Determination of α-keto acids including phenylpyruvic acid in human plasma by high-performance liquid chromatography with chemiluminescence detection

Abstract

A highly sensitive method for the determination of α-keto acids including phenylpyruvic acid in human plasma is investigated. The method employs high-performance liquid chromatography with chemiluminescence detection. The acids and α-ketocaproic acid (internal standard) in human plasma are isolated by anion-exchange chromatography on a Toyopak DEAE cartridge, and then converted into the corresponding chemiluminescent derivatives with 4,5-diaminophthalhydrazide dihydrochloride, a chemiluminescence derivatization reagent for α-keto acids. The derivatives are separated within 50 min on a reversed-phase column, TSKgel ODS-120T, with isocratic elution, followed by chemiluminescence detection; the chemiluminescence is produced by the reaction of the derivatives with hydrogen peroxide in the presence of potassium hexacyanoferrate(III). The detection limits for the acids are in the range 9–92 pmol/ml in plasma (signal-to-noise ratio = 3). This sensitivity permits precise determination of several α-keto acids including phenylpyruvic acid, which cannot be determined by other HPLC methods, in 10 μl of normal human plasma. The chemiluminescent product from phenylpyruvic acid was characterized as 3-benzyl-7,8-dihydropyridazino[4,5- g]quinoxaline-2,6,9(1 H)-trione.