Qualitative confirmation of 9 synthetic cannabinoids and 20 metabolites in human urine using LC-MS/MS and library search

Abstract

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 XB-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.

Figure 1

Structures of synthetic cannabinoid parent compounds included in the method.

Figure 2

Total ion chromatograms of an LOD control at 5 ng/mL with (A) sMRM and EPI triggering and (B) unscheduled MRM. 1a, JWH-018; 1b, JWH-018 5-hydroxypentyl; 1c, JWH-018 6-hydroxyindole; 1d, JWH-018 5-hydroxyindole; 1e, JWH-018 pentanoic acid; 2a, JWH-073; 2b, JWH-073 4-hydroxybutyl; 2c, JWH-073 6-hydroxyindole; 2d, JWH-073 5-hydroxyindole; 2e, JWH-073 butanoic acid; 3a, JWH-081; 3b, JWH-081 5-hydroxypentyl; 4a, JWH-122; 4b, JWH-122 5-hydroxypentyl; 5a, JWH-200 5-hydroxyindole; 5b, JWH-200 6-hydroxyindole; 6a, JWH-210; 6b, JWH-210 5-hydroxypentyl; 6c, JWH-210 4-hydroxypentyl; 6d, JWH-210 5-hydroxyindole; 6e, JWH-210 pentanoic acid; 7a, JWH-250; 7b, JWH-250 5-hydroxypentyl; 7c, JWH-250 4-hydroxypentyl; 7d, JWH-250 5-hydroxyindole; 7e, JWH-250 pentanoic acid; 8a, RCS-4; 8b, RCS-4 5-hydroxypentyl; 8c, RCS-4 pentanoic acid; 9a, AM2201; 9b, AM2201 4-hydroxypentyl; 9c, AM2201 6-ydroxyindole; 10a, MAM2201.

Figure 3

Library search with LibraryView for AM2201 4-hydroxypentyl (4.86 min retention time) in an LOD control sample at 2.5 ng/mL and in an authentic urine sample.