Journal of Mass Spectrometry - Peer review proof only. Spice and other herbal blends: harmless incense or cannabinoid designer drugs?

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1 Spice and other herbal blends: harmless incense or cannabinoid designer drugs? Journal: Journal of Mass Spectrometry Manuscript ID: JMS-0-0 Wiley - Manuscript type: JMS Letters Date Submitted by the Author: -Dec-00 Complete List of Authors: Auwärter, Volker; Institute of Forensic Medicine, Forensic Toxicology Dresen, Sebastian; Institute of Forensic Medicine, Forensic Toxicology Weinmann, Wolfgang; Institute of Forensic Medicine, Forensic Toxicology Müller, Michael; Institute of Pharmaceutical Sciences, Pharmaceutical and Medicinal Chemistry Puetz, Michael; Bundeskriminalamt, KT -Forensic Toxicology Ferreirós Bouzas, Nerea; Institute of Forensic Medicine, Forensic Toxicology Keywords: cannabinoids, designer drugs, herbal drugs, spice, blood analysis

2 Page of Journal of Mass Spectrometry - Peer review proof only Dear Sir, Spice and other herbal blends: harmless incense or cannabinoid designer drugs? Since 00 herbal mixtures like Spice are sold in Switzerland, Austria, Germany and other European countries mainly via internet shops. Although declared as incense they are smoked as bio-drugs by the consumers. In corresponding blogs drug users reported cannabis-like effects after smoking. These products enjoy great popularity particularly among younger people, as up to now the mixtures are sold legally in many countries without age restriction. Moreover, commonly used drug tests are not able to detect use of these drugs so far. The list of ingredients indicates potentially bioactive herbs containing alkaloids (e.g. aucubin in Pedicularis densiflora, declared as Indian Warrior or leonurine in Leonotis leonurus declared as Wild Dagga or Lion s Tail ) which could in general produce cannabis-like effects. However, the suspicion was raised that synthetic compounds added underhand may be the main cause for pharmacological activity. To prove pharmacological activity and to gain drug positive blood and urine samples, a selfexperiment was conducted by two of the authors. One cigarette containing 0. g of Spice diamond was smoked together and several blood and urine samples were collected. Approximately minutes post application first noticeable effects occurred in the form of considerably reddened conjunctivae, significant increase of pulse rates, xerostomia and an alteration of mood and perception. In objective psychomotor tests, no abnormalities were detected, although the subjects had the impression of being moderately impaired. The effects continued for about hours under slow attenuation. The whole next day some minor aftereffects were still noticeable. These findings were consistent with the majority of reports available on the internet and confirmed the presence of pharmacologically active compounds. For isolation of active compounds, ethanolic extracts of the materials (0. g of Spice silver, Spice gold, Spice diamond, Smoke, Sence, Skunk and Yucatan Fire each, with ml ethanol) were prepared. Routine qualitative drug analysis procedures including GC-MS- Screening with library search, multi target screening by LC-MS/MS [] and immunological screening procedures revealed no evidence for the presence of any illegal drug or known active pharmaceutical ingredient. Only alpha-, beta- and gamma-tocopherol as well as some phytosterols were identified. Furthermore three abundant signals were detected in GC-MS with unknown mass spectra (fig. ). The signals at. and. min showed similar EI mass spectra with a nominal molecular mass of amu (compound and ). The third signal (compound ) was detected at. min. Compound was identified by a laboratory in Frankfurt as JWH 0, a synthetic cannabimimetic aminoalkylindole (news item dpa, December th 00) with an about -fold affinity to the CB receptor and about -fold affinity to the CB receptor compared to -THC () [,]. The fragmentation as proposed in fig. explains the main fragments seen in the EI/MS spectrum. Thin layer chromatography (TLC) of the extracts of three commercially available variants of Spice ( silver, gold and diamond ) showed an increase in intensity of the spot corresponding to compound and after dyeing (Fast Blue RR Salt, NaOH), in parallel to an increase in price of product (fig. ). The UV-spectrum confirmed the presence of a phenolic chromophore. High-accuracy mass measurement (monoisotopic mass of [M+H] + :. Da ± ppm) led to the molecular formula C H O (calculated monoisotopic mass of [M+H] + :. Da). For isolation of compound and, g Spice diamond were extracted with n-heptane and separated on a preparative silica gel column (00 mm x 0 mm i.d., silica 0, mm particle size; solvent for elution: cyclohexane:ethyl acetate from 0: (v/v) to : (v/v)). The fractions containing and/or were dried under a nitrogen stream. Approximately 0 mg of raw product were obtained, corresponding to a concentration of roughly %. NMR studies and interpretation of EI-MS spectra

