LC-MS/MS Quantification of Total Milk Protein Allergens: A Dual-Target Approach Using β-Lactoglobulin/β-Casein Standards and Stable Isotope-Labeled Peptide Calibration

Accurate quantification of total milk protein allergens in foods remains challenging due to processing-induced alterations in whey-to-casein ratios and matrix interference effects. Herein, a LC-MS/MS method was developed and validated for accurate quantification of milk allergens in foods using a novel dual-target approach. This method simultaneously quantifies whey and casein fractions using high-purity protein standards of β-lactoglobulin and β-casein through marker peptides LIVTQTMK and VLPVPQK, respectively, coupled with stable isotope-labeled peptide internal standards containing flanking sequences. The dual-target strategy overcomes limitations of conventional single-target methods by providing accurate total milk protein determination regardless of processing-induced alterations in protein ratios. Method validation demonstrated excellent performance with detection limits of 0.1 mg/kg for β-lactoglobulin and β-casein, recovery rates of 80.6%–92.4%, and precision below 12.7% RSD. This dual-target approach provides a robust analytical tool for accurate milk allergens determination in processed foods, significantly advancing allergen control and regulatory compliance.