Name: integration of miniaturized solid phase extraction and lc-ms/ms detection of 3-nitrotyrosine in human urine for clinical applications
Uploaded: 2017-07-14T12:30:00
Description: Watch this Scientific Journal Video about Integration of Miniaturized Solid Phase Extraction and LC-MS/MS Detection of 3-Nitrotyrosine in Human Urine for Clinical Applications at JoVE.com

作者将承认斯科特·霍华德（Scott Howard）和阿比盖尔·马里纳克（Abigail Marinack

所有涉及人体尿液样本的研究都遵循了Pharmasan / Neuroscience Institutional Review Board（IRB）批准的手术。 尿液样品收集和肌酐（Cr）测定<ol><li>第二天早晨收集5 mL尿样。 10小时过夜禁食在含有250μL3 N HCl作为防腐剂的5 mL输送管A中，储存于-20°C直至使用。 </li><li>解冻并涡旋5 mL运输尿A管和离心机离心机（ 如 Sorvall）（2297 xg，10分钟）。 </li><li>从5 mL运输管A等分1 mL尿液两次</li><...

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在浓度实质变化先前在文献中内源性游离3-NT报导在人尿样品揭示了与可用的测定8，9，10，11相关联的方法问题。准确测定人尿中3-NT的低基础水平仍然是一项具有挑战性的任务，需要对样品制备和LC-MS / MS分析采取特殊预防措施。该协议概述了一种新型SPE程序，结合选择性LC-MS / MS检测，允许以高通量对...

氧化应激的临床表现的影响已被推到前列，近年来1。正在探索的生物标志物之一是3-硝基酪氨酸（3-NT），当活性氮物质（RNS）与酪氨酸，儿茶酚胺神经递质前体相互作用时形成最终稳定的产物。而3-NT可以具有临床价值作为体内 RNS的生物标志物，其属性和酪氨酸的功能的显着变化可能不利地相应的蛋白质和细胞功能1,2影响。新兴的研究表明，3-NT可以在炎症条件下发挥重要作用3，神经退行性疾病4，5，心血管疾病6和7糖尿病，以及与氧化应激的条件。但是，这些都是这样的rvations基于来自缺乏灵敏度和/或选择性8，9，10，11的方法的结果。以前在文献中报道的生物样品的3-NT浓度范围巨大，显示出严重的分析问题与这些检测有关，需要进行技术改进来准确量化3-NT的水平，并验证其在这些病症的病理学中的作用。 免费3-NT生物基质的定量呈现人与仪器8，9，10，11一个特殊的挑战。首先，内源性3-NT的痕量水平要求超敏感检测;第二，存在众多结构相似的类似物，特别是酪氨酸大量过剩，需要高度的选择性;第三，通过酪氨酸硝化与无处不在的硝酸盐和亚硝酸盐的3-NT的人工形成需要在样品制备过程中特别考虑，以避免过度估计3-NT。 中的各种各样的用于测量3-NT方法的，MS / MS一直被认为是金标准方法由于其优越的灵敏度和选择性11，12，13，14。气相色谱（GC），其耦合MS / MS提供最佳的灵敏度，然而，必不可少的样品衍生步骤过于繁琐和耗时是有效的临床效用15,16。 LC-MS / MS不需要复杂的样品衍生化，使其成为更有希望的选择。尽管如此，还有几个要克服的障碍的文献中报道的LC-MS / MS方法nsitivity需要改善的测量低丰度3-NT 7，17，18和相对长的处理时间必须缩短用于高通量应用12，13，17，19 。 另外，在考虑临床应用时，所用的生物矩阵起着重要的作用。它应该是容易且廉价获得和非侵入性的如果可能的话20，21，22。等离子体是文献中传统使用的样品，不是临床上理想的基质，因此寻求利用非侵入性和成本效益的尿液的方法。 几个尝试发展reliable和特定LC-MS / MS方法已经使用尿9，10，11制成。然而，它们的选择性，可靠性或效率都不足以临床使用。已经提出使用传统反相柱（C18型）作为3-NT分析的样品清洗的主要SPE的有效性，并提出了强阳离子交换（SCX）和反相C18-OH的顺序SPE 6 ， 7，19。最近开发的LC-MS / MS方法采用手动C18 SPE，制备型高压液相色谱（HPLC）和在线SPE的多步纯化方法，用于3-NT 23的分析。尽管这种方法对于临床目的来说足够敏感，LLOQ为0.041nM，但是清除过程是密集和繁琐的，红色3 mL尿液，限制了其高通量的可行性。采用分子印迹聚合物作为SPE吸附剂，以提高清洗过程14的效率，但是所得到的LLOQ（0.7μg/ mL）对于临床标本不够低。另一种方法需要二维（2D）LC-MS / MS和免疫亲和层析来进行样品净化，以达到0.022nM 24的检测限（LOD）。虽然所有这些方法在3-NT的评估方面取得了进步，但没有一个已经实现了临床应用所需的灵敏度，可靠性和效率。 为了研究的自由3-NT病理和其在临床环境中的氧化应激的生物标志物的作用，我们已经开发出一种方法，它是简单，高效，准确，精确，可以实现高通量临床应用25。小型化混合模式阳离子实施了hange（MCX）96孔提取微孔板，以实现单次提取中简单有效的3-NT样品清除和富集，绕过了需要衍生化，蒸发和2D-LC的现有方法中看到的缺点。具有0.01％HCOOH作为流动相添加剂的液相色谱提供了快速循环时间的增强的信号响应。通过使用温和的NH 4 OAc洗脱溶液用于3-NT的选择性洗脱以及使用3-NT和内标（IS）的MRM转换，进一步改善了选择性。通过使用减少量的优选13 C-标记的同位素IS进行定量来补偿基质效应。随着这种方法的出现，研究人员和临床医生将能够验证3-NT在临床条件下的作用，并进一步探索氧化应激的影响。

