Standardization of Fecal Metabolomics Using Microbiome Preservation Kits: Implications for Multiomics Integration

With the advancement of multiomics technologies and cohort study designs, integrative omics research is increasingly applied to human health and nutrition. However, optimal storage and preprocessing of labile biological samples, particularly feces, remain challenging. In this study, we systematically evaluated three normalization methods-wet weight, dry weight, and protein quantification-for quantitative metabolomic profiling of fecal samples, using 41 metabolites. Fresh fecal samples from three healthy individuals showed high reproducibility, with 24 metabolites exhibiting a coefficient of variation (CV) below 30 for both wet and dry weight normalization. Fecal samples from 20 obese patients collected using the OMNIgene·GUT kit demonstrated improved reproducibility with wet weight normalization (20 metabolites, CV < 30) and protein quantification normalization (19 metabolites, CV < 30), whereas dry weight normalization yielded no metabolites meeting the CV < 30 criterion. Direct analysis of the kit solution without a drying step further enhanced chromatographic clarity, highlighting practical considerations for large-scale studies. Overall, wet weight normalization consistently minimized variation across sample types, providing a robust and standardized framework for fecal metabolite profiling. These findings demonstrate that the OMNIgene·GUT kit is compatible with broad-spectrum metabolomic analyses and support its integration into multiomics workflows. By establishing reproducible normalization protocols, this study provides the foundation for accurate, comparable, and scalable fecal metabolomics in both clinical and nutritional research settings.