UPLC-MS/MS测定鱼肉中4种硝基呋喃类药物代谢物残留量的方法研究

Determination of four metabolites of nitrofurans in fish by UPLC-MS/MS

建立了基于UPLC-MS/MS分析技术的,能够快速检测鱼肉中硝基呋喃类3-氨基-2-恶唑烷酮(AOZ)、5-甲基吗啉-3-氨基-2-唑烷基酮(AMOZ)、1-氨基-2-乙内酰(AHD)、氨基脲(SEM)4种残留检测方法。实验样品经盐酸溶液水解后,邻苯基甲醛衍生16 h,用氢氧化钠溶液将提取液调至弱碱性,用正己烷去除脂肪等杂质,高速离心除去蛋白质等杂质后,加入氯化钠、乙腈萃取提取液,将目标物转移至乙腈层,加入等量的0.1%甲酸水溶液稀释后过滤膜,滤液上UPLC-MS/MS分析,以多离子反应检测模式(MRM)方式进行数据采集,采用同位素内标法进行定量。结果表明,4种硝基呋喃类代谢物在浓度0.1~2.0 ng/ml呈良好线性关系,相关系数(R^2)均不小于0.995,检出限为0.5μg/kg。在0.5、1.0、5.0μg/kg添加水平下,目标分析物的加标回收率为94.3%~96.4%,相对标准偏差为3.0%~4.5%(n=5)。该方法无需采用乙酸乙酯提取,无需将提取液吹干,无需采用固相萃取柱净化,减少操作步骤,节省实验时间、实验成本,具有操作简单、快捷、低成本、高效、灵敏度高等特点,可作为鱼肉中4种硝基呋喃类兽药代谢物残量同时分析的快速检测方法。

In this paper,UPLC-MS/MS analysis technology was established to detect nitrofuran-3-amino-2-oxazolidinone(AOZ),5-methylmorpholine-3-amino-2-zolidinone(AMOZ),1-amino-2-ethyllactone(AHD)and semicarbazide(SEM)residues in fish.After hydrolysis in hydrochloric acid solution,o-phenylformaldehyde derivatized for 16 hours,the extract was adjusted to weak alkalinity with sodium hydroxide solution,fat and other impurities were removed with n-hexane,protein and other impurities were removed by high-speed centrifugation,sodium chloride and acetonitrile extract were added,the target was transferred to acetonitrile layer,diluted by 0.1%formic acid aqueous solution(m/m)and filtered on UPLC.The filtrate was analyzed by UPLC-MS/MS,and the data were collected by means of multi-ion reaction detection mode(MRM),and the isotopic internal standard method was used for quantitative analysis.The results showed that there was a good linear relationship between the fournitrofuran metabolites in the concentration range of 0.1-2.0 ng/ml.The correlation coefficient(R^2)was not less than 0.995,and the detection limit was 0.5μg/kg.The recovery of target analyte was 94.3%-96.4%with the addition levels of 0.5,1.0 and 5.0μg/kg,and the relative standard deviation was 3.0%-4.5%(n=5).The method without using ethyl acetate extraction,don't need to blow dry the extract,without using solid phase extraction column purification,reduce the operation steps,save the experiment time and cost.It has the characteristics of simple operation,fast,low cost,high efficiency and high sensitivity,and can be used as a rapid detection method for simultaneous analysis of metabolites residues of four nitrofurans in fish.