鸡组织中氢溴酸常山酮残留的UPLC-MS/MS检测法及其消除规律
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摘要
为探索鸡组织中氢溴酸常山酮残留量及其消除规律,选取25～30日龄AA肉鸡100只,体重约1.0kg,按氢溴酸常山酮推荐剂量3mg/kg给药。于最后一次给药后第0d、6h、1d、2d、3d、5d和8d等每个时间点处死10只鸡,并采集每只鸡的胸肌、皮肤+脂肪、全肝和双肾,分别装入密封袋,做好标记-20℃保存。鸡组织试样经胰蛋白酶酶解,乙酸乙酯提取,正己烷脱脂,固相萃取净化,采用超高效液相色谱-串联质谱检测,外标法定量,并采用WT1.4软件处理,从而推算其休药期。结果表明:该方法检测限为1.5μg/kg,定量限为5.0μg/kg;4种组织的添加回收率为74.8%～91.1%,批内、批间变异系数均<11.5%;残留检测结果表明肌肉和皮肤+脂肪组织的休药期为0d,肾脏组织的休药期为4d,肝脏组织的休药期为5d。因此,本研究中检测方法操作简单,灵敏度较高,可用于鸡组织中氢溴酸常山酮残留的检测和分析。考虑到饲养环境和个体差异等因素的影响,建议氢溴酸常山酮的休药期暂定为5d。
The purpose of this research was to determinate the halofuginone residue and its depletion in chicken tissue.A total of 100 AA broilers aged 25-30 dand weighted about 1.0 kg were selected and fed with recommended halofuginone dosage of 3 mg/kg.Ten chickens of each timepoint were sampled at 0 d,6 h,1 d,2 d,3 d,5 dand 8 d after the last time administration.Their pectoral muscle,skin+fat,liver and kidney were then respectively collected and stored at-20 ℃.The samples were extracted with ethyl acetate after enzymatic hydrolysis of trypsin solution,degreased with n-hexane,and purified on solid phase extraction column.UPLC-MS/MS was used to detect the halofugimone residue.External standard method was used for quantitative analysis,and the data of the residues were processed using WT1.4 software to calculate the withdrawal time.The results showed that:The detection limit and quantitative limit of this method were 1.5 and 5.0μg/kg,respectively.The recovery rates of these four different tissues were ranged from 74.8%to 91.1%,and the coefficients of variation were less than 11.5%.The residues test results showed that withdrawal time was 0 dfor muscle and skin+fat tissues,4 dfor kidney and 5 dfor liver.In conclusion,this method was simple for operation with higher sensitivity,and could be used for the detection and analysis of halofuginone residues in chicken tissues.Considering the influence of environment and individual differences,the withdrawal period of halofuginone was determined 5 dtentatively.
The purpose of this research was to determinate the halofuginone residue and its depletion in chicken tissue.A total of 100 AA broilers aged 25-30 dand weighted about 1.0 kg were selected and fed with recommended halofuginone dosage of 3 mg/kg.Ten chickens of each timepoint were sampled at 0 d,6 h,1 d,2 d,3 d,5 dand 8 d after the last time administration.Their pectoral muscle,skin+fat,liver and kidney were then respectively collected and stored at-20 ℃.The samples were extracted with ethyl acetate after enzymatic hydrolysis of trypsin solution,degreased with n-hexane,and purified on solid phase extraction column.UPLC-MS/MS was used to detect the halofugimone residue.External standard method was used for quantitative analysis,and the data of the residues were processed using WT1.4 software to calculate the withdrawal time.The results showed that:The detection limit and quantitative limit of this method were 1.5 and 5.0μg/kg,respectively.The recovery rates of these four different tissues were ranged from 74.8%to 91.1%,and the coefficients of variation were less than 11.5%.The residues test results showed that withdrawal time was 0 dfor muscle and skin+fat tissues,4 dfor kidney and 5 dfor liver.In conclusion,this method was simple for operation with higher sensitivity,and could be used for the detection and analysis of halofuginone residues in chicken tissues.Considering the influence of environment and individual differences,the withdrawal period of halofuginone was determined 5 dtentatively.
