We would like to thank the Ministry of Science and Technology of Taiwan (grant number: MOST 104-2628-M-009-003-MY4) for the financial support of this work.

This protocol assumes that all the steps are performed according to the relevant laboratory safety regulations. Some of the steps use commercial instruments - in those cases, manufacturer guidelines need to be followed. When handling toxic chemicals, MSDS guidelines need to be followed. The custom-made equipment4 must be operated cautiously; especially, when handling pressurized gases and live electric wiring.
1. Preparation of Standard Solution
<ol>
	<li>Prepare 6.2 x 10-2 M stock solution of limonene in ethanol by mixing 10 &#181;L limonene with 990 &#181;L ethanol.</li>
	<li>Prepare 10 mL of 6.2 x 10-5 M limonene solution by mixing 10 &#181;L 6.2 x 10-2 limonene, 490 &#181;L ethanol, and adding pure water to the final volume of 10 mL. Shake the volumetric flask thoroughly.</li>
	<li>Transfer the prepared standard solution to a 20-mL screw top headspace glass vial with septum cap. The diluted standard solution can be used for testing the system.</li>
</ol>
2. Preparation of Real Sample
<ol>
	<li>Obtain lime juice by squeezing fresh lime fruit (cut in half) on a kitchen squeezer.</li>
	<li>Prepare 10 mL of diluted lime juice by mixing 2 mL lime juice, 500 &#181;L ethanol, and adding pure water to the final volume of 10 mL. Shake the volumetric flask thoroughly.</li>
	<li>Transfer the prepared sample to a 20-mL screw top headspace glass vial with septum cap.</li>
</ol>
3. Spiking the Real Sample with Standard Solution
<ol>
	<li>First standard addition: Prepare 10 mL of spiked sample by mixing 2 mL lime juice, 10 &#181;L 6.2 x 10-2 M limonene solution, 490 &#181;L ethanol, and adding pure water to the final volume of 10 mL. Shake the volumetric flask thoroughly.</li>
	<li>Second standard addition: Prepare 10 mL of spiked sample by mixing 2 mL lime juice, 20 &#181;L 6.2 x 10-2 M limonene solution, 480 &#181;L ethanol, and adding pure water to the final volume of 10 mL. Shake the volumetric flask thoroughly.</li>
</ol>
4. Setting Up the Fizzy Extraction System
<ol>
	<li>Put the fizzy extraction system (Figure 1)4 next to the APCI source of the triple quadrupole mass spectrometer.</li>
	<li>Connect the carbon dioxide gas cylinder to the gas supply inlet of the fizzy extraction system. Open the valve in the gas regulator. Set the output pressure to 1.5 bar (150 kPa).</li>
	<li>Connect the extraction chamber outlet to the ion source inlet.</li>
	<li>Connect the fizzy extraction system to the 12-V power supply.</li>
	<li>Set up the data acquisition software of the triple quadrupole mass spectrometer (Figure 2). Operate the instrument with the APCI source, in the positive-ion multiple reaction monitoring (MRM) mode, with argon as collision gas.
	<ol>
		<li>Run the data acquisition software.</li>
		<li>Select the option &#34;LCMS8030 only&#34;.</li>
		<li>Select the option &#34;MS On/Off&#34;.</li>
		<li>Set the desolvation line temperature to 250 &#176;C, and the flow rate of drying gas to 15 L min-1. Wait until the value of every instrument parameter becomes the same as the preset value.</li>
		<li>Select the MS data acquisition method file.</li>
		<li>Make sure the collision voltage is -20 V, the precursor ion m/z is 137, and the fragment ion m/z are 81 and 95</li>
		<li>Click on the &#34;Start Single Run&#34; button.</li>
		<li>Type the file name.</li>
		<li>Select the file path.</li>
		<li>Move to section 5 (&#34;Performing fizzy extraction&#34;).</li>
		<li>Select the option &#34;MS On/Off&#34;.</li>
		<li>Close the software window.</li>
