超高效液相色谱-串联质谱法测定鲟鱼中庆大霉素和新霉素残留量
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摘要
目的建立超高效液相色谱-串联质谱法(ultra performance liquid chromatography-tandemmass spectrometric,UPLC-MS/MS)检测鲟鱼中庆大霉素和新霉素的分析方法。方法样品经10mmol/L磷酸盐缓冲溶液提取, 0.1%甲酸沉淀蛋白, HLB固相萃取柱净化。采用CORTECS HILIC(100 mm×2.1 mm, 1.6μm)色谱柱分离,以0.1%甲酸溶液(A)和乙腈(B)作为流动相进行梯度洗脱,通过电喷雾正离子扫描(electronic spray ion,ESI+),多反应监测模式(multiplereaction monitoring,MRM)对庆大霉素和新霉素的定量离子和定性离子进行测定。结果本方法在5 min内完成2种目标化合物的分离分析。庆大霉素在25～300μg/L浓度范围内呈现良好的线性关系,相关系数r2>0.995;方法检出限为10μg/kg,定量限为25μg/kg;添加量为25、50、100μg/kg时回收率在76.7%～106.3%之间,相对标准偏差(relative standard deviation, RSD)在0.18%～2.15%之间(n=6)。新霉素在50～600μg/L浓度范围内呈现良好的线性关系,相关系数r2>0.996;方法检出限为20μg/kg,定量限为50μg/kg;在50、100、200μg/kg添加水平的回收率为96.1%～109.0%之间,相对标准偏差(RSD)在0.72%～2.84%之间(n=6)。结论该方法精密度好、灵敏度高,能简便、准确地测定鲟鱼中庆大霉素和新霉素的药物残留。
Objective To establish a method for the determination of gentamycin and neomycin residues in sturgeon by ultra performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS).Methods The samples were extracted with 10 mmol/L phosphate buffer solution,precipitating protein with 0.1% formic acid,and purified by HLB solid phase extraction column.And then gentamycin and neomycin residues in the extracts were separated by CORTECS HILIC chromatographic column using a gradient elution program of 0.1% formic acid aqueous solution(A) and acetonitrile solution(B).The quantitative and qualitative ions of gentamicin and neomycin were determined by electronic spray ion(ESI+) and multiple reaction monitoring(MRM).Results The method completed the separation analysis of 2 target compounds within 5 min.Gentamycin had good linear relationships in the range of 25-300 μg/L,r2>0.995.The limits of detection were 10 μg/kg and the limits of quantitation were 25 μg/kg.The recoveries at 3 spiked levels(25,50,100 μg/kg) were 76.7%-106.3%,with the relative standard deviations of 0.18%-2.15%(n=6).Neomycin had good linear relationships in the range of 50-600 μg/L,r2>0.996.The limits of detection were 20 μg/kg and the limits of quantitation were 50 μg/kg.The recoveries at 3 spiked levels(50、100、200 μg/kg) were 96.1%-109.0%,with the relative standard deviations of 0.72%-2.84%(n=6).Conclusion This method is accurate and sensitive,which is convenient for determination of gentamycin and neomycin residues in sturgeon.
Objective To establish a method for the determination of gentamycin and neomycin residues in sturgeon by ultra performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS).Methods The samples were extracted with 10 mmol/L phosphate buffer solution,precipitating protein with 0.1% formic acid,and purified by HLB solid phase extraction column.And then gentamycin and neomycin residues in the extracts were separated by CORTECS HILIC chromatographic column using a gradient elution program of 0.1% formic acid aqueous solution(A) and acetonitrile solution(B).The quantitative and qualitative ions of gentamicin and neomycin were determined by electronic spray ion(ESI+) and multiple reaction monitoring(MRM).Results The method completed the separation analysis of 2 target compounds within 5 min.Gentamycin had good linear relationships in the range of 25-300 μg/L,r2>0.995.The limits of detection were 10 μg/kg and the limits of quantitation were 25 μg/kg.The recoveries at 3 spiked levels(25,50,100 μg/kg) were 76.7%-106.3%,with the relative standard deviations of 0.18%-2.15%(n=6).Neomycin had good linear relationships in the range of 50-600 μg/L,r2>0.996.The limits of detection were 20 μg/kg and the limits of quantitation were 50 μg/kg.The recoveries at 3 spiked levels(50、100、200 μg/kg) were 96.1%-109.0%,with the relative standard deviations of 0.72%-2.84%(n=6).Conclusion This method is accurate and sensitive,which is convenient for determination of gentamycin and neomycin residues in sturgeon.
