The main objective of this work was to develop an analytical method that can be used in a dairy manufacturing facility for the quantitation of phospholipids in dairy products. Total lipids from a dairy matrix were obtained first by Folch extraction. The total lipid extract was then applied to a silica gel-based solid-phase extraction column, and triglycerides and other nonpolar lipids were separated from the phospholipids and sphingolipids. Quantitation was performed by hydrophilic interaction HPLC coupled to evaporative light-scattering detection using a quaternary separation method. The method was validated using a commercial whey protein phospholipid concentrate and was used to analyze phospholipid and sphingolipid composition in buttermilk, whey protein concentrate, whey protein phospholipid concentrate, and several other dairy ingredients. This method was sensitive and reproducible and can be used in the dairy industry as a research tool to develop new value-added dairy phospholipid products, then later as a standard protocol for quality assurance analysis of current and future products.

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%.