Internal Standard Addition System for Online Breath Analysis.

Breath analysis with secondary electrospray ionization (SESI) coupled to mass spectrometry (MS) is a sensitive method for breath metabolomics. To enable quantitative assessments using SESI-MS, a system was developed to introduce controlled amounts of gases into breath samples and carry out standard addition experiments. The system combines gas standard generation through controlled evaporation, humidification, breath dilution, and standard injection with the help of mass-flow controllers. The system can also dilute breath, which affects the signal of the detected components. This response can be used to filter out contaminating compounds in an untargeted metabolomics workflow. The system's quantitative capabilities have been shown through standard addition of pyridine and butyric acid into breath in real time. This system can improve the quality and robustness of breath data.

Alternative electrolyte solutions for untargeted breath metabolomics using secondary-electrospray ionization high-resolution mass spectrometry.

RATIONALE: Secondary-electrospray ionization (SESI) coupled with high-resolution mass spectrometry is a powerful tool for the discovery of biomarkers in exhaled breath. A primary electrospray consisting of aqueous formic acid (FA) is currently used to charge the volatile organic compounds in breath. To investigate whether alternate electrospray compositions could enable different metabolite coverage and sensitivities, the electrospray dopants NaI and AgNO3 were tested. METHODS: In a proof-of-principle manner, the exhaled breath of one subject was analyzed repeatedly with different electrospray solutions and with the help of a spectral stitching technique. Capillary diameter and position were optimized to achieve proper detection of exhaled breath. The detected features were then compared using formula annotation. Using an evaporation-based gas standard system, the signal response of the different solutions was probed. RESULTS: Principal component analysis revealed a substantial difference in features detected with AgNO3 . With silver, more sulfur-containing features and more unsaturated hydrocarbon compounds were detected. Furthermore, more primary amines were potentially ionized, as indicated by van Krewelen diagrams. In total, twice as many features were unique to AgNO3 than for other electrospray dopants. Using gas standards at known concentrations, the high sensitivity of FA as a dopant was demonstrated but also indicated alternate sensitivities of the other electrospray solutions. CONCLUSIONS: This work demonstrated the potential of AgNO3 as a complementary dopant for further biomarker discovery in SESI-based breath analysis.

Membrane inlet mass spectrometry method for food intake impact assessment on specific volatile organic compounds in exhaled breath.

This research work describes the development of a novel bioanalytical method for the assessment of food impact on selected exhaled breath volatile organic compounds (VOCs) using a fast and portable screening VOC prototype sensor based on membrane inlet mass spectrometry (MIMS). Method and sensor prototype functionality was verified by obtaining good response times, linearity in the examined concentration ranges, and sensitivity and repeatability for several breath VOCs-acetone, ethanol, n-pentane, and isoprene. A new VOC sensor prototype was also proven to be sensitive enough for selected breath VOC quantification with limits of detection at low part per billion (ppb) levels-5 ppb for n-pentane, 10 ppb for acetone and ethanol, and 25 ppb for isoprene. Food impact assessment was accomplished by tracking the levels of acetone, ethanol, n-pentane, and isoprene in exhaled breath samples collected from 50 healthy participants before the meal and 60 min and 120 min after the meal. For acetone, isoprene, and n-pentane, a larger impact was noticed 120 min after the meal, while for ethanol, it was after 60 min. Obtained VOC levels were in the expected concentration ranges. Mean values at all time points were ~ 500-900 ppb for acetone and ~ 400-600 ppb for ethanol. Most of the results for n-pentane were below 5 ppb, but the mean value for those which were detected was ~ 30 ppb. Along with samples, data about participants' lifestyle were collected via a short questionnaire, which were compared against obtained VOC levels in order to reveal some significant correlations between habits of participants and their breath VOC levels. Portable MS: monitoring of food impact on the levels of selected VOCs from exhaled breath.

Real-Time Detection of Aerosol Metals Using Online Extractive Electrospray Ionization Mass Spectrometry.

