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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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

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 &#62; 5.0x104) and 173 peaks (peak intensity &#62; 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 detail...

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

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

8.8K Views.  Jinan University. 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.

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

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry (UPLC-HRMS)

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first separated into polar and non-polar fractions by a liquid-liquid phase extraction, and partially purified to facilitate downstream analysis. Both aqueous (polar metabolites) and organic (non-polar metabolites) phases of the initial extraction are processed to survey a broad range of metabolites. Metabolites are separated by different liquid chromatography methods based upon their partition properties. In this method, we present microflow ultra-performance (UP)LC methods, but the protocol is scalable to higher flows and lower pressures. Introduction into the mass spectrometer can be through either general or compound optimized source conditions. Detection of a broad range of ions is carried out in full scan mode in both positive and negative mode over a broad m/z range using high resolution on a recently calibrated instrument. Label-free differential analysis is carried out on bioinformatics platforms. Applications of this approach include metabolic pathway screening, biomarker discovery, and drug development.

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry UPLC-HRMS

Untargeted metabolomics provides a hypothesis generating snapshot of a metabolic profile. This protocol will demonstrate the extraction and analysis of metabolites from cells, serum, or tissue. A range of metabolites are surveyed using liquid-liquid phase extraction, microflow ultraperformance liquid chromatography/high-resolution mass spectrometry (UPLC-HRMS) coupled to differential analysis software.

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first ...

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

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 ...

Leaf Spray Mass Spectrometry: A Rapid Ambient Ionization Technique to Directly Assess Metabolites from Plant Tissues

Plants produce thousands of small molecules that are diverse in their chemical properties. Mass spectrometry (MS) is a powerful technique for analyzing plant metabolites because it provides molecular weights with high sensitivity and specificity. Leaf spray MS is an ambient ionization technique where plant tissue is used for direct chemical analysis via electrospray, eliminating chromatography from the process. This approach to sampling metabolites allows for a wide range of chemical classes to be detected simultaneously from intact plant tissues, minimizing the amount of sample preparation needed. When used with a high-resolution, accurate mass MS, leaf spray MS facilitates the rapid detection of metabolites of interest. It is also possible to collect tandem mass fragmentation data with this technique to facilitate a compound identification. The combination of accurate mass measurements and fragmentation is beneficial in confirming compound identities. The leaf spray MS technique requires only minor modifications to a nanospray ionization source and is a useful tool to further expand the capabilities of a mass spectrometer. Here, fresh leaf tissue from Sceletium tortuosum (Aizoaceae), a traditional medicinal plant from South Africa, is analyzed; numerous mesembrine alkaloids are detected with leaf spray MS.

Leaf spray mass spectrometry is a direct chemical analysis technique that minimizes the sample preparation and eliminates chromatography, allowing for the rapid detection of small molecules from plant tissues.

Plants produce thousands of small molecules that are diverse in their chemical properties. Mass spectrometry (MS) is a powerful technique for analyzing ...

Biochemistry

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).

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 ...

Sample Preparation for Probe Electrospray Ionization Mass Spectrometry

Mass spectrometry (MS) is a powerful tool in analytical chemistry because it provides very accurate information about molecules, such as mass-to-charge ratios (m/z), which are useful to deduce molecular weights and structures. While it is essentially a destructive analytical method, recent advancements in the ambient ionization technique have enabled us to acquire data while leaving tissue in a relatively intact state in terms of integrity. Probe electrospray ionization (PESI) is a so-called direct method because it does not require complex and time-consuming pretreatment of samples. A fine needle serves as a sample picker, as well as an ionization emitter. Based on the very sharp and fine property of the probe tip, destruction of the samples is minimal, allowing us to acquire the real-time molecular information from living things in situ. Herein, we introduce three applications of PESI-MS technique that will be useful for biomedical research and development. One involves the application to solid tissue, which is the basic application of this technique for the medical diagnosis. As this technique requires only 10 mg of the sample, it may be very useful in the routine clinical settings. The second application is for in vitro medical diagnostics where human blood serum is measured. The ability to measure fluid samples is also valuable in various biological experiments where a sufficient volume of sample for conventional analytical techniques cannot be provided. The third application leans toward the direct application of probe needles in living animals, where we can obtain real-time dynamics of metabolites or drugs in specific organs. In each application, we can infer the molecules that have been detected by MS or use artificial intelligence to obtain a medical diagnosis.

This article introduces sample preparation methods for a unique real-time analytical method based on the ambient mass spectrometry. This method lets us perform real-time analysis of the biological molecules in vivo without any special pretreatments.

Mass spectrometry (MS) is a powerful tool in analytical chemistry because it provides very accurate information about molecules, such as mass-to-charge ...

Breath Collection from Children for Disease Biomarker Discovery

Breath collection and analysis can be used to discover volatile biomarkers in a number of infectious and non-infectious diseases, such as malaria, tuberculosis, lung cancer, and liver disease. This protocol describes a reproducible method for sampling breath in children and then stabilizing breath samples for further analysis with gas chromatography-mass spectrometry (GC-MS). The goal of this method is to establish a standardized protocol for the acquisition of breath samples for further chemical analysis, from children aged 4-15 years. First, breath is sampled using a cardboard mouthpiece attached to a 2-way valve, which is connected to a 3 L bag. Breath analytes are then transferred to a thermal desorption tube and stored at 4-5 &#176;C until analysis. This technique has been previously used to capture breath of children with malaria for successful breath biomarker identification. Subsequently, we have successfully applied this technique to additional pediatric cohorts. The advantage of this method is that it requires minimal cooperation on part of the patient (of particular value in pediatric populations), has a short collection period, does not require trained staff, and can be performed with portable equipment in resource-limited field settings.

