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 A liquid chromatography-mass spectrometry method for quantitation of casein was validated using incurred samples and a matrix-matched external standard curve. METHODS 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 a limit of quantitation (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.