The authors would acknowledge Scott Howard and Abigail Marinack for general support and coordination of this work.

All studies involving human urine samples were conducted adherence to the procedure approved by Pharmasan/Neuroscience Institutional Review Board (IRB).
1. Urine Sample Collection and Creatinine (Cr) Determination
<ol>
	<li>Collect 5 mL of the next morning urine samples after ca. 10 h overnight fasting in a 5 mL transport tube A containing 250 &#181;L of 3 N HCl as preservative and store at -20 &#176;C until use.</li>
	<li>Thaw and vortex 5 mL transport urine A tube and centrifuge in a centrifuge (e.g. Sorvall) (2297 x g, 10 min).</li>
	<li>Aliquot 1 mL of urine twice from the 5 mL transport tube A.</li>
	<li>Determine Cr by a urinary creatinine method26.</li>
</ol>
2. Preparation of Standard, IS and Quality Control (QC) Samples
<ol>
	<li>Prepare a stock solution of 3-NT at 100 &#181;g/mL in water with 0.01% HCOOH mobile phase A (MA) and store at -20 &#176;C.</li>
	<li>Make a 3-NT working standard solution at concentration of 100 ng/mL by diluting the 100 &#181;g/mL 3-NT stock solution with MA.</li>
	<li>Generate standards ranging from 5 to 2500 pg/mL by dilution of the 100 ng/mL working standard with 0.15 M HCl acidified blank urine free of 3-NT along with blank and double blank samples.</li>
	<li>Prepare a stock solution of IS 13C9-3-NT at 100 &#181;g/mL in water with MA and store at -20 &#176;C.</li>
	<li>Make an IS working solution at 500 pg/mL by dilution of the 100 &#181;g/mL 13C9-3-NT IS stock solution with MA.</li>
	<li>Establish five levels of QC samples covering the LLOQ, low, medium and high levels (i.e., 10, 25, 100, 500 and 1,250 pg/mL), by dilution of 3-NT working standard in the acidified blank urine.</li>
</ol>
3. Solid Phase Extraction Procedure
<ol>
	<li>Thaw and vortex urine samples, standards and QC samples.</li>
	<li>Add urine samples, standards and QC samples (250 &#181;L each) to 32&#160;wells of a clean 2 mL 96-well collection plate.</li>
	<li>Introduce the 500 pg/mL IS working solution (50 &#181;L) to each well except double blank sample well. Add 0.01% HCOOH&#160;(50 &#181;L) to the double blank sample well.</li>
	<li>Add LC-MS/MS water with 0.1% HCOOH (250 &#181;L).</li>
	<li>Mix the above mixture with an 8-channel pipette three times.</li>
	<li>Cover the plate until SPE loading.</li>
	<li>Place an MCX 96-well extraction plate and a collection reservoir on a positive pressure processor.</li>
	<li>Condition the extraction plate with flowing MeOH (200 &#181;L) through the sorbent.</li>
	<li>Equilibrate by flowing water with 2% HCOOH (200 &#181;L) through the sorbent.</li>
	<li>Load the entire volume of each of the pre-mixed samples onto the pre-conditioned extraction plate carefully with an 8-channel pipette.</li>
	<li>Set low positive pressure (e.g., 3 psi) on the positive pressure processor to allow the mixture to flow through the sorbent slowly, adjust the pressure if needed.</li>
	<li>Wash the wells by flowing water with 2% HCOOH (200 &#181;L) through the sorbent.</li>
	<li>Wash the wells by flowing methanol (200 &#181;L) through the sorbent.</li>
	<li>Wash the wells by flowing water (200 &#181;L) through the sorbent.</li>
