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><li>通过尿肌酐法测定Cr 26 。 </li></ol> 2.准备标准，IS和质量控制（QC）样品<ol><li>在含有0.01％HCOOH流动相A（MA）的水中以100μg/ mL制备3-NT的储备溶液，并储存在-20℃。 </li><li>使通过用MA稀释100μg/ mL 3-NT储备溶液，浓度为100ng / mL的3-NT工作标准溶液。 </li><li>通过用不含3-NT的0.15M HCl酸化空白尿以及空白和双倍空白样品稀释100ng / mL工作标准，产生5至2500pg / mL的标准品。 </li><li>用MA在水中以100μg/ mL制备IS 13 C 9 -3-NT的储备溶液，并储存在-20℃。 </li><li>通过用MA稀释100μg/ mL 13 C 9 -3-NT IS储备溶液，使其成为500 pg / mL的IS工作溶液。 </li><li>通过在酸化的空白尿中稀释3-NT工作标准，建立覆盖LLOQ，低，中和高水平（ 即 10,25,100,5500和1,250pg / mL）的五个等级的QC样品。 </li></ol>固相萃取法<ol><li>解冻和涡流尿样，标准品和QC样品。 </li><li>添加尿样，标准品和QC（每个250μL）至清洁的2mL 96孔收集板的32个孔中。 </li><li>将500pg / mL IS工作溶液（50μL）引入每个孔，除了双空白样品孔。向双层样品孔中加入0.01％HCOOH（50μL）。 </li><li>加入0.1％HCOOH（250μL）的LC-MS / MS水。 </li><li>将上述混合物与8通道移液管混合三次。 </li><li>盖板，直到SPE加载。 </li><li>将MCX 96孔提取板和收集容器放在正压处理器上。 </li><li>通过吸附剂使流动的MeOH（200μL）对提取板进行调节。 </li><li>用2％HCOOH（200μL）通过吸附剂进行平衡。 </li><li>将每个预混合样品的整个体积用8通道移液管小心地装入预处理提取板上。 </li><li>在正压处理器上设置低正压（ 例如 3 psi），以使混合物流过缓慢吸收剂，如果需要，调整压力。 </li><li>用2％HCOOH（200μL）将水冲洗通过吸附剂清洗孔。 </li><li>通过使甲醇（200μL）通过吸附剂清洗孔。 </li><li>用水（200μL）通过吸附剂清洗孔。 </li><li>在正压处理器上以高正压设置（ 例如 40 psi）完全干燥油井。 </li><li>用干净的2 mL 96孔收集板更换储存器。 </li><li>应用pH 9（25μL）的25 mM NH 4 OAc，从萃取板中洗脱保留的分析物和IS。 </li><li>移取5％HCOOH（50μL）的LC-MS水中和洗脱液。 </li><li>与8通道移液管混合三次，并提交给LC-MS / MS站进行分析。 </li></ol> LC-MS / MS分析<ol><li>用量筒精确测量2,000 mL LC-MS水，并将其转移到2升瓶中。 </li><li> Pipette将200μL纯HCOOH装入含有LC-MS水的上述瓶中。 </li><li>充分混合并标记为流动相A（MA）。包括缩写，准备日期和到期日。 </li><li>取2升瓶的LC-MS甲醇，标记为流动相B（MB），开始日期和有效期。 </li><li>将自动进样器的温度设置为4°C。 </li><li>将带有准备样品的收集板放入自动进样器。 </li><li>将PFP柱（150 mm x 2.1 mm id，3μm）和保护柱放在烤箱中。 </li><li>将烤箱温度设置为30°C。 </li><li>使用采集方法用LC梯度洗脱平衡10分钟，如表1所示。 </li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 时间（分钟） </td><td> 模 </td><td> 活动 </td><td> 参数 </td></tr><tr><td> 0 </td><td>水泵</td><td>泵B浓度</td><td>五</td></tr><tr><td> 0.5 </td><td>水泵</td><td>泵B浓度</td><td> 20 </td></tr><tr><td> 1 </td><td>水泵</td><td>泵B浓度</td><td> 50 </td></tr><tr><td> 3 </td><td>水泵</td><td>泵B浓度</td><td> 80 </td></tr><tr><td> 4 </td><td>水泵</td><td>泵B浓度</td><td> 90 </td></tr><tr><td> 4.01 </td><td>水泵</td><td>泵B浓度</td><td> 95 </td></tr><tr><td> 5.5 </td><td>水泵</td><td>泵B浓度</td><td> 95 </td></tr><tr><td> 5.6 </td><td>水泵</td><td>泵B浓度</td><td>五</td></tr><tr><td> 7 </td><td>调节器</td><td>停止</td><td></td></tr></tbody></table> 表1: 液相色谱梯度洗脱条件 <ol start="10"><li>创建一个批处理列表，其中包括标准品，QC和尿样。 </li><li>通过注射准备的样品（12μL）开始批次。 </li></ol>峰值识别，集成和数据处理<ol><li>使用该软件控制数据采集和处理。 </li><li>识别并整合所有样品的3-NT和IS峰。 </li><li>通过3-NT和IS与标称3-NT浓度的峰面积比以1 / x的线性回归线性回归，建立10-2,500pg / mL范围的3-NT定量的标准曲线。 </li><li>使用标准曲线量化所有样品。 </li><li>确定QC样品是否落在确定的范围内。 </li><li>将检测到的尿样浓度以nM或nmol / mmol Cr为单位转换为最终结果。 </li></ol>

