Determination of hidden milk allergens in meat-based foodstuffs by liquid chromatography coupled to electrospray ionization and high-resolution tandem mass spectrometry

The issue of deliberate addition of antigenic proteins to foodstuffs for ameliorating bulk properties or the unintentional cross-contamination poses potentially life-threatening health problems to susceptible subjects. Even the intake of food products declaring the absence of allergens on their labels could lead to severe risks for sensitive consumers due to the presence of the so-called "hidden allergens". Thus, the quantification of low-abundant proteins as putative allergens has become mandatory. Herein, we present a sensitive and selective analytical method based on reversed-phase liquid chromatography coupled to electrospray ionization and hybrid orbitrap high-resolution mass spectrometry (RPLC-ESI-HRMS) and tandem MS, identifying, and quantifying allergenic milk proteins in complex meat-based foodstuffs from direct measurement of tryptic peptides. Two signature peptides of alpha-S1-casein and beta-lactoglobulin, i.e., FFVAPFPEVFGK (m/z 692.868(2+)) and TPEVDDEALEK (m/z 623.295(2+)), respectively, were chosen to search for hidden allergens in meat-based samples such as cooked meat, sausages, and sterilised pate. The marker peptides were identified and were exploited for method validation including recovery, matrix effect, precision, linearity, method variation, limit of detection, and limit of quantification. The undeclared occurrence of milk allergens as total milk protein content (TCMP) was verified in commercial meat products; beef and pork pate were meat-based products which require a major alert because up to 22 mu g(TCMP)/g of matrix i.e. more than 10 times the action level was determined.