An improved mass spectrometry-based measurement of NO metabolites in biological fluids

Assessment of NO metabolism in vivo relies on the accurate measurement of its metabolites nitrite (NO2-), nitrate (NO3-), 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 N-15-labeled internal standards by selected-ion monitoring at m/z 46 for fragment NO2- and m/z 47 for its isotope analogue, (NO2-)-N-15, and m/z 62 for NO3- and m/z 63 for (NO3-)-N-15. 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 (I-3(-)-CL) assay indicated that the GC/MS assay validated the lower physiological RSNO and nitrite levels reported using I-3(-)-CL detection compared with values obtained using UV-photolysis methods. Plasma levels of RSNOs determined by GC/MS and I-3(-)-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-NO2- or a high-NO3- meal. (C) 2012 Elsevier Inc. All rights reserved.