Purpose: Synthetic Cannabinoid Agonists (SCA) (Spice) are high potency ligands for cannabis CB1 and CB2 receptors. They produce psychoactive effects by binding to CB1 receptors. SCA are also the most popular class of Novel Psychoactive Substances (NPS) by seizures. They are responsible for many severe intoxications and some fatalities. Their pharmaceutical analysis is complicated by various factors including: many analytes in the sample which are typically present in only sub-milligram (~0.1 mg) amounts, matrix/plant material components.
Methods: Extraction solvents (methanol, chloroform, acetonitrile) 99.9% anhydrous, were purchased from Fisher Scientific and ACROS Organics. Deuterated solvent (CDCl3) was purchased from Cambridge Isotopes Laboratories (Goss Scientific). Herbal blend SCA samples (1.0 g) were provided from recent seizures by the Avon and Somerset Constabulary (K2 black edition). Samples were weighed using a SE2F Sartorius analytical balance, extracted into methanol (2 x 30.0 mL) with sonication (30 min), and then centrifuged (5 min). The supernatant extract was evaporated to dryness under reduced pressure and reconstituted in deuterated solvent (1.0 mL). NMR spectra were recorded on a Bruker AVANCE III 500 MHz spectrometer, 1H, 13C, and 19F frequencies are 500.130, 125.758, and 470.592 MHz respectively. QTOF-UHPLC-MS analysis used a MaXis HD Quadrupole electrospray Time-of-Flight (ESI-QTOF) mass spectrometer (Bruker Daltonik GmbH, Bremen, Germany), operated in ESI positive mode.
Results: Using UHPLC/QTOF/MS/MS six base-line resolved peaks were achieved. Five of them are SCA and one is a possible reaction precursor (PX1 acid) for the synthesis of PX1 amide (Fig. 1). All [M+H]+ are within 5 ppm of mass accuracy limits and have distinctive fragmentation patterns in MS/MS. 1D/2D NMR spectra of the crude extracts are complex. Nevertheless, signals for integration in 1H NMR were identified for each SCA analyte and then used to calculate the molar ratio of each SCA in the herbal blend: 5F-PB-22 6%, MDMB-CHMICA 7%, 5F-AKB-48 9%, PX1 amide 11%, and 5F-ADB 23%, and an excipient propylene glycol 44%.
Conclusion: We have determined an analytical platform for the separation and characterization of closely related SCA from a seized herbal blend sample containing: PX1 amide, 5-F-PB-22, 5-F-ADB, MDMB-CHMICA, and 5-F-AKB-48 using LC-TOF, LC/MS/MS, and 2D NMR spectroscopy. LC/MS/MS allowed the identification of a possible reaction precursor, PX1 acid, not previously reported. This approach will provide drug analysts with quantitative methods to tackle complex mixtures of SCA in herbal matrices. It also provides health service and Emergency Room responders with details of the mixtures of SCA with potent CB1 activities. We thank the Government of Kuwait for a scholarship (to HAN) and DEAT, the Avon and Somerset Constabulary, for the seized samples.
Stephen Husbands– Head of Medicinal Chemistry at the dept of Pharmacy and Pharmacology University of Bath, University of Bath
Ian Blagbrough– Senior lecturer in the department of pharmacy and pharmacology, University of Bath