Determination of Major Bovine Milk Proteins by Reversed Phase High Performance Liquid Chromatography

A reversed-phase high-performance liquid chromatography (RP-HPLC) for the quantification of six major bovine milk proteins (κ-casein (κ-CN), αs2-casein (αs2-CN), αs1-casein (αs1-CN), β-casein (β-CN), immunoglobulins (IgG) and whey) was developed. The separation and quantification of these major proteomic components were achieved with an Agilent Zorbax 300SB-C8 analytical column (250 mm × 4.6 mm I.D., 5 μm) at 45 °C with detection wavelength at 214 nm. The gradient elution was carried out with solvents of 0.1% TFA in water and 0.1% TFA in acetonitrile at a flow rate of 0.8 ml min−1. The method validation consisted of testing the linearity, repeatability, reproducibility and accuracy. The recoveries of six target proteins spiked in milk were in the range from 74.8% to 132.5%. Nine different commercial milk brands were used, the difference of the concentration and relative ration of κ-CN, αs2-CN, αs1-CN, β-CN and whey were found.