Analyzing human breath for pharmacokinetics

Exhaled breath analysis is a potential method for pharmacokinetic investigations aimed at understanding the transformations, distribution and destiny of exogenous compounds in the human body.

Analyzing human breath for pharmacokinetics

Image Credit: TOFWERK

The measurement of volatile and semi-volatile organic molecules in breath allows non-invasive and real-time monitoring of metabolic processes, as opposed to traditional blood or urine collection, which requires extensive processing periods.

With the widespread use of “breathalyzers” for ethanol detection, law enforcement has already recognized the benefits of real-time breath analysis. A considerably more chemically thorough study can also benefit medical studies.

The Vocus Chemical Ionization Time-of-Flight Mass Spectrometer (CI-TOF) can monitor hundreds of substances in a human breath at ultra-low concentrations (ppt) concurrently and quantitatively, making it a potential online tool for detecting fast changes in the human metabolome.

Experimental design

The breath of three healthy adult participants was measured after they took a capsule containing 300 mg of eucalyptol oil in a proof-of-concept study (Gelomyrtol, Alpinamed). During an inflammation, eucalyptol oil is a popular active medicinal component that helps to clear the airways.

The capsule is broken in the gastrointestinal tract after intake, and eucalyptol is transported and circulated through the bloodstream. It crosses the blood-air barrier in the lungs and is expelled during breathing.

Eucalyptol-derived volatile molecules can also pass through the blood-air barrier and be exhaled. As a result, online monitoring of volatiles concentrations in the breath allows for the assessment of changes in volatiles concentrations in the blood.

The Vocus CI-TOF was used to measure each of the three individuals’ breath profiles 15 minutes before and every 15–30 minutes following capsule intake. A customized heated breath intake was used to directly sample human breath into the Vocus.

For optimal detection of oxygenated molecules, the instrument was switched between typical PTR (H3O+) mode and NH4+ reagent ions. The data was collected at a time resolution of 2 Hz.


The concentrations of acetone and eucalyptol in thirteen exhaled breaths are shown in Figure 1. Acetone, a ketone produced by lipolysis, is the most common ketone found in human breath.

The primary ingredient in eucalyptol oil is eucalyptol (1,8-cineole). The concentrations were obtained using a liquid calibration system and direct calibration of 1,8-cineole solution.

Analyzing human breath for pharmacokinetics

Figure 1. Example of a measurement set of exhalations sampled via breath inlet coupled to VOCUS CI-TOF. Concentrations (ppb) of acetone and eucalyptol are shown. The dashed area region highlights one single breath exhalation. Time zero corresponds to ~3 hours after the capsule ingestion. Image Credit: TOFWERK

Figure 2 shows mass spectra taken in PTR and NH4+ modes before and after capsule consumption. Acetone and isoprene are the most common VOCs in the breath before taking a capsule. In the NH4+ mode, strong peaks at the mass-to-charge ratio (m/Q) 172.17, 154.16, and 137.13 are recorded five hours after consumption.

These are eucalyptol (C10H18O) and monoterpenes (C10H16) molecules such as α-pinene, β-pinene, Camphene, or Limonene, which are present in eucalyptus oil to a lesser amount or generated during eucalyptol molecule metabolism.

As the amount of C10H16NH4+ ion found at m/Q 154 in a pure eucalyptol standard in NH4+ mode is less than 2%, the contribution of eucalyptol fragmentation to the monoterpene signal may be ignored.

In PTR mode, the most significant peak is seen at m/Q 137 (C10H17+), with just a small quantity of parent ion signals for eucalyptol at m/Q 155 (C10H19O+). As a result, the NH4+ mode has a major advantage over PTR in terms of fragmentation suppression and hence the simpler detection of eucalyptol and its metabolites.

Analyzing human breath for pharmacokinetics

Figure 2. Mass spectra recorded in NH4+ and PTR mode for one individual before and after eucalyptol oil ingestion. Image Credit: TOFWERK

Figure 3 shows the amount of eucalyptol measured in one subject’s exhaled breath throughout a 10-hour period. Using a simple exponential fit, the greatest value attained was ~700 ppb with an elimination rate constant of 0.33 h-1.

The next day, the same person was tested using the same measuring protocol: the highest value was ~650 ppb, with an elimination rate constant of 0.25 h-1. Using the average value, this person’s eucalyptol half-life in breath would be ~2.3 hours.

Analyzing human breath for pharmacokinetics

Figure 3. Washout of eucalyptol from one of the subjects over 10 h after an ingestion of a eucalyptol oil containing capsule. Three exhalations were measured each 15-30 minutes for better statistics. Image Credit: TOFWERK

The venous blood levels are reflected in the breath concentration, and after 4–5 half-lives, the drug level should be ~3% of its maximal value, with no impact predicted.

In this situation, the concentration is still ~45 ppb (~7% of maximum value) after 8 half-lives. This shows that a two-phase model containing fast and slow exponential decay would be more realistic in describing the elimination of eucalyptol from the body. Simple and double exponential fits for two individuals are illustrated in Figure 4.

Analyzing human breath for pharmacokinetics

Figure 4. Washout kinetics for eucalyptol from two individuals after an ingestion of a eucalyptol oil containing capsule. Image Credit: TOFWERK

The removal of eucalyptol is slower in both circumstances than in the single-exponential distribution. It can take up to two days for eucalyptol concentration to restore to its initial level.

Figure 5 depicts the temporal profiles of the described compounds in the breath of three people. Depending on the test person, the Tmax for eucalyptol in the breath ranges from 2 to 4 hours. Dehydrocineol (C10H16O), oxocineol (C10H16O2), and hydroxycineole (C10H18O2) are the primary metabolites of eucalyptol (1-3).

A delay in the kinetics was seen for these compounds in exhaled breath, demonstrating the use of Vocus CI-TOF in pharmacokinetics research.

Analyzing human breath for pharmacokinetics

Figure 5. Temporal profiles of signal intensities normalized to its maxima for eucalyptol (C10H18O), monoterpene (C10H16) and in breath elevated oxygenated species (C10H16-18O1-2). Solid lines represent eucalyptol present in the original capsule whereas dashed lines represent possible metabolites. Image Credit: TOFWERK


  1. M. Duisken, F. Sandner, B. Blomeke, J. Hollender, Metabolism of 1,8-cineole by human cytochrome P450 enzymes: identification of a new hydroxylated metabolite. Biochim Biophys Acta 1722, 304-311 (2005).
  2. K. Horst, M. Rychlik, Quantification of 1,8-cineole and of its metabolites in humans using stable isotope dilution assays. Mol Nutr Food Res 54, 1515-1529 (2010).
  3. G. J. Pass, S. McLean, I. Stupans, N. Davies, Microsomal metabolism of the terpene 1,8-cineole in the common brushtail possum (Trichosurus vulpecula), koala (Phascolarctos cinereus), rat and human. Xenobiotica 31, 205-221 (2001).


TOFWERK is a global leader in time-of-flight mass spectrometry, delivering sensitive instruments for laboratory, industrial, and field analyses. Our customers’ interests range from materials science and geochemistry to metabolomics and trace-gas

TOFWERK engineers and scientists collaborate with research laboratories and OEM customers to develop custom MS solutions based on our modular design platform. This platform enables rapid design and manufacturing of novel instrumentation for research laboratories and OEM customers.

Our end-user product line includes the icpTOF, Vocus PTR-TOF, IMS-TOF, and EI-TOF for GC. These mass spectrometers bring the speed and sensitivity of TOFMS to many disciplines and sample types.

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Last updated: Jun 21, 2022 at 6:50 AM


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