Fluorometric Anqlysis of α-Keto Acids

概要

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4'-Hydrazino-2-stilbazole (4H2S) was discovered as aspecific and sensitive reagent for α-dicarbonyl compounds. 4H2S readily produced hydrazones of carbonyl compounds, among which only α-dicarbonyl compounds strongly fluoresced yellow in acid media as shown in Fig. 1 and TABLE I, hence the reagent becomes specific for a-keto acids in biological materials (TABLE II). A micro-determination procedure was established as follows: One ml of deproteinized and neutralized sample solution is mixed with 1 ml of 5×10-4M aqueous solution of 4H2S and 1 ml of 0.1M acetate buffer solution at pH 4. After standing for 1 hour in a dark at 20°C-30°C, 2 ml of N HCl are added, and fluorescence intensity of the solution is measured immediately at 550 mμ, excited by light of 450 mμ. As shown in Fig. 3, the intensity showed a linear relationship with the concentration of α-keto acid in the sample over the range of 2×10-7 to 2×10-5M. With 4H2S, each α-keto acid was detected on a filter paper after electrophoretic separation at pH 4, as shown in Fig. 4. The detection limit was 0.01 μg of a-keto acid.
Japan Society of Clinical Chemistryの論文