Jonas Herth, Felix Schmidt, Sarah Basler, Noriane A Sievi and Malcolm Kohler

Exhaled breath analysis has emerged as a non-invasive and promising method for early detection of lung cancer, offering a novel approach for diagnosis through the identification of specific biomarkers present in a patient's breath…

M. Z. Islam, S. E. Räisänen, A. Schudel, K. Wang, T. He, C. Kunz, Y. Li, X. Ma, A. M. Serviento, Z. Zeng, F. Wahl, R. Zenobi, S. Giannoukos, and M. Niu.

Previously, we used secondary electrospray ionization-mass spectrometry (SESI-MS) to investigate the diurnal patterns and signal intensities of exhaled (EX) volatile fatty acids (VFA) of dairy cows. The current study aimed to validate the potential of an exhalomics approach for evaluating rumen fermentation. The experiment was conducted in a switchback design, with 3 periods of 9 d each, including 7 d for adaptation and 2 d for sampling. Four rumen-cannulated original Swiss Brown (Braunvieh) cows were randomly assigned to 1 of 2 diet sequences (ABA or BAB): (A) low starch (LS; 6.31% starch on a dry matter basis) and (B) high starch (HS; 16.2% starch on a dry matter basis). Feeding was once per day at 0830 h. Exhalome (with the GreenFeed System), and rumen samples were collected 8 times to represent every 3 h of a day, and EX-VFA and ruminal (RM)-VFA were analyzed using SESI-MS and HPLC, respectively. Furthermore, the VFA concentration in the gas phase (HR-VFA) was predicted based on RMVFA and Henry’s Law (HR) constants….

Mo Awchi, Kapil Dev Singh, Sara Bachmann Brenner, Marie-Anne Burckhardt, Melanie Hess, Jiafa Zeng, Alexandre N Datta, Urs Frey, Urs Zumsteg, Gabor Szinnai, Pablo Sinues

Purpose: This feasibility study aimed to investigate the use of exhaled breath analysis to capture and quantify relative changes of metabolites during resolution of acute diabetic ketoacidosis under insulin and rehydration therapy…

Cedric Wüthrich, Renato Zenobi, Stamatios Giannoukos

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….

Mateusz Fido, Simone Hersberger, Andreas Güntner, Renato Zenobi, Stamatios Giannoukos

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...

Rosa A Sola-Martínez, Jiafa Zeng, Mo Awchi, Amanda Gisler, Kim Arnold, Kapil Dev Singh, Urs Frey, Manuel Cánovas Díaz, Teresa de Diego Puente, Pablo Sinues

Secondary electrospray ionization-high resolution mass spectrometry (SESI-HRMS) is an established technique in the field of breath analysis characterized by its short analysis time, as well as high levels of sensitivity and selectivity. Traditionally, SESI-HRMS has been used for real-time breath analysis, which requires subjects to be at the location of the analytical platform. Therefore, it limits the possibilities for an introduction of this methodology in day-to-day clinical practice. However, recent methodological developments have shown feasibility on the remote sampling of exhaled breath in Nalophan® bags prior to measurement using SESI-HRMS…

Fouad Choueiry, Rui Xu, Kelly Meyrath, Jiangjiang Zhu.

Secondary electrospray ionization high-resolution mass spectrometry (SESI-HRMS) is an innovative analytical technique for the rapid and non-invasive analysis of volatile organic compounds (VOCs). However, compound annotation and ion suppression in the SESI source has hindered feature detection, stability and reproducibility of SESI-HRMS in untargeted volatilomics.

Fouad Choueiry, Andrew Gold, Rui Xu, Shiqi Zhang and Jiangjiang Zhu.

The symbiotic relationship between the gut microbial population is capable of regulating numerous aspects of host physiology, including metabolism. Bacteria can modulate the metabolic processes of the host by feeding on nutritional components within the lumen and releasing bioactive components into circulation. Endogenous volatile organic compound (VOC) synthesis is dependent on the availability of precursors found in mammalian metabolism

Cedric Wüthrich, Stamatios Giannoukos, Renato Zenobi

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…

Mo Awchi, Kapil Dev Singh, Patricia E Dill, Urs Frey, Alexandre N Datta, and Pablo Sinues.

Therapeutic drug monitoring (TDM) of medications with a narrow therapeutic window is a common clinical practice to minimize toxic effects and maximize clinical outcomes. Routine analyses rely on the quantification of systemic blood concentrations of drugs. Alternative matrices such as exhaled breath are appealing because of their inherent non-invasive nature. This is especially the case for pediatric patients. We have recently

Bettina Streckenbach

Metabolomics, or the comprehensive study of metabolites and involved processes, provides insights into the current physiological state of humans. In the clinical setting, the detection and quantification of specific metabolites has proven to be invaluable in diagnostic testing. Herein, body samples that are non-invasively accessible are of particular interest…

M.Z. Islam, S. Giannoukos, Räisänen, K. Wang, X. Ma, F. Wahl, R. Zenobi, M. Niu.