		<li>Tick the items &#34;Nebulizing Gas Off&#34;, &#34;DL Heater Off&#34;, &#34;Heat Block Off&#34;, and &#34;Dry Gas Off&#34;. Click &#34;OK&#34;.</li>
	</ol>
	</li>
</ol>
5. Performing Fizzy Extraction
<ol>
	<li>Place a sample vial in the fizzy extraction system by using the screw mount. The extraction system is operated at room temperature (~ 25 &#176;C).</li>
	<li>Press the &#34;Start&#34; button on the LCD shield of the fizzy extraction system.</li>
	<li>Wait while the automated fizzy extraction process is executed (Figure 3). Observe development of ion signals on the screen of the triple quadrupole mass spectrometer. 
	NOTE: The following steps are executed automatically: Sample headspace is flushed with carbon dioxide during 60 s. Sample is pressurized with carbon dioxide during 60 s. Stirrer motor is on. Sample is depressurized. Multiple bubbles are formed. In the later phase, stirrer motor is on to enhance bubbling.</li>
	<li>Take out (unscrew) the sample vial.</li>
	<li>Wipe the sample stirring spindle with cellulose tissue.</li>
	<li>Wash the stirring spindle with ethanol, and wipe it with cellulose tissue again.</li>
	<li>The system is ready for analysis of another sample (repeat steps 5.1-5.6).</li>
	<li>Switch off the power supply.</li>
	<li>Disconnect the fizzy extraction outlet tube from the ion source.</li>
	<li>Close the valve of the gas cylinder, and disconnect the gas tubing.</li>
</ol>
6. Data Analysis
<ol>
	<li>Export extracted ion currents for the m/z 81 from the triple quadrupole mass spectrometer&#39;s data acquisition software to ASCII files (Figure 4). 
	NOTE: The ion current at the m/z 95 is not used in this demonstration.
	<ol>
		<li>Run the data acquisition software. Select the option &#34;Postrun&#34;.</li>
		<li>Select the option &#34;Select Project (Folder)&#34;, and choose the data file.</li>
		<li>Click on the &#34;File&#34; menu, and select &#34;Export Data&#34; / &#34;Export Data as ASCII&#34;.</li>
		<li>Select &#34;Output File&#34;, and select the file path. Select &#34;MS Chromatogram (MC)&#34;.</li>
	</ol>
	</li>
	<li>Import the raw data sets into peak integration software, and measure peak areas (Figure 5). Settings: linear baseline; HVL function.
	<ol>
		<li>Run the peak integration software.</li>
		<li>Select the option &#34;Import&#34; from the &#34;File&#34; menu. Click &#34;Yes&#34; button.</li>
		<li>Select the data in X and Y column. Click &#34;OK&#34; button. Select the option &#34;AutoFit Peaks I Residuals&#34;.</li>
		<li>Fit the extraction peak semi-automatically. Make sure the fitted curve follows the experimental data points. Select the option &#34;List Peak Estimates&#34;. Select the option &#34;ASCII Editor&#34;.</li>
		<li>Copy the fitting results to &#34;Clipboard&#34;.</li>
	</ol>
	</li>
	<li>Input the measured peak areas into a spreadsheet in data analysis software (Figure 6).
	<ol>
		<li>Run the data analysis software.</li>
		<li>Input the concentration values in X column and peak area values in Y column. Select the option &#34;Symbol&#34; / &#34;Scatter&#34; from the &#34;Plot&#34; menu. Select the option &#34;Fitting&#34; / &#34;Fit Linear&#34; from the &#34;Analysis&#34; menu.</li>
	</ol>
	</li>
	<li>Calculate the concentration of limonene in the diluted real sample based on the formula: 
	<img alt="Equation 1" src="/files/ftp_upload/56008/56008eq1.jpg" /> 
	where I is the intercept of the linear function, while S is the slope.</li>
	<li>Compute the concentration of limonene in the original real sample (before dilution) based on the formula: 
	<img alt="Equation 2" src="/files/ftp_upload/56008/56008eq2.jpg" /> 
	where DF is the dilution factor (here, 5).</li>
</ol>

