title: 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.
abstract: 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(2)O(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.
authors.affiliation: Institute of Clinical Pharmacology, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany. tsikas.dimitros@mh-hannover.de
authors.forename: Dimitrios
authors.initials: D
authors.lastname: Tsikas
mesh_terms: D002021:Buffers; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005464:Fluorobenzenes / Q000737:chemistry; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D006861:Hydrogen Peroxide / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D009566:Nitrates / Q000737:chemistry*; D009573:Nitrites / Q000737:chemistry*; D030421:Peroxynitrous Acid / Q000032:analysis* / Q000138:chemical synthesis / Q000737:chemistry; D026422:S-Nitrosoglutathione / Q000737:chemistry*; D012996:Solutions / Q000737:chemistry; D013481:Superoxides / Q000737:chemistry*; D013997:Time Factors; D014867:Water / Q000737:chemistry; D005466:Fluorocarbons
chemical_list: D002021:Buffers; D005464:Fluorobenzenes; D006861:Hydrogen Peroxide; D009566:Nitrates; D009573:Nitrites; D030421:Peroxynitrous Acid; D026422:S-Nitrosoglutathione; D012996:Solutions; D013481:Superoxides; D014867:Water; C039316:potassium superoxide; C025063:pentafluorobenzyl bromide; D005466:Fluorocarbons