Robust ultra-trace quantification of multi-class antibiotics in environmental waters using automated 96-well SPE and LC-HRMS.

Growing concerns regarding the spread of antibiotic resistance, together with increasing regulatory demands for advanced wastewater treatment, necessitate sensitive high-throughput analytical methods for monitoring antibiotic residues in the environment. In this study, an automated workflow for the determination of multi-class antibiotics in environmental waters was developed by combining a miniaturized 96-well solid-phase extraction (SPE) protocol with LC-HRMS detection. The SPE procedure enabled enrichment of 5 mL water samples to final extract volumes of 50 µL, corresponding to an enrichment factor of 100, while allowing direct injection of the SPE eluates into the LC-HRMS system without additional sample treatment. The automated workflow enabled parallel processing of up to 48 samples within 45 min while requiring <2 mL of organic solvent per sample. Comprehensive method validation demonstrated excellent analytical performance, including high linearity and method limits of quantification ranging from 0.07 to 0.68 ng L-1 for all investigated antibiotics. Matrix effects, particularly in wastewater effluent, as well as variations in extraction recovery were effectively compensated using analyte-specific isotopically labeled internal standards. The applicability of the workflow for high-throughput environmental monitoring was demonstrated by analyzing wastewater effluent and river water samples, revealing the widespread occurrence of sulfamethoxazole, trimethoprim, ciprofloxacin, levofloxacin, azithromycin, clarithromycin, erythromycin, and roxithromycin, ranging from ultra-trace levels in river water to substantially elevated concentrations in wastewater effluent. Thus, the developed workflow represents a robust and sensitive high-throughput approach for the analysis of antibiotic residues in environmental waters.