3 Page of (underivatised, trimethylsilylated and acetylated, cp. fig. and ) and ESI-IonTrap-MS n experiments (fig. ) lead to the structure of the synthetic cannabinoid (cp. fig. ). The isotopic patterns of molecular ions and main fragments of all GC-MS spectra were in good agreement with the proposed structures, too. Compound is a homolog of a non-classical cannabinoid called CP, (compound ) and is formally derived from by prolonging the dimethylheptyl side chain to dimethyloctyl. C-NMR data of from Xie et al. [] are shown together with data of compound in table. Both and are potent CB and CB agonists with being the most potent in the row of homologs []. Compound was found to be the trans-diastereomer of which is a much weaker cannabinoid receptor agonist and may be a side product of the synthesis procedure which according to Melvin et al. [] includes a reduction of a racemic ketone precursor with NaBH. The analgesic potency of CP, () in various mouse models is about to -fold higher compared to -THC []. Compton et al. found compound and to have similar pharmacological effects as -THC, starting at considerably lower doses []. A further study showed that qualitatively resembles -THC pharmacology, showing a to times greater potency compared to -THC depending on the applied model []. In table the compounds found in the different herbal mixtures are listed. Structures are shown in figure. In the preparations named Smoke and Skunk, besides JWH 0 () a larger quantity of oleamide () was found. Oleamide exhibits cannabinoid-like behavioural responses when ingested [] and may be an additional adulterant in these products. Sence contained primarily compound and, but small amounts of and its less potent transdiastereomer were detected alongside with benzophenone. The different Spice variants and Yucatan Fire contained mainly and, only in some of the tested packets also JWH 0 was detectable. Further products are currently under investigation in our labs. Compound was detected in the blood samples obtained from the self-experiment ( Spice diamond ) using C solid phase extraction, trimethylsilylation and GC-EI/MS (modified routine method for cannabinoid analysis in blood []). All samples showed signals in SIM mode (targets ions m/z,, ) at the expected retention time and with the correct relative ion intensities. Three blank serum samples were analysed in the same series and showed negative results. Figure shows typical XIC s for a positive and a negative sample. Screening with an immunoassay based on an antibody specific for -nor--carboxy-delta- THC (major oxidative metabolite of THC) was negative for all blood and urine samples. Obviously, compounds,, and and their metabolites do not show significant cross reactivity to the used assay. This is so far the first time cannabinoid-like designer drugs were used as adulterants in commercially available products designed for inhalative application. Compounds and are strong cannabinoid receptor agonists and therefore account for the cannabis-like effects of Spice and further herbal blends. So far nothing is known about metabolism of these compounds. Some of the metabolites may be toxic and/or pharmacologically active. Furthermore, it can be assumed that from batch to batch there are differences in the amounts of drugs applied, resulting in the risk of accidental overdosing, which in the last weeks occurred several times in our region with hospitalisation of the patients. There are hundreds of compounds more with cannabinoid receptor activity and it can be assumed that further substances will appear on the market soon, which will be an ongoing challenge for toxicologists as well as for law enforcement. Yours,

