A 2,4-Dinitrophenylhydrazine Derivative of Dehydroascorbic Acid and the Estimation of Vitamin G

Abstract

When dehydroascorbic acid is treated with a saturated solution of 2,4-dinitrophenylhydrazine in N HC1, a reddish crystalline derivative is formed. The reaction is catalyzed by acetic acid. Analysis of the product obtained showed it contains two 2,4-dinitrophenylhydrazine groups in its molecule. The derivative is apparently an osazone of dehydroascorbic acid with the hydrazine groups attached to carbon atoms 2 and 3. The compound melted at 257-259° (corrected) and microanalysis showed it has the following composition: C₁₈ H₁₄ O₁₂ N₈

Calculated: C 40.43, H 2.64, N 20.98.

Found: ″40.40, ″ 2.88, 21.11.

The evidence indicates the reaction is as follows:

This compound was prepared as follows: 0.2 gm. of pure ascorbic acid in 200 cc. of 10% acetic acid was shaken with norit and filtered. Two volumes of 2,4-dinitrophenylhydrazine in N HC1 were added. The mixture was allowed to stand at room temperature for 72 hours and then centrifuged. The precipitate was washed 6 times with distilled water.

Advantage was taken of this reaction to make it the basis of a specific method for the determination of vitamin C. The dehydroascorbic acid osazone is reduced to a colorless compound by warming with 12% HC1 containing 10% SnCl₂ and the compound is hydro-lyzed by heating with these reagents in an autoclave at 15 lb. pressure. Under the latter conditions the released dehydroascorbic acid is converted into furfural, which, upon cooling, is determined by the colorimetric aniline acetate method 1 previously published. Use is made of a standard solution of pure ascorbic acid which is treated in the same way as the acid extract of tissue.

Substances most likely to interfere in this method are free pen-toses which will give a centrifugable precipitate in concentrations greater than one mg. per cc. As the vitamin is extracted from tissues by a procedure which usually involves a 10-fold dilution, and greater dilutions may be made, this possible difficulty with tissues containing free pentose is readily overcome. The method appears to be entirely specific for the estimation of vitamin C.

Osazone formation does not occur in the first few hours after mixing 2,4-dinitrophenylhydrazine with ascorbic acid but later crystals appear slowly. This behavior is interpreted as indicating that ascorbic acid does not react with the hydrazine under these conditions, but upon standing it is oxidized to dehydroascorbic acid which reacts to produce the compound described.

This procedure is particularly advantageous for the determination of vitamin C in urine, for which the indophenol and other oxidation-reduction methods lack specificity. Ascorbic acid added to urine is recovered quantitatively by this method.

In preparing this paper the report of Stewart, Scarborough and Drumm 2 came to our attention. These authors have made a 2,4-dinitrophenylhydrazine derivative from the ascorbic acid in urine. As the method of preparation was not stated and no analyses were reported, a comparison of their derivative with ours cannot be made. The melting point reported (269-271 uncorrected) is about 20 degrees higher than that of our product.

The details of our procedure for the estimation of vitamin C will be published later.