GC-ECNICI-MS analysis of S-nitrosothiols and nitroprusside after treatment with aqueous sulphide (S(2-)) and derivatization with pentafluorobenzyl bromide: Evidence of S-transnitrosylation and formation of nitrite and nitrate.

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-)). (15)N-Labelled analogs (RS(15)NO) or (15)N-labelled nitrite and nitrate were used as internal standards. The nitrite ((14)NO2(-) and (15)NO2(-)) and nitrate (O(14)NO2(-) and O(15)NO2(-) 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 (14)NO2(-), m/z 47 for (15)NO2(-), m/z 62 for O(14)NO2(-), and m/z 63 for O(15)NO2(-). The expected thionitrites ((-)S(14)NO and (-)S(15)NO) 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/Cu(2+) 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.