Integrating liquid chromatography–high resolution mass spectrometry based suspect screening with ionization efficiency prediction to expand semi-quantitative coverage in wastewater-based epidemiology: application to environmental chemicals

Wastewater-based epidemiology (WBE) is increasingly used to assess population-level chemical exposure, yet its analytical scope is often limited by target-based methods and the availability of reference standards. In this study, we developed an integrated analytical workflow that expanded the coverage of environmental chemicals and enabled standard-free semi-quantitative analysis under a realistic post hoc WBE scenario. Influent wastewater samples from nine wastewater treatment plants in Taiwan were re-examined using a single additional liquid chromatography-high resolution mass spectrometry (LC-HRMS) analysis in full-scan and data-dependent acquisition modes. A total of 189 environmental compound signals were structurally identified, representing 79 unique chemical structures. Semi-quantitative concentration estimation was achieved using a single-point calibration strategy based on isotope-labeled internal standards originally included for illicit drug analysis, combined with ionization efficiency (IE) prediction tools. Internal cross-validation yielded median prediction errors of 2.3-fold in positive ionization mode and 5.0-fold in negative ionization mode. Furthermore, cross-method validation against historical targeted datasets for six illicit drugs within the same sample cohort revealed a median underestimation of 6.7-fold. To evaluate accuracy under controlled conditions, an independent spiking experiment using authentic standards in groundwater demonstrated an overall median error of 4.3-fold, with 81.3 % of the quantified analytes falling within one order of magnitude of their nominal concentrations. Population-normalized mass load analysis revealed regional differences in chemical exposure profiles, with elevated mass loads of smoking-related biomarkers, including cotinine and nornicotine, observed in wastewater from southern Taiwan.