引文
[1]刘晓东,林洪,江洁.动物源性食品中氨基糖苷类抗生素检测技术的研究进展[J].安徽农业科学,2010,38(8):4128-4132.Liu XD,Lin H,Jiang J.Detection technology for aminoglycosidesin animal origin foods[J].J Anhui Agric Sci,2010,38(8):4128-4132.
[2]刘皋林,盛海峰,邓渝林,等.庆大霉素给药间隔与豚鼠耳、肾毒性的关系[J].第二军医大学学报,2001,22(1):61-64.Liu GL,Sheng HF,Deng YL,et al.The relation between single or multiple dose gentamycin daily and nephrotoxicity and ototoxicity in guinea pigs[J].Acad J Sec Mil Med Univ,2001,22(1):61-64.
[3]李昱,王爱梅.氨基糖苷类抗生素耳毒性机理与防治的研究[J].医学综述,2008,14(3):459-461.Li Y,Wang AM.Ototoxic research progress of aminoglycoside antibiotics[J].Med Recap,2008,14(3):459-461.
[4]刘晓东.水产品中氨基糖苷类抗生素高效液相检测方法的建立[D].青岛:中国海洋大学,2010.Liu XD.Studies on establishment of HPLC determination methods of aminoglycosides residue in seafood[D].Qingdao:Ocean University of China,2010.
[5]GB/T 21323-2007动物组织中氨基糖苷类药物残留量的测定高效液相色谱-质谱/质谱法[S].GB/T 21323-2007 Determination of aminoglycosides residues in animal tissues-HPLC-MS/MS method[S].
[6]龙朝阳,许秀敏.动物源性食品中氨基糖苷类抗生素兽药残留分析[J].中国食品卫生杂志,2006,18(2):148-152.Long CY,Xu XM.Determination of aminoglycosides veterinary residues in animal foods[J].Chin J Food Hyg,2006,18(2):148-152.
[7]蔡春燕,杨芳,闫爱国,等.UPLC-MS/MS法同时检测饲料中5种氨基糖苷类抗生素[J].广东化工,2014,41(10):144-145.Cai CY,Yang F,Yan AG,et al.Determination of five aminogly coside antibiotics in fodder by UPLC-MS/MS[J].Guangdong Chem Ind,2014,41(10):144-145.
[8]高玲,张丹,高峰,等.水产品中氨基糖苷类药物残留的高效液相色谱-串联质谱检测方法研究[J].中国兽药杂志,2012,46(11):27-30.Gao L,Zhang D,Gao F,et al.Determination of aminoglycoside residue in aquatic products using high performance liquid chromatography tandem mass spectrometry[J].Chin J Vet Drug,2012,46(11):27-30.
[9]刘永涛,李乐,徐春娟,等.固相萃取-高效液相色谱/串联质谱法测定水产品中硫酸新霉素残留量[J].分析科学学报,2017,33(1):6-10.Liu YT,Li L,Xu CJ,et al.Determination of neomycin sulfate in aquatic products by high performance liquid chromatography-tandem mass spectrometry with solid phase extraction[J].J Anal Sci,2017,33(1):6-10.
[10]王强,王旭峰,赵东豪,等.免疫亲和柱净化-UPLC-MS/MS测定鱼虾肉中的三种氨基糖苷类抗生素[J].http://kns.cnki.net/kcms/detail/11.2206.TS.20180209.1041.068.html.2018-02-09.Wang Q,Wang XF,Zhao DH,et al.Determination of three aminoglycosides in fish and shrimp by ultra performance liquid chromatography-tandem mass spectrometry with immunoaffinity column clean-up[J].http://kns.cnki.net/kcms/detail/11.2206.TS.20180209.1041.068.html.2018-02-09.
