Unveiling the pharmaceutical footprint in freshwater and seawater: HRMS method optimization, validation, and uncertainty estimation

• ESI-HRMS parameters were optimized for accurate & sensitive analysis of 25 pharmaceuticals. • SPE-LC-LTQ/Orbitrap MS performance was optimized thoroughly. • Method validation showed good recoveries and precision as well as low quantification limits. • Expanded measurement uncertainty was estimated (2.4–48.1 %). • Five compounds were detected with carbamazepine showing the highest concentration (11 ng/L). A thorough investigation was performed to optimize and validate an analytical approach for the targeted analysis of multi-class pharmaceuticals in aqueous matrices using high-resolution mass spectrometry (HRMS). Five pharmaceuticals were selected based on their potential detrimental effects on aquatic environments and humans. To identify and quantify the target analytes in seawater and river water, solid-phase extraction (SPE) combined with an optimized liquid chromatography-high resolution mass spectrometry (LC–HRMS) method was employed. The extraction efficiencies of three SPE protocols were evaluated within the established operational range (MQL-500/1000 ng/L).The improved extraction method was rigorously validated, showing excellent linearity (r 2 > 0.997), precision (RSD < 15 %), and accuracy (recoveries 55.9–110.3 %). Limits of quantification ranged from 0.8 to 25 ng/L. Furthermore, the calculation of the method measurement uncertainty (expanded MU < 48.1 %) offered a robust estimation of the reliability of results, thereby enhancing confidence in environmental monitoring and risk assessment applications. The method was applied to samples from the water bodies of the Louros and Arachthos rivers located in NW Greece, as well as in seawater impacted by aquaculture facilities. Five compounds were detected, with carbamazepine exhibiting the highest concentration (11 ng/L). Venlafaxine and paracetamol were the most frequently detected compounds, although they were present in trace amounts. The proposed approach is a sensitive and selective tool for the trace-level quantification of pharmaceuticals across diverse water matrices. Thus, it can support environmental monitoring efforts to reveal the pharmaceutical footprint and risks involved in aquatic ecosystems which may be affected by human activities.