Spice

Anal Bioanal Chem DOI 10.1007/s00216-012-6462-0

ORIGINAL PAPER

A fast and inexpensive procedure for the isolation of synthetic cannabinoids from ‘Spice’ products using a flash chromatography system Bjoern Moosmann & Stefan Kneisel & Ariane Wohlfarth & Volker Brecht & Volker Auwärter Received: 17 July 2012 / Revised: 11 September 2012 / Accepted: 26 September 2012 # Springer-Verlag Berlin Heidelberg 2012

Abstract In the age of the Internet, the variety of drugs offered online is constantly increasing, and new drugs emerge every month. One group of drugs showing such an enormous increase is that of synthetic cannabinoids. Since their first identification in ‘herbal mixtures’, new structural modifications continue to appear on the market. In order to keep up with this process, toxicological screening methods need to be up to date. This can become extremely difficult if no reference material is available. In this article, a fast and effective way to extract and purify synthetic cannabinoids from ‘herbal mixtures’ is presented. This method opens a new opportunity for a timely reaction by obtaining reference material straight out of the ‘herbal mixtures’ ordered via the Internet. Isolation was carried out on a flash chromatography system with gradient

elution on a C18 column using methanol and 0.55 % formic acid as mobile phases. The obtained purity of all compounds exceeded 99 %. In addition to the isolation of single compounds, the method proved to be suitable for the separation of various synthetic cannabinoids in one mixture, including the diastereomers cis- and trans-CP-47,497-C8. This approach for obtaining pure standards of new drugs proved to be effective, inexpensive and much quicker than waiting for the substances to be commercially available as reference material. Keywords Flash chromatography . Spice . Synthetic cannabinoids . Reference compounds

Introduction Published in the special paper collection Forensic Toxicology with guest editors Kazuhito Watanabe and Satoshi Chinaka. Presented at the Young Scientist Symposium at the 50th Annual TIAFT meeting in Hamamatsu, Japan. B. Moosmann : S. Kneisel : A. Wohlfarth : V. Auwärter (*) Institute of Forensic Medicine, Department of Forensic Toxicology, University Medical Center Freiburg, Albertstr. 9, 79104 Freiburg, Germany e-mail: [email protected] B. Moosmann : S. Kneisel Hermann Staudinger Graduate School, University of Freiburg, Hebelstr. 27, 79104 Freiburg, Germany A. Wohlfarth Chemistry and Drug Metabolism, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Baltimore, MD 21224, USA V. Brecht Institute of Pharmaceutical Sciences, University of Freiburg, Albertstr. 25, 79104 Freiburg, Germany

Since the detection of JWH-018 and CP-47,497-C8 in the ‘herbal mixture’ ‘Spice’ in 2008 in Germany [1] and CP-47,497-C8 in Japan [2], new products have appeared on this lucrative market almost on a weekly basis. Many of them contain synthetic cannabinoids already identified in hundreds of products before. However, often new substances are added due to changes in the narcotics law [3–19]. In 2011, 23 new synthetic cannabinoids were reported through the European Early Warning System of the European Monitoring Centre for Drugs and Drug Addiction (EMCDDA) [20], raising the number of identified compounds in mixtures to more than 35. Some of these compounds have never been described in the literature before [10, 13, 15, 16, 21, 22] making identification even more difficult. In addition to that, some substances disappeared from the market after a couple of months, bearing the risk that by the time reference standards are available, the product is not available on the market anymore, and people have already switched to other products. This ‘creativity’ of the producers makes it difficult for analytical laboratories to implement new substances into their screening methods

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[23–31] soon after people start consuming them. When a substance is detected for the very first time, NMR techniques need to be applied for structure confirmation. Often, there is more than just one synthetic cannabinoid present in a product, and sometimes, traces of other synthetic cannabinoids can be found. These impurities are most probably due to contamination of the equipment used for manufacturing the products. These additional synthetic cannabinoids along with other impurities extracted from the plant material can complicate and prolong substance identification. Therefore, an efficient method to separate the analyte of choice from other components can facilitate analysis and save precious time. Another crucial point is that little or sometimes nothing is known about the toxicity of these compounds, and toxicity screening assays need to be carried out. In the case of the octyl homolog of CP-47,497, most ‘herbal mixtures’, e.g. the original ‘Spice’, contain the pharmacologically active 3-cis-CP47,497-C8 [1, 32] alongside the 3-trans-CP-47,497-C8 as a synthesis by-product [33]. Little is known about the 3-transCP-47,497-C8, and in order to test the pharmacological activity and toxicity of each diastereomer individually, separation has to be performed. For these screening assays, larger quantities of the pure isomer of the substances are necessary. However, this can raise the costs of such a project if the drug has to be obtained through a commercial supplier, even though larger quantities of seized ‘herbal mixtures’ may be available to extract the compounds. On the other hand, it may be impossible to purchase them from any wholesaler, because they do not cover them with their product range yet. A separation technique which allows for fast separation and is at the same time inexpensive enough can be found in

