Identification and Quantification of αS1, αS2, β, and κ-Caseins in Water Buffalo Milk by Reverse Phase-High Performance Liquid Chromatography and Mass Spectrometry

A method for the simultaneous quantitation of αS1, αS2, ?, and ?-caseins in water buffalo (Bubalus bubalis) milk using reverse phase high-performance liquid chromatography was developed. The molecular masses of the peaks separated by the described chromatographic protocol were determined by ESI-MS. αS1- and ?-caseins were found to be heteromorphic in several individual milk samples. In particular, αS1-casein showed two peaks with a molecular mass of 23,490 Da and 23,516 Da, and ?-casein showed three peaks with molecular masses of 19,165 Da, 19,177 Da, and 19,247 Da. Only one form for ?-casein (24,033 Da) and αS2-casein (22,741 Da) were detected. The mean values of casein fraction concentration observed throughout the individual samples were 8.89 gL?1 with a relative standard deviation (RSD) of 20% for αS1-casein, 5.08 gL?1 with a RSD of 25% for αS2-casein, 20.91 gL?1 with a RSD of 16% for ?-casein, and 4.13 gL?1 with a RSD of 24% for ?-casein. Linear and second-order polynomial correlations with total nitrogen were calculated for all casein fractions.