Metal emissions are of major environmental and practical concern because of their highly toxic effects on human health and ecosystems. Current technologies available in the market for their detection are typically limited by a time resolution of 1 h or longer (e.g., via semicontinuous X-ray fluorescence measurements) or are nonquantitative (e.g., laser ablation mass spectrometry). In this work, we report the development of a novel technique for the real-time detection and monitoring of metal particles in situ using an extractive electrospray ionization (EESI) source coupled to a high-resolution time-of-flight mass spectrometer (TOF-MS). The experiments were conducted in negative ionization mode using disodium ethylenediamine tetraacetic acid (EDTA) dihydrate to chelate with metals and form stable metal complexes. Results for water-soluble metal compounds were obtained. The following representative metal ions were examined: Pb, Cd, Zn, Ce (III), Ba, Ni, Fe(II), Fe(III), Cu(II), Cr, Mo, Co(II), Mg, Nd, Li, Ti, Ca, Cs, Ag, Tm, Er(III), La(III), Yb(III), Eu(III), Pr(III), Gd(III), Lu(III), Dy(III), Tb(III), Ho, and Ru(III). The results showed a very good linear mass response (R2 = 0.9983), low ng/m3 limits of detection (LoD), and a fast response time (1 s). The stability and repeatability of the developed EESI-TOF-MS were tested under complex dynamic and periodic experimental conditions, and negligible matrix effects were measured for internally and externally mixed metal particles. Benchmark testing against inductively coupled plasma-mass spectrometry (ICP-MS) was also performed, highlighting the online measurement capabilities of aerosol metals with a LoD lower than those of ICP-MS. Proof-of-concept ambient measurements were performed in New Delhi, India, and very promising results were obtained, allowing further exploitation elsewhere.

A Chemical Alphabet for Macromolecular Communications.

Molecular communications in macroscale environments is an emerging field of study driven by the intriguing prospect of sending coded information over olfactory networks. For the first time, this article reports two signal modulation techniques (on-off keying-OOK, and concentration shift keying-CSK) which have been used to encode and transmit digital information using odors over distances of 1-4 m. Molecular transmission of digital data was experimentally investigated for the letter "r" with a binary value of 01110010 (ASCII) for a gas stream network channel (up to 4 m) using mass spectrometry (MS) as the main detection-decoding system. The generation and modulation of the chemical signals was achieved using an automated odor emitter (OE) which is based on the controlled evaporation of a chemical analyte and its diffusion into a carrier gas stream. The chemical signals produced propagate within a confined channel to reach the demodulator-MS. Experiments were undertaken for a range of volatile organic compounds (VOCs) with different diffusion coefficient values in air at ambient conditions. Representative compounds investigated include acetone, cyclopentane, and n-hexane. For the first time, the binary code ASCII (American Standard Code for Information Interchange) is combined with chemical signaling to generate a molecular representation of the English alphabet. Transmission experiments of fixed-width molecular signals corresponding to letters of the alphabet over varying distances are shown. A binary message corresponding to the word "ion" was synthesized using chemical signals and transmitted within a physical channel over a distance of 2 m.

An optimised quadrupole mass spectrometer with a dual filter analyser for in-field chemical sniffing of volatile organic compounds.

We report a novel portable 17 kg system based on a quadrupole mass spectrometer (QMS) with an electronic power consumption of 24 W. The system can be used for the in-field identification of gases and volatile/semivolatile organic compounds (VOCs/SVOCs). The mass analyser is a custom-made quadrupole mass filter with a Brubaker pre-filter that gives a mass range of m/z 1-500. It is an upgrade of the previous m/z 1-200 range triple filter analyser system. Analyser design was optimized using 3D numerical simulations as a performance trade-off between single and triple filter designs while maintaining high sensitivity and ease of integration. This also required enhanced design of the electronic control unit (ECU) compared to the previous triple filter ECU designs with lower power consumption, size, weight and cost of the overall system. Another major ECU improvement includes high stability of DC voltage control and ultra-low RF drift, which is important for in-field applications that require stable mass peaks for reliable quantitative analysis and continuous monitoring. Experimental results are presented for the perfluorotributylamine (PFTBA) calibrant and acetone to assess the functionality of the instrument. Performance comparison between the dual and triple filter quadrupole analysers has also been done. Mass spectra are given for methyl benzoate (cocaine simulant), piperidine (phencyclidine simulant), cyclohexanone (C4 simulant) and 2-nitrotoluene (TNT simulant) to assess potential capability for the identification of threat compounds. All spectral results show good correlation with the NIST library mass spectra with unit resolution obtained for spectral peaks within a m/z 1-400 mass range.

