超高效液相色谱串联质谱法检测水产品中四环素类药物及其差向异构体
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摘要
建立了同时检测水产品中四环素母体及其差向异构体四环素、土霉素、金霉素、强力霉素、4-差向四环素、4-差向土霉素、4-差向金霉素共7种药物的超高效液相色谱-串联质谱(UPLC-MS/MS)方法。样品采用0.1mol/L Na2EDTA-Mcllvaine缓冲溶液提取2次,经滤纸过滤和高速离心除去提取液中大颗粒物质,取提取液的一半体积上HLB固相萃取柱净化,应用Waters Acquity UPLC BEH C18柱(100 mm×2.1 mm,1.7μm)高效分离,以0.1%甲酸水溶液-乙腈为流动相梯度洗脱,电喷雾正离子多反应监测模式监测,质谱分段扫描方式扫描。结果表明,7种四环素药物呈现良好的线性关系(R2>0.995),4种四环素母体的定量限(S/N=10)为2.0μg/kg,3种代谢物的定量限为5.0μg/kg。各分析物的加标回收率为70.3%～90.5%,RSD为5.5%～11%。本方法分析时间短,8min内即完成7种药物的分离,且分离效果好,峰形尖锐。方法的灵敏度、准确度高,适用于水产品中四环素类母体和代谢物的同时测定。
An ultra high performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS) method was developed for the simultaneous determination of 7 kinds of tetracyclines and their epimers including tetracycline,chlortetracycline,oxytetracycline,doxycycline,4-epi-tetracycline,4-epi-chlortetracycline,4-epi-oxytetracycline in edible parts of aquatic products.The samples were extracted with 0.1 mol/L EDTA-Mcllvaine buffer solution(pH 4.0).The extract was filtered and high-speed centrifuged in order to remove some large particulate matters before further purification.Then Half of the volume was cleaned-up and pre-concentrated using HLB cartridge.All the target compounds were separated on an ACQUITY UPLC BEH C18 column(100 mm×2.1 mm,1.7 μm) with gradient elution by using acetonitrile and 0.1%formic acid aqueous as eluent,and detected by UPLC-MS/MS under ESI+ionization,MRM mode and section scanning mode.Under optimized conditions,this method had a good linearity(R2>0.995) and the limits of quantification were in the range of 2.0-5.0 μg/kg(S/N=10).The recoveries of the method for the 7 TCs spiked at three different levels were70.3%-90.5% with the relative standard deviations(RSDs) of 5.5%-11%.The analysis was rapid and can be achieved in less than 8 min with good separation and sharp peak shapes.The method had the characteristics is of high accuracy and good precision,was suitable for simultaneous determination of tetracycline antibiotics and their metabolites in aquatic products.
An ultra high performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS) method was developed for the simultaneous determination of 7 kinds of tetracyclines and their epimers including tetracycline,chlortetracycline,oxytetracycline,doxycycline,4-epi-tetracycline,4-epi-chlortetracycline,4-epi-oxytetracycline in edible parts of aquatic products.The samples were extracted with 0.1 mol/L EDTA-Mcllvaine buffer solution(pH 4.0).The extract was filtered and high-speed centrifuged in order to remove some large particulate matters before further purification.Then Half of the volume was cleaned-up and pre-concentrated using HLB cartridge.All the target compounds were separated on an ACQUITY UPLC BEH C18 column(100 mm×2.1 mm,1.7 μm) with gradient elution by using acetonitrile and 0.1%formic acid aqueous as eluent,and detected by UPLC-MS/MS under ESI+ionization,MRM mode and section scanning mode.Under optimized conditions,this method had a good linearity(R2>0.995) and the limits of quantification were in the range of 2.0-5.0 μg/kg(S/N=10).The recoveries of the method for the 7 TCs spiked at three different levels were70.3%-90.5% with the relative standard deviations(RSDs) of 5.5%-11%.The analysis was rapid and can be achieved in less than 8 min with good separation and sharp peak shapes.The method had the characteristics is of high accuracy and good precision,was suitable for simultaneous determination of tetracycline antibiotics and their metabolites in aquatic products.
