COMPREHENSIVE TWO-DIMENSIONAL GAS CHROMATOGRAPHY-TIME OF FLIGHT MASS SPECTROMETRY (GC×GC-TOF MS) AND IMAGE PATTERN RECOGNITION: VOLATILOMICS UNREVEAL METABOLIC SYNERGIES IN FECAL MICROBIOME

Comprehensive two-dimensional gas chromatography-time of flight mass spectrometry (GC×GC-TOF MS) represents significant advancement in analytical techniques, providing exceptional resolution and sensitivity, especially for characterizing complex samples with high chemical dimensionality. Unlike one-dimensional gas chromatography, GC×GC-TOF MS offers a more precise analysis of volatile organic compounds (VOCs), effectively addressing issues of co-elution and covering a wide dynamic range of concentrations, a key feature in metabolomic studies. This study explores the application of GC×GC-TOF MS for analyzing fecal volatile metabolites, focusing on profiling the fecal volatilome, an underexplored area in metabolomics. Fifty individuals with suspected non-celiac gluten sensitivity (NCGS) underwent a double-blind placebo-controlled gluten challenge. Twenty-seven participants who were gluten responsive were randomized into two groups: one receiving daily probiotics (L. plantarum, L. paracasei, L. salivarius) and the other receiving a placebo, both under a gluten-free diet for 4 weeks, after which gluten was reintroduced for 2 weeks. Using combined untargeted and targeted fingerprinting (UT fingerprinting), the study identified over 830 volatile features, with around 200 being putatively identified and correlated with study variables using chemometric analyses. Results showed significant changes in the fecal volatilome after probiotic treatment, with Partial-Least Squares Discriminant Analysis (PLS-DA) providing classification models with 89% accuracy at 4 weeks and 90% at 6 weeks. Key features included butanoic and propanoic acid esters, alcohols, aldehydes and terpenoids. Despite the limited sample size, this study underscores the potential of GC×GC-TOF MS in advancing personalized nutrition and enhancing our understanding of fecal metabolomics.