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

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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.

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The authors thank Guanjun Chen and Shulong Li of the Center for Scientific Research of Anhui Medical University for valuable help in our experiment. We are deeply grateful for the help provided by all the members in the experimental center platform for physical and chemical of Anhui Medical University.


This work was supported by the National Natural Science Foundation of China (81202209), the Key Projects introduced and funded by leading talent teams of colleges and universities of Anhui province (0303011224) and Key Projects of Natural Science Research in Colleges and Universities of Anhui province (KJ2018A0164).

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Correspondence to Fang-biao Tao.

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Ethical approval

All procedures involving human participants in this study were in accordance with the Helsinki declaration. This study was approved by the Anhui medical university human research ethics committee (Ethical approval code: 20131195).

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Oral and written consents were acquired from all participants in the current study.

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Liu, K., Zhang, J., Geng, M. et al. A Stable Isotope Dilution Assay for Multi-class Antibiotics in Pregnant Urines by LC–MS/MS. Chromatographia (2020).

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  • Liquid chromatography-triple quadrupole tandem mass spectrometry
  • Bio-monitoring
  • Antibiotics
  • China-Anhui Birth Cohort
  • Urine