Evaluating off-line exhaled breath sampling techniques: a comparative study of practices for clinical implementation

Exhaled breath analysis represents a promising non-invasive approach for disease monitoring through volatile organic compounds (VOCs) detection. However, the lack of standardized sampling methods do not enable direct clinical translation. This study compared three widely used offline breath sampling techniques (Tedlar® bags, BioVOC-2®, and ReCIVA®) using the established peppermint benchmarking protocol and comprehensive two-dimensional gas chromatography coupled to mass spectrometry (GC × GC-MS). Seven healthy participants completed the peppermint experiment, with breath samples collected at multiple time points following capsule ingestion. Washout curves for targeted terpenoid compounds were analyzed to assess analytical performance, reproducibility, and background contamination across devices. Clinical feasibility was evaluated through focus groups with clinicians, researchers, and study participants. Tedlar® bags demonstrated reliable performance with lowest overall pooled relative standard deviations, though sensitive to exogenous contamination. ReCIVA® showed higher overall variability, superior selectivity and reduced background interference compared to Tedlar® bags (p< 0.01). However, ReCIVA® showed higher complexity, cost, reduced comfort and potential for saliva contamination during extended sampling. BioVOC-2® offered operational simplicity but was limited by small sampling volume (129 ml) reducing its sensitivity and manual handling variability. No single device emerged as universally optimal. Tedlar® bags, when accompanied by rigorous standard operating procedures, remain most suitable for large-scale studies, BioVOC-2® for rapid targeted screening, and ReCIVA® for controlled research requiring high selectivity. Successful clinical implementation will require balancing analytical performance with practical considerations including patient comfort, cost-effectiveness, and workflow integration. These findings support ongoing standardization efforts within the breathomics community and extend peppermint database for exhaled breath sampling.