A Practical Framework for Wastewater-Based Monitoring of Substance Use in Public Health Settings.

The ongoing substance use crisis in the United States involves a broad range of illicit and prescription drugs, including opioids, stimulants, sedatives, and various psychoactive and non-psychoactive compounds. Traditional surveillance methods rely on self-reported data, which could lead to bias and recall inconsistencies. Wastewater-based epidemiology has emerged as a powerful, non-invasive tool for monitoring community-level drug use, offering near real-time estimates and the potential to serve as an early warning system. However, challenges such as analyte degradation, wastewater variability, and matrix effects can affect data quality and comparability across regions. This study presents a standardized, practical workflow for multi-drug (n = 52) detection in wastewater, aiming to minimize analyte loss and improve reproducibility. Composite samples were collected from multiple U.S. cities, transported on ice, and extracted using solid-phase extraction. Extraction efficiencies were compared using Oasis Hydrophilic-Lipophilic-Balanced and Mixed-mode Cation-Exchange (MCX) cartridges, with the MCX sorbent providing complementary reversed-phase and cation-exchange interactions that enabled the retention of chemically diverse compounds across multiple drug classes. Analysis was performed with an Ultra-High-Performance Liquid Chromatography system coupled to a triple quadrupole mass spectrometer, in which the instrument parameters and critical methodological considerations, including sample handling, transport, column selection, and method validation, are detailed. This work contributes to the development of a robust, scalable protocol for multi-drug surveillance in wastewater, supporting timely, data-driven public health responses and informing national drug policy efforts.