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High‐performance liquid chromatography/chemiluminescence determination of biological thiols with n ‐[4‐(6‐dimethylamino‐2‐benzofuranyl)phenyl]maleimide
Author(s) -
Nakashima Kenichiro,
Umekawa Chiemi,
Nakatsuji Shin'Ichi,
Akiyama Shuzo,
Givens Richard S.
Publication year - 1989
Publication title -
biomedical chromatography
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.4
H-Index - 65
eISSN - 1099-0801
pISSN - 0269-3879
DOI - 10.1002/bmc.1130030110
Subject(s) - maleimide , chemistry , chemiluminescence , chromatography , thiol , high performance liquid chromatography , organic chemistry
The peroxyoxalate chemiluminescence detection of biological thiols combined with high‐performance liquid chromatography (HPLC) is described. SH groups of the thiol compounds including glutathione (GSH), cysteine, N ‐acetylcysteine, cysteamine, and D‐penicillamine were labelled with N ‐[4‐(6‐dimethylamino‐2‐benzofuranyl)phenyl]maleimide (DBPM), a specific fluorogenic reagent for SH 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 × 4.6mm ID; 5 μm) and detected in the ranges from 500 fmol to 2 pmol/100 μL (cysteamine and N ‐acetylcysteine), to 3 pmol/100 μL (cysteine) and to 5 pmol/100 μL (GSH and D‐penicillamine). The lower detection limits were from 7 fmol (cysteamine) to 113 fmol (GSH) per 100 μL (S/N = 2). The method was applied to the determination of thiols in a rat liver. The amounts of glutathione and cysteine were 1.23±0.15 μmol/g ( n = 5) and 0.15±0.04 μmol/g ( n = 5), respectively.