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作者宣称他们没有利益冲突。

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。

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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

微型固相萃取与 LC-MS/MS 检测人尿液中 3-硝基酪氨酸的集成在临床应用中的应用化学|视频

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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9.2K 次观看.  Pharmasan Labs, Inc.. 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。 

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

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

Plasma enhanced chemical vapor deposition (PECVD) of perfluoroalkanes has long been studied for tuning the wetting properties of surfaces. For high surface area microporous materials, such as metal-organic frameworks (MOFs), unique challenges present themselves for PECVD treatments. Herein the protocol for development of a MOF that was previously unstable to humid conditions is presented. The protocol describes the synthesis of Cu-BTC (also known as HKUST-1), the treatment of Cu-BTC with PECVD of perfluoroalkanes, the aging of materials under humid conditions, and the subsequent ammonia microbreakthrough experiments on milligram quantities of microporous materials. Cu-BTC has an extremely high surface area (~1,800 m2/g) when compared to most materials or surfaces that have been previously treated by PECVD methods. Parameters such as chamber pressure and treatment time are extremely important to ensure the perfluoroalkane plasma penetrates to and reacts with the inner MOF surfaces. Furthermore, the protocol for ammonia microbreakthrough experiments set forth here can be utilized for a variety of test gases and microporous materials.

Herein the procedures for plasma enhanced chemical vapor deposition of perfluoroalkanes on microporous materials such as metal-organic frameworks to enhance their stability and hydrophobicity are described. Furthermore, breakthrough testing of milligram quantities of samples is described in detail.

增强型氟烷烃的化学气相沉积等离子通过疏水金属 - 有机骨架的制备氨的去除

Plasma enhanced chemical vapor deposition (PECVD) of perfluoroalkanes has long been studied for tuning the wetting properties of surfaces. For high ...

