Quantitative detection of food allergens is important in assessing the risk of eliciting an allergic response in susceptible individuals in the event of an accidental exposure and to ensure compliance with allergen management and labeling regulations. Targeted mass spectrometry-based quantitative methods provide an opportunity to analyze allergens from complex food matrices which have undergone food processing. However, probable interferences of diverse food matrices mandates preparation of matrix-matched calibration curves, challenging method adaptation to multiple matrices. In this study, we have demonstrated that a novel matrix-independent calibration strategy employing an exogenous carrier protein as background in food matrices and calibrants, enables quantitative detection of amounts of milk allergen at concentrations relevant for risk assessment applications. Matrix-independent calibration curves prepared with egg white powder in background yielded comparable quantitative values with a matrix-matched calibration curve. Performance of matrix-independent calibration strategy with egg white powder was evaluated for nonfat dry milk (NFDM) incurred baked cookie, beverages containing pasteurized milk, and casein-incurred chocolate. Peptides representing both casein and whey fractions of milk were detectable from concentrations as low as 1 ppm NFDM in cookie matrix (46-99% recovery across the peptides), 0.5 ppm total milk protein (TMP) in pasteurized beverages (94-138% recovery), or 1 ppm casein in chocolate (35-51% recovery). Although the quantitative accuracy was low from traditionally difficult chocolate and baked cookie matrices, variance in quantification was within 30% across the inter-day replicates indicating method robustness for high throughput analysis of samples required in testing labs.

Manuscript Figures and Corresponding Skyline Files: Figure 1: Matrix-independent Calibration Curve Replicates_EggWhite BG; Figure 2A & 2B: Wheat cookie and Cookie Dough: Milk Allergen Detection from Cookie-Dough_EggWhiteBG; Figure 2C: MoniQA Cookie: Milk Allergen Detection from Incurred Gluten-free Cookie Reference Material; Figure 2D & 2E: Milk Allergen Detection from HTST-UHT Beverage Matrix; Figure 2F: Chocolate: Milk Allergen Detection from Casein Chocolate; Table 1: Probability of Detection Assay; Figure 3: Method Robustness Assay_3x3x3_Interday; Figure 4: Probability of Detection Assay; Supplementary Figures and Corresponding Skyline Files: Figure S1: Impact of matrix in analyte recovery; Figure S2, S3: Matrix-independent Calibration Curve Replicates_EggWhite BG; Figure S4: Matrix-matched Calibration_Dough Matrix; Matrix-matched calibration_Cookie Matrix; Milk Allergen Detection from Cookie-Dough_PorcineGelatinBG; Milk Allergen Detection from Cookie-Dough_EggWhiteBG; Figure S5: Wheat cookie and Cookie Dough: Milk Allergen Detection from Cookie-Dough_EggWhiteBG; Milk Allergen Detection from Incurred Gluten-free Cookie Reference Material; Milk Allergen Detection from HTST-UHT Beverage Matrix; Milk Allergen Detection from Casein Chocolate; Figure S6: Matrix-matched Calibration_Dough Matrix; Matrix-matched calibration_Cookie Matrix; Milk Allergen Detection from Cookie-Dough_PorcineGelatinBG; Milk Allergen Detection from Cookie-Dough_EggWhiteBG; Figure S7: Milk Allergen Detection from Spiked vs Incurred Cookie Matrix; Figure S8: Milk Allergen Detection from HTST-UHT Beverage Matrix; Figure S9: Milk Allergen Detection from Spiked Chocolate Matrix; Figure S10: Milk Allergen Detection from Cookie-Dough_EggWhiteBG; Milk Allergen Detection from HTST-UHT Beverage Matrix; Milk Allergen Detection from Casein Chocolate; Figure S11, S1, S132: QC Tracking; Figure S14: Milk Allergen Detection from Dough Matrix by Standard Addition Calibration