Suspect and non‐target screening workflows to investigate the in vitro and in vivo metabolism of the synthetic cannabinoid 5Cl‐THJ‐018

The use of synthetic cannabinoids causes similar effects as Δ9‐tetrahydrocannabinol and long‐term (ab)use can lead to health hazards and fatal intoxications. As most investigated synthetic cannabinoids undergo extensive biotransformation, almost no parent compound can be detected in urine, which hampers forensic investigations. Limited information about the biotransformation products of new synthetic cannabinoids makes the detection of these drugs in various biological matrices challenging. This study aimed to identify the main in vitro biotransformation pathways of 5Cl‐THJ‐018 and to compare these findings with an authentic urine sample of a 5Cl‐THJ‐018 user. The synthetic cannabinoid was incubated with pooled human liver microsomes and cytosol to simulate phase I and phase II biotransformations. Resulting extracts were analyzed with liquid chromatography coupled to quadrupole time‐of‐flight mass spectrometry (LC–QTOF–MS). Three different data analysis workflows were applied to identify biotransformation products. A suspect screening workflow used an in‐house database built from literature data and in silico biotransformation predictions. Two non‐target screening workflows used a commercially available software and an open‐source software for mass spectrometry data processing. A total of 23 in vitro biotransformation products were identified, with hydroxylation, oxidative dechlorination, and dihydrodiol formation pathways as the main phase I reactions. Additionally, five glucuronidated and three sulfated phase II conjugates were identified. The predominant in vivo pathway was through oxidative dechlorination and in total six metabolites of 5Cl‐THJ‐018 were identified. Biotransformation products both in vitro and in vivo were successfully identified using complementary suspect and non‐target screening workflows. The synthetic cannabinoid 5Cl‐THJ‐018 was incubated with pooled human liver microsomes and cytosol to simulate phase I and phase II biotransformations. Using complementary suspect and non‐target screening workflows a total of 23 in vitro biotransformation products were identified including hydroxylation, oxidative dechlorination, dihydrodiol formation, glucuronidation and sulfation pathways. The results were compared with an authentic urine sample of a 5Cl‐THJ‐018 user. The predominant in vivo pathway was through oxidative dechlorination and in total six metabolites of 5Cl‐THJ‐018 were identified.