Tsikas D, Denker K, Frölich J C
Institute of Clinical Pharmacology, Hannover Medical School, Germany.
J Chromatogr A. 2001 Apr 27;915(1-2):107-16. doi: 10.1016/s0021-9673(01)00634-3.
The endogenous potent vasodilators and inhibitors of platelet aggregation S-nitrosoglutathione (GSNO) and S-nitroglutathione (GSNO2) are frequently analyzed by high-performance liquid chromatography (HPLC) using mobile phases of acidic pH. These systems are associated with problems stemming from rapid and considerable artifactual formation of GSNO from glutathione (GSH) and ubiquitous nitrite. We describe a novel ion-pairing HPLC method with UV absorbance detection at 334 nm for the highly specific and interference-free analysis of GSNO and GSNO2 in the presence of high GSH and nitrite concentrations. Complete avoidance of artifactual formation of GSNO was accomplished by using the anion-pairing agent tetrabutylammoniumhydrogen sulphate in the mobile phase that enables analysis of GSNO at neutral pH, at which GSH and nitrite do not react to form GSNO. This HPLC system was used to study formation of GSNO2 from GSH and peroxynitrite under physiological conditions. We found by this HPLC system that peroxynitrite (0-300 microM) reacts with GSH (0-5 mM) to form GSNO2 at a mean yield of 2%. Analysis of the same samples by a cation-pairing HPLC system with acidic mobile phase (pH 2.0) revealed, however, GSNO plus GSNO2 formation of the order of 20% due to on column reaction of GSH with peroxynitrite-derived nitrite to form GSNO. Ammonium sulfamate is frequently used to remove nitrite from thiol-containing solutions under acidic conditions. By means of the anion-pairing HPLC system it is demonstrated that nitrite removal by this method is incomplete even when ammonium sulfamate is used at high concentrations. These findings underscore the absolute requirement of neutral pH conditions for the analysis of GSNO. The novel anion-pairing HPLC method should be useful to provide reliable data on formation, reaction and metabolism of GSNO and GSNO2 in biological fluids using various detectors including mass spectrometers.
内源性强效血管舒张剂及血小板聚集抑制剂S-亚硝基谷胱甘肽（GSNO）和S-硝基谷胱甘肽（GSNO2）常通过高效液相色谱法（HPLC）进行分析，使用酸性pH值的流动相。这些系统存在一些问题，源于谷胱甘肽（GSH）和普遍存在的亚硝酸盐会快速且大量地人为生成GSNO。我们描述了一种新型离子对HPLC方法，在334 nm处进行紫外吸收检测，用于在高GSH和亚硝酸盐浓度存在的情况下对GSNO和GSNO2进行高特异性且无干扰的分析。通过在流动相中使用阴离子对试剂硫酸氢四丁铵，实现了完全避免GSNO的人为生成，这使得能够在中性pH条件下分析GSNO，此时GSH和亚硝酸盐不会反应生成GSNO。该HPLC系统用于研究在生理条件下GSH和过氧亚硝酸根生成GSNO2的情况。通过该HPLC系统我们发现，过氧亚硝酸根（0 - 300 microM）与GSH（0 - 5 mM）反应生成GSNO2，平均产率为2%。然而，使用酸性流动相（pH 2.0）的阳离子对HPLC系统分析相同样品时，由于GSH与过氧亚硝酸根衍生的亚硝酸盐在柱上反应生成GSNO，显示出GSNO加GSNO2的生成量约为20%。氨基磺酸铵常用于在酸性条件下从含硫醇的溶液中去除亚硝酸盐。通过阴离子对HPLC系统证明，即使使用高浓度的氨基磺酸铵，该方法去除亚硝酸盐也不完全。这些发现强调了分析GSNO时对中性pH条件的绝对要求。这种新型阴离子对HPLC方法应有助于使用包括质谱仪在内的各种检测器，提供有关生物流体中GSNO和GSNO2的生成、反应和代谢的可靠数据。
