In-depth in vivo and in vitro investigation of metabolism and detectability of three novel methylprolintane psychostimulants by hyphenated mass spectrometry

Amphetamine-type stimulants (ATS) remain a major group among internationally misused psychostimulants. Prolintane (1-(1-phenylpentan-2-yl)pyrrolidine) is one representative historically applied in clinical settings. Recently, 2-, 3-, and 4-methylprolintane, structurally related prolintane analogs, have been identified as inhibitors of monoamine reuptake, prompting the need for detailed toxicological and analytical characterization. To explore their metabolic transformation and assess their detectability in urine, we analyzed rat samples collected over 24 h following oral administration (2 mg/kg) of each isomer, as well as human liver S9 incubations (25 μM; 1 h and 6 h). Metabolites were tentatively assigned using liquid chromatography-high-resolution tandem mass spectrometry (LC-HRMS/MS). Complementary isoenzyme-specific incubations with 11 human phase I monooxygenases enabled mapping of the primary biotransformation routes. The same in vivo samples were subsequently evaluated with commonly used urine screening strategies, including gas chromatography-MS, LC-ion trap MS, and LC-HRMS/MS. Across all isomers, metabolism was extensive, yielding 95 distinct metabolites. Initial hydroxylation and oxidation reactions were mainly linked to the activity of CYP1A2, CYP2B6, CYP2C19, and CYP3A4, followed by conjugation through glucuronidation, sulfation, or O -methylation. Each screening platform successfully detected the compounds via their metabolites; however, the high degree of overlap in the metabolic profiles limits the ability to distinguish between the three isomers. These results provide essential analytical reference data and contribute to toxicological risk assessment of these emerging ATS derivatives.