氨基糖苷类药物残留检测筛选和确证方法的研究
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摘要
随着抗体制备技术、荧光示踪标记技术、色谱质谱技术的不断发展,小分子化合物的新的检测模式和检测方法也不断涌现。氨基糖普类(AGs)抗生素是一类含有氨基糖与氨基环醇结构的药物,在我国畜牧养殖中广泛用于奶牛乳房炎的治疗和促生长作用,对该类抗生素在动物性食品中的残留监控也是我国食品安全工作的重要组成部分。因此,以现有检测方法为基础,结合新的检测模式或新的示踪材料,开展AGs类抗生素快速筛选和仪器确证新方法的探索研究具有非常重要的意义。
本研究制备了安普霉素、庆大霉素和卡那霉素的酶标抗原,建立了可视化凝胶ELSA快速检测方法。其中,安普霉素可视化凝胶ELSA方法灵敏度为0.5μg/L,对猪肉、鸡肉的Cut-off值为3μg/kg,对牛奶的Cut-off值为3μg/L,对猪肝、鸡肝的Cut-off值为10μg/kg,方法采用二步法,检测时间为20min；同步检测庆大霉素和卡那霉素的可视化凝胶ELISA方法的灵敏度为2.0μg/L,对牛奶的Cut-off值为6μg/L,方法采用一步法,检测时间为15min。
制备了庆大霉素和卡那霉素荧光微球示踪物,建立了荧光微球免疫层析检测方法。其中,庆大霉素检测方法为定性方法,方法灵敏度为100μg/L,牛奶样品Cut-off值为100μg/L,方法采用微孔垂直上样法,检测时间为20min；卡那霉素检测方法为定量方法,方法的IC50值为24.8μg/L,线性范围(以20%～80%抑制率对应的浓度计算)为9.5～100μg/L,最低检出限(以10%抑制率对应的浓度计算)为5μg/L。以25、50和100μg/L浓度添加,牛奶中的平均添加回收率在78.4%～92.7%之间,变异系数在10.8%～12.4%之间。
本研究尝试采用亲水作用色谱(HILIC)模式,利用低密度键合C18柱对AGs类抗生素进行分离测定,建立了牛奶中12个AGs类抗生素的UPLC-MS/MS检测方法。方法采用常见的乙腈和甲酸水作为流动相,梯度洗脱条件,12种AGs类抗生素均得到良好的保留和洗脱,出峰时间在1min以后,峰型尖锐对称,整个反应条件中均没有使用离子对试剂。该方法的LOD为为10～50μg/L,LOQ为20～100μ/L,在20～5000pg/L范围内线性关系良好。以20～1000μg/L浓度添加试验表明,样品中的平均添加回收率在60.9%～109.2%之间,日内变异系数在4.6%～14.8%之间,日间变异系数在6.0%～16.2%之间。综上所述,本研究建立的AGs类抗生素残留检测快速筛选和仪器确证的方法,其方法性能符合残留检测的需求,丰富了AGs类残留检测方法体系,为动物性食品中AGs类抗生素的残留监测提供了有效的技术手段。
With the development of antibody preparation, fluorescent tracer marker, chromatography and mass spectrometry technologies, there are many novel and rapid methods emerging for detection of small molecule compounds. Aminoglycosides (AGs) are a class of antibiotic drugs containing amino sugars and amino alcohols ring structure, which are widely used to treat mastitis in dairy and to promote growth in our country. At the same time, the monitoring for AGs antibiotics residue in animal foods is an important part in China's food safety system. Therefore, it is very important significance to explore and develop new rapid screening methods and validation methods for detection of AGs antibiotics residue, based on the combination of existing residue detection method with new analytical technologies.
In this study, Antigen-enzyme tracers were prepared, and visual gel-ELISA methods were established. For detection of apramycin, the sensitivity of the gel-ELISA method was0.5μg/L, and the Cut-off values for raw milk, muscles and livers was3μg/L,3μg/kg, and10μg/kg, respectively.It was a two-steps method with the detection time of20min. For detection of gentamicin and kanamycin simultaneously, the sensitivity of the gel-ELISA method was2.0μg/L, and the Cut-off values for raw milk was5μg/L. It was a one-step method with the detection time of15min.
Antibody-fluorescent microspheres tracers were prepared, and Fluorescent immunochromatographic methods were established. For detection of gentamicin, it was a yes/no method, with the Cut-off value of100μg/L in solution and milk. Vertical chromatography mode in wells was applied, and the detection time was within20min. For detection of kanamycin, it was a qualitative method, with the LOD of5μg/L. The dynamic range IC20～IC80was calculated as9.5-100μg/L, and the IC5o was24.8μg/L. When the blank samples were spiked at25,50and100μg/L, the mean recovery ranged from78.4～92.7%, with intra-assay CVs ranging from10.8～12.4%.
In the study, low-density bonded C18column was selected for determination of AGs based on hydrophilic interaction chromatography (HILIC) mode, and a UPLC-MS/MS method for determination of12AGs antibiotics residue in milk was developed successfully. The common reagents (such as acetonitrile, formic acid and water) were used as mobile phase, and gradient elution mode was used. The results showed sharp symmetrical peaks and good retention for12AGs antibiotics, with the retention time of more than1min. There is no ion-pair reagents used in the method.
