Merci à Pandréa sur bourso.
Conférence Nicox - Pfizer !!
Auj. à 11:34
Ce matin aux US (heure locale):
Determination of Exogenous Nitric Oxide Using Stable Labeled Nitrogen Coupled with Liquid Chromatography-Mass Spectrometry
Tracks: Contributed Papers: Analytical Methodology / Hyphenated Methods - Small Molecule (e.g., GC-MC, LC-MS, Etc) / Biological Matrix
Date/Time: Tuesday, November 10, 2009
8:00 AM - 12:00 PM
Location: T2047
Description:
Nitric oxide (NO) is involved in many signals of the human body and can influence processes such as vasodilation and inflammation. There is potential to regulate these processes using exogenous NO therapy. NO can be quantified indirectly via oxidative metabolites, nitrite and nitrate, using fluorometric (2,3-diaminonapththalene [DAN] reaction) and colorimetric (Griess reaction) analysis. While useful, these methods cannot distinguish between endogenous levels of NO and levels generated from exogenous therapy. Here we describe the use of stable-labeled [15N]NO-donating moiety coupled with the mass selectivity of LC-MS/MS to quantify exogenous NO in biological samples. [15N]Nitrite was derivatized with DAN using literature methods to form [15N]-naphthotriazole (NAT). Conversion of nitrate to nitrite was achieved using nitrate reductase. LC-MS/MS methods were developed to quantify concentrations of [14N]NAT and [15N]NAT. Time course incubations were performed using rat liver S9 with a [15N]NO-donating substrate, and NO release was monitored. Levels of [15N]nitrite were calculated based on natural abundance of nitrogen isotopes. The mass unit selectivity of the LC-MS/MS method allows for separation between [14N]NAT and [15N]NAT. This improved sensitivity significantly reduced background contribution of endogenous NO. Low nanomolar changes in NO levels can be detected over conventional fluorometric and colorimetric methods. The efficiency of nitrate conversion and derivatization was assessed against a [15N]NAT standard, and efficiency was found to be greater than 80%. Following incubation of a [15N]NO-donating substrate in rat liver S9, an increase in NO formation over time was demonstrated. A sensitive, selective LC/MS/MS method was developed to quantify exogenous NO levels in biological samples resulting from NO-donating therapeutics. The selectivity of this stable-label-LC/MS/MS method offers a marked advantage over traditional colorimetric methods with respect to elucidation of NO levels resulting from exogenous sources.
Speakers:
Author
Miss Minerva R. Batugo
Scientist, Pfizer Global Research & Development
Author
Stefano Biondi
NicOx Research Institute
Author
Claudio De Nardi
NicOx Research Institute
Author
Francesco Impagnatiello
NicOx Research Institute
Author
Dr. Theodore R. Johnson
Senior Principal Scientist, Pfizer Global Research & Development