Uncovering the hidden: Method validation for analysis of 36 psychoactive substances in Türkiye’s wastewater

Identifying trends in psychoactive substance use is essential for developing effective public health policies. Wastewater-based epidemiology (WBE) offers an objective and rapid approach to monitoring illicit drugs and new psychoactive substances (NPS). Given the continuous evolution of the drug market, it is crucial to establish analytical methods that can simultaneously analyze both conventional illicit drugs and emerging NPS. In this work, we present a novel simultaneous solid-phase extraction procedure coupled with liquid chromatography-tandem mass spectrometry for the qualitative and quantitative determination of 36 psychoactive drugs (30 NPS and 6 illicit drugs) in influent wastewater. The methodology was fully validated, with linearity, accuracy, precision, and robustness meeting all the required standards. Additional assessments were conducted to evaluate filtration loss, matrix effects, and analyte stability. The validated method was applied to 63 influent wastewater samples collected in Türkiye, revealing the presence of several NPS, including 4F-MDMB-BUTICA (detection frequency, 3 %), 4F-MDMB-BINACA-N-4-hydroxybutyl (2 %), ADB-BUTINACA (57 %), mephedrone (5 %), and ketamine (87 %). Biomarkers of illicit drugs (amphetamine, methamphetamine, MDMA, cocaine [i.e., benzoylecgonine], and cannabis [i.e., THCCOOH]) were detected in most samples, with detection frequencies between 65 % and 100 %, although some values were below the limit of quantification. This study represents the first validation of CUMYL-4CN-B7AICA, BZOHEXOXIZID, 5F-BZO-POXIZID, 4F-MDMB-BINACA butanoic acid, and 4F-MDMB-BINACA-N-4-hydroxybutyl in influent wastewater, marking a pioneering effort in Türkiye to monitor NPS and their metabolites through WBE. Considering the lack of historical data on NPS consumption in Türkiye, these findings provide a novel insight into the patterns of NPS use across the country.