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

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

Analytical Methods

Royal Society of Chemistry Publishing

Lugar: Londres; 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 mM (or 1.2 pmol) and the signal is linear with concentration beyond 50 mM. 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 neutrophilsincubated 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.