Nitric oxide (˙NO) and superoxide (O2−˙) are ubiquitous in nature. Their reaction product peroxynitrite (ONOO−) and notably its conjugated peroxynitrous acid (ONOOH) are highly unstable in aqueous phase. ONOO−/ONOOH (referred to as peroxynitrite) isomerize and decompose to NO3−, NO2− and O2. Here, we report for the first time GC-MS and HPLC methods for the analysis of peroxynitrite in aqueous solution. For GC-MS analysis peroxynitrite in alkaline solution was derivatized to a pentafluorobenzyl derivative using pentafluorobenzyl bromide. O15NOO− was synthesized from H2O2 and 15NO2− and used as internal standard. HPLC analysis was performed on stationary phases consisting of Nucleosil® 100–5C18AB or Nucleodur® C18 Gravity. The mobile phase consisted of a 10 mM aqueous solution of tetrabutylammonium hydrogen sulfate and had a pH value of 11.5. UV absorbance detection at 300 nm was used. HPLC allows simultaneous analysis of ONOO−, NO2− and NO3−. The GC-MS and HPLC methods were used to study stability, synthesis, formation from S-[15N]nitrosoglutathione (GS15NO) and KO2, and isomerization/decomposition of peroxynitrite to NO2− and NO3− in aqueous buffer.
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