Monitoring peppermint washout in the breath metabolome by secondary electrospray ionization-high resolution mass spectrometry

In this study, a secondary electrospray ionization-high resolution mass spectrometer (SESI-HRMS) system was employed to profile the real-time exhaled metabolome of ten subjects who had ingested a peppermint oil capsule. In total, six time points were sampled during the experiment. Using an untargeted way of profiling breath metabolome, 2333 m/z unique metabolite features were determined in positive mode, and 1322 in negative mode. To benchmark the performance of the SESI-HRMS setup, several additional checks were done, including determination of the technical variation, the biological variation of one subject within three days, the variation within a time point, and the variation across all samples, taking all m/z features into account. Reproducibility was good, with the median technical variation being similar to 18% and the median variation within biological replicates being similar to 34%. Both variations were lower than the variation across individuals. Washout profiles of compounds from the peppermint oil, including menthone, limonene, pulegone, menthol and menthofuran were determined in all subjects. Metabolites of the peppermint oil were also determined in breath, for example, cis/trans-carveol, perillic acid and menthol glucuronide. Butyric acid was found to be the major metabolite that reduce the uptake rate of limonene. Pathways related to limonene metabolism were examined, and meaningful pathways were identified from breath metabolomics data acquired by SESI using an untargeted analysis.