Metabolic profiling of synthetic cannabinoid 5F‐ADB and identification of metabolites in authentic human blood samples via human liver microsome incubation and ultra‐high‐performance liquid chromatography/high‐resolution mass spectrometry

Abstract

Rationale Indazole carboxamide synthetic cannabinoids, a prevalent class of recreational drugs, are a major clinical, forensic and public health challenge. One such compound, 5F‐ADB, has been implicated in fatalities worldwide. Understanding its metabolism and distribution facilitates the development of laboratory assays to substantiate its consumption. Synthetic cannabinoid metabolites have been extensively studied in urine; studies identifying metabolites in blood are limited and no data on the metabolic stability (half‐life, clearance and extraction ratio) of 5F‐ADB have been published prior to this report. Methods The in vitro metabolism of 5F‐ADB was elucidated via incubation with human liver microsomes for 2 h at 37°C. Samples were collected at multiple time points to determine its metabolic stability. Upon identification of metabolites, authentic forensic human blood samples underwent liquid–liquid extraction and were screened for metabolites. Extracts were analyzed via ultra‐high‐performance liquid chromatography/quadrupole time‐of‐flight mass spectrometry (UHPLC/QTOFMS) operated in positive electrospray ionization mode. Results Seven metabolites were identified including oxidative defluorination (M1); carboxypentyl (M2); monohydroxylation of the fluoropentyl chain (M3.1/M3.2) and indazole ring system (M4); ester hydrolysis (M5); and ester hydrolysis with oxidative defluorination (M6). The half‐life (3.1 min), intrinsic clearance (256.2 mL min −1 kg −1 ), hepatic clearance (18.6 mL min −1 kg −1 ) and extraction ratio (0.93) were determined for the first time. In blood, M1 was present in each sample as the most abundant substance; two samples contained M5; one contained 5F‐ADB, M1 and M5. Conclusions 5F‐ADB is rapidly metabolized in HLM. 5F‐ADB, M1 and M5 are pharmacologically active at the cannabinoid receptors (CB 1 /CB 2 ) and M1 and M5 may contribute to a user's impairment profile. The results demonstrate that it is imperative that synthetic cannabinoid assays screen for pharmacologically active metabolites, especially for drugs with short half‐lives. The authors propose that M1 and M5 are appropriate markers to include in laboratory blood tests screening for 5F‐ADB.