Nitric Oxide pp 113-129 | Cite as

Measurement of S -Nitrosoglutathione in Plasma by Liquid Chromatography–Tandem Mass Spectrometry

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1747)

Abstract

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 words

Carbonic anhydrase Electrospray ionization GSNO LC-MS/MS Nitric oxide Nitrite Plasma Stable isotopes 

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Institute of Toxicology, Core Unit ProteomicsHannover Medical SchoolHannoverGermany

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