科研

化学

Activating Molecules, Ions, and Solid Particles with Acoustic Cavitation

The chemical and physical effects of ultrasound arise not from a direct interaction of molecules with sound waves, but rather from the acoustic cavitation: the nucleation, growth, and implosive collapse of microbubbles in liquids submitted to power ultrasound. The violent implosion of bubbles leads to the formation of chemically reactive species and to the emission of light, named sonoluminescence. In this manuscript, we describe the techniques allowing study of extreme intrabubble conditions and chemical reactivity of acoustic cavitation in solutions. The analysis of sonoluminescence spectra of water sparged with noble gases provides evidence for nonequilibrium plasma formation. The photons and the "hot" particles generated by cavitation bubbles enable to excite the non-volatile species in solutions increasing their chemical reactivity. For example the mechanism of ultrabright sonoluminescence of uranyl ions in acidic solutions varies with uranium concentration: sonophotoluminescence dominates in diluted solutions, and collisional excitation contributes at higher uranium concentration. Secondary sonochemical products may arise from chemically active species that are formed inside the bubble, but then diffuse into the liquid phase and react with solution precursors to form a variety of products. For instance, the sonochemical reduction of Pt(IV) in pure water provides an innovative synthetic route for monodispersed nanoparticles of metallic platinum without any templates or capping agents. Many studies reveal the advantages of ultrasound to activate the divided solids. In general, the mechanical effects of ultrasound strongly contribute in heterogeneous systems in addition to chemical effects. In particular, the sonolysis of PuO2 powder in pure water yields stable colloids of plutonium due to both effects.

Acoustic cavitation in liquids submitted to power ultrasound creates transient extreme conditions inside the collapsing bubbles, which are the origin of unusual chemical reactivity and light emission, known as sonoluminescence. In the presence of noble gases, nonequilibrium plasma is formed. The "hot" particles and the photons generated by collapsing bubbles are able to excite species in solution.

活化分子，离子和固体颗粒与声空化

The chemical and physical effects of ultrasound arise not from a direct interaction of molecules with sound waves, but rather from the acoustic ...

Synthetic Methodology for Asymmetric Ferrocene Derived Bio-conjugate Systems via Solid Phase Resin-based Methodology

Early detection is a key to successful treatment of most diseases, and is particularly imperative for the diagnosis and treatment of many types of cancer. The most common techniques utilized are imaging modalities such as Magnetic Resonance Imaging (MRI), Positron Emission Topography (PET), and Computed Topography (CT) and are optimal for understanding the physical structure of the disease but can only be performed once every four to six weeks due to the use of imaging agents and overall cost. With this in mind, the development of &#8220;point of care&#8221; techniques, such as biosensors, which evaluate the stage of disease and/or efficacy of treatment in the clinician&#8217;s office and do so in a timely manner, would revolutionize treatment protocols.1 As a means to exploring ferrocene based biosensors for the detection of biologically relevant molecules2, methods were developed to produce ferrocene-biotin bio-conjugates described herein. This report will focus on a biotin-ferrocene-cysteine system that can be immobilized on a gold surface.

The synthesis of asymmetric species of ferrocene is challenging using solution techniques. This report focuses on the methods carried out to produce a ferrocene-biotin bioconjugate using facile and clean reactions accomplished via solid-phase synthesis. Incorporation of a thiolate moiety is shown to impart the ability for immobilization on gold surfaces.

合成方法通过固相树脂为基础的方法不对称二茂铁产生的生物共轭系统

Early detection is a key to successful treatment of most diseases, and is particularly imperative for the diagnosis and treatment of many types of cancer. ...

X-ray Powder Diffraction in Conservation Science: Towards Routine Crystal Structure Determination of Corrosion Products on Heritage Art Objects

The crystal structure determination and refinement process of corrosion products on historic art objects using laboratory high-resolution X-ray powder diffraction (XRPD) is presented in detail via two case studies.
The first material under investigation was sodium copper formate hydroxide oxide hydrate, Cu4Na4O(HCOO)8(OH)2&#8729;4H2O (sample 1) which forms on soda glass/copper alloy composite historic objects (e.g., enamels) in museum collections, exposed to formaldehyde and formic acid emitted from wooden storage cabinets, adhesives, etc. This degradation phenomenon has recently been characterized as &#34;glass induced metal corrosion&#34;.
For the second case study, thecotrichite, Ca3(CH3COO)3Cl(NO3)2&#8729;6H2O (sample 2), was chosen, which is an efflorescent salt forming needlelike crystallites on tiles and limestone objects which are stored in wooden cabinets and display cases. In this case, the wood acts as source for acetic acid which reacts with soluble chloride and nitrate salts from the artifact or its environment.
The knowledge of the geometrical structure helps conservation science to better understand production and decay reactions and to allow for full quantitative analysis in the frequent case of mixtures.