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The authors have nothing to disclose.

Several smart ways to deliver samples to a mass spectrometer were developed in the studies conducted during the past three decades (e.g., references8,9,10,11,12,13,14). One of the goals of those studies was to simplify preparation of samples for analysis. To achieve that goal, various modifications were introduced to the ion source design. In some cases, the newly developed ion sources allowed chemists to analyze matrix-rich samples, which could normally produce spectral interferences. In an alternative approach, modifications to the conventional ionization schemes are minimized, while the sample preparation (extraction) is conducted on-line and automated (e.g., references15,16,17,18). The measurement system presented here4 exemplifies that notion because the extraction process is combined with a conventional ionization technique (i.e., APCI19,20, cf. Figure 1). However, in the future, coupling fizzy extraction with other ion sources or detectors should not be excluded, which could potentially increase the range of detectable species.
The fizzy extraction apparatus is simple to operate, and can be used in quantitative analyses. In this demonstration, we show the possibility to detect and quantify a volatile compound present in a real sample (lime juice) by implementing a double standard addition method. The obtained concentration of limonene (along with its isomers) in fresh lime juice was estimated to 4.26 x 10-4 M, which is very close to the concentration range of limonene in this kind of matrix, as reported in the literature (4.4 x 10-4-5.1 x 10-4 M)21. Certainly, the concentration of limonene in lime fruit is expected to vary depending on the cultivar, growth conditions, harvest time, storage conditions, and the method of obtaining juice for analysis - to name just a few factors. It would be ideal to implement an isotopically labeled internal standard to compensate for the experimental variability. However, such isotopologue standards are expensive, and they are not available for most analytes of interest.
The critical steps in the presented fizzy extraction protocol include: (i) setting up the fizzy extraction device (connecting power supplies, carrier gas cylinder, triple quadrupole mass spectrometer); (ii) adjusting pressure of the carrier gas; (iii) setting up the software of the triple quadrupole mass spectrometer for data acquisition; (iv) placing the sample vial in the fizzy extraction system; (v) cleaning the stirrer spindle; and (vi) data processing (peak integration).
For example, if the stirrer spindle is not cleaned well, this may lead to carryover of the analyte, and decrease analytical accuracy. Moreover, attention should be paid to the level of bubble foam formed during extraction. If some of this foam accidentally gets into the carrier gas tubing or extract tubing, the system may become contaminated. In such cases, the tubing has to be cleaned thoroughly with ethanol.
Carbon dioxide was the first-choice carrier gas because it has high solubility in water. It is also used in the production of fizzy drinks. In fact, fizzy extraction was inspired by observation of bubbles-and the release of aroma-from fizzy drinks. However, in a follow-up study, we will verify the possibility to use other gases as carrier gas.
Overall, the advantages of fizzy extraction include: simplicity, speed, and limited use of chemicals (i.e., solvents for extraction or dilution). One disadvantage of the experimental system presented here is the use of a low-resolution quadrupole mass spectrometer. The possible spectral interferences limit the applicability of the technique. Thus, it is appealing to couple fizzy extraction with a high-resolution mass spectrometer equipped with ion cyclotron resonance or orbital ion trap analyzer. In the present demonstration, the low resolution of the mass analyzer (quadrupole) is compensated for (to some extent) by applying multiple reaction monitoring, which slightly increases selectivity of the mass spectrometric detection.
We foresee that fizzy extraction will find new applications in the near future. For example, it may be suitable for detection of volatile organic compounds in foodstuffs, beverages, cosmetic and household products, as well as environmental samples.

Various phenomena observed in nature and daily life are linked to gas-liquid phase equilibriums. Carbon dioxide is dissolved in soft and alcoholic drinks under elevated pressure. When a bottle of such a fizzy drink is opened, the pressure drops down, and gas bubbles rush to the liquid surface. In this case, effervescence improves organoleptic properties of beverages. The release of gas bubbles is also the main cause of decompression sickness ("the bends")1. Due to sudden decompression, bubbles form in divers' bodies. The persons suffering from the decompression sickness are treated in hyperbaric chambers.
Gas bubbles have various applications in analytical chemistry. Notably, sparging methods rely on passing gas bubbles through liquid samples to extract volatile compounds2. For example, a method called "purge-closed loop" is combined with gas chromatography to enable rapid analysis of dissolved volatiles3. While sparging can continuously extract volatiles over time, it does not confine them in space or time. The released gas-phase species need to be trapped, and-in some cases-concentrated by applying a temperature program or using sorbents. Thus, there is a need to introduce new on-line sample treatment strategies, which could reduce the number of steps, and-at the same time-concentrate volatile analytes in space or time.
To address the challenge of extracting volatile compounds from liquid samples, and performing analysis on-line, we recently introduced "fizzy extraction"4. This new technique takes advantage of the effervescence phenomenon. Briefly, a carrier gas (here, carbon dioxide) is first dissolved in the sample by applying overpressure and stirring the sample. Then, the sample chamber is decompressed abruptly. The sudden decompression leads to formation of numerous carrier gas bubbles in the sample liquid. These bubbles assist the release of dissolved analyte species from the liquid to the gas phase. The released analytes are immediately transferred to the mass spectrometer, producing signals in the time domain. Because the release of the analyte species is confined to a short period of time (a few seconds), the temporal signals have high amplitudes and high signal-to-noise ratios.
The pressures involved in the fizzy extraction process are very low (~150 kPa)4; much lower than in supercritical fluid extraction5 (e.g., &#8805;10 MPa). The technique does not require the use of any special consumable items (columns, cartridges). Only small volumes of solvents are used for dilution and cleaning. The extraction device can be assembled by chemists with medium technical skills using widely available parts4; for example, open-source electronic modules6,7. Fizzy extraction can be coupled on-line with modern mass spectrometers equipped with atmospheric pressure chemical ionization (APCI) interface. Because gas-phase extracts are transferred to the ion source, operation of fizzy extraction does not substantially contaminate vulnerable parts of the mass spectrometer.
The purpose of this visualized experiment article is to guide the viewers on how to implement fizzy extraction in a simple analytical task. While the core of the fizzy extraction system is as described in our previous report4, several improvements have been introduced to make the operation more straightforward. A microcontroller equipped with an LCD screen shield has been incorporated into the system to display the key extraction parameters in real time. All the functions are programmed in the microcontroller scripts, and there is no longer a need to use an external computer to control the extraction system.