Chemical Sniffing Instrumentation for Security Applications.

Border control for homeland security faces major challenges worldwide due to chemical threats from national and/or international terrorism as well as organized crime. A wide range of technologies and systems with threat detection and monitoring capabilities has emerged to identify the chemical footprint associated with these illegal activities. This review paper investigates artificial sniffing technologies used as chemical sensors for point-of-use chemical analysis, especially during border security applications. This article presents an overview of (a) the existing available technologies reported in the scientific literature for threat screening, (b) commercially available, portable (hand-held and stand-off) chemical detection systems, and (c) their underlying functional and operational principles. Emphasis is given to technologies that have been developed for in-field security operations, but laboratory developed techniques are also summarized as emerging technologies. The chemical analytes of interest in this review are (a) volatile organic compounds (VOCs) associated with security applications (e.g., illegal, hazardous, and terrorist events), (b) chemical "signatures" associated with human presence, and

Monitoring of human chemical signatures using membrane inlet mass spectrometry.

This work is an attempt to assist border security crackdown on illegal human immigration, by providing essential results on human chemical signatures. Data was obtained using a portable quadrupole mass spectrometer coupled with a membrane probe for volunteers of both genders and under different conditions in a container simulator. During experiments, participants were asked to follow various protocols while volatile organic compounds emitted from their breath, sweat, skin, and other biological excretes were continuously being monitored. Experimental setups using different membrane materials (both hydrophilic and hydrophobic) including heating of the sampling probe and sampling flow rates were examined. From our measurements, significant information was obtained for NH3, CO2, water, and volatile organic compounds levels, illustrating a human chemical profile and indicating human presence in a confined space.

Comparative analysis of feature annotation methods for SESI-HRMS in exhaled breath analysis.

Secondary electrospray ionization coupled to high-resolution mass spectrometry (SESI-HRMS) is a powerful method for the analysis of exhaled breath in real time. However, feature annotation is challenging due to the flow-injection nature of the technique. To evaluate alternative methods for enhancing feature annotation, a study was conducted where the exhaled breath of sixteen subjects was condensed and analyzed using dynamic headspace vacuum in-trap extraction gas chromatography-mass spectrometry (DHS-V-ITEX-GC-MS) and liquid chromatography coupled to mass spectrometry (LC-MS) using polar and reverse-phase conditions along with a data-independent MS2-acquisition method based on multiple injections. The annotation results obtained from these methods were compared to those from SESI-HRMS. The use of these techniques on breath condensate is unprecedented. The GC-MS method primarily detected compounds of exogenous origin, particularly additives in oral hygiene products like menthol. On the other hand, LC-MS detected a vast number of features, especially with the utilized data-independent acquisition method. Chemical classes to these features were assigned in-silico. In positive ion mode, mostly amino acids and amines were detected, while the largest group in negative ion mode consisted of carboxylic acids. Approximately 25% and 5% of SESI features had a corresponding match with LC-MS and GC-MS. While both GC-MS and LC-MS methods partially overlapped with the SESI features, there was limited overlap of both in the mass-to-charge range from 150 to 200. In conclusion, both GC-MS and LC-MS analysis of breath condensate can serve as supplementary tools for annotating features obtained from SESI-MS. However, to increase confidence in the annotation results, combining these methods with additional on-line fragmentation techniques is recommended.

Systematic study of polymer gas sampling bags for offline analysis of exhaled breath.

