Validation and application of an UPLC–MS/MS method for the quantification of synthetic cannabinoids in urine samples and analysis of seized materials from the Portuguese market

• Synthetic cannabinoids analysis in urine samples through an UPLC–MS/MS method.
• The method shows small run time, high sensitivity, very good precision and accuracy.
• Allows the simultaneous analysis of THC, THC-COOH and 11-OH-THC.
• Analysis of 17 seized materials from the Portuguese market.

An UPLC–MS/MS method using ESI + ionization and MRM was developed and fully validated according to international guidelines for the qualitative and quantitative analysis of nine synthetic cannabinoids and/or their metabolites in urine samples (1 mL). Prior to extraction the samples were subjected to an enzymatic hydrolysis using β-glucuronidase followed by a SPE procedure using Oasis® HLB 3 cc (60 mg) columns. The chromatographic separation was performed with an Acquity UPLC® HSS T3 (50 mm × 2.1 mm i.d., 1.8 μm) reversed-phase column using a gradient with methanol–ammonium formate 2 mM (0.1% formic acid) and with a run time of 9.5 min. The method was validated in terms of selectivity, capacity of identification, limits of detection (0.01–0.5 ng/mL) and quantification (0.05–0.5 ng/mL), recovery (58–105%), carryover, matrix effect, linearity (0.05–50 ng/mL), intra-assay precision, inter-assay accuracy and precision (CV < 20%). The method was applied to 80 authentic samples, five of them (6.2%) were confirmed or suspected to be positive for the metabolites JWH-018 N-hydroxypentyl and JWH-018 N-pentanoic acid of JWH-018 and for the metabolite JWH-122 N-(5-hydroxypentyl) of JWH-122, and three of them in association with THC and/or THCCOOH (substances included in the method, together with the 11-OH-THC). Additionally, 17 spice products were analyzed, for which were confirmed the presence of the following substances: AM-2201, JWH-018, JWH-022 JWH-073, JWH-122, JWH-203, JWH-210, JWH-250, HU-210 and RCS-4, according to the comparison with authentic reference material and published data. The analytical method developed allowed the analysis of synthetic cannabinoids and the notification of the first cases in Portugal.