引文
[1]李伟明,鲍艳宇,周启星.四环素类抗生素降解途径及其主要降解产物研究进展[J].应用生态学报,2012,23(8):2300-2308.
[2]贺德春,许振成,吴根义,等.四环素类抗生素的环境行为研究进展[J].动物医学进展,2011,32(4):98-102.
[3]郭晓,李国良,刘孝利,等.梅江流域沉积物中四环素类抗生素的空间分布特征及其迁移转化规律[J]环境科学学报,2015,35(10):3202-3209.
[4]吴晓凤,郑嘉熹,魏源送,等.超高效液相色谱串联质谱法同时检测复杂基质中四环素类抗生素及其代谢产物[J].农业环境科学学报,2013,32(11):2293-2301.
[5]HALLING-SORENSEN B,SENGELOV G,TJOR-NELUND J.Toxicity of tetracyclines and tetracycline degradation products to environmentally relevant bacteria,including selected tetracycline-resistant bacteria[J].Archives of Environmental Contamination and Toxicology,2002,42(3):263-271.
[6]DAGHRIR R,DROGUI P.Tetracycline antibiotics in the environment:A review[J].Environmental Chemistry Letters,2013,11(3):209-227.
[7]周启星,罗义,王美娥.抗生素的环境残留、生态毒性及抗性基因污染[J].生态毒理学报,2007,2(3):243-251.
[8]ADRIAN J,FERN魣NDEZ F,S魣NCHEZBAEZA F,et al.Preparation of antibodies and development of an enzyme-linked immunosorbent assay(ELISA)for the determination of doxycycline antibiotic in milk samples[J].Journal of Agricultural&Food Chemistry,2012,57(60):3837-3846.
[9]QIN J Y,XIE L J,YING Y B.Feasibility of terahertz time-domain spectroscopy to detect tetracy clines hydrochloride in infant milk powder[J].Anal Chem,2014,86(23):11750-11757.
[10]SONG J,ZHANG Z H,ZHANG Y Q,et al.Ionic liquid dispersive liquid-liquid microextraction combined with high performance liquid chromatography for determination of tetracycline drugs in eggs[J].Analytical Methods,2014,6(6):6459-6466
[11]LIU Y,YANG H,YANG S,et al.High-performance liquid chromatography using pressurized liquid extraction for the determination of seven tetracyclines in egg,fish and shrimp[J].Journal of Chromatography B Analytical Technologies in the Biomedical&Life Sciences,2013,917-918(4):11-17.
[12]ZHENG W L,ZHANG L F,ZHANG K Y,et al Determination of tetracyclines and their epimers in agricultural soil fertilized with swine manure by ultra-high-performance liquid chromatography tandem mass spectrometry[J].Journal of Integrative Agriculture,2012,11(7):1189-1198.
[13]PAILLER J Y,KREIN A,PFISTER L,et al.Solid phase extraction coupled to liquid chromatographytandem mass spectrometry analysis of sulfon amides,tetracyclines,analgesics and hormones in surface water and wastewater in Luxembourg[J].Science of the Total Environment,2009,407(16):4736-4743
[14]BITTENCOURT M S,MARTINS M T,DE ALBU-QUERQUE F G S,et al.High-throughput multiclass screening method for antibiotic residue analysis in meat using liquid chromatography-tandem mass spectrometry:A novel minimum sample preparation procedure[J].Food Additives&Contaminants.Part A,Chemistry,Analysis,Control,Exposure&Risk Assessment,2012,29(4):508-516.
[15]林荆,张金虎,郑宇,等.水产品中四环素类药物残留的超高效液相色谱-串联质谱法测定[J].食品科学,2010,31(20):286-289.
[16]中华人民共和国深圳出入境检验检疫局,中国检验检疫科学研究院.动物源性食品中四环素类兽药残留量检测方法液相色谱-质谱/质谱法与高效液相色谱法:GB/T 21317-2007[S].北京:中国标准出版社,2008.
