Volatile emissions in human health applications: sampling and standardisation endeavours in relation to exogenous compounds in inhaled and exhaled breath

The early diagnosis of health issues at disease onset is of particular importance to ensure an effective and timely patient treatment and improve the resulting outcome success. The discovery and exploitation of biomarkers that act as indicators of deleterious pathophysiological processes, medication efficacy or exposures to environmental stressors not only provides an opportunity for diagnosis and treatment monitoring, but also for prediction and prevention of adverse health outcomes. Exhaled breath represents a biofluid that is of potential use for biomarker exploitation via its rich mixture of volatile organic compounds (VOCs). The premise of breath-based diagnostics is to utilise representative VOC constituents or their patterns in the exhaled breath of an individual as indicators of physiological imbalances. A handful of volatile breath biomarkers are already utilised in practical and/or clinical applications, demonstrating the potential of this approach, yet there are a number of obstacles that limit the development and routine adoption of breath tests. These include disparate sampling and analytical methodologies, as well as poor reproducibility of independent study outcomes. The main objective of the present research was to address current limitations and uncertainties in breath sampling and analysis by developing novel techniques to sample and analyse volatiles in breath and from sampling interface materials, as well as to assess standardisation procedures by comparing common analytical techniques in widespread use in the field of breath research. In the first part of this work, a novel breath sampling interface was designed as a compatible alternative to the conventional silicon mask used in a commercial breath sampling system. The new interface was designed to overcome specific shortcomings of the mask and consisted of a 3D-printed adapter mouthpiece and a pulmonary function filter. This interface represents a costeffective, reusable and sustainable solution for breath sampling. The suitability of this interface for use in breath sampling applications was assessed in terms of volatiles emitted from the material in relation to biocompatibility (inhalation toxicology) and potential biomarker confounders, as well as the degree of adsorption of potential biomarkers by the adapter materials. These assessments were made in comparison with the conventional mask. Volatile emissions from the different materials were determined after (hydro)thermal treatment, whereby a reduction of total VOC emissions in the adapter (by 99%) and in the filter (by 89%) compared to the mask (62%) could be achieved. In terms of compound uptake, the degree of losses by adsorption to the wetted surfaces of the interfaces was found to be compound-dependent, with negligible uptake of some compounds but marked losses of others across all materials. Generally, uptake levels were lowest for the adapter and most pronounced in the mask. The need to characterise volatile emissions from the materials motivated the task of developing a suitable novel online extraction and analysis method that allowed a rapid assessment of such emissions. This was achieved through use of a microchamber/thermal extractor (μ-CTE) coupled to a proton transfer reaction-mass spectrometer (PTR-MS). This configuration enabled the dynamic VOC emissions to be monitored online and provided data to determine emission rates and evaluate these in relation to their toxicological relevance. These analyses were complemented by thermal desorption comprehensive two-dimensional gas chromatography-mass spectrometry (TDGC × GC-MS) analyses to confirm VOC identities. Results showed that two of the materials investigated were of no concern, whereas the third material emitted compounds at a rate that would exceed the daily tolerance if it were used in breath sampling. As well as characterising the aforementioned sampling components, other aspects of breath testing in relation to standardisation and quality assurance were also explored in this work. Specifically, the degree of variability across a range of sampling and analytical platforms when simultaneously analysing common breath samples was assessed by means of the Peppermint Experiment benchmarking protocol. This study was conducted using a PTR-MS, a gas chromatography-ion mobility spectrometer (GC-IMS) and a GC×GCMS in combination with the respective dedicated breath sampling interfaces. The experiments with a cohort of 11 healthy volunteers followed the washout profile of peppermint compounds in breath within six hours after ingestion of a peppermint oil capsule. It was demonstrated that intra- and inter-individual differences were the many drivers for variability in the datasets compared to differences in instrument performance, which played only a minor role. Overall, this work contributes to a number of aspects of the field of breath research. The novel breath sampling interface developed in this work represents a configuration that reduces the risks associated with disruption of breath sample integrity and minimises the health hazards related to cross-contamination during the sampling procedure and exposure to toxic substances from sampling materials. Further, the newly developed analysis configuration to evaluate the dynamic VOC emissions from materials in real-time will facilitate quicker screening of potential toxicological and confounding volatiles, which represent critical aspects of consumer protection and sample integrity, respectively. Ultimately, standardisation of sampling and analysis methods will help to increase the reproducibility of research outcomes and pave the way towards the widespread applicability of breath-based tests in human health research.