稳定同位素稀释-超高效液相色谱-三重四极杆质谱法测定牛奶及牛源性婴幼儿配方乳粉中A1型和A2型β-酪蛋白含量

Determination of A1 and A2 β-casein in milk and bovine-derived infant formula by purified protein-isotope internal standard dilution-ultra performance liquid chromatography coupled triple quadrupole mass spectrometry

中文关键词:  β-酪蛋白  A2β-酪蛋白  A2牛奶  婴幼儿配方乳粉  三重四极杆质谱

英文关键词:β-casein  A2 β-casein  A2 milk  infant formula  ultra performance liquid chromatography coupled triple quadrupole mass spectrometry

中文摘要:

      目的 建立基于纯化蛋白和稳定同位素稀释-超高效液相色谱-三重四极杆质谱法(ultra performance liquid chromatography coupled triple quadrupole mass spectrometry, UPLC-MS/MS)定性及定量测定牛奶及牛源性婴幼儿配方乳粉中A1和A2 β-酪蛋白变异体的分析方法。方法 首先将β-酪蛋白标准品中A1和A2 β-酪蛋白变异体在高效液相色谱仪上分离, 通过各自的纯化蛋白确定两种变异体在标准品中的含量, 再将试样中的β-酪蛋白经胰蛋白酶酶解成肽, 经UPLC-MS/MS检测。通过筛选的特异肽段, 经β-酪蛋白标准品及稳定同位素稀释内标法计算, 获得试样中β-酪蛋白A1变异体和A2变异体的含量。结果 本研究采用的β-酪蛋白标准对照品中A1和A2 β-酪蛋白变异体含量分别为20.8%和55.1%, 且在检测中标准曲线的线性相关系数均大于0.99, 方法重现性中相对标准偏差为2.290%~4.720%。结论 本方法溯源性及精密度好, 适用于牛奶及牛源性婴幼儿配方乳粉中两种β-酪蛋白变异体的测定。

英文摘要:

      Objective To establish a method for the qualitative and quantitative determination of A1 and A2 β-casein in milk and bovine-derived infant formula by purified protein-isotope internal standard dilution-ultra performance liquid chromatography coupled triple quadrupole mass spectrometry (UPLC-MS/MS). Methods Firstly, the A1 and A2 β-casein variants in the β-casein standard were separated by high performance liquid chromatography. The content of 2 kinds of variants in the standard was determined by purifying their respective proteins. Then, the β-casein in the sample was hydrolyzed into peptides by trypsin and detected by UPLC-MS/MS. By selecting specific peptide segments, the content of A1 and A2 β-casein variants in the sample was calculated using casein standard and stable isotope dilution internal standard methods. Results The A1 and A2 β-casein variant content in the β-casein standard reference substance used in this study were 20.8% and 55.1%, respectively. The linear correlation coefficients of the standard curve in the detection were all greater than 0.99, and the relative standard deviations in the reproducibility of the method were 2.290%-4.720%. Conclusion This method has good traceability and precision, and can be applied for determination of 2 kinds of β-casein variants in milk and cow’s milk derived infant formula.