A Stable Isotope Dilution Assay for Multi-class Antibiotics in Pregnant Urines by LC–MS/MS

An accurate assay suitable for human biomonitoring of multiclass antibiotics preferred for veterinary use is currently lacking. In this study, we developed a liquid chromatography-triple quadrupole tandem mass spectrometry (LC–QqQ–MS/MS) method for analysis of 41 representative antibiotics (eight categories) and their two metabolites in human urine samples. Additionally, along with matrix-matched calibration and solid-phase extraction (SPE), use of 36 stable isotopically labeled internal standards (SIL-ISs) compensated for matrix effects and helped in the accuracy of quantitation. The absolute matrix effects (MEs), the linearity of limits of detection (LODs) and limits of quantitation (LOQs), stability, accuracy and precision of this method were validated. The validation parameters obtained for the targeted compounds are as follows: MEs (68.0–129.2%), LODs (0.016–1.481 ng mL−1), LOQs (0.050–4.812 ng mL−1), and higher recoveries (80.3–112.5%) except cyadox and cefaclor. The regression coefficients (r2) of targeted compounds at a concentration range of 0.05–100 ng mL−1 ranged from 0.9958 to 0.9998. The intra- and inter-precisions were obtained with a relative standard deviation (RSD%) < 13%. The method was successfully applied to analyze 302 urine samples of pregnant women from the China-Anhui Birth Cohort (C-ABC), and 36 of the selected antibiotics and two metabolites were detected. The total detection frequencies were 52.6% (fluoroquinolones), 40.4% (tetracyclines), 33.1% (sulfonamides), 16.9% (chloramphenicols), 11.6% (macrolides), 10.9% (β-lactams), 2.3% (lincosamides), and 0.3% (quinoxalines), respectively. The developed method has been successfully employed to obtain reliable quantitative results in the urine of pregnant women.