In vitro metabolism of the synthetic cannabinoids PX‐1, PX‐2, and PX‐3 by high resolution mass spectrometry and their clearance rates in human liver microsomes

RATIONALE: N-(1-Amino-1-oxo-3-phenylpropan-2-yl)-1-(5-fluoropentyl)-1H-indole-3-carboxamine (PX-1), N-(1-amino-1-oxo-3-phenylpropan-2-yl)-1-(5-fluoropentyl)-1H-indazole-3-carboxamid e (PX-2), and N-(1-amino-1-oxo-3-phenylpropan-2-yl)-1-(cyclohexylmethyl)-1H-indazole-3-carboxam ide (PX-3) are scheduled synthetic cannabinoids (SCs). Due to the lack of metabolism data and the extensive metabolism of SCs, consumption of these illicit substances is challenging to prove. The aim of this study was to investigate the metabolism of PX-1, PX-2, and PX-3 and propose marker metabolites to confirm their use. METHODS: PX-1, PX-2, and PX-3 were incubated in pooled human liver microsomes (HLM) to evaluate the phase I metabolites which were identified using a Q-Exactive hybrid quadrupole-Orbitrap mass spectrometer. Metabolic stability studies were also performed to investigate the half-life and clearance rates using an Agilent 6470A triple quadrupole mass spectrometer. RESULTS: The calculated half-lives were 15.1 +/- 1.02, 3.4 +/- 0.27, and 5.2 +/- 0.89 min for PX-1, PX-2, and PX-3, respectively, in HLM. The calculated intrinsic clearance values were 0.046, 0.202, and 0.133 mL/min/mg for PX-1, PX-2, and PX-3, respectively. Four metabolites of PX-1, six metabolites of PX-2, and five phase I metabolites of PX-3 were detected. Oxidative deamination was the common biotransformation between the three compounds and there were no common metabolites. CONCLUSIONS: The metabolic profiles of PX-1, PX-2, and PX-3 provide valuable information for the detection of their metabolites in forensic samples.