【作者】 王波；

【导师】 曾东平； 曾振灵；

【作者基本信息】 华南农业大学 ， 兽医硕士（专业学位）， 2017， 硕士

【摘要】 近年来,兽药的安全使用越来越受到人们的重视,动物性食品中的兽药残留,已经引起了社会的普遍关注。酮洛芬具有消炎作用强、疗效高、毒副作用小、使用方便等诸多优点。为了制定酮洛芬在牛奶中的弃奶期,确保动物性食品质量安全,本文建立了酮洛芬在牛奶中残留的液相色谱-串联质谱法（liquid chromatography-tandem mass spectrometry,LC-MS/MS）,开展了酮洛芬注射液在健康荷斯坦奶牛乳中的残留消除规律研究,为科学合理用药提供依据。建立酮洛芬在牛奶中残留的LC-MS/MS测定方法。牛奶样品采用甲醇提取,浓缩后用0.1%甲酸水-乙腈溶液（90/10,v/v）溶解,高速离心后过滤膜,LC-MS/MS测定,内标法定量。色谱柱为Agilent EcliPse XDB-C18（5μm,4.6×150 mm）不锈钢色谱柱,流动相为0.1%甲酸水溶液（A）和色谱纯乙腈（B）,梯度洗脱;流速为0.25 mL/min;洗脱程序流动相A的比例变化:0¹.0分钟为90%,1.0¹.5分钟为90¹0%,1.5⁷.5分钟为10%,7.5⁸.0分钟为10⁹0%,8.0¹3.0分钟为90%;进样量为5μL。质谱仪为Agilent 6410B型电喷雾—串联四级杆质谱仪,采用多反应监测（multiple-reaction monitoring,MRM）正离子模式对酮洛芬进行定性与定量分析,酮洛芬的定量离子对为255.1>209.2,酮洛芬-D₃的定量离子对为258.1>212.1。在5μg/kg²00μg/kg浓度范围内,酮洛芬和酮洛芬-D₃的比值与浓度呈现良好的线性关系,相关系数大于0.99。在5μg/kg、25μg/kg、50μg/kg和100μg/kg四个添加水平下,平均回收率为100.53%¹03.05%,批内变异系数（coefficient of variation,C.V.）为1.72%⁷.82%,批间变异系数为1.13%¹0.84%,酮洛芬在牛奶中的检测限（Limit of detection,LOD）为1μg/kg,定量限为（limit of quantification,LOQ）5μg/kg。20头健康荷斯坦奶牛,高产、低产各10头,颈部肌内注射酮洛芬注射液,剂量为3 mg/kg.b.w.,一天一次,连用三天。分别于首次给药后2、15、26、39、50、64、74、87、98 h采集奶样,进行残留消除规律研究。结果表明,酮洛芬在机体内消除快,首次给药后2 h,奶样中酮洛芬的最高浓度为（38.43±3.90）μg/kg,低于最高残留限量（maximum residue limit,MRL）50μg/kg,停药后15 h,奶样中均检测不到酮洛芬。本研究建立了酮洛芬在牛奶中残留的LC-MS/MS测定方法,该方法重现性好、灵敏度高、特异性强、前处理方法简单易行,符合兽药残留分析的要求。开展了酮洛芬注射液在健康荷斯坦奶牛乳中的残留消除规律研究,弃奶期为0 d。更多还原

【Abstract】 In recent years,the safe use of veterinary drugs has attracted more and more attention the veterinary drug residues in the animal product food has caused global concern.Ketoprofen has many advantages,such as anti-inflammatory effect,high curative effect,little side effect,convenient use and so on.To establish the milk discard time of ketoprofen,ensure the quality of animal food safety and provide the basis for scientific rational drug use,the study presented a sensitive and rapid liquid chromatography tandem mass spectrometry（LC-MS/MS）method for the determination of ketoprofen in milk,and milk residue depletion profiles of ketoprofen in lactating dairy cows after intramuscular administration were determined.The determination of ketoprofen in milk was conducted using LC-MS/MS.Target analyte could be extracted by methanol and taken to dryness under a gentle flow of nitrogen at40℃.The residues were redissolved by 0.1%formic acid in acetonitrile（90/10,v/v）and filtered through a 0.22μm nylon filter into an autosampler vial for LC-MS/MS analysis.Chromatographic separation for ketoprofen was performed on an Agilent HPLC system with the column oven temperature maintained at 30℃via an Agilent EcliPse XDB-C18（4.6×15mm i.d.,0.5μm particle size）.The mobile phase of 0.1%formic acid aqueous solution（A）and acetonitrile（B）with a flow rate of 0.25 mL/min.Gradient elution program was performed as follows:0¹.0 min,90%A;1.0¹.5min,90¹0%A;1.5⁷.5min,10%A;7.5⁸.0min,10⁹0%A;8.0¹3.0 min,90%A.The injection volume was 5μL.The MS instrument was a triple quadrupole 6410B（Agilent Technologies）,fitted with an electrospray ionization（ESI）source.The ESI interface was operated in positive ionization mode.The triple quadrupole was used in multiple reaction monitoring（MRM）mode.The quantitative ion for ketoprofen and ketoprofen-D₃ were 255.1>209.2,258.1>212.1,respectively.At 5μg/kg²00μg/kg concentration range showed a good linear relationship with the concentration ratio of ketoprofen and ketoprofen-D3,correlation coefficient is greater than 0.99.In 5μg/kg,25μg/kg,50μg/kg and 100μg/kg four concentration,the average recovery rate was 100.53%¹03.05%,CV was 1.72%⁷.82%,coefficient of variation between groups was 1.13%¹0.48%,ketoprofen in milk detection limit was 1μg/kg,the limit of quantification was 5μg/kg.20 healthy Holstein dairy cows（10 high yield,10 low yield）were intramuscular injected of ketoprofen injection at a dose of 3 mg/kg.b.w.,once daily,for three days.At 2,15,26,39,50,64,74,87,98 h after the first injection,milk samples were collected and immediately frozen at-20°C until further analysis.The highest levels residue concentration was38.43±3.95μg/kg at 2h after the first injection,which was lower than the MRL（50μg/kg）.No milk level of ketoprofen was detected 15 h after the last injection.This study established a method for the determination of ketoprofen residues in milk by LC-MS/MS with good reproducibility,high sensitivity,specificity,simple and easy pretreatment.The analysis meets the requirements of veterinary drug residues.The exploring of ketoprofen residue elimination through injection of in healthy Holstein cows proved that the milk disposed for a period of 0 day.更多还原