flash chromatography systems. This technique was first published in 1978 [34] and has gained popularity in the separation of natural substances and in the field of synthetic chemistry for sample cleanup. Also in toxicology, flash chromatography techniques have become more and more interesting. In 2011, Wohlfarth et al. [35] published two different flash chromatography methods to separate the two natural cannabinoids Δ9-tetrahydrocannabinolic acid A and Δ9-tetrahydrocannabinol with one of them using a C18 column with methanol and formic acid, raising the interest in applying a flash chromatography system for the separation of synthetic cannabinoids. In this article, a procedure is presented which allows to isolate synthetic cannabinoids from ‘Spice’ products and separate various synthetic cannabinoids (Fig. 1) from one another in order to obtain reference material of high purity for timely development or completion of analytical methods. A similar approach was used in 2011 by our group to isolate MAM-2201 from a ‘herbal mixture’ [15].

Experimental Chemicals and reagents Methanol (HPLC grade) was purchased from J.T. Baker (Deventer, Netherlands); formic acid (analytical grade), chloroform (CHCl3) and silica powder were obtained from Carl Roth GmbH (Karlsruhe, Germany). Ethanol (analytical grade), ethyl acetate (analytical grade), hydrochloric acid (fuming 37 %, ACS) as well as tert-butyl methyl ether (TBME,

Fig. 1 Structural formulas of the synthetic cannabinoids isolated from various ‘herbal mixtures’ and of the relatively polar synthetic cannabinoids JWH-200 and cannabipiperidiethanone

Isolation of synthetic cannabinoids from ‘Spice’ products

anhydrous) were purchased from Sigma-Aldrich (Steinheim, Germany). Sodium hydroxide was purchased from Riedel-de Haen (Seelze, Germany), and sodium sulphate was obtained from Merck (Darmstadt, Germany). Deionized water was prepared with a cartridge deionizer from Memtech (Moorenweis, Germany), and deuterated chloroform (CDCl3) was purchased from Euriso-Top (Saint-Aubin, France).

CP-47,497-C8. For the other compounds, the time was reduced to 1 min with shaking. The extract was filtrated through a 0.45 μm Rotilabo filter (Carl Roth GmbH, Karlsruhe, Germany), and the remaining herbal residue was rinsed once with 5 mL of ethanol. The filtrated solution was added to the extract. Flash chromatography

Samples Various ‘herbal mixtures’ containing AM-2201, CP-47,497C8, JWH-015, JWH-018, JWH-073, JWH-081, JWH-122, JWH-200, JWH-203, JWH-210, JWH-250, JWH-251, RCS-4 and ortho-RCS-4 were bought over the Internet from different online shops selling ‘legal highs’ and used for extraction. To simulate a combination of four synthetic cannabinoids in one ‘herbal mixture’, 300 mg each of three different products were mixed to result in a mixture containing JWH-015, JWH-018, JWH-073 and JWH-210. Screening of the ‘herbal mixtures’ All the ‘herbal mixtures’ were analysed by GC-MS after purchase as described before [3, 14]. In short, 1 mL of ethanol was added to 100 mg of plant material. After brief vortexing and centrifugation at 2,860×g for 5 min (Heraeus Megafuge 1.0, Thermo Scientific, Schwerte, Germany), 10 μL of the supernatant was evaporated to dryness in a glass vial under a gentle stream of nitrogen. One millilitre of ethyl acetate was added to reconstitute the sample prior to the injection of 1 μL into the GC-MS system. Analysis was performed on a 6890 series gas chromatography system with a 5973 series mass selective detector, a 7683 B series injector and a Chemstation G1701GA version D.03.00.611 (Agilent, Waldbronn, Germany). The GC parameters were as follows: splitless injection; column, HP-5-MS (30 m×0.25 mm I.D., 0.25 μm film thickness) (Agilent, Waldbronn, Germany); injection port temperature, 270 °C; carrier gas, helium; flow rate, 1 mL/min; oven temperature, initially 100 °C for 3 min, ramped to 310 °C at 30 °C/min, 310 °C for 10 min. The MS conditions were as follows: transfer line heater, 280 °C; ion source temperature, 230 °C; electron ionization (EI) mode; ionization energy, 70 eV. Analysis was performed in scan mode, from 50 to 550 amu at a speed of 1.5 scans/s. Solvent delay was set to 3.5 min. The obtained mass spectra were compared to commonly used EI-MS spectra libraries (Maurer Pfleger Weber (MPW) 2007, NIST 08, Wiley 6th) and an in-house library of previously identified synthetic cannabinoids. Sample preparation for flash chromatography One gram of the ‘herbal mixture’ was extracted with 10 mL of ethanol over a period of 24 h with occasional shaking for

