Beyond Structural Similarity: Metabolic and Analytical Approaches to Differentiate α-PHP and α-PiHP

Synthetic cathinones (SCs) pose significant challenges to forensic and clinical toxicology. Among these, α-pyrrolidinohexanophenone (α-PHP) and α-pyrrolidinoisohexanophenone (α-PiHP) are closely related structural isomers whose similar structures and overlapping metabolic pathways complicate their differentiation in biological specimens. This critical review examines their pharmacological and toxicological properties, metabolism, biological matrices, sample preparation, analytical methods, and biomarkers of exposure. Emphasis is placed on distinguishing computationally predicted and in vitro-generated metabolites from those confirmed in human samples, and on evaluating their suitability as exposure markers. Conventional and emerging analytical approaches, including GC–MS, LC–MS/MS, high-resolution mass spectrometry, and NMR, are critically assessed, alongside the emerging role of toxicometabolomics in identifying endogenous metabolic perturbations beyond xenobiotic metabolism. The review highlights key challenges in isomer discrimination, biomarker validation, and analytical harmonization, and proposes an evidence-based framework for selecting biological matrices, analytical targets, and instrumental platforms according to forensic and clinical needs.