Name: Real-time Breath Analysis by Using Secondary Nanoelectrospray Ionization Coupled to High Resolution Mass Spectrometry (Video) | JoVE Journal
Uploaded: 2018-03-09T12:30:00.000+00:00
Duration: 8 min 23 s
Description: Learn real-time breath analysis using secondary nanoelectrospray ionization coupled to high-resolution mass spectrometry. This sensitive method enables non-invasive lung cancer biomarker detection and therapeutic monitoring.

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5 minutes

This work has been financially supported by National Natural Science Foundation of China (No. 91543117).

Caution: Please consult all relevant material safety data sheets (MSDS) before use. Please use appropriate personal protective equipment, e.g., lab coat, gloves, goggles, full length pants and closed-toe shoes).
1. Set up the Sec-nanoESI source
<ol>
	<li>Set up a Sec-nanoESI source according to the SESI process, i.e., the breath gas is introduced to intersect an electrospray plume and ionized by the charged droplets (Figure 1). The sources built in individual labs depend on the interface of the mass spectrometer used24,25. Here, set up the Sec-nanoESI source based on a commercial nanoESI source (Figure 2) and implement to a benchtop quadrupole orbitrap mass spectrometer. 
	NOTE: The main body of the source is a cubic stainless-steel chamber (Length 25 mm, I.D. 13 mm) (Figure 2b) with an inlet (I.D. 4 mm) to introduce the nanoESI capillary into the chamber. Thus, the chamber is not completely sealed (Figure 2b).</li>
	<li>Install two stainless-steel tubes (Length 8 mm, O.D. 5 mm, I.D. 3 mm) on each side of the chamber for gas delivery.</li>
	<li>Equip two quartz windows (I.D. 14 mm) on the top and bottom of the chamber to check the position of the tip of the nanoESI capillary and nanoESI spray either by eyes or a digital microscope.</li>
	<li>Weld the chamber to the sweep cone of the mass spectrometer. 
	NOTE: The design may change depending on the particular geometry of the atmospheric pressure interface of the mass spectrometer used in individual labs.</li>
</ol>
2. Instrument optimization
<ol>
	<li>Calibrate the mass spectrometer in both positive and negative ion detection modes according to manufacturer&#39;s instructions. By applying calibration, mass spectrometer parameters, such as lens potentials and detection conditions, are optimized to give good sensitivity and peak shape at a specified resolution value. The mass resolution of 70000 is used here.
	<ol>
		<li>Perform complete Q Exactive calibrations by using the commercial ESI source; however, mass calibration can be performed with any compatible sources, including customized ones.</li>
	</ol>
	</li>
	<li>Set the temperature of the ion transfer tube (ITT) of the mass spectrometer &#62; 100 &#176;C. Though the highest temperature can be set at 350 &#176;C, it may result in the decomposition of some compounds. Thus, 150&#176;C is used in this experiment. 
	NOTE: For the mass spectrometers featuring a sampling orifice instead of ITT, the temperature of the sampling orifice is set &#62; 100 &#176;C.</li>
	<li>For the ESI solvent and flow rate, select the appropriate ESI solvent on the basis of properties of solvent (e.g., polarity and volatility) and targeted compounds (e.g., proton affinity). A mixture of water and methanol in various ratios has been commonly used as ESI solvent25. In this experiment, use water containing 0.1% (v/v) formic acid, for high ionization efficiency has been reported this solvent13,19,23. Set the flow rate of ESI solvent in the range of 0-1.5 &#956;L/min and 200 nL/min. 
	NOTE: Degas ESI solvent for 30 min before use.</li>
	<li>Optimize Sec-nanoESI source parameters, mainly nanoESI voltage and nanoESI capillary tip position. The voltage commonly ranges from 2.0 to 4.5 kV. Use 2.5 kV here. 
	NOTE: Higher ESI voltage is applied as the flow rate increases. The distance between the tip and the mass spectrometer orifice can be adjusted from 1 to 5 mm. After optimization, the normalized intensity level (NL) observed in a mass spectrum should be &#62; 1x106 and the variation of total ion chromatogram (TIC) should be &#60; 10% in both positive and negative ion detection modes. The mass spectrum and TIC are obtained in the mass range of m/z 50-750.</li>
