Separation and determination of denatured caseins by hydrophobic interaction chromatography - Part II. Method validation and applications

A method recently described for the separation of denatured alpha-, beta- a nd kappa -caseins by hydrophobic interaction chromatography was validated b y the analysis of reference skim milk powder (BCR-063R) certificated for to tal nitrogen content. The method is based on fast and easy solubilization o f commercial and real samples by 4.0 M guanidine thiocyanate and elution on a TSK-Gel (R) Phenyl-5PW column (TosoHaas) in the presence of 8.0 M urea i n the mobile phase. No preliminary precipitation or separation of the casei n fraction is required. A linear relationship between the concentration of casein and peak area (UV absorbance detector at 280 nm) was obtained over t he concentration range 0.5-60 muM. The detection limits for alpha-, beta- a nd kappa -caseins ranged between 0.30 and 0.65 muM. The precision of the me thod was evaluated; the relative standard deviation for alpha-, beta- and k appa -casein determination ranged between 2.2 and 2.7% for standard solutio ns and between 3.5 and 6.2% for real sample solutions. The mean casein cont ent found in 10 aliquots of BCR-063R calculated with respect to the total p rotein content (estimated on the basis of certified total nitrogen content) was 79.1 +/- 2.7%. Results of linear fitting of standard additions data fo r alpha-, beta- and kappa -caseins to BCR-063R were compared with linear fi tting of alpha-, beta- and kappa -casein calibration data. The method was a pplied to commercial caseins and to 31 real, raw samples [processed cow's m ilk (pasteurised, UHT-treated), follow-up milk powders, cream, cheeses, cas ein-free infant formulae, cookies for babies containing milk proteins] with the aim of showing the wide applicability of the method in order to determ ine alpha-, beta- and kappa -caseins.