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GC-ECNICI-MS analysis of S-nitrosothiols and nitroprusside after treatment with aqueous sulphide (S2−) and derivatization with pentafluorobenzyl bromide: Evidence of S-transnitrosylation and formation of nitrite and nitrate B Analytical technologies in the biomedical and life sciences

Tsikas, Dimitrios, Schmidt, Mario, Hanff, Erik, Böhmer, Anke
Journal of chromatography 2017 v.1043 pp. 209-218
acetone, acetylcysteine, anions, bromides, cysteine, derivatization, gas chromatography-mass spectrometry, glutathione, hemoglobin, ionization, isotope labeling, mercuric chloride, monitoring, nitrates, nitrites, nitroprusside, organobromine compounds, pH, quantitative analysis, sodium sulfide, stable isotopes, thiols
A GC–MS method is reported for the quantitative analysis of S-nitrosothiols (RSNO) derived from endogenous low- and high-molecular mass thiols (RSH) including hemoglobin, cysteine, glutathione, N-acetylcysteine, and the exogenous N-acetylcysteine ethyl ester. The method is based on the conversion of RSNO to nitrite by aqueous Na2S (S²⁻). ¹⁵N-Labelled analogs (RS¹⁵NO) or ¹⁵N-labelled nitrite and nitrate were used as internal standards. The nitrite (¹⁴NO2⁻ and ¹⁵NO2⁻) and nitrate (O¹⁴NO2⁻ and O¹⁵NO2⁻ anions were derivatised by pentafluorobenzyl (PFB) bromide (PFB-Br) in aqueous acetone and their PFB derivatives were separated by gas chromatography. After electron-capture negative-ion chemical ionization, the anions were separated by mass spectrometry and detected by selected-ion monitoring of m/z 46 for ¹⁴NO2⁻, m/z 47 for ¹⁵NO2⁻, m/z 62 for O¹⁴NO2⁻, and m/z 63 for O¹⁵NO2⁻. The expected thionitrites (⁻S¹⁴NO and ⁻S¹⁵NO) were not detected, suggesting that they are intermediates and rapidly exchange their S by O from water, presumably prior to PFB-Br derivatization. The reaction of S²⁻ with RSNO and sodium nitroprusside (SNP) resulted in the formation of nitrite and nitrate as the major and minor reaction products, respectively. The novel Na2S procedure was compared with established procedures based on the use of aqueous HgCl2 or cysteine/Cu²⁺ reagents to convert the S-nitroso group to nitrite. Our results provide evidence for an equilibrium S-transnitrosylation reaction between S²⁻ with RSNO in buffered solutions of neutral pH. Use of Na2S in molar excess over RSNO shifts this reaction to the right, thus allowing almost complete conversion of RSNO to nitrite and nitrate. The Na2S procedure should be useful for the quantitative determination of RSNO as nitrite and nitrate after PFB-Br derivatization and GC–MS analysis. The Na2S procedure may also contribute to explore the complex reactions of S²⁻ with RSNO, SNP and other NO-containing compounds.