The Vitamin C Content of Human Urine and Its Determination through the 2,4-dinitro- Phenylhydrazine Derivative of Dehydroascorbic

It is well known that the determination of ascorbic acid in urine by titration with 2,6dichlorophenol indophenol in acid solution is not a specific method. Various interfering substances have been reported, such as sulfhydryl compounds (l), thiosulfate (2), and pyridium (medication) (3). The same limitations apply to other oxidation-reduction methods, except when the reducing substance is determined before and after oxidation by ascorbic acid “oxidase” (4). The latter procedure is unsatisfactory for urine for two reasons: It has been found that the “oxidase” is not specific for the oxidation of ascorbic acid (5); and the enzyme may not be effective in oxidizing the vitamin, as shown by Bezssonoff and Vertruyen (6) who found that ascorbic acid “oxidase” prepared from pumpkin pulp did not act upon ascorbic acid in human urine, spinal fluid, and cow’s milk. To remove interfering reducing and colored substances from urine, Emmerie and van Eekelen (7) used mercuric acetate as a precipitant of the interfering material. Later, van Eekelen and Emmerie (8) showed that mercuric acetate precipitation must be carried out rapidly, and excesses of the reagent must be avoided to prevent irreversible oxidation of ascorbic acid. Reducing the severity of treatment minimizes, but does not remove, the error from this procedure. Furthermore, when mercuric acetate is used,