Comparison of three breath sampling methods for volatile organic compounds analysis in healthy adults: End-Tidal, Whole, and Continuous

Abstract Breath volatile organic compounds (VOCs) are promising biospecimens to be potentially utilized as non-invasive biomarkers. In this study, we analyzed breath samples from 90 non-smoking healthy adults using three sampling methods—Whole, Continuous, and End-tidal—to compare and evaluate patterns of breath VOCs detected by each. A total of 1,346 VOCs were identified, with 34 consistently detected across all methods, forming a core set of robust compounds. While principal component analysis showed no distinct clustering by method, several VOCs, including methylcyclohexane, toluene, D-Limonene, ethylbenzene and 1,1,2-trichloro-1,2,2-trifluoroethane, displayed method-dependent intensity differences. The Continuous method tended to yield lower values, but overall VOC profiles remained comparable to those from End-tidal samples. Participant surveys indicated that Whole breath was perceived as most convenient, whereas the Continuous method achieved consistent acceptability. These findings suggest that although End-tidal sampling remains the reference method, the Continuous method may serve as a practical and user-friendly alternative, particularly when alveolar air collection is not feasible. By establishing healthy reference VOCs and comparing the feasibility of different approaches, this study provides a foundation for applying breath analysis more flexibly in future clinical and translational research.