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)

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.

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.

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 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).

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.

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.

Li Chen, Fanyi Zhong and Jiangjiang Zhu

This mini-review aims to discuss the development and applications of mass spectrometry (MS)-based hybrid approaches in metabolomics.

Lukas Bregy, Constanze Hirsigerb, Stefanie Gartenmann, Tobias Bruderer, Renato Zenobi, Patrick R.Schmidlinb

It has been shown that bacteria in periodontally diseased patients can be recognized by the detection of volatile metabolites in the headspace of saliva by real-time ambient mass spectrometry. The aim of this study was to use this detection method to analyze the oral metabolome in diseased periodontitis patients before and after therapy to monitor disease evolution and healing events.

Haorong Li, Mengyang Xu, and Jiangjiang Zhu

This study aimed to sensitively detect volatile metabolites from the headspace of in vitro gut microbial culture in a human colonic model (HCM). Two SESI-tandem mass spectrometry panels with a comparable number of targeted metabolites/features were established.

Haorong Li and Jiangjiang Zhu

SESIMS/MS was applied to examine the VOC metabolome of a pair of isogenic methicillin-susceptible and resistant Staphylococcus aureus (MSSA and MRSA) strains. Our results indicated that MSSA and MRSA strains can be clearly differentiated Furthermore, we studied the stress response of MSSA and MRSA to antibiotics treatment.

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.

A.Tejero Rioseras, K.D. Singh, N. Nowak, M. T. Gaugg, T. Bruderer, R. Zenobi, P. M-L Sinues

The tricarboxylic acid (TCA) cycle is one of the most important metabolic pathway for cellular respiration in aerobic organisms. It provides and collects intermediates for many other interconnecting pathways and acts as a hub connecting metabolism of carbohydrates, fatty acids, and amino acids.

A. Tejero-Rioseras, D. Garcia-Gomez, B. E. Ebert, L. M. Blank, A. J. Ibanez and P. M-L Sinues

While yeast is one of the most studied organisms, its intricate biology remains to be fully mapped and understood. This is especially the case when it comes to capture rapid, in vivo fluctuations of metabolite levels. According to the authors, the results suggest that a large number of metabolites produced by yeast from glucose neither are reported in the literature nor are their biochemical origins deciphered.