Ultra-High-Performance Liquid Chromatography-High Resolution Mass Spectrometry and Gas Chromatography-Mass Spectrometry Screening of Classical Psychoactive Substances, Novel Psychoactive Substances, and Metabolites in Postmortem Urine Samples

The use of new psychoactive substances (NPSs) is increasing, and their chemical diversity together with potential postmortem changes demands accurate screening and confirmatory analysis in biological matrices. This study highlights the analytical value of combining ultra-high-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-ESI(+/−)-Orbitrap-HRMS) and gas chromatography-mass spectrometry (GC/MS) for the broad-coverage screening of classical psychoactive substances (CPSs), NPSs, and their metabolites in a selected cohort of 25 postmortem urine samples with suspected NPS consumption; given this limited size, results should be regarded as a proof-of-concept rather than epidemiologically representative. Forty-four drugs/metabolites were identified, most confirmed against authentic standards (Level 1), while a subset of minor or emerging metabolites received tentative/probable identifications (Level 2) based on accurate mass, isotopic pattern, and fragmentation. Ketamine, one of the most prevalent drugs (88% LC/MS, 84% GC/MS), followed by cocaine (68% LC/MS, 12% GC/MS); amphetamine-type substances (ATS) showed marked technique-dependent detection (e.g., 3,4-methylenedioxyamphetamine (MDA): 48% LC/MS, 8% GC/MS). LC/MS offered broader polarity coverage. GC/MS reliably detected parent drugs and derivatizable xenobiotics. Together, both platforms provided complementary confirmed and tentative-level coverage for forensic urine screening; quantitative validation and larger cohorts are needed before diagnostic, impairment, or epidemiological extrapolation can be made.