with thebaine and oripavine (not morphine) as the major constituents. Routine screening by Tasmanian Alkaloids estimates the major alkaloid content of the Papaver somniferum N. (PSN) cultivar as a function of dry weight to be; thebaine (2.0%), oripavine (0.8%), codeine (0.01%) and morphine (0.05%) compared with the traditional Papaver somniferum L. (PSL) cultivar with morphine (2.4%), codeine (0.1%), oripavine (0.03%) and thebaine (0.1%) by dry weight. Morphine is present as a by-product from PSL infestation as a weed in the PSN crop and there is significant season-to-season variation in these minor alkaloids (morphine and codeine) [14]. Since initial plantings in 1996/7 (500 ha), PSN now accounts for 60% of the crop under cultivation [15]. Isolation of morphine from PSN is via harvesting and extraction of the poppy straw (PSN does not ‘bleed’ opium sap). In 1998–99, sufficient flower pods (66, 013) were stolen with the potential to produce ca 500 kg of heroin. The distribution of PSL versus PSN in these stolen flower pods was not reported. Accordingly, there is a need to identify HSP markers that will specifically identify heroin of PSN origin. In this regard, researchers have identified the alkaloid, oripavine (1) (Fig. 1) as being Tasmanian PSN specific [12,13,15–20]. Consequently, the presence of oripavine and its reaction by-products derived from refluxing in acetic anhydride in seized heroin samples identifies Tasmanian origin heroin derived from PSN. Herein we wish to report the isolation and identification of oripavine derived byproducts from the heroin from Tasmanian PSN opium.
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Unless otherwise stated all other reagents were used as supplied.
2.2. Fourier transform infrared and nuclear magnetic resonance spectroscopy Fourier Transform Infrared Spectra (FTIR) were recorded at 4 cm 1 resolution as NaCl films or casts using a Perkin-Elmer Paragon 1000 FT-IR Spectrophotometer and chloroform as the solvent. 1 H and 13C NMR spectra were recorded on a Bruker Avance-300DPX 300 MHz NMR spectrometer, using either deuterated chloroform (CDCl3) or methanol (CD3OD) as the solvent. For spectra recorded in CDCl3 the residual signals at d7.24 ppm for 1H and the triplet at d77.0 ppm for 13C were used as internal standards. When recorded in CD3OD the residual quintet at d3.35 ppm for 1H and the septet at d49.0 ppm for 13C were used as internal standards.
2.3. Thin layer and preparative chromatography Thin layer chromatography (TLC) was performed on Merck aluminum backed sheets pre-coated with silica gel 60 F254 (0.2 mm layer thickness). Visualization was achieved through exposure to UV light, followed by dipping in a phosphomolybdic and ceric sulfate mixture (2.5% H2SO4, 2% H3PO3MoO4, 1% Ce(SO4)2), and subsequent charring. Short column (‘speedy’) chromatography was performed using a sintered filter funnel (70 mm 55 mm) packed with approximately 30 g of silica gel HF254 (Merck). A gradient solvent system was employed commencing with 3 20 ml portions of dichloromethane:hexane (60:40). The polarity of the system was then increased maintaining a 20 ml portion size throughout: dichloromethane:hexane, 80:20; 100:0, dichloromethane:ethyl acetate; 60:40; 40:60; and finally the addition of three portions of ethyl acetate:methanol: 80:20; 60:40; and 40:60. The gel was then washed with 4 50 ml portions of acetone. Centrifugal chromatography (Chromatotron) separations were carried out on a Harrison Research Chromatotron using glass rotors coated with 2 mm of gypsum binder containing silica gel 60 PF254.
