Gliadin analysis by reversed-phase high performance liquid chromatography

Abstract

In order to develop a method for rapid, efficient and accurate detection and quantitative determination of gliadin proteins from wheat flour, the usage of different solvents and different separation conditions were investigated by reversed-phase high performance liquid chromatography (RP-HPLC). After albumin and globulins were removed from wheat flour (100 mg) with a salt solution, the gliadin extraction was achieved with ethanol, 1-propanol and isopropanol (50% v/v, 60% v/v and 70% v/v each). Then, the glutenin fractions were extracted using nitrogen and 50% (v/v) aqueous solution of 1-propanol containing Tris-HCl (0.05 mol/dm 3 , pH = 7.5) at 60 °C, urea (2 mol/dm 3 ) and dithioerythritol (1%). The separation and quantitative gliadin determination were carried out by RP-HPLC chromatography on C3 column maintained at different temperatures: 40 °C, 45 °C and 50 °C. In order to determine the absolute amounts of the protein type, gliadin protein standard was used. The obtained results in terms of repeatability, linearity and accuracy showed that RP-HPLC chromatography could be used as a fast, relatively simple and reliable way to quantify all types of gluten proteins in wheat flour and can be applied to assess the quality of cereals as raw materials or in cereal products.