High-performance liquid chromatography and fluorometric detection of biologically important thiols, derivatized with ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate (SBD-F).

Thiol compounds which are derivatized with a fluorogenic reagent, ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate (SBD-F), are separated on a high-performance liquid chromatography column (muBondapak C18) and detected fluorometrically at 515 nm with excitation at 385 nm. Two kinds of gradient elution system were adopted for the separation of SBD cysteine, homocysteine, cysteamine, glutathione and N-acetylcysteine. The detection limits were in the range 0.07-1.4 pmole. Utilizing the proposed method, only a reduced form of glutathione was found in human whole blood at the level of 1.6 +/- 0.4 mM. However, in the plasma, both the reduced and oxidized (tri-n-butylphosphine-treated) L-cysteine, glutathione and an unknown substance were detected. The amounts of L-cysteine and total L-cysteine (reduced plus oxidized) in plasma were 27 +/- 2 and 72 +/- 7 microM, respectively.