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.

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.

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.

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.

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

Identifying and differentiating bacteria based on their emitted volatile organic compounds (VOCs) opens vast opportunities for rapid diagnostics. Secondary electrospray ionization high-resolution mass spectrometry (SESI-HRMS) is an ideal technique for VOC-biomarker discovery because of its speed, sensitivity towards polar molecules and compound characterization possibilities. Here, an in vitro SESI-HRMS workflow to find biomarkers for cystic fibrosis (CF)-related pathogens P. aeruginosa, S. pneumoniae, S. aureus, H. influenzae, E. coli and S. maltophilia is described.

Fouad Choueirya, Jiangjiang Zhu

Lung cancer is one of the leading causes of cancer related deaths in the United States. A novel volatile analysis platform is needed to complement current diagnostic techniques and better elucidate chemical signatures of lung cancer and subsequent treatments. A systems biology bottom-up approach using cell culture volatilomics was employed to identify pathological volatile fingerprints of lung cancer in real time. An advanced secondary electrospray ionization (SESI) source, named SuperSESI was used in this study and directly attached to a Thermo Q-Exactive high-resolution mass spectrometer (HRMS).

Fabienne Decrue, Kapil Dev Singh, Amanda Gisler, Mo Awchi, Jiafa Zeng, Jakob Usemann, Urs Frey and Pablo Sinues

Breath analysis by secondary electrospray ionization-high resolution mass spectrometry (SESI-HRMS) offers the possibility to measure comprehensive metabolic profiles. The technology is currently being deployed in several clinical settings in Switzerland and China. However, patients are required to exhale directly into the device located in a dedicated room. Consequently, clinical implementation in patients incapable of performing necessary exhalation maneuvers (e.g., infants) or immobile (e.g., too weak, elderly, or in intensive care) remains a challenge. The aim of this study was to develop a method to extend such breath analysis capabilities to this subpopulation of patients by collecting breath samples remotely (offline) and promptly (within 10 min) transfer them to SESI-HRMS for chemical analysis.

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.

Francisco G. Blanco and Guillermo Vidal-de-Miguel

Here, we review the SESI technology development, the different SESI configurations developed, and the standardization procedures described to translate SESI into the clinical environment. Finally, SESI main applications described in the literature with prompt translation into the clinical environment, namely, biomarker discovery or pharmacokinetics and drug monitoring are revised.

Kseniya Dryahina, Miroslav Polášek, David Smith, Patrik Španěl

Secondary electrospray ionization (SESI) is currently only semi-quantitative. In the Zspray™ arrangement of SESI-MS, the transfer of ions from near atmospheric pressure to a triple quadrupole is achieved by guiding E-fields that partially desolvate both reagent and analyte ions which must be understood.