<table><tbody><tr><td>Water</td><td>Fisher</td><td>W6212</td><td>Diluent</td></tr><tr><td>Ethanol</td><td>Sigma-Aldrich</td><td>32221-2.5L</td><td>Diluent</td></tr><tr><td>(R)-(+)-Limonene</td><td>Sigma-Aldrich</td><td>183164-100ML</td><td>Standard</td></tr><tr><td>Carbon dioxide</td><td>ChiaLung</td><td>n/a</td><td>Carrier gas</td></tr><tr><td>Cellulose tissue, Kimwipes Kimtech</td><td>Kimberly-Clark</td><td>34120</td><td>Used for cleaning</td></tr><tr><td>Triple quadrupole mass spectrometer</td><td>Shimadzu</td><td>LCMS-8030</td><td>Detection system</td></tr><tr><td>Atmospheric pressure chemical ionization interface</td><td>Shimadzu</td><td>Duis</td><td>Ion source</td></tr><tr><td>20-mL screw top headspace glass vial with septum cap</td><td>Thermo Fisher Scientific</td><td>D-52379</td><td>Sample vial</td></tr><tr><td>LabSolutions software</td><td>Shimadzu</td><td>n/a</td><td>version 5.82</td></tr><tr><td>PeakFit software</td><td>Systat Software</td><td>n/a</td><td>version 4.12</td></tr><tr><td>OriginPro software</td><td>OriginLab</td><td>n/a</td><td>version 8</td></tr></tbody></table>

fizzy extraction of volatile organic compounds combined with atmospheric pressure chemical ionization quadrupole mass spectrometry

Chemical analysis of volatile and semivolatile compounds dissolved in liquid samples can be challenging. The dissolved components need to be brought to the gas phase, and efficiently transferred to a detection system. Fizzy extraction takes advantage of the effervescence phenomenon. First, a carrier gas (here, carbon dioxide) is dissolved in the sample by applying overpressure and stirring the sample. Second, the sample chamber is decompressed abruptly. Decompression leads to the formation of numerous carrier gas bubbles in the sample liquid. These bubbles assist the release of the dissolved analyte species from the liquid to the gas phase. The released analytes are immediately transferred to the atmospheric pressure chemical ionization interface of a triple quadrupole mass spectrometer. The ionizable analyte species give rise to mass spectrometric signals in the time domain. Because the release of the analyte species occurs over short periods of time (a few seconds), the temporal signals have high amplitudes and high signal-to-noise ratios. The amplitudes and areas of the temporal peaks can then be correlated with concentrations of the analytes in the liquid samples subjected to fizzy extraction, which enables quantitative analysis. The advantages of fizzy extraction include: simplicity, speed, and limited use of chemicals (solvents).

At the beginning, the fizzy extraction system is tested with a standard solution. Subsequently, the real sample and real sample spiked with standard are analyzed. The areas of the temporal peaks of extraction events are correlated with concentrations of the analytes in the liquid samples subjected to fizzy extraction, which enables quantitative analysis. Here, we performed double standard addition to demonstrate quantitative capabilities of the technique (Figure 7). The linear regression led to the following function (Figure 8):
Peak_area = (3.25 x 107 &#177; 0.36 x 107)Cdiluted + (2,770 &#177; 276)
The variable Cdiluted refers to the concentration of the added limonene standard (in moles per liter, M). Based on the obtained slope and intercept values, the concentration of limonene in the diluted lime juice sample was: 8.51 x 10-5 &#177; 1.26 x 10-5 M. After multiplying that value by the dilution factor (5), the concentration of limonene in the original lime juice sample was: 4.26 x 10-4 &#177; 0.63 x 10-4 M.
The main descriptors of analytical performance such as limits of detection and quantification of this method were reported previously4. For example, the analytical repeatability (RSD) was 6-19%. The limit of detection for limonene was ~10-4 M4. We believe that the imperfections of the fizzy extraction prototype system contribute to the signal fluctuations. These imperfections can be eliminated when the fizzy extraction apparatus is developed further and commercialized.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56008/56008fig1.jpg" /> 
Figure 1: Photographs of the fizzy extraction system (with labels). (A) top view; (B) front view. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56008/56008fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56008/56008fig2.jpg" /> 
Figure 2: Setting up the data acquisition software of the triple quadrupole mass spectrometer. The consecutive steps are shown in the panels A and B. The numbers refer to the protocol step 4.5. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56008/56008fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56008/56008fig3.jpg" /> 
Figure 3: Workflow of typical fizzy extraction experiment. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56008/56008fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56008/56008fig4.jpg" /> 
Figure 4: Exporting extracted ion currents from the data acquisition software to ASCII files. The numbers refer to the protocol step 6.1. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56008/56008fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56008/56008fig5.jpg" /> 
Figure 5: Importing the raw data sets into the peak integration software, and measuring peak areas. The consecutive steps are shown in the panels A and B. The numbers refer to the protocol step 6.2. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56008/56008fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56008/56008fig6.jpg" /> 
Figure 6: Inputting the measured peak areas into a spreadsheet in the data analysis software, and conducting linear regression. The numbers refer to the protocol step 6.3. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56008/56008fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/56008/56008fig7.jpg" /> 
Figure 7: Typical raw data for limonene standard solution and lime juice sample. 
Extracted ion currents recorded at the m/z 81, following fragmentation of the parent ion at the m/z 137 by collision-induced dissociation. Collision gas: argon. Collision voltage: -20 V. The raw data for: diluted lime juice; diluted lime juice after the first addition of limonene standard (concentration increase: 6.20 x 10-5 M); diluted lime juice after the second addition of limonene standard (concentration increase: 1.24 x 10-4 M). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56008/56008fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/56008/56008fig8.jpg" /> 
Figure 8: Plot relating temporal peak areas with concentration of limonene standard added to diluted lime juice sample. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56008/56008fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Fizzy Extraction of Volatile Organic Compounds Combined with Atmospheric Pressure Chemical Ionization Quadrupole Mass Spectrometry at JoVE.com

