Longitudinal Monitoring of Breath Biomarkers

Rapid, Reproducible Breath Biomarker Analysis

Longitudinal VOC monitoring graphic

Exhaled breath contains thousands of volatile organic compounds (VOCs), valuable biomarkers that reflect metabolic processes taking place within a person’s cells and tissues, within their microbiome, and from their response to environmental exposures. As VOCs in the bloodstream are efficiently exchanged with air in the lung’s alveoli, measuring VOCs in exhaled breath allows metabolic processes occurring throughout the body to be analyzed non-invasively (Figure 1).

Sources of VOCs in Exhaled Breath
Figure 1. VOCs originating from the entire body can be accessed by continuously sampling breath over a few minutes. Capturing breath VOCs onto the Breath Biopsy cartridge allows pre-concentration of volatile metabolites originating from the entire body via the circulatory system.

Measuring VOC biomarkers over time offers a completely non-invasive way of monitoring health, diagnosing disease and assessing response to therapy. This case study describes how the Breath Biopsy platform can be used to capture multiple breath samples over time, uncovering detailed changes in the concentration of VOCs present in breath.

Rationale for Longitudinal Measurements of Breath VOC Biomarkers

Single measurements of any biomarker provide only a snapshot of an individual’s current state. While this provides useful information, point measurements do not give information about the baseline abundance of biomarkers for that individual. Making multiple measurements over time allows detection of changes in biomarker concentration that can indicate deviation from a baseline state and may point to the onset of disease or a successful response to therapy.

Breath Biopsy can be used to monitor longitudinal change in exhaled VOCs, and discover biomarkers for monitoring:

  • Drug activity
  • Drug compliance
  • Disease burden
  • Disease recurrence
  • Therapy response
  • Changes pre- vs. post-treatment or surgery

An Experiment in Longitudinal VOC Analysis Using Breath Biopsy

Successful longitudinal measurements require highly reproducible sampling and analysis techniques. Our Breath Biopsy platform includes the ReCIVA Breath Sampler, which was designed in collaboration with experts in the breathomics field to provide a standardized method to collect exhaled breath samples.

By measuring VOCs in breath following ingestion of a peppermint capsule we show that Breath Biopsy can be used to observe the decrease in target compounds over time using repeated, robust breath collection and analysis over a period of 8 hours.

Breath Biopsy Workflow

After ingestion of the peppermint capsule, breath samples were collected from an individual onto a Breath Biopsy Cartridge every 30 minutes for 8 hours using a ReCIVA Breath Sampler and CASPER Air Supply. For comparison, two breath collections were made from the same individual prior to ingestion to provide a baseline concentration for the VOCs of interest. Breath samples were analyzed in the Breath Biopsy Clinical Lab by FAIMS and mass spectrometry.

VOCs in breath following capsule ingestion

Analysis of breath captured 30 minutes after consumption of the peppermint capsule shows a large increase in the VOCs ɑ-pinene, β-pinene, limonene, eucalyptol and (±)-menthol compared to baseline pre-ingestion controls captured immediately prior to taking the capsule (Figure 2). The most abundant of these peppermint-related compounds are ɑ-pinene, β-pinene and limonene. Limonene was present at part-per-trillion (ppt) concentrations.

Breath collections made every 30 minutes after this initial capture show a consistent decrease in the target VOCs over time. Captures made from 6.5 hours after consumption show the levels of the target VOCs decreasing to baseline levels. All of the target compounds display a similar washout curve over time.

Longitudinal monitoring of VOCs in exhaled breath
Figure 2. Peppermint-related VOCs in breath before and at 30 minute intervals after consumption of a peppermint capsule. Data from a single individual is shown. Breath was collected using ReCIVA and analyzed using TD-GC-TOF mass spectrometry, as described in Methods.

In this study, standard deviations were calculated for the 4 replicate samples collected on the Breath Biopsy Cartridge at each breath collect (Table 1). This gives an indication of the high intra-sample reproducibility of breath sampling and analysis using the Breath Biopsy platform.

%RSD peppermint capsule longitudinal data
Table 1. Mean average and range of %relative standard deviation (%RSD) of peak area for compounds shown in Figure 4. Note high max %RSDs are for points close to baseline where VOC concentrations were much lower.


This study demonstrates that the Breath Biopsy platform can be used to reproducibly capture and analyze breath samples during a longitudinal study. Using a peppermint capsule as a surrogate pharmaceutical, the levels of peppermint-related compounds in breath were found to increase rapidly after ingestion, and subsequently decrease following a washout curve over time.

Further Information

More detailed results of this study were presented as a poster at the Metabolomics 2018 Conference in Seattle, USA in June 2018.  To download the poster please click the link below.


ReCIVA Breath Sampler device

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