Evaluating off-line exhaled breath sam-pling 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 (VOC) detection. However, the lack of standardized sam-pling 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 estab-lished peppermint benchmarking protocol and comprehensive two-dimensional gas chroma-tography coupled to mass spectrometry (GC×GC-MS). Seven healthy participants completed the peppermint experiment, with breath samples col-lected at multiple time points following capsule ingestion. Washout curves for targeted terpe-noid compounds were analyzed to assess analytical performance, reproducibility, and back-ground 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 vari-ability, 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 (SOPS), 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 breathomics community and extend peppermint database for exhaled breath sampling. .