M. Fido

Expanding high-resolution mass spectrometry applications in metabolomics

This doctoral thesis explores the use of high-resolution mass spectrometry to address diverse challenges in metabolomics, ranging from volatile compound analysis to targeted quantification and computational data processing. Across four studies, the work investigates polymer sampling bags for offline breath analysis, applies volatilomics to study the metabolic effects of gut microbiota and pathogen infection in honey bees, develops an HRMS-based method for the detection and quantification of nitrogen-containing metabolites, and introduces LCMSpector, an open-source tool for targeted mass spectrometry data analysis. Together, these contributions demonstrate the versatility of high-resolution mass spectrometry for studying complex biological matrices, improving analytical workflows, and supporting more reliable metabolomic interpretation.

M. Richard, K.D. Singh, D. Sezer, S. Buergler, L. Palermo, Y. Schulz, Z. Tang, X. Luo, U. Frey, P. C. Cattin, X. Li, J. Gaab, P. Sinues

Breath metabolomics for real-time detection of acute pain responses

Cold-induced pain produces rapid physiological changes that are difficult to assess objectively, especially in vulnerable or non-communicative patients. This study investigates whether real-time breath metabolomics using SESI-HRMS can detect metabolic responses to the cold pressor test, a standardized model of acute nociceptive and sympathetic activation. Across two independent cohorts from Switzerland and China, the intervention induced reproducible shifts in the exhaled metabolome, with more than 400 upregulated features shared between both populations. Pathway analyses linked these changes mainly to amino acid metabolism, energy regulation, oxidative stress, and vascular responses. In addition, a neural network model classified pre- and post-pain breath fingerprints with an AUC of 0.856 and 78% accuracy. Overall, the study supports exhaled breath analysis as a promising non-invasive and observer-independent approach for monitoring short-term physiological responses related to pain.

S. Palmisano, A. R. Anderson, Z. J. Sasiene, E. Schaller, R. M. Taylor, R. Chitale1, P. T. McMahon, P. M. Mach, T. G. Glaros, J. Baca and E. M. McBride

Linking breath and blood metabolomics: advancing noninvasive biomarker discovery

Breath analysis offers a promising noninvasive alternative to blood-based diagnostics, but the relationship between breath and plasma metabolites remains poorly understood. This study integrates real-time SESI-MS breath analysis with LC-MS plasma profiling to systematically compare volatile and semi-volatile compounds across both matrices. By optimizing sample preparation and data processing workflows, including methanol-based extraction and the BreathXplorer pipeline, the study enhances feature detection and overlap between breath and plasma. The identification of shared metabolic signatures highlights the potential of breath analysis as a surrogate for systemic metabolomics, paving the way for more reliable, noninvasive diagnostic strategies.

D. García, A. Ballester, M. E. Fernández

SESI-MS for volatile analysis: overcoming challenges toward clinical and industrial adoption
Secondary electrospray ionization mass spectrometry (SESI-MS) is a powerful platform for real-time, noninvasive analysis of volatile compounds in complex matrices such as breath and microbial systems. This review examines the key methodological challenges limiting its broader adoption,including quantification, compound identification, and standardization, and highlights emerging solutions such as advanced fragmentation strategies, spectral stitching, and improved quality control approaches. By addressing these critical gaps, SESI-MS is positioned to transition from a research tool to a robust technology for clinical diagnostics, volatilomics, and industrial applications.

F. Schmidt, D. M. Baur, P. Baumgartner, J. Herth, K. Fricke, N.A. Sievi, K. Dev Singh, T. Gaisl, A. Huang, D. Franzen, S. Ulrich, P Sinues, M. Kohler

Real-time breath metabolomics: a new frontier in personalized lung cancer diagnostics
Real-time breath analysis is emerging as a powerful, non-invasive strategy for lung cancer detection and metabolic phenotyping. In this prospective matched case-control study, SESI-HRMS enabled rapid breath profiling and identified distinct metabolic signatures that differentiated lung cancer patients from matched controls with promising accuracy. Beyond detection, the study revealed subtype-specific metabolic patterns, highlighting the potential of breath metabolomics to complement imaging and genomic profiling in a more personalized, multi-omics approach to lung cancer diagnosis and care.

H.S. Shepard, J.C. May, J.A. McLean

Ambient mass spectrometry: rapid, in situ insights into microbial systems
Ambient ionization mass spectrometry is transforming microbial analysis by enabling rapid, high-throughput, and in situ chemical characterization with minimal sample preparation. This overview highlights key ambient MS platforms, including DESI-MS, DART-MS, paper spray MS, and SESI-MS,and their growing impact on microbial profiling and imaging of complex, fragile substrates. By capturing microbial biochemistry in real time, ambient MS is opening new avenues for fast diagnostics, spatially resolved analysis, and advanced microbiological research.

