A rapid, simple method for the determination of reduced, dehydro-, and total ascorbic acid in biological material.

Chemical methods for the determination of reduced ascorbic acid (Zascorbic acid) are based upon its reducing properties. The indophenol method of Bessey (1, 2) and the perinaphthindantrione hydrate method of Moubasher (3) are examples of these methods. Modifications to increase the specificity of the indophenol method have been presented by Robinson and Stotz (4), Lowry, Lopez, and Bessey (5), and Huelin (6). However, all of these procedures require tedious photometric measurements in 5 and 10 second intervals to determine the rate of reduction of indophenol by Iascorbic acid. El Ridi, Moubasher, and Hassan (7) demonstrated that perinaphthindantrione reacts with l-ascorbic acid to form a specific, stable reddish color. Total ascorbic acid (reduced ascorbic acid plus dehydroascorbic acid) is not measured by this reaction, and its maximal light absorption is in the ultraviolet region of the spectrum. Roe and Kuether (8) used the coupling reaction of dehydroascorbic acid with 2,4dinitrophenylhydrazine for the determination of total ascorbic acid in body fluids. Diketogulonic acid, a biologically inactive oxidation product of l-ascorbic acid, reacts in the same manner with 2,4-dinitrophenylhydrazine as does dehydroascorbic acid. This method also requires a 3 hour incubation period for completion of the coupling reaction. Roe et al. (9) have modified this procedure to determine l-ascorbic acid, dehydroascorbic acid, and diketogulonic acid in tissue extracts by laborious chemical procedures and a 6 hour incubation which are not suitable for routine analyses. The determination of “free” and “bound” ascorbic acid in liver tissue has been reported by Sumerwell and Sealock (10). Geschwind, Williams, and Li (11) obtained good recoveries of l-ascorbic acid added to adrenal glands, using higher temperatures of incubation with 2,4-dinitrophenylhydrazine, but charring of the samples increased as the temperature approached 100”. An investigation of the total ascorbic acid method of Roe and Kuether was undertaken to shorten the 3 hour incubation period and to improve