Automated data exploration of α-pyrrolidinoisohexiophenone metabolites in Upcyte human hepatocytes reveals potential individual-specific metabolic profiles

Abstract

Synthetic cathinones, a major class of new psychoactive substances, pose significant public health challenges due to their toxicity and frequent involvement in overdoses. However, detecting them in biological samples remains challenging because of their structural similarities and a lack of metabolic data. This study analyses the in vitro toxicity and metabolism of the synthetic cathinone α-pyrrolidinoisohexiophenone (α-PiHP) using Upcyte Human Hepatocytes (UHH) as hepatic cell model. Liquid chromatography coupled with high-resolution tandem mass spectrometry (LC-HRMS/MS) was employed to analyze both cell media and intracellular contents. An automatic data mining strategy was developed to identify putative metabolites based on predicted biotransformations, MS/MS spectral similarities among putative metabolites and the parent compound, and unique signals in treated samples. This workflow led to the identification of 10 Phase I metabolites. The most abundant metabolite in cell extracts was M1 (C₁₆H₂₅NO, α-PiHP ketone β-reduction), while metabolite M8 (C₁₆H₂₃NO₃, α-PiHP dihydroxylation) predominated in the cell medium, suggesting their potential as biomarkers for α-PiHP consumption. Notably, interindividual differences in in vitro hepatic metabolism were observed across two donors, indicating idiosyncratic metabolic profiles, and underscoring the importance of human variability in drug metabolism studies and in forensic and clinical applications. In summary, these findings enhance our understanding of α-PiHP metabolism and may improve the detection of synthetic cathinones in biological and environmental samples.