A benchmarking protocol for breath analysis: the peppermint experiment

In the preparatory TD-GC-MS analysis of breath, eight compounds were attributed to ingestion of a peppermint oil capsule, see table 1. All of these were observed in breath for at least 6 h after ingestion of a peppermint capsule. All of these compounds, except p-Menthadien-7-ol and Dehydro-1,8-cineole, are peppermint oil constituents identified with GC-MS headspace analysis in previous work carried out by consortium members, where over 20 compounds were reported with their relative concentration levels [13].

Some participants reported mild indigestion-related discomfort from swallowing a peppermint oil capsule, which passed within 1 h after capsule ingestion. No participant asked to withdraw from the study.

Figure 2 presents the peppermint oil washout profiles (fold change, compared to the reference sample, over time) of menthone as determined using an MCC-IMS with online breath sampling during the pilot study. The absolute levels of the washout profiles varied between participants, as demonstrated by the high relative standard deviation (% RSD), but the kinetics of the washouts were similar. The different profiles may also reflect differences in metabolism. Factors such as body mass index (BMI; kg m⁻²), age, and diet were collected to explore possible sources of variability in the peppermint washout profiles. Presenting data as a fold change ($I/{I_0}$) enables data sets from different platforms to be compared in a straightforward way.

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Washout profiles were modelled with a power relationship:

$$\begin{equation}I = {B_0}{t\,^{{B_1}}}\end{equation} \tag{ 1 }$$

where I—intensity (counts), t—time (h), and B coefficients transformed to a linear form, as follows:

$$\begin{equation}{\text{lo}}{{\text{g}}_{{\text{10}}}}\left( I \right) = {\text{lo}}{{\text{g}}_{{\text{10}}}}\left( {{B_0}} \right) + {B_1}{\text{lo}}{{\text{g}}_{{\text{10}}}}\left( t \right).\end{equation} \tag{ 2 }$$

The descriptive statistics of the washout profiles provided useful information about the precision and performance of the analytical platform. A plot of the fold-change log₁₀ ($I/{I_0}$) vs log₁₀ ($t$) of the washout of peppermint oil VOCs, in this case menthone, yielded a linear plot with R² values between 0.979 and 0.997 (figure 3). The x-axis intercept indicates the estimated time to return to initial concentrations. The curves also provide information on the sensitivity of a method (or limit of detection).

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Extrapolation of the power relationships in the pilot data shown in figure 3 provides a prediction of how long it will take menthone in breath to return to its initial (pre-ingestion, reference) concentration. (Note that these data are illustrative and not intended to be used as benchmarks; the benchmarks require the consolidated data from the full-scale peppermint studies with larger numbers of participants.) For the pilot data plotted in figure 3 the mean (±99% confidence level) washout time was determined to be 39.3 (±18.6) h, as indicated by the solid horizontal bar and arrow. Figure 2 highlights the biological variability between the participants with different individual uptake and elimination rates. Combining the data from all the peppermint studies will provide a larger sample size to enable a detailed examination of the nature of this variability. This will be addressed in a future synoptic paper.

It is important to note that variability in an individual's uptake and elimination of peppermint volatiles also extends to how long it takes for the exhaled concentration of peppermint volatiles to reach its maximum. Some participants will have a washout profile that results in less than four data points obtained after the maximum exhaled peppermint concentrations. Such profiles do not yield enough data to generate a reliable washout model, and should be excluded from benchmarking evaluation. Consequently, it appears that a precautionary approach of standardizing ranges of the BMI, age, and diet in the participant cohort may reduce the effect of variability in the participants' peppermint uptake and washout behaviour on the benchmarking evaluation. The influence of these factors on the washout of ingested volatiles is not well defined, and this aspect of the Peppermint Initiative will be studied and disseminated once all the benchmark datasets become available and have been analysed; see the synoptic paper comment above.

An example of how potential differences in data from different laboratories may be evaluated rapidly is also included in figure 3 with a dataset of a single washout curve from an alternative instrument, with a different sampling approach, obtained at a different laboratory (labelled 'F'). The data from experiment F is clearly different to the other pilot data. However, a complete Peppermint experiment dataset would be needed from the analytical system used for the F data to provide sufficient data on variability before an informed comparative evaluation could be reached. However, at face-value, the differences in datasets between the two techniques is sufficient to justify proceeding with caution when combining data from the two systems and running a full Peppermint experiment to establish a more rigorous comparison.

While the washout data (figures 2 and 3) reveal much about the precision and overall sensitivity of a technique towards a targeted compound, they do not disclose the complete range of attributes of all the data obtained from the study. The washout data tracks a peppermint marker but discloses little else about the sample being analysed. Further information about the characteristics of all the data acquired during the benchmark study has significant utility in evaluating the overall attributes of the method being used. The approach proposed below can be especially useful for the breathomics studies, where sensitivity, resolution, and richness of the information contained within the samples are crucial aspects for marker discoveries.

