{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,11,9]],"date-time":"2026-11-09T09:16:12Z","timestamp":1794215772055,"version":"4.8.0"},"reference-count":42,"publisher":"MDPI AG","issue":"4","license":[{"start":{"date-parts":[[2024,4,19]],"date-time":"2024-04-19T00:00:00Z","timestamp":1713484800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Microorganisms"],"abstract":"<jats:p>Short chain fatty acids (SCFAs) are primarily produced in the caecum and proximal colon via the bacterial fermentation of undigested carbohydrates that have avoided digestion in the small intestine. Increasing evidence supports the critical role that SCFAs play in health and homeostasis. Microbial SCFAs, namely butyric acid, serve as a principal energy source for colonocytes, and their production is essential for gut integrity. A direct link between SCFAs and some human pathological conditions, such as inflammatory bowel disease, irritable bowel syndrome, diarrhea, and cancer, has been proposed. The direct measurement of SCFAs in feces provides a non-invasive approach to demonstrating connections between SCFAs, microbiota, and metabolic diseases to estimate their potential applicability as meaningful biomarkers of intestinal health. This study aimed to adapt a robust analytical method (liquid\u2013liquid extraction, followed by isobutyl chloroformate derivatization and GC\u2013MS analysis), with comparable performances to methods from the literature, and to use this tool to tackle the question of pre-analytical conditions, namely stool processing. We focused on the methodology of managing stool samples before the analysis (fresh stool or dilution in either ethanol\/methanol, lyophilized stool, or RNAlater\u00ae), as this is a significant issue to consider for standardizing results between clinical laboratories. The objective was to standardize methods for future applications as diagnostic tools. In this paper, we propose a validated GC\u2013MS method for SCFA quantification in stool samples, including pre- and post-analytical comparison studies that could be easily used for clinical laboratory purposes. Our results show that using lyophilization as a stool-processing method would be the best method to achieve this goal.<\/jats:p>","DOI":"10.3390\/microorganisms12040828","type":"journal-article","created":{"date-parts":[[2024,4,19]],"date-time":"2024-04-19T10:53:17Z","timestamp":1713523997000},"page":"828","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":13,"title":["Effect of Stool Sampling on a Routine Clinical Method for the Quantification of Six Short Chain Fatty Acids in Stool Using Gas Chromatography\u2013Mass Spectrometry"],"prefix":"10.3390","volume":"12","author":[{"given":"Tarek","family":"Mahdi","sequence":"first","affiliation":[{"name":"H\u00f4pital Piti\u00e9 Salp\u00eatri\u00e8re-Charles Foix, AP-HP, Service de Biochimie Endocrinienne et Oncologique, 75000 Paris, France"},{"name":"Sorbonne Universit\u00e9, Inserm, UMR_S 1166, Research Institute of Cardiovascular Disease, Metabolism and Nutrition, 75000 Paris, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Aurore","family":"Desmons","sequence":"additional","affiliation":[{"name":"Centre de Recherche Saint-Antoine, Sorbonne Universit\u00e9, INSERM, AP-HP, D\u00e9partement Metomics, H\u00f4pital Saint Antoine, 75000 Paris, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Pranvera","family":"