Polymeric bags are a widely applied, simple, and cost-effective method for the storage and offline analysis of gaseous samples. Various materials have been used as sampling bags, all known to contain impurities and differing in their cost, durability, and storage capabilities. Herein, we present a comparative study of several well-known bag materials, Tedlar (PVF), Kynar (PVDF), Teflon (PTFE), and Nalophan (PET), as well as a new material, ethylene vinyl copolymer (EVOH), commonly used for storing food. We investigated the influences of storage conditions, humidity, bag cleaning, and light exposure on volatile organic compound concentration (acetone, acetic acid, isoprene, benzene, limonene, among others) in samples of exhaled human breath stored in bags for up to 48 h. Specifically, we show high losses of short-chain fatty acids (SCFAs) in bags of all materials (for most SCFAs, less than 50% after 8 h of storage). We found that samples in Tedlar, Nalophan, and EVOH bags undergo changes in composition when exposed to UV radiation over a period of 48 h. We report high initial impurity levels in all the bags and their doubling after a period of 48 h. We compare secondary electrospray ionization and proton transfer reaction mass spectrometry in the context of offline analysis after storage in sampling bags. We provide an analytical perspective on the temporal evolution of bag contents by presenting the intensity changes of all significantm/zfeatures. We also present a simple, automated, and cost-effective offline sample introduction system, which enables controlled delivery of collected gaseous samples from polymeric bags into the mass spectrometer. Overall, our findings suggest that sampling bags exhibit high levels of impurities, are sensitive to several environmental factors (e.g. light exposure), and provide low recoveries for some classes of compounds, e.g. SCFAs.

Elucidating the Role of Ion Suppression in Secondary Electrospray Ionization.

Ion suppression is a known matrix effect in electrospray ionization (ESI), ambient pressure chemical ionization (APCI), and desorption electrospray ionization (DESI), but its characterization in secondary electrospray ionization (SESI) is lacking. A thorough understanding of this effect is crucial for quantitative applications of SESI, such as breath analysis. In this study, gas standards were generated by using an evaporation-based system to assess the susceptibility and suppression potential of acetone, deuterated acetone, deuterated acetic acid, and pyridine. Gas-phase effects were found to dominate ion suppression, with pyridine exhibiting the most significant suppressive effect, which is potentially linked to its gas-phase basicity. The impact of increased acetone levels on the volatiles from exhaled breath condensate was also examined. In humid conditions, a noticeable decrease in intensity of approximately 30% was observed for several features at an acetone concentration of 1 ppm. Considering that this concentration is expected for breath analysis, it becomes crucial to account for this effect when SESI is utilized to quantitatively determine specific compounds.

Food and lifestyle impact on breath VOCs using portable mass spectrometer-pilot study across European countries.

In the modern world, many people are changing old dietary and lifestyle habits to improve the quality of their living-to treat or just prevent possible diseases. The main goal of this pilot study was to assess the food and lifestyle impact on exhaled breath volatile organic compounds (VOCs) in various population groups. It was done by employing a recently validated portable membrane-inlet mass spectrometer-MIMS. Thus, the obtained results would also represent the additional confirmation for the employment of the new instrument in the breath analysis. The pilot study involved 151 participants across Europe, including people with overweight, obesity, type 2 diabetes mellitus, cardiovascular disease, people with poor-quality diet and professional athletes. Exhaled breath acetone, ethanol, isoprene, and n-pentane levels were determined in samples before the meal, and 120 min after the meal. Obtained basal ppbvvalues were mainly in accordance with previously reported, which confirms that MIMS instrument can be used in the breath analysis. Combining the quantified levels along with the information about the participants' lifestyle habits collected via questionnaire, an assessment of the food and lifestyle impact was obtained. Notable alteration in examined VOC levels upon meal consumption was detected in more than 70% of all participants, with exception for isoprene, which was affected in about half of participants. Lifestyle parameters impact was examined using statistical analysis of variance (ANOVA) on ranks test. Statistically significant differences in basal breath VOC levels were observed among all examined population groups. Also, n-pentane and ethanol levels significantly differed in people of different ages, as well as acetone levels in people with different physical activity habits. These findings are promising for further, more focused research using MIMS technique in breath analysis.

Analysis of volatile short-chain fatty acids in the gas phase using secondary electrospray ionization coupled to mass spectrometry.