To date, the commonly used methods to assess rumen fermentation are invasive. Exhaled breath contains hundreds of volatile organic compounds (VOC) that can reflect animal physiological processes. In the present study, for the first time, we aimed to use a non-invasive metabolomics approach based on high-resolution mass spectrometry to identify rumen fermentation parameters in dairy cows

Ronja Weber, Bettina Streckenbach, Lara Welti, Demet Inci, Malcolm Kohler, Nathan Perkins, Renato Zenobi, Srdjan Micic , and Alexander Moeller.

There is a need to improve the diagnosis and management of pediatric asthma. Breath analysis aims to address this by non-invasively assessing altered metabolism and disease-associated processes.

Patrik Španěl, Kseniya Dryahina, Maroua Omezzine Gnioua, David Smith

The detection sensitivity of secondary electrospray ionisation mass spectrometry (SESI-MS) is much lower for saturated aldehydes than for unsaturated aldehydes. This needs to be understood in terms of gas phase ion-molecule reaction kinetics and energetics to make SESI-MS analytically more quantitative.

Kim Arnold, Philippe Dehio, Jonas Lötscher, Kapil Dev Singh, Diego García-Gómez, Christoph Hess, Pablo Sinues, and Maria L. Balmer

Dendritic cells (DCs) actively sample and present antigen to cells of the adaptive immune system and are thus vital for successful immune control and memory formation. Immune cell metabolism and function are tightly interlinked, and a better understanding of this interaction offers potential to develop immunomodulatory strategies. However, current approaches for assessing the immune cell metabolome are often limited by endpoint measurements, may involve laborious sample preparation, and may lack unbiased, temporal resolution of the metabolome. In this study, we present a novel setup coupled to a secondary electrospray ionization-high resolution mass spectrometric (SESI-HRMS) platform allowing headspace analysis of immature andactivated DCs in real-time with minimal sample preparation and intervention, with high technical reproducibility and potential for automation.

Mo Awchi, Pablo Sinues, Alexandre N. Datta, Diego García-Gómez, and Kapil Dev Singh

Real-time breath analysis using secondary electrospray ionization coupled with high-resolution mass spectrometry is a fast and noninvasive method to access the metabolic state of a person. However, it lacks the ability to unequivocally assign mass spectral features to compounds due to the absence of chromatographic separation. This can be overcomed by using exhaled breath condensate and conventional liquid chromatography–mass spectrometry (LC–MS) systems.

Bettina Streckenbach, Justinas Sakas, Nathan Perkins, Malcolm Kohler, Alexander Moeller and Renato Zenobi.

Applications for direct breath analysis by mass spectrometry (MS) are rapidly expanding. One of the more recent mass spectrometry-based approaches is secondary electrospray ionization coupled to high-resolution mass spectrometry (SESI-HRMS). Despite increasing usage, the SESI methodology still lacks standardization procedures for quality control and absolute quantification.

Cedric Wüthrich, Zhiyuan Fan, Guy Vergères, Fabian Wahl, Renato Zenobi and Stamatios Giannoukos.s

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.

Amanda Gisler, Kapil Dev Singh, Jiafa Zeng, Martin Osswald, Mo Awchi, Fabienne Decrue, Felix Schmidt, Noriane A. Sievi, Xing Chen, Jakob Usemann, Urs Frey, Malcolm Kohler, Xue Li, Pablo Sinues

Exhaled breath contains valuable information at the molecular level and offers promising potential for precision medicine. However, few breath tests transition to routine clinical practice, partly because of the missing validation in multicenter trials. Therefore, we developed and applied an interoperability framework for standardized multicenter data acquisition and processing for breath analysis with secondary electrospray ionization-high resolution mass spectrometry.

Ronja Weber, Jérôme Kaeslin, Sophia Moeller, Nathan Perkins, Srdjan Micic, and Alexander Moeller.

Environmental volatile organic compounds (VOCs) from the ambient air potentially influence on-line breath analysis measurements by secondary electrospray ionization high-resolution mass spectrometry (SESI-HRMS). The aim of this study was to investigate how inhaling through a VOC filter affects the detected breath profiles and whether it is feasible to integrate such filters into routine measurements..

T. Zivkovic Semren*, S. Majeed, M. Fatarova, C. Laszlo, C. Pak, S. Steiner, G. Vidal, A. Kuczaj, A. Mazurov, M. C. Peitsch, N. V. Ivanov, J. Hoeng, P. A. Guy

Inhalation as a route for administering drugs and dietary supplements has garnered significant attention over the last decade. We performed real-time analysis of aerosols using secondary electrospray ionization (SESI) technology interfaced with high-resolution mass spectrometry (HRMS)

R Weber, B Streckenbach, J Kaeslin, L Welti, D Inci, N Perkins, R Zenobi, S Micic, A Möller.
We hypothesized that the breath of children with allergic asthma contains a unique signature of disease specific metabolites. Using secondary electrospray ionization high-resolution mass spectrometry (SESI-HRMS), we aimed to identify relevant VOCs to assess underlying interconnections between biomarkers belonging to common metabolic pathways in the pathophysiology of asthma.

