METABOLITE PROFILING OF SYNTHETIC CANNABINOIDS AND IDENTIFICATION IN HUMAN BLOOD VIA HUMAN LIVER MICROSOME INCUBATION AND HIGH RESOLUTION TANDEM MASS SPECTROMETRY

Abstract

Synthetic cannabinoids are recreational drugs designed to mimic the effects of Δ9-tetrahydrocannabinol (THC), the main psychoactive component present in cannabis. These drugs exhibit severe toxic effects upon consumption due to their high binding affinity and potency at the cannabinoid receptors (CB1 and CB2). Synthetic cannabinoids have proliferated over the last decade and become a major public health and analytical challenge, critically impacting the clinical and forensic communities. Indazole carboxamide and indole carboxamide class synthetic cannabinoids have been particularly rampant, and are the compound classes most frequently reported to governmental agencies worldwide. However, the metabolic and pharmacological properties of many of these compounds remains unknown. Elucidating these characteristics allows members of the clinical and forensic communities to identify causative agents in patient samples, as well as render conclusions regarding their toxic effects. The aim of this research study was to assess the in vitro Phase I metabolic profile of five synthetic cannabinoids and report the major metabolites identified; compounds evaluated included MDMB-CHNINACA; APP-CHMINACA (PX-3); 5F-APP-PICA (PX-1); 5F-MDMB-PINACA (5F-ADB); and FUB-AMB. These analytes were incubated for 120 minutes with human liver microsomes, followed by analysis of the extracts via ultra high performance liquid chromatography – tandem mass spectrometry (UHPLC-MS/MS). The high-resolution mass spectrometry tool utilized (quadrupole-time of flight mass spectrometry, QTOF) allowed for a thorough characterization of the metabolites, including the assignment of a chemical formula and structure, and accurate mass. The metabolic stability and kinetic profiles of 5F-ADB and FUB-AMB were evaluated by aliquoting the incubation samples at various time points throughout the procedure. It was observed that these compounds were metabolized rapidly, resulting in short half-lives and relatively elevated metabolic clearances. A variety of metabolites were identified for most of the species studied, and this was dependent on the chemical structure of the parent molecule. The major metabolites identified overall for the species were products of amide or ester hydrolysis; hydroxylation (including polyhydroxylation) of the pentyl side chain or cyclohexylmethyl moiety; and oxidative defluorination. It is proposed that these metabolites (especially analyte-specific metabolite) be included in laboratory assay panels to facilitate unequivocal identification of the synthetic cannabinoid agent of interest. For select compounds (5F-ADB and FUB-AMB), authentic forensic human blood samples which screened positive for these analytes were provided by a renowned forensic toxicology laboratory. These samples were tested to verify that the major metabolites identified in the in vitro studies were also present in blood in vivo; the resultant data from the 5F-ADB and FUB-AMB samples showed that the major hydroxylated and hydrolysis metabolite, respectively, were present in greater abundance than the parent molecule, which was most often absent or not present in an appreciable quantity. Additionally, it was observed in the time studies of 5F-ADB and FUB-AMB that the metabolites containing carboxylic acid functional groups were detected in incubation samples longer than the hydroxylated metabolites, potentially indicative of longer detection windows in human samples. These findings have important toxicological implications; many synthetic cannabinoid metabolites, including those identified in this study may have pharmacological activity and contribute to a drug user’s overall impairment profile; identifying them in blood in the absence of parent compound can point to the causative agent. The results demonstrate that it is imperative that synthetic cannabinoid assays screen for known pharmacologically active metabolites; this is particularly important for drugs with short half-lives. The results of this research can be applied to the prediction of metabolic pathways for synthetic cannabinoids as well as non-drug substances with similar structural elements whose metabolic profile has not yet been elucidated, and whose pharmacological activity is currently unknown. Additionally, the results provide reference standard manufacturers and research scientists with further insight into the metabolic products of synthetic cannabinoids and related compounds for the synthesis of materials for the development of laboratory assays.