The in-sample stability of selected pharmaceuticals, illicit drugs, and their metabolites in wastewater was assessed under six different conditionsuntreated, addition of hydrochloric acid or sodium metabisulfite solution, combined with or without sterile filtration, and at four representative temperatures, at 35 °C for up to 28 days, 22 °C for 56 days, and 4 °C and −20 °C for 196 days, or freeze/thaw cycles for 24 weeks. Paracetamol, 6-monoacetylmorphine, morphine, and cocaine were poorly stable in untreated wastewatere.g., with 50% transformation within 1.2−8.1 days at 22 °C, and acidification reduced their in-sample transformations. Acesulfame, carbamazepine, cotinine, methamphetamine, 3,4methylenedioxy-methamphetamine (MDMA), ketamine, norfentanyl, 3,4-methylenedioxy-N-ethylamphetamine (MDEA), and norbuprenorphine were highly or moderately stable over the observed period, even in untreated wastewater. Fitting of pseudofirst-order kinetics and the Arrhenius equation was used to develop a multistage transformation estimation model combined with an interactive tool to evaluate possible transformation scenarios of selected biomarkers for the processes from sampling to preanalysis. However, as the wastewater composition can vary between sites and over time, the variability of in-sample stability requires further exploration.

Wastewater analysis shows promise in tracking community use of performance- and image-enhancing drugs, yet their post-sampling stability remains unknown. To understand and minimize post-sampling analyte degradation, knowledge of stability and optimal preservation methods is essential. A selection of 52 performance-enhancing substance biomarkers relevant to sports doping and image enhancement were subjected to a series of stability experiments in wastewater, testing several conditions including two pretreatments, filtered and unfiltered, and three sample preservation methods, unpreserved, sodium metabisulfite addition, and hydrochloric acid addition to pH 2, and were assessed at three temperatures, 22, 4, and −20 °C, for up to 200 days. Results indicated that the optimum preservation techniques were filtration, with or without preservatives, and acidification to pH 2 without filtration, with the times to 50% transformation indicating that the majority had medium (40–160%) to high (80–120%) stability at 4 °C for up to 92 days and at −20 °C for up to 200 days. A typical sample collection and archiving scenario was assumed and evaluated for each biomarker using an interactive calculator made available online from this study. This study provides, for the first time, post-sampling/archiving stability assessments for 52 biomarkers of performance- and image-enhancing drugs in wastewater for six pretreatment methods.