GC-MS and HPLC methods for peroxynitrite (ONOO- and O15NOO-) analysis: a study on stability, decomposition to nitrite and nitrate, laboratory synthesis, and formation of peroxynitrite from S-nitrosoglutathione (GSNO) and KO2

Nitric oxide ((NO)-N-center dot) and superoxide (O-2(-center dot)) 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 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. (ONOO-)-N-15 was synthesized from H2O2 and (NO2-)-N-15 and used as internal standard. HPLC analysis was performed on stationary phases consisting of Nucleosil (R) 100-5C(18)AB or Nucleodur (R) 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-, NO2- and NO3-. The GC-MS and HPLC methods were used to study stability, synthesis, formation from S-[N-15] nitrosoglutathione (GS 15 NO) and KO2, and isomerization/decomposition of peroxynitrite to NO2- and NO3- in aqueous buffer.