Comparative analyses for ascorbic acid by the 2,4-dinitrophenylhydrazine method with the coupling reaction at different temperatures: a procedure for determining specificity.

The dinitrophenylhydrazine method for the determination of ascorbic acid is based upon treatment with 85% HzS04 of the chromogen formed by the coupling of 2,4-dinitrophenylhydrazine with oxidized ascorbic acid. In the original publication of this method, Roe and Kuether (1) set the temperature of the coupling reaction at 37” and used a 3-hour incubation period. These authors showed that a low temperature for this reaction, together with proper dilution, would avoid interference from glucose, fructose, xylose, and glucuronic acid. Schaffert and Kingsley (2) published a modification of this method in which the coupling reaction is carried out at 100” for 10 minutes. It is the purpose of this paper to show that the Schaffert and Kingsley modification yields values markedly higher than the true content, because of the formation of chromogen from nonascorbic acid substances. The amount of interference that may be expected from glucose, fructose, and glucuronic acid when the coupling reaction is carried out at 37” and 100” is also reported. Further studies of the specificity of the dinitrophenylhydrazine method are presented and a convenient procedure for testing the specificity of this procedure is proposed.