Rapid and sensitive gradient liquid chromatography method for the quantitation of 2-hydroxyethidium ion from neutrophils

Abstract

We developed an improved reversed-phase high-performance liquid chromatography(RPLC) assay for the rapid separation and quantitation of the 2-hydroxyethidium ion and achieved a high resolution between the ethidium and 2-hydroxyethidium ions, the latter being the specific product of the reaction between hydroethidine and the superoxide radical within a practicable analysis time. The RPLC-fluorescence method can reliably detect 2-hydroxyethidium-ion oncentrations down to 0.12 µM (or 1.2 pmol) and the signal is linear with concentration beyond 50 µM. An application of the method to neutrophil samples demonstrated that intracellular quantification of 2-hydroxyethidium was reproducible, as evidenced by low values of the relative standard deviations. The resulting analytical method combines a rapid separation of the relevant peaks with the degree of sensitivity required for use in routine biological analyses.