Rapid and sensitive determination of dehydroascorbic acid in addition to ascorbic acid by reversed-phase high-performance liquid chromatography using a post-column reduction system

An improved procedure for the direct determination of l-dehydroascorbic acid (DHAA), in addition to l-ascorbic acid (AA), has been developed. The two biologically active forms of vitamin C were separated using reversed-phase high-performance liquid chromatography. DHAA was reduced to AA with dithiothreitol (DTT) in a post-column reaction system. Complete conversion was achieved at 50°C using a 1-ml reaction coil. Recoveries were in the range of 95–99% and both forms could be detected spectrophotometrically at 267 nm with high sensitivity. Reproducible results [relative standard deviations 2.4% (DHAA) and 1.0% (AA), n = 5] were obtained when the method was applied to the analysis of rose hip samples.