Targeted Metabolomic Approach for Assessing Human Synthetic Cannabinoid Exposure and Pharmacology

Abstract

Designer synthetic cannabinoids like JWH-018 and AM2201 have unique clinical toxicity. Cytochrome-P450-mediated metabolism of each leads to the generation of pharmacologically active (ω)- and (ω-1)-monohydroxyl metabolites that retain high affinity for cannabinoid type-1 receptors, exhibit Δ(9)-THC-like effects in rodents, and are conjugated with glucuronic acid prior to excretion in human urine. Previous studies have not measured the contribution of the specific (ω-1)-monohydroxyl enantiomers in human metabolism and toxicity. This study uses a chiral liquid chromatography-tandem mass spectroscopy approach (LC-MS/MS) to quantify each specific enantiomer and other nonchiral, human metabolites of JWH-018 and AM2201 in human urine. The accuracy (average % RE = 18.6) and reproducibility (average CV = 15.8%) of the method resulted in low-level quantification (average LLQ = 0.99 ng/mL) of each metabolite. Comparisons with a previously validated nonchiral method showed strong correlation between the two approaches (average r(2) = 0.89). Pilot data from human urine samples demonstrate enantiospecific excretion patterns. The (S)-isomer of the JWH-018-(ω-1)-monohydroxyl metabolite was predominantly excreted (>87%) in human urine as the glucuronic acid conjugate, whereas the relative abundance of the corresponding AM2201-(ω-1)-metabolite was low (<5%) and did not demonstrate enantiospecificity (approximate 50:50 ratio of each enantiomer). The new chiral method provides a comprehensive, targeted metabolomic approach for studying the human metabolism of JWH-018 and AM2201. Preliminary evaluations of specific enantiomeric contributions support the use of this approach in future studies designed to understand the pharmacokinetic properties of JWH-018 and/or AM2201.

Altered metabolism of synthetic cannabinoid JWH-018 by human cytochrome P450 2C9 and variants

“…Quantification of analytes of interest was performed using positive ion electrospray ionization LC-MS/MS methods developed previously for chiral analysis of synthetic cannabinoid metabolites [17]. For these studies, analytical standards (0.50–100 ng/ml) were matrix-matched in the final in vitro reaction mixture (ethanol-water-0.1 M phosphate buffer, pH 7.4, 50:30:20%).…”

“…All metabolic products were confirmed by matching retention times of analytical standards and deuterated internal standards. Mass spectra and NMR data for each metabolite have been reported previously [8,17,18]. …”

Impaired Driving Associated with the Synthetic Cannabinoid 5F-ADB

“…Previous liquid-chromatography tandem mass-spectrometry (LC-MS/MS) methods validated for detecting synthetic cannabinoids and other designer drugs were modified to detect 5F-ADB and 5F-ADB metabolite 7 (Cayman Chemical Company, Ann Arbor, MI) [3–5]. In brief, 0.25 mL of sample was pretreated with 0.25 mL of 0.5M ammonium hydroxide and extracted with two 0.90 mL fractions of ethyl acetate after loading on Biotage ISOLUTE® SLE+ Supported Liquid Extraction 96-well plates (Biotage, LLC, Charlotte, NC).…”

Annual banned‐substance review: analytical approaches in human sports drug testing

“…Predominantly, oxygenation/ hydroxylation of the alkyl side chain and the phenyl moiety were postulated; however, though providing a comprehensive set of information, more data (e.g. [154] While the differentiation of urinary metabolites of cannabinoids as to their enantiomeric composition is (yet) irrelevant for doping controls, the need for comprehensive and fast analytical assays is of great Gandhi et al used human hepatocytes and LC-MS(/MS) with high resolution/high accuracy mass spectrometry to generate a metabolic scheme for AKB-48 [N-(1-adamantyl)-1-pentylindazole-3carboxamide], demonstrating the generation of 15 phase-I plus two glucuronic acid conjugated metabolites.…”