高效液相色谱法检测牛奶中免疫球蛋白 G 含量

Determination of immunoglobulin G content in milk by high performance liquid chromatography

中文关键词:  高效液相色谱  免疫球蛋白G  检测  牛奶

英文关键词:high performance liquid chromatography  immunoglobulin G  milk

中文摘要:

      目的 建立高效液相色谱法检测牛奶中免疫球蛋白G含量的分析方法。方法 样品加pH为7.0的磷酸盐缓冲液提取, 于12000 r/min下离心10 min, 经玻璃纤维滤纸过滤, 取滤液用1 mL规格的Protein G亲和柱净化, 用4 mL pH为2.5的洗脱液定容, 过滤膜, 以BEH C4为色谱柱, 柱温65℃, 以三氟乙酸水溶液、三氟乙酸乙腈溶液为流动相, 流速设置为1.5 mL/min, 进样量30 L, 在波长为280 nm下用高效液相色谱仪进行测定。结果 免疫球蛋白G质量浓度在5.0~200.0 mg/L范围内线性关系良好, 相关系数r2大于0.999, 方法定量限(limits of quantification, LOQs)为10.0 mg/kg。当加标浓度在100.0~400.0 mg/kg范围内, 加标回收率在97.2%~103.6%之间, 相对标准偏差在2.5%~5.9%之间。结论 依据化学分析方法验证指南(GB/T 27417—2017《合格评定 化学分析方法确认和验证指南》), 确认所建立的分析方法具有足够的准确性和重复性。该方法可为准确检测牛奶中免疫球蛋白G含量提供技术支持。

英文摘要:

      Objective To establish an analytical method for the determination of immunoglobulin G content in milk by high performance liquid chromatography. Methods Samples were to be extracted using phosphate buffers with pH 7.0, centrifuged at 12000 r/min for 10 min and filtered by fiberglass filter paper. Then 1 mL Protein G affinity column and 4 mL eluent at pH 2.5 were used to purify and filter the membrane. The high performance liquid chromatography column was BEH C4 and set at 65℃. Mobile phases were trifluoroacetic acid solution and acetonitrile trifluoroacetic acid solution with a flow rate of 1.5 mL/min. The volume of injection was 30 μL, and the detection wavelength was 280 nm. Results The content of immunoglobulin G was to range from 5.0 to 200.0 mg/L, the standard curve was expected to show high linearity (r2>0.999), the limits of quantification (LOQs) were 10.0 mg/kg. When the concentration was in the range of 100.0 to 400.0 mg/kg, the recovery rate was in the range of 97.2%–103.6%, and the relative standard deviation was in the range of 2.5%–5.9%. Conclusion Based chemical analysis method validation guidelines (GB/T 27417—2017 Conformity assessment—Guidance on validation and verification of chemical analytical methods), it is confirmed that the developed analytical method is to have sufficient accuracy and repeatability. The method can provide technical support for the accurate determation of immunoglobulin G in milk.