2.4. HPLC and GCMS instrumentation and conditions
2. Methods and materials
Gas chromatography/mass spectrometry was carried out using a Shimadzu QP–5050A GCMS. Chromatographic separation was achieved on a HP-5MS (5% phenyl methyl siloxane) capillary column (length 30 m, I.D 0.25 mm, film thickness 0.25 mm) with helium carrier gas at a flow of 1.2 ml/min (pressure 88.9 kPa). The injection temperature was maintained at 250 8C, with 1 ml splitless injection. The column was heated at 100 8C for 1 min, then increased at a rate 28 8C/min to 240 8C, then 2 8C/min to 280 8C, followed by 6 8C/min to 320 8C. High performance liquid chromatography (HPLC) was performed on a Waters 600 series system equipped with a Waters 996 diode array detector measuring at 290 nm. Chromatographic separation was achieved using a semipreparative steel column (10 mm silica gel, 250 mm 10 mm, Phenomenex) at a flow rate of 5 ml/min with a mixture of hexane and ethyl acetate as the mobile phase.
2.1. Sample purity and resourcing
2.5. Heroin signature analysis
All solvents used were HPLC grade or bulk solvents re-distilled from glass before use. Oripavine was obtained as a gift from the National Analytical Reference Laboratory (NARL), Pymble NSW.
Heroin hydrochloride (70%, 50 mg) was taken up in 5% H2SO4 (4 ml) and a 60:40 mixture of CH2Cl2 and diethyl ether (4 ml) added. The mixture was vortexed for 10 min and centrifuged for 10 min. The upper solvent layer (4 ml) was carefully removed and evaporated to dryness under a stream of nitrogen at 60 8C. The residue was derivatised prior to analysis with the addition of 250 mM of a derivatising solution containing 50% CH2Cl2 and 50% BSTFA (containing 1% trimethylchlorosilane (TMCS). Finally 50 ml of the internal standard (500 ppm benzopinacolone) was added and the mixture was heated at 60 8C for 30 min, and subjected to GC–MS analysis (as described in Section 2) [5].
2.6. Acetylation of Tasmanian poppy straw
Fig. 1. Oripavine (1).
Acetic anhydride (600 ml) was added slowly to Tasmanian PSN poppy straw concentrate (200 mg) and the resulting solution was stirred and heated to 80 8C. After 2.25 h the reaction mixture was allowed to cool to room temperature, and
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then to 0 8C. The pH was adjusted with cold 10% sodium carbonate and the mixture was then extracted with CHCl3 (3 10 ml), dried over MgSO4, and evaporated to afford 177.2 mg (87%) of crude heroin base.
2.7. Purification and identification of oripavine acetylation product 2.7.1. Oripavine 3-acetate (2) Crude (2) was obtained from the MeOH:EtOAc ‘speedy’ column fractions and purified by flash chromatography (silica gel, 4 g) using CHCl3:MeOH (9:1) as the mobile phase to yield (2) (22%) as a brown oil. 1 H NMR (CDCl3): dH 1.8–1.85 (1H, m), 2.2–2.26 (1H, m), 2.26 (3H, s), 2.57 (3H, s), 2.76–2.84 (1H, m), 2.89 (1H, dd, J = 6.9, 18.9 Hz), 3.02 (1H, dt, J = 3.5, 13.2 Hz), 3.45 (1H, d, J = 18.6 Hz), 3.62 (3H, s), 3.82 (1H, d, J = 6.9 Hz), 5.06 (1H, d, J = 6.5 Hz), 5.31 (1H, s), 5.66 (1H, d, J = 6.5 Hz), 6.64 (1H, d, J = 8.2 Hz), 6.78 (1H, d, J = 8.2 Hz). 