Direct injection liquid chromatography-tandem mass spectrometry as a sensitive and high-throughput method for the quantitative surveillance of antimicrobials in wastewater

Environmental antimicrobial pollution and antimicrobial resistance pose a threat to environmental and human health. Wastewater analysis has been identified as a promising tool for antimicrobial monitoring and the back-estimation of antimicrobial consumption, but current pretreatment methods are tedious and complicated, limiting their scope for high-throughput analysis. A sensitive direct injection method for the quantification of 109 antimicrobials and their metabolites in wastewater samples was developed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The method was validated for both wastewater influent and effluent in terms of specificity, calibration range, matrix effect, filtration loss, accuracy, precision, limit of detection (LOD), and limit of quantification (LOQ). Most analytes achieved calibration of R² > 0.99, and the calibration range was from 0.0002 to 150 μg L⁻¹. Recoveries ranged consistently between ~50 % and ~100 % and losses were attributed to sample filtration. Method LOQs were determined as low as 0.0003 μg L⁻¹, and acceptable accuracy (75 %–125 %) and precision (within 25 %) were achieved for >90 % of the analytes. The method was subsequently further assessed using wastewater of raw influent and treated effluent collected from 6 Australian wastewater treatment plants in 2021. In total, 37 analytes were detected in influent and 22 in effluent. Most of them could be quantified at concentrations ranging from 0.0053 to 160 μg L⁻¹, with benzalkonium chloride-C12, amoxicilloic acid, and cephalexin detected at the highest concentrations. The current study provides a straightforward analytical method for antimicrobial monitoring in wastewater with a fast and simple pretreatment procedure.