Diketogulonic acid, determination

Vitamin C can also be determined colorimetrically, after oxidation to dehydroascorbate, by reaction with dinitrophenylhydrazine. Under appropriate conditions, neither ascorbic acid itself nor potentially interfering sugars react with dinitrophenylhydrazine. However, diketogulonate, which has no vitamin activity, also reacts with dinitrophenylhydrazine under the same conditions. Unless diketogulonate is determined separately after reduction of dehydroascorbate to ascorbate, this method overestimates the vitamin. 

The formation of phenylhydrazones and osazones from dehydroas-corbic acid is the basis of the most commonly used method of ascorbic acid analysis. The reactions are described in the section on determinations. There is no great specificity to this reaction, which is given by most compounds containing keto groups. However, most phenylhydrazones become intensely colored only in alkaline solution, while the dehy-droascorbic acid type of compound is intensely colored in acid solution. Both dehydroascorhic acid and 2,3-diketogulonic acid are converted to the same osazone under the conditions used. 

Roe, Joseph H., Chemical Determinations of Ascorbic, Dehydro-ascurbic, and Diketogulonic Acids. 

Chemical Determination of ASCORBIC, DEHYDROASCORBIC, AND DIKETOGULONIC ACIDS... 

Differential Method for Determination of Ascorbic Acid, Dehydro-ascorbic Acid, and Diketogulonic Acid. Method of Roe, Mills, Oesterling,... 

The methods of Roe and Kuether (21) and of Roe and Oesterling (22) for the determination of vitamin C do not differentiate between ascorbic acid (AsA), dehydroascorbic acid (DHA), and diketogulonic acid (DKA). The same basic principle is applied to the determination of the three substances (23), the differentiation being made through the use of H2S which reduces DHA to AsA and does not reduce DKA under the conditions used. In analyzing an extract which contains all three forms of vitamin C, one aliquot is reduced with H2S, a second aliquot is left untreated, and a third aliquot is treated with bromine. After coupling with 2,4-dinitrophenyl-hydrazine the derivative in the first aliquot is that of DKA, the one in the second aliquot is that of DHA and DKA, and the one in the third aliquot is that of AsA, DHA, and DKA. Appropriate subtraction of the values obtained by colorimetric measurements will give the value for each component. 

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