Improving wastewater-based epidemiology for new psychoactive substance surveillance by combining a high-throughput in vitro metabolism assay and LC−HRMS metabolite identification

One of the primary criteria for a suitable drug biomarker for wastewater-based epidemiology (WBE) is having a unique source representing human metabolism. For WBE studies, this means it is important to identify and monitor metabolites rather than parent drugs, to capture consumption of drugs and not fractions that could be directly disposed. In this study, a high-throughput workflow based on a human liver S9 fraction in vitro metabolism assay was developed to identify human transformation products of new chemicals, using α-pyrrolidino-2-phenylacetophenone (α-D2PV) as a case study. Analysis by liquid chromatography coupled to high resolution mass spectrometry identified four metabolites. Subsequently, a targeted liquid chromatography – tandem mass spectrometry method was developed for their analysis in wastewater samples collected from a music festival in Australia. The successful application of this workflow opens the door for future work to better understand the metabolism of chemicals and their detection and application for wastewater-based epidemiology.