Simultaneous quantification of 20 synthetic cannabinoids and 21 metabolites, and semi-quantification of 12 alkyl hydroxy metabolites in human urine by liquid chromatography–tandem mass spectrometry

Abstract

Clandestine laboratories constantly produce new synthetic cannabinoids to circumvent legislative efforts, complicating toxicological analysis. No extensive synthetic cannabinoid quantitative urinary methods are reported in the literature. We developed and validated a liquid chromatography–tandem mass spectrometric (LC–MS/MS) method for simultaneously quantifying JWH-018, JWH-019, JWH-073, JWH-081, JWH-122, JWH-200, JWH-210, JWH-250, JWH-398, RCS-4, AM-2201, MAM-2201, UR-144, CP 47,497-C7, CP 47,497-C8 and their metabolites, and JWH-203, AM-694, RCS-8, XLR-11 and HU-210 parent compounds in urine. Non-chromatographically resolved alkyl hydroxy metabolite isomers were considered semi-quantitative. β-Glucuronidase hydrolyzed urine was extracted with 1 ml Biotage SLE+ columns. Specimens were reconstituted in 150 μL mobile phase consisting of 50% A (0.01% formic acid in water) and 50% B (0.01% formic acid in 50:50 methanol:acetonitrile). 4 and 25 μL injections were performed to acquire data in positive and negative ionization modes, respectively. The LC–MS/MS instrument consisted of a Shimadzu UFLCxr system and an ABSciex 5500 Qtrap mass spectrometer with an electrospray source. Gradient chromatographic separation was achieved utilizing a Restek Ultra Biphenyl column with a 0.5 ml/min flow rate and an overall run time of 19.5 and 11.4 min for positive and negative mode methods, respectively. Quantification was by multiple reaction monitoring with CP 47,497 compounds and HU-210 ionized via negative polarity; all other analytes were acquired in positive mode. Lower and upper limits of linearity were 0.1–1.0 and 50–100 μg/l (r² > 0.994). Validation parameters were evaluated at three concentrations spanning linear dynamic ranges. Inter-day analytical recovery (bias) and imprecision (N = 20) were 88.3–112.2% and 4.3–13.5% coefficient of variation, respectively. Extraction efficiencies and matrix effect (N = 10) were 44–110 and −73 to 52%, respectively. We present a novel LC–MS/MS method for simultaneously quantifying 20 synthetic cannabinoids and 21 metabolites, and semi-quantifying 12 alkyl hydroxy metabolites in urine.

Introduction

Synthetic cannabinoids bind CB1 and/or CB2 receptors and were originally developed for studying endocannabinoid pharmacology; however, now are abused drugs smoked or inhaled for psychoactive effects, but deceptively marketed as herbal incenses and air fresheners. Synthetic cannabinoid abuse resulted in increases in emergency room visits and occasional deaths [1], [2], [3]. Synthetic cannabinoid subclasses include napthoylindoles (JWH-015, JWH-018, JWH-019, JWH-073, JWH-081, JWH-122, JWH-200, JWH-210 and JWH-398), phenylacetylindoles (JWH-203, JWH-250, JWH-251, and RCS-8), benzoylindoles (RCS-4 and AM694), cyclohexylphenols (CP 47,497 C7 and C8 analogs) and dibenzopyrans (HU-210).

In July 2012 the United States Drug Enforcement Agency classified JWH-018, JWH-019, JWH-073, JWH-081, JWH-122, JWH-200, JWH-203, JWH-250, JWH-398, AM694, AM2201, RCS-4, RCS-8, HU-210, CP 47,497-C7, CP 47,497-C8 and their analogs as schedule I controlled substances [4], [5]. Recently, UR-144, XLR11 and AKB48 were temporarily added to the Schedule I controlled substance list [6]. Most countries enacted similar legislation. Clandestine laboratories constantly synthesize new compounds in response to legislative efforts, complicating drug testing.

New synthetic cannabinoid structures may not cross-react in antibody-based techniques, leading laboratorians to consider mass spectrometric screening [7], [8], [9], [10]. Mass spectrometry is flexible, allowing incorporation of new analytes as rapidly as reference standards become available. We recently published a liquid chromatography–tandem mass spectrometric (LC–MS/MS) qualitative screening method employing spectral library searching simultaneously targeting 9 synthetic cannabinoids and 20 metabolites in urine [8]. Urinary quantitative methods were only published for single parent analytes and metabolites [11], [12] or for metabolites of JWH-018 and JWH-073 [13], [14], [15]. The most comprehensive urine quantification method reported to-date targets 8 parent analyte families [16]. A comprehensive, up-to-date quantitative confirmatory synthetic cannabinoid method is required for confirming presumptive positive and negative screening results, comparing screening techniques and evaluating optimal cutoff concentrations. We present a fully validated LC–MS/MS method targeting 53 analytes: JWH-018, JWH-019, JWH-073, JWH-081, JWH-122, JWH-200, JWH-210, JWH-250, JWH-398, RCS-4, AM2201, MAM2201, UR-144, CP 47,497-C7, CP 47,497-C8 and their metabolites, and JWH-203, AM694, RCS8, XLR11 and HU210 parent compounds in urine. Non-chromatographically resolved alkyl hydroxyl metabolite isomers were semi-quantitative.