A benchmarking protocol for breath analysis: The peppermint experiment

Ben Henderson, Dorota M. Ruszkiewicz*, Max Wilkinson, Jonathan D. Beauchamp, Simona M. Cristescu, Stephen J. Fowler, Dahlia Salman, Fabio Di Francesco, Gudrun Koppen, Jens Langejürgen, Olaf Holz, Andria Hadjithekli, Sergi Moreno, Michele Pedrotti, Pablo Sinues, Gitte Slingers, Michael Wilde, Tommaso Lomonaco, Delphine Zanella, Renato ZenobiJean François Focant, Stanislas Grassin-Delyle, Flavio Antonio Franchina, Michaela Malásková, Pierre Hugues Stefanuto, Giovanni Pugliese, Christopher Mayhew, C.L.P. Thomas

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

34 Citations (Scopus)


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.

Original languageEnglish
Article number046008
JournalJournal of Breath Research
Issue number4
Publication statusPublished - Oct 2020


  • benchmark
  • breathomics
  • peppermint
  • repeated sampling8pharmacokinetics
  • standardization


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