Quantitative Determination of Gluten Protein Types in Wheat Flour by Reversed-Phase High-Performance Liquid Chromatography

A combined extraction-HPLC procedure was developed on a microscale to determine the amounts of the different gluten protein types (ω5-, ω1,2-, α- and γ-gliadins; high molecular weight [HMW] and low molecular weight [LMW] glutenin subunits) in wheat flour. After preextraction of albumins and globulins from flour (100 mg) with a salt solution (2 × 1.0 mL), extraction of gliadins was achieved with 60% aqueous ethanol (3 × 0.5 mL). Subsequently, the glutenin subunits were extracted under nitrogen and at 60°C with 50% aqueous 1-propanol containing Tris-HCl (0.05 mol/L, pH 7.5), urea (2 mol/L) and dithioerythritol (1%). The separation and quantitative determination of gliadins and glutenin subunits was then performed by reversed-phase HPLC on C₈ silica gel at 50°C using a gradient of increasing acetonitrile concentration in the presence of 0.1% trifluoroacetic acid. The flow rate was 1.0 mL/min, and the detection wavelength was 210 nm. Temperature and flow rate were modified for the quantitation of single underivatized HMW subunits. To determine the absolute amounts of protein types, different protein standards (gliadin, LMW and HMW subunits, bovine serum albumin) with known protein contents were compared to HPLC absorbance areas. The calibration curves were almost identical and linear over a broad range (20–220 μg). This extraction-HPLC procedure allows an accurate, reproducible, sensitive, and relatively fast quantitative determination of all gluten protein types in wheat flour, and can be applied to quality evaluation of cereals as raw materials or in processed products.