盐析液液萃取-超高效液相色谱-串联质谱法测定奶粉中四环素类抗生素
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摘要
建立了盐析液液萃取-超高效液相色谱-串联质谱法检测婴儿配方奶粉中四环素、土霉素、金霉素和强力霉素4种四环素类抗生素的方法。奶粉样品经Na_2EDTA-Mcllkaine缓冲液提取,在硫酸铵辅助下采用少量乙腈萃取浓缩后,以体积分数0. 1%甲酸水溶液和乙腈为流动相梯度洗脱,经BEHC18柱分离,ESI+电喷雾模式扫描,MRM多反应模式检测,外标法进行定量。结果显示,在硫酸铵作用下,乙腈对目标化合物的提取净化效果较好;该方法下4种四环素类抗生素的最低检出限为0. 36μg/kg,远远低于国标固相萃取法的50μg/kg;质量浓度为1～100 ng/mL时,4种化合物的线性相关性好,相关系数都在0.9990以上;加标浓度为1、5、50μg/kg时的回收率为71.88%～107. 13%,相对标准偏差都小于8%。研究结果可为婴儿配方奶粉中四环素类抗生素的定性定量分析提供参考。
The use of salting-out assisted liquid-liquid extraction(SALLE) combined with UPLC-MS/MS has been evaluated for the determination of tetracyclines in milk powder. The samples were extracted with Na_2 EDTA-Mcllvaine buffer, and then the target analytes were extracted with acetonitrile assisted with(NH_4)_2 SO_4. After concentrated, the target analytes were separated on a BEH C18 column with gradient elution using acetonitrile and water containing volume fraction 0. 1% formic acid as mobile phase. MS detection was performed with the positive ion mode in multi-reaction monitoring. The samples were quantified with external standard method. The result showed that, under the assisted with( NH_4)_2 SO_4, the extraction and purification effect of acetonitrile was improved. The limit of detection was 0. 36 μg/kg,which was much lower than 50 μg/kg for the solid-phase extraction method. The linear range was from 1 to 100 μg/kg with the correlation coefficient R>0. 999. The recoveries of the method were ranged from71. 88% to 107. 13% at the spiked levels of 1, 5, 50 μg/kg and the relative standard deviation was less than 8%. This method was simple,sensitive, economical, reliable and environmentally friendly. The method was suitable for the qualitative and quantitative of tetracyclines in milk powder.
The use of salting-out assisted liquid-liquid extraction(SALLE) combined with UPLC-MS/MS has been evaluated for the determination of tetracyclines in milk powder. The samples were extracted with Na_2 EDTA-Mcllvaine buffer, and then the target analytes were extracted with acetonitrile assisted with(NH_4)_2 SO_4. After concentrated, the target analytes were separated on a BEH C18 column with gradient elution using acetonitrile and water containing volume fraction 0. 1% formic acid as mobile phase. MS detection was performed with the positive ion mode in multi-reaction monitoring. The samples were quantified with external standard method. The result showed that, under the assisted with( NH_4)_2 SO_4, the extraction and purification effect of acetonitrile was improved. The limit of detection was 0. 36 μg/kg,which was much lower than 50 μg/kg for the solid-phase extraction method. The linear range was from 1 to 100 μg/kg with the correlation coefficient R>0. 999. The recoveries of the method were ranged from71. 88% to 107. 13% at the spiked levels of 1, 5, 50 μg/kg and the relative standard deviation was less than 8%. This method was simple,sensitive, economical, reliable and environmentally friendly. The method was suitable for the qualitative and quantitative of tetracyclines in milk powder.
引文
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[3] European Commission. European Commission Regulation(EU)No 37/2010 of 22 December 2009 on pharmacologically active substances and their classification regarding maximum residue limits in foodstuffs of animal origin[J].Official Journal of the European Communities, 2010(37):1-72.
[4]中华人民共和国农业部.中华人民共和国农业部公告第235号[EB/OL].(2011-07-30)[2018-06-20]. http://jiuban. moa. gov. cn/zwllm/tzgg/gg/200302/t20030226_59300. htm.
[5]中华人民共和国国家质量监督检验检疫总局,中国国家标准化管理委员会.牛奶和奶粉中土霉素,四环素,金霉素,强力霉素残留量的测定液相色谱-紫外检测法:GB/T22990-2008[S].北京:中国标准出版社,2008.