Fizzy Extraction of Volatile Organic Compounds Combined with Atmospheric Pressure Chemical Ionization Quadrupole Mass Spectrometry

Fizzy extraction is a new laboratory technique for analysis of volatile and semivolatile compounds. A carrier gas is dissolved in the liquid sample by applying overpressure and stirring the sample. The sample chamber is then decompressed. The analyte species are liberated to the gas phase due to effervescence.

Chemical analysis of volatile and semivolatile compounds dissolved in liquid samples can be challenging. The dissolved components need to be brought to ...

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Nanyang Technological University

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Analytical Chemistry

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Unit 1

Mass Spectrometry Fragmentation Methods

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7.6K Views.  National Chiao Tung University.  Fizzy extractie is een nieuwe laboratorium techniek voor de analyse van vluchtige en semivolatiele verbindingen. Een draaggas wordt opgelost in het vloeibare monster door overdruk toe te passen en het monster te roeren. De monsterkamer wordt dan gedecomprimeerd. De analysesoort wordt bevrijd aan de gasfase door bruising. 

Video: Fizzy Extraction of Volatile Organic Compounds Combined with Atmospheric Pressure Chemical Ionization Quadrupole Mass Spectrometry

Characterizing Bacterial Volatiles using Secondary Electrospray Ionization Mass Spectrometry (SESI-MS)

Secondary electrospray ionization mass spectrometry (SESI-MS) is a method developed for the rapid detection of volatile compounds, without the need for sample pretreatment. The method was first described by Fenn and colleagues1 and has been applied to the detection of illicit drugs2 and explosives3-4, the characterization of skin volatiles5, and the analysis of breath6-7.
SESI ionization occurs by proton transfer reactions between the electrospray solution and the volatile analyte, and is therefore suitable for the analysis of hetero-organic molecules, just as in traditional electrospray ionization (ESI). However, unlike standard ESI, the proton transfer process of SESI occurs in the vapor phase rather than in solution (Fig. 1), and therefore SESI is best suited for detecting organic volatiles and aerosols.
We are expanding the use of SESI-MS to the detection of bacterial volatiles as a method for bacterial identification and characterization8. We have demonstrated that SESI-MS volatile fingerprinting, combined with a statistical analysis method, can be used to differentiate bacterial genera, species, and mixed cultures in a variety of growth media.8 Here we provide the steps for obtaining bacterial volatile fingerprints using SESI-MS, including the instrumental parameters that should be optimized to ensure robust bacterial identification and characterization.

Characterizing Bacterial Volatiles using Secondary Electrospray Ionization Mass Spectrometry SESI-MS

Secondary electrospray ionization mass spectrometry (SESI-MS) enables the detection of volatile organic compounds (VOCs) without the need for any sample pretreatment. This protocol provides instructions for the rapid (within minutes) characterization of bacterial VOCs using SESI-MS.

Secondary electrospray ionization mass spectrometry (SESI-MS) is a method developed for the rapid detection of volatile compounds, without the need for ...

Bioengineering

Analysis of Volatile and Oxidation Sensitive Compounds Using a Cold Inlet System and Electron Impact Mass Spectrometry

This video presents a protocol for the mass spectrometrical analysis of volatile and oxidation sensitive compounds using electron impact ionization. The analysis of volatile and oxidation sensitive compounds by mass spectrometry is not easily achieved, as all state-of-the-art mass spectrometric methods require at least one sample preparation step, e.g., dissolution and dilution of the analyte (electrospray ionization), co-crystallization of the analyte with a matrix compound (matrix-assisted laser desorption/ionization), or transfer of the prepared samples into the ionization source of the mass spectrometer, to be conducted under atmospheric conditions. Here, the use of a sample inlet system is described which enables the analysis of volatile metal organyls, silanes, and phosphanes using a sector field mass spectrometer equipped with an electron impact ionization source. All sample preparation steps and the sample introduction into the ion source of the mass spectrometer take place either under air-free conditions or under vacuum, enabling the analysis of compounds highly susceptible to oxidation. The presented technique is especially of interest for inorganic chemists, working with metal organyls, silanes, or phosphanes, which have to be handled using inert conditions, such as the Schlenk technique. The principle of operation is presented in this video.