4 Page of Journal of Mass Spectrometry - Peer review proof only Volker Auwärter, Sebastian Dresen, Wolfgang Weinmann, Michael Müller, Michael Pütz and Nerea Ferreirós Institute of Forensic Medicine, Forensic Toxicology, University Medical Centre Freiburg, Albertstr., Freiburg, Germany Institute of Pharmaceutical Sciences, Albert-Ludwigs-University Freiburg, Albertstr., Freiburg, Germany Bundeskriminalamt, Federal Criminal Police Office, Forensic Science Institute, KT- Toxicology, Wiesbaden, Germany References [] Mueller CA, Weinmann W, Dresen S, Schreiber A, Gergov M. Development of a multitarget screening analysis for 0 drugs using a QTrap liquid chromatography/tandem mass spectrometry system and automated library searching. Rapid Commun Mass Spectrom (00) - [] Huffman JW, Zengin G, Wu MJ, Lu J et al. Structure-activity relationships for -alkyl-- (-naphthoyl)indoles at the cannabinoid CB() and CB() receptors: steric and electronic effects of naphthoyl substituents. New highly selective CB() receptor agonists. Bioorg Med Chem (00) - [] Aung MM, Griffin G, Huffman JW, Wu M, Keel C, Yang B, Showalter VM, Abood ME, Martin BR. Influence of the N- alkyl chain length of cannabimimetic indoles upon CB() and CB() receptor binding. Drug Alcohol Depend 0 (000) - [] Xie XQ, Yang DP, Melvin LS, Makriyannis A. Conformational analysis of the prototype nonclassical cannabinoid CP-,, using D NMR and computer molecular modeling. J Med Chem () - [] Melvin LS, Milne GM, Johnson MR, Subramaniam B, Wilken GH, Howlett AC. Structure-activity relationships for cannabinoid receptor-binding and analgesic activity: studies of bicyclic cannabinoid analogs. Mol Pharmacol () 0- [] Melvin LS, Johnson MR, Harbert CA, Milne GM, Weissman A. A cannabinoid derived prototypical analgesic. J Med Chem () - [] Koe BK, Milne GM, Weissman A, Johnson MR, Melvin LS. Enhancement of brain [H]flunitrazepam binding and analgesic activity of synthetic cannabimimetics. Eur J Pharmacol () 0- [] Compton DR, Johnson MR, Melvin LS, Martin BR. Pharmacological profile of a series of bicyclic cannabinoid analogs: classification as cannabimimetic agents. J Pharmacol Exp Ther 0 () 0-0 [] Weissman A, Milne GM, Melvin LS Jr. Cannabimimetic activity from CP-,, a derivative of -phenylcyclohexanol. J Pharmacol Exp Ther () -

5 Page of [] Thakur GA, Nikas SP, Makriyannis A. CB cannabinoid receptor ligands. Mini Rev Med Chem (00) -0 [] Stumptner D, Kempf J, Weinmann W. Increase of extraction efficiency for THC in serum by larger SPE cartridges (German). In: GTFCH-Symposium 00: Ausgewählte Aspekte der Forensischen Toxikologie. Hrsg.: F. Pragst, R. Aderjan, Verlag Dr. Dieter Helm, Heppenheim 00

6 Page of Journal of Mass Spectrometry - Peer review proof only Table : C NMR chemical shift assignments of CP, ( MHz, data taken from Xie et al. []) and compound (0. MHz, isolated from Spice diamond ) in CDCl solution at K. Chemical shifts are reported in ppm relative to CDCl (δ =.0) as internal standard. Carbon (cp. fig., compound ) Chemical shift (ppm) for CP, Carbon (cp. fig., compound ) Chemical shift (ppm) for compound

7 Page of Table : List of compounds found in the different herbal mixtures. The number of + indicates relative content of the respective compound as estimated from signal intensities in the corresponding GC-MS chromatograms. Product Compounds and Compound Compound Compound Other substances Spice silver + + (?) * - - ethylvanillin Spice gold ++ + (?) * - - ethylvanillin Spice diamond (?) * - - ethylvanillin Yucatan Fire eucalyptol Smoke eugenol Skunk eugenol Sence benzophenone * Compound (JWH 0) was only found in some of the investigated packets and in much lower quantity compared to Yucatan Fire, Smoke and Skunk.

