Tsikas Dimitrios, Dehnert Sabine, Urban Karin, Surdacki Andrzej, Meyer Hartmut H
Institute of Clinical Pharmacology, Hannover Medical School, Hannover, Germany.
J Chromatogr B Analyt Technol Biomed Life Sci. 2009 Oct 15;877(28):3442-55. doi: 10.1016/j.jchromb.2009.06.032. Epub 2009 Jun 30.
S-Nitrosothiols from low-molecular-mass and high-molecular-mass thiols, including glutathione, albumin and hemoglobin, are endogenous potent vasodilators and inhibitors of platelet aggregation. By utilizing the S-transnitrosation reaction and by using the lipophilic (pK(L) 0.78) and strong nucleophilic synthetic thiol N-acetyl cysteine ethyl ester (NACET) we have developed a GC-MS method for the analysis of S-nitrosothiols and their (15)N- or (2)H-(15)N-labelled analogs as S-nitroso-N-acetyl cysteine ethyl ester (SNACET) and S(15)NACET or d(3)-S(15)NACET derivatives, respectively, after their extraction with ethyl acetate. Injection of ethyl acetate solutions of S-nitrosothiols produced two main reaction products, compound X and compound Y, within the injector in dependence on its temperature. Quantification was performed by selected-ion monitoring of m/z 46 (i.e., NO(2)) for SNACET and m/z 47 (i.e., (15)NO(2)) for S(15)NACET/d(3)-S(15)NACET for compound X, and m/z 157 for SNACET and m/z 160 for d(3)-S(15)NACET for compound Y. In this article we describe the development, validation and in vitro and in vivo applications of the method to aqueous buffered solutions, human and rabbit plasma. Given the ester functionality of SNACET/S(15)NACET/d(3)-S(15)NACET, stability studies were performed using metal chelators and esterase inhibitors. The method was found to be suitable for the quantitative determination of various S-nitrosothiols including SNACET externally added to human plasma (0-10microM). Nitrite contamination in ethyl acetate was found to interfere. Our results suggest that the concentration of endogenous S-nitrosothiols in human plasma does not exceed about 200nM in total. Oral administration of S(15)NACET to rabbits (40-63micromol/kg body weight) resulted in formation of ALB-S(15)NO, [(15)N]nitrite and [(15)N]nitrate in plasma.
来自低分子量和高分子量硫醇（包括谷胱甘肽、白蛋白和血红蛋白）的S-亚硝基硫醇是内源性强效血管舒张剂和血小板聚集抑制剂。通过利用S-转亚硝化反应，并使用亲脂性（pK(L) 0.78）且强亲核性的合成硫醇N-乙酰半胱氨酸乙酯（NACET），我们开发了一种气相色谱-质谱联用（GC-MS）方法，用于分析S-亚硝基硫醇及其（15）N或（2）H-（15）N标记的类似物，分别为S-亚硝基-N-乙酰半胱氨酸乙酯（SNACET）和S（15）NACET或d（3）-S（15）NACET衍生物，在它们用乙酸乙酯萃取之后进行分析。注入S-亚硝基硫醇的乙酸乙酯溶液后，根据进样器温度，在进样器内产生了两种主要反应产物，化合物X和化合物Y。通过对化合物X的SNACET的m/z 46（即[NO(2)]-）和S（15）NACET/d（3）-S（15）NACET的m/z 47（即[(15)NO(2)]-）进行选择离子监测，以及对化合物Y的SNACET的m/z 157和d（3）-S（15）NACET的m/z 160进行选择离子监测来进行定量分析。在本文中，我们描述了该方法在水缓冲溶液、人血浆和兔血浆中的开发、验证以及体外和体内应用。鉴于SNACET/S（15）NACET/d（3）-S（15）NACET的酯官能团，使用金属螯合剂和酯酶抑制剂进行了稳定性研究。发现该方法适用于定量测定各种S-亚硝基硫醇，包括外部添加到人血浆（0 - 10微摩尔）中的SNACET。发现乙酸乙酯中的亚硝酸盐污染会产生干扰。我们的结果表明，人血浆中内源性S-亚硝基硫醇的总浓度不超过约200纳摩尔。给兔子口服S（15）NACET（40 - 63微摩尔/千克体重）导致血浆中形成ALB-S（15）NO、[(15)N]亚硝酸盐和[(15)N]硝酸盐。
