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.

NCT02050425 (registered at ClinicalTrials.gov).

Sampling of volatile organic compounds (VOCs) has shown promise for detection of a range of diseases but results have proved hard to replicate due to a lack of standardization. In this work we introduce the ‘Peppermint Initiative’. The initiative seeks to disseminate a standardized experiment that allows comparison of breath sampling and data analysis methods. Further, it seeks to share a set of benchmark values for the measurement of VOCs in breath. Pilot data are presented to illustrate the standardized approach to the interpretation of results obtained from the Peppermint experiment. This pilot study was conducted to determine the washout profile of peppermint compounds in breath, identify appropriate sampling time points, and formalise the data analysis. Five and ten participants were recruited to undertake a standardized intervention by ingesting a peppermint oil capsule that engenders a predictable and controlled change in the VOC profile in exhaled breath. After collecting a pre-ingestion breath sample, five further samples are taken at 2, 4, 6, 8, and 10 h after ingestion. Samples were analysed using ion mobility spectrometry coupled to multi-capillary column and thermal desorption gas chromatography mass spectrometry. A regression analysis of the washout data was used to determine sampling times for the final peppermint protocol, and the time for the compound measurement to return to baseline levels was selected as a benchmark value. A measure of the quality of the data generated from a given technique is proposed by comparing data fidelity. This study protocol has been used for all subsequent measurements by the Peppermint Consortium (16 partners from seven countries). So far 1200 breath samples from 200 participants using a range of sampling and analytical techniques have been collected. The data from the consortium will be disseminated in subsequent technical notes focussing on results from individual platforms.

Online breath analysis is an attractive approach to track exhaled compounds without sample preparation. Current commercially available real-time breath analysis platforms require the purchase of a full mass spectrometer. Here we present an ion source compatible with virtually any preexisting atmospheric pressure ionization mass spectrometer that allows real-time analysis of breath. We illustrate the capabilities of such technological development by upgrading an orbitrap mass spectrometer. As a result, we detected compounds in exhaled breath between 70 and 900 Da, with a mass accuracy of typically <1 ppm; resolutions between m/Δm 22,000 and 70,000 and fragmentation capabilities. The setup was tested in a pilot study, comparing the breath of smokers (n  =  9) and non-smokers (n  =  10). Exogenous compounds associated to smoking, as well as endogenous metabolites suggesting increased oxidative stress in smokers, were detected and in some cases identified unambiguously. Most of these compounds correlated significantly with smoking frequency and allowed accurate discrimination of smokers and non-smokers.