Felix Schmidt, Nora Nowak, Patrick Baumgartner, Thomas Gaisl, Stefan Malesevic, Bettina Streckenbach, Noriane A. Sievi, Esther I. Schwarz, Renato Zenobi, Steven A. Brown, and Malcolm Kohler.

The direct pathophysiological effects of obstructive sleep apnea (OSA) have been well described. However, the systemic and metabolic consequences of OSA are less well understood.

A. Gómez-Mejia, K. Arnold, J. Bär, K. Dev Singh, T. C. Scheier, S. D. Brugger, A. S. Zinkernagel, P. Sinues

Early detection of pathogenic bacteria is needed for rapid diagnostics allowing adequate and timely treatment of infections.

Ronja Weber, Nathan Perkins, Tobias Bruderer, Srdjan Micic and Alexander Moeller.

The early detection of inflammation and infection is important to prevent irreversible lung damage in cystic fibrosis. Novel and non-invasive monitoring tools would be of high benefit for the quality of life of patients.

F. Schmidt ; M. Osswald ; R. Zenobi ; M. Kohler

Monitoring of drug pharmacokinetics is used in personalized therapy, therapeutic drug monitoring (TDM), toxicology assessments, doping controls and clinical drug development. Drugs are predominantly measured in plasma, serum or urine. Monitoring of volatile organic compounds (VOCs) in breath has so far not received much attention, but nevertheless it has many advantages over conventional approaches

B. Streckenbach, M. Osswald, S. Malesevic, R. Zenobi, and M. Kohler

Chemical analysis of exhaled breath have suggested the existence of an OSA-specific metabolic signature. Here, we validated this diagnostic approach and the proposed marker compounds, as well as their potential to reliably diagnose OSA.

Jérôme Kaeslin, Cedric Wüthrich, Stamatios Giannoukos, and Renato Zenobi

Secondary electrospray ionization (SESI) is a soft ionization method, which is important to avoid interference from in-source fragments and to simplify compound annotation. In this work, it is shown that SESI is softer than electrospray ionization (ESI), and therefore, SESI indeed qualifies as a soft ionization technique. However…

Jérôme Kaeslin, Cedric Wüthrich, Stamatios Giannoukos, Renato Zenobi

It is a set of accompanying files for the publication "How soft is secondary electrospray ionization?".

A. Gómez-Mejia, K. Arnold, J. Bär, K. Dev Singh, T. C. Scheier, S. D. Brugger, A. S. Zinkernagel, P. Sinues

Early detection of pathogenic bacteria is needed for rapid diagnostics allowing adequate and timely treatment of infections.

C. Wüthrich, M.de Figueiredo, K. J. Burton-Pimentel, G. Vergères, F. Wahl, R. Zenobi and S. Giannoukos

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. Tentative compounds include fatty acids, amino acids, and amino acid derivatives, some of them likely derived from nutrients by the gut microbiome, 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.

Kim Arnold, Xing Chen, Hui Zhang, Kapil Dev Singh, Zhihong Yin, Yao Yao, Tiangang Luan, Pablo Sinues, and Xue Li

Secondary electrospray ionization-high resolution mass spectrometry allows monitoring in vivo 2H-incorporation of metabolites in a non-invasive and real-time setup and opens new opportunities to use 2H tracing to extend current metabolic studies, especially those with a focus on anaerobic glycolysis, lysine methylation and gut microbiome via monitoring of short-chain fatty acids.

Charles D.Smith, Ashley C.Fulton, Mark Romanczyk, Braden C. Giordano, Christopher J. Katilie, Lauryn E. DeGreeffe

Determination of fentanyl vapor signature enables the identification of target analytes for the indirect detection of the parent opioid without direct sample handling.

Hendrik G. Mengers, Christina Schier, Martin Zimmermann, Martin C. H. Gruhlke, Eric Block, Lars M. Blank*, Alan J. Slusarenko

Using SESI-Orbitrap MS, we measured gas phase concentrations of allicin evaporating from a pure solution. The SESI-Orbitrap MS was used to follow the known chemistry of alliin, isoallin and methiin conversion in garlic, onion and ramsons. Allicin and its metabolites were also measured in human breath after garlic consumption. These results demonstrate the utility of SESI-Orbitrap MS for analysis of sulfur-containing volatiles from plants and for capturing volatilomes of foodstuffs in general.

