Metabolic profile of the synthetic drug 4,4′-dimethylaminorex in urine by LC–MS-based techniques: selection of the most suitable markers of its intake

Purpose: In this study, the phase I and II metabolic pathways of 4,4′-dimethylaminorex were characterized to select the marker(s) of intake allowing the unequivocal identification of this novel psychoactive substance in urine samples. Methods: The metabolic profile of 4,4′-dimethylaminorex was characterized using both in vitro and in vivo models. In detail, for the in vitro experiments, either pooled human liver microsomes or recombinant cytochrome P450 isoforms were selected, whereas the in vivo investigation was performed on male mice ICR (CD-1®). Sample preparation included enzymatic hydrolysis followed by liquid/liquid extraction. The instrumental analysis was performed by ultra-high-performance liquid chromatography coupled to either high- or low-resolution tandem mass spectrometry. Results: Five metabolic products were isolated only for the cis-isomer: the phase I metabolic reactions included hydrolysis, carboxylation, hydroxylation, and carbonylation. CYP2D6 was the principal isoenzyme involved, and the incubation in the presence of different allelic variants showed significant alteration on the metabolic profile. Once formed, the phase I metabolites underwent extensive conjugation. Not only the most abundant compounds detected, but also those with the most extended window of detection, were the carboxylated and the hydroxylated metabolites. These analytes together with the parent compound were selected as the most suitable markers of intake. Conclusions: Knowledge of the metabolic profiles of the new drugs is essential for their fast identification. Phase I and phase II metabolites of 4,4′-dimethylaminorex were identified and selected as markers of intake, to be considered as the most suitable analytical targets in forensic toxicology.