INTRODUCTION: Faecal metabolomics investigates small molecules in stool to elucidate metabolic pathways and identify potential biomarkers. However, topographical heterogeneity within individual stool specimens poses a challenge for data consistency. Although this issue is well recognised, quantitative comparisons across defined stool regions, sampling depths, and whole-stool homogenates remain limited. OBJECTIVES: H NMR) spectroscopy. METHODS: Seventy-six metabolites were quantified across defined faecal regions and depths. Comparative analyses determined spatial differences in metabolite abundance and assessed the similarity between spot samples and bulk homogenates. RESULTS: Distinct metabolic profiles were observed between the two ends of the faecal bulk. The tail region, representing more recently discharged material, exhibited higher levels of 3-(3-hydroxyphenyl)propionate (+ 57-58%) and 3-phenylpropionate (+ 50%), whereas the head region showed greater abundance of proteolysis-related metabolites such as isovalerate (+ 30%) and methionine (+ 54%). No consistent differences were detected across sampling depths. Despite these metabolite-specific differences, overall deviations from the homogenised profile were small, with mean absolute differences remaining approximately 1% or less across regions. CONCLUSION: Pronounced longitudinal heterogeneity exists in faecal metabolite composition along the stool axis. In this pilot cohort, tail-region spot sampling most closely represents the overall faecal metabolome and may serve as a practical, less labour-intensive alternative to whole-stool homogenisation for metabolomic studies.
Jenickova et al. (2026) studied this question.