For sample loading onto the flash chromatography system, the ‘dried solid sample loading’ technique was used. For this purpose 3 g of silica powder was added to the extract obtained as described above. Ethanol was removed from the suspension with a rotary evaporator at 40 °C under reduced pressure. After removal of the ethanol, the dried extract was loaded to the silica powder, and the sample was transferred into an empty universal Rf cartridge® (Teledyne Isco, Lincoln, USA). The flash chromatography system used was a CombiFlash® Rf apparatus with a 15.5 g RediSep® Rf Gold C18 column (Teledyne Isco, Lincoln, USA). Mobile phase A consisted of 0.55 % formic acid in water (pH2.3) and mobile phase B of methanol. Runtime was 25 min with a flow of 15 mL/min and a gradient starting at 60 % B, running to 100 % B in 20 min followed by a 5 min hold at 100 % B. Fraction collection was performed by measuring the absorbance at 207 nm as well as the average absorbance over the wavelength range from 200 to 360 nm. To obtain the highest possible purity, only the fractions (12 mL per fraction) representing approximately the centre 80 % of the absorbance peak area were further processed. Final sample cleanup The fractions assumed to contain the desired synthetic cannabinoid were pooled, and the methanol was removed using a rotary evaporator at 40 °C under vacuum. The compounds were extracted from the remaining aqueous solution with TBME, while, depending on the structure of the compound, the conditions were either acidic through the addition of HCl for the cyclohexylphenols or alkaline after the addition of NaOH for the aminoalkylindoles. Any remaining water was removed by the addition of water-free sodium sulphate. As the final step, the TBME was removed under a gentle stream of nitrogen at 40 °C. Purity To determine the purity and to verify that the collected fraction contained the desired synthetic cannabinoid, the dried substance was reconstituted in methanol leading to a concentration of 1 mg/mL. This solution was analysed by GC-MS as described for the screening of ‘herbal mixtures’. Purity was estimated using the percentage report of the software Enhanced ChemStation G1701GA version D.03.00.611 (Agilent, Waldbronn, Germany).

B. Moosmann et al. Fig. 2 Flash chromatogram of one ‘herbal mixture’ containing the cis- and trans-isomer of CP47,497-C8. Shown in red/dark grey is the absorbance at 207 nm; in orange/light grey, the average absorbance over the wavelength range from 200 to 360 nm. The blue/black line indicates the gradient used for separation

NMR analysis For the synthetic cannabinoids cis- and trans-CP47,497-C8, NMR analysis was performed. The spectra

Fig. 3 GC-EI-MS of the cis(lower diagram) and the transisomer (upper diagram) of CP47,497-C8 after their separation using a flash chromatography system

were recorded in CDCl3 at room temperature using a DRX 400 (Bruker Physik AG, Germany). The obtained 1 D-1H- and 13C-NMR (400 and 100 MHz) were compared to the chemical shifts found in the literature [1].

