Evaluation of Methods to Quantify Sialic Acid on Glycomacropeptide

Glycomacropeptide (GMP) is isolated from whey and used as an ingredient in phenylketonuria-safe foods because it does not contain phenylalanine. GMP is highly glycosylated and has several sites where N-acetylneuraminic acid (NANA) is bound. In the dairy industry, quantification of NANA from dairy proteins is accomplished by colorimetric, fluorometric, enzymatic, and chromatographic procedures; there is no uniformly accepted industry-wide standard method. In this investigation, NANA quantification methods were evaluated using GMP, and a comparison was made based on the length of time to complete the assay, protein-specificity, linearity, precision, and accuracy. From the methods evaluated, the chromatography protocol was determined to have the greatest benefit for use as a dairy industry standard to measure NANA on GMP. The average mass percent of NANA in 10 statistically independent replicates from a commercial GMP product was measured to be 6.18% ± 0.12%, with a relative standard deviation of 1.94%, which was the lowest of all the methods tested. The accuracy of the chromatographic approach was validated using spike and recovery experiments that provided an average recovery of 90.25%.