An Analytical Method to Quantify Osteopontin in Dairy Powders and Infant Formulas by Signature Peptide Quantification with UHPLC-MS/MS.

Abstract

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.