Simultaneous identification and quantitative determination in urine of the more significant metabolites of synthetic cannabinoids JWH-018, JWH-073, JWH-122 and JWH-250 using authentic references and deuterated isotopologues as internal standards

Introduction Synthetic cannabinoids (SC) are substances displaying a high affinity for cannabinoid receptor CB1 and represent the psychoactive agents in herbal mixtures called “Spice” or “K2” which are sold as an incense or smoking material mainly through the Internet. Because of its great abusive potential, several SC are banned in many countries, but despite this, the widespread use of herbal smoking mixtures containing SC may be partially explained by the fact that post-ingestion urines are known to produce negative results in standard toxicological screening methods for cannabis. As a consequence, an increasing number of analytical methods have been developed in forensic and doping control laboratories to enable the detection of illegal intake of these psychoactive substances in human fluids originating from psychiatric patients, emergency units or assessment of fitness to drive. Due to rapid metabolic transformation, the native SC are not usually detectable in urine samples and then the analytical methods must be based on the identification and quantization of their metabolites. Aims The aim of our study is to set-up, using synthesized reference standards, a LC-MS/MS method for routine screening procedures to assess the assumption of JWH-018, JWH-073, JWH 122 and JWH-250, the SC included in Table 1 of narcotic and psychotropic substances banned in Italy. The method gives the simultaneous identification of the three more significant metabolites of each cannabinoid and adequate sensitivity, precision and accuracy are assured by the use of deuterated internal standards. Methods For each of the four cannabinoids were synthesized the three more significant metabolites, the ω- and (ω-1)-hydroxyl and the ω-carboxyl derivatives (ω position represent the terminal carbon of the N-alkyl side chain) while as internal standards were synthesized the (ω-1)-hydroxyl metabolites trideuterated on the terminal methyl of the side chain. Urine samples were subjected to deconjugation using 30% hydrochloric acid at 90-95°C for 60 min, followed by a solvent extraction procedure with n-hexane-ethyl acetate (9/1 v/v). The LC-MS/MS analysis was performed in positive mode on an API 4000 Triple Quadrupole Mass Spectrometer (AB Sciex) equipped with a 1,8µm Acquity C-18 HSS T3 100 x 2 mm HPLC column (Waters) with isocratic elution (55 % of 10 mM HCOONH4 in water containing 0.1% HCOOH and 45 % of acetonitrile) at 45 °C and at flow rate of 0.4 mL/min. Two transitions in ‘multiple reaction monitoring’ mode and the retention time have permitted the unambiguous identification of each metabolite which, through the presence of a suitable internal standard, was quantified. Result and discussion All the synthesized compounds were fully characterized with regard to the structure and purity, by 1H,13C NMR and GC-MS (after esterification with CH2N2 for the ω-carboxylic metabolites). For sample treatment, the recovery of the metabolites was evaluated at different pH (1, 3, 5, 9 and 10). The validation of the method was performed testing linearity (0.5-100 ng/mL), reproducibility and accuracy (ranged between -15% and + 15%) at three levels. The method developed was applied to the analysis of urine samples from individuals who have taken SC. This LC-MS/MS method can be used for routine screening of urine specimens from subjects suspected of using “herbal incense” or “Spice” products spiked with the synthetic cannabinoids JWH-018, JWH-073, JWH 122 or JWH-250.