Development and validation of a targeted mass spectrometry method for the quantification of milk protein allergens in multiple complex food matrices using matrix-independent calibration

Quantitative detection of food allergens is crucial in assessing the risk of allergic responses in susceptible individuals and ensuring regulatory compliance. Targeted mass spectrometry methods provide an opportunity to analyze allergens from complex, processed food matrices. However, probable interferences of diverse food matrices mandates preparation of matrix-matched calibration curves, challenging adaptation of methods into multiple matrices. In this study, we have developed a novel matrix-independent calibration strategy employing exogenous carrier proteins as background and demonstrated its adaptability in quantification of milk from multiple food matrices at concentrations relevant for risk assessment applications. Peptides representing casein or whey fractions of milk were detectable from concentrations as low as 1 ppm nonfat dry milk (NFDM) in baked cookie matrix at 46–99% accuracy, 0.5 ppm total milk protein (TMP) in pasteurized beverages at 94–138% accuracy, and 1 ppm casein in chocolate at 35–51% accuracy. Quantitative estimations of milk from matrix-independent calibration curves in the background of egg white powder as exogenous carrier were comparable to those from a matrix-matched calibration curve, and the quantitative precision was within 30% CV across the inter-day replicates. The sensitivity, accuracy, and precision of this matrix-independent approach ensure robustness of the method for high throughput food allergen analysis as required in routine testing labs.