Chapter title |
Measurement of S -Nitrosoglutathione in Plasma by Liquid Chromatography–Tandem Mass Spectrometry
|
---|---|
Chapter number | 10 |
Book title |
Nitric Oxide
|
Published in |
Methods in molecular biology, January 2018
|
DOI | 10.1007/978-1-4939-7695-9_10 |
Pubmed ID | |
Book ISBNs |
978-1-4939-7694-2, 978-1-4939-7695-9
|
Authors |
Dimitrios Tsikas, Erik Hanff, Tsikas, Dimitrios, Hanff, Erik |
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. |
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