[11]黄原飞,娄晓祎,周哲,等.分子印迹聚合物固相萃取-超高效液相色谱-串联质谱法检测水产品中11种氨基糖苷类药物残留[J].分析化学,2018,46(3):454-461.Huang YF,Lou XW,Zhou Z,et al.Determination of 11 kinds of aminoglycosides in aquatic products by ultra performance liquid chromatography-tandem mass spectrometry with molecularly imprinted polymers solid phase extraction[J].Chin J Anal Chem,2018,46(3):454-461.
[12]苏晶,汤立忠,陈长毅,等.高效液相色谱串联质谱法同时测定9种龙虾中氨基糖苷类和四环素类抗生素残留[J].食品工业科技,2016,37(2):60-63,67.Su J,Tang LZ,Chen CY,et al.Simultaneous determination of nine aminoglycosides and tetracyclines antibioyics residues in crayfish by high performance liquid chromatography-tandem mass spectrometry[J].Sci Technol Food Ind,2016,37(2):60-63,67.
[13]刘雪红,张秀芹,侯颖,等.超高效液相色谱-串联质谱法检测牛奶中7种氨基糖苷类药物残留[J].中国兽药杂志,2015,49(3):48-52.Liu XH,Zhang XQ,Hou Y,et al.Detection of seven aminoglycoside residues in milk by UPLC-MS-MS[J].Chin J Vet Drug,2015,49(3):48-52.
[14]龚强,丁利,朱绍华,等.高效液相色谱串联质谱法检测乳制品中10种氨基糖苷类抗生素残留[J].色谱,2012,30(11):1143-1147.Gong Q,Ding L,Zhu SH,et al.Determination of ten aminoglycoside residues in milk and dairy products using high performance liquid chromatography-tandem mass spectrometry[J].Chin J Chromatogr,2012,30(11):1143-1147.
[15]王立琦,贺利民,曾振灵,等.液相色谱-串联质谱检测兽药残留中的基质效应研究进展[J].质谱学报,2011,32(6):321-332.Wang LQ,He LM,Zeng ZL,et al.Progress in matrix effect of veterinary drug residues analysis by high performance liquid chromatography tandem mass spectrometry[J].J Chin Mass Spectrom Soc,2011,32(6):321-332.
[2]刘皋林,盛海峰,邓渝林,等.庆大霉素给药间隔与豚鼠耳、肾毒性的关系[J].第二军医大学学报,2001,22(1):61-64.Liu GL,Sheng HF,Deng YL,et al.The relation between single or multiple dose gentamycin daily and nephrotoxicity and ototoxicity in guinea pigs[J].Acad J Sec Mil Med Univ,2001,22(1):61-64.
[3]李昱,王爱梅.氨基糖苷类抗生素耳毒性机理与防治的研究[J].医学综述,2008,14(3):459-461.Li Y,Wang AM.Ototoxic research progress of aminoglycoside antibiotics[J].Med Recap,2008,14(3):459-461.
[4]刘晓东.水产品中氨基糖苷类抗生素高效液相检测方法的建立[D].青岛:中国海洋大学,2010.Liu XD.Studies on establishment of HPLC determination methods of aminoglycosides residue in seafood[D].Qingdao:Ocean University of China,2010.
[5]GB/T 21323-2007动物组织中氨基糖苷类药物残留量的测定高效液相色谱-质谱/质谱法[S].GB/T 21323-2007 Determination of aminoglycosides residues in animal tissues-HPLC-MS/MS method[S].
[6]龙朝阳,许秀敏.动物源性食品中氨基糖苷类抗生素兽药残留分析[J].中国食品卫生杂志,2006,18(2):148-152.Long CY,Xu XM.Determination of aminoglycosides veterinary residues in animal foods[J].Chin J Food Hyg,2006,18(2):148-152.
