Introduction

Breath volatile organic compounds (VOCs) are promising non-invasive biomarkers for clinical purposes. In digestive health, gut microbiome VOCs enter the bloodstream and diffuse into exhaled breath, offering a real-time snapshot of gut activity and metabolism. Translating these biomarkers into clinical practice depends on precise identification and robust validation—challenges that require standardized protocols and data integration. Our previously developed breath collection and GC-MS analysis method identified 148 on-breath VOCs against ambient background that were catalogued into an Atlas using purified chemical standards. Building on this success, further method development was undertaken, with a focus on enhancing the detection of previously underrepresented compound classes.

Aims & Methods

Our aim was to create a high-resolution TD-GC-MS method using a PEG-phase column to detect and confidently identify gut microbial VOCs—fatty acyls, esters, phenols, indoles, and sulfones. By combining enhanced sample preparation and compound enrichment with rigorous spectral and retention-time matching, plus improved quality-control and identity-confirmation protocols, we could reliably assign chemical identities to candidate VOCs in breath samples.

Results

Using three metrics; standard deviation analysis, paired t-test, and ROC-AUC evaluation, we identified 621 VOCs as potential breath biomarkers, 223 of which met all three on-breath criteria. These tools effectively distinguished exhaled VOCs from ambient background, allowing us to select a focused subset for confident chemical identification. Notably, over 25% of these are metabolites derived from the gut microbiome, and their detection offers mechanistic insights into microbial activity. Butyric acid, for example, serves as a crucial energy source for colonocytes and plays a key role in regulating intestinal inflammation. Branched-chain fatty acids—including isobutyric acid, isovaleric acid, 2-methylbutyric acid, and 4-methylvaleric acid—are products of protein fermentation and have been associated with inflammatory responses. The phenolic compound p-cresol, linked to dysbiosis, may affect gut barrier integrity, while 2-methylindole, derived from altered tryptophan metabolism, has implications for immune modulation and gut homeostasis. Dimethyl sulfone, involved in sulphur metabolism, may contribute to inflammatory signalling. Alongside previously identified short-chain fatty acids such as acetic and propionic acid, these compounds represent a broad and functionally diverse profile of gut microbial metabolism. Their detection across all on-breath metrics supports their endogenous origin in exhaled breath.

Conclusion

The high-confidence identification of these gut-derived VOCs reinforces their potential as non-invasive biomarkers for monitoring gastrointestinal health. With the updated methodology, our Atlas now includes a total of 186 confirmed on-breath VOCs, paving the way for improved diagnostic breath tests tailored to gut health and disease.

Disclosure

No conflict of interest