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.
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.