Hendrik G. Mengers, Martin Zimmermann & Lars M. Blank

We use secondary electrospray ionisation high-resolution Orbitrap mass spectrometry (SESI-Orbitrap MS) to monitor the complete yeast volatilome every 2.3 s. Over 200 metabolites were identified during growth in shake flasks and bioreactor cultivations, all with their unique intensity profile. Special attention was paid to ethanol as biotech largest product and to acetaldehyde as an example of a low-abundance but highly volatile metabolite. Volatilome shifts are visible, e.g. after glucose depletion, fatty acids are converted to ethyl esters in a detoxification mechanism after stopped fatty acid biosynthesis. This work showcases the SESI-Orbitrap MS system for tracking microbial physiology without the need for sampling and for time-resolved discoveries during metabolic transitions.

Wetter Slack, Emma (ETH Zurich)

Dataset used in Non-invasive monitoring of microbiota and host metabolism using secondary electrospray ionization.

Jiayi Lan, Giorgia Greter, Bettina Streckenbach, Markus Arnoldini, Renato Zenobi & Emma Slack

We demonstrate that secondary electrospray ionization mass spectrometry can be used to non-invasively monitor metabolic activity of the intestinal microbiome of a live, awake mouse. This was achieved via analysis of the headspace volatile and semi-volatile metabolome of individual gut microbiota bacterial species growing in pure culture, as well as from live gnotobiotic mice specifically colonized with these microbes. The microbial origin of these compounds was confirmed by feeding of heavy-isotope labeled microbiota-accessible sugars. This reveals that the microbiota is a major contributor to the released metabolites of a whole live mouse, and that it is possible to capture the catabolism of sugars and cross-feeding within the gut microbiota of a living animal using volatile/semi-volatile metabolite monitoring.

R. Weber, B. Streckenbach, L. Welti, D. Inci, M. Kohler, N. Perkins, R. Zenobi, S. Micic⋆, A. Moeller

Breath analysis was performed via a SESI source linked to a high-resolution mass spectrometer. 48 allergic asthmatics and 56 healthy controls were included in the study. We found several pathways that are well-represented by the significant metabolites, for example lysine degradation elevated in the asthmatic group and two arginine pathways in the healthy group. For the first time, a large number of breath-derived volatile organic compounds (VOCs) that discriminate children with allergic asthma from healthy controls were identified. Many are linked to well-described metabolic pathways and chemical families involved in pathophysiological processes of asthma. Furthermore, a subset of these VOCs showed high potential for clinical diagnostic applications.

Chiara Veronese Francesco Segrado Riccardo Caldarella Roberto Boffi Rosaria Orlandi

Breath analysis for the identification of volatile organic compounds by mass spectrometry is a very innovative and non-invasive technology, which represents a great opportunity for an early and personalised diagnosis. In this pilot study we recruited a series of volunteers, smokers and non-smokers, characterized from the respiratory point of view, and profiled their exhaled breath through SESI- HRMS technology. The aim of the study is to identify a volatile molecular signature associated with tobacco use. The supervised analysis highlighted 32 features that discriminate the breath of smokers and non-smoker subjects, at the baseline. We therefore identified a volatile molecular signature closely related to tobacco smoke, which will be characterized in subsequent studies

Fouad Choueiry, Addison Barham, Jiangjiang Zhu

Lung cancer is one of the leading causes of cancer incidence and cancer-related deaths in the world. Early diagnosis of pulmonary tumors results in improved survival compared to diagnosis with more advanced disease, yet early disease is not reliably indicated by symptoms. Despite of the improved testing and monitoring techniques for lung cancer in the past decades, most diagnostic tests, such as sputum cytology or tissue biopsies, are invasive and risky, rendering them unfeasible for large population screening. The non-invasive analysis of exhaled breath has gained attentions as an innovative screening method to measure chemical alterations within the human volatilome profile as a result of oncogenesis. More importantly, volatile organic compounds (VOCs) have been correlated to the pathophysiology of disease since the source of volatile compounds relies mostly on endogenous metabolic processes that are altered as a result of disease onset.

Bettina Streckenbach

Exhaled breath can reveal insights about the metabolic state of the human body through the endo- and exogenous compounds it contains. The extent of detectable compounds, however, was revolutionized by the application of mass spectrometry. More specifically, secondary electrospray ionization high-resolution mass spectrometry (SESI-HRMS) enables the detection of a broad range of breath-derived compounds simultaneously and with high sensitivity. Together with its rapid and non-invasive nature, direct breath analysis by SESI-HRMS raised particular interest for clinical applications. Over the past years, various clinical trials successfully demonstrated the technology´s capability for biomarker discovery in exhaled breath in adults and more recently in children. Current challenges lie within the potential translation of SESI-HRMS into clinical settings and the requirements therein, such as biomarker identification and validation, which became a focus of more recent studies..

