TitleStable isotope-assisted LC-MS/MS monitoring of glyceryl trinitrate bioactivation in a cell culture model of nitrate tolerance.
Publication TypeJournal Article
Year of Publication2016
AuthorsAxton ER, Hardardt EA, Stevens JF
JournalJ Chromatogr B Analyt Technol Biomed Life Sci
Date Published2016 Apr 15
KeywordsCell Line, Chromatography, Liquid, Endothelial Cells, Humans, Isotope Labeling, Nitrates, Nitrites, Nitrogen Isotopes, Nitroglycerin, Tandem Mass Spectrometry

The nitric oxide (NO) metabolites nitrite (NO2(-)) and nitrate (NO3(-)) can be quantified as an endpoint of endothelial function. We developed a LC-MS/MS method of measuring nitrite and nitrate isotopologues, which has a lower limit of quantification (LLOQ) of 1 nM. This method allows for isotopic labeling to differentiate newly formed nitrite and nitrate from nanomolar to micromolar background levels of nitrite and nitrate in biological matrices. This method utilizes 2,3-diaminonaphthalene (DAN) derivatization, which reacts with nitrite under acidic conditions to produce 2,3-naphthotriazole (NAT). NAT was chromatographically separated on a Shimadzu LC System with an Agilent Extend-C18 5 μm 2.1 × 150 mm column and detected using a multiple reaction monitoring (MRM) method on an ABSciex 3200 QTRAP mass spectrometer operated in positive mode. Mass spectrometry allows for the quantification of (14)N-NAT (m/z 170.1) and (15)N-NAT (m/z 171.1). Both nitrite and nitrate demonstrated a linear detector response (1 nM - 10 μM, 1 nM - 100 nM, respectively), and were unaffected by common interferences (Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS), phenol red, and NADPH). This method requires minimal sample preparation, making it ideal for most biological applications. We applied this method to develop a cell culture model to study the development of nitrate tolerance in human endothelial cells (EA.hy926).

Alternate JournalJ. Chromatogr. B Analyt. Technol. Biomed. Life Sci.
PubMed ID26796748
PubMed Central IDPMC4909631
Grant ListP30 ES000210 / ES / NIEHS NIH HHS / United States
S10 RR022589 / RR / NCRR NIH HHS / United States
T32 ES007060 / ES / NIEHS NIH HHS / United States
T32ES007060 / ES / NIEHS NIH HHS / United States