Department of Chemistry, University of British Columbia, Vancouver, BC, Canada.
Electrophoresis. 2012 Feb;33(4):697-704. doi: 10.1002/elps.201100368.
We have developed a simple, sensitive, and robust liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) method to determine free 3-nitrotyrosine concentrations in human plasma of patients receiving on-pump coronary artery bypass grafting surgery. A one-step solid-phase extraction protocol was optimized to enrich the analyte at low nanomolar concentrations. The processed samples were analyzed by LC-MS/MS with a 2.1 × 100 mm Kinetex PFP column and a triple quadrupole mass spectrometer. The method was validated for 3-nitrotyrosine concentrations close to real patient plasma levels. The relative standard deviations or relative errors of the intraday and interday determinations were all within 10%. Limit of detection and limit of quantitation were determined to be 0.034 nM and 0.112 nM, respectively, while lower limit of quantitation was below 0.625 nM. No deterioration of the column performance was noticed after running a large number of patient samples. The results showed that the 3-nitrotyrosine concentrations in coronary sinus plasma samples were elevated after cardiopulmonary bypass (CPB) procedure. The pre-CPB and post-CPB concentrations of 3-nitrotyrosine in patient plasmas were 1.494 ± 0.107 nM and 2.167 ± 0.177 nM (mean ± SEM), respectively. Application of this method to more patients in clinical studies may help validate 3-nitrotyrosine as a meaningful biomarker for nitrosative stress and link patient characteristics, clinical outcomes, and cardioprotective treatments to endogenous nitrosative stress levels.
我们开发了一种简单、灵敏、稳健的液相色谱-质谱/质谱（LC-MS/MS）方法，用于测定接受体外循环冠状动脉旁路移植术患者血浆中游离 3-硝基酪氨酸的浓度。优化了一步固相萃取方案，以在纳摩尔低浓度下富集分析物。处理后的样品通过 LC-MS/MS 进行分析，使用 2.1×100mm Kinetex PFP 柱和三重四极杆质谱仪。该方法针对接近真实患者血浆水平的 3-硝基酪氨酸浓度进行了验证。日内和日间测定的相对标准偏差或相对误差均在 10%以内。检测限和定量限分别确定为 0.034 nM 和 0.112 nM，而定量下限低于 0.625 nM。运行大量患者样本后，未发现柱性能恶化。结果表明，心肺旁路（CPB）手术后冠状窦血浆样本中 3-硝基酪氨酸的浓度升高。患者血浆中 CPB 前和 CPB 后的 3-硝基酪氨酸浓度分别为 1.494±0.107 nM 和 2.167±0.177 nM（平均值±SEM）。在更多的患者中应用该方法进行临床研究，可能有助于验证 3-硝基酪氨酸作为硝化应激的有意义的生物标志物，并将患者特征、临床结局和心脏保护治疗与内源性硝化应激水平联系起来。