	<li>Dry the wells completely with high positive pressure setting (e.g., 40 psi) on the positive pressure processor.</li>
	<li>Replace the reservoir with a clean 2 mL 96-well collection plate.</li>
	<li>Apply 25 mM NH4OAc at pH 9 (50 &#181;L) to elute the retained analyte and IS from the extraction plate.</li>
	<li>Pipette LC-MS water with 5% HCOOH (50 &#181;L) to neutralize the eluate.</li>
	<li>Mix with the 8-channel pipette three times and submit to LC-MS/MS station for analysis.</li>
</ol>
4. LC-MS/MS Analysis
<ol>
	<li>Accurately measure 2,000 mL of LC-MS water with a graduated cylinder and transfer it into a 2 L bottle.</li>
	<li>Pipette 200 &#181;L pure HCOOH into the above bottle containing LC-MS water.</li>
	<li>Mix thoroughly and label as mobile phase A (MA). Include initials, preparation date and expiration date.</li>
	<li>Take a 2 L bottle of LC-MS methanol and label as mobile phase B (MB) with starting date and expiration date.</li>
	<li>Set temperature of auto-sampler to 4 &#176;C.</li>
	<li>Place the collection plate with prepared samples into the autosampler.</li>
	<li>Place a PFP column (150 mm x 2.1 mm i.d., 3 &#181;m) and guard column in the oven.</li>
	<li>Set the oven temperature to 30 &#176;C.</li>
	<li>Equilibrate 10 min using the acquisition method with the LC gradient elution as shown in Table 1.</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Time (min)</td>
			<td>Module</td>
			<td>Events</td>
			<td>Parameter</td>
		</tr>
		<tr>
			<td>0</td>
			<td>Pumps</td>
			<td>Pump B Conc.</td>
			<td>5</td>
		</tr>
		<tr>
			<td>0.5</td>
			<td>Pumps</td>
			<td>Pump B Conc.</td>
			<td>20</td>
		</tr>
		<tr>
			<td>1</td>
			<td>Pumps</td>
			<td>Pump B Conc.</td>
			<td>50</td>
		</tr>
		<tr>
			<td>3</td>
			<td>Pumps</td>
			<td>Pump B Conc.</td>
			<td>80</td>
		</tr>
		<tr>
			<td>4</td>
			<td>Pumps</td>
			<td>Pump B Conc.</td>
			<td>90</td>
		</tr>
		<tr>
			<td>4.01</td>
			<td>Pumps</td>
			<td>Pump B Conc.</td>
			<td>95</td>
		</tr>
		<tr>
			<td>5.5</td>
			<td>Pumps</td>
			<td>Pump B Conc.</td>
			<td>95</td>
		</tr>
		<tr>
			<td>5.6</td>
			<td>Pumps</td>
			<td>Pump B Conc.</td>
			<td>5</td>
		</tr>
		<tr>
			<td>7</td>
			<td>Controller</td>
			<td>Stop</td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 1: Liquid Chromatography Gradient Elution Conditions
<ol start="10">
	<li>Create a batch list including standards, QC and urine samples.</li>
	<li>Start the batch by injection of the prepared samples (12 &#181;L).</li>
</ol>
5. Peak Identification, Integration and Data Process
<ol>
	<li>Control data acquisition and processing using the software.</li>
	<li>Identify and integrate 3-NT and IS peaks for all the samples.</li>
	<li>Establish a standard curve with the range of 10-2,500 pg/mL for 3-NT quantitation by linear regression of the peak area ratio of 3-NT and IS versus the nominal 3-NT concentration with a weighting factor of 1/x.</li>
	<li>Quantify all the samples using the standard curve.</li>
	<li>Determine if the QC samples fall in the established range.</li>
	<li>Convert the detected concentrations of urine samples to final results in the unit of nM or nmol/mmol Cr.</li>
</ol>

