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.
Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
