• 研究论文 •

### 超高效液相色谱-串联质谱法同时测定土壤中30种抗生素

1. 1.中国科学院生态环境研究中心, 环境化学与生态毒理学国家重点实验室, 北京 100085
2.中国科学院大学资源与环境学院, 北京 100049
3.国科大杭州高等研究院环境学院, 浙江 杭州 310000
• 收稿日期:2021-02-19 出版日期:2021-08-08 发布日期:2021-06-29
• 通讯作者: 廖春阳
• 作者简介:*E-mail: cyliao@rcees.ac.cn.
• 基金资助:
国家重点研发计划项目(2018YFC1801602);国家重点研发计划项目(2019YFC1604802);国家自然科学基金项目(21806172)

### Simultaneous determination of 30 antibiotics in soil by ultra-high performance liquid chromatography-tandem mass spectrometry

HU Yu1,2, ZHU Qingqing1,2, HU Ligang1,2,3, LIAO Chunyang1,2,3,*()

1. 1. State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China
2. College of Resources and Environment, University of Chinese Academy of Sciences (UCAS), Beijing 100049, China
3. Institute of Environment and Health, Hangzhou Institute for Advanced Study, UCAS, Hangzhou 310000, China
• Received:2021-02-19 Online:2021-08-08 Published:2021-06-29
• Contact: LIAO Chunyang
• Supported by:
National Key Research and Development Program of China(2018YFC1801602);National Key Research and Development Program of China(2019YFC1604802);National Natural Science Foundation of China(21806172)

Abstract:

The complexity of the soil matrix, as well as the wide spectrum and trace levels of antibiotic residues in soil, make highly sensitive instrumental methods, efficient purification and enrichment methods, and simultaneous determination of multiple antibiotics key and challenging aspects in the analysis of antibiotics in soil. In this study, a solid phase extraction-ultra-high performance liquid chromatography-tandem mass spectrometry (SPE-UHPLC-MS/MS) method was developed for the simultaneous determination of 30 antibiotics (grouped into seven categories: sulfonamides, fluoroquinolones, tetracyclines, macrolides, β-lactams, amphenicols, and lincosamides) in soil samples.
In the UHPLC-MS/MS experiment, florfenicol and chloramphenicol were analyzed in the negative ionization multiple-reaction monitoring (MRM) mode, and the other 28 target analytes were analyzed in the positive MRM mode. Sensitive MS conditions were realized by optimizing the instrumental parameters such as collision energy and declustering potential. The effects of the injection solvent (proportion of methanol to water) and mobile phase (types and compositions of the solvents) on the shape and intensity of the chromatographic peaks were studied. The optimized UHPLC conditions were as follows: injection solvent, 10%(v/v) methanol aqueous solution; chromatographic column, BEH-C18 column; mobile phase, methanol and water both containing 0.1%(v/v) formic acid; flow rate, 0.4 mL/min; sample injection volume, 5.0 μL. The effects of the extraction solution (the types and compositions of solvents) and clean-up processes (pH of the loading solution, as well as the types and compositions of the rinse solution and elution solvent) on the method performance were investigated. The acetonitrile/Na2EDTA-McIlvaine buffer showed better extraction efficiency for fluoroquinolones than did the methanol/Na2EDTA-McIlvaine buffer. Improved recoveries of sulfonamides, macrolides, tetracyclines, and β-lactams were observed when the pH of the loading solution was set to 8.0. The recoveries of sulfadiazine and amoxicillin increased with a decrease in the proportion of methanol to water for the rinse solution. Compared to individual methanol or acetonitrile, the methanol-acetonitrile (1∶1, v/v) mixture showed better elution efficiency for the target analytes. The optimized pretreatment conditions were determined as follows: the soil sample was spiked with mixed internal standards, and then extracted with 10 mL of acetonitrile/Na2EDTA-McIlvaine buffer (1∶1, v/v) by shaking for 30 min and ultrasonication for 15 min. The extraction was repeated three times. The sample extract was adjusted to pH 8.0 and loaded onto an Oasis HLB cartridge for purification. The cartridge was rinsed with 10 mL of water to remove impurities and eluted with 10 mL of methanol-acetonitrile (1∶1, v/v). Quantitative analysis was conducted using the isotope internal standard method. The method limits of detection and quantification were in the range of 0.013-1.21 and 0.043-4.04 μg/kg, respectively. The correlation coefficients of the calibration curve were 0.992-1.00, suggesting good linearity of the method. At three spiked levels (20, 100, and 200 μg/kg), the average recoveries of most target antibiotics were 44.8% to 164%, and the relative standard deviations were 0.700% to 14.8%. The method was successfully applied to the analysis of the 30 antibiotics in six soil samples. Seventeen antibiotics were detected in the soil samples, and the total contents of the antibiotics in each sample ranged from 73.4 to 184 μg/kg. Twelve antibiotics with a detection frequency of 100% included roxithromycin, clarithromycin, ciprofloxacin, norfloxacin, enrofloxacin, ofloxacin, fleroxacin, lomefloxacin, oxytetracycline, doxycycline, tetracycline, and penicillin G. Ciprofloxacin and norfloxacin were the predominant antibiotics in the soils, with contents in the range of 13.7-32.1 and 15.6-43.6 μg/kg, respectively.
The developed method is simple, rapid, and solvent-saving, and it shows promise for use in the simultaneous determination of trace levels of the 30 antibiotics in soil.