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The authors declare that they have no conflict of interest.

Substantial variations in concentrations previously reported in the literature for the endogenous free 3-NT in human urine samples reveal methodological problems associated with available assays8,9,10,11. Accurate determination of the low basal level of 3-NT in human urine remains a challenging task that requires special precautions for sample preparation and LC-MS/MS analysis. This protocol ou...

The effects of oxidative stress on clinical presentation have been thrust into the forefront in recent years1. One of the biomarkers being explored is 3-nitrotyrosine (3-NT), an end stable product formed when reactive nitrogen species (RNS) interact with tyrosine, a catecholamine neurotransmitter precursor. While 3-NT may have clinical value as a biomarker for RNS in vivo, the substantial changes of the properties and functions of tyrosine may adversely affect corresponding proteins and cellular functions1,2. Emerging research has suggested that 3-NT may play an important role in inflammatory conditions3, neurodegenerative disorders4,5, cardiovascular disease6 and diabetes7 as well as conditions related to oxidative stress. However, these observations are based on results from methodologies lacking in sensitivity and/or selectivity8,9,10,11. The enormous 3-NT concentration ranges for the biological samples previously reported in the literature reveal that serious analytical problems are associated with these assays and technical improvement is needed to accurately quantify the levels of 3-NT and verify its role in the pathology of these conditions.
The quantitation of free 3-NT in biological matrices presents a special challenge to man and instrument8,9,10,11. First, the trace level of endogenous 3-NT demands an ultra-sensitive detection; second, the existence of numerous structurally similar analogues, especially tyrosine, which is present in vast excess, requires a high degree of selectivity; third, the artefactual formation of 3-NT by tyrosine nitration with ubiquitous nitrate and nitrite requires special consideration during sample preparation to avoid false overestimation of 3-NT.
Among a wide variety of methodologies employed to measure 3-NT, MS/MS has been considered the gold standard method due to its superior sensitivity and selectivity11,12,13,14. Gas chromatography (GC) coupled MS/MS offers the best sensitivity, however, the indispensable sample derivatization steps are too tedious and time-consuming to be efficient for clinical utility15,16. LC-MS/MS does not require complex sample derivatization, making it the more promising option. Nonetheless, there are several obstacles to overcome such as the sensitivity of LC-MS/MS methods reported in the literature needs to improve for the measurement of low abundant 3-NT7,17,18 and the relatively long turnaround time must be shortened for high-throughput applications12,13,17,19.
Additionally, when considering clinical applications, the biological matrix used plays a significant role. It should be easy and inexpensive to obtain and non-invasive if possible20,21,22. Plasma, the traditionally used sample in the literature, is not a clinically desirable matrix, so a methodology utilizing urine which is non-invasive and cost-effective, was sought.
Several attempts to develop reliable and specific LC-MS/MS methodologies have been made using urine9,10,11. However, they have all fallen short of being either selective, reliable or efficient enough for clinical use. The effectiveness of the predominant SPE using traditional reversed-phase cartridge (C18 type) as sample cleanup for the 3-NT analysis has been questioned and a sequential SPE of strong cation exchange (SCX) and reversed phase C18-OH has been proposed6,7,19. One recently developed LC-MS/MS method utilized a multi-step purification process of manual C18 SPE, preparative high pressure liquid chromatography (HPLC), and online SPE for analysis of 3-NT23. Although this method was sensitive enough for clinical purposes, with an LLOQ of 0.041 nM, the cleanup process was intensive and tedious and required 3 mL of urine, limiting its feasibility for high-throughput. A molecularly imprinted polymer was employed as the SPE sorbent to improve the efficiency of the cleanup process14, but the resulting LLOQ (0.7 &#181;g/mL) was not low enough for clinical specimens. Another method required two-dimensional (2D) LC-MS/MS and immunoaffinity chromatography for sample cleanup in order to achieve a limit of detection (LOD) of 0.022 nM24. While all these methods have made advancements in the assessment of 3-NT, none have achieved the sensitivity, reliability, and efficiency necessary for clinical applications.
In order to investigate the pathology of free 3-NT and its role as a biomarker of oxidative stress in clinical settings, we have developed a methodology that is simple, efficient, accurate and precise, enabling for high-throughput clinical applications25. A miniaturized mixed-mode cation exchange (MCX) 96-well extraction microplate was implemented to achieve simple and effective sample cleanup and enrichment of 3-NT in a single extraction bypassing the drawbacks seen in the existing methods that require derivatization, evaporation and 2D-LC. Liquid chromatography with 0.01% HCOOH as an additive in mobile phase offered an enhanced signal response with a rapid cycle time. Selectivity was further improved through application of a mild NH4OAc elution solution for selective elution of 3-NT, and use of MRM transition for both 3-NT and the internal standard (IS). The matrix effect was compensated for by using a reduced amount of a preferred 13C-labeled isotopic IS for quantification. With the advent of this methodology, researchers and clinicians will be able to verify the role of 3-NT in clinical conditions and further explore the impact of oxidative stress.

