Tsikas Dimitrios, Hanff Erik
Institute of Toxicology, Core Unit Proteomics, Hannover Medical School, Hannover, Germany.
Methods Mol Biol. 2018;1747:113-129. doi: 10.1007/978-1-4939-7695-9_10.
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-[N]Nitrosoglutathione (GSNO) 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 GSNO (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-NO]) for GSNO and m/z 338 ([M+H]) → m/z 307 ([M+H-NO]) for GSNO. Matrix effects are outweighed by the internal standard GSNO. The lower limit of quantitation (LOQ) is 2.8 nM.
本章介绍了一种用于定量测定人血浆中S-亚硝基谷胱甘肽（GSNO）的超高效液相色谱-串联质谱（UPLC-MS/MS）方法。S-[N]亚硝基谷胱甘肽（GSNO）用作内标。该方案包括用丝氨酸-硼酸盐使血浆γ-谷氨酰转移酶失活，用EDTA稳定GSNO，并通过用N-乙基马来酰亚胺（NEM）封闭SH基团来避免S-转亚硝基化反应。新鲜血液用NEM/丝氨酸-硼酸盐/EDTA处理，向血浆中加入GSNO（50 nM），超滤（截留分子量10 kDa），取10 μL超滤等分试样通过UPLC-MS/MS在正电喷雾电离（ESI+）模式下进行分析。液相色谱在Nucleoshell柱上进行，使用乙腈-20 mM甲酸铵（70:30，v/v）等度洗脱（0.5 mL/min），pH 7。通过选择反应监测GSNO的质量跃迁m/z 337（[M+H]）→m/z 307（[M+H-NO]）和GSNO的质量跃迁m/z 338（[M+H]）→m/z 307（[M+H-NO]）进行定量。内标GSNO抵消了基质效应。定量下限（LOQ）为2.8 nM。