Modern high resolution X-ray powder diffraction (XRPD) in the laboratory is used as an efficient tool to determine crystal structures of long-known corrosion products on historic objects.

X射线粉末衍射在保护科学:迈向文物艺术品腐蚀产物的常规晶体结构测定

The crystal structure determination and refinement process of corrosion products on historic art objects using laboratory high-resolution X-ray powder ...

Measurement of Particle Size Distribution in Turbid Solutions by Dynamic Light Scattering Microscopy

A protocol for measuring polydispersity of concentrated polymer solutions using dynamic light scattering is described. Dynamic light scattering is a technique used to measure the size distribution of polymer solutions or colloidal particles. Although this technique is widely used for the assessment of polymer solutions, it is difficult to measure the particle size in concentrated solutions due to the multiple scattering effect or strong light absorption. Therefore, the concentrated solutions should be diluted before measurement. Implementation of the confocal optical component in a dynamic light scattering microscope1 helps to overcome this barrier. Using such a microscopic system, both transparent and turbid systems can be analyzed under the same experimental setup without a dilution. As a representative example, a size distribution measurement of a temperature-responsive polymer solution was performed. The sizes of the polymer chains in an aqueous solution were several tens of nanometers at a temperature below the lower critical solution temperature (LCST). In contrast, the sizes increased to more than 1.0 &#181;m when above the LCST. This result is consistent with the observation that the solution turned turbid above the LCST.

A protocol for the direct measurement of particle size distribution in concentrated solutions using dynamic light scattering microscopy is presented.

通过动态光散射显微镜混浊解决方案粒度分布的测定

A protocol for measuring polydispersity of concentrated polymer solutions using dynamic light scattering is described. Dynamic light scattering is a ...

Protocol for the Solid-phase Synthesis of Oligomers of RNA Containing a 2'-O-thiophenylmethyl Modification and Characterization via Circular Dichroism

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 methodology for applications in various fields and for different research purposes. The procedure described herein focuses on the synthesis, purification, and characterization of dodecamers of RNA 5&#39;-[CUA CGG AAU CAU]-3&#39; containing zero, one, or two modifications located at the C2&#39;-O-position. The probes are based on 2-thiophenylmethyl groups, incorporated into RNA nucleotides via standard organic synthesis and introduced into the corresponding oligonucleotides via their respective phosphoramidites. This report makes use of phosphoramidite chemistry via the four canonical nucleobases (Uridine (U), Cytosine (C), Guanosine (G), Adenosine (A)), as well as 2-thiophenylmethyl functionalized nucleotides modified at the 2&#39;-O-position; however, the methodology is amenable for a large variety of modifications that have been developed over the years. The oligonucleotides were synthesized on a controlled-pore glass (CPG) support followed by cleavage from the resin and deprotection under standard conditions, i.e., a mixture of ammonia and methylamine (AMA) followed by hydrogen fluoride/triethylamine/N-methylpyrrolidinone. The corresponding oligonucleotides were purified via polyacrylamide electrophoresis (20% denaturing) followed by elution, desalting, and isolation via reversed-phase chromatography (Sep-pak, C18-column). Quantification and structural parameters were assessed via ultraviolet-visible (UV-vis) and circular dichroism (CD) photometric analysis, respectively. This report aims to serve as a resource and guide for beginner and expert researchers interested in embarking in this field. It is expected to serve as a work-in-progress as new technologies and methodologies are developed. The description of the methodologies and techniques within this document correspond to a DNA/RNA synthesizer (refurbished and purchased in 2013) that uses phosphoramidite chemistry.