This protocol describes a simple method for the acquisition of breath samples from children. Briefly, samples of mixed air are pre-concentrated in sorbent tubes prior to gas chromatography-mass spectrometry analysis. Breath biomarkers of infectious and non-infectious diseases can be identified using this breath collection method.

Breath collection and analysis can be used to discover volatile biomarkers in a number of infectious and non-infectious diseases, such as malaria, ...

Fast and Accurate Exhaled Breath Ammonia Measurement

This exhaled breath ammonia method uses a fast and highly sensitive spectroscopic method known as quartz enhanced photoacoustic spectroscopy (QEPAS) that uses a quantum cascade based laser. The monitor is coupled to a sampler that measures mouth pressure and carbon dioxide. The system is temperature controlled and specifically designed to address the reactivity of this compound. The sampler provides immediate feedback to the subject and the technician on the quality of the breath effort. Together with the quick response time of the monitor, this system is capable of accurately measuring exhaled breath ammonia representative of deep lung systemic levels. 
Because the system is easy to use and produces real time results, it has enabled experiments to identify factors that influence measurements. For example, mouth rinse and oral pH reproducibly and significantly affect results and therefore must be controlled. Temperature and mode of breathing are other examples. As our understanding of these factors evolves, error is reduced, and clinical studies become more meaningful. This system is very reliable and individual measurements are inexpensive. 
The sampler is relatively inexpensive and quite portable, but the monitor is neither. This limits options for some clinical studies and provides rational for future innovations.

Ammonia is an important physiologic metabolite relevant to various disease and wellness states. It is also a difficult molecule to measure in breath, which demands particular precautions be taken to obtain accurate results. Not all factors influencing ammonia are known, but progress can be difficult without accounting for these factors.

This exhaled breath ammonia method uses a fast and highly sensitive spectroscopic method known as quartz enhanced photoacoustic spectroscopy (QEPAS) that ...

Medicine

Versatile Dual-Inlet Sample Introduction System for Multi-Mode Single Particle Inductively Coupled Plasma Mass Spectrometry Analysis and Validation

Metal-containing nanoparticles (NP) can be characterized with inductively coupled plasma mass spectrometers (ICP-MS) in terms of their size and number concentration by using the single-particle mode of the instrument (spICP-MS). The accuracy of measurement depends on the setup, operational conditions of the instrument and specific parameters that are set by the user. The transport efficiency of the ICP-MS is crucial for the quantification of the NP and usually requires a reference material with homogenous size distribution and a known particle number concentration.
Currently, NP reference materials are available for only a few metals and in limited sizes. If particles are characterized without a reference standard, the results of both size and particle number may be biased. Therefore, a dual-inlet setup for characterizing nanoparticles with spICP-MS was developed to overcome this problem. This setup is based on a conventional introduction system consisting of a pneumatic nebulizer (PN) for nanoparticle solutions and a microdroplet generator (&#181;DG) for ionic calibration solutions. A new and flexible interface was developed to facilitate the coupling of &#181;DG, PN and the ICP-MS system. The interface consists of available laboratory components and allows for the calibration, nanoparticle (NP) characterization and cleaning of the arrangement, while the ICP-MS instrument is still running.
Three independent analysis modes are available for determining particle size and number concentration. Each mode is based on a different calibration principle. While mode I (counting) and mode III (&#181;DG) are known from the literature, mode II (sensitivity), is used to determine the transport efficiency by inorganic ionic standard solutions only. It is independent of NP reference materials. The &#181;DG based inlet system described here guarantees superior analyte sensitivities and, therefore, lower detection limits (LOD). The size dependent LODs achieved are less than 15 nm for all NP (Au, Ag, CeO2) investigated.

Here we provide a protocol for the use of a dual-inlet system for single particle inductively coupled mass spectrometry which allows for a standard independent nanoparticle characterization.

Metal-containing nanoparticles (NP) can be characterized with inductively coupled plasma mass spectrometers (ICP-MS) in terms of their size and number ...

Atmospheric-pressure Molecular Imaging of Biological Tissues and Biofilms by LAESI Mass Spectrometry

Ambient ionization methods in mass spectrometry allow analytical investigations to be performed directly on a tissue or biofilm under native-like experimental conditions. Laser ablation electrospray ionization (LAESI) is one such development and is particularly well-suited for the investigation of water-containing specimens. LAESI utilizes a mid-infrared laser beam (2.94 &mu;m wavelength) to excite the water molecules of the sample. When the ablation fluence threshold is exceeded, the sample material is expelled in the form of particulate matter and these projectiles travel to tens of millimeters above the sample surface. In LAESI, this ablation plume is intercepted by highly charged droplets to capture a fraction of the ejected sample material and convert its chemical constituents into gas-phase ions. A mass spectrometer equipped with an atmospheric-pressure ion source interface is employed to analyze and record the composition of the released ions originating from the probed area (pixel) of the sample. A systematic interrogation over an array of pixels opens a way for molecular imaging in the microprobe analysis mode. A unique aspect of LAESI mass spectrometric imaging is depth profiling that, in combination with lateral imaging, enables three-dimensional (3D) molecular imaging. With current lateral and depth resolutions of ~100 &mu;m and ~40 &mu;m, respectively, LAESI mass spectrometric imaging helps to explore the molecular structure of biological tissues. Herein, we review the major elements of a LAESI system and provide guidelines for a successful imaging experiment.

Laser ablation electrospray ionization (LAESI) is an atmospheric-pressure ion source for mass spectrometry. In the imaging mode, a mid-infrared laser probes the distributions of molecules across a tissue section or a biofilm. This technique presents a new approach for diverse bioanalytical studies carried out under native experimental conditions.