Development of a perfusion reversed‐phase HPLC method for the separation of soybean and cereal (wheat, corn, and rice) proteins in binary mixtures. Application to the detection of soybean proteins in commercial bakery products

Abstract

A perfusion reversed-phase HPLC method enabling the simultaneous separation of soybean and cereal (wheat, corn, and rice) proteins in commercial bakery products has been proposed for the first time. The method utilises an acetonitrile-water gradient containing an ion-pairing agent. Different ion-pairing agents were tried, 0.3% (v/v) acetic acid being observed to enable the separation of soybean from wheat, rice, and corn proteins while with 0.1% (v/v) trifluoroacetic acid only the separation of soybean and corn proteins was possible. Optimisation of the solubilisation conditions for proteins was achieved by testing different acetonitrile concentrations for the simultaneous extraction of soybean and cereal proteins: best recoveries were found with 25% (v/v) acetonitrile + 0.3% (v/v) acetic acid and with 40% (v/v) acetonitrile + 0.1% (v/v) trifluoroacetic acid. Chromatographic conditions such as gradient, temperature, and wavelength detection were also optimised. The method enabled the separation of soybean and cereal proteins in binary mixtures (soybean and wheat, soybean and corn, or soybean and rice proteins) in less than 5 minutes in a total analysis time of 20 min.

High performance liquid chromatography and capillary electrophoresis in the analysis of soybean proteins and peptides in foodstuffs

“…Table 7 groups the HPLC methodologies developed with this aim. Thus, the possibility of applying HPLC techniques to the analysis of soybean proteins in bakery products was opened by Castro-Rubio A. et al [60]. They carried out a chromatographic separation of soybean proteins from wheat, corn and rice proteins in binary mixtures, by using a perfusion stationary phase.…”

High-performance liquid chromatography and capillary electrophoresis for the analysis of maize proteins

“…Despite the fact that more sophisticated supports such as monolithic or perfusion columns are being used in the separation of amino acids, peptides, and proteins in very short analysis times, no articles were found dealing with the application of these new supports to the separation of maize proteins. Only the separation of soybean proteins from maize proteins has recently been reported using perfusion chromatography but the separation of maize proteins was not the aim of this work [47].…”

Development of a capillary electrophoresis method for the determination of soybean proteins in soybean–rice gluten‐free dietary products

“…In all cases, the injection of soybean and rice proteins was performed using as sample solvent a 25:75 v/v ACN-water mixture containing 0.3% v/v acetic acid. These conditions were optimized in a previous work and enabled the simultaneous solubilization of soybean proteins (water-soluble) and rice proteins (mostly insoluble in water) [19]. The separation media consisted of 80 mM phosphate buffer at different pH values (2.0, 2.5, and 7.0) and 80 mM borate buffer at pH 8.5, all of them modified with 20% v/v ACN.…”

“…Only recently, our research group has proposed an analytical methodology for the separation of soybean and cereal (wheat, rice, and maize) proteins in binary mixtures by perfusion RP-HPLC. This method has also been applied to the determination of the soybean protein content in cereal-based products [19,20]. However, there is no reference in the literature dealing with the simultaneous separation of soybean and rice proteins by CE.…”