Accurate β-Casein Allergen Quantification in Processed Foods via an Isotope-Labeled Full-Length Protein Internal Standard

Accurate quantification of cow’s milk allergens in processed foods by UPLC-MS/MS is challenged by enzymatic digestion variability and complex matrix effects. β-Casein, a major milk allergen, requires reliable quantification. Herein, we developed a novel method for quantifying bovine β-casein using a recombinant, isotope-labeled, full-length protein (β-casein-I) as an internal standard. With a high isotopic labeling rate and sequence homology to the native analyte, β-casein-I corrects errors from digestion, sample preparation, and MS analysis. The validated method showed excellent linearity from 0.1 to 50 ng/mL. In three processed food matrices, mean recoveries were 89–116% with RSD < 13% at three spiking levels, and LODs were 0.2–0.5 μg/g. Specificity was ensured by simultaneous monitoring of two signature peptides, minimizing false positives from cross-reactivity with goat milk. β-Casein was detected in all 11 commercial samples analyzed. This study provides an accurate, reliable approach for milk allergen quantification, significantly advancing traditional peptide-based methods.