Use of UPLC-HRMS/MS for In Vitro and In Vivo Metabolite Identification of Three Methylphenidate-derived New Psychoactive Substances.

The distribution of so-called new psychoactive substances (NPS) as substitute for common drug of abuse was steadily increasing in the last years, but knowledge about their toxicodynamic and toxicokinetic properties is lacking. However, a comprehensive knowledge of their toxicokinetics, particularly their metabolism, is crucial for developing reliable screening procedures and to verify their intake, e.g., in case of intoxications. The aim of this study was therefore to tentatively identify the metabolites of the methylphenidate-derived NPS isopropylphenidate (isopropyl 2-phenyl-2-(2-piperidyl) acetate, IPH), 4-fluoromethylphenidate (methyl 2-(4-fluorophenyl)-2-(piperidin-2-yl) acetate, 4-FMPH) and 3,4-dichloromethylphenidate (methyl 2-(3,4-dichlorophenyl)-2-(piperidin-2-yl) acetate, 3,4-CTMP) using different in vivo and in vitro techniques and ultra-high performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS/MS). Urine samples of male rats were analyzed, and the transfer to human metabolism was done by using pooled human S9 fraction (pS9), which contains the microsomal fraction of liver homogenisate as well as its cytosol. UHPLC-HRMS/MS analysis of rat urine revealed 17 metabolites for IPH (14 phase I and 3 phase II metabolites), 13 metabolites were found for 4-FMPH (12 phase I metabolites and 1 phase II metabolite) and 7 phase I metabolites and no phase II metabolites were found for 3,4-CTMP. pS9 incubations additionally indicated that all investigated substances were primarily hydrolyzed, resulting in the corresponding carboxy metabolites. Finally, these carboxy metabolites should be used as additional analytical targets besides the parent compounds for comprehensive mass spectrometry-based screening procedures.