Quantification of metabolites present within exhaled breath is a major challenge for on-line breath analysis. It is also important for gauging the analytical performance, accuracy, reproducibility, reliability, and stability of the measuring technology. Short-chain fatty acids (SCFAs) are of high interest for nutrition and health. Their quantification enables a deep mechanistic understanding of a wide range of biological processes and metabolic pathways, while their high volatility makes them an attractive target for breath analysis. This article reports, for the first time, the development and testing of a modular, dynamic vapor generator for the qualitative and quantitative analysis of volatile SCFAs in the gaseous phase using a secondary electrospray ionization (SESI) source coupled to a high-resolution mass spectrometer. Representative compounds tested included acetic acid, propionic acid, butyric acid, pentanoic acid and hexanoic acid. Gas-phase experiments were performed both in dry and humid (95% relative humidity) conditions from ppt to low ppb concentrations. The results obtained exhibited excellent linearity within the examined concentration range, low limits of detection and quantification down to the lower ppt area. Mixture effects were also investigated and are presented.

How Soft Is Secondary Electrospray Ionization?

Secondary electrospray ionization (SESI) mass spectrometry (MS) is a direct infusion technique often used for untargeted metabolomics, e.g., for online breath analysis. SESI is thought to be a soft ionization method, which is important to avoid interference from in-source fragments and to simplify compound annotation. In this work, benzylammonium ions, formed from volatile benzylamines, with known bond dissociation enthalpies were used as thermometer ions to investigate the internal energy distribution of ions that are produced by SESI. It is shown that SESI is softer than electrospray ionization (ESI), and therefore, SESI indeed qualifies as a soft ionization technique. However, we also found that the standard MS instrument settings used in the SESI community are relatively harsh. Proper soft tuning of the instrument is essential to fully benefit from the softness that SESI can provide. Moreover, there is evidence from in-source collision-induced dissociation (CID) experiments that analytes can be solvated in SESI under soft conditions, which supports a recently proposed SESI mechanism referred to as ligand switching.

Breath response following a nutritional challenge monitored by secondary electrospray ionization high-resolution mass spectrometry.

On-line breath analysis using secondary electrospray ionization coupled to high-resolution mass spectrometry (SESI-HRMS) is a sensitive method for biomarker discovery. The strengths of this technology have already been demonstrated in the clinical environment. For the first time, this study demonstrates the application of SESI-HRMS in the field of nutritional science using a standardized nutritional intervention, consisting of a high-energy shake (950 kcal, 8% protein, 35% sugar and 57% fat). Eleven subjects underwent the intervention on three separate days and their exhaled breath was monitored up to six hours postprandially. In addition, sampling was performed during equivalent fasting conditions for selected subjects. To estimate the impact of inter- and intra-individual variability, analysis of variance simultaneous component analysis was conducted, revealing that the inter-individual variability accounted for 30% of the data variation. To distinguish the effect of the intervention from fasting conditions, partial least squares discriminant analysis was performed. Candidate compound annotation was performed with pathway analysis and collision-induced dissociation (CID) experiments. Pathway analysis highlighted, among others, features associated with the metabolism of linoleate, butanoate and amino sugars. Tentative compounds annotated through CID measurements include fatty acids, amino acids, and amino acid derivatives, some of them likely derived from nutrients by the gut microbiome (e.g. propanoate, indoles), as well as organic acids from the Krebs cycle. Time-series clustering showed an overlap of observed kinetic trends with those reported previously in blood plasma.

Analysis of volatile emissions from grape berries infected with Aspergillus carbonarius using hyphenated and portable mass spectrometry.

Mycotoxins represent a serious risk for human and animal health. Οchratoxin A (OTA) is a carcinogenic mycotoxin produced by A. carbonarius that constitutes a severe problem for viticulture. In this study, we investigate the development of novel detection and on-line monitoring approaches for the detection of OTA in the field (i.e. out of the chemical laboratory) using advanced molecular sensing. Both stand-alone and hyphenated mass spectrometry (MS) based systems (e.g. Time-of-Flight ToF-MS and gas chromatography GC combined with MS) and compact portable membrane inlet MS (MIMS) have been employed for the first time to detect and monitor volatile emissions of grape berries infected by the fungus Aspergillus carbonarius. In vacuo (electron impact-EI) and ambient ionisation (electrospray ionisation-ESI) techniques were also examined. On-line measurements of the volatile emissions of grape berries, infected by various strains of A. carbonarius with different toxicity levels, were performed resulting in different olfactory chemical profiles with a common core of characteristic mass fragments, which could be eventually used for on-site detection and monitoring allowing consequent improvement in food security.

