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

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

  • Ok, Chi-Wan (College of Pharmacy, Won Kwang University) ;
  • Kim, Dae-Ki (College of Pharmacy, Won Kwang University) ;
  • Park, Song-Ja (Dopping Control Center, Korea Insitute of Science and Technology) ;
  • Park, Jong-Sei (Dopping Control Center, Korea Insitute of Science and Technology)
  • 옥치완 (원광대학교 약학대학) ;
  • 김대기 (원광대학교 약학대학) ;
  • 박송자 (한국과학기술원 도핑콘트롤센터) ;
  • 박종세 (한국과학기술원 도핑콘트롤센터)
  • Published : 1992.08.29

Abstract

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 $100^{\circ}C$, 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 wavelengh of the fluorescence detector are ${\lambda}_{ex}=364\;nm$ and ${\lambda}_{em}=445\;nm$. The quantitation of the individual ${\alpha}-Keto$ acids was reproducible with relative standard deviation of $3.0{\sim}7.9%$ and had a detection limits of $10{\sim}60$ fmol, except for p-hydroxyphenylpyruvic acid (960 fmol).