title: An improved mass spectrometry-based measurement of NO metabolites in biological fluids.
abstract: Assessment of NO metabolism in vivo relies on the accurate measurement of its metabolites nitrite (NO(2)(-)), nitrate (NO(3)(-)), and nitrosothiols (RSNOs) in biological fluids. We report a sensitive method to simultaneously determine NO(2)(-) and NO(3)(-) in biological matrixes. Tetraoctylammonium was used to catalyze the complete conversion of NO(2)(-) and NO(3)(-) 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 NO(2)(-) (4.5 times) and NO(3)(-) (55 times) compared to noncatalyzed derivatization methods. Analysis was performed using (15)N-labeled internal standards by selected-ion monitoring at m/z 46 for fragment NO(2)(-) and m/z 47 for its isotope analogue, (15)NO(2)(-), and m/z 62 for NO(3)(-) and m/z 63 for (15)NO(3)(-). This method allowed specific detection of both PFB derivatives over a wide dynamic range with a limit of detection below 4.5 pg for NO(2)(-) and 2.5 pg for NO(3)(-). After the specific conversion of RSNOs by HgCl(2) to NO(2)(-), 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 NO(2)(-) and NO(3)(-) as well as plasma RSNOs in humans after either a low-NO(3)(-) or a high-NO(3)(-) meal.
authors.affiliation: College of Food Engineering and Nutritional Science, Shaanxi Normal University, Xi'an 710062, China.
authors.forename: Xingbin
authors.initials: X
authors.lastname: Yang
authors.forename: Catherine P
authors.initials: CP
authors.lastname: Bondonno
authors.forename: Adeline
authors.initials: A
authors.lastname: Indrawan
authors.forename: Jonathan M
authors.initials: JM
authors.lastname: Hodgson
authors.forename: Kevin D
authors.initials: KD
authors.lastname: Croft
mesh_terms: D001826:Body Fluids / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry; D008875:Middle Aged; D009566:Nitrates / Q000032:analysis* / Q000378:metabolism; D009569:Nitric Oxide / Q000737:chemistry / Q000378:metabolism*; D009573:Nitrites / Q000032:analysis* / Q000378:metabolism; D012016:Reference Values; D026403:S-Nitrosothiols / Q000032:analysis* / Q000378:metabolism
chemical_list: D009566:Nitrates; D009573:Nitrites; D026403:S-Nitrosothiols; D009569:Nitric Oxide