A benchmarking protocol for breath analysis: The peppermint experiment

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. The Marie Sklodowska-Curie Actions Innovative Training Network: Ion-Molecule Processes for Analytical Chemistry Technologies (IMPACT) (www.impact-h2020itn.com), which has supported BH, MM and GP as Early Stage Researchers through the European Commission's HORIZON 2020 Programme under Grant Agreement Number 674911. The EMBER consortium for support in the dissemination of the Peppermint Initiative; researchers involved in the application of the protocol to specific techniques (to be published in due course) Abigail Joyce, Amisha Manek, Bellagambi Francesca Giuseppa, Bonsignori Alessio, Ghimenti Silvia and Biagini Denise, Liesl Carr, Luke Bryant and Rebecca Cordell. MW and SJF are supported by the NIHR Manchester Biomedical Research Centre. The group from Li`ege University is supported by the FNRS through the EOS and the FRIA initiatives. Ruszkiewicz, DM (, orresponding author), Loughborough Univ, Ctr Analyt Sci, Dept Chem, Loughborough, Leics, England ; Univ Birmingham, Sch Phys & Astron, Mol Phys Grp, Birmingham, W Midlands, England. D.M.Ruszkiewicz2@lboro.ac.uk