	<li>Apply pure gas to the source. This is an optional step, aiming at reducing the influence of VOCs from indoor air. High purity nitrogen gas (N2, 99.99%) or pure air can be used. With the presence of pure gas, the NL observed in a mass spectrum should be &#62; 1x105 and the variation of TIC should be &#60; 10% in both positive and negative ion detection modes. High purity N2 is used here and delivered at 0.8 L/min. 
	NOTE: The total flow rate of pure gas and breath gas should be higher than the flow rate through the orifice of the mass spectrometer.</li>
</ol>
3. Measurement of exhaled breath
<ol>
	<li>Inhale the indoor air and perform a normal exhalation to breathe out all the air in the lungs at a constant flow rate. Monitor the exhalation flow rate either by a manometer or a flow meter visible to the subject. Use Teflon (PTFE) tubing to deliver the breath gas23.
	<ol>
		<li>To prevent the condensation of water vapor inside the tubing, heat the tubing at a temperature of 80-100 oC7,23,27 or use a Nafion dryer28,29. In this experiment, the subject exhaled at 0.4 L/min controlled by a flow meter.</li>
		<li>Connect the inlet of the flow meter to a Nafion tubing (Length 60 cm) to remove the water vapor in the exhaled breath and connect the outlet of the flow meter to a PTFE tubing (length 13 cm, I.D. 4 mm). It takes &#60; 30 s for one exhalation measurement.</li>
		<li>Perform 4-6 replicated measurements28,29.</li>
		<li>To minimize confounding effects, have participants from eating, drinking, and brushing their teeth at least 30 min prior to the measurements23. 
		NOTE: To minimize the influence of VOCs from indoor air, it has been reported to inhale pure gas instead of indoor air26. When a Nafion tubing is used, some polar compounds may be lost.</li>
		<li>During measurement, keep checking if the ion intensity exceeds the linear detection limit of the instrument or not. The saturation of signal can lead to an artifact peak that does not practically result from the compound in the sample. By inhaling through the nose, part of ambient VOCs and particles would be removed; however, it is noteworthy that compounds in the nasal passages may also be detected.</li>
	</ol>
	</li>
</ol>
4. Obtain a breath fingerprint and a time trace of a compound
<ol>
	<li>Obtain chromatograms and mass spectra. Use software (e.g., Xcalibur) to record chromatograms and mass spectra. Because this is direct MS analysis and no chromatographic separation is performed, the total ion chromatogram (TIC) actually indicates the time trace of all the signals detected in the mass spectra and the extracted ion chromatogram (EIC) shows the time trace of a specified compound. 
	NOTE: For other commercial mass spectrometers, the chromatograms and mass spectra can be obtained by the corresponding data acquisition software.</li>
	<li>Obtain an exhaled breath fingerprint by selecting a number of scans in the TIC when exhaled breath is measured. Obtain a mass spectrum representing an average of these scans by the software.
	<ol>
		<li>To eliminate background peaks from the breath fingerprint, use the Subtract Background tool in the software. Please refer to the user guide provided by the manufacturer. In brief, select the same number of scans when no breath sample is introduced, and subtract the background mass spectrum from the breath fingerprint. 
		NOTE: In this method, the threshold to identify the features in the breath fingerprint is defined as three times the standard deviation of the background signal. For other commercial mass spectrometers, background subtraction can be performed by the corresponding data acquisition software.</li>
	</ol>
	</li>
	<li>Obtain a time trace of a specified compound. Select the peak of a targeted compound in the breath fingerprint, and the time trace of the compound is acquired subsequently by the software.</li>
</ol>

