Separation and quantification of the major casein fractions by reverse-phase high-performance liquid chromatography and urea–polyacrylamide gel electrophoresis: Detection of milk adulterations

The separation and quantification of bovine κ-, α- and β-caseins by HPLC–UV using an RP column which contained polystyrene–divinilbenzene copolymer based packing was optimized and validated. Gradient elution was carried out at a flow-rate of 1 ml/min and a temperature of 46 °C, using a mixture of two solvents. Solvent A was 0.1% trifluoroacetic acid in water and solvent B was acetonitrile–water–trifluoroacetic acid (95:5:0.1). The effluent was monitored by a UV detector at 280 nm. The determinations were performed in the linear range of 0.038–0.377 mg/ml for κ-casein, 0.188–1.883 mg/ml for α-casein and 0.151–1.506 mg/ml for β-casein. The detection limits were 0.006, 0.019 and 0.015 mg/ml for κ-casein, α-casein and β-casein, respectively. The validity of the method was verified. The recoveries ranged from 91 to 100% for bovine milk. The precision of the method was also evaluated, the RSD being less than 3.67%. The same HPLC procedure was used for the separation of caprine and ovine caseins. Different chromatographic profiles were obtained for bovine, ovine and caprine milks, although it was only possible to detect and quantify additions of 5% or more of bovine milk to caprine milk. With respect to detection of milk adulterations, electrophoresis using urea–polyacrylamide gel electrophoresis (PAGE) analysis was more sensitive. The evolution of casein proteolysis in cheeses made from bovine milk and cheeses made from ovine milk, during 30 days of ripening was followed by HPLC–UV and urea–PAGE methodologies. The results obtained by these techniques were similar.