| Summary: | Nitrite (O=N-O<sup>−</sup>, NO<sub>2</sub><sup>−</sup>) and nitrate (O=N(O)-O<sup>−</sup>, NO<sub>3</sub><sup>−</sup>) are ubiquitous in nature. In aerated aqueous solutions, nitrite is considered the major autoxidation product of nitric oxide (<sup>●</sup>NO). <sup>●</sup>NO is an environmental gas but is also endogenously produced from the amino acid L-arginine by the catalytic action of <sup>●</sup>NO synthases. It is considered that the autoxidation of <sup>●</sup>NO in aqueous solutions and in O<sub>2</sub>-containing gas phase proceeds via different neutral (e.g., O=N-O-N=O) and radical (e.g., ONOO<sup>●</sup>) intermediates. In aqueous buffers, endogenous <i>S</i>-nitrosothiols (thionitrites, RSNO) from thiols (RSH) such as L-cysteine (i.e., <i>S</i>-nitroso-L-cysteine, CysSNO) and cysteine-containing peptides such as glutathione (GSH) (i.e., <i>S</i>-nitrosoglutathione, GSNO) may be formed during the autoxidation of <sup>●</sup>NO in the presence of thiols and dioxygen (e.g., GSH + O=N-O-N=O → GSNO + O=N-O<sup>−</sup> + H<sup>+</sup>; p<i>K</i><sub>a</sub><sup>HONO</sup>, 3.24). The reaction products of thionitrites in aerated aqueous solutions may be different from those of <sup>●</sup>NO. This work describes in vitro GC-MS studies on the reactions of unlabeled (<sup>14</sup>NO<sub>2</sub><sup>−</sup>) and labeled nitrite (<sup>15</sup>NO<sub>2</sub><sup>−</sup>) and RSNO (RS<sup>15</sup>NO, RS<sup>15</sup>N<sup>18</sup>O) performed in pH-neutral aqueous buffers of phosphate or t<i>ris</i>(hydroxyethylamine) prepared in unlabeled (H<sub>2</sub><sup>16</sup>O) or labeled H<sub>2</sub>O (H<sub>2</sub><sup>18</sup>O). Unlabeled and stable-isotope-labeled nitrite and nitrate species were measured by gas chromatography–mass spectrometry (GC-MS) after derivatization with pentafluorobenzyl bromide and negative-ion chemical ionization. The study provides strong indication for the formation of O=N-O-N=O as an intermediate of <sup>●</sup>NO autoxidation in pH-neutral aqueous buffers. In high molar excess, HgCl<sub>2</sub> accelerates and increases RSNO hydrolysis to nitrite, thereby incorporating <sup>18</sup>O from H<sub>2</sub><sup>18</sup>O into the SNO group. In aqueous buffers prepared in H<sub>2</sub><sup>18</sup>O, synthetic peroxynitrite (ONOO<sup>−</sup>) decomposes to nitrite without <sup>18</sup>O incorporation, indicating water-independent decomposition of peroxynitrite to nitrite. Use of RS<sup>15</sup>NO and H<sub>2</sub><sup>18</sup>O in combination with GC-MS allows generation of definite results and elucidation of reaction mechanisms of oxidation of <sup>●</sup>NO and hydrolysis of RSNO.
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