This video presents a protocol for the mass spectrometrical analysis of volatile and oxidation sensitive compounds using electron impact ionization. The presented technique is especially of interest for inorganic chemists, working with metal organyls, silanes, or phosphanes which have to be handled using inert conditions, such as the Schlenk technique.

This video presents a protocol for the mass spectrometrical analysis of volatile and oxidation sensitive compounds using electron impact ionization. The ...

Quantitative Detection of Trace Explosive Vapors by Programmed Temperature Desorption Gas Chromatography-Electron Capture Detector

The direct liquid deposition of solution standards onto sorbent-filled thermal desorption tubes is used for the quantitative analysis of trace explosive vapor samples. The direct liquid deposition method yields a higher fidelity between the analysis of vapor samples and the analysis of solution standards than using separate injection methods for vapors and solutions, i.e., samples collected on vapor collection tubes and standards prepared in solution vials. Additionally, the method can account for instrumentation losses, which makes it ideal for minimizing variability and quantitative trace chemical detection. Gas chromatography with an electron capture detector is an instrumentation configuration sensitive to nitro-energetics, such as TNT and RDX, due to their relatively high electron affinity. However, vapor quantitation of these compounds is difficult without viable vapor standards. Thus, we eliminate the requirement for vapor standards by combining the sensitivity of the instrumentation with a direct liquid deposition protocol to analyze trace explosive vapor samples.

Trace explosive vapors of TNT and RDX collected on sorbent-filled thermal desorption tubes were analyzed using a programmed temperature desorption system coupled to GC with an electron capture detector. The instrumental analysis is combined with direct liquid deposition method to reduce sample variability and account for instrumentation drift and losses.

The direct liquid deposition of solution standards onto sorbent-filled thermal desorption tubes is used for the quantitative analysis of trace explosive ...

PTR-ToF-MS Coupled with an Automated Sampling System and Tailored Data Analysis for Food Studies: Bioprocess Monitoring, Screening and Nose-space Analysis

Proton Transfer Reaction (PTR), combined with a Time-of-Flight (ToF) Mass Spectrometer (MS) is an analytical approach based on chemical ionization that belongs to the Direct-Injection Mass Spectrometric (DIMS) technologies. These techniques allow the rapid determination of volatile organic compounds (VOCs), assuring high sensitivity and accuracy. In general, PTR-MS requires neither sample preparation nor sample destruction, allowing real time and non-invasive analysis of samples. PTR-MS are exploited in many fields, from environmental and atmospheric chemistry to medical and biological sciences. More recently, we developed a methodology based on coupling PTR-ToF-MS with an automated sampler and tailored data analysis tools, to increase the degree of automation and, consequently, to enhance the potential of the technique. This approach allowed us to monitor bioprocesses (e.g. enzymatic oxidation, alcoholic fermentation), to screen large sample sets (e.g. different origins, entire germoplasms) and to analyze several experimental modes (e.g. different concentrations of a given ingredient, different intensities of a specific technological parameter) in terms of VOC content. Here, we report the experimental protocols exemplifying different possible applications of our methodology: i.e. the detection of VOCs released during lactic acid fermentation of yogurt (on-line bioprocess monitoring), the monitoring of VOCs associated with different apple cultivars (large-scale screening), and the in vivo study of retronasal VOC release during coffee drinking (nosespace analysis).

Proton Transfer Reaction Time of Flight Mass Spectrometry allows high-sensitivity, rapid and non-invasive analysis of volatile organic compounds. To demonstrate its potential, we give three examples: lactic acid fermentation of yogurt (on-line bioprocess monitoring), different apple genotypes (large-scale screening), and retronasal space after drinking coffee (nosespace analysis).

Proton Transfer Reaction (PTR), combined with a Time-of-Flight (ToF) Mass Spectrometer (MS) is an analytical approach based on chemical ionization that ...

Extraction of Aqueous Metabolites from Cultured Adherent Cells for Metabolomic Analysis by Capillary Electrophoresis-Mass Spectrometry

Metabolomic analysis is a promising omics approach to not only understand the specific metabolic regulation in cancer cells compared to normal cells but also to identify biomarkers for early-stage cancer detection and prediction of chemotherapy response in cancer patients. Preparation of uniform samples for metabolomic analysis is a critical issue that remains to be addressed. Here, we present an easy and reliable protocol for extracting aqueous metabolites from cultured adherent cells for metabolomic analysis using capillary electrophoresis-mass spectrometry (CE-MS). Aqueous metabolites from cultured cells are analyzed by culturing and washing cells, treating cells with methanol, extracting metabolites, and removing proteins and macromolecules with spin columns for CE-MS analysis. Representative results using lung cancer cell lines treated with diamide, an oxidative reagent, illustrate the clearly observable metabolic shift of cells under oxidative stress. This article would be especially valuable to students and investigators involved in metabolomics research, who are new to harvesting metabolites from cell lines for analysis by CE-MS.

