SELECTIVE BIOCOULOMETRY OF VITAMIN-C USING DITHIOTHREITHOL, N-ETHYLMALEIMIDE, AND ASCORBATE OXIDASE

Controlled-potential coulometry of dehydroascorbic acid using dithiothreithol as a reducing agent has been proposed. The reduction of dehydroascorbic acid to L-ascorbic acid is complete in a few minutes at pH 7.0 and room temperature. N-Ethylmaleimide was added after the reduction was finished, and excess dithiothreithol was converted to an electroinactive species. This solution was then completely electrooxidized and the total charge (Q(s)) was obtained. Next, ascorbate oxidase was added to this solution and an electroactive L-ascorbic acid was converted to electroinactive dehydroascorbic acid. The total charge (Q(b)) was then obtained and also for the previously described solution without enzyme. Hence, the difference between two charges (Q(s) - Q(b)) corresponds to the charge due to the oxidation of dehydroascorbic acid. The current efficiencies of dehydroascorbic acid were attained to be more than 98%. The coulometric results of L-ascorbic acid and dehydroascorbic acid in fruit juices obtained by the present method were in fairly good agreement with those obtained by spectrophotometry.