A comprehensive chromatographic fingerprinting study shows that dietary intervention and probiotic supplementation produce detectable, coordinated changes in the human fecal volatilome. The research demonstrates how analytical strategies that emphasize global patterns, rather than individual markers, can resolve metabolic variation in complex biological samples.
The researchers examined fecal samples from individuals with non-celiac gluten/wheat sensitivity (NCGWS) undergoing a controlled dietary protocol. Participants followed a gluten-free diet before gluten reintroduction, while receiving either a multi-strain probiotic formulation or a placebo. Samples collected at defined time points were analyzed to assess how dietary phase and supplementation influenced volatile organic compound (VOC) profiles linked to gut microbial activity.
To capture these changes, the researchers combined comprehensive two-dimensional gas chromatography with time-of-flight mass spectrometry (GC×GC-TOFMS) and an image-based data analysis workflow. Rather than focusing on a limited set of predefined targets, the approach was designed to detect broad, pattern-level shifts in the fecal volatilome, where many compounds co-elute and vary simultaneously.
Volatile compounds were extracted using headspace solid-phase microextraction and separated under orthogonal GC×GC conditions to increase separation capacity. The resulting two-dimensional chromatograms were treated as chemical “fingerprints,” allowing samples to be compared at the level of overall volatile patterns rather than individual analytes. This strategy enabled the reproducible detection of around 1000 features per sample, with approximately 270 VOCs subsequently annotated based on retention behavior and mass spectral matching.
The detected volatiles spanned several chemical classes, including short-chain fatty acids, esters, aldehydes, ketones, alcohols, terpenes, and aromatic hydrocarbons, many of which are linked to microbial fermentation and host–diet interactions. The authors report that their analysis expanded the catalog of known human fecal volatiles by roughly 20 percent.
Multivariate analysis showed structured shifts in volatilome profiles across dietary phases and between probiotic and placebo groups. These differences reflected coordinated changes across multiple VOC classes rather than single dominant markers.
According to the authors, integration of GC×GC-TOFMS with image-based fingerprinting “proved effective in capturing subtle metabolic variations within the fecal volatilome,” supporting its use in dietary intervention studies. They note that the approach “offers a robust analytical platform for biomarker discovery and for investigating gut metabolic responses in personalized nutrition research.”
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