Advancing fecal volatilome profiling by comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry and image pattern recognition

Comprehensive two-dimensional gas chromatography coupled with time-of-flight mass spectrometry (GC×GC-TOFMS) is a powerful technique suited for resolving complex mixtures of volatile organic compounds (VOCs). In this study, GC×GC-TOFMS was combined with an image-based chromatographic fingerprinting strategy to characterize the fecal volatilome of subjects affected by non-celiac gluten/wheat sensitivity (NCGWS) under dietary intervention. Fifty participants underwent a dietary protocol consisting of a gluten-free diet followed by gluten reintroduction while receiving probiotics or placebo. Headspace solid-phase microextraction (HS-SPME) conditions were specifically optimized to maximize the extraction coverage of volatiles, while a combined untargeted–targeted fingerprinting approach (UT fingerprinting) enabled the mapping of approximately 1000 volatile features in fecal samples. Chemometric analyses, including principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA), highlighted informative differences in VOC profiles induced by gluten reintroduction and probiotic administration. Around 270 VOCs were annotated, expanding the existing knowledge on the human fecal volatilome by 20%, predominantly comprising microbial fermentation-derived metabolites such as short-chain fatty acid esters, aldehydes, alcohols, terpenes, and aromatic hydrocarbons. PLS-DA models discriminated probiotic-treated individuals from placebo recipients with 89–90% accuracy, confirming the impact of probiotic supplementation on gut metabolic signatures. The integration of GC×GC-TOFMS and image-based chromatographic fingerprinting effectively captured subtle metabolic variations, demonstrating its suitability in biomarker discovery for dietary interventions and personalized nutrition studies.