Validation of a new reversed-phase high-performance liquid chromatography method for separation and quantification of bovine milk protein genetic variants

A new RP-HPLC method for the separation and quantification of the most common genetic variants of bovine milk proteins is described. A reversed-phase analytical column C8 (Zorbax 300SB-C8 RP, 3.5 μm, 300 Å, 150 × 4.6 I.D.) was used. All the most common casein (CN) and whey protein genetic variants, including β-CNI, were detected and separated simultaneously in less then 40 min, with the exception of αS1-CNB and CNC variants. Purified protein genetic variants were employed in calibration and showed different absorbances at 214 nm. The procedure was developed using 40 raw individual milk samples of cows belonging to four different breeds and certified skim milk powder BCR-063R. Method validation consisted in testing linearity, repeatability, reproducibility and accuracy. A linear relationship (R2 > 0.99) between the concentrations of proteins and peak areas was observed over the concentration range, with low detection limits. Repeatability and reproducibility were satisfactory for both retention times and peak areas. The RSD of peak areas ranged from 0.92 to 4.32% within analytical day and from 0.85 to 9.52% across analytical days. The recoveries, calculated using mixtures of samples previously quantified, ranged from 98.1 to 103.7%.