Spice up your life: screening the illegal components of ‘Spice’ herbal products

Abstract

‘Spice’ is a herbal blend which was freely available in the UK until December 2009 after which it was classified as a Class B drug. The Spice product range includes ‘Gold’ and ‘Diamond’ and a very limited number of reports have identified that these contain non-traditional cannabinoids. We have determined for the first time the components of Spice ‘Gold Spirit’ using Gas Chromatography-Mass Spectroscopy and also explored a potential screening approach using Solid Probe Mass Spectroscopy which requires no liquid–liquid extraction. This methodology has the potential to facilitate the accelerated screening of illegal components in ‘Spice’ and other related herbal-type products.

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Introduction

Spice is a herbal blend sold predominantly in the European community and is well known by users to have effects similar to that of Cannabis.¹ Spice was declared as a ‘bio-drug’ since the listed ingredients indicate bioactive herbs which contain alkaloids and generally produce cannabis-like effects.^1–3 Accordingly the popularity of these products dramatically increased and was given the tag line of ‘legal drugs’.

In 2008 THC Pharma reported the active ingredients of Spice to be ‘JWH-018’ (see Scheme 1) which is a cannabinoid agonist from the aminoalkylindole family which has been shown to have a binding affinity for CB₁ receptors at low nanomolar levels (∼9 nM). Note that this is the same cannabinoid receptor that is linked to behaviours affected by tetrahydrocannabinol. Very recently reports by Atwood et al. have demonstrated that JWH-018 is a potent and effective CB₁ receptor agonist that activates multiple CB₁ receptor signalling pathways.⁴ Thus it is highly likely that the psycho-activity of ‘Spice’ is due to the JWH-018 agonist.⁴

Scheme 1 Structures of compounds commonly found in Spice herbal products. (1) CP 47,497-C8; (2) CP 47,497-C8 isomer and (3) JWH-018.

Following the report by THC Pharma, Auwarter et al.¹ and Uchiyama et al.⁵ identified and characterized the CP 47,497-C8 (see Scheme 1) homolog and its isomer, a synthetic by-product, in Spice Silver, Gold and Diamond as well as ‘Yucatan Fire’ and ‘Sence’. Note that it has been reported that the analgesic potency of CP 47,497-C8 is 5 to 10 times higher compared with tetrahydrocannabinol.⁶ Lindigkeit and co-workers² have analysed Spice Gold in Germany with mass spectrometry and found that the samples contained CP 47,497-C8 and JWH-018 but after the German Health Authorities prohibited the active components of Spice on January 22^nd 2009, JWH-018 was found to be absent in Spice products.² However, a new analogue JWH-073 was found.² In the UK, Spice was legal until December 2009 where the synthetic cannabinoids, which are sprayed onto herbal smoking products, are now classified as a Class B drug.^7,8 Given that the manufacturers can readily change the components of Spice, a methodology that can readily identify the presence of prohibited compounds in these complex mixtures would be highly welcome.

In this communication we explore for the first time the components of the Spice product ‘Gold Spirit’ via Gas Chromatography-Mass Spectrometry and explore the possibility of using Solid Probe Mass Spectrometry which precludes the need for liquid–liquid extraction.

Experimental section

The Gas Chromatography-Mass Spectroscopy was a Hewlett Packard 5890 Series 2 Gas Chromatogram. This machine is equipped with a 30 m × 0.25 mm film = 0.25 µm Restek column (5% diphenyl–95% dimethyl polysiloxane) with helium as the carrier gas (flow rate 1 ml min⁻¹). Temperature program: 50 °C (2 min) to 12 °C min⁻¹ to 290 °C (6 min). The GC was coupled directly to a Hewlett Packard 5972 series mass spectrometer with an electron ionization mode at 70 eV. The acquisition rate was 1.7 scans per second with a mass range of 50–500 amu, using MSDChemstation software.

A Finnigan trace mass spectrometer coupled with a trace gas chromatogram 2000 series was also used which is equipped with a J & W Scientific 30 m × 25 mm film = 0.25 µm analytical column and helium gas carrier (flow rate 1.0 ml min⁻¹). Temperature program: 50 °C (2 min) to 12 °C min⁻¹ to 285 °C (15 min). Mass spec. parameters electron ionization mode at 70 eV using Excalibur software. The emission current was 300 micro-amps, detector voltage 500 °C, the source temperature 280 °C. The acquisition rate was 2.5 scans per second, mass range 45–460 amu. The Solid Probe Mass Spectrometer uses a DIP (direct insertion probe) equipped with a vial containing a small amount of sample was inserted into the mass spectrometer. Analysis settings: detector voltage 500.0 V, source temperature 250 °C the interface temperature 200 °C, emission current 300 µV. The temperature was set at 30 °C for 1 min followed by a temperature ramp of 800 °C min⁻¹ taking the temperature to 400 °C. The temperature was then held at 750 °C for 2 min. The acquisition type: full scan at 1.8 scans per second scan range 50–640 amu. Mass spectra were obtained and compared with recent literature reports.^1,2

Spice ‘Gold’, ‘Diamond’ and ‘Gold Spirit’ were purchased from the Internet in 2009. The ingredients listed by the supplier for Spice Gold are: bay bean, blue lotus, Lion's Tail, Indian Warrior, Dwarf skullcap, Maconha brava, Pink Lotus, Marshmallow, Red Clover, Rose, Siberian motherwort, Vanilla and honey. Ingredients listed for Spice Gold Spirit are: Leonurus, Cardiaca, Pedicularis, Canadensis, Scutellaria, Latero flora, Althaea officinalis, Rosa damascena, Vanilla planifolia. Finally, the ingredients listed for Spice Diamond are: Bay bean, Blue Lotus, Lion's Tail, Indian Warrior, Dwarf skullcap, Maconha brava, Pink Lotus, Marshmallow, Red Clover, Rose, Siberian motherwort, vanilla, honey, aroma.