引文
[1]王贤玉.抗球虫药氢溴酸常山酮的研究进展[J].养禽与禽病防治,2012(1):39-40Wang X Y.Progress in the study of the coccidiostats halofuginone[J].Poultry Husbandry and Disease Control,2012(1):39-40(in Chinese)
[2]曹莹,黄士新,沈富林,孙亚云,王蓓.高效液相色谱法测定鸡肝组织中的氢溴酸常山酮残留[J].中国兽药杂志,2004,38(4):11-13Cao Y,Huang S X,Shen F L,Sun Y Y,Wang B.Determination of halofuginone hydrobromide residues in chicken liver by high performance liquid chromatography[J].Chinese Journal of Veterinary Drug,2004,38(4):11-13(in Chinese)
[3]Analytical Methods Committee.Collaborative study of a method for the determination of residues of halofuginone in chicken tissue[J].Analyst,1984,109(2):171-174
[4]Galarini R,Fioroni L,Moretti S,Pettinacci L,Dusi G.Development and validation of a multiresidue liquid chromatography-tandem mass spectrometry confirmatory method for eleven coccidiostats in eggs[J].Analytica Chimica Acta,2011,700(1-2):167-176
[5]李丹,孙雷,毕言锋,徐倩,王鹤佳,徐士新.超高效液相色谱-串联质谱法测定鸡肉和牛肉中五种常用抗球虫药[J].中国兽药杂志,2013,47(4):38-41Li D,Sun L,Bi Y F,Xu Q,Wang H J,Xu S X.Determination of five coccidiostats in chicken and beef by UPLC-MS/MS[J].Chinese Journal of Veterinary Drug,2013,47(4):38-41(in Chinese)
[6]Anderson A,Christopher D H,Woodhouse R N.Analysis of the anti-coccidial drug,halofuginone,in chicken feed using gasliquid chromatography and high-performance liquid chromatography[J].Journal of Chromatography A,1979,168(2):471-480
[7]Yamamoto Y,Kondo F.Determination of halofuginone and amprolium in chicken muscle and egg by liquid chromatography[J].Journal of AOAC International,2001,84(1):43-46
[8]Dubois M,Pierret G,Delahaut P.Efficient and sensitive detection of residues of nine coccidiostats in egg and muscle by liquid chromatography-electrospray tandem mass spectrometry[J].Journal of Chromatography B,2004,813(1-2):181-189
[9]贾涛.液相色谱-串联质谱法检测饲料中氢溴酸常山酮的探讨[J].饲料广角,2015(6):16-19Jia T.Study on the determination of halofuginone in feed by liquid chromatography-tandem mass spectrometry[J].Feed China,2015(6):16-19(in Chinese)
[10]吴宁鹏,王建平,杜向党,沈建忠.鸡组织中常山酮残留的ELISA与HPLC检测方法比较[J].中国兽医杂志,2007,43(9):68-69Wu N P,Wang J P,Du X D,Shen J Z.Determination of halofuginone residues in chicken tissues by ELISA and HPLCassays[J].Chinese Journal of Veterinary Medicine,2007,43(9):68-69(in Chinese)
[11]Beier R C,Dutko T J,Buckley S A,Muldoon M T,Holtzapple C K,Stanker L H.Detection of halofuginone residues in chicken liver tissue by HPLC and a monoclonal-based immunoassay[J].Journal of Agricultural and Food Chemistry,1998,46(3):1049-1054
[12]李俊锁,邱月明,王超.兽药残留分析[M].上海:上海科学技术出版社,2002:20-22Li J S,Qiu Y M,Wang C.Veterinary Drug Residue Anailysis[M].Shanghai:Shanghai Science and Technology Press,2002:20-22(in Chinese)
[13]Opinion S,Efsagmouk R,Seeds S,Panel E.European food safety authority(EFSA)[J].Nutrition&Food Science,2010(6)
[14]Ding S Y,Hou Y L,Wu N P,Shen J Z.Residue analysis for halofuginone in sturgeon muscle by immunoaffinity cleanup and liquid chromatography[J].Journal of AOAC International,2005,88(6):1644-1648
[15]么红霞.休药期与动物性食品安全[J].畜牧兽医科技信息,2014(4):29-29Yao H X.Withdraw time and animal food safety[J].Chinese Journal of Animal Husbandry and Veterinary Medicine,2014(4):29-29(in Chinese)
