Qualitative Confirmation of 9 Synthetic Cannabinoids and 20 Metabolites in Human Urine Using LC-MS/MS and Library Search

Introduction: Synthetic cannabinoids are an emerging illicit drug class. The variety of available substances is large and ever-changing, making it difficult for laboratories to remain current. We present a qualitative LC-MS/MS method identifying urinary metabolites of JWH-018, JWH-073, JWH-081, JWH-122, JWH-200, JWH-210, JWH-250, RCS-4, and AM2201 and the parent compounds JWH-018, JWH-073, JWH-081, JWH-122, JWH-210, JWH-250, RCS-4, AM2201, and MAM2201. Methods: After enzymatic hydrolysis, urinary proteins were precipitated with acetonitrile. Chromatography utilized a 10 min gradient on a Kinetex XII-C18 column with 0.1% formic acid in water and acetonitrile. Scheduled multiple reaction monitoring "survey scans" were followed by information-dependent acquisition-enhanced product ion scan experiments on an ABSciex 5500 QTRAP mass spectrometer. Analytes were identified by software-assisted library searching against reference spectra. Results: The method was fully validated, including proof of selectivity (no exogenous or endogenous interferences were observed), assessment of matrix effects (95-122%) and recovery (53-95%), determination of limits of detection (0.5-10 ng/mL), carry-over studies (thresholds between 100 and 1000 ng/mL), and determination of autosampler stability (samples were stable for at least 3 days). Hydrolysis efficiency was thoroughly investigated for a wide range of glucuronides and for the reference standard, JWH-018 5-hydroxypentyl glucuronide