Speciation and determination of thiols in biological samples using high performance liquid chromatography-inductively coupled plasma-mass spectrometry and high performance liquid chromatography-Orbitrap MS
Speciation and determination of thiols in biological samples using high performance liquid chromatography-inductively coupled plasma-mass spectrometry and high performance liquid chromatography-Orbitrap MS
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DOI：
10.1016/j.aca.2010.09.023
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发表时间：
2010-11-08
影响因子：
6.2
通讯作者：
Mester, Zoltan
中科院分区：
文献类型：
--
作者：
Bakirdere, Sezgin;Bramanti, Emilia;Mester, Zoltan
开发了一种测定生物样品中硫醇的分析方法。采用反相色谱法与 ICP 四极杆 MS 或 Orbitrap MS 联用来分离和检测硫醇。为了测定总硫醇，使用二硫苏糖醇 (DTT) 还原氧化硫醇。发现感兴趣物种的减少效率接近 100%。还原的硫醇通过对羟基汞苯甲酸酯 (PHMB) 衍生化，然后在 C8 柱上分离。对 HPLC-ICP-MS 和 HPLC-Orbitrap MS 方法的提取、分离和检测步骤进行了优化。使用 HPLC-Orbitrap MS 发现半胱氨酸、同型半胱氨酸、硒代半胱氨酸、谷胱甘肽、硒代蛋氨酸和半胱氨酰甘氨酸的检测限分别为 18、34、39、12、128 和 103 fmol，半胱氨酸的检测限分别为 730、1110、440、1110 和 580 fmol。使用 HPLC-ICP-MS 分析同型半胱氨酸、硒代半胱氨酸、谷胱甘肽和半胱氨酰甘氨酸。与预期相反，HPLC-ICP-MS 仪器的 LOD 和 RSD 较高，因此 HPLC-Orbitrap MS 用于测定酵母样品中的硫醇。对三种不同品牌的面包酵母和硒化酵母进行了分析。这些样品中GSH和半胱氨酸的水平分别为4.45至17.87 mu mol g(-1)和0.61至1.32 mu mol g(-1)。 (C) 2010 Elsevier B.V. 保留所有权利。
An analytical method was developed for the determination of thiols in biological samples. Reverse phase chromatography coupled to ICP quaduropole MS or Orbitrap MS was employed for the separation and detection of thiols. For the determination of total thiols, oxidized thiols were reduced using dithiothreitol (DTT). Reduction efficiencies for species of interest were found to be close to 100%. Reduced thiols were derivatized by p-hydroxymercuribenzoate (PHMB) and then separated on a C8 column. Optimization of the extraction, separation and detection steps of the HPLC-ICP-MS and HPLC-Orbitrap MS methods was carried out. Detection limits for cysteine, homocysteine, selenocysteine, glutathione, selenomethionine and cysteinyl-glycine were found to be 18,34,39,12,128 and 103 fmol, respectively, using HPLC-Orbitrap MS and 730, 1110, 440, 1110 and 580 fmol for cysteine, homocysteine, selenocysteine, glutathione, and cysteinyl-glycine using HPLC-ICP-MS. Contrary to expectation, the LODs and RSDs are higher for the HPLC-ICP-MS instrument, therefore HPLC-Orbitrap MS was used for the determination of thiols in yeast samples. Three different brands of baker's yeast and a selenized yeast were analyzed. The GSH and cysteine levels found in these samples ranged from 4.45 to 17.87 mu mol g(-1) and 0.61 to 1.32 mu mol g(-1), respectively. (C) 2010 Elsevier B.V. All rights reserved.