Assessment of NO metabolism in vivo relies on the accurate measurement of its metabolites nitrite (NO2 -), nitrate (NO 3 -), and nitrosothiols (RSNOs) in biological fluids. We report a sensitive method to simultaneously determine NO2 - and NO3 - in biological matrixes. Tetraoctylammonium was used to catalyze the complete conversion of NO2 - and NO3 - to stable pentafluorobenzyl (PFB) derivatives directly from aqueous acetone medium before gas chromatography and negative-ion chemical ionization mass spectrometry (GC/NICI/MS). This catalyst dramatically improved the yield of PFB derivatives for NO2 - (4.5 times) and NO3 - (55 times) compared to noncatalyzed derivatization methods. Analysis was performed using 15N-labeled internal standards by selected-ion monitoring at m/z 46 for fragment NO 2 - and m/z 47 for its isotope analogue, 15NO2 -, and m/z 62 for NO3 - and m/z 63 for 15NO3 -. This method allowed specific detection of both PFB derivatives over a wide dynamic range with a limit of detection below 4.5 pg for NO2 - and 2.5 pg for NO3 -. After the specific conversion of RSNOs by HgCl2 to NO2 -, this GC/NICI/MS analysis was used to measure RSNOs in plasma. A further comparison with the widely used tri-iodide chemiluminescence (I3 --CL) assay indicated that the GC/MS assay validated the lower physiological RSNO and nitrite levels reported using I3 --CL detection compared with values obtained using UV-photolysis methods. Plasma levels of RSNOs determined by GC/MS and I3 --CL were well correlated (r = 0.8). The improved GC/MS method was successfully used to determine the changes in plasma, urinary, and salivary NO2 - and NO3 - as well as plasma RSNOs in humans after either a low-NO3 - or a high-NO3 - meal. © 2012 Elsevier Inc.
|Journal||Free Radical Biology and Medicine|
|Publication status||Published - 2013|