An integrated calibration strategy for the development and validation of an LC-MS/MS method for accurate quantification of egg allergens (Gal d 1–6) in foods

•A LC-MS/MS method was developed and validated for the quantification of egg allergens (Gal d 1–6) in foods.•VMVLC[+57]NR (Gal d 1) and GTDVQAWIR (Gal d 5) were employed for analyzing egg allergens using LC-MS/MS.•Best practice: employing a matrix-matched calibration curve with allergen ingredients and internal standards for precise food allergen quantification. Accurate determination of egg allergens in food is vital for allergen management and labeling. However, quantifying egg allergens with mass spectrometry poses challenges and lacks validation methods. Here, we developed and validated an LC-MS/MS method for quantifying egg allergens (Gal d 1–6) in foods. Sample extraction, enzymatic digestion, purification, proteins/peptides selection, and calibration curves were optimized. VMVLC[+57]NR (Gal d 1) and GTDVQAWIR (Gal d 5) exhibited outstanding sensitivity and stability, serving as quantitation markers for egg white and yolk. Using a matrix-matched calibration curve with allergen ingredients as calibrants and labeled peptides as standards, we achieved highly accurate quantitation. Validation involved spiking egg protein into egg-free foods, showing excellent sensitivity (LOQ: 1–5 mg/kg), accuracy (62.4 %–88.5 %), and reproducibility (intra-/inter-day precision: 3.5 %–14.2 %/8.2 %–14.6 %). Additionally, we successfully applied this method to commercial food analysis. These findings demonstrate optimal allergen selection, peptides, and calibration strategy are crucial parameters for food allergen quantification via MS-based methods.