Jiafa Zeng, Alexandra Christen, Kapil Dev Singh, Urs Frey, and Pablo Sinues

Real-time breath analysis by high-resolution mass spectrometry (HRMS) is a promising method to non-invasively retrieve relevant biochemical information. In this work, we conducted a head-to-head comparison of two ionization techniques: Secondary electrospray ionization (SESI) and plasma ionization (PI), for the analysis of exhaled breath. Two commercially available SESI and PI sources were coupled to the same HRMS device to analyze breath of two healthy individuals in a longitudinal study. We analyzed 58 breath specimens in both platforms, leading to 2,209 and 2,296 features detected by SESI-HRMS and by PI-HRMS, respectively. 60% of all the mass spectral features were detected in both platforms. However, remarkable differences were noted in terms of the signal-to-noise ratio (S/N), whereby the median (interquartile range, IQR) S/N ratio for SESI-HRMS was 115 (IQR = 408), whereas for PI-HRMS it was 5 (IQR = 5). Differences in the mass spectral profiles for the same samples make the inter-comparability of both techniques problematic. Overall, we conclude that both techniques are excellent for real-time breath analysis because of the very rich mass spectral fingerprints. However, further work is needed to fully understand the exact metabolic insights one can gather using each of these platforms.

Kolli, A.R., Semren, T.Z., Bovard, D. et al.

In vitro screening for pharmacological activity of existing drugs showed chloroquine and hydroxychloroquine to be effective against severe acute respiratory syndrome coronavirus 2. Oral administration of these compounds to obtain desired pulmonary exposures resulted in dose-limiting systemic toxicity in humans. However, pulmonary drug delivery enables direct and rapid administration to obtain higher local tissue concentrations in target tissue. In this work, inhalable formulations for thermal aerosolization of chloroquine and hydroxychloroquine were developed, and their physicochemical properties were characterized. Thermal aerosolization of 40 mg/mL chloroquine and 100 mg/mL hydroxychloroquine formulations delivered respirable aerosol particle sizes with 0.15 and 0.33 mg per 55 mL puff, respectively. In vitro toxicity was evaluated by exposing primary human bronchial epithelial cells to aerosol generated from Vitrocell. An in vitro exposure to 7.24 μg of chloroquine or 7.99 μg hydroxychloroquine showed no significant changes in cilia beating, transepithelial electrical resistance, and cell viability. The pharmacokinetics of inhaled aerosols was predicted by developing a physiologically based pharmacokinetic model that included a detailed species-specific respiratory tract physiology and lysosomal trapping.

Cedric Wüthrich, Stamatios Giannoukos

The mass spectrometer used in this study was an Orbitrap Q-Exactive Plus (Thermo Scientific) operated with the manufacturer’s standard control software (ExactiveTune, version 2.9, Thermo Scientific) and Xcalibur (version 4.1. 31.9, Thermo Scientific). Mass calibration was done according to the instrument manual and was always more recent than seven days according to specifications.

Jérôme Kaeslin, Srdjan Micic, Ronja Weber, Simona Müller, Nathan Perkins, Christoph Berger, Renato Zenobi, Tobias Bruderer, and Alexander Moeller

Continuous monitoring of metabolites in exhaled breath has recently been introduced as an advanced method to allow non-invasive real-time monitoring of metabolite shifts during rest and acute exercise bouts. The purpose of this study was to continuously measure metabolites in exhaled breath samples during a graded cycle ergometry cardiopulmonary exercise test (CPET), using secondary electrospray high resolution mass spectrometry (SESI-HRMS).We also sought to advance the research area of exercise metabolomics by comparing metabolite shifts in exhaled breath samples with recently published data on plasma metabolite shifts during CPET.

Qi, Lu ; Lee Chuan, Ping ; Giannoukos, Stamatios ; Wang, Dongyu ; Li, Zhiyu ; Ran, Weikang ; Ye, Penglin ; Wang, Liwei ; Wang, Meng ; Lin, Yue ; Han, Yuemei ; Wang, Qiyuan ; Baltensperger, Urs ; El-Haddad, Imad ; Cao, Junji ; Prevot, Andre; Slowik, Jay

Despite extensive study, the sources and processes governing the formation and chemical evolution of haze pollution episodes in urban China remain unclear. Real-time molecular characterization of organic aerosol (OA) represents a promising approach to this problem. Extractive electrospray ionization (EESI) mass spectrometry allows real-time detection of individual OA components, while avoiding thermal decomposition and ionization-induced fragmentation. In previous field measurements, the EESI source was coupled with time-of-flight (TOF) mass analyzers, which are limited to a mass resolution of ~12,000 (at 200 m/z)…

N.Nowak, T. Gaisl, D. Miladinovic, R. Marcinkevics, M. Osswald, S. Bauer, J. Buhmann, R. Zenobi, P. Sinues, S. A. Brown, M. Kohler

Using exhaled breath analysis by secondary  high-resolution mass spectrometry, we measured the human exhaled metabolome at 10-s resolution across a night of sleep in combination with conventional polysomnography. Our subsequent analysis of almost 2,000 metabolite features demonstrates rapid, reversible control of major metabolic pathways by the individual vigilance states. A switch to wake reduces fatty acid oxidation, a switch to slow-wave sleep increases it, and the transition to rapid eye movement sleep results in elevation of tricarboxylic acid (TCA) cycle intermediates. Thus, in addition to daily regulation of metabolism, there exists a surprising and complex underlying orchestration across sleep and wake. Both likely play an important role in optimizing metabolic circuits for human performance and health.

