A NOVEL HPLC METHOD FOR THE DETERMINATION OF ALPHA-KETO ACIDS IN HUMAN SERUM USING MESO STILLBENEDIAMINE AS DERIVATIZATION REAGENT

A simple and rapid isocratic high-performance liquid chromatographic (HPLC) method has been developed for the determination of seven alpha-keto acids, glyoxylic acid, pyruvic acid, 2-oxobutyric acid, 3-methyl-2-oxobutyric acid, 2-oxoglutaric acid, 2-oxohexanoic acid, and phenylpyruvic acid using meso silbenediamine as derivatizing reagent in the blood samples. The chromatographic separation was achieved with methanol-water-acetonitrile-tetrahydrofuran (38.4:60:1:0.6, v/v/v/v) as the mobile phase, a C18 column, and UV detection at 255 nm. Linear calibration curves were obtained with 0.2 to 100 µg/mL. The limits of detection and quantitation were obtained within the range 0.07–0.2 µg/mL and 0.21–0.6 µg/mL, respectively. The method was applied for the determination of alpha-keto acids from human serum and urine of uremic and diabetic patients before hemodialysis, after hemodialysis, and healthy volunteers.