High-performance liquid chromatography/chemiluminescence determination of biological thiols with n-[4-(6-dimethylamino-2-benzofuranyl)phenyl]maleimide

✍ Scribed by Kenichiro Nakashima; Chiemi Umekawa; Shin'Ichi Nakatsuji; Shuzo Akiyama; Richard S. Givens

• Publisher: John Wiley and Sons
• Year: 1989
• Tongue: English
• Weight: 359 KB
• Volume: 3
• Category: Article
• ISSN: 0269-3879
• DOI: 10.1002/bmc.1130030110

✦ Synopsis

The peroxyoxalate chemiluminescence detection of biological thiols combined with high-performance liquid chromatography (HPLC) is described. S H groups of the thiol compounds including glutathione (GSH), cysteine, N-acetylcysteine, cysteamine, and D-penicillamine were labelled with N-[4-(6-dimethylamino-2benzofuranyl)phenyllmaleimide (DBPM), a specific fluorogenic reagent for S H group. The labelling reaction was carried out at 60 "C for 30 min and at pH 8.5 and a sample of the resulting reaction mixture was subjected to HPLC. Five kinds of labelled thiols were separated within 12 min on ODs-80 column (150 X 4.6 m m ID; 5 pm) and detected in the ranges from 500 fmol to 2 pmo1/100 p L (cysteamine and N-acetylcysteine), to 3 pmo1/100 p L (cysteine) and to 5 pmoV100 p L (GSH and D-penicillamine). The lower detection limits were from 7 fmol (cysteamine) to 113 fmol (GSH) per 100 p L (S/N = 2). The method was applied to the determination of thiols in a rat liver. The amounts of glutathione and cysteine were 1.23i0.15 ymol/g (n =5) and 0.15i0.04 pmol/g ( n =5), respectively.