Abstract: An ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed to simultaneously detect the three major milk allergens β-lactoglobulin, αs1-casein and αs2-casein in food, which could avoid false negatives caused by factors such as the degradation of single allergens during processing and low sensitivity. The standard protein hydrolysates were analyzed by nanoflow liquid chromatography-tandem orbitrap high resolution mass spectrometry, obtaining eight signature peptides. Wheat flour was selected as the blank matrix for methodological validation in the multi-reaction monitoring (MRM) mode by triple quadrupole mass spectrometry. This method showed a good linear relationship in the concentration range of 1.6–30 000 ng/mL with correlation coefficient (R2) > 0.999. The limits of quantitation (LODs) of β-lactoglobulin, αs1-casein, and αs2-casein were 50, 3.2, and 40 μg/g, respectively. The average recoveries were between 86.41% and 98.60% with relative standard deviations (RSDs) ≤ 8.95%, and the matrix effects were 86.04%–97.48%. This method could be applied to actual products with and without milk in order to identify whether there is any inconsistence between the allergen label and the actual allergens in food, and more broadly to reduce the potential health risks for milk-sensitive consumers.

Key words: ultra-high performance liquid chromatography-tandem mass spectrometry; milk; allergen; signature peptide; detection