Institute of Clinical Pharmacology, Hannover Medical School, Carl-Neuberg-Strasse 1, D-30625 Hannover, Germany.
J Chromatogr B Analyt Technol Biomed Life Sci. 2013 May 15;927:147-57. doi: 10.1016/j.jchromb.2013.01.023. Epub 2013 Feb 4.
We developed and validated a fast UPLC-MS/MS method with positive electrospray ionization (ESI+) for the quantitative determination of S-nitrosoglutathione (GSNO) in human plasma. We used a published protocol for the inactivation of plasma γ-glutamyltransferase (γGT) activity by using the γGT transition inhibitor serine/borate and the chelator EDTA for the stabilization of GSNO, and N-ethylmaleimide (NEM) to block SH groups and to avoid S-transnitrosylation reactions which may diminish GSNO concentration. S-[(15)N]Nitrosoglutathione (GS(15)NO) served as internal standard. Fresh blood was treated with NEM/serine/borate/EDTA, plasma spiked with GS(15)NO (50nM) was ultrafiltered (cut-off 10kDa) and 10μL aliquots of the ultrafiltrate were analyzed by UPLC-MS/MS. Five HILIC columns and an Acquity UPLC BH amide column were tested. The mobile phase was acetonitrile-water (70:30, v/v), contained 20mM ammonium formate, had a pH value of 7, and was pumped isocratically (0.5mL/min). The Nucleoshell column allowed better LC performance and higher MS sensitivity. The retention time of GSNO was about 1.1min. Quantification was performed by selected-reaction monitoring the mass transition m/z 337 (M+H)→m/z 307 (M+H(14)NO) for GSNO (i.e., GS(14)NO) and m/z 338 (M+H)→m/z 307 (M+H(15)NO) for GS(15)NO. NEM/serine/borate/EDTA was found to stabilize GSNO in human plasma. The method was validated in human plasma (range, 0-300nM) using 50nM GS(15)NO. Accuracy and precision were in generally acceptable ranges. A considerable matrix effect was observed, which was however outweighed by the internal standard GS(15)NO. In freshly prepared plasma from heparinized blood donated by 10 healthy subjects, no endogenous GSNO was determined above 2.8nM, the limit of quantitation (LOQ) of the method. This study challenges previously reported GSNO plasma concentrations being far above the present method LOQ value and predicts that the concentration of low-molecular-mass and high-molecular-mass S-nitrosothiols are in the upper pM- and lower nM-range, respectively.
我们开发并验证了一种快速的 UPLC-MS/MS 方法，采用正电喷雾电离（ESI+）定量测定人血浆中的 S-亚硝基谷胱甘肽（GSNO）。我们使用已发表的方案，通过使用 γGT 过渡抑制剂丝氨酸/硼酸和螯合剂 EDTA 来使血浆 γ-谷氨酰转移酶（γGT）失活，并用 N-乙基马来酰亚胺（NEM）来封闭 SH 基团并避免 S-转亚硝化为可能会降低 GSNO 浓度的反应。S-[(15)N]亚硝基谷胱甘肽（GS(15)NO）用作内标。新鲜血液用 NEM/丝氨酸/硼酸/EDTA 处理，用 GS(15)NO（50nM）对血浆进行超滤液过滤（截止值 10kDa），用 UPLC-MS/MS 分析 10μL 等分试样。测试了五个亲水作用色谱柱和 Acquity UPLC BH 酰胺柱。流动相为乙腈-水（70:30，v/v），含有 20mM 甲酸铵，pH 值为 7，并以等度（0.5mL/min）泵送。Nucleoshell 柱允许更好的 LC 性能和更高的 MS 灵敏度。GSNO 的保留时间约为 1.1min。通过对 GSNO（即 GS(14)NO）的质量转移 m/z 337(M+H)→m/z 307(M+H(14)NO）和 m/z 338(M+H)→m/z 307(M+H(15)NO）进行选择反应监测来进行定量分析。发现 NEM/丝氨酸/硼酸/EDTA 可稳定人血浆中的 GSNO。使用 50nM GS(15)NO 在人血浆（范围 0-300nM）中验证了该方法。准确度和精密度通常在可接受范围内。观察到相当大的基质效应，但被内标 GS(15)NO 所抵消。在 10 名健康受试者捐赠的肝素化血液的新鲜制备血浆中，方法的定量限（LOQ）未超过 2.8nM，无法测定内源性 GSNO。本研究对先前报道的 GSNO 血浆浓度远高于本方法 LOQ 值提出了挑战，并预测低分子量和高分子量 S-亚硝硫醇的浓度分别在 pM 级和 nM 级以上。