<table border="0" cellpadding="0" cellspacing="0" width="912">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">3-Nitro-L-tyrosine</td>
			<td style="width:161px;">Sigma</td>
			<td style="width:115px;">N7389-5g</td>
			<td style="width:263px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">3-Nitro-L-tyrosine-13C9</td>
			<td>Sigma</td>
			<td>652296-5.0mg</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mass Spec Gold Urine</td>
			<td>Golden West Biologicals</td>
			<td>MSG 5000-1L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Oasis MCX 96-well &#181;Elution plate</td>
			<td style="width:161px;">Waters</td>
			<td>186001830BA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2mL 96 well collection plate</td>
			<td style="width:161px;">Phenomenex&#160;&#160;</td>
			<td style="width:115px;">AH0-7194</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">96 positive processor</td>
			<td style="width:161px;">Waters&#160;</td>
			<td style="width:115px;">186005521</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LC-MS Ultra CHROMASOLV methanol&#160;&#160;</td>
			<td style="width:161px;">Sigma</td>
			<td style="width:115px;">14262-2L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LC-MS Ultra CHROMASOLV water</td>
			<td style="width:161px;">Sigma</td>
			<td style="width:115px;">14263-2L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Formic acid for mass spectrometry</td>
			<td style="width:161px;">Sigma</td>
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integration of miniaturized solid phase extraction and lc-ms/ms detection of 3-nitrotyrosine in human urine for clinical applications

游离的3-硝基酪氨酸（3-NT）已广泛用作氧化应激的可能生物标志物。在各种病理状况下报道了3-NT的水平升高。然而，现有方法缺乏足够的灵敏度和/或特异性，可靠地测量3-NT的低内源水平，对于临床应用来说太麻烦了。因此，迫切需要进行分析改进，以准确量化3-NT的水平，并验证3-NT在病理状况中的作用。该方案介绍了新型液相色谱串联质谱（LC-MS / MS）检测与小型化固相萃取（SPE）的开发，用于快速准确测量人尿中3-NT作为非侵入性生物标志物用于氧化应激。使用96孔板的SPE通过组合样品净化和分析物富集显着简化了工艺，而无需繁琐的衍生化和蒸发步骤，减少溶剂消耗，废物处理，污染风险和整体处理时间。使用pH为9的25mM乙酸铵（NH 4 OAc）作为SPE洗脱溶液，显着提高了选择性。通过调整多重反应监测（MRM）转换，改善了质谱信号的响应。在五氟苯基（PFP）柱（150mm×2.1mm，3μm）上使用0.01％HCOOH作为添加剂将信号响应提高2.5倍，并将总运行时间缩短至7分钟。实现了10 pg / mL（0.044 nM）的定量下限（LLOQ），相对于所报告的测定显示出显着的灵敏度改善。这种简化，快速，选择性和敏感性方法允许在24小时的时间段内处理两个尿液样本（n = 192）。考虑到明显改善的分析性能和非侵入性和便宜的尿液采样，所提出的检测方法有利于临床前和临床学习。

图1说明，在优化的LC条件下，3-NT与其它结构相似的酪氨酸类似物完全色谱分离，这消除了由于这些极度过量的化合物引起的共洗脱干扰，从而提高了测定选择性的程度。此外，以0.01％HCOOH作为添加剂在MA和甲醇中的流动速率为0.45 mL / min的梯度洗脱可以快速洗脱3-NT（ 即 3分钟，周转时间为7分钟）。 <p class="jove_content" fo:keep-together.within-page="1...

Watch this Scientific Journal Video about Integration of Miniaturized Solid Phase Extraction and LC-MS/MS Detection of 3-Nitrotyrosine in Human Urine for Clinical Applications at JoVE.com

Integration of Miniaturized Solid Phase Extraction and LC-MS/MS Detection of 3-Nitrotyrosine in Human Urine for Clinical Applications

开发了选择性和灵敏的液相色谱串联质谱（LC-MS / MS）方法与混合模式阳离子交换（MCX）96孔微量培养板上的有效固相萃取相结合，用于测量游离的3-硝基酪氨酸（ 3-NT），具有高通量，适用于临床应用。

微型固相萃取和LC-MS / MS检测人尿中3-硝基酪氨酸的临床应用

Free 3-nitrotyrosine (3-NT) has been extensively used as a possible biomarker for oxidative stress. Increased levels of 3-NT have been reported in a wide ...

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Preparation of Hydrophobic Metal-Organic Frameworks via Plasma Enhanced Chemical Vapor Deposition of Perfluoroalkanes for the Removal of Ammonia

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化学

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用于固相合成含有2&#39;-<em&gt; 0</em通过圆二色性修饰和表征

Solid-phase synthesis has been used to obtain canonical and modified polymers of nucleic acids, specifically of DNA or RNA, which has made it a popular ...

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The extraction and analysis of catecholamine neurotransmitters in biological fluids is of great importance in assessing nervous system function and ...

Surface Properties of Synthesized Nanoporous Carbon and Silica Matrices

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In this work, we report the synthesis and characterization of ordered nanoporous carbon material (also called ordered mesoporous carbon material [OMC]) ...

Solid-phase Synthesis of [4.4] Spirocyclic Oximes

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A convenient synthetic route for spirocyclic heterocycles is well sought after due to the molecule&#39;s potential use in biological systems. By means of ...

Development and Validation of Chromium Getters for Solid Oxide Fuel Cell Power Systems

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Degradation of cathode in solid oxide fuel cells (SOFC) remains a major concern for the long-term performance stability and operational reliability. The ...