Advancing analytical methodologies for the determination of lifestyle and dietary biomarkers in municipal wastewater.

Wastewater-based epidemiology (WBE) is increasingly used to assess the consumption of illicit drugs and other lifestyle chemicals, while its potential for evaluating population dietary habits has been scarcely investigated. In this study, a multi-residue analytical method was developed to determine more than 40 lifestyle and dietary biomarkers in raw municipal wastewater. In addition to biomarkers commonly included in WBE studies, this method incorporates several food biomarkers not previously analysed in this context. The target biomarkers were analysed using ultra-high-performance liquid chromatography-tandem mass spectrometry. Optimal separation was achieved on an ACQUITY UPLC HSS T3 column using gradient elution with 0.1% acetic acid and acetonitrile, while multiple reaction monitoring mode was used for detection and quantification of the target compounds. Sample preparation involved solid-phase extraction (SPE) on hydrophilic-lipophilic cartridges for biomarkers present at lower concentrations in wastewater (mainly food biomarkers), while direct injection of diluted wastewater samples was used for other analytes, including alcohol and tobacco/nicotine biomarkers, artificial sweeteners, antidiabetic metformin, certain vitamins, and a few other food biomarkers. The method validation parameters, including SPE recovery and accuracy, were acceptable in most cases, and the method sensitivity was sufficient to enable reliable determination of the target biomarkers in real samples. Regarding stability in wastewater during sample collection and storage, most biomarkers remained stable at 4 °C for 24 h, but moderate to high loss of some analytes occurred during the freeze-thaw cycle. The method's applicability was verified using 24-h wastewater samples from four Croatian cities, confirming its suitability for WBE.