GC-MS and HPLC methods for peroxynitrite (ONOO- and O ~(15)NOO-) analysis: A study on stability, decomposition to nitrite and nitrate, laboratory synthesis, and formation of peroxynitrite from S-nitrosoglutathione (GSNO) and KO_2

摘要

Nitric oxide (NO) and superoxide (O_2~-) 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 NO_3~-, NO_2~- and O_2. 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. O~(15)NOO- was synthesized from H_2O_2 and ~(15)NO_2~- and used as internal standard. HPLC analysis was performed on stationary phases consisting of Nucleosil? 100-5C_(18)AB or Nucleodur? C _(18) 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 ~-, NO_2~- and NO_3~-. The GC-MS and HPLC methods were used to study stability, synthesis, formation from S-[~(15)N]nitrosoglutathione (GS~(15)NO) and KO_2, and isomerization/decomposition of peroxynitrite to NO_2~- and NO_3~- in aqueous buffer.