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Wastewater-based epidemiology applies the analysis of human metabolic excretion products of xenobiotics in wastewater to estimate the community-wide use of these compounds. A new bioanalytical method was developed, optimised and validated for the analysis of a broad range of antidepressants and their metabolites at trace concentrations in influent wastewater. The assay was based on solid-phase extraction and liquid chromatography coupled to tandem mass spectrometry. For most compounds, Oasis® HLB cartridges were used for sample preparation. Oasis® MCX cartridges were used for extraction of normirtazapine, moclobemide, sertraline, and melitracen in particular. The Kinetex XBC18 column with a gradient elution resulted in appropriate separation for the analytes under investigation. Validation was done according to the European Medicines Agency guidelines on bioanalytical method validation. For 27 compounds, the performance criteria met the requirements for method validation. For these analytes, the lower limit of quantification (LLOQ) ranged between 1 and 25 ng/L. Furthermore, all targeted biomarkers showed high in-sample stability during 24 h, with the exception of mianserin. The validated assay was applied to influent wastewater samples collected from four wastewater treatment plants in Belgium. Among these four locations, a total of 18 out of 27 biomarkers for antidepressant use were present in the samples in concentrations above the LLOQ. Additionally, the proposed methodology proved capable of analysing high resolution spatio-temporal trends. Mann-Kendall trend analyses showed that antidepressant use is stable throughout the week, except for trazodone which increased throughout the week. fx1 • Fully validated method for 27 antidepressants analysis in wastewater. • Different matrix effects for 5 antidepressants vs deuterated standard. • High in-sample stability for all targeted biomarkers. • Successfully applied to influent wastewater samples. [ABSTRACT FROM AUTHOR]