[6]岳振峰,邱月明,林秀云,等.高效液相色谱串联质谱法测定牛奶中四环素类抗生素及其代谢产物[J].分析化学,2006,34(9):1255-1259.YUE Z F, QIU Y M, LIN X Y, et al. Determination of multi-residues of tetracyclines and their metabolites in milk by high performance liquid chromatography-tandem positive-ion electrospray ionization mass spectrometry[J]. Analytical Chemistry,2006,34(9):1255-1259.
[7]连英杰,吴敏,黎翠玉,等.直接提取-超高压液相色谱-电喷雾串联质谱法检测原料奶及奶制品中的四环素类抗生素[J].食品安全质量检测学报,2013,4(2):489-495.LIAN Y J,WU M,LI C Y,et al. Determination of tetracycline antibiotics residues in milk and dairy products by direct extraction-UPLC-ESI-MS/MS[J]. Journal of Food Safety and Quality,2013,4(2):489-495.
[8] GURE A, LARA F J, MORENO-GONZALEZ D, et al.Salting-out assisted liquid-liquid extraction combined with capillary HPLC for the determination of sulfonylurea herbicides in environmental water and banana juice samples[J]. Talanta, 2014, 127:51-58.
[9] WEN Y.LI J,YANG F,et al. Salting-out assisted liquidliquid extraction with the aid of experimental design for determination of benzimidazole fungicides in high salinity samples by high-performance liquid chromatography[J].Talanta, 2013, 106:119-126.
[10] JAIN A, GUPTA M,VERMA K K. Salting-out assisted liquid-liquid extraction for the determination of biogenicamines in fruit juices and alcoholic beverages after derivatization with 1-naphthylisothiocyanate and high performance liquid chromatography[J]. Journal of Chromatography A, 2015, 1422:60-72.
[11]王东,侯传金,赵尔成,等.盐析辅助均相液液萃取/分散固相萃取-超高效液相色谱串联质谱法测定蜂蜜中新烟碱类农药残留[J].分析测试学报,2015,34(6):681-685.WANG D,HOU C J,ZHAO E C,et al. Determination of neonicotinoid residues in honey by ultra performance liquid chromatography-tandem mass spectrometry combined with salting-out homogeneous liquid-liquid extraction and dispersive solid-phase extraction[J].Journal of Instrumental Analysis, 2015,34(6):681-685.
[12]白宝清,王祺.盐析-涡旋辅助分散液液微萃取-液相色谱质谱联用法测定茶叶和果汁中有机磷农药残留[J].食品安全质量检测学报,2017,8(3):935-941.BAI B Q,WANG Q. Determination of organophosphorus pesticides residues in tea and fruit juice by salting-out and vortex assisted dispersive liquid-liquid microextraction-liquid chromatography-tandem mass spectrometry[J]. Journal of Food Safety and Quality, 2017,8(3):935-941.
[13] WU H,ZHANG J,NOREM K,et al. Simultaneous determination of a hydrophobic drug candidate and its metabolite in human plasma with salting-out assisted liquid/liquid extraction using a mass spectrometry friendly salt[J]. Journal of Pharmaceutical and Biomedical Analysis, 2008, 48(4):1243-1248.
[14]中华人民共和国国家质量监督检验检疫总局,中国国家标准化管理委员会.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,2007.
[15]粟有志,刘俊,李芳,等.高效液相色谱-串联质谱法测定可食性包装材料中22种酸性染料[J].色谱,2015,33(4):363-370.SU Y Z, LIU J, LI F, et al. Determination of 22 acidic dyes in edible packagings by high performance liquid chromatography-tandem mass spectrometry[J]. Chinese Journal of Chromatography, 2015, 33(4):363-370.
[16] INCZEDY J, LENGYEL T, URE A M. Compendium of analytical nomenclature[M]. 3rd edition, IUPAC:Blackwell Scientific Publication, 1998.
[2] FRITZ J W,ZUO Y E. Simultaneous determination of tetracycline,oxytetracycline, and 4-epitetracycline in milkby high-performance liquid chromatography[J]. Food Chemistry, 2007, 105(3):1297-1301.
[3] European Commission. European Commission Regulation(EU)No 37/2010 of 22 December 2009 on pharmacologically active substances and their classification regarding maximum residue limits in foodstuffs of animal origin[J].Official Journal of the European Communities, 2010(37):1-72.