Isolation of synthetic cannabinoids from ‘Spice’ products Table 1 Retention times of the various synthetic cannabinoids tested on the flash chromatography system

Compound

ortho-RCS-4 JWH-015 AM-2201 RCS-4 JWH-250 JWH-073 JWH-251 JWH-203 JWH-018 JWH-081 JWH-122 JWH-019 JWH-210 cis-CP-47,497-C8 trans-CP-47,497-C8

Retention time [min] 8.7 9.8 9.9 11.1 12.7 13.0 13.8 14.2 14.9 15.9 16.5 16.5 18.0 18.7 20.2

Results and discussion The GC-EI-MS data of 3-cis-CP-47,497-C8 and 3-transCP-47,497-C8 can only be differentiated by comparing the retention time as well as the intensity of the two fragments m/z 215 and 233. With the above-described flash chromatography method, we were able to separate the two diastereomers (Fig. 2). For both isomers, we obtained purities greater than 99 % (Fig. 3). Similar to the GC-MS analysis, the NMR spectra did not show any impurities (data not shown). The separation of the two diastereomers provides the opportunity to obtain larger quantities of each isomer at low cost for testing the toxicity of each isomer separately, e.g. in cell cultures. The chosen wavelength of 207 nm, where the UV spectra published in the literature [5, 9, 11, 12] showed a relatively high absorbance, proved to be suitable for all the compounds. In addition to that, measuring the average absorbance over a wavelength range prevents new substances Fig. 4 Flash chromatogram of a mixture containing four different synthetic cannabinoids. Shown in red/ dark grey is the absorbance at 207 nm; in orange/light grey, the average absorbance over the wavelength range from 200 to 360 nm. The blue/black line indicates the gradient used for separation

with a poor absorbance at 207 nm from going undetected [36]. For the various synthetic cannabinoids tested, purities greater than 99 % were reached as well. Since the purity for these compounds was tested by GC-MS analysis, it has to be taken into consideration that non-volatile compounds as well as compounds with poor chromatographic properties in GC would not be detected. Even though these impurities seem unlikely, a HPLC-DAD method could be applied to gain assurance. Table 1 lists the retention times of the tested compounds on the flash chromatography system. With the exception of the pairs JWH-122/019, JWH-250/073, JWH 251/203 and JWH-015/AM-2201, separation can be achieved with this system. For these pairs, modification of the gradient and/or the mobile phase could lead to better separation. Since all the analytes are highly soluble in ethanol and a lower percentage of extracted plant ingredients was preferred over a 100 % extraction of the synthetic cannabinoids from the plant material, the extraction time was reduced from 24 h under occasional shaking (as applied for CP-47,497-C8) to 1 min with shaking for the various synthetic cannabinoids. If an unknown compound needs to be extracted, the authors recommend the use of ethanol for 1 min, as all of the synthetic cannabinoids tested in our laboratory so far were highly soluble in this solvent. The separation of the mixture was successful, and the desired synthetic cannabinoids were received in high purity (Fig. 4). This is particularly important, since very often not only one but several synthetic cannabinoids are added to one product. While in these cases mostly two to three synthetic cannabinoids can be found, there are rare exceptions where products contained up to six different active compounds (own unpublished data). Therefore, the compounds need to be separated not only from the impurities and extracted plant material but also from each other. When a new synthetic cannabinoid appears on the market alongside another one, this separation method facilitates the recording of the NMR spectra for its identification.

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Although overall yield was not the main focus and the concentration of the respective cannabinoids in the mixture was not determined, a minimum of 1 mg substance per 100 mg ‘herbal mixture’ could be obtained for most substances. This means that one package is sufficient to obtain the quantities needed for NMR analysis and an adequate amount of sample for use as reference. If larger quantities are needed, e.g. for toxicity screening assays, the yield could be enhanced by recycling the fractions collected at the peak shoulder and repetition of the extraction process from the plant material. It should be noted that if a synthetic cannabinoid is relatively polar, like, e.g. JWH-200 or cannabipiperidiethanone [10] (Fig. 1), it will elute together with other polar compounds extracted from the plant material. In these cases, preliminary results show that a second run using normalphase chromatography and ethyl acetate/methanol as mobile phase could lead to a sufficient separation.

Conclusions The described method provides a fast, easy and inexpensive way to isolate synthetic cannabinoids from ‘Spice’ products. This approach makes it possible to obtain high-purity reference material in a short time, which facilitates the timely development or enhancement of analytical methods in forensic toxicology. It is particularly helpful when new synthetic compounds with yet unknown structures emerge. Additionally, the flash chromatography system offers easy upscaling if larger quantities for toxicological testing are needed. Acknowledgments The authors thank the EU Commission (JUST/ 2009/DPIP/AG/0948), the German Ministry of Health and the city of Frankfurt (Main) for funding the project ‘Spice and synthetic cannabinoids’.

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