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<li>Martínez-Lozano, P., Fernandez de la Mora, J. <a target="_blank" href="http://www.sciencedirect.com/science/article/pii/S1387380607002254">Electrospray ionization of volatiles in breath.</a> Int. J. Mass Spectrom. 265 (1), 68-72 (2007).</li>
<li>Zeng, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Detection+of+creatinine+in+exhaled+breath+of+humans+with+chronic+kidney+disease+by+extractive+electrospray+ionization+mass+spectrometry%5BTitle%5D)+AND+%22J.+Breath+Res%22%5BJournal%5D)">Detection of creatinine in exhaled breath of humans with chronic kidney disease by extractive electrospray ionization mass spectrometry.</a> J. Breath Res. 10 (1), 016008-016015 (2016).</li>
<li>Chen, H. W., Wortmann, A., Zhang, W. H., Zenobi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rapid+in+vivo+fingerprinting+of+nonvolatile+compounds+in+breath+by+extractive+electrospray+ionization+quadrupole+time-of-flight+mass+spectrometry%5BTitle%5D)+AND+%22Angew.+Chem.+Int.+Ed%22%5BJournal%5D)">Rapid in vivo fingerprinting of nonvolatile compounds in breath by extractive electrospray ionization quadrupole time-of-flight mass spectrometry.</a> Angew. Chem. Int. Ed. 46 (4), 580-583 (2007).</li>
<li>Benoi, F. M., Davldson, W. R., Lovett, A. M., Nacson, S., Ngo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Breath+analysis+by+atmospheric+pressure+ionization+mass+spectrometry%5BTitle%5D)+AND+%22Anal.+Chem%22%5BJournal%5D)">Breath analysis by atmospheric pressure ionization mass spectrometry.</a> Anal. Chem. 55 (4), 805-807 (1983).</li>
<li>Bregy, L., Martínez-Lozano Sinues, P., Nudnova, M. M., Zenobi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Real-time+breath+analysis+with+active+capillary+plasma+ionization-ambient+mass+spectrometry%5BTitle%5D)+AND+%22J.+Breath+Res%22%5BJournal%5D)">Real-time breath analysis with active capillary plasma ionization-ambient mass spectrometry.</a> J. Breath Res. 8 (2), 027102-027110 (2014).</li>
<li>Gaugg, M. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Expanding+metabolite+coverage+of+real-time+breath+analysis+by+coupling+a+universal+secondary+electrospray+ionization+source+and+high+resolution+mass+spectrometry-a+pilot+study+on+tobacco+smokers%5BTitle%5D)+AND+%22J.+Breath+Res%22%5BJournal%5D)">Expanding metabolite coverage of real-time breath analysis by coupling a universal secondary electrospray ionization source and high resolution mass spectrometry-a pilot study on tobacco smokers.</a> J. Breath Res. 10 (1), 016010-016020 (2016).</li>
<li>Martínez-Lozano, P., Zingaro, L., Finiguerra, A., Cristoni, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Secondary+electrospray+ionization-mass+spectrometry%3A+breath+study+on+a+control+group%5BTitle%5D)+AND+%22J.+Breath+Res%22%5BJournal%5D)">Secondary electrospray ionization-mass spectrometry: breath study on a control group.</a> J. Breath Res. 5 (1), 016002-016012 (2011).</li>
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</ol>

The authors have nothing to disclose.

Constructing the Sec-nanoESI source based on a commercial nanoESI source, the ionization efficiency is higher than that of using an ESI source30. In addition, the ionization efficiency is further improved in a closed chamber, as it isolates the process from the ambient background air, and at the same time facilitates the mixing between the gas sample and the spray plume. By using a Sec-nanoESI, less parameters need to be optimized compared to an ESI source, making it easier for installation, appli...