Protocol for the Solid-phase Synthesis of Oligomers of RNA Containing a 2'-O-thiophenylmethyl Modification and Characterization via Circular Dichroism

This article provides a detailed procedure on the solid-phase synthesis, purification, and characterization of dodecamers of RNA modified at the C2&#39;-O-position. UV-vis and circular dichroism photometric analyses are used to quantify and characterize structural aspects, i.e., single-strands or double-strands.

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 ...

A Convenient Method for Extraction and Analysis with High-Pressure Liquid Chromatography of Catecholamine Neurotransmitters and Their Metabolites

The extraction and analysis of catecholamine neurotransmitters in biological fluids is of great importance in assessing nervous system function and related diseases, but their precise measurement is still a challenge. Many protocols have been described for neurotransmitter measurement by a variety of instruments, including high-pressure liquid chromatography (HPLC). However, there are shortcomings, such as complicated operation or hard-to-detect multiple targets, which cannot be avoided, and presently, the dominant analysis technique is still HPLC coupled with sensitive electrochemical or fluorimetric detection, due to its high sensitivity and good selectivity. Here, a detailed protocol is described for the pretreatment and detection of catecholamines with high pressure liquid chromatography with electrochemical detection (HPLC-ECD) in real urine samples of infants, using electrospun composite nanofibers composed of polymeric crown ether with polystyrene as adsorbent, also known as the packed-fiber solid phase extraction (PFSPE) method. We show how urine samples can be easily precleaned by a nanofiber-packed solid phase column, and how the analytes in the sample can be rapidly enriched, desorbed, and detected on an ECD system. PFSPE greatly simplifies the pretreatment procedures for biological samples, allowing for decreased time, expense, and reduction of the loss of targets. 
Overall, this work illustrates a simple and convenient protocol for solid-phase extraction coupled to an HPLC-ECD system for simultaneous determination of three monoamine neurotransmitters (norepinephrine (NE), epinephrine (E), dopamine (DA)) and two of their metabolites (3-methoxy-4-hydroxyphenylglycol (MHPG) and 3,4-dihydroxy-phenylacetic acid (DOPAC)) in infants' urine. The established protocol was applied to assess the differences of urinary catecholamines and their metabolites between high-risk infants with perinatal brain damage and healthy controls. Comparative analysis revealed a significant difference in urinary MHPG between the two groups, indicating that the catecholamine metabolites may be an important candidate marker for early diagnosis of cases at risk for brain damage in infants.

We present a convenient solid-phase extraction coupled to high-pressure liquid chromatography (HPLC) with electrochemical detection (ECD) for simultaneous determination of three monoamine neurotransmitters and two of their metabolites in infants' urine. We also identify the metabolite MHPG as a potential biomarker for the early diagnosis of brain damage for infants.

用高压液相色谱法提取和分析儿茶酚胺神经递质及其代谢物的简便方法

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

In this work, we report the synthesis and characterization of ordered nanoporous carbon material (also called ordered mesoporous carbon material [OMC]) with a 4.6 nm pore size, and ordered silica porous matrix, SBA-15, with a 5.3 nm pore size. This work describes the surface properties of nanoporous molecular sieves, their wettability, and the melting behavior of D2O confined in the differently ordered porous materials with similar pore sizes. For this purpose, OMC and SBA-15 with highly ordered nanoporous structures are synthesized via impregnation of the silica matrix by applying a carbon precursor and by the sol-gel method, respectively. The porous structure of investigated systems is characterized by an N2 adsorption-desorption analysis at 77 K. To determine the electrochemical character of the surface of synthesized materials, potentiometric titration measurements are conducted; the obtained results for OMC shows a significant pHpzc shift toward the higher values of pH, relative to SBA-15. This suggests that investigated OMC has surface properties related to oxygen-based functional groups. To describe the surface properties of the materials, the contact angles of liquids penetrating the studied porous beds are also determined. The capillary rise method has confirmed the increased wettability of the silica walls relative to the carbon walls and an influence of the pore roughness on the fluid/wall interactions, which is much more pronounced for silica than for carbon mesopores. We have also studied the melting behavior of D2O confined in OMC and SBA-15 by applying the dielectric method. The results show that the depression of the melting temperature of D2O in the pores of OMC is about 15 K higher relative to the depression of the melting temperature in SBA-15 pores with a comparable 5 nm size. This is caused by the influence of adsorbate/adsorbent interactions of the studied matrices.

