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

Abstract

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 (S^2-). 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 S^2- 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 S^2- 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 S^2- with RSNO, SNP and other NO-containing compounds.

Keywords: Derivatization; Nitric oxide; Polysulfides; S-Nitrosothiols; Sulfide; Thiols.