Development and validation of an RP-HPLC DAD method for the simultaneous quantification of minor and major whey proteins.

Whey represents a valuable protein source for human nutrition. Whey composition varies with respect to process characteristics during milk processing. For efficient exploitation of this dairy side stream, reliable analytical methods are essential. The aim of this study was to develop and validate an RP-HPLC-DAD method for the simultaneous quantification of the minor (lactoferrin, lactoperoxidase, bovine serum albumin) and major (α-lactalbumin, β-lactoglobulin) whey proteins. Seven RP-columns were compared and the composition of the mobile phase was optimized to achieve baseline separation. In validation experiments the limits of detection (LOD < 8 mg/L) and quantification (LOQ < 24 mg/L) were determined. Validity was proofed by precision (>96%), accuracy (95% - 103%) and recovery (96% - 102%) measurements. Peak homogeneity was confirmed by SDS-PAGE. The individual working ranges were adjusted to the estimated protein concentrations in whey, allowing direct analysis without sample preparation at a method runtime of 23 min.
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