Wheat gluten amino acid composition analysis by high-performance anion-exchange chromatography with integrated pulsed...

A simple accurate method for determining amino acid composition of wheat gluten proteins and their gliadin and glutenin fractions using high-performance anion-exchange chromatography with integrated pulsed amperometric detection is described. In contrast to most conventional methods, the analysis requires neither pre- or post-column derivatization, nor oxidation of the sample. It consists of hydrolysis (6.0M hydrochloric acid solution at 110 degrees C for 24h), evaporation of hydrolyzates (110 degrees C), and chromatographic separation of the liberated amino acids. Correction factors (f) accounted for incomplete cleavage of peptide bonds involving Val (f=1.07) and Ile (f=1.13) after hydrolysis for 24h and for Ser (f=1.32) losses during evaporation. Gradient conditions including an extra eluent (0.1M acetic acid solution) allowed multiple sequential sample analyses without risk of Glu contamination on the anion-exchange column. While gluten amino acid compositions by the present method were mostly comparable to those obtained by a conventional method involving oxidation, acid hydrolysis and post-column ninhydrin derivatization, the latter method underestimated Tyr, Val and Ile levels. Results for the other amino acids obtained by the different methods were linearly correlated (r>0.99, slope=1.03).