On-line analysis of exhaled breath offers insight into a person's metabolism without the need for sample preparation or sample collection. Due to its non-invasive nature and the possibility to sample continuously, the analysis of breath has great clinical potential. The unique features of this technology make it an attractive candidate for applications in medicine, beyond the task of diagnosis. We review the current methodologies for on-line breath analysis, discuss current and future applications, and critically evaluate challenges and pitfalls such as the need for standardization. Special emphasis is given to use of the technology in diagnosing respiratory diseases, potential niche applications, and the promise of breath analysis for personalized medicine. The analytical methodologies used range from very small and low-cost chemical sensors, which are ideal for continuous monitoring of disease status, to optical spectroscopy and state-of-the-art, high-resolution mass spectrometry. The latter can be utilized for untargeted analysis of exhaled breath, with the capability to identify hitherto unknown molecules. The interpretation of the resulting big data sets is complex and often constrained due to a limited number of participants. Even larger data sets will be needed for assessing reproducibility and for validation of biomarker candidates. In addition, molecular structures and quantification of compounds are generally not easily available from on-line measurements and require complementary measurements, for example, a separation method coupled to mass spectrometry. Furthermore, lack of standardization still hampers using the technique for screening larger cohorts of patients. The present review summarizes the present status and continuous improvements of the main on-line breath analysis methods, and evaluates obstacles for its wider application.

Omega-oxidation is a fatty acid degradation pathway that can occur alternatively to the dominant β-oxidation. The dysregulation of fatty acid oxidation has been related with a variety of diseases, termed fatty acid oxidation disorders. This work shows evidence for real-time detection in exhaled breath of the complete series of saturated linear ω-hydroxyalkanoic acids, ω-oxoalkanoic acids, and alkanedioic acids with carbon chain lengths of 5-15. We present a comprehensive analytical workflow using online and subsequent offline methods: secondary electrospray ionization mass spectrometry of exhaled breath and UHPLC-HRMS/MS experiments using exhaled breath condensate, respectively. By analyzing online breath measurements of 146 healthy individuals, we were able to obtain strong evidence for the correlation of these metabolite families. This enabled us to monitor the full ω-oxidation pathway in human exhaled breath. We could unambiguously identify these compounds, many of which have never been reported in breath so far. This comprehensive study on breath metabolites reinforces the notion of breath as a valuable source of information, which is underexploited in metabolomics.

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. Alteration in intracellular levels of its intermediates has been linked with a wide range of illnesses ranging from cancer to cellular necrosis or liver cirrhosis. Therefore, there exists an intrinsic interest in monitoring such metabolites. Our goal in this study was to evaluate whether, at least the most volatile metabolites of the TCA cycle, could be detected in breath in vivo and in real time. We used secondary electrospray ionization coupled with high-resolution mass spectrometry (SESI-HRMS) to conduct this targeted analysis. We enrolled six healthy individuals who provided full exhalations into the SESI-HRMS system at different times during 3 days. For the first time, we observed exhaled compounds that appertain to the TCA cycle: fumaric, succinic, malic, keto-glutaric, oxaloacetic, and aconitic acids. We found high intraindividual variability and a significant overall difference between morning and afternoon levels for malic acid, oxaloacetic acid, and aconitic acid, supporting previous studies suggesting circadian fluctuations of these metabolites in humans. This study provides first evidence that TCA cycle could conveniently be monitored in breath, opening new opportunities to study in vivo this important metabolic pathway.

I125 radioimmunoassay (RIA) is currently the standard technique for quantifying cerebrospinal fluid (CSF) orexin-A/hypocretin-1, a biomarker used to diagnose narcolepsy type 1. However, orexin-A RIA is liable to undergo cross-reactions with matrix constituents generating interference, high variability between batches, low precision and accuracy, and requires special radioactivity precautions. Here we developed the first quantitative mass spectrometry assay of orexin-A based on a multiple reaction monitoring (MRM) approach. This method was tested in keeping with the Clinical and Laboratory Standards Institute (CLSI) guidelines and its clinical relevance was confirmed by comparing patients with narcolepsy type 1 versus patients with other neurological conditions. The results obtained using MRM and RIA methods were highly correlated, and Bland–Altman analysis established their interchangeability. However, the MRM values had a wider distribution and were 2.5 time lower than the RIA findings. In conclusion, this method of assay provides a useful alternative to RIA to quantify orexin-A, and may well replace it not only in narcolepsy type 1, but also in the increasing number of pathologies in which the quantification of this analyte is relevant.