[2]曹莹,黄士新,沈富林,孙亚云,王蓓.高效液相色谱法测定鸡肝组织中的氢溴酸常山酮残留[J].中国兽药杂志,2004,38(4):11-13Cao Y,Huang S X,Shen F L,Sun Y Y,Wang B.Determination of halofuginone hydrobromide residues in chicken liver by high performance liquid chromatography[J].Chinese Journal of Veterinary Drug,2004,38(4):11-13(in Chinese)
[3]Analytical Methods Committee.Collaborative study of a method for the determination of residues of halofuginone in chicken tissue[J].Analyst,1984,109(2):171-174
[4]Galarini R,Fioroni L,Moretti S,Pettinacci L,Dusi G.Development and validation of a multiresidue liquid chromatography-tandem mass spectrometry confirmatory method for eleven coccidiostats in eggs[J].Analytica Chimica Acta,2011,700(1-2):167-176
[5]李丹,孙雷,毕言锋,徐倩,王鹤佳,徐士新.超高效液相色谱-串联质谱法测定鸡肉和牛肉中五种常用抗球虫药[J].中国兽药杂志,2013,47(4):38-41Li D,Sun L,Bi Y F,Xu Q,Wang H J,Xu S X.Determination of five coccidiostats in chicken and beef by UPLC-MS/MS[J].Chinese Journal of Veterinary Drug,2013,47(4):38-41(in Chinese)
[6]Anderson A,Christopher D H,Woodhouse R N.Analysis of the anti-coccidial drug,halofuginone,in chicken feed using gasliquid chromatography and high-performance liquid chromatography[J].Journal of Chromatography A,1979,168(2):471-480
[7]Yamamoto Y,Kondo F.Determination of halofuginone and amprolium in chicken muscle and egg by liquid chromatography[J].Journal of AOAC International,2001,84(1):43-46
[8]Dubois M,Pierret G,Delahaut P.Efficient and sensitive detection of residues of nine coccidiostats in egg and muscle by liquid chromatography-electrospray tandem mass spectrometry[J].Journal of Chromatography B,2004,813(1-2):181-189
[9]贾涛.液相色谱-串联质谱法检测饲料中氢溴酸常山酮的探讨[J].饲料广角,2015(6):16-19Jia T.Study on the determination of halofuginone in feed by liquid chromatography-tandem mass spectrometry[J].Feed China,2015(6):16-19(in Chinese)
[10]吴宁鹏,王建平,杜向党,沈建忠.鸡组织中常山酮残留的ELISA与HPLC检测方法比较[J].中国兽医杂志,2007,43(9):68-69Wu N P,Wang J P,Du X D,Shen J Z.Determination of halofuginone residues in chicken tissues by ELISA and HPLCassays[J].Chinese Journal of Veterinary Medicine,2007,43(9):68-69(in Chinese)
[11]Beier R C,Dutko T J,Buckley S A,Muldoon M T,Holtzapple C K,Stanker L H.Detection of halofuginone residues in chicken liver tissue by HPLC and a monoclonal-based immunoassay[J].Journal of Agricultural and Food Chemistry,1998,46(3):1049-1054
[12]李俊锁,邱月明,王超.兽药残留分析[M].上海:上海科学技术出版社,2002:20-22Li J S,Qiu Y M,Wang C.Veterinary Drug Residue Anailysis[M].Shanghai:Shanghai Science and Technology Press,2002:20-22(in Chinese)
[13]Opinion S,Efsagmouk R,Seeds S,Panel E.European food safety authority(EFSA)[J].Nutrition&Food Science,2010(6)
[14]Ding S Y,Hou Y L,Wu N P,Shen J Z.Residue analysis for halofuginone in sturgeon muscle by immunoaffinity cleanup and liquid chromatography[J].Journal of AOAC International,2005,88(6):1644-1648
[15]么红霞.休药期与动物性食品安全[J].畜牧兽医科技信息,2014(4):29-29Yao H X.Withdraw time and animal food safety[J].Chinese Journal of Animal Husbandry and Veterinary Medicine,2014(4):29-29(in Chinese)
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