Use of direct injection UHPLC-MS/MS for antibiotic population use assessment by wastewater analysis - application to laboratory and monitoring studies.

Antimicrobial resistance (AMR) is a global human health threat and wastewater analysis of antibiotics can help inform AMR policies. Accurate quantification of antibiotic use in urban populations is needed. This requires analytical methods capable of high sample throughput to support short-term laboratory antibiotic fate investigations where large sample numbers are generated, and sub-µg L-1 quantification limits for wastewater monitoring studies. Therefore, a direct injection ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed for 11 representative antibiotics and metabolites with a 13 min run time and quantitation limits in the range of 0.0012-0.69 µg L-1. Avoiding solid phase extraction simplified sample preparation whilst mitigating potential contamination or analyte loss, additional solvent use and single-use plastics. High throughput application of the method was demonstrated by assessing antibiotic stability in laboratory-scale gravity sewers (n = 6) with sampling intervals of 0, 0.5, 1, 2, 4, 6, 8, and 24 hours. Amoxicilloic acid, hydroxymetronidazole, trimethoprim and clarithromycin showed satisfactory short-term stability (4 h) for sampling influent wastewater at treatment plants for population use assessment. The sensitivity of the direct injection methodology was demonstrated by random influent wastewater monitoring (n = 5) at three treatment plants in North-East Scotland during August by 24 h pseudo flow-weighted composite sampling. This revealed amoxicillin, metronidazole, trimethoprim and clarithromycin use ranged from 263 to 3559 mg/1000 people per day. This was 0.5 to 1.8 times the corresponding average daily prescribed quantity (calculated from total August prescribing) within the treatment plant's catchment area, highlighting differences in prescription and consumption levels across locations. Findings demonstrated direct injection UHPLC-MS/MS to be ideal for supporting population use assessment of antibiotics through application to laboratory and monitoring wastewater studies.