[4]中华人民共和国农业部.中华人民共和国农业部公告第235号[EB/OL].(2011-07-30)[2018-06-20]. http://jiuban. moa. gov. cn/zwllm/tzgg/gg/200302/t20030226_59300. htm.
[5]中华人民共和国国家质量监督检验检疫总局,中国国家标准化管理委员会.牛奶和奶粉中土霉素,四环素,金霉素,强力霉素残留量的测定液相色谱-紫外检测法:GB/T22990-2008[S].北京:中国标准出版社,2008.
[6]岳振峰,邱月明,林秀云,等.高效液相色谱串联质谱法测定牛奶中四环素类抗生素及其代谢产物[J].分析化学,2006,34(9):1255-1259.YUE Z F, QIU Y M, LIN X Y, et al. Determination of multi-residues of tetracyclines and their metabolites in milk by high performance liquid chromatography-tandem positive-ion electrospray ionization mass spectrometry[J]. Analytical Chemistry,2006,34(9):1255-1259.
[7]连英杰,吴敏,黎翠玉,等.直接提取-超高压液相色谱-电喷雾串联质谱法检测原料奶及奶制品中的四环素类抗生素[J].食品安全质量检测学报,2013,4(2):489-495.LIAN Y J,WU M,LI C Y,et al. Determination of tetracycline antibiotics residues in milk and dairy products by direct extraction-UPLC-ESI-MS/MS[J]. Journal of Food Safety and Quality,2013,4(2):489-495.
[8] GURE A, LARA F J, MORENO-GONZALEZ D, et al.Salting-out assisted liquid-liquid extraction combined with capillary HPLC for the determination of sulfonylurea herbicides in environmental water and banana juice samples[J]. Talanta, 2014, 127:51-58.
[9] WEN Y.LI J,YANG F,et al. Salting-out assisted liquidliquid extraction with the aid of experimental design for determination of benzimidazole fungicides in high salinity samples by high-performance liquid chromatography[J].Talanta, 2013, 106:119-126.
[10] JAIN A, GUPTA M,VERMA K K. Salting-out assisted liquid-liquid extraction for the determination of biogenicamines in fruit juices and alcoholic beverages after derivatization with 1-naphthylisothiocyanate and high performance liquid chromatography[J]. Journal of Chromatography A, 2015, 1422:60-72.
[11]王东,侯传金,赵尔成,等.盐析辅助均相液液萃取/分散固相萃取-超高效液相色谱串联质谱法测定蜂蜜中新烟碱类农药残留[J].分析测试学报,2015,34(6):681-685.WANG D,HOU C J,ZHAO E C,et al. Determination of neonicotinoid residues in honey by ultra performance liquid chromatography-tandem mass spectrometry combined with salting-out homogeneous liquid-liquid extraction and dispersive solid-phase extraction[J].Journal of Instrumental Analysis, 2015,34(6):681-685.
[12]白宝清,王祺.盐析-涡旋辅助分散液液微萃取-液相色谱质谱联用法测定茶叶和果汁中有机磷农药残留[J].食品安全质量检测学报,2017,8(3):935-941.BAI B Q,WANG Q. Determination of organophosphorus pesticides residues in tea and fruit juice by salting-out and vortex assisted dispersive liquid-liquid microextraction-liquid chromatography-tandem mass spectrometry[J]. Journal of Food Safety and Quality, 2017,8(3):935-941.
[13] WU H,ZHANG J,NOREM K,et al. Simultaneous determination of a hydrophobic drug candidate and its metabolite in human plasma with salting-out assisted liquid/liquid extraction using a mass spectrometry friendly salt[J]. Journal of Pharmaceutical and Biomedical Analysis, 2008, 48(4):1243-1248.
[14]中华人民共和国国家质量监督检验检疫总局,中国国家标准化管理委员会.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,2007.
[15]粟有志,刘俊,李芳,等.高效液相色谱-串联质谱法测定可食性包装材料中22种酸性染料[J].色谱,2015,33(4):363-370.SU Y Z, LIU J, LI F, et al. Determination of 22 acidic dyes in edible packagings by high performance liquid chromatography-tandem mass spectrometry[J]. Chinese Journal of Chromatography, 2015, 33(4):363-370.
[16] INCZEDY J, LENGYEL T, URE A M. Compendium of analytical nomenclature[M]. 3rd edition, IUPAC:Blackwell Scientific Publication, 1998.