Simultaneous Determination of Seven Antibiotics and Five of Their Metabolites in Municipal Wastewater and Evaluation of Their Stability under Laboratory Conditions

The selection and spread of antibiotic resistance poses risks to public health by reducing the therapeutic potential of antibiotics against human pathogens. Wastewater-based epidemiology (WBE) is potentially the most reliable approach to estimate antibiotics use. Previous WBE studies used parent antibiotics as biomarkers, which may lead to overestimation since parent antibiotics may be directly disposed of. Using metabolites as biomarkers can avoid this drawback. This study developed a simultaneous solid-phase extraction coupled with ultra-high-performance liquid chromatography tandem mass spectrometry method for analyzing 12 antibiotics and human metabolites in wastewater to help assess health risk. Optimum conditions were achieved using a PEP cartridge at pH 3.0. The extraction efficiencies were 73.3~95.4% in influent and 72.0~102.7% in effluent for most of the target analytes. Method detection limit ranged from 0.1 to 1.5 ng/L for influent wastewater and 0.03 to 0.7 ng/L for effluent wastewater. A stability experiment showed that sulfonamide parents and their metabolites were stable at 4 °C, −20 °C and −80 °C, while macrolides metabolites were more stable than their corresponding parents at 4 °C and −20 °C. Finally, the method was applied to measure these analytes in wastewater samples collected from three Beijing WWTPs and to derive apparent removal rates. All metabolites were detected in wastewater samples with concentrations ranging from 1.2 to 772.2 ng/L in influent, from <MDL to 235.6 ng/L in effluent. The apparent removal rates of five metabolites were above 72.6%. These results set a solid foundation for applying WBE to evaluate antibiotics use and its public health effects.