T. Živković Semren, S. Majeed, M. Fatarova, C. Laszlo, C. Pak, S. Steiner, A. Kuczaj, A. Mazurov, M. Peitsch, N. Ivanov, J. Hoeng, and P. A. Guy

We used a programmable dual syringe pump (PDSP) connected to a super secondary electrospray ionisation (SESI) system interfaced with a Q Exactive HF mass spectrometer (MS) to detect the main constituents of thermally generated aerosols in real time. The benefits of Super SESI–HRMS for detecting drug aerosolisation from variably prepared in-house formulations with anatabine, azithromycin, chloroquine, favipiravir, and hydroxychloroquine were evaluated.

Chuan Ping Lee, Mihnea Surdu, David M. Bell, Josef Dommen, Mao Xiao, Xueqin Zhou, Andrea Baccarini, Stamatios Giannoukos, Günther Wehrle, Pascal André Schneider, Andre S. H. Prevot, Jay G. Slowik, Houssni Lamkaddam, Dongyu Wang, Urs Baltensperger, and Imad El Haddad

To elucidate the sources and chemical reaction pathways of organic vapors and particulate matter in the ambient atmosphere, real-time detection of both the gas and particle phase is needed. State-of-the-art techniques often suffer from thermal decomposition, ionization-induced fragmentation, high cut-off size of aerosols or low time resolution. In response to all these limitations, we developed a new technique that uses extractive electrospray ionization (EESI) for online gas and particle chemical speciation, namely the dual-phase extractive electrospray ionization time-of-flight mass spectrometer (Dual-Phase-EESI-TOF or Dual-EESI for short)….

Nora Kristina Nowak

Most physiological processes in humans are synchronized with their environment by socalled circadian clocks. These molecular time-keeping machineries are present in almost every cell. While light is the most important external stimulus to reset the circadian clocks, they can also be entrained by other stimuli, such as feeding or rest/activity cycles. Sleep is both, one of the major outputs of circadian clocks and also an independently regulated recuperative neurobiological process.

Stamatios Giannoukos, Mohamed Tarik, Christian Ludwig, Serge Biollaz, Jay Slowik, Urs Baltensperger, Andre Stephan Henry Prevot

On-line measurements of metal emissions in energy conversion systems at very low concentrations are difficult to perform using existing techniques. Metals are of high importance due to their detrimental impact on human health, the environment and various industrial processes and/or equipment. Herewith and for the first time, we report the real-time detection and characterization of metals and trace elements in a Swiss biogas production plant using a novel technology based on an extractive electrospray ionization (EESI) source coupled to a high-resolution time-of-flight mass spectrometer (TOF-MS)…

Ronja Weber, Naemi Haas, Astghik Baghdasaryan, Tobias Bruderer, Demet Inci, Srdjan Micic, Nathan Perkins, Renate Spinas, Renato Zenobi, Alexander Moeller

Early pulmonary infection and inflammation result in irreversible lung damage and are major contributors to cystic fibrosis (CF)-related morbidity. An easy to apply and noninvasive assessment for the timely detection of disease-associated complications would be of high value. We aimed to detect volatile organic compound (VOC) breath signatures of children with CF by real-time secondary electrospray ionisation high-resolution mass spectrometry (SESI-HRMS).
A total of 101 children, aged 4–18 years (CF=52; healthy controls=49) and comparable for sex, body mass index and lung function were included in this prospective cross-sectional study. Exhaled air was analysed by a SESI-source linked to a high-resolution time-of-flight mass spectrometer. Mass spectra ranging from m/z 50 to 500 were recorded.
Out of 3468 m/z features, 171 were significantly different in children with CF (false discovery rate adjusted p-value of 0.05). The predictive ability (CF versus healthy) was assessed by using a support-vector machine classifier and showed an average accuracy (repeated cross-validation) of 72.1% (sensitivity of 77.2% and specificity of 67.7%).
This is the first study to assess entire breath profiles of children with SESI-HRMS and to extract sets of VOCs that are associated with CF. We have detected a large set of exhaled molecules that are potentially related to CF, indicating that the molecular breath of children with CF is diverse and informative.

