Development and Validation of an In-House LC-MS/MS Method for the Qualitative and Quantitative Determination of 23 New Psychoactive Substances in Human Urine

The rapid diversification of new psychoactive substances (NPS) has generated a persistent analytical burden for forensic toxicology laboratories, particularly when biologically relevant concentrations are low, urinary matrices are complex, and the chemical life cycle of target compounds changes faster than routine laboratory menus can be updated. In response to this problem, an in-house LC-MS/MS method for the qualitative and quantitative determination of 23 NPS in human urine was developed and validated. An experimental method-development design was used. Blank urine matrices were collected from 20 healthy volunteers aged 18-65 years, matrix suitability was verified, enzymatic hydrolysis was applied, and solid-phase extraction was used for synthetic cathinones. The analytical panel included 16 synthetic cannabinoids, 3 synthetic opioids, and 4 synthetic cathinones. Validation was conducted in accordance with international analytical guidance, and precision, accuracy, matrix effect, selectivity, carry-over, linearity, limit of detection (LOD), limit of quantification (LOQ), and measurement uncertainty were evaluated. Across the validated panel, linearity was found to be excellent (R2 = 0.9940-0.9993), carry-over and analytical interference were not observed, and expanded uncertainty remained within acceptable limits. Sensitivity was especially strong for several high-priority analytes, including 5-Fluoro ADB, 5-Fluoro ADB-PINACA, CUMYL-4CN-BINACA, MDMB-4-EN-PINACA, pentylone, methylone, carfentanil, isotonitazene, and protonitazene. Matrix effects were dominated by ion suppression, particularly among lipophilic synthetic cannabinoids, yet the optimized workflow preserved analytical robustness. The thesis findings further indicated that 5F-AMB, ADB-BUTINACA, MDMB-4-EN-PINACA, 3-MMC, methylone, pentylone, and AMB-FUBINACA represented the compounds most salient in urine analyses. It was concluded that the proposed method provides a defensible and operationally useful platform for both screening and confirmatory toxicology, while also strengthening the analytical basis of early warning and forensic decision-making in the Turkish context.