Rapid separation of soybean and cereal (wheat, corn, and rice) proteins in complex mixtures: Application to the selective determination of the soybean protein content in commercial cereal-based products

A perfusion reversed-phase high-performance liquid chromatography method is proposed for the rapid and simultaneous separation of soybean and cereal (wheat, rice, and corn) proteins in complex mixtures of these vegetable proteins. The method consisted of a linear binary gradient in two steps, from 5 to 10% B in 0.42 min, and from 10 to 50% B in 5.71 min being mobile phase A, 0.3% (v/v) acetic acid in Milli-Q water and mobile phase B, 0.3% (v/v) acetic acid in acetonitrile. A temperature of 50 °C and UV detection at 254 nm were used. Samples and standards were prepared in a 25:75 acetonitrile–water mixture containing 0.3% (v/v) acetic acid. The method was successfully applied to the determination of the soybean protein content in commercial cereal-based products manufactured with binary, ternary, and quaternary soybean–cereal mixtures. Moreover, the method was also valid when samples contained, in addition, other cereals such as barley, rye, and millet and other non-vegetal sources of proteins such as milk and egg proteins. Unlike the Kjeldahl method, this method enabled the selective determination of soybean proteins in these complex mixtures being possible the detection and quantitation of up to 12.5 and 42.5 g/kg, respectively, of soybean proteins (related to 0.1 g of sample).