13 C NMR (CDCl3): dC 168.2 (s), 152.5 (s), 147.1 (s), 133.1 (s), 131.8 (s), 131.5 (s), 128.4 (s), 121.8 (d), 119.0 (d), 113.7 (d), 95.5 (d), 88.8 (d), 60.5 (d), 54.6 (q), 44.7 (t), 45.2 (s), 41.7 (q), 34.9 (t), 30.7 (t), 20.9 (q). FTIR (cm 1): 3018 (m), 1758 (s), 1604 (m), 1445 (m), 1214 (s). MS: m/z 339 (M+, 99), 297 (M–CH2CO, 100), 282 (20), 240 (20), 165 (8), 43 (65). 2.7.2. 3-Acetyl-N-acetyldesthebaine (3) A mixture of (1) and (6) was obtained from the DCM:EtOAc ‘speedy’ column fractions. Separation from (6) was achieved using the Chromatotron (centrifugal chromatography) (2 mm plate thickness) and a five-step gradient elution system (EtOAc:hexane (a) 6:4; (b) 8:2; (c) 1:0; (d) EtOAc:MeOH 9.5:0.5; (e) 9:1). (3) (147 mg, 30%) degraded upon storage in EtOAc and was repurified by flash chromatography (Redisep, Silica Gel, 4 g) using ether as the mobile phase to yield (3) (35 mg, 7% of oripavine mass) as a yellow oil. The 1H and 13C NMR spectra of (3) indicated the presence of two conformers. 1 H NMR (CD3OD): dH 1.5–1.87 (2H, m), 1.87 and 1.95 (3H, s), 2.29 and 2.30 (3H, s), 2.75 and 2.84 (3H, s), 3.19–3.36 (2H, m), 3.36–3.62 (2H, m), 3.9
(3H, s), 5.8 and 5.83 (1H, d, J = 10.4 Hz), 6.1–6.15 (1H, m), 6.15 and 6.18 (1H, d, J = 10.3 Hz), 6.91 (1H, d, J = 8.9 Hz), 6.92 (1H, d, J = 9 Hz). 13 C NMR (CD3OD): dC 171.0 and 170.9 (s), 167.9 (s), 148.1 and 148.0 (s), 139.8 and 139.6 (s), 138.9 and 138.8 (s), 131.5 and 131.0 (s), 130.75 and 130.7 (s), 130.62 and 130.6 (s), 130.5 and 130.48 (s), 127.0 and 126.6 (d), 123.2 and 123.1 (d), 121.3 and 121.1 (d), 120.8 and 120.65 (d), 120.6 and 120.1 (d), 57.7 (q), 46.2 (s), 45.3 and 42.5 (t), 35.5 and 34.1 (q), 36.2 and 34.9 (t), 29.3 and 29.2 (t), 19.7 and 18.9 (q), 18.5 and 18.4 (q). FTIR (cm 1): 2979 (m), 1766 (s), 1651 (s), 1634 (s), 1490 (m), 1214 (s), 751 (s). MS: m/z 381 (M+, 36), 339 (M–CH2CO, 10), 266 (18), 238 (16), 101 (82), 86 (58), 44 (100). 2.7.3. 3-Acetyl-6-methoxy-4,5-epoxyphenanthrene (4) A mixture of (4) and (5) (83 mg) was obtained from the DCM:hexane ‘speedy’ column fractions. Separation from (5) was achieved using the Chromatotron (1 mm plate thickness) and EtOAc:hexane (6:4) as the mobile phase. (4) was obtained as a yellow solid (27 mg, 5.4% of oripavine mass). M.p. 110– 112 8C. 1 H NMR (CD3OD): dH 2.47 (3H, s), 4.34 (3H, s), 7.47 (1H, d, J = 8.3 Hz), 7.53 (1H, d, J = 8.2 Hz), 7.78 (1H, d, J = 8.2 Hz), 7.79 (1H, d, J = 8.3 Hz), 7.82 (1H, d, J = 8.9 Hz), 7.91 (1H, d, J = 8.9 Hz). 13 C NMR (CD3OD): dC 167.0 (s), 144.3 (s), 141.1 (s), 140.8 (s), 129.5 (s), 125.6 (s), 125.1 (s), 124.7 (s), 121.9 (s), 125.0 (d), 123.3 (d), 122.9 (d), 121.5 (d), 120.7 (d), 116.7 (d), 58.5 (q), 20.0 (q). FTIR (cm 1): 1766 (s), 1626 (m), 1500 (m), 1435 (m), 1199 (s), 1149 (m), 1011 (m), 887 (w), 756 (m). MS: m/z 280 (M+, 25), 238 (M–CH2CO, 100), 223 (70), 139 (15), 75 (28). 2.7.4. 3,4-Diacetyl-6-methoxyphenathrene (5) Was obtained as a dark orange solid (52 mg, 10.5% of oripavine mass) with m.p. 156–160 8C, as outlined above.
Fig. 2. Synthesis and purification of acetylated oripavine products.
