羊乳婴幼儿配方粉中牛乳成分鉴别与测定模型的建立

Identification and quantitative model establishment for cow milk components in infant and young children goat formulas

目的 基于超高效液相色谱-串联质谱法(high performance liquid chromatography-tandem mass spectrometry,UPLC-MS/MS)建立羊乳婴幼儿配方粉中牛乳的鉴别与测定模型,实现羊乳婴幼儿配方粉中牛乳成分的快速分析。方法 样品经胰蛋白酶水解,采用四极杆/静电场轨道阱高分辨质谱法(quadrupole-orbitrap high resolution mass spectrometry, Q-Orbitrap-HRMS)结合蛋白数据库筛选特征肽段。选择具有代表性的羊全脂乳粉、牛全脂乳粉、羊乳清粉、牛乳清粉,分别按不同的比例进行混合, UPLC-MS/MS测定,通过换算系数构建特征肽与牛全脂奶粉和牛乳清粉的鉴别和定量分析模型。结果 牛β-酪蛋白与牛全脂奶粉的换算系数k1为2.8343,牛β-乳球蛋白与牛全脂奶粉的换算系数k2为1.6542,牛β-乳球蛋白与牛乳清粉的换算系数k3为27.8598。牛β-乳球蛋白和牛β-酪蛋白在20~1000 nmol/L范围内,线性关系良好;方法的检出限为1.9 g/kg,定量限为6.4g/kg;日内精密度为3.2%~8.1%,日间精密度为3.4%~7.3%,准确度为99.0%~103.0%;经3家实验室验证,方法准确度为91.5%~103.0%;重复性相对标准偏差为4.3%~5.1%,再现性相对标准偏差为4.7%~5.7%。结论 该模型可分别对羊乳婴幼儿配方粉中牛乳清粉和牛全脂乳粉进行鉴别与测定,两者之和即为牛乳含量。该模型极大地简化了前处理,具有操作简便、快速、方法实用性强等优点。

Objective To develop an identification and quantitative model for the rapid analysis of cow milk components in infant and young children goat formulas based on ultra performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS).Methods The samples were hydrolyzed by trypsin,and the marker peptides were screened by combining protein database with quadrupole-orbitrap high resolution mass spectrometry(Q-Orbitrap-HRMS).The representative goat whole milk powder,bovine whole milk powder,goat whey powder and bovine whey powder were selected and mixed in different proportions,respectively.Identification and quantitative analysis model was established for peptide marker and bovine whole milk powder and bovine whey powder by the conversion coefficient.Results The conversion coefficient k1 of bovineβ-casein and bovine whole milk powder was 2.8343,and the conversion coefficient k2 of bovineβ-lactoglobulin and bovine whole milk powder was 1.6542.The conversion coefficient k3 of bovineβ-lactoglobulin and bovine whey powder was 27.8598.Theβ-lactoglobulin andβ-casein had a good linear relationship in the range of 20‒1000 nmol/L,the limit of detection was 1.9 g/kg and the limit of quantitation was 6.4 g/kg.The recoveries ranged from 99.0%to 103.0%,and the precision of intra-and inter-day precision were 3.2%to 8.1%and 3.4%to 7.3%,respectively.The method was verified by 3 laboratories,the accuracy of the method were 91.5%‒103.0%.The repeatability relative standard deviations were 4.3%‒5.1%.The reproducibility relative standard deviations were 4.7%‒5.7%.Conclusion This model can be used for the identification and determination of bovine whey powder and bovine whole milk powder in infant and young children goat formulas,respectively,the sum of the 2 is the content of bovine milk.This model greatly simplifies the preprocessing,which is simple,fast and practical.