Ronja Weber, Srdjan Micic, Bettina Streckenbach, Lara Welti, Tobias Bruderer, Nathan Perkins, Demet Inci, Jakob Usemann, Alexander Möller

The diagnosis of asthma in children is still a clinical challenge. Breath-analysis has the potential to overcome this challenge. Our goal is to show that secondary electrospray ionization high-resolution mass-spectrometry (SESI-HRMS) can be used to detect asthma-specific metabolites in exhaled breath.
We are conducting an exploratory observational study comparing the molecular composition of exhaled breath from school children (5-18 years) with allergic asthma (confirmed by objective tests) to healthy controls. Patients are taken off their asthma medication two weeks prior to breath measurements. Breath analysis is performed on an AB SCIEX TripleTOF 5600+ HRMS coupled to a Super SESI ion source, detecting m/z features between 50 and 500 Da (mass accuracy <1ppm). A combination of data extraction and machine learning models is used to isolate the most discriminative features and assess the predictive power of breath profiles.
We acquired data from 47 children (21 with allergic asthma, 26 healthy controls). In our preliminary data analysis, we identified 193 m/z features which differed significantly between the two groups (adjusted p value < 0.05), which showed an average predictive power (asthma vs. healthy) of 78.7% (leave-one-out cross-validation with Random Forests algorithm). We could allocate molecular formulas to most significant m/z peaks. Compound identification is currently ongoing but some of the compounds have previously been reported in a biological context.
For the first time we identified exhaled molecules that differ between children with allergic asthma and healthy controls by real-time SESI-HRMS. Such a discovery has the potential to improve the early diagnosis of asthma.

Stamatios Giannoukos, Chuan Ping Lee, Mohamed Tarik, Christian Ludwig, Serge Biollaz, Houssni Lamkaddam, Urs Baltensperger, Andre Stephan Henry Prevot, and Jay Slowik

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)…

Martin Thomas Gaugg

Respiratory diseases are among the leading causes of death worldwide and pose a great financial burden on the health care system. During the last decades the medical community has started to recognize that a patient’s individual set of genes, along with environmental factors, are immensely important for the diagnosis and treatment of diseases. This has led to a strong drive towards further developments in personalized and evidencebased medicine. Understanding the underlying metabolic fundamentals of diseases is crucial to provide the appropriate patient care.One of the fastest methods to obtain new insights in this regard is to analyze metabolites in exhaled breath, which offers a non-invasive window into human metabolism, and which can be monitored in real time.

Katharina Heschl, Naemi Haas-Baumann, Ronja Weber, Astghik Baghdasaryan, Srdjan J. Micic, Florian Singer, Renato Zenobi, Tobias Bruderer, Alexander Möller

We present data of 41 children, 4-16yrs (23 CF and 17 healthy controls) from an ongoing cross-sectional study. Children were breathing via a mouthpiece into a heated sampling tube connected to the SESI-HRMS-analyser. Carefully pre-processed MS data was used for biomarker detection and classification. Mann-Whitney U tests together with FDR adjusted p-values were applied to isolate mass-to-charge ratios (m/z: representing potential biomarkers) Subsequent variable reduction through principal component analysis was used to perform binary logistic regression.
CF and HC had comparable BMI (17.7±3.0 vs 16.9±2.3; p=0.17) and FEV1 (z-scores -0.45±1.18 vs -0.27±1.13; p=0.37). 4 children were P.aeruginosa positive. We were able to isolate 43 m/z peaks with elevated intensity signals in CF vs HC below an FDR control level of q = 0.15. Classification applied to first principal components resulted in significant outcomes (CF vs. HC) with an average accuracy of 81.3% and a Cohen’s Kappa value of 0.61.
We could annotate the detected m/z peaks with molecular formula and identify different sets of related molecules. Structure elucidation is ongoing based on comparison of fragment spectra with reference chemicals for unequivocal confirmation of the involved molecular markers.
Non-invasive breath analysis with SESI-HRMS identifies CF specific compounds in children. This will allow further phenotyping of CF lung disease with the potential for early detection of airway infection.

Astghik Baghdasaryan, Tobias Bruderer, Simona Mueller, Ronja Weber, Naemi Haas-Baumann, Srdjan J. Micic, Christoph Berger, Renato Zenobi, Alexander Möller

Microbial infection entails recruitment of inflammatory cells and release of proteases and contributes to morbidity in CF. The non-invasive early detection of airway colonization and infection would be a great step forward in the management of children with CF. The Zurich Exhalomics program aims to describe disease state specific breath-prints to assess airway inflammation and infection in children with CF.
We used headspace secondary electrospray Ionisation high-resolution mass spectrometry (SESI-HRMS) to analyze three biological replicates in randomized order for strain specific volatile metabolites of the following CF pathogens: S. aureus, S. maltophilia, H. influenzae, S. pneumoniae, E. coli, B. cepacia, P. aeruginosa and growth medium.
We found good repeatability between measurements and minimal variation between biological replicates. We performed ANOVA on 130 m/z features with intensities over 1000 cps. 61 showed significant difference in signal intensity between strains (under the FDR adjusted significance level of 5%). This resulted in unique sets of 4-12 volatile markers for P. aeruginosa, H. influenzae, S. pneumoniae, S. aureus and E. coli. Several markers for P. aeruginoas, B. cepacia and S. maltophilia were closely related. Compound identification has started by comparison of headspace fragment spectra for cultures with proposed reference compounds. So far, we could identify molecules from the following compound classes: heterocyclic organic compounds, aromatic aldehydes and aromatic heterocyclic organic compounds.
We report unique sets of volatile markers for CF relevant bacteria detected with SESI-HRMS.

