Public Health and Forensic Toxicology: the role in preventing and managing risks of New Psychoactive Substances.

New Psychoactive Substances (NPS) represent a rapidly evolving class of synthetic compounds designed to mimic the effects of controlled drugs while evading regulatory frameworks. Their increasing chemical diversity, high potency, and unpredictable toxicity pose major challenges for public health and forensic toxicology. NPS are associated with severe clinical outcomes, including neuropsychiatric disturbances, cardiotoxicity, and hepatotoxicity, often driven by reactive metabolites, oxidative stress, and cytochrome P450 interactions. Detection and identification remain difficult due to the constant emergence of new analogues, limited availability of reference standards, and rapid metabolic transformation leading to low parent-drug detectability in biological matrices. This thesis aims to improve the analytical and toxicological understanding of NPS through two complementary studies. The first focuses on the development and validation of an HPLC–MS/MS chiral method capable of simultaneous chemo-selective and enantio-selective separation of the positional isomers 2-, 3-, and 4-CMC. The method was successfully applied to authentic oral fluid samples, demonstrating its relevance for forensic casework. The second study investigates the in vitro and in vivo metabolism of MDPiHP using HPLC-QToF-MS, supported by in silico prediction tools. A comprehensive panel of phase I and phase II metabolites was identified, enabling the proposal of specific biomarkers suitable for forensic and clinical detection. Together, these findings contribute to enhanced early warning, toxicological interpretation, and analytical monitoring of emerging NPS.