S.J. Swift, K. Dryahina, P. Španěl, F. Kontopidou, C. Kokkotis, R.Zenobi, S. Giannoukos

Breath analysis in Switzerland: advancing real-time, noninvasive clinical diagnostics
Breath analysis is emerging as a powerful, noninvasive approach to clinical diagnostics, with Switzerland at the forefront of recent advances. This perspective highlights progress since 2019, focusing on secondary electrospray ionization high-resolution mass spectrometry (SESI-HR-MS) for real-time metabolic profiling. By identifying key methodological gaps and clinical challenges, it outlines the path toward translating breath analysis into routine clinical practice and precision medicine.

M. Niu, U. Arshad, M.Z. Islam, M.A. Barrientos-Blanco, E. Slack, S. Giannoukos, R. Zenobi

Exhalomics: advancing noninvasive monitoring in livestock health and welfare
This review highlights exhaled breath analysis (“exhalomics”) as an emerging noninvasive approach for monitoring livestock health, metabolism, and welfare. It synthesizes advances in sampling and analytical techniques, outlining applications in disease detection, nutrition assessment, and physiological and microbial profiling across animal species. Despite promising progress, the field faces challenges in standardization, metabolite identification, and scalability for farm use. The integration of exhalomics with multi-omics and AI-driven analytics is poised to transform precision livestock farming by enabling earlier disease detection, improved productivity, and enhanced animal welfare.

Z. Yin, W. Xiong, K. Zhang, X. Luo, M. Wei, K.D. Singh1,2, U. Frey, X. Li, X. Feng, and P. Sinues

Breath metabolomics under lidocaine-SGB
This pilot study provides the first real-time metabolic profiling of lidocaine-based stellate ganglion block (SGB) using high-resolution breath analysis (SESI-HRMS). In 28 participants, more than 300 metabolites were significantly altered after lidocaine injection, including both drug-derived compounds and endogenous metabolites linked to fatty-acid oxidation and amino-acid metabolism. The findings demonstrate that breath metabolomics can simultaneously capture drug metabolism and host physiological responses, while revealing notable inter-individual variability. This work positions breath-based pharmacometabolomics as a promising tool for personalized monitoring and optimization of SGB interventions in clinical practice.

N.A. Sievi, F. Schmidt, K. Fricke, D.M. Baur, S. Basler, J. Herth, M. Kohler

Breath metabolomics in COPD exacerbations
This study examined whether real-time breath analysis can detect metabolic changes during acute exacerbations of COPD (AECOPD) compared to the stable state in patients receiving triple inhaled therapy. Breath profiles from 28 patients revealed alterations in aminosugar, linoleate, and butanoate pathways. A prediction model discriminated AECOPD from stable state with high accuracy (AUC = 0.84, sensitivity and specificity 86%). These findings suggest that exhaled breath analysis may provide a rapid, non-invasive tool for detecting exacerbations and reveal aminosugar metabolism as a potential therapeutic target.

Z. Tang, J. Yang, B. Su, X. Xu, X. Luo, H. Wang, K. Zhang, T. Huan, P. Sinues, M. Fang, X. Li

Breath Metabolomics Beyond Blood
This study demonstrates how exhaled breath can complement blood in metabolomics by retaining volatile compounds often lost in blood analysis. Researchers combined a controlled exercise protocol, a custom breath metabolomics database, and metabolite fusion across breath and blood to boost identification accuracy. High-resolution MS revealed 66 unique breath metabolites, 59 unique blood metabolites, and only 4 shared. Integrating both matrices expanded pathway coverage and highlighted breath-specific markers of exercise-induced changes, paving the way for novel biomarkers and future smart wearable applications.

S. Giannoukos, K. J.e Burton-Pimentel, R. Guillod, G. Vergères, D. Pohl

The Lactobreath study explores real-time breath analysis as a novel, non-invasive tool to detect lactose intolerance. In this double-blind trial, 120 participants undergo postprandial testing with lactose or glucose. Using Super SESI-HRMS, the study captures detailed exhalome profiles and correlates them with symptoms, hydrogen levels, gut transit (via ingestible gas capsules), bowel sounds, urine metabolites, and genetic markers. This integrative approach aims to move beyond standard hydrogen breath tests and validate molecular breath signatures as clinical biomarkers for food intolerance.

H. G. Mengers, F. Völker, L. M. Blank

Decoding Bacillus subtilis’ Volatilome Under Acid Stress, in Real Time with SESI-Orbitrap
This study reveals the acid stress volatilome of Bacillus subtilis using real-time SESI-Orbitrap MS, offering unprecedented temporal resolution (0.3 Hz) during pH shock in resting cells. Over 450 compounds were tracked across 16,000 data points, uncovering both immediate and delayed VOC responses. Acetoin dominated, as expected, but a novel signal (C₄H₆O) also emerged. This work highlights SESI’s power to monitor microbial stress responses via off-gas VOCs, non-invasively, instantly, and at the molecular scale.

S.M. Ashbacher D.C. Muddiman

Heat-Stress VOC Profiling in Basil Using TP-SESI
This study showcases temperature-programmed secondary electrospray ionization (TP-SESI) as a powerful tool for probing plant responses to heat stress. By applying controlled heating to Ocimum basilicum leaves, TP-SESI enabled real-time, non-destructive monitoring of volatile organic compounds (VOCs). The results revealed clear, temperature-dependent changes in VOC composition and abundance, offering insights into thermal stress signaling pathways. TP-SESI emerges as a sensitive, orthogonal method for exploring plant resilience mechanisms and stress-induced metabolism.

S. Basler, K. Fricke, N.A. Sievi1, A. Arvaji, F.Schmidt, J. Herth, D.M. Baur, M.Kohler, S.Ulrich, and M. Lichtblau

Breath metabolome profiling for pulmonary vascular disease detection
This study evaluated whether real-time breath analysis could identify metabolic differences between patients with pulmonary vascular disease (PVD) and healthy controls, and distinguish between two major subtypes: pulmonary arterial hypertension (PAH) and chronic thromboembolic pulmonary hypertension (CTEPH). Using metabolomic prediction models, breath profiles from 75 patients and 115 controls were compared. The models achieved high accuracy in detecting PVD (AUC = 0.917) and moderate accuracy in differentiating PAH from CTEPH (AUC = 0.764). Pathway analysis revealed alterations in fatty acid metabolism. These findings highlight the potential of breath analysis as a non-invasive, real-time diagnostic tool for early detection and subtype differentiation in PVD.

M.A. Barrientos-Blanco, U. Arshad, S. Giannoukos, M.Z. Islam, C. Kunz, R. Peng, S.E. Räisänen, R. Zenobi, M. Niu

Separating Breath from Eructation: Advancing SESI-Based Breathomics in Dairy Cows
This study establishes a validated sampling method to distinguish true breath (BR) from ruminal eructation (RE) in dairy cows—an essential step for applying breathomics in ruminant metabolic research. Using a CH₄-based threshold and the GreenFeed system, researchers collected BR and RE samples from Holstein cows and analyzed them via SESI-HRMS and GC. CH₄ levels were 80% lower in BR, confirming successful separation. SESI-MS detected hundreds of VOC features, with distinct profiles between BR and RE. RE was enriched in VFAs like acetate and butyrate, while BR showed unique endogenous signals. This approach unlocks non-invasive, real-time breath metabolomics for metabolic phenotyping in ruminants, overcoming a long-standing barrier in the field.

X.Luo, H. Wang, X. Hu, Sa. Gligorovski, X.Liu, P.Sinues

SESI and EESI: Two Decades of Real-Time Volatile Analysis
Since their resurgence during the ambient mass spectrometry revolution, secondary electrospray ionization (SESI) and extractive electrospray ionization (EESI) have transformed real-time analysis of gas and aerosol mixtures. Originally discovered in the 1980s, these techniques now underpin a wide range of applications—from drug detection to environmental monitoring and metabolomics. Over the past two decades, the field has seen rapid expansion, thanks in part to technological advances driven by innovators such as Fossiliontech (FIT). This review summarizes key milestones, showcases diverse applications, and outlines emerging opportunities and challenges.

H. G. Mengers, F. Völker, L. M. Blank

Validating VOC Quantification in Breath: MS1 vs MS2 Approaches in SESI-MS
This study critically evaluates the accuracy of compound identification and quantification in SESI-MS breath analysis, comparing MS1-based methods to targeted MS2 techniques. Using C5–C10 aldehydes, limonene, and pyridine as known breath markers, researchers tested full scan, selected ion monitoring, and parallel reaction monitoring across 12 volunteers. While high-abundance VOCs like limonene and pyridine were reliably detected, low-abundance aldehydes posed significant challenges due to isomeric interference (e.g., from ketones), leading to misassignments—even with MS2. The study underscores the need for robust MS2 validation in SESI workflows to avoid false positives and ensure quantitative accuracy in clinical and diagnostic breath analysis.

J. Zeng, N. Stankovic, K. D. Singh, R. Steiner, U. Frey, T. Erb, P. Sinues

Breath Pharmacometabolomics of Propofol Anesthesia in Children
This pilot study demonstrates that on-site SESI-HRMS breath analysis can robustly track propofol and its metabolites in pediatric patients, with exhaled signals showing strong correlation to serum concentrations (R² ≥ 0.65). Beyond pharmacokinetics, SESI revealed surgery-induced metabolic responses, including elevated fatty aldehydes—markers of oxidative stress and lipid peroxidation. Conducted in children undergoing IV anesthesia, this work highlights real-time, non-invasive breath profiling as a promising tool for individualized anesthetic monitoring and metabolic assessment during surgery.

A. Gisler, K. D. Singh, A. Marten, F. Decrue, U. Frey, P. Sinues and J. Usemann

Real-time breath analysis via secondary electrospray ionization high-resolution mass spectrometry (SESI-HRMS) shows promise as a non-invasive tool for assessing oxidative stress. In a study involving 128 children (25 tobacco smoke-exposed, 103 non-exposed), 71 breath features significantly correlated with urinary levels of the oxidative stress marker 8-iso-prostaglandin F2α (8-iso-PGF2α). Breath analysis moderately predicted urinary 8-iso-PGF2α (concordance correlation: 0.37 ± 0.05), suggesting potential clinical applicability