Development, validation, and application of an HPLC/UV method for quantification of casein in infant formulae and follow-up milks

A simple, rapid and accurate HPLC method was used to determine the casein content in infant formulae and follow-up milks. The proposed methodology used a Chrompack 300 RP chromatographic column and an UV detector. Gradient elution was carried out with a mixture of two solvents. Solvent A was trifluoroacetic acid in water and solvent B was acetonitrile-water-trifluoroacetic acid. A linear relationship between the concentration of casein and the UV absorbance at 280 nm was obtained. This linearity was maintained over the concentration range 0.04-2.0 mg/mL. Preliminary studies made on processed milks showed that casein content in those products was not affected by heat treatment when compared with raw milk casein content. Hence, the quantification of casein in other milk products could be possible using this chromatographic column. The detection limit value was 0.02 mg/mL. The validity of the method was verified. For recovery studies several determinations were carried out. Four samples were analysed before and after the addition of known amounts of casein. Recovery values ranged between 80 and 98%. The precision of the method was also evaluated, and the %CV found was less than 2%. The developed methodology was applied to the monitorization of casein in twelve samples of infant formulae and follow-up milks. The mean values obtained were 7.5 +/- 2.8 (n=14) and 16.6 +/- 3.7 (n= 10), respectively.