Pharmaceutically active compounds (PhACs) are recognized as important emerging pollutants in aquatic environments. Despite growing concern, an increasing number of new pharmaceuticals are being authorized each year, reaching the environment and compromising the quality and health of ecosystems. However, comprehensive multi-matrix investigations integrating water, sediments, biota, and wastewater systems remain scarce. In this study, a comprehensive HRMS-based suspect screening workflow was implemented using ultra-high performance liquid chromatography coupled with high-resolution mass spectrometry (UPLC-HRMS). Through a multi-compartment approach involving water (n = 89), sediments (n = 28), fish (n = 24), wastewater (n = 16), and sewage sludge (n = 16) matrices collected during 2020-2022, this study identified 153 PhACs (136 active ingredients and 17 metabolites or transformation products). Cardiovascular, psychotropic, pain management, and other nervous system drugs were the dominant therapeutic families, accounting for 63% of all identifications. Surface water contained the highest number of compounds (122), followed by effluents (85). Although influents presented fewer compounds, they showed significantly higher total input (p < 0.05). Solid matrices such as sediments and sludge accumulated highly hydrophobic substances (e.g., telmisartan, citalopram), whereas aqueous matrices contained more polar compounds (e.g., tramadol) and metabolites. Fish exhibited the lowest number of PhACs (11). Regarding spatial distribution, sites more strongly influenced by human activities displayed higher (p < 0.05) normalized areas and number of compounds. Overall, this study provides one of the most comprehensive multi matrix suspect screening assessments of PhACs to date. The holistic evaluation of the riverine ecosystem highlights the value of HRMS based workflows for identifying and prioritizing emerging contaminants, supporting the development of regulatory strategies to control pharmaceutical pollution and enabling early detection of newly marketed drugs.
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