High-performance liquid chromatography/chemiluminescence determination of biological thiols with N-[4-(6-dimethylamino-2-benzofuranyl)phenyl]maleimide.
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 degrees 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 mm ID; 5 microns) and detected in the ranges from 500 fmol to 2 pmol/100 microL (cysteamine and N-acetylcysteine), to 3 pmol/100 microL (cysteine) and to 5 pmol/100 microL (GSH and D-penicillamine). The lower detection limits were from 7 fmol (cysteamine) to 113 fmol (GSH) per 100 microL (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 mumol/g (n = 5) and 0.15 +/- 0.04 mumol/g (n = 5), respectively.
Nakashima
K
K
Faculty of Pharmaceutical Sciences, Nagasaki University, Japan.
Umekawa
C
C
Nakatsuji
S
S
Akiyama
S
S
Givens
R S
RS
D000818:Animals; D001572:Benzofurans; D002851:Chromatography, High Pressure Liquid; D005456:Fluorescent Dyes; D008099:Liver / Q000032:analysis; D008163:Luminescent Measurements; D008297:Male; D008301:Maleimides; D051381:Rats; D011919:Rats, Inbred Strains; D013438:Sulfhydryl Compounds / Q000032:analysis*
D001572:Benzofurans; D005456:Fluorescent Dyes; D008301:Maleimides; D013438:Sulfhydryl Compounds; C049623:N-(4-(2-(6-dimethylamino)benzofuranyl)phenyl)maleimide