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Fig. 3. Chemical structures of compounds 2–6. 1
H NMR (CDCl3): dH 2.38 (3H, s), 2.54 (3H, s), 3.97 (3H, s), 7.28 (1H, dd, J = 2.2, 8.8 Hz), 7.47 (1H, d, J = 8.7 Hz), 7.57 (1H, d, J = 8.9 Hz), 7.66 (1H, d, J = 8.9 Hz), 7.8 (1H, d, J = 8.7 Hz), 7.81 (1H, d, J = 8.8 Hz), 8.57 (1H, d, J = 2.2 Hz). 13 C NMR (CDCl3): dC 168.5 (s), 167.8 (s), 158.2 (s), 158.2 (s), 140.7 (s), 139.7 (s), 132.5 (s), 130.0 (s), 129.9 (d), 127.7 (s), 127.5 (d), 127.4 (s), 127.0 (d), 124.5 (d), 121.6 (d), 116.6 (d), 108.5 (d), 55.3 (q), 21.0 (q). FTIR (cm 1): 1772 (s), 1617 (m), 1512 (m), 1452 (m), 1206 (s). MS: m/z 324 (M+, 16), 282 (M–CH2CO, 13), 240 M – 2 CH2CO, 100), 139 (11), 43 (37). 2.7.5. 3,4-Diacetyl-6-methoxy-5-[2-(N-methylacetamido)]ethylphenanthrene (6) Separation of (6) from (3) was achieved as outlined above. Final purification of (6) was carried out via HPLC (normal phase) with EtOAc:hexane (Gradient, Start 9:1, End 1:0) as the mobile phase to obtain a yellow oil (5.8 mg, 1.2% of oripavine mass). 1H and 13C NMR spectra of (6) showed the presence of two conformers. 1 H NMR (CDCl3): dH 1.42 and 1.64 (3H, s), 2.1 and 2.12 (3H, s), 2.35 (3H, s), 2.17 and 2.54 (3H, s), 3.2–3.38 (2H, br m), 3.45–3.55 (2H, br m), 3.99 and 4.02 (3H, s), 7.26 (1H, d, J = 8.7 Hz), 7.36 (1H, d, J = 8.5 Hz), 7.41 (1H, d, J = 8.8 Hz), 7.47 (1H, d, J = 8.8 Hz), 7.68 (1H, d, J = 8.7 Hz), 7.73 (1H, d, J = 8.5 Hz). 13 C NMR (CDCl3): dC 169.9 (s), 169.9 (s), 168.0 (s), 156.5 (s), 140.2 (s), 137.8 (s), 131.8 (s), 127.9 (s), 127.4 (s), 124.1 (s), 122.0 (s), 126.85 and 126.8 (d), 126.6 and 126.4 (d), 125.3 and 124.9 (d), 122.3 and 122.2 (d), 121.0 and 120.7 (d), 111.9 and 111.4 (d), 55.8 and 55.4 (q), 50.5 and 46.5 (t), 34.6 and 32.5 (t), 28.9 and 27.8 (t), 20.1 (q), 20.05 and 20.0 (q), 19.9 and 19.6 (q). FTIR (cm 1): 2936 (m), 1770 (s), 1633 (s), 1591 (m), 1500 (m), 1434 (m), 1201 (s). MS: m/z 423 (M+, 12), 381 (M–CH2CO, 13), 339 (M – 2 CH2CO, 16), 266 (39), 251 (43), 248 (36), 100 (12), 44 (100).
