Towards Standards for Human Fecal Sample Preparation in Targeted and Untargeted LC-HRMS Studies

Metabolites

Amy C. Harms¹  Naama Karu¹  Farideh Hosseinkhani¹  Anne-Charlotte Dubbelman¹  Thomas Hankemeier¹ 

[1] Leiden Academic Center for Drug Research, Division of System Biomedicine and Pharmacology, Leiden University, 2300 RA Leiden, The Netherlands;

关键词: fecal metabolites;    gut microbiota;    metabolomics;    sample preparation;    LCMS (liquid chromatography mass spectrometry);   

DOI  :  10.3390/metabo11060364

来源: DOAJ

【 摘 要 】

Gut microbiota and their metabolic products are increasingly being recognized as important modulators of human health. The fecal metabolome provides a functional readout of the interactions between human metabolism and the gut microbiota in health and disease. Due to the high complexity of the fecal matrix, sample preparation often introduces technical variation, which must be minimized to accurately detect and quantify gut bacterial metabolites. Here, we tested six different representative extraction methods (single-phase and liquid–liquid extractions) and compared differences due to fecal amount, extraction solvent type and solvent pH. Our results indicate that a minimum fecal (wet) amount of 0.50 g is needed to accurately represent the complex texture of feces. The MTBE method (MTBE/methanol/water, 3.6/2.8/3.5, v/v/v) outperformed the other extraction methods, reflected by the highest extraction efficiency for 11 different classes of compounds, the highest number of extracted features (97% of the total identified features in different extracts), repeatability (CV < 35%) and extraction recovery (≥70%). Importantly, optimization of the solvent volume of each step to the initial dried fecal material (µL/mg feces) offers a major step towards standardization, which enables confident assessment of the contributions of gut bacterial metabolites to human health.

【 授权许可】

Unknown