High-throughput and cost-effective quantification of psychoactive pharmaceuticals in wastewater with comprehensive in-sample stability evaluation for wastewater surveillance purpose

There is a growing need for efficient methods to quantify a wide range of psychoactive pharmaceuticals in wastewater and to comprehensively evaluate their in-sample stability for both population-level drug use monitoring and environmental risk assessment. Traditional analytical approaches using solid-phase extraction (SPE) incur high time and cost burdens and generally lack systematic stability assessment, hindering research progress in high-throughput monitoring. In this study, a cost-effective and sensitive method using direct injection LC-MS/MS was developed for the simultaneous quantification of 30 psychoactive pharmaceutical biomarkers, with a comprehensive assessment of their in-sample stability under six storage conditions across multiple freeze-thaw cycles. The method met all required performance criteria and achieved quantification limits of 0.2–100 ng/L, comparable to or lower than SPE methods, while saving 5.5 h and 15 Australian dollars per sample processing. This method also reduced solvent and material consumption, contributing to improved analytical sustainability. In our stability assessment, acidification of samples reduced in-sample transformation for most target biomarkers, with <20 % degradation over 28 days at 22 °C, even after multiple freeze-thaw cycles. However, certain benzodiazepines exhibited higher stability in untreated wastewater. The method was applied to 68 influent wastewater samples collected from ten wastewater treatment plants from Australia in 2021. Nineteen chemicals from five pharmacological classes were detected at concentration ranging from 1.0 to 7900 ng/L. This high-throughput, cost-effective, and environmentally sustainable method can thus be applied for large-scale wastewater-based epidemiology and long-term monitoring of psychoactive pharmaceutical use. • New rapid LC-MS/MS method for analysing psychoactive pharmaceuticals in wastewater. • The sensitive method has LOD and LOQ comparable or lower than previous SPE methods. • Acidification preserves antidepressants over time, enduring multiple freeze-thaw cycles. • Psychoactive pharmaceutical biomarkers were quantified in Australian wastewater. • Bupropion and methylphenidate were detected with concentrations as low as 1 ng/L.