Metabolic fate and detectability of three novel fluoroprolintane psychostimulants: in vitro and in vivo characterization by hyphenated mass spectrometry

Amphetamine-type stimulants (ATS) represent a major segment of the global drugs of abuse market. Prolintane (1-(1-phenylpentan-2-yl)pyrrolidine), a substituted phenylethylamine ATS, was historically utilized as medication for several therapeutic indications. This study aimed to investigate the metabolic fate and urinary detectability of the three novel derivatives 2-, 3-, and 4-fluoroprolintane, which were recently shown to act as monoamine reuptake inhibitors. Liquid chromatography (LC)-high-resolution (HR) tandem mass spectrometry (MS/MS) was used for tentative identification of metabolites in rat urines (collected over a 24 h period following oral administration of 2 mg/kg fluoroprolintane isomer, each) or incubations with pooled human liver S9 fraction (1 h and 6 h, 25 µM fluoroprolintane isomer, each). Isozyme mapping was performed using individual incubations with 11 human phase I monooxygenases. The same rat urines were used for detectability studies using standard urine screening approaches (SUSA) by gas chromatography (GC)-mass spectrometry (MS), LC-ion trap MS, and LC-HRMS/MS. The fluoroprolintanes were extensively metabolized, with a total of 79 metabolites identified. Hydroxylations and subsequent oxidations were primarily mediated by CYP1A2, CYP2B6, CYP2C19, and CYP2D6, while glucuronidation and O-methylation were observed as main follow-up phase II reactions. All three SUSA allowed detection, primarily via metabolites. However, the high degree of metabolic overlap will make isomer differentiation challenging. These findings contribute to a comprehensive risk assessment and provide critical reference data for clinical and forensic laboratories to identify these substances in biological specimens.