Differential rapid analysis of ascorbic acid and ascorbic acid 2-sulfate by dinitrophenylhydrazine method

Abstract

Ascorbic acid 2-sulfate (AAS) has recently been detected in animals (1,2), and the suggestion has been made that it is involved in some physiological functions (3–5). AAS has been determined by spectrophotometer measurements at 254 nm, ϵ = 17,000, after isolation from animal tissues (1,6,7). This procedure, however, is complicated and time consuming. Baker et al. (8) reported a rapid assay for AAS from biological samples based on a dinitrophenylhydrazine (DNPH) method which is a standard method for ascorbic acid (AA) determination in biological materials. In this method, AA is oxidized to the osazone by incubation with DNPH in a dilute sulfric acid solution. According to Baker et al. (8), AAS reacts with DNPH during a 3-hr incubation at 60°C but not during a 1-hr incubation at 37°C, and the difference in the reading at 540 nm between the two temperatures corresponds to AAS. This report is concerned with a more rapid and specific method with DNPH for differential measurement of AA and AAS, that is, the differential oxidation of AA and AAS with 2,6-dinitrophenolindophenol (2,6-Dye) and KBrO 3 and the determination of the osazone produced with the original method by Roe and Kuether (9).