Solid-Phase Extraction and Quantitative Measurement of Omega and Omega-1 Metabolites of JWH-018 and JWH-073 in Human Urine

Abstract

The aminoalkylindole agonists JWH-018 and JWH-073 are contained in “K2/SPICE” products sold as “legal marijuana”. Previous human metabolic studies have identified (ω)-hydroxyl and (ω)-carboxyl metabolites as biomarkers indicative of product use. However, other primary metabolites exhibiting similar chromatographic properties and mass spectra are also excreted in human urine. Analytical standards were used in this study to identify new primary metabolites as (ω-1)-hydroxyl derivatives of JWH-018 and JWH-073. The liquid chromatography tandem mass spectrometry (LC-MS/MS) procedure coupled with an automated solid-phase extraction procedure incorporating deuterium labeled internal standards provides rapid resolution of the (ω)- and (ω-1) metabolites with adequate sensitivity, precision, and accuracy for trace analysis in human urine. Results from four urine specimens collected after individuals reportedly self-administered either JWH-018 or a mixture of JWH-018 and JWH-073 showed the following: 1) all tested metabolites were excreted in high concentrations, 2) (ω)- and (ω-1)-hydroxyl metabolites were exclusively excreted as glucuronic acid conjugates, and 3) approximately 5–80% of the (ω)-carboxyl metabolites were excreted as glucuronic acid conjugates. This is the first report to identify and quantify (ω-1)-hydroxyl metabolites of JWH-018 and JWH-073 and the first to incorporate automated extraction procedures using deuterium labeled internal standards. Full clinical validation awaits further testing.

Cytochrome P450-Mediated Oxidative Metabolism of Abused Synthetic Cannabinoids Found in K2/Spice: Identification of Novel Cannabinoid Receptor Ligands

“…at ASPET Journals on May 7, 2018 dmd.aspetjournals.org previous reports, JWH-018 and AM2201 appear to be metabolized similarly to produce several ring and alkyl side chain oxidized metabolites that are excreted in human urine as glucuronic acid conjugates (Chimalakonda et al, 2011b;Moran et al, 2011). It also appears that AM2201 readily undergoes cytochrome P450-mediated oxidative dehalogenation to produce common metabolites with JWH-018, whereas the -1-OH metabolites of each synthetic cannabinoid are distinct.…”

“…We and others have identified several hydroxylated metabolites of JWH-018 and AM2201 in humans with a known history of K2 use, implicating the role of mixedfunction oxidases, such as cytochromes P450, in the biotransformation of synthetic cannabinoids (Zhang et al, 2002;Sobolevsky et al, 2010;Wintermeyer et al, 2010;Chimalakonda et al, 2011b;Hutter et al, 2012). Previous reports showed that JWH-018 undergoes hepatic metabolism, leading to the formation of several naphthalene, indole, and alkyl side chain hydroxylated and carboxylated metabolites (Fig.…”

“…Quantification of analytes of interest was performed using positive ion electrospray ionization LC-MS/MS methods developed previously for trace analysis of synthetic cannabinoid metabolites (Chimalakonda et al, 2011b;Moran et al, 2011). For these studies, analytical standards (0.20 -100 ng/ml) were matrix-matched in pooled human urine void of synthetic cannabinoid contamination or in the final in vitro reaction mixture (ethanol-water-0.1 M phosphate buffer, pH 7.4, 50:30:20%).…”

“…Analytes of interest were chromatographically separated as described previously under isocratic conditions using an Zorbax Eclipse XDB-C18 analytical column (150 ϫ 4.6 mm, 5 m; Agilent Technologies) heated to 40°C. Mobile phases consisted of 45% A (0.1% formic acid in water) and 55% B (0.1% formic acid in acetonitrile (Chimalakonda et al, 2011b;Moran et al, 2011). The XDB-C18 analytical column was washed and reequilibrated between each injection by ramping mobile phase B to 95% and then returning the system to initial conditions.…”

“…The SRM-IDA transition threshold that triggered EPI experiments was fixed at 500 cps. Resulting EPI mass spectra for quality control and unknown specimens were library-matched against stored EPI mass spectra as reported previously (Chimalakonda et al, 2011b;Moran et al, 2011) or obtained from newly synthesized analytical standards to ensure that similar urinary metabolites that may interfere with analysis were fully resolved. Supplemental Figure 1 shows the mass spectra used to confirm the presence of the -1-OH metabolite of AM2201 in each reaction and identifies the appropriate molecular ion ([MH ϩ ], m/z 376) and expected product ions characteristic of aminoalkylindoles (m/z 127 and 155).…”