Untargeted Metabolomics via UPLC-HRMS and ASCA-Based Statistical Modeling to Characterize Metabolic Alterations...

Background: Novel Synthetic Opioids (NSOs) currently represent the third most prevalent group among New Psychoactive Substances (NPS), yet they represent the category associated with the highest health risk. Their extreme pharmacological potency compared to traditional opioids, coupled with structural heterogeneity and low concentrations in biological matrices, poses significant analytical challenges for detection. Methods: This study proposes an indirect screening approach based on untargeted metabolomics to monitor systemic responses following brorphine administration. Urinary metabolic profiling was conducted using a sex-balanced murine cohort (n = 10); metabolic profile variations were analyzed following acute and repeated administrations. Samples were collected at 0-12 h and 12-24 h intervals and analyzed by liquid chromatography coupled with high resolution mass spectrometry (LC-HRMS); data were processed using ANOVA-Simultaneous Component Analysis (ASCA), for the isolation of metabolic fingerprints associated with distinct experimental factors (sex, treatment, sampling time). Results: The results revealed a notable metabolic divergence upon repeated administration, linked to sexual diergism: while females exhibited a high recovery capacity supported by a more efficient metabolic biotransformation, males showed a stronger energetic deficit, likely due to greater substance persistence and accumulation. Male vulnerability is characterized by neuro-metabolic dysfunction, inhibition of γ-aminobutyric acid (GABA) release and altered dopaminergic turnover. This systemic imbalance is further defined by massive amino acid and nucleotide catabolism, alongside an energetic deficit exacerbated by inefficient β-oxidation. Conclusions: This study demonstrates that metabolomics may provide interesting insights for the study of NPS by recognizing the biochemical signatures of drug consumption and it is essential for developing sex-specific strategies within the broader context of opioid pharmacology.