The purpose of this article is to describe a protocol for extraction of aqueous metabolites from cultured adherent cells for metabolomic analysis, particularly, capillary electrophoresis-mass spectrometry.

Metabolomic analysis is a promising omics approach to not only understand the specific metabolic regulation in cancer cells compared to normal cells but ...

Cancer Research

Temperature-programmed Deoxygenation of Acetic Acid on Molybdenum Carbide Catalysts

Temperature programmed reaction (TPRxn) is a simple yet powerful tool for screening solid catalyst performance at a variety of conditions. A TPRxn system includes a reactor, furnace, gas and vapor sources, flow control, instrumentation to quantify reaction products (e.g., gas chromatograph), and instrumentation to monitor the reaction in real time (e.g., mass spectrometer). Here, we apply the TPRxn methodology to study molybdenum carbide catalysts for the deoxygenation of acetic acid, an important reaction among many in the upgrading/stabilization of biomass pyrolysis vapors. TPRxn is used to evaluate catalyst activity and selectivity and to test hypothetical reaction pathways (e.g., decarbonylation, ketonization, and hydrogenation). The results of the TPRxn study of acetic acid deoxygenation show that molybdenum carbide is an active catalyst for this reaction at temperatures above ca. 300 &#176;C and that the reaction favors deoxygenation (i.e., C-O bond-breaking) products at temperatures below ca. 400 &#176;C and decarbonylation (i.e., C-C bond-breaking) products at temperatures above ca. 400 &#176;C.

Presented here is a protocol for the operation of a micro-scale temperature-programmed reactor for evaluating the catalytic performance of molybdenum carbide during acetic acid deoxygenation.

Temperature programmed reaction (TPRxn) is a simple yet powerful tool for screening solid catalyst performance at a variety of conditions. A TPRxn system ...

Real-time Breath Analysis by Using Secondary Nanoelectrospray Ionization Coupled to High Resolution Mass Spectrometry

Exhaled volatile organic compounds (VOCs) have aroused considerable interest, since they can serve as biomarkers for disease diagnosis and environmental exposure in a non-invasive manner. In this work, we present a protocol to characterize the exhaled VOCs in real time by using secondary nanoelectrospray ionization coupled to high resolution mass spectrometry (Sec-nanoESI-HRMS). The homemade Sec-nanoESI source was readily set up based on a commercial nanoESI source. Hundreds of peaks were observed in the background-subtracted mass spectra of exhaled breath, and the mass accuracy values are -4.0-13.5 ppm and -20.3-1.3 ppm in the positive and negative ion detection modes, respectively. The peaks were assigned with accurate elemental composition according to the accurate mass and isotopic pattern. Less than 30 s is used for one exhalation measurement, and it takes approximately 7 min for six replicated measurements.

A protocol for characterizing chemical composition of exhaled breath in real time by using secondary nanoelectrospray ionization coupled to high resolution mass spectrometry is demonstrated.

Exhaled volatile organic compounds (VOCs) have aroused considerable interest, since they can serve as biomarkers for disease diagnosis and environmental ...

Combustion Chemistry of Fuels: Quantitative Speciation Data Obtained from an Atmospheric High-temperature Flow Reactor with Coupled Molecular-beam Mass Spectrometer

This manuscript describes a high-temperature flow reactor experiment coupled to the powerful molecular beam mass spectrometry (MBMS) technique. This flexible tool offers a detailed observation of chemical gas-phase kinetics in reacting flows under well-controlled conditions. The vast range of operating conditions available in a laminar flow reactor enables access to extraordinary combustion applications that are typically not achievable by flame experiments. These include rich conditions at high temperatures relevant for gasification processes, the peroxy chemistry governing the low temperature oxidation regime or investigations of complex technical fuels. The presented setup allows measurements of quantitative speciation data for reaction model validation of combustion, gasification and pyrolysis processes, while enabling a systematic general understanding of the reaction chemistry. Validation of kinetic reaction models is generally performed by investigating combustion processes of pure compounds. The flow reactor has been enhanced to be suitable for technical fuels (e.g. multi-component mixtures like Jet A-1) to allow for phenomenological analysis of occurring combustion intermediates like soot precursors or pollutants. The controlled and comparable boundary conditions provided by the experimental design allow for predictions of pollutant formation tendencies. Cold reactants are fed premixed into the reactor that are highly diluted (in around 99 vol% in Ar) in order to suppress self-sustaining combustion reactions. The laminar flowing reactant mixture passes through a known temperature field, while the gas composition is determined at the reactors exhaust as a function of the oven temperature. The flow reactor is operated at atmospheric pressures with temperatures up to 1,800 K. The measurements themselves are performed by decreasing the temperature monotonically at a rate of -200 K/h. With the sensitive MBMS technique, detailed speciation data is acquired and quantified for almost all chemical species in the reactive process, including radical species.

