Advances in Liquid Chromatography-Mass Spectrometry Analysis of Non-Fentanyl Opioids in Biological Samples
Non-fentanyl opioids, a subclass of new synthetic opioids (NSOs), have emerged as the fastest-growing category of new psychoactive substances (NPS) globally, driven by regulatory tightening on fentanyl analogs. Their structural diversity, rapid in vivo metabolism, and multiple metabolic pathways complicate detection in biological matrices, posing significant challenges for forensic toxicology and environmental monitoring. Liquid chromatography-mass spectrometry (LC-MS) remains the gold standard for trace-level quantification due to its high sensitivity, specificity, and accuracy. This review systematically examines the classification, toxicological profiles, and metabolic routes of non-fentanyl opioids, including AH-7921, MT-45, U-47700, brorphine, and nitazenes. It critically evaluates sample preparation techniques—solid-phase extraction (SPE), liquid-liquid extraction (LLE), and protein precipitation (PPT)—highlighting their efficiency, recovery rates, and matrix effects. Furthermore, it synthesizes recent advances in LC-MS methodologies, including high-resolution mass spectrometry (HRMS) and tandem mass spectrometry (MS/MS), with emphasis on multiplex detection capabilities, limits of detection (LODs) reaching sub-ng/mL levels, and validation parameters. The review underscores the necessity for continuous analytical innovation to keep pace with emerging NSOs and provides a technical framework for accurate identification in forensic and environmental contexts.