Determination of trace amounts of sulphide in human red blood cells by high-performance liquid chromatography with fluorimetric detection after derivatization with p-phenylenediamine and iron(III)

Abstract

A high-performance liquid chromatographic method based on pre-column fluorescence derivatization has been developed for the determination of trace amounts of sulphide. After the sulphide had been converted into a fluorescent derivative, thionine, by the reaction with p-phenylenediamine and Fe³⁺, it was separated on a reversed-phase column and detected fluorimetrically (excitation, 600 nm; emission, 623 nm). Sulphide ion can be determined in the range from 0.01 to 3.0 µmol dm^–3 with a relative standard deviation (n= 5) of 2.54% at 0.02 µmol dm^–3 and 1.74% at 1.0 µmol dm^–3. The proposed method was applied to the determination of sulphide in human red blood cells from ten healthy subjects, by generating sulphide in a microdiffusion apparatus; the concentrations found ranged from 0.123 to 0.189 µmol dm^–3.