(500 mg) was treated with acetic anhydride (2 ml) at 80 8C for 2 h, and then the reaction mixture was evaporated in vacuo to leave a dark orange residue (613 mg). Subsequent chromatographic fractionation techniques yielded five pure compounds 2–6 (Figs. 2 and 3). Of these 3–6 have not previously been reported and 2 has only been reported, without spectral data, as an oxidation product of heterocodeine 3-acetate [16]. Separately, we utilized the same experimental condition, and identified compounds 3–6 in the resulting heroin product (Fig. 4). 4. Discussion The isolation and structural elucidation of 2–6 provides a simple and effective method for the detection of Tasmanian PSN opium derived heroin. These compounds are well resolved and present in sufficient quantities to allow detection using the current Australian National Heroin Signature Program GC–MS procedure for trace organic analysis (see Section 2) [5]. Fig. 4 shows the appearance of 3–6 in a typical ‘Tasmanian PSN’ heroin sample, i.e. a sample of heroin prepared by the
3. Results In order to simplify the identification and isolation of oripavine-derived species, we commenced our studies with pure oripavine (supplied by the National Analytical Reference Laboratory) and subjected it to the standard Thiboumery and Mohr methodology for heroin synthesis [21]. Thus, oripavine
Fig. 4. Identification of oripavine derived products 3–6 in a typical Tasmanian heroin sample. Note: the PSN does not yield high quantities of morphine, hence low heroin yields.
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Fig. 5. GC–MS traces of illicit heroin seized with various geographical origins. Key marker compounds have been highlighted: heroin; acetyl codeine (AC); 3monoacetylmorphine (3-MAM); and 6-monoacetylmorphine (6-MAM). (a) GC–MS trace of heroin identified as being of SWA (Turkey) origin. (b) GC–MS trace of heroin identified as being of SWA (Pakistan) origin. (c) GC–MS trace of heroin identified as being of SA (Colombia) origin. (d) GC–MS trace of heroin identified as being of SWA (Myanmar) origin. (e) GC–MS trace of heroin identified as being of Tas PSL origin.
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Fig. 5. (Continued ).
acetylation of opium extracted from Tasmanian PSN poppies. The advantage of this technique is that, unlike other profiling methods, which rely on differences in production processes that result in quantitative differences in the ratios of commonly encountered compounds, it is based on ‘marker’ impurities that are unique to Tasmanian grown PSN poppies [15,22]. Thus, a heroin sample containing 3–6 can only be explained by the presence of oripavine in the original opium extract, which occurs only in Tasmanian grown PSN poppies. These findings were consistent across a number of Tasmanian PSN heroin samples examined (n = 5). In all HSP except the U.S., distinction between SEA, SWA, South American, or Mexican heroin samples is based solely on the morphine extraction and acetylation procedures employed in its production (see Section 2), referred to as ‘processing’ origin. Although these methods may be accurate, to a degree, in determining processing origin, they do not guarantee that a sample made using a typical procedure actually originated from that geographical origin. Consequently, as long as oripavine remains unique to Tasmanian PSN poppies, the detection of 3–6 in seized samples will provide unequivocal evidence of its origin. The
U.S. HSP procedures use a combination of impurity profiling, trace solvent analysis, and isotopic ratio mass spectrometry, and are typically 95% accurate in assigning geographical origin. The GC–MS trace for heroin based on Tasmanian PSN poppies (Fig. 4) clearly shows 3–6. Our initial assignments of these compounds were tentative via mass spectral analysis prior to analysis and complete characterization of these analogues in this work. The acetylation of oripavine afforded sufficient quantities of compounds 3–6 for identification purposes. The presence of 3–6 in typical ‘clan-lab’ preparations (Fig. 4) clearly shows that our efforts to maximize retrieval for identification did not contribute to the formation of these impurities. It is noteworthy that Tasmanian PSN derived heroin samples actually have low levels of heroin. This is a consequence of the low morphine concentration in the PSN poppy, typically 0.05%, however this is often contaminated with the more traditional PSL with a morphine content of 2.4%. Given that the thieves are not (normally) aware which cultivar they steal, PSN derived heroin is often contaminated with PSL derived heroin, and this is further exacerbated by inappropriate collection of
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both PSN and PSL cultivar by seasonal harvesters. Fig. 5 shows the corresponding GC–MS traces for heroin seized from a variety of geographical origins; Turkey, Pakistan, Columbia, Myanmar, and Tasmania (PSL cultivar). In all of these instances the GC–MS traces display significant differences from those observed with that of Tasmanian PSN origin. Most notably, Fig. 5 shows no evidence of 3–6, whilst these compounds are readily identified in the Tasmanian PSN sample. Acknowledgements LO wishes to acknowledge generous financial support from the Australian Forensic Drug Laboratory (Pymble, NSW, Australia). We also acknowledge the generous donation of oripavine from the National Analytical Reference Laboratory (Pymble, NSW, Australia). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.forsciint.2007.07.002. References [1] United Nations Drug Control Program, World Illicit Drugs Report 2006, New York, 2001. http://www.unodc.org/unodc/world_drug_report.html. [2] Australian Crime Commission, Illicit Drug Report 2005-6; http:// www.crimecommission.gov.au/html/pg_iddr2005-06.html. [3] Australian Institute of Health and Welfare, 2004 National Drugs Strategy Household Survey 2004: First Results; http://www.aihw.gov.au/publications/index.cfm/title/10122. [4] R. Dams, T. Benijts, W.E. Lambert, D.L. Massart, A.P. De Leenheer, Heroin impurity profiling: trends throughout a decade of experimenting, Forensic Sci. Int. 123 (2001) 81–88. [5] R.-B. Myors, P.-T. Crisp, S.-V. Skopec, R.-J. Wells, Investigation of heroin profiling using trace organic impurities, Analyst 126 (2001) 679– 689.