An investigation of the oxidative combustion chemistry of novel biofuels, fuel components, or jet fuels by comparison of quantitative speciation data is presented. The data can be used for kinetic model validation and enables fuel assessment strategies.This manuscript describes the atmospheric high-temperature flow reactor and demonstrates its capabilities.

This manuscript describes a high-temperature flow reactor experiment coupled to the powerful molecular beam mass spectrometry (MBMS) technique. This ...

Profiling Volatile Compounds in Blackcurrant Fruit using Headspace Solid-Phase Microextraction Coupled to Gas Chromatography-Mass Spectrometry

There is an increasing interest in measuring volatile organic compounds (VOCs) emitted by ripe fruits for the purpose of breeding varieties or cultivars with enhanced organoleptic characteristics and thus, to increase consumer acceptance. High-throughput metabolomic platforms have been recently developed to quantify a wide range of metabolites in different plant tissues, including key compounds responsible for fruit taste and aroma quality (volatilomics). A method using headspace solid-phase microextraction (HS-SPME) coupled with gas chromatography-mass spectrometry (GC-MS) is described here for the identification and quantification of VOCs emitted by ripe blackcurrant fruits, a berry highly appreciated for its flavor and health benefits.
Ripe fruits of blackcurrant plants (Ribes nigrum) were harvested and directly frozen in liquid nitrogen. After tissue homogenization to produce a fine powder, samples were thawed and immediately mixed with sodium chloride solution. Following centrifugation, the supernatant was transferred into a headspace glass vial containing sodium chloride. VOCs were then extracted using a solid-phase microextraction (SPME) fiber and a gas chromatograph coupled to an ion trap mass spectrometer. Volatile quantification was performed on the resulting ion chromatograms by integrating peak area, using a specific m/z ion for each VOC. Correct VOC annotation was confirmed by comparing retention times and mass spectra of pure commercial standards run under the same conditions as the samples. More than 60 VOCs were identified in ripe blackcurrant fruits grown in contrasting European locations. Among the identified VOCs, key aroma compounds, such as terpenoids and C6 volatiles, can be used as biomarkers for blackcurrant fruit quality. In addition, advantages and disadvantages of the method are discussed, including prospective improvements. Furthermore, the use of controls for batch correction and minimization of drift intensity have been emphasized.

A headspace solid-phase microextraction-gas-chromatography platform is described here for fast, reliable, and semi-automated volatile identification and quantification in ripe blackcurrant fruits. This technique can be used to increase knowledge about fruit aroma and to select cultivars with enhanced flavor for the purpose of breeding.

There is an increasing interest in measuring volatile organic compounds (VOCs) emitted by ripe fruits for the purpose of breeding varieties or cultivars ...

Biochemistry

Capturing Actively Produced Microbial Volatile Organic Compounds from Human-Associated Samples with Vacuum-Assisted Sorbent Extraction

Volatile organic compounds (VOCs) from biological samples have unknown origins. VOCs may originate from the host or different organisms from within the host&#39;s microbial community. To disentangle the origin of microbial VOCs, volatile headspace analysis of bacterial mono- and co-cultures of Staphylococcus aureus, Pseudomonas aeruginosa, and Acinetobacter baumannii, and stable isotope probing in biological samples of feces, saliva, sewage, and sputum were performed. Mono- and co-cultures were used to identify volatile production from individual bacterial species or in combination with stable isotope probing to identify the active metabolism of microbes from the biological samples.
Vacuum-assisted sorbent extraction (VASE) was employed to extract the VOCs. VASE is an easy-to-use, commercialized, solvent-free headspace extraction method for semi-volatile and volatile compounds. The lack of solvents and the near-vacuum conditions used during extraction make developing a method relatively easy and fast when compared to other extraction options such as tert-butylation and solid phase microextraction. The workflow described here was used to identify specific volatile signatures from mono- and co-cultures. Furthermore, analysis of the stable isotope probing of human associated biological samples identified VOCs that were either commonly or uniquely produced. This paper presents the general workflow and experimental considerations of VASE in conjunction with stable isotope probing of live microbial cultures.

This protocol describes the extraction of volatile organic compounds from a biological sample with the vacuum-assisted sorbent extraction method, gas chromatography coupled with mass spectrometry using the Entech Sample Preparation Rail, and data analysis. It also describes culture of biological samples and stable isotope probing.

Volatile organic compounds (VOCs) from biological samples have unknown origins. VOCs may originate from the host or different organisms from within the ...

Fizzy Extraction of Volatile Organic Compounds Combined with Atmospheric Pressure Chemical Ionization Quadrupole Mass Spectrometry

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