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 α-S1-casein and β-lactoglobulin, i.e., FFVAPFPEVFGK (m/z 692.8682+) and TPEVDDEALEK (m/z 623.2952+), respectively, were chosen to search for hidden allergens in meat-based samples such as cooked meat, sausages, and sterilised pâté. 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 pâté were meat-based products which require a major alert because up to 22 μgTCMP/g of matrix i.e. more than 10 times the action level was determined. A schematic description of the developed method: proteins are extracted from meat, purified and digested. Then, LC-HRMS analysis is used to search for selected quantifier and qualifier peptides of α-S1-casein and β-lactoglobulin: two orthogonal information, i.e., RT and accurate m/z, allow to recognize the presence of those two allergenic proteins. Then, confirmation of putative attributions by MS/MS experiments permits to identify the presence of milk hidden allergens. [Display omitted] •Simple method for quantitative analysis of hidden food allergens•Two signature peptides of α-S1-casein and β-lactoglobulin were selected•LOD down to 3.8 μging/gmatrix and LOQ of 13 μging/gmatrix were calculated in spiked samples•Caseinates were easily identified and quantified in sausages, hamburger, and pâté samples•Milk protein contents up to 10 times the action level in samples declared