Stable isotope-assisted LC–MS/MS monitoring of glyceryl trinitrate bioactivation in a cell culture model of nitrate tolerance

• We quantified 15N-nitrite and 15N-nitrate with LC–MS/MS.
• Nitrite and nitrate have a LLOQ of 1 nM in water and culture media.
• Glyceryl trinitrate is metabolized into nitrite and nitrate by endothelial cells.
• Nitrite production is reduced in response to continuous exposure to GTN.
• Nitrate tolerance may be caused by reduced bioactivation of glyceryl trinitrate.

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 14N-NAT (m/z 170.1) and 15N-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).