High-performance liquid chromatographic determination of α-keto acids in human serum and urine using 1,2-diamino-4,5-methylenedioxybenzene as a precolumn fluorescence derivatization reagent

Abstract

A simple and highly sensitive high-performance liquid chromatographic (HPLC) method for the determination of α-keto acids in human serum and urine is described. In an acidic solution, twelve species of α-keto acids examined were converted by reaction with 1,2-diamino-4,5-methylenedioxybenzene into highly fluorescent derivatives. The derivatives were separated isocratically by reversed-phase HPLC on a TSK gel ODS-80TM column and detected fluorimetrically. Eight α-keto acids in human serum and eleven α-keto acids in human urine can be determined simultaneously. The detection limits (signal-to-noise ratio = 5) are 6–44 fmol in an injection volume of 5 μl. The intra-assay relative standard deviations for both serum and urine sample analyses are usually ca. 5%.