Ascorbic acid (AA) functions as an electron donor and scavenges free radicals such as superoxide radicals 1) and hydroxyl radicals 2) in vitro. Moreover, AA is essential for posttranslational proline hydroxylation of collagen molecules, 3) after which hydroxyproline residues play a critical role in stabilizing the triple helical structure of collagen.4) Dehydroascorbic acid (DHA), the oxidized form of AA, is generated as a result of these reactions. The biological activity of DHA has been considered equivalent to that of AA, because intracellular DHA is rapidly reduced to AA by dehydroascorbic acid reductase. 5) However, Ogiri et al. reported that DHA had less than 10% of AA's biological efficiency on a molar basis, judging from experiments with the inherently scorbutic ODS rat, which is a convenient animal model for investigating the metabolism of AA in humans. 6) Moreover, May et al. noted that the human mature red blood cells they studied did not have sodium-dependent vitamin C transporters (SVCT1 and SVCT2) yet manifested a very low late uptake of radiolabeled AA. 7) In contrast, human mature red blood cells efficiently took up DHA from blood via a glucose transporter 1 (Glut1).8) Erythrocyte Glut1 and associated DHA uptake are unique traits of humans and the few other mammals that have lost the ability to synthesize AA in vivo. Furthermore, DHA is known to enter mitochondria via Glut1 and undergo reduction to AA in the mitochondria. 9) AA, which enters mitochondria as DHA, also protects mitochondria from oxidative injury. Thus, because DHA possesses unique physiological features, distinct from those of AA, establishment of a simple and accurate method of assessing the presence of DHA is very important.AA in biological samples is routinely evaluated by using HPLC systems. AA's maximum absorbance at 265 nm in HPLC coupled with detection by UV light are widely used for such analyses.10) Other means of AA determination subsequently developed are the ketone derivatization method with 2,4-dinitrophenylhydrazine (DNPH) or o-phenylenediamine and HPLC coupled with electrochemical detection (ECD) system. 11-13) HPLC-ECD is widely used at present, because of this assay's great sensitivity and specificity. 14)However, for DHA determination, the UV detector is not suitable, because DHA's maximum absorbance at 220 nm provides only insensitive and nonselective results. DHA is also difficult to detect with HPLC-ECD because of its low electroactivity. Therefore, a DHA concentration is typically calculated by subtracting AA concentration from the total AA (AAϩDHA) concentration determined in a different chromatographic run. The total AA concentration is usually calculated by reducing the DHA to AA with sulfhydryl compounds including dithiothreitol (DTT) (Fig. 1), mercaptoethanol and homocysteine.15) However, the reducing ability of these sulfhydryl compounds is limited to a narrow neutral pH range. In general, metaphosphoric acid (MPA) and EDTA are used to stabilize the AA and DHA in biological samples.16) Therefore, raising...