HPLC법에 의한 1,2-디아미노-4,5-메틸렌디옥시벤젠을 형광유도체화제로 한 혈청 및 뇨 중의 alpha-케토산의 분석

Determination of alpha-Keto Acids in Serum and Urine Using 1,2-Diamino-4,5-methylendioxybenzene as a Fluorescent Derivatizating Agent by High Performance Liquid Chromatography

A simple and sensitive high performance liquid chromatographic method to quantitate alpha-keto acids in serum and urine was investigated. alpha-Keto acids react with 1,2-diamino-4,5-methylenedioxybenzene (DMB) in the presence of 2-mercapto-ethanol and sodium hydrogen sulfite to form highly fluorescent derivatives, substituted 6,7-methylenedioxyquinoxalinol. The derivatization procedure was performed in water bath at 100oC , and completed within 50 min. By the use of a reversed-phase column and multi-step gradient with two solvents, a mixture containing twelve of these derivatives were efficiently resolved within 35 minutes. The optimal wavelength of the fluorescence detector are lambdaex = 364nm and lambdaem = 445nm. The quantitation of the individual alpha-Keto acids was reproducible with relative standard deviation of 3.0~7.9% and had a detection limits of 10~60 fmol, except for p-hydroxyphenylpyruvic acid (960 fmol).