Methods for the detection and quantification of food allergens in complex matrices
are necessary to ensure compliance with labeling regulations and assess the effectiveness
of food allergen preventive controls. Liquid chromatography-tandem mass spectrometry
(LC-MS/MS) has emerged as an orthogonal technique in complement to immunochemical-based
assays. However, the absence of established guidelines for MS-based quantification
of allergens in food has limited harmonization among the method development community.
In this study, different quantification strategies were evaluated using a previously
developed multiplexed LC-MS/MS method for the detection of egg, milk, and peanut.
Peptide performance criteria (retention time, signal-to-noise ratio, and ion ratio
tolerance) were established and quantification approaches using varying calibrants,
internal standards, background matrices, and calibration curve preparation schemes
were systematically evaluated to refine the previous method for routine laboratory
use. A matrix-matched calibration curve using allergen ingredients as calibrants and
stable isotope-labeled peptides as internal standards provided the most accurate quantitative
results. The strategy was further verified with commercially available reference materials
and allowed for the confident detection and quantification of food allergens. This
work highlights the need for transparency in calibration strategy and peptide performance
requirements for effective evaluation of mass spectrometric methods for the quantification
of food allergens.