A liquid–liquid extraction of the chosen Spice product was undertaken with ethanol with the sample placed into a laboratory ultrasonic bath for 15 minutes. The sample was then filtered using a Buchner filter. The solution was diluted further with the addition of ethanol.

Results and discussion

The GC-MS chromatograms of the three Spice products are depicted in Fig. 1. The spectra obtained from Spice Gold reveal the presence of ethyl vanillin, α-tocopherol and γ-tocopherol. Also found present was CP 47,497-C8 which has been reported to be a cannabinoid receptor agonist.¹ These constituents of Spice Gold compare well with previous literature reports.^1,2 In the case of Spice Diamond, caffeine, α-tocopherol and γ-tocopherol and palmitic acid are found to be present with the latter being saturated fatty acids commonly found in plants and given the herbal origin of Spice products its presence is expected. Interestingly CP 47,497-C8 is found to be present which has been observed before in Spice Silver, Gold and Diamond¹ with its analgesic potency reported to be 5 to 10 times higher compared with tetrahydrocannabinol.⁶ We also find that JWH-018 (341, 324, 284, 214 m/z) is present in Spice Diamond which has only been reported once before by Auwarter et al.¹ Recently Atwood et al.⁴ have proven that JWH-018 is a potent and effective CB₁ receptor agonist that activates multiple CB₁ receptor signalling pathways.⁴

Fig. 1 GC-MS chromatograms for various Spice products.

Next we turn to exploring the components of Spice Gold Spirit which is a new addition to the Spice herbal range and until now, its components have never been quantified. Fig. 1 depicts a typical chromatogram where α-tocopherol and JWH-018 are found to be present. Clearly this new range of Spice has the highly active cannabinoid mimic which has attracted attention from consumers as a legal high. A recent study by Lindigkeit et al.² has demonstrated that Spice Gold purchased before the prohibition by German Authorities contained CP 47,497-C8 and JWH-018 but samples analysed after the prohibition were found not to contain the highly active JWH-018 compound. Our work has shown that a simple liquid–liquid extraction followed by GC-MS can quantify, without doubt, the presence of recently illegal compounds. Given that the manufacturers of Spice herbal blends are continuously changing the compounds,² there is a need for the quantification of these banned substances, for example, in a Forensic situation. Consequently we turn to exploring the concept of using Solid Probe Mass Spectrometry for quantifying the components of Spice herbal products and associated herbal type products.

Fig. 2A displays a typical spectra obtained using the Solid Probe MS where it is evident that the fragmentation pattern of α-tocopherol (430, 205, 165 m/z) and CP 47,497-C8 (332, 314, 233, 215 m/z) is present. The Solid Probe MS also functions separately as a stand alone GC-MS and using the chromatograph obtained in Fig. 1 the mass spectra resulting from α-tocopherol were determined and subtracted from that of the spectra in Fig. 2A. The resulting spectrum is shown in Fig. 2B which allows the fragmentation pattern of CP 47,497-C8 to be clearly visible and quantifiable. Note that it is entirely reasonable to subtract the spectra of the usually obtained α-tocopherol which is almost entirely found in Spice herbal products. Last we consider the case of Spice Gold Sprit. Fig. 3 depicts the mass spectra before and after subtraction from α-tocopherol with the latter allowing the quantification of JWH-018 (341, 324, 284, 214), the potent and effective CB₁ receptor agonist that is currently banned in a plethora of countries.

Fig. 2 Mass spectra obtained using the Solid Probe MS before (A) and after (B) subtraction for Spice Gold.

Fig. 3 Mass spectra obtained using the Solid Probe MS before (A) and after (B) subtraction for Spice Gold Spirit.

Conclusions

We have quantified via GC-MS for the first time the components of the herbal product Spice ‘Gold Spirit’ which is found to contain the recently banned compounds JWH-018 and CP 47,497-C8. Additionally we have shown that Solid Probe Mass Spectrometry can be used precluding the need for liquid–liquid extraction allowing the fast screening of Spice herbal products. This screening methodology is useful for the rapid analysis of any possible prohibited substances which should be followed up fully with quantification via GC-MS.

References

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2. R. Lindigkeit, A. Boehme, I. Eiserloh, M. Luebbecke and M. Wiggermann, Forensic Sci. Int., 2009, 191, 58.
3. M. A. Neukamm, T. E. Muerdter, C. Knabbe, H.-D. Wehner and F. Wehner, Blutalkohol, 2009, 46, 373.
4. B. K. Atwood, J. Huffman, A. Straiker and K. Mackie, Br. J. Pharmacol., 2010 DOI:10.1111/j.1476-5381.2009.00582.x.
5. N. Uchiyama, R. Kikura-Hanajiri, N. Kawahara, Y. Haishima and Y. Goda, Chem. Pharm. Bull., 2009, 57, 439.
6. B. K. Koe, G. M. Milne, A. Weissman, M. R. Johnson and L. S. Melvin, Eur. J. Pharmacol., 1985, 109, 201.
7. http://drugs.homeoffice.gov.uk/publication-search/acmd/acmd-report-agonists.html .
8. http://news.bbc.co.uk/1/hi/uk/8427439.stm .

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