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 (omega 5-, omega 1,2-, alpha- and gamma-gliadins; high molecular weight [HMW] an d low molecular weight [LMW] glutenin subunits) in wheat flour. After preextraction of albumins and globulins from flour (100 mg) with a sal t solution (2 x 1.0 mL), extraction of gliadins was achieved with 60% aqueous ethanol (3 x 0.5 mL). Subsequently, the glutenin subunits were extracted under nitrogen and at 60 degrees C with 50% aqueous 1-propa nol containing Tris-HCl (0.05 mol/L, pH 7.5), urea (2 mol/L) and dithi oerythritol (1%). The separation and quantitative determination of gli adins and glutenin subunits was then performed by reversed-phase HPLC on C-8 silica gel at 50 degrees C using a gradient of increasing aceto nitrile concentration in the presence of 0.1% trifluoroacetic acid. Th e flow rate was 1.0 mL/min, and the detection wavelength was 210 nm. T emperature and flow rate were modified for the quantitation of single underivatized HMW subunits. To determine the absolute amounts of prote in types, different protein standards (gliadin, LMW and HMW subunits, bovine serum albumin) with known protein contents were compared to HPL C absorbance areas. The calibration curves were almost identical and l inear over a broad range (20-220 mu g). This extraction-HPLC procedure allows an accurate, reproducible, sensitive, and relatively fast quan titative determination of all gluten protein types in wheat flour, and can be applied to quality evaluation of cereals as raw materials or i n processed products.