[7]蔡春燕,杨芳,闫爱国,等.UPLC-MS/MS法同时检测饲料中5种氨基糖苷类抗生素[J].广东化工,2014,41(10):144-145.Cai CY,Yang F,Yan AG,et al.Determination of five aminogly coside antibiotics in fodder by UPLC-MS/MS[J].Guangdong Chem Ind,2014,41(10):144-145.
[8]高玲,张丹,高峰,等.水产品中氨基糖苷类药物残留的高效液相色谱-串联质谱检测方法研究[J].中国兽药杂志,2012,46(11):27-30.Gao L,Zhang D,Gao F,et al.Determination of aminoglycoside residue in aquatic products using high performance liquid chromatography tandem mass spectrometry[J].Chin J Vet Drug,2012,46(11):27-30.
[9]刘永涛,李乐,徐春娟,等.固相萃取-高效液相色谱/串联质谱法测定水产品中硫酸新霉素残留量[J].分析科学学报,2017,33(1):6-10.Liu YT,Li L,Xu CJ,et al.Determination of neomycin sulfate in aquatic products by high performance liquid chromatography-tandem mass spectrometry with solid phase extraction[J].J Anal Sci,2017,33(1):6-10.
[10]王强,王旭峰,赵东豪,等.免疫亲和柱净化-UPLC-MS/MS测定鱼虾肉中的三种氨基糖苷类抗生素[J].http://kns.cnki.net/kcms/detail/11.2206.TS.20180209.1041.068.html.2018-02-09.Wang Q,Wang XF,Zhao DH,et al.Determination of three aminoglycosides in fish and shrimp by ultra performance liquid chromatography-tandem mass spectrometry with immunoaffinity column clean-up[J].http://kns.cnki.net/kcms/detail/11.2206.TS.20180209.1041.068.html.2018-02-09.
[11]黄原飞,娄晓祎,周哲,等.分子印迹聚合物固相萃取-超高效液相色谱-串联质谱法检测水产品中11种氨基糖苷类药物残留[J].分析化学,2018,46(3):454-461.Huang YF,Lou XW,Zhou Z,et al.Determination of 11 kinds of aminoglycosides in aquatic products by ultra performance liquid chromatography-tandem mass spectrometry with molecularly imprinted polymers solid phase extraction[J].Chin J Anal Chem,2018,46(3):454-461.
[12]苏晶,汤立忠,陈长毅,等.高效液相色谱串联质谱法同时测定9种龙虾中氨基糖苷类和四环素类抗生素残留[J].食品工业科技,2016,37(2):60-63,67.Su J,Tang LZ,Chen CY,et al.Simultaneous determination of nine aminoglycosides and tetracyclines antibioyics residues in crayfish by high performance liquid chromatography-tandem mass spectrometry[J].Sci Technol Food Ind,2016,37(2):60-63,67.
[13]刘雪红,张秀芹,侯颖,等.超高效液相色谱-串联质谱法检测牛奶中7种氨基糖苷类药物残留[J].中国兽药杂志,2015,49(3):48-52.Liu XH,Zhang XQ,Hou Y,et al.Detection of seven aminoglycoside residues in milk by UPLC-MS-MS[J].Chin J Vet Drug,2015,49(3):48-52.
[14]龚强,丁利,朱绍华,等.高效液相色谱串联质谱法检测乳制品中10种氨基糖苷类抗生素残留[J].色谱,2012,30(11):1143-1147.Gong Q,Ding L,Zhu SH,et al.Determination of ten aminoglycoside residues in milk and dairy products using high performance liquid chromatography-tandem mass spectrometry[J].Chin J Chromatogr,2012,30(11):1143-1147.
[15]王立琦,贺利民,曾振灵,等.液相色谱-串联质谱检测兽药残留中的基质效应研究进展[J].质谱学报,2011,32(6):321-332.Wang LQ,He LM,Zeng ZL,et al.Progress in matrix effect of veterinary drug residues analysis by high performance liquid chromatography tandem mass spectrometry[J].J Chin Mass Spectrom Soc,2011,32(6):321-332.