Determination of Casein Allergens in Extensively Hydrolyzed Casein Infant Formula by Liquid Chromatography-Tandem Mass Spectrometry.

Background: The use of hypoallergenic infant formulas and the need for reliable tests to determine the presence of residual antigens have increased in parallel.

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.