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

P. J. LEBED, J. OSORIO GRISALES, S. KEUNCHKARIAN, J. GOTTA, M. GIAMBELLUCA, C. B. CASTELLS

Analytical Methods

Springer

Año: 2011 vol. 3 p. 593 - 598

1759-9660

We developed an improved reversed-phase high-performance liquid chromatography (RPLC) assay for the rapid separation and determination of the 2-hydroxyethidium ion. The 2-hydroxyethidium ion is the specific product of the redox reaction between hydroethidine with superoxide radical. High resolution between the chromatographic bands corresponding to ethidium and 2-hydroxyethidium ions was achieved within a practicable analysis time. The RPLC-fluorescence method can reliably detect 2-hydroxyethidium ion concentrations down to 0.12 M (or 1.2 pmol) and the signal is linear with concentration beyond 50 M. 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: 0.016 for non-stimulated cells, and 0.056 and 0.0125 for neutrophils incubated with agonists phorbol myristate acetate (PMA) and N-formyl-methionyl-leucyl- phenylalanine (fMLP), respectively. The resulting analytical method combines a rapid separation of the relevant peaks with the degree of sensitivity required for use in routine biological analyses. © 2011 The Royal Society of Chemistry.