Analysis of β-Methylphenethylamine (BMPEA) and Its Novel Metabolites in Rat Blood Using MMSPE and UPLC-qTOF-MS

β-Methylphenethylamine (BMPEA), a positional isomer of amphetamine increasingly detected in dietary supplements and weight-loss products, poses significant analytical challenges in forensic and doping control due to its structural similarity to amphetamine. This study presents a validated analytical workflow combining mixed-mode solid-phase extraction (MMSPE) with ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-qTOF-MS) for the selective quantification of BMPEA and identification of its metabolites in rat cardiac blood. Blood was taken at 20 and 90 min after injection from twelve adult male Sprague-Dawley rats that were randomly assigned to four groups (n = 3): an untreated control, a low-dose cohort (10 mg/kg, i.p.), and two high-dose cohorts (30 mg/kg, i.p.). The technique demonstrated strong differentiation between BMPEA and amphetamine isomers, excellent linearity over 20-1000 ng/mL (R² > 0.99), and quantification limits appropriate for forensic applications. A short biological half-life and quick elimination kinetics are consistent with related phenethylamines, as evidenced by the peak BMPEA concentrations of 899 ng/mL at 20 min and 22 ng/mL at 90 min. Comprehensive low- and high-energy mass spectrometric analyses revealed a novel BMPEA metabolite, characterized as 1-amino-2-phenylpropan-2-ol, based on fragmentation patterns and retention time comparison with reference standards. This work delivers a rigorous, high-sensitivity analytical tool for BMPEA detection in biological matrices and enhances understanding of its metabolic fate, offering critical biomarkers for forensic toxicology and anti-doping investigations.