With fast development of modern analytical techniques, hundreds of volatile organic compounds (VOCs) have been identified in human exhaled breath1. These VOCs mostly result from alveolar air (~350 mL for a healthy adult) and anatomical dead space air (~150 mL)2, which are affected by body metabolism3,4,5,6,7,8 and environmental pollution9, respectively. As a result, if identified, these VOCs are promising to be used as biomarkers for disease diagnosis and environmental exposure in a non-invasive manner.
Though gas chromatography mass spectrometry (GC-MS) is the most widely used technique for qualitative and quantitative analysis of exhaled VOCs2, direct MS techniques, which have been developed for real-time breath analysis, have the advantages of high time resolution and simple sample pre-preparation. Direct MS techniques, such as proton transfer reaction MS (PTR-MS)10, selected ion flow tube MS (SIFT-MS)11, secondary electrospray ionization MS (SESI-MS)12,13 (also named as extractive electrospray ionization MS, EESI-MS14,15), trace atmospheric gas analyzer (TAGA)16 and plasma ionization MS (PI-MS)17 have been investigated in recent years.
Among all the direct MS techniques, SESI is well-known as a universal soft ionization technique19,20,21; and the source is easy to be customized and coupled to different types of mass spectrometers, e.g., time of flight mass spectrometer8,15, ion trap mass spectrometer14 and orbitrap mass spectrometer12,18. Up to now, SESI-MS has been successfully used in diagnosing respiratory diseases22, gauging circadian rhythm3,6,23, pharmacokinetics7,8, and revealing metabolic pathways4, etc. Most recently, a commercial SESI source has become available.
In this study, a facile and compact secondary nanoelectrospray ionization source (Sec-nanoESI) was set up and coupled to a high-resolution mass spectrometer. Real-time measurements of exhaled VOCs in breath were presented.

<table><tbody><tr><td>Ultrapure water</td><td>Merck Millipore, USA</td><td>MPGP04001</td><td>Resistance &gt;18.2 M&amp;Omega;&amp;middot;cm</td></tr><tr><td>Formic acid</td><td>Sigma-Aldrich, USA</td><td>F0507</td><td>Corrosive to the respiratory tract.</td></tr><tr><td>Nitrogen gas</td><td>Guangzhou Shiyuan Gas Co. Ltd., China</td><td>N.A.&lt;sup&gt;a&lt;/sup&gt;</td><td>Purity &gt;99.99%</td></tr><tr><td>Q Exactive hybrid quadrupole-orbitrap mass spectrometer</td><td>Thermo Scientific, USA</td><td>02634L(S/N)</td><td>Beware of high voltage and high temperature</td></tr><tr><td>NanoESI source</td><td>Thermo Scientific, USA</td><td>ES002373(S/N); ES071(P/N)</td><td>Beware of high voltage and high temperature</td></tr><tr><td>Nano LC pump</td><td>Thermo Scientific, USA</td><td>5041.0010A(P/N)</td><td>/</td></tr><tr><td>Xcalibur software (Version 3.0)</td><td>Thermo Scientific, USA</td><td>BRE0008596</td><td>/</td></tr><tr><td>Dino-Lite Digital Microscope</td><td>Tech Video System (SuZhou) Co.Ltd., China</td><td>CQ401833R(S/N)</td><td>/</td></tr><tr><td>Nafion tubing</td><td>Perma Pure LLC, USA</td><td>ME60</td><td>/</td></tr><tr><td>PTFE tubing (I.D. 4 mm)</td><td>Dongguan Hongfu Insulating Material Co. Ltd., China</td><td>N.A.</td><td>Beware of the possible loss of polar compounds</td></tr><tr><td>Mass flow controller</td><td>Line-Tech, Korea</td><td>M15122007 (S/N)</td><td>/</td></tr><tr><td>Flow meter</td><td>Yuyao Industrial Automation Meter Factory, China</td><td>40784</td><td>/</td></tr><tr><td>&lt;sup&gt;a&lt;/sup&gt;N.A.: not available.</td><td></td><td></td><td></td></tr></tbody></table>

real-time breath analysis by using secondary nanoelectrospray ionization coupled to high resolution mass spectrometry

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.

Figure 3 shows the breath fingerprints in the mass range of m/z 50-750 recorded under both positive and negative ion detection modes. 291 peaks (peak intensity &gt; 5.0x104) and 173 peaks (peak intensity &gt; 3.0x104) have been observed in background-subtracted breath fingerprints in the positive and negative ion detection modes, respectively. To identify peaks in the mass spectra, please refer to prior publications for details<...

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 ...

real-time-breath-analysis-using-secondary-nanoelectrospray-ionization

Research

JoVE Journal

Chemistry

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