Here we report the synthesis and characterization of ordered nanoporous carbon (with a 4.6 nm pore size) and SBA-15 (with a 5.3 nm pore size). The work describes the surface and textural properties of nanoporous molecular sieves, their wettability, and the melting behavior of D2O confined in the materials.

合成纳米碳和二氧化硅矩阵的表面性能

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

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 solid-phase synthesis, regenerating Michael (REM) linker strategies, and 1,3-dipolar cycloaddition, a library of structurally similar heterocycles, both with and without a spirocyclic center, can be constructed. The main advantages of the solid-support synthesis are as follows: first, each reaction step can be driven to completion using a large excess of reagents resulting in high yields; next, the use of commercially available starting materials and reagents keep the costs low; finally, the reaction steps are easy to purify via simple filtration. The REM linker strategy is attractive because of its recyclability and traceless nature. Once a reaction scheme is completed, the linker can be reused multiple times. In a typical solid-phase synthesis, the product contains either a part of or the whole linker, which can prove undesirable. The REM linker is &#34;traceless&#34; and the point of attachment between the product and the polymer is indistinguishable. The high diastereoselectivity of the intramolecular 1,3-dipolar cycloaddition is well documented. Limited by the insolubility of the solid support, the reaction progression can only be monitored by a change in the functional groups (if any) via infrared (IR) spectroscopy. Thus, the structural identification of intermediates cannot be characterized by conventional nuclear magnetic resonance (NMR) spectroscopy. Other limitations to this method stem from the compatibilities of the polymer/linker to the desired chemical reaction scheme. Herein we report a protocol that allows for the convenient production of spirocyclic heterocycles that, with simple modifications, can be automated with high-throughput techniques.

Here we present a protocol to demonstrate an efficient method for the synthesis of spirocyclic heterocycles. The five-step process utilizes solid-phase synthesis and regenerating Michael linker strategies. Generally difficult to synthesize, we present a customizable method for the synthesis of spirocyclic molecules otherwise inaccessible to other modern approaches.

[4.4] 刺环氧基的固相合成

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 ...

Natural Product Discovery with LC-MS/MS Diagnostic Fragmentation Filtering: Application for Microcystin Analysis

Natural products are often biosynthesized as mixtures of structurally similar compounds, rather than a single compound. Due to their common structural features, many compounds within the same class undergo similar MS/MS fragmentation and have several identical product ions and/or neutral losses. The purpose of diagnostic fragmentation filtering (DFF) is to efficiently detect all compounds of a given class in a complex extract by screening non-targeted LC-MS/MS datasets for MS/MS spectra that contain class specific product ions and/or neutral losses. This method is based on a DFF module implemented within the open-source MZmine platform that requires sample extracts be analyzed by data-dependent acquisition on a high-resolution mass spectrometer such as quadrupole Orbitrap or quadrupole time-of-flight mass analyzers. The main limitation of this approach is the analyst must first define which product ions and/or neutral losses are specific for the targeted class of natural products. DFF allows for the subsequent discovery of all related natural products within a complex sample, including new compounds. In this work, we demonstrate the effectiveness of DFF by screening extracts of Microcystis aeruginosa, a prominent harmful algal bloom causing cyanobacteria, for the production of microcystins.

Diagnostic fragmentation filtering, implemented into MZmine, is an elegant, post-acquisition approach to screen LC-MS/MS datasets for entire classes of both known and unknown natural products. This tool searches MS/MS spectra for product ions and/or neutral losses that the analyst has defined as being diagnostic for the entire class of compounds.

LC-MCYMS 诊断碎裂过滤的天然产物发现: 微囊藻毒素分析的应用

Natural products are often biosynthesized as mixtures of structurally similar compounds, rather than a single compound. Due to their common structural ...