In-Situ Analysis of Essential Fragrant Oils Using a Portable Mass Spectrometer.

A portable mass spectrometer was coupled to a direct inlet membrane (DIM) probe and applied to the direct analysis of active fragrant compounds (3-methylbutyl acetate, 2-methyl-3-furanthiol, methyl butanoate, and ethyl methyl sulfide) in real time. These fragrant active compounds are commonly used in the formulation of flavours and fragrances. Results obtained show that the portable mass spectrometer with a direct membrane inlet can be used to detect traces of the active fragrant compounds in complex mixtures such as essential fragrant oils and this represents a novel in-situ analysis methodology. Limits of detection (LOD) in the sub-ppb range (< 2.5 pg) are demonstrated. Standard samples in the gaseous phase presented very good linearity with RSD % at 5 to 7 for the selected active fragrant compounds (i.e., isoamyl acetate, 2-methyl-3-furanthiol, methyl butanoate, and methyl ethyl sulphide). The rise and fall times of the DIM probe are in the ranges from 15 to 31 seconds and 23 to 41 seconds, respectively, for the standard model compounds analysed. The identities of the fragrance active compounds in essential oil samples (i.e., banana, tangerine, papaya, and blueberry muffin) were first identified by comparison with a standard fragrance compounds mixture using their major fragment peaks, the NIST standard reference library, and gas chromatography mass spectrometry (GC-MS) analysis. No sample preparation is required for analysis using a portable mass spectrometer coupled to a DIM probe, so the cycle time from ambient air sampling to the acquisition of the results is at least 65 seconds.

Molecular Communication over Gas Stream Channels using Portable Mass Spectrometry.

The synthetic generation/coding and transmission of olfactory information over a gas stream or an odor network is a new and unexplored field. Application areas vary from the entertainment or advertisement industry to security and telemedicine. However, current technological limitations frustrate the accurate reproduction of decoded and transmitted olfactory data. This study describes the development, testing, and characterization of a novel odor emitter (OE) that is used to investigate the generation-encoding of gaseous standards with odorous characteristics with a regulatable way, for scent transmission purposes. The calibration and the responses of a developed OE were examined using a portable quadrupole mass spectrometer (MS). Experiments were undertaken for a range of volatile organic compounds (VOCs) at different temperatures and flow rates. Individual compounds and mixtures were tested to investigate periodic and dynamic transmission characteristics within two different size tubular containers for distances up to 3 m. Olfactory information transmission is demonstrated using MS as the main molecular sensor for odor detection and monitoring and for the first time spatial encryption of olfactory information is shown. Graphical Abstract ᅟ.

Membrane inlet mass spectrometry for homeland security and forensic applications.

A man-portable membrane inlet mass spectrometer has been built and tested to detect and monitor characteristic odors emitted from the human body and also from threat substances. In each case, a heated membrane sampling probe was used. During human scent monitoring experiments, data were obtained for inorganic gases and volatile organic compounds emitted from human breath and sweat in a confined space. Volatile emissions were detected from the human body at low ppb concentrations. Experiments with compounds associated with narcotics, explosives, and chemical warfare agents were conducted for a range of membrane types. Test compounds included methyl benzoate (odor signature of cocaine), piperidine (precursor in clandestine phencyclidine manufacturing processes), 2-nitrotoluene (breakdown product of TNT), cyclohexanone (volatile signature of plastic explosives), dimethyl methylphosphonate (used in sarin and soman nerve agent production), and 2-chloroethyl ethyl sulfide (simulant compound for sulfur mustard gas). Gas phase calibration experiments were performed allowing sub-ppb LOD to be established. The results showed excellent linearity versus concentration and rapid membrane response times.