Chemiluminescence detection of thiols by high-performance liquid chromatography using o-phthalaldehyde and N-(4-aminobutyl)-N-ethylisoluminol as precolumn derivatization reagents

A method for the determination of thiols by high-performance liquid chromatography (HPLC) with chemiluminescence detection has been developed. The method is based on the precolumn derivatization of thiols with o-phthalaldehyde and N-(4-aminobutyl)-N-ethylisoluminol to form isoindole derivatives and the reversed-phase HPLC separation of the derivatives, followed by successive postcolumn chemiluminescence reactions with hydrogen peroxide and hematin. The limits of the detection of glutathione (GSH) and cysteine were 1.5 nM (15 fmol/injection) and 3.5 nM (35 fmol/injection), respectively. The calibration curve for GSH was linear in the range 5 - 1000 nM (1 - 200 pmol/200 mu l). The method was applied to the determination of GSH in human blood. The recovery of GSH added to the blood at the concentration level of 500 mu M was about 70%. The GSH concentrations in the blood of three healthy male persons were 502 - 770 mu M.