8 Page of Journal of Mass Spectrometry - Peer review proof only Figure captions Figure : Chemical structures of cannabinoid receptor ligands. : JWH 0, : CP,, : -THC, : oleamide. Figure : GC-MS chromatogram (TIC) of an ethanolic Spice extract with mass spectra of the suspicious peaks. Main fragments are indicated for compound and. Figure : TLC plate (silica gel 0, n-hexane/diethylether [:(v/v)]) with extracts of three variants of Spice and a cannabis control. The orange spots show compounds and after dyeing with Fast Blue RR Salt. Quotation of prices from on October th 00. Figure : EI/MS spectra of compound after derivatisation (chromatograms not shown) with illustration of the main fragments. The upper mass spectrum corresponds to compound after silylation, lower after acetylation. Figure : ESI-IonTrap-MS n spectra of compound after TLC of an extract of Spice diamond with indicated fragmentation. Instrument: Bruker HCTplus Iontrap with sheath liquid coaxial ESI interface (Agilent Technologies), sheath liquid: isopropanol/h O [: (v/v)], flow µl/min. Figure : Extracted ion chromatograms (GC-EI/MS) for a drug positive sample from the selfexperiment (a) and a blank serum sample (b). SIM mode with m/z, and for detection of compound, internal standard -hydroxy- -THC-D.

9 Page of Chemical structures of cannabinoid receptor ligands. : JWH 0, : CP,, : -THC, : oleamide. xmm (00 x 00 DPI)

10 Page of Journal of Mass Spectrometry - Peer review proof only GC-MS chromatogram (TIC) of an ethanolic Spice extract with mass spectra of the suspicious peaks. Main fragments are indicated for compound and. xmm (00 x 00 DPI)

11 Page of TLC plate (silica gel 0, n-hexane/diethylether [:(v/v)]) with extracts of three variants of Spice and a cannabis control. The orange spots show compounds and after dyeing with Fast Blue RR Salt. Quotation of prices from on October th 00. xmm (00 x 00 DPI)

12 Page of Journal of Mass Spectrometry - Peer review proof only TLC plate (silica gel 0, n-hexane/diethylether [:(v/v)]) with extracts of three variants of Spice and a cannabis control. The orange spots show compounds and after dyeing with Fast Blue RR Salt. Quotation of prices from on October th 00. xmm (00 x 00 DPI)

13 Page of EI/MS spectra of compound after derivatisation (chromatograms not shown) with illustration of the main fragments. The upper mass spectrum corresponds to compound after silylation, lower after acetylation. xmm (00 x 00 DPI)

14 Page of Journal of Mass Spectrometry - Peer review proof only ESI-IonTrap-MSn spectra of compound after TLC of an extract of Spice diamond with indicated fragmentation. Instrument: Bruker HCTplus Iontrap with sheath liquid coaxial ESI interface (Agilent Technologies), sheath liquid: isopropanol/ho [: (v/v)], flow µl/min. xmm (00 x 00 DPI)

15 Page of ESI-IonTrap-MSn spectra of compound after TLC of an extract of Spice diamond with indicated fragmentation. Instrument: Bruker HCTplus Iontrap with sheath liquid coaxial ESI interface (Agilent Technologies), sheath liquid: isopropanol/ho [: (v/v)], flow µl/min. xmm (00 x 00 DPI)

16 Page of Journal of Mass Spectrometry - Peer review proof only Extracted ion chromatograms (GC-EI/MS) for a drug positive sample from the self-experiment (a) and a blank serum sample (b). SIM mode with m/z, and for detection of compound, internal standard -hydroxy- -THC-D. xmm (00 x 00 DPI)

17 Page of Extracted ion chromatograms (GC-EI/MS) for a drug positive sample from the self-experiment (a) and a blank serum sample (b). SIM mode with m/z, and for detection of compound, internal standard -hydroxy- -THC-D. xmm (00 x 00 DPI)