Figure 4 is an example of how the cumulative frequency distribution and intensity distributions of all analytical features may be combined usefully; obtained from an MCC-IMS method run on a participant's sample taken at 60 min, when the exhaled abundances of peppermint markers were at their highest. The nature of the 'breathome' recorded may be discerned by combining the distribution of the intensity of the features with the cumulative frequency distribution against retention time. Highlighting the peppermint washout markers, α-pinene, an unidentified feature, and eucalyptol (indicated with solid markers with increasing retention times, respectively), along with their extracted signals, places the peppermint washout data in the broader context of the individual's breathome for this sample. Consolidating all the data from a peppermint experiment will provide a systematic overview of the performance of the methodology under controlled conditions with a panel of peppermint volatiles providing a frame of reference.

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Experiment F was a comparative peppermint breath experiment undertaken with different instrumentation and a different sampling method to the pilot data. The difference between the observed washout in experiment F and the pilot data illustrates why multiple peppermint washout experiments are needed, and how data from this test may be used to compare the analytical performance of different breath analysis techniques, methods, and studies.

Combining and summarising the cumulative frequency and feature intensity distributions with the washout data from a peppermint experiment provides an overview of the performance of a technique for breath analysis and enables inter-/intra-laboratory differences in the fidelity of breath data to be ascertained. Ultimately, such data enable inter-technique comparisons based on a common approach to monitoring a controlled intervention, which considers the inherent biological variability present in all breath data.

All partners in the Peppermint Initiative received institutional ethics board approvals to undertake these studies. Each contributing group obtained approval from their local ethical review board to participate in this study. Written informed consent was obtained from all volunteer participants. All study protocols complied with the Declaration of Helsinki.

The 16 participating research groups and a summary of the initial techniques used for the first phase of the Peppermint study are listed in table 2. The Peppermint Initiative consortium is coordinated by Loughborough University, with an oversight team additionally comprising Radboud University, Fraunhofer IVV, and the University of Manchester. Regular (teleconference) meetings between the consortium partners have been held to coordinate the organization, management and operation of the study, data curation methods, data management, dissemination of results, publication strategy, and recruitment of new participating laboratories.

4.1.1. Study documentation.

Thirty-six bottles containing 60 × 200 mg peppermint oil capsules, obtained from Boots UK Ltd. (product number: 10 115 320) were acquired from the same production batch (batch no. 200 207) and distributed to the participating groups of the Peppermint Initiative.

To account for inter-participant variability a study group of ten participants is recommended. Participants are requested to exclude peppermint and peppermint-associated products from their diet and personal care routines for 24 h prior to participation in the study and until completion of the experiment.

Each participant provides a reference breath sample for analysis and then ingests a peppermint capsule washed down with 100 to 150 cm³ of tap water (t = 0 min). It is important that rigorous time-management is maintained, and that the peppermint capsule is taken within 30 min of the reference sample.

Each participant provides five additional breath samples at 60 min, 90 min, 165 min, 285 min, and 360 min post-ingestion.

In addition to the breath samples, at least one environmental air sample should be collected after one of the sampling points (selected at random) for each participant while both researcher and participant were still present in the room. An air supply/instrument blank sample should also be taken after completion of a sampling series (i.e. after 360 min).

This protocol was based on a 1D central-composite experimental design applied to a representative washout profile, obtained from a preparatory investigation; see below (figure 1).

Evaluation of the washout data of the peppermint oil constituents combined with a global summary of the VOC features isolated under these conditions provides a standardized test of a methodology, and enables comparison against benchmark data.

The Peppermint Initiative relies on data evaluation and sharing. Within this study, each participating team acquired different types of datasets that included: raw data; processed data; summary results; informed consent forms; and participant metadata. Each group was provided with a secure file-store in a cloud repository by the study coordinator (see table 3). No personal information was shared or stored to ensure anonymity of participants. All data were assigned a study code by the study coordinator and anonymized before release. Consolidated data will be available in *.csv file format for wider use upon request to the corresponding author once the research results have been published.

The Peppermint Initiative aims to establish a benchmark to ascertain performance of breath sampling and analytical methods, and to survey comparative performances of current breath research across the community. The pilot study presented here enabled the preparation and development of the methodology for the Peppermint experiment. Further, these initial trials highlighted the challenges associated with creating a benchmarking method and accompanying data work-up for breath analysis. Comparing different sampling approaches and analytical techniques requires sufficient sample numbers to account for both technical and biological variability. Alignment of data processing as well as quality assurance and control approaches across the Peppermint Initiative consortium are essential.

To date, 16 teams have provided data from 200 individual volunteer participants. Two hundred sets of six samples of comparative intra-subject variability profiles, 200 sets of comparative inter-subject variation, and 200 ambient air VOC profiles provide an objective multi-centre verified review of comparative analytical characteristics of different techniques and sampling approaches.

Moreover, this initial dataset and the accompanying protocol provides a benchmark standard for assessments that will support and verify quality assurance and control activities undertaken in the field of breath research.