Measurement of S -Nitrosoglutathione in Plasma by Liquid Chromatography–Tandem Mass Spectrometry
This chapter describes an ultraperformance liquid chromatographic–tandem mass spectrometric (UPLC-MS/MS) method for the quantitative determination of S-nitrosoglutathione (GSNO) in human plasma. S-[15N]Nitrosoglutathione (GS15NO) serves as the internal standard. The protocol involves inactivation of plasma γ-glutamyltransferase activity by serine-borate, stabilization of GSNO with EDTA, and avoidance of S-transnitrosylation reactions by blocking SH groups with N-ethylmaleimidide (NEM). Fresh blood is treated with NEM/serine-borate/EDTA, plasma is spiked with GS15NO (50 nM), ultrafiltered (cutoff 10 kDa) and 10-μL aliquots of ultrafiltrate are analyzed by UPLC-MS/MS in the positive electrospray ionization (ESI+) mode. LC is performed on a Nucleoshell column using isocratic (0.5 mL/min) elution with acetonitrile-20 mM ammonium formate (70:30, v/v), pH 7. Quantification is performed by selected-reaction monitoring the mass transition m/z 337 ([M+H]+) → m/z 307 ([M+H−14NO]+●) for GSNO and m/z 338 ([M+H]+) → m/z 307 ([M+H−15NO]+●) for GS15NO. Matrix effects are outweighed by the internal standard GS15NO. The lower limit of quantitation (LOQ) is 2.8 nM.
Key wordsCarbonic anhydrase Electrospray ionization GSNO LC-MS/MS Nitric oxide Nitrite Plasma Stable isotopes
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