Abstract
Background: Wastewater-based epidemiology (WBE) enables the population-level surveillance of molecular and chemical targets. Despite the high prevalence of respiratory diseases, there is a lack of sensitive analytical methods for detecting associated medications in complex wastewater matrices. Methods: We developed and validated a liquid chromatography-mass spectrometry (LC-MS)/MS method using multiple reaction monitoring for 10 common respiratory pharmaceuticals. The workflow integrated freeze-drying for preconcentration, online solid-phase extraction for cleanup, and stable isotope-labeled internal standards (SILs) to compensate for matrix effects. Results: Detection and quantification limits ranged from 0.7 to 19 ng L(-1) and 3 to 125 ng L(-1), respectively, with recoveries of 82-194% and precision within 0.14-7.2% relative standard deviation. Matrix effects (64-228%) were effectively corrected using SILs. Application to 12 neighborhood-level wastewater samples detected 9 of the 10 target compounds, with 6 (albuterol, amoxicillin, azithromycin, cetirizine, diphenhydramine, and fexofenadine), detected above their quantification limits. Fexofenadine was the most abundant, reaching 3309 ng L(-1). Conclusion: This robust, low-volume, high-throughput LC-MS/MS method enables the reliable detection of respiratory pharmaceuticals in wastewater, supporting WBE applications for pharmaceutical use surveillance.