Alex R. Hill, Mark Edgar, Maria Chatzigeorgiou, James C. Reynolds, Paul F. Kelly* and Colin S. Creaser*

The complexation of triacetone triperoxide (TATP) with a range of alkali metals has been studied by electrospray ionisation mass spectrometry to yield [M + Cat]+ ions for all of the alkali metals. The formation of [2TATP + Li + LiX]+ (X = Br, Cl) sandwich complexes was also observed. Collision cross-sections for the lithium-containing complexes of TATP were measured by travelling wave ion mobility spectrometry mass spectrometry and compared well with computationally determined structures. Extractive electrospray ionisation (EESI) using a lithium-doped electrospray is demonstrated for the detection of TATP vapours desorbed from a metal surface. The limit of detection for EESI was shown to be 20 ng using the [TATP + Li]+ ion.

James C Reynolds, Modupe A Jimoh, Cristina Guallar-Hoyas, Colin S Creaser, Salman Siddiqui and C L Paul Thomas

A two-stage thermal desorption/secondary electrospray ionization/time-of-flight mass spectrometry for faster targeted breath profiling has been studied. A new secondary electrospray ionization (SESI) source was devised to constrain the thermal desorption plume and promote efficient mixing in the ionization region. Further, a chromatographic preseparation stage was introduced to suppress interferences from siloxanes associated with thermal desorption profiles of exhaled breath samples…

Helen J. Martin,a James C. Reynolds,a Svetlana Riazanskaiab and C. L. Paul Thomas*a

The non-invasive nature of volatile organic compound (VOC) sampling from skin makes this a priority in the development of new screening and diagnostic assays. Evaluation of recent literature highlights the tension between the analytical utility of ambient ionisation approaches for skin profiling and the practicality of undertaking larger campaigns (higher statistical power), or undertaking research in remote locations. This study describes how VOC may be sampled from skin and recovered from a polydimethylsilicone sampling coupon and analysed by thermal desorption (TD) interfaced to secondary electrospray ionisation (SESI) time-of-flight mass spectrometry (MS) for the high throughput screening of volatile fatty acids (VFAs) from human skin…

Neil A. Devenport, Daniel J. Blenkhorn, Daniel J. Weston, James C. Reynolds, and Colin S. Creaser

A direct, ambient ionization method has been developed for the determination of creatinine in urine that combines derivatization and thermal desorption with extractive electrospray ionization and ion mobility-mass spectrometry. The volatility of creatinine was enhanced by a rapid on-probe aqueous acylation reaction, using a custom-made thermal desorption probe, allowing thermal desorption and ionization of the monoacylated derivative..

Neil A. Devenport, Laura C. Sealey, Faisal H. Alruways, Daniel J. Weston, James C. Reynolds, and Colin S. Creaser.

A direct, ambient ionization method has been developed using atmospheric pressure thermal desorption–extractive electrospray–mass spectrometry (AP/TD-EESI-MS) for the detection of the genotoxic impurity (GTI) methyl p-toluenesulfonate (MTS) in a surrogate pharmaceutical matrix. A custom-made thermal desorption probe was used to the desorb and vaporize MTS from the solid state, by rapid heating to 200 °C then cooling to ambient temperature, with a cycle time of 6 min…

Lukas Christian Meier

It has been known for years that breath analysis has the potential of becoming a powerful tool to acquire information on the health status of individuals. Using mass spectrometry (MS) for breath analysis would allow for the detection of hundreds of compounds simultaneously and even render real time (online) monitoring of the individual’s health status possible. Unfortunately, the efficient transfer of breath into mass spectrometers is difficult to achieve. Therefore, only very volatile organic compounds (VOCs) being present in breath at high concentrations have been measured so far. The work presented in this thesis intends to extend the range of compounds being detected in breath towards higher masses, less volatile and less concentrated compounds…

J. C. Reynolds,* G. J. Blackburn, C. Guallar-Hoyas, V. H. Moll, V. Bocos-Bintintan, G. Kaur-Atwal, M. D. Howdle, E. L. Harry, L. J. Brown, C. S. Creaser, and C. L. P. Thomas

A thermal desorption unit has been interfaced to an electrospray ionization-ion mobility-time-of-flight mass spectrometer. The interface was evaluated using a mixture of six model volatile organic compounds which showed detection limits of <1 ng sample loaded onto a thermal desorption tube packed with Tenax, equivalent to sampled concentrations of 4 μg L−1. Thermal desorption profiles were observed for all of the compounds, and ion mobility-mass spectrometry separations were used to resolve the probe compound responses from each other…