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 degrees 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 mu g/g of powdered infant formula with an LOQ of 1.8 mu g/g. The determination coefficients (R-2) 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 mu g/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.

Automated summary

An LC-MS method was developed and validated for quantitation of casein in hypoallergenic infant formula, with a linear range of 1.8-42 μg/g. The method demonstrated good repeatability and precision, with recovery of casein ranging from 78% to 118% from incurred material.