Simultaneous Determination of Five Major Proteins in Whey Protein Powder by Reversed-Phase High-Performance Liquid Chromatography

In this paper, a reversed-phase high-performance liquid chromatography method was proposed to determine five major proteins including α-lactalbumin, β-lactoglobulin, glycomacropeptide, bovine serum albumin, and immunoglobulin G in whey protein powder. After dissolution and ultrasonication, samples were separated on ChromCore 300 C4-T chromatographic column (4.6 mm × 150 mm, 5 μm) with gradient elution using a mobile phase consisting of 0.1% (V/V) aqueous trifluoroacetic acid (TFA) and 90% acetonitrile (containing 0.09% TFA). These analytes were detected using a diode array detector scanning in the wavelength range from 200 to 700 nm at 214 nm. Under the optimal experimental conditions, the five major bovine whey proteins were well separated within 30 min. The average recoveries from spiked samples were in the range of 83.5%–95.3% with relative standard deviations (RSDs) of 2.03%–7.49%. The limit of detection was 0.25% (m/m) for all analytes. The established method is rapid, accurate and sensitive that enables the simultaneous detection of the major bovine whey proteins in whey protein powder.