Abstract: Thermal processing induces the Maillard reaction, which can specifically modify lysine and arginine residues. This modification interferes with trypsin digestion efficiency and compromises the accuracy of allergen quantification. To address this problem, we used chymotrypsin instead of trypsin for proteolysis, effectively circumventing the impact of thermal modifications. Using milk and egg as model foods, we successfully identified one highly specific and thermally stable signature peptide for each as a quantitative marker. We then established a quantitative method combining liquid chromatography-tandem mass spectrometry (LC-MS/MS) with stable isotope-labeled internal standard calibration. Method validation demonstrated high sensitivity with limits of quantification of 5 and 10 mg/kg for allergen of milk and egg in a biscuit matrix, respectively. The method exhibited excellent linearity (determination coefficient R2 > 0.996), reliable accuracy with spiked recoveries ranging from 73.0% to 94.7%, and good stability with precision (relative standard deviations (RSDs)) below 12.6%. These results indicate that the strategy reduces the interference of thermal processing with the efficiency of enzymatic digestion and the quantitative accuracy, providing a new approach for accurate allergen quantification, risk assessment and management in complex thermally processed food matrices.

Key words: food allergens; allergenic proteins; Maillard reaction; chymotrypsin; mass spectrometric analysis