Determination of alpha-keto acids in serum and urine by high-performance liquid chromatography with fluorescence detection.

A simple, rapid and highly sensitive high-performance liquid chromatographic method for the determination of alpha-keto acids in serum and urine is described. In dilute hydrochloric acid, alpha-keto acids are converted by 1,2-diamino-4,5-dimethoxybenzene into highly fluorescent quinoxalinone derivatives. The derivatives are isocratically separated simultaneously within 14 min by reversed-phase chromatography on a Radial-Pak cartridge C18 and detected fluorimetrically. The limits of detection are 10-300 fmol in an injection volume of 10 microliters (40-1200 pmol/ml of serum or urine). This sensitivity permits precise determination of several alpha-keto acids in 5 microliter of serum or urine from healthy persons, and also the determination of phenylpyruvic acid in normal urine which cannot be simultaneously determined by other methods.