BackgroundAssessing antimicrobial chemicals from wastewater source to recipient water systems is crucial in planning effective, policy-related interventions for antimicrobial resistance (AMR) risk mitigation. However, the capability of related analytical methods for AMR assessment has not been explored previously. There is also a lack of knowledge on the effectiveness of alternative extraction sorbents with ion-exchange functions, and little information on chemical stability from sampling to analysis as well as preservative options. Hence, our study aims to address the clear need for advanced, broad-range and microbiologically-sensitive methodologies, paired with thorough stability assessments. ResultsOasis® WCX ion-exchange was for the first time employed in solid-phase extraction (SPE) for antibacterials, antifungals, antivirals and human metabolites in various water matrices. Analysis was performed using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) on a biphenyl analytical column. The optimized and validated method provided satisfactory accuracy, precision, and recovery for 53 compounds via LC-MS/MS direct injection and for up to 35 compounds via SPE-LC-MS/MS. Method quantification limits (MQLs) were determined in groundwater (0.33–54 ng L−1), surface water (0.53–75 ng L−1), effluent wastewater (2.5–470 ng L−1), and influent wastewater (11–650 ng L−1). As a novel approach, MQLs were compared with minimum inhibitory concentrations, to confirm our method's microbiological sensitivity for studying AMR. Stability assessment revealed that most compounds remained stable in standard solution at −80 °C for six months, in various waters at −20 °C for eight weeks, and during 24-h sampling at 4 °C. Sodium azide was a better preservative than sodium metabisulfite. SignificanceOur study is an added value to the analytical methodology for water measurements of antimicrobial chemicals, in which it provides a novel, alternative method that is robust and overall more sensitive than others using generic Oasis® HLB sorbents and C18 analytical columns in SPE-LC-MS/MS. Also, the comprehensive data on antimicrobial stability helps reduce methodological uncertainty for future studies. Our method shows sufficient microbiologically-sensitive and thus is suitable for future (inter)national regulatory water monitoring of AMR.