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

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

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Analytical Methods Vol - 3 Issue - 2

Analytical Methods Vol -2

Analytical Methods Vol -1

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

Content Provider	Royal Society of Chemistry (RSC)
Author	Grisales, Jaiver Osorio Castells, Cecilia Gotta, Javier Giambelluca, Miriam Lebed, Pablo J. Keunchkarian, Sonia
Copyright Year	2011
Abstract	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 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: 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.
Starting Page	593
Ending Page	598
Page Count	6
File Format	HTM / HTML PDF
ISSN	17599660
Volume Number	3
Issue Number	3
Journal	Analytical Methods
DOI	10.1039/c0ay00577k
Language	English
Publisher	Royal Society of Chemistry
Access Restriction	Open
Subject Keyword	PMA Redox Ion Superoxide Neutrophil High-performance liquid chromatography Reversed-phase chromatography N-Formylmethionine-leucyl-phenylalanine Chromatography
Content Type	Text
Resource Type	Article
Subject	Analytical Chemistry Engineering Chemical Engineering

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

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