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Thin-layer Chromatographic Determination of L-Ascorbic, L-Dehydroascorbic and 2.3-Diketo-L-gulonic Acids in Animal Tissues, Blood and Urine

A new method for the determination of ascorbic, dehydroascorbic and 2.3-diketogulonic acids in animal tissues, blood and urine is described. The material for analysis is homogenized in the presence of stannous chloride in trichloracetic acid and the extract is divided into three aliquots: in one part the ascorbic acid is converted into dehydroascorbic acid by oxidation with bromine water, in the second part the original content of dehydroascorbic and diketogulonic acids is stabilized by the addition of thiourea and in the third part dehydroascorbic acid is reduced to ascorbic acid by thioglycolic acid. Reaction with 2.4-dinitrophenylhydrazine produces a mixture of osazones, from which the bis-hydrazone of diketogulonic acid is isolated by thin-layer chromatography on Silicagel (solvent, ethyl aeetate-chloroform. acetic acid 50:50:5 v/v). The amount of bis-hydrazone is determined photometrically at 502 nm after dissolution in ethyl-acetate-toluene (1:3 v/v). The content of ascorbic, dehydroascorbic and diketogulonic acids is calculated by comparison of the results from the three aliquots of the extract. The described method is more specific and more sensitive than the former colorimetric methods and is suitable for the isolation and evaluation of small amounts of ascorbic, dehydroascorbic and diketogulonic acids labelled with carbon C.