Background
The use of hypoallergenic infant formulas and the need for reliable tests to determine the presence of residual antigens have increased in parallel.
Conclusions
An LC-MS/MS method was developed and validated for confirmation of casein allergens in hypoallergenic infant formula. Highlights: A method was developed to accurately and reliably quantify casein allergens in extensively hydrolyzed casein infant formula by LC-MS without the need for custom peptide standards.
Objective
An LC-MS method for quantitation of casein was validated using incurred samples and a matrix-matched external standard curve. Method: Powdered infant formula samples were extracted in a buffer of sodium deoxycholate and ammonium bicarbonate at 60°C and filtered through 7 kDa desalting columns. Samples were digested overnight with trypsin and precipitated with acid prior to analysis of marker peptides by tandem mass spectrometry.
Results
Based on three marker peptides, the linear range for casein was 1.8-42 μg/g of powdered infant formula with an LOQ of 1.8 μg/g. The determination coefficients (R2) for each curve were ≥0.99 for casein peptides. Method repeatability was ≤22% RSD and intermediate precision was ≤23% RSD; recovery of casein from incurred material (2-20 µg/g) ranged from 78% to 118%. Conclusions: An LC-MS/MS method was developed and validated for confirmation of casein allergens in hypoallergenic infant formula. Highlights: A method was developed to accurately and reliably quantify casein allergens in extensively hydrolyzed casein infant formula by LC-MS without the need for custom peptide standards.