1,2-Diamino-4,5-methylenedioxybenzene as a highly sensitive fluorogenic reagent for .ALPHA.-keto acids.

Abstract

The reactivity of pyruvic acid with 1, 2-diaminobenzenes was investigated as part of a survey of fluorogenic reagents for α-keto acids of biological importance. Of eight 1, 2-diaminobenzenes tested, 1, 2-diamino-4, 5-methylenedioxybenzene was found to be the best reagent in terms of sensitivity and reactivity. The reagent reacts with α-keto acids in an acidic solution to produce the corresponding fluorescent derivatives, which fluorescent most intensely in a neutral medium. The derivatives of ten α-keto acids can be separated within 18 min by reversed-phase high-performance liquid chromatography with isocratic elution. The detection limits for the acids are at femtomole levels for an injection volume of 10 μl. The fluorescent product from pyruvic acid was characterized as 3-methyl-6, 7-methylenedioxy-2 (1H) -quinoxalinone. The fluorescence properties of the product are also described.