同位素稀释超高效液相色谱串联质谱法同时测定牛奶中的克伦特罗、氯霉素与己烯雌酚

Simultaneous Determination of Clenbuterol，Chloramphenicol and Diethylstilbestrol in Bovine Milk by Isotope Dilution Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry

中文关键词:克伦特罗  氯霉素  己烯雌酚  液相色谱串联质谱法  同位素稀释  牛奶  残留

Key Words:clenbuterol  chloramphenicol  diethylstilbestrol  liquid chromatography-tandem mass spectrometry  isotope dilution  bovine milk  residue

中文摘要:建立了同时测定牛奶中克伦特罗、氯霉素和己烯雌酚残留量的同位素稀释超高效液相色谱-串联质谱（UPLC-MS/MS）分析方法。牛奶样品无需蛋白沉淀，直接经HLB小柱净化及水和正己烷淋洗，由乙酸乙酯洗脱后进行分析。采用Acquity UPLC BEH C18色谱柱进行分离，以乙酸铵溶液-乙腈作为流动相进行梯度洗脱，MRM方式测定，同位素稀释内标法定量。药物分别在0.2~20（克伦特罗）、1.2~100（氯霉素）、2.0~200 μg?L-1（己烯雌酚）质量浓度范围内呈良好线性，相关系数均不低于0.998 5，克伦特罗、氯霉素和己烯雌酚在牛奶样品中的检出限分别为 0.009、0.007、0.04 ng?g-1。牛奶中3种药物在0.05~0.10（克伦特罗）、0.30~0.60（氯霉素）、0.50~1.0 ng?g-1（己烯雌酚）加标水平下的回收率为94%~107%，相对标准偏差均小于10%。

Abstract:A method for the determination of clenbuterol，chloramphenicol and diethylstilbestrol in bovine milk was developed by isotope dilution ultra high performance liquid chromatography-tandem mass spectrometry.Samples were directly purified through HLB cartridge with 5 mL water and 5 mL hexane as washing solution.The analytes were eluted with 15 mL ethyl acetate.Then the eluate was evaporated to dryness at 40 ℃ under nitrogen and redissolved with 5 mmol?L-1 of ammonium acetate solution-acetonitrile（90∶10，by volume）.The analytes were analyzed by LC-MS/MS on an Acquity UPLC BEH C18 column using acetonitrile -5 mmol?L-1 ammonium acetate as mobile phase by gradient elution.The mass spectrometer was operated under multiple reaction monitoring（MRM） mode.The samples were quantified by the internal standards labeled with stable isotopes.The calibration curves of clenbuterol，chloramphenicol and diethylstilbestrol were linear in the ranges of 0.2-20，1.2-100,2.0-200 μg?L-1，respectively，with correlation coefficients more than 0.998 5.The limits of detection of clenbuterol，chloramphenicol and diethylstilbestrol were 0.009，0.007,004 ng?g-1，respectively.The recoveries of three drugs in bovine milk sample at the fortified levels of 0.05-0.10（clenbuterol），0.30-0.60（chloramphenicol），0.50-1.0 ng?g-1（diethylstilbestrol） were in the range of 94%-107% with RSDs less than 10%.The method was suitable for the determination of clenbuterol，chloramphenicol and diethylstibestrol residues in bovine milk.