Nestle Development Centre Nutrition, Askeaton, Limerick, Ireland.
CAPPA, Centre for Advanced Photonics & Process Analysis, Cork Institute of Technology, Cork, Ireland.
J AOAC Int. 2020 Nov 1;103(6):1646-1653. doi: 10.1093/jaoacint/qsaa058.
Osteopontin (OPN) is an important protein in human milk, and is of growing interest to infant formula (IF) manufacturers. OPN is present at low quantities in bovine milk and its derived ingredients, and there is a need for an accurate quantitative method in complex matrixes such as IF and growing-up milks (GUMs).
The objective of this work was to validate a method to quantify OPN in several dairy powders produced from bovine milk, including skimmed milk powder (SMP), whey protein concentrate (WPC), demineralized WPC and α-lactalbumin-enriched WPC (α-lac WPC). The method was further validated in intact-protein IF and GUM powders produced using combinations of these ingredients.
Test samples were digested using trypsin, and the most appropriate peptide fragmentation transitions were identified by UHPLC-MS/MS. Quantification was made against a standard curve constructed from OPN reference material, and isotopically-labelled peptide standards were used as internal standards. Curve linearity was assessed, and samples were spiked at two OPN levels.
The validation parameters were met in almost all cases, with precision RSDr and RSDiR values ranging from 0.26-7.43% and 1.22-12.70%, respectively, and spike recoveries ranging from 88-102%. The method was used to accurately measure OPN in bovine milk-based IF and GUM powders with intact protein systems, based on comparisons with mass balance calculations.
The results from this study show that the method is fit-for-purpose to support IF and GUM manufacturers in evaluating OPN contents of raw materials and products containing whole, intact protein systems from bovine milk.
An LC-MS/MS method was developed to measure OPN in dairy powders, IF and GUMs containing whole, intact protein systems from bovine milk.
骨桥蛋白（OPN）是人乳中的一种重要蛋白质，越来越受到婴儿配方奶粉（IF）制造商的关注。牛初乳及其衍生成分中的 OPN 含量较低，因此需要在 IF 和成长奶（GUM）等复杂基质中使用准确的定量方法。
本研究旨在验证一种定量检测牛乳来源的几种乳制品中 OPN 的方法，包括脱脂乳粉（SMP）、乳清蛋白浓缩物（WPC）、去矿化 WPC 和富含α-乳白蛋白的 WPC（α-lac WPC）。该方法还进一步在使用这些成分组合生产的完整蛋白 IF 和 GUM 粉末中得到验证。
采用胰蛋白酶对测试样品进行消化，通过 UHPLC-MS/MS 鉴定最适合的肽片段转换。通过 OPN 标准物质构建标准曲线进行定量，并使用同位素标记的肽标准品作为内标。评估曲线的线性，在两个 OPN 水平下对样品进行加标。
在几乎所有情况下，验证参数都得到满足，精密度 RSDr 和 RSDiR 值分别在 0.26-7.43%和 1.22-12.70%之间，加标回收率在 88-102%之间。该方法用于准确测量含有完整蛋白体系的牛乳基 IF 和 GUM 粉末中的 OPN，结果与质量平衡计算相比较。
本研究结果表明，该方法适用于支持 IF 和 GUM 制造商评估含有完整蛋白体系的牛乳原料和产品中的 OPN 含量。
开发了一种 LC-MS/MS 方法，用于测量含有完整蛋白体系的乳制品、IF 和 GUM 中的 OPN。
