High Performance Liquid Chromatographic Determination of Bound Sulfide and Sulfite and Thiosulfate at Their Low Levels in Human Serum by Pre-column Fluorescence Derivatization with Monobromobimane

Abstract

A sensitive and specific high performance liquid chromatographic method for the determination of sulfide, sulfite, and thiosulfate was established. Inorganic sulfur anions were converted into fluorescent derivatives with monobromobimane. The derivatives were separated on a coupled column chromatography with a reversed-phase octadecyl silica column connected with a weakly basic anion exchanger column by isocratic elution with acetic acid solution (pH 3)-acetonitrile (13 : 3, v/v) containing 25 mM NaClO₄. The method was applied to the determination of bound sulfide and sulfite and thiosulfate in normal human serum. Thiosulfate could be determined directly by use of an ultrafiltered sample. For the determination of bound sulfide and sulfite, the pretreatment step with continuous flow gas dialysis was effective for the sample after releasing sulfide and sulfite by reduction with dithiothreitol. The limits of quantification by the present method were 0.05 μM for thiosulfate, 0.5 μM for bound sulfide, and 0.2 μM for bound sulfite.