The dataset was generated as part of a study focused on the development, validation, and application of an SPE-LC-MS/MS method for the determination of antibiotics belonging to the Reserve group of the WHO AWaRe classification in hospital wastewater. The Reserve group includes last-resort antibiotics used primarily for the treatment of infections caused by multidrug-resistant microorganisms. Monitoring their occurrence in hospital wastewater is important both from the perspective of pharmaceutical pollution and because antibiotic residues may contribute to the selection and spread of antimicrobial resistance. Eight compounds were included in the study: aztreonam (ATM), linezolid (LZD), meropenem (MEM), tigecycline (TGC), ceftazidime (CFD), fosfomycin (FOF), vaborbactam (VBR), and cilastatin (CIL).
Hospital wastewater samples were collected from 16 public hospitals in Poland, one from each voivodeship, between February and March 2024. Samples were collected directly from hospital outflows, transported to the laboratory, centrifuged, filtered, and subjected to solid-phase extraction. The developed sample preparation procedure included acidification of 1 L of wastewater to pH 2.5, extraction using Oasis HLB 500 mg/6 mL cartridges, conditioning of the sorbent with methanol, 0.1 M HCl, and water, followed by elution of the analytes with methanol and 0.1% formic acid in water. The extracts were concentrated to a final volume of 1 mL, filtered, and analyzed by LC-MS/MS.
Method validation included, among other parameters, the assessment of linearity, precision, accuracy, matrix effect, and recovery. Calibration solutions were prepared at ten concentration levels: 0.5, 2, 5, 25, 75, 100, 150, 250, 350, and 500 ng L⁻¹. Quality control samples used in validation studies were prepared at three concentration levels: LQC – 10 ng L⁻¹, MQC – 250 ng L⁻¹, and HQC – 400 ng L⁻¹. The matrix effect was evaluated by comparing the analytical response obtained for analytes in the wastewater matrix with that obtained for the corresponding standard solutions, whereas recovery was determined by comparing the response for samples spiked before the SPE step with that for samples spiked at the same concentration level after matrix preparation.
The data deposited in RepOD constitute a supplement to and a more detailed extension of the data presented in the publication “Development SPE-LC-MS/MS method for determination of WHO AWaRe Reserve antibiotics in hospital wastewater”, published in Scientific Reports (2025, 15:22163). In the article, validation results and concentrations determined in real samples were presented mainly as aggregated values, summary tables, and final method parameters. The repository provides more detailed experimental data, allowing the user to trace how the reported results were obtained, reanalyze the data, and assess the variability of individual measurements.
The dataset consists of four separate files:
- Calibration-curve-areas – contains detailed data related to the calibration curves and the analytical responses obtained for individual antibiotics at the respective concentration levels. These data form the basis for establishing calibration relationships and evaluating method linearity.
- Matrix-effect – contains data used to assess the influence of the wastewater matrix on detector response for individual analytes. The reported values allow comparison of analytical responses obtained in the matrix and in the corresponding standard solutions, and enable evaluation of ion suppression or ion enhancement.
- Procedures-recovery – contains detailed recovery data generated during the development of the SPE sample preparation procedure. The data document the extraction efficiency of individual antibiotics and provide a more detailed account of the results used to select the optimal extraction procedure.
- Concentration-repeat – contains concentration values of the determined Reserve antibiotics obtained for individual replicate analyses of hospital wastewater samples. The data represent individual determinations for the respective samples and analytes and were used to calculate the mean concentration values and their variability reported in the publication. In the article, the final results were presented for 16 hospital wastewater samples as concentrations accompanied by standard deviations.
The structure of the dataset reflects the successive stages of the analytical workflow: method calibration, assessment of the matrix effect, evaluation of sample preparation efficiency, and determination of analytes in real samples. Providing the data in a more detailed form than that presented in the publication increases the transparency and reproducibility of the study and enables further reuse of the data, including independent analysis of method performance and hospital wastewater monitoring results.
