Concentration, purification and quantification of milk protein residues following cleaning processes using a combination of SPE and RP-HPLC

Detection and quantification of milk protein residues can be of utmost importance for validation of cleaning process efficiency in removing even traces of residues as well as quality assurance and product safety. However, currently available assays cannot provide a combination of high sensitivity and a simultaneous quantification of the individual milk proteins. Furthermore, a low protein-to-protein-variability and high compatibility with other reagents such as residual cleaning agents (e.g. surfactants) cannot be ensured. Therefore, a new method was developed comprised of a pre-concentration of proteins by solid-phase extraction and optimisation of the sensitivity of an existing reversed-phase high performance liquid chromatography method for the separate quantification of bovine milk proteins κ-Casein, α(S2)-Casein, α(S1)-Casein, β-Casein, α-Lactalbumin, and β-Lactoglobulin. Hereby, solid-phase extraction enables robust and reproducible purification and concentration of protein residues with a high protein recovery rate and flexible adjustment of concentration factors. The increased sensitivity of the reversed-phase high performance liquid chromatography method was achieved by changes in the measurement wavelength and guanidine buffer concentration. This new method enables reproducible concentration, purification and quantification of protein concentrations below 7 ng mL(−1) • Concentration, purification and quantification of milk protein residues with a high recovery rate of proteins (> 94%) and high reproducibility (coefficient of variation (CV) < 3.0%); • Flexible adjustment of sample volumes allows the utilisation of high concentration factors (≤ 500) without compromising the recovery rate of proteins (recovery rate of proteins decreases by 2.74% per 100 CF).