[6] Office on drugs and crime, Methods for Impurity Profiling of Heroin and Cocaine, United Nations, New York, 2005. [7] T. Bora, M. Merdivan, C. Hamamci, Levels of trace and major elements in illicit heroin, J. Forensic Sci. 47 (2002) 959–963. [8] A.C. Allen, D.A. Cooper, J.M. Moore, M. Gloger, H. Neumann, Illicit heroin manufacturing by products: capillary gas chromatography, determination and structural elucidation of narcotine and norlaudanosine related compounds, Anal. Chem. 56 (1984) 2940–2947. [9] P. Esseiva, L. Dujourdy, F. Anglada, F. Taroni, P. Margot, A methodology for illicit heroin seizures comparison in a drug intelligence perspective using large databases, Forensic Sci. Int. 132 (2003) 139–152. [10] V. Dufey, L. Dujourdy, F. Besacier, H. Chaudron, A quick and automated method for profiling heroin samples for tactical intelligence purposes, Forensic Sci. Int. 169 (2007) 108–117. [11] International Narcotics Control Board, Narcotic Drugs, estimated world requirements for 2007, United Nations, New York 2007; http://www.incb.org/incb/en/narcotic_drugs.html. [12] J.C. Laughlin, B. Chung, B.M. Beattie, in: J. Bernath (Ed.), Poppy – The Genus Papaver, Harwood Academic Publishers, Amsterdam, 1998, pp. 249–277. [13] U. Nymann, O. Hall, Some varieties of Papaver somniferum L. with changed morphine alkaloid content, Hereditas 84 (1976) 69–76. [14] A.J. Fist, Tasmanian Alkaloids, Westbury, Tasmania, 7303, Australia, Personal communication. [15] A.J. Fist, The Tasmania poppy industry: a case study of the application of science and technology, in: Proceedings 10th Agronomy Conference, Hobart, Australia, 2001. [16] R.B. Barber, H. Rapport, Synthesis of thebaine and oripavine from codeine and morphine, J. Med. Chem. 18 (1975) 1074–1077. [17] A. Allen, D. Cooper, J. Moore, C. Teer, Thebaine rearrangements: nonclassical ring migrations, J. Org. Chem. 49 (1984) 3462–3465. [18] G.A. Cordell, Introduction to Alkaloids, 1st ed., John Wiley and Sons, Brisbane Australia, 1981. [19] Merck and Co., The Merck Index, 12th ed., Merck Research Laboratories, Whitehouse Station, 1996. [20] C.V. Trenerry, R.J. Wells, Analysis of drugs of abuse by micellar electrokinetic capillary chromatography, Progress in HPLC 5 (1996) 1–55. [21] D.A. Cooper, in: M. Klein, F. Sapienza, H. McClain, I. Khan (Eds.), Clandestinely Produced Drugs and Precursors, United States Department of Justice, Drug Enforcement Administration, Washington, DC, 1989. [22] B. Neilsen, J. Roe, Oripavine – a new opium alkaloid, Planta Med. 48 (1983) 205–207.