In vitro metabolomics for the characterization of drugs of abuse and new psychoactive substances in SH-SY5Y cells.

New psychoactive substances (NPS) continuously emerge on unregulated drug markets, while in vitro pharmacological profiling at primary targets (e.g., cannabinoid receptor 1) often remains the only means of effect characterization. In this proof-of-concept study, we evaluated a complementary untargeted in vitro metabolomics approach for its ability to distinguish centrally active drugs based on drug-specific alterations of the cellular metabolome. SH-SY5Y neuroblastoma cells were exposed for 18 h to two NPS (MDMB-4en-PINACA and 4-methylmethcathinone) as well as alprazolam, cocaine, and tramadol. Cell extracts were analyzed using normal- and reverse-phase high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (HPLC-QToF). Multivariate analyses revealed distinct metabolic signatures and clear separation from controls for all compounds except tramadol. These findings demonstrate the feasibility and discriminatory capacity of the established workflow, highlighting its potential as a complementary tool for pharmacological and toxicological characterization of emerging psychoactive substances.