Analysis of free reduced and free oxidised glutathione in wheat flour

Abstract

A rapid and sensitive high-performance liquid chromatography (HPLC) method is described for the determination of reduced and oxidised glutathione (GSH and GSSG, respectively) in wheat flour. The procedure, in which the various steps were optimised, involves extraction with 5% (w/v) perchloric acid (PCA) so as to prevent sulphydryl-disulphide (SH/SS) interchange reactions and to separate PCA-extractable peptides from proteins, the latter being insoluble in PCA. After S-carboxymethylation of free sulphydryl groups by alkylation with iodoacetic acid (IAA) and dinitrophenylation of free amino groups with 1-fluoro-2,4-dinitrobenzene (FDNB), the derivatives are analysed by HPLC on an amino-bonded phase silica column. Recoveries of GSH and GSSG standards added during extraction averaged 98% and 86%, respectively, and detector responses were linear up to 100 nmol GSH/g flour and 50 nmol GSSG/g flour. GSH levels in ‘straight-run’ Brabender Quadrumat Junior-milled white flours from single samples of grain of 11 U.K.-grown wheat cultivars ranged from 18 to 89 nmol/g flour and GSSG levels from 12 to 22 nmol/g flour. The ratio of GSH/GSSG varied between 0·9 and 5·9. No clear relationships were noted for this limited sample set between either the flour GSH or GSSG contents or the GSH/GSSG ratios and the bread making classifications of these cultivars. Storage of white flour samples at 4 °C for 90 days resulted in substantially reduced values for both GSH and GSSG necessitating standardisation of the time between milling and subsequent analysis.