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			<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>
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		<tr height="25">
			<td height="25" style="height:25px;">3-Nitro-L-tyrosine-13C9</td>
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			<td>652296-5.0mg</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Mass Spec Gold Urine</td>
			<td>Golden West Biologicals</td>
			<td>MSG 5000-1L</td>
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			<td height="21" style="height:21px;">Oasis MCX 96-well &#181;Elution plate</td>
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			<td height="21" style="height:21px;">LC-MS Ultra CHROMASOLV methanol&#160;&#160;</td>
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			<td height="21" style="height:21px;">LC-MS Ultra CHROMASOLV water</td>
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			<td height="21" style="height:21px;">Formic acid for mass spectrometry</td>
			<td style="width:161px;">Sigma</td>
			<td>94318-50ML-F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ammonium hydroxide solution</td>
			<td style="width:161px;">Sigma</td>
			<td style="width:115px;">338818-1L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultra PFP propyl columns</td>
			<td style="width:161px;">Restek</td>
			<td style="width:115px;">9179362</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5500 Triple quad</td>
			<td style="width:161px;">AB Sciex&#160;</td>
			<td style="width:115px;">/</td>
			<td style="width:263px;">Contact manufacture for more detail</td>
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			<td height="21" style="height:21px;">UFLC-XR</td>
			<td style="width:161px;">Shimadzu&#160;</td>
			<td style="width:115px;">/</td>
			<td style="width:263px;">Contact manufacture for more detail</td>
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			<td height="21" style="height:21px;">Integra 400 Plus&#160;</td>
			<td style="width:161px;">Roche</td>
			<td style="width:115px;">/</td>
			<td style="width:263px;">Urinary Creatinine Jaff&#233; Gen 2 method</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LCMS certified 12 x 32mm screw neck vial</td>
			<td style="width:161px;">Waters</td>
			<td>600000751CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LCGC certified 12 x 32mm screw neck total recovery vial</td>
			<td style="width:161px;">Waters</td>
			<td>186000384C</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">5 mL transport tube</td>
			<td style="width:161px;">Phenix</td>
			<td>TT-3205</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">50 mL Centrifuge tube</td>
			<td style="width:161px;">Crystalgen&#160;</td>
			<td style="width:115px;">23-2263</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">15 mL Centrifuge tube</td>
			<td style="width:161px;">Crystalgen&#160;</td>
			<td style="width:115px;">23-2266</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">eLine electronic pipette</td>
			<td style="width:161px;">Sartorius</td>
			<td style="width:115px;">730391</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microfuge centrifuge&#160;</td>
			<td style="width:161px;">Beckman Coulter</td>
			<td style="width:115px;">A46474</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">OHAUS balance&#160;&#160;</td>
			<td style="width:161px;">Kennedy Scales, inc.</td>
			<td style="width:115px;">735</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vortex mixer&#160;</td>
			<td style="width:161px;">Bernstead Thermolyne</td>
			<td style="width:115px;">M16715</td>
			<td></td>
		</tr>
	</tbody>
</table>

integration of miniaturized solid phase extraction and lc-ms/ms detection of 3-nitrotyrosine in human urine for clinical applications

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 variety of pathological conditions. However, existing methods lack the sufficient sensitivity and/or specificity necessary to measure the low endogenous level of 3-NT reliably and are too cumbersome for clinical applications. Hence, analytical improvement is urgently needed to accurately quantify the levels of 3-NT and verify the role of 3-NT in pathological conditions. This protocol presents the development of a novel liquid chromatography tandem mass spectrometry (LC-MS/MS) detection combined with a miniaturized solid phase extraction (SPE) for the rapid and accurate measurement of 3-NT in human urine as a non-invasive biomarker for oxidative stress. SPE using a 96-well plate markedly simplified the process by combining sample cleanup and analyte enrichment without tedious derivatization and evaporation steps, reducing solvent consumption, waste disposal, risk of contamination and overall processing time. The employment of 25 mM ammonium acetate (NH4OAc) at pH 9 as the SPE elution solution substantially enhanced the selectivity. Mass spectrometry signal response was improved through adjustment of the multiple reaction monitoring (MRM) transitions. Use of 0.01% HCOOH as additive on a pentafluorophenyl (PFP) column (150 mm x 2.1 mm, 3 &#181;m) improved signal response another 2.5-fold and shortened the overall run time to 7 min. A lower limit of quantitation (LLOQ) of 10 pg/mL (0.044 nM) was achieved, representing a significant sensitivity improvement over the reported assays. This simplified, rapid, selective and sensitive method allows two plates of urine samples (n = 192) to be processed in a 24 h time-period. Considering the markedly improved analytical performance, and non-invasive and inexpensive urine sampling, the proposed assay is beneficial for pre-clinical and clinical studies.

Figure 1 illustrates that 3-NT is completely chromatographically separated from other structurally similar tyrosine analogues under the optimized LC condition, which eliminates the co-eluting interferences due to these vastly excessive compounds and consequently enhances the degree of assay selectivity. In addition, the gradient elution with 0.01% HCOOH as additive in MA and methanol at a flow rate of 0.45 mL/min allows rapid elution of 3-NT (i.e., 3...

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 | Chemistry | Video

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

A selective and sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method coupled with an efficient solid phase extraction on a mixed-mode cation-exchange (MCX) 96-well microplate was developed for the measurement of free 3-nitrotyrosine (3-NT) in human urine with high throughput, which is suitable for clinical applications.

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 Views.  Pharmasan Labs, Inc.. A selective and sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method coupled with an efficient solid phase extraction on a mixed-mode cation-exchange (MCX) 96-well microplate was developed for the measurement of free 3-nitrotyrosine (3-NT) in human urine with high throughput, which is suitable for clinical applications.

Video: 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.

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

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.

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.

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.

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.

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.

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.

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.

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.

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.

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

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