The result showed good linear relationships for analytes ranged from20-5000μg/L, and the LOD and LOQ for the method were10～50μg/L and20～100μg/L, respectively. Fortification at the range of20～1000μg/L, mean recoveries were60.9%～109.2%with intra-and inter-assay CVs of4.6%-14.8%and6.0%～16.2%in milk.
In summary, the methods established in the study were rapid and effective, which could provide effective techniques and enrich the detection system for AGs antibiotics residue monitoring.
本研究制备了安普霉素、庆大霉素和卡那霉素的酶标抗原,建立了可视化凝胶ELSA快速检测方法。其中,安普霉素可视化凝胶ELSA方法灵敏度为0.5μg/L,对猪肉、鸡肉的Cut-off值为3μg/kg,对牛奶的Cut-off值为3μg/L,对猪肝、鸡肝的Cut-off值为10μg/kg,方法采用二步法,检测时间为20min；同步检测庆大霉素和卡那霉素的可视化凝胶ELISA方法的灵敏度为2.0μg/L,对牛奶的Cut-off值为6μg/L,方法采用一步法,检测时间为15min。
制备了庆大霉素和卡那霉素荧光微球示踪物,建立了荧光微球免疫层析检测方法。其中,庆大霉素检测方法为定性方法,方法灵敏度为100μg/L,牛奶样品Cut-off值为100μg/L,方法采用微孔垂直上样法,检测时间为20min；卡那霉素检测方法为定量方法,方法的IC50值为24.8μg/L,线性范围(以20%～80%抑制率对应的浓度计算)为9.5～100μg/L,最低检出限(以10%抑制率对应的浓度计算)为5μg/L。以25、50和100μg/L浓度添加,牛奶中的平均添加回收率在78.4%～92.7%之间,变异系数在10.8%～12.4%之间。
本研究尝试采用亲水作用色谱(HILIC)模式,利用低密度键合C18柱对AGs类抗生素进行分离测定,建立了牛奶中12个AGs类抗生素的UPLC-MS/MS检测方法。方法采用常见的乙腈和甲酸水作为流动相,梯度洗脱条件,12种AGs类抗生素均得到良好的保留和洗脱,出峰时间在1min以后,峰型尖锐对称,整个反应条件中均没有使用离子对试剂。该方法的LOD为为10～50μg/L,LOQ为20～100μ/L,在20～5000pg/L范围内线性关系良好。以20～1000μg/L浓度添加试验表明,样品中的平均添加回收率在60.9%～109.2%之间,日内变异系数在4.6%～14.8%之间,日间变异系数在6.0%～16.2%之间。综上所述,本研究建立的AGs类抗生素残留检测快速筛选和仪器确证的方法,其方法性能符合残留检测的需求,丰富了AGs类残留检测方法体系,为动物性食品中AGs类抗生素的残留监测提供了有效的技术手段。
With the development of antibody preparation, fluorescent tracer marker, chromatography and mass spectrometry technologies, there are many novel and rapid methods emerging for detection of small molecule compounds. Aminoglycosides (AGs) are a class of antibiotic drugs containing amino sugars and amino alcohols ring structure, which are widely used to treat mastitis in dairy and to promote growth in our country. At the same time, the monitoring for AGs antibiotics residue in animal foods is an important part in China's food safety system. Therefore, it is very important significance to explore and develop new rapid screening methods and validation methods for detection of AGs antibiotics residue, based on the combination of existing residue detection method with new analytical technologies.
In this study, Antigen-enzyme tracers were prepared, and visual gel-ELISA methods were established. For detection of apramycin, the sensitivity of the gel-ELISA method was0.5μg/L, and the Cut-off values for raw milk, muscles and livers was3μg/L,3μg/kg, and10μg/kg, respectively.It was a two-steps method with the detection time of20min. For detection of gentamicin and kanamycin simultaneously, the sensitivity of the gel-ELISA method was2.0μg/L, and the Cut-off values for raw milk was5μg/L. It was a one-step method with the detection time of15min.
Antibody-fluorescent microspheres tracers were prepared, and Fluorescent immunochromatographic methods were established. For detection of gentamicin, it was a yes/no method, with the Cut-off value of100μg/L in solution and milk. Vertical chromatography mode in wells was applied, and the detection time was within20min. For detection of kanamycin, it was a qualitative method, with the LOD of5μg/L. The dynamic range IC20～IC80was calculated as9.5-100μg/L, and the IC5o was24.8μg/L. When the blank samples were spiked at25,50and100μg/L, the mean recovery ranged from78.4～92.7%, with intra-assay CVs ranging from10.8～12.4%.
In the study, low-density bonded C18column was selected for determination of AGs based on hydrophilic interaction chromatography (HILIC) mode, and a UPLC-MS/MS method for determination of12AGs antibiotics residue in milk was developed successfully. The common reagents (such as acetonitrile, formic acid and water) were used as mobile phase, and gradient elution mode was used. The results showed sharp symmetrical peaks and good retention for12AGs antibiotics, with the retention time of more than1min. There is no ion-pair reagents used in the method.
The result showed good linear relationships for analytes ranged from20-5000μg/L, and the LOD and LOQ for the method were10～50μg/L and20～100μg/L, respectively. Fortification at the range of20～1000μg/L, mean recoveries were60.9%～109.2%with intra-and inter-assay CVs of4.6%-14.8%and6.0%～16.2%in milk.
In summary, the methods established in the study were rapid and effective, which could provide effective techniques and enrich the detection system for AGs antibiotics residue monitoring.
引文
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