[17]陈毓芳.肌肉组织中四环素类抗生素的固相萃取-液相色谱法测定[J].分析测试学报,2003,22(3):97-99.
[2]贺德春,许振成,吴根义,等.四环素类抗生素的环境行为研究进展[J].动物医学进展,2011,32(4):98-102.
[3]郭晓,李国良,刘孝利,等.梅江流域沉积物中四环素类抗生素的空间分布特征及其迁移转化规律[J]环境科学学报,2015,35(10):3202-3209.
[4]吴晓凤,郑嘉熹,魏源送,等.超高效液相色谱串联质谱法同时检测复杂基质中四环素类抗生素及其代谢产物[J].农业环境科学学报,2013,32(11):2293-2301.
[5]HALLING-SORENSEN B,SENGELOV G,TJOR-NELUND J.Toxicity of tetracyclines and tetracycline degradation products to environmentally relevant bacteria,including selected tetracycline-resistant bacteria[J].Archives of Environmental Contamination and Toxicology,2002,42(3):263-271.
[6]DAGHRIR R,DROGUI P.Tetracycline antibiotics in the environment:A review[J].Environmental Chemistry Letters,2013,11(3):209-227.
[7]周启星,罗义,王美娥.抗生素的环境残留、生态毒性及抗性基因污染[J].生态毒理学报,2007,2(3):243-251.
[8]ADRIAN J,FERN魣NDEZ F,S魣NCHEZBAEZA F,et al.Preparation of antibodies and development of an enzyme-linked immunosorbent assay(ELISA)for the determination of doxycycline antibiotic in milk samples[J].Journal of Agricultural&Food Chemistry,2012,57(60):3837-3846.
[9]QIN J Y,XIE L J,YING Y B.Feasibility of terahertz time-domain spectroscopy to detect tetracy clines hydrochloride in infant milk powder[J].Anal Chem,2014,86(23):11750-11757.
[10]SONG J,ZHANG Z H,ZHANG Y Q,et al.Ionic liquid dispersive liquid-liquid microextraction combined with high performance liquid chromatography for determination of tetracycline drugs in eggs[J].Analytical Methods,2014,6(6):6459-6466
[11]LIU Y,YANG H,YANG S,et al.High-performance liquid chromatography using pressurized liquid extraction for the determination of seven tetracyclines in egg,fish and shrimp[J].Journal of Chromatography B Analytical Technologies in the Biomedical&Life Sciences,2013,917-918(4):11-17.
[12]ZHENG W L,ZHANG L F,ZHANG K Y,et al Determination of tetracyclines and their epimers in agricultural soil fertilized with swine manure by ultra-high-performance liquid chromatography tandem mass spectrometry[J].Journal of Integrative Agriculture,2012,11(7):1189-1198.
[13]PAILLER J Y,KREIN A,PFISTER L,et al.Solid phase extraction coupled to liquid chromatographytandem mass spectrometry analysis of sulfon amides,tetracyclines,analgesics and hormones in surface water and wastewater in Luxembourg[J].Science of the Total Environment,2009,407(16):4736-4743
[14]BITTENCOURT M S,MARTINS M T,DE ALBU-QUERQUE F G S,et al.High-throughput multiclass screening method for antibiotic residue analysis in meat using liquid chromatography-tandem mass spectrometry:A novel minimum sample preparation procedure[J].Food Additives&Contaminants.Part A,Chemistry,Analysis,Control,Exposure&Risk Assessment,2012,29(4):508-516.
[15]林荆,张金虎,郑宇,等.水产品中四环素类药物残留的超高效液相色谱-串联质谱法测定[J].食品科学,2010,31(20):286-289.
[16]中华人民共和国深圳出入境检验检疫局,中国检验检疫科学研究院.动物源性食品中四环素类兽药残留量检测方法液相色谱-质谱/质谱法与高效液相色谱法:GB/T 21317-2007[S].北京:中国标准出版社,2008.
[17]陈毓芳.肌肉组织中四环素类抗生素的固相萃取-液相色谱法测定[J].分析测试学报,2003,22(3):97-99.