Determination of endothelium-derived nitrite/nitrate by gas chromatography/tandem mass spectrometry using (15N)NaNO2 as internal standard

Abstract

A sensitive gas chromatographic/tandem mass spectrometric method for the determination of nitrite and nitrate in endothelial cell column perfusates was developed. Nitrite/nitrate were converted in the presence of the internal standard (15N)NaNO2 to the corresponding 1-nitro-2,4,6-trimethoxybenzene (NTMB) derivatives which were subsequently analysed by gas chromatography/tandem mass spectrometry, monitoring m/z = 46 and m/z = 47, respectively. The good reproducibility, the wide linear range of the calibration curve from 25 ng ml-1 to 2500 ng ml-1 nitrite and the low detection limit of 200 pg NTMB allowed a sensitive and accurate quantification of nitrite/nitrate in perfusates of endothelial cell columns. Bradykinin had a stimulating effect on human and bovine endothelial cells and led to a threefold increase of nitrite/nitrate in endothelial cell column perfusates compared to those of unstimulated cells.