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Article

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

Leiden Academic Center for Drug Research, Division of System Biomedicine and Pharmacology, Leiden University, 2300 RA Leiden, The Netherlands
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Academic Editor: Helen G. Gika
Metabolites 2021, 11(6), 364; https://doi.org/10.3390/metabo11060364
Received: 31 March 2021 / Revised: 31 May 2021 / Accepted: 2 June 2021 / Published: 7 June 2021
(This article belongs to the Special Issue Metabolomics Methodologies and Applications II)
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. View Full-Text
Keywords: fecal metabolites; gut microbiota; metabolomics; sample preparation; LCMS (liquid chromatography mass spectrometry) fecal metabolites; gut microbiota; metabolomics; sample preparation; LCMS (liquid chromatography mass spectrometry)
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MDPI and ACS Style

Hosseinkhani, F.; Dubbelman, A.-C.; Karu, N.; Harms, A.C.; Hankemeier, T. Towards Standards for Human Fecal Sample Preparation in Targeted and Untargeted LC-HRMS Studies. Metabolites 2021, 11, 364. https://doi.org/10.3390/metabo11060364

AMA Style

Hosseinkhani F, Dubbelman A-C, Karu N, Harms AC, Hankemeier T. Towards Standards for Human Fecal Sample Preparation in Targeted and Untargeted LC-HRMS Studies. Metabolites. 2021; 11(6):364. https://doi.org/10.3390/metabo11060364

Chicago/Turabian Style

Hosseinkhani, Farideh, Anne-Charlotte Dubbelman, Naama Karu, Amy C. Harms, and Thomas Hankemeier. 2021. "Towards Standards for Human Fecal Sample Preparation in Targeted and Untargeted LC-HRMS Studies" Metabolites 11, no. 6: 364. https://doi.org/10.3390/metabo11060364

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