Paper Electrophoresis of Urinary α-Keto Acids using 4'-Hydrazino-2-stilbazole (4 H 2 S) as a Fluorescence Reagent

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4'-Hydrazino-2-stilbazole (4 H 2 S) had been discovered as a selective and sensitive fluorescence reagent for α-keto acids in biological materials. Method of fluorometric determination2) and paperelectrophoretic detection3) of α-keto acids with 4H2S had also been developed.<BR>We reinvestigated the conditions of paper electrophoretic detection of urinary α-keto acids and improved the procedure as follows: A piece of Toyo filter paper,(No.51A 0.2 ×13cm) spotted with 10-20 μl of urine is placed at 10cm position from cathode on the same paper (35×13cm), is electropl9oresed in a phosphate buffer (pH 6.7, μ=0.16) at 400 volt constat potential for 1.5hr. After drying sufficiently, the paper is sprayecd with 0.01% solution of 4 H 2 S in formate buffer (1 M, pH 3), allowed to stand for 5 minute to form hydrazone and sprayed with 5 N HC1. Under a low prsssure mercury lamp equipped with Toshiba V-V44 filter, the yellowish fluorescent spot of α-keto acid is detected on the dark blue background.<BR>As shown in Table 1, separation of the α-keto acids was fairly good, although KIV, KIC and KMV could not be separated.<BR>The present method was applied to urinary specimens which were positive to 2, 4-dinitrophenylhydrazine test (see Table II).