A. M.Casas, M. del Nogal, E.Herrero, D. Garcia, E. Rodriguez, J. L. Pérez

Real-Time Pharmaceutical Aerosol Analysis

This study explores the application of secondary electrospray ionization mass spectrometry (SESI-MS) for the real-time characterization of pharmaceutical aerosols from metered-dose inhalers (MDIs). The analysis includes one of the most widely used inhalers worldwide, Ventolin®, a simple formulation containing salbutamol and norflurane for which salbutamol was reliably detected, while norflurane remained undetectable due to its low proton affinity; and Ventoduo®, a multicomponent formulation also including beclomethasone dipropionate, ethanol, and oleic acid for which all major components were successfully detected. The coupling of SESI with high-resolution MS enabled unambiguous identification of all analytes, with excellent mass accuracy, confirming the identity of both active ingredients through characteristic fragmentation patterns. These results demonstrate that SESI-MS, particularly when coupled with high-resolution instrumentation (SESI-HRMS), is a powerful and reliable tool for the comprehensive and real-time analysis of pharmaceutical aerosols, including those with multiple active ingredients and co-solvents.

Z. Yin, K. D. Singh, Z. Tang, M. Richard, J. Zeng, U. Frey, P. Sinues, X. Li

Real-Time Metabolic Tracing Through Breath

This proof-of-concept study explores the use of SESI-HRMS for noninvasive, real-time metabolic isotopic tracing through exhaled breath after deuterated water ingestion. By monitoring deuterium incorporation in breath metabolites, the approach enables dynamic tracking of metabolic responses across fasting and postprandial states. The study revealed distinct labeling patterns associated with short-chain fatty acids, energy metabolism, and host-microbial metabolic activity, demonstrating the potential of breath-based SESI-HRMS as a powerful tool for metabolic phenotyping, nutritional studies, and future clinical research.

S. Giannoukos, D. Raptopoulos, M. Konstantopoulou

Monitoring Insect Pheromones in Real Time

This study explores the use of SESI-HRMS and portable membrane inlet mass spectrometry for the rapid detection and monitoring of insect sex pheromones. By measuring pheromone levels in the gas phase, these technologies can support more precise pest management strategies, helping verify pheromone coverage, identify gaps, and optimize reapplication in mating disruption programs. The work demonstrates sensitive detection from low ppt to low ppb levels, fast response times, and successful identification of key pheromone components in real Plutella xylostella headspace samples, highlighting a promising path toward real-time and field-deployable pheromone monitoring.

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.