Abstract
Breath volatile organic compound analysis can non-invasively detect diseases, with short-chain fatty acids (SCFAs) identified as key biomarkers. However, SCFA quantification is technically challenging due to chemical instability during thermal desorption (TD) tube analysis. Esterification with methanol may cause methyl ester formation, which impairs diagnostic sensitivity and reproducibility. We hypothesised that methanol-driven esterification of SCFAs is temperature- and time-dependent and can occur under common solvent handling and storage conditions used in TD-based analysis, even without the addition of acid or base catalyst. Here we show that methanol-driven SCFA esterification occurs in the liquid phase but not in the gas phase. Esterification rates increase with higher methanol-to-SCFA ratios and elevated temperatures. Furthermore, prolonged storage at higher temperatures accelerated SCFA esterification, reducing recovery by up to 70% after two months at room temperature and refrigerated conditions. Addressing these artefacts is crucial for ensuring the diagnostic accuracy of SCFA-based breath tests.
Data availability
All data generated or analysed during this study are included in this article, with numerical source data for all figures included under supplementary data 1. Further enquiries can be directed to the corresponding author.
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Acknowledgements
This project was supported by Royal Society of Chemistry through the Research Fund grant (R21-0038618851) awarded to IB. PKHL is funded by a Cancer Research UK programme grant awarded to GBH (EDDPGM-May21\100007). IB is funded on an Epilepsy Research Institute UK Emerging Leader Fellowship. MB was supported through an International Research Mobility Grant CEU–Santander. The lab of G.B.H. is supported by programme grants from Cancer Research UK (EDDPGM-May21\100007), Pancreatic Cancer UK (BT2022_Hanna, BT2025_Hanna) and National Institute for Health Research (NIHR207551). Additional laboratory funding originates from the Rosetrees and Stoneygate Trusts, HCA Healthcare UK and infrastructure funding support from the NIHR Imperial Biomedical Research Centre (BRC), and NIHR HealthTech Research Centre in In Vitro Diagnostics (formerly London Medtech and In vitro diagnostic Co-operative).
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Conceptualization & methodology: PKHL, AP, PS, GBH, IB. Investigation: PKHL, AHKW, YM, JJY, VC, MB, IB. Formal analysis: PKHL, AHKW, YM, IB. Project administration: PKHL, IB. Supervision: PS, GBH, IB. Writing – original draft: PKHL. Writing – review & editing: AP, PS, GBH, IB. Funding acquisition & resources: GBH, IB.
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GBH is a founder of a cancer diagnostic company. The remaining authors disclose no conflicts.
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Communications Chemistry thanks Christopher Mayhew and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. A peer review file is available.
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Leung, P.K.H., Wong, A.H.K., Ma, Y. et al. Methanol-driven esterification of volatile short-chain fatty acids in thermal desorption-based analysis. Commun Chem (2026). https://doi.org/10.1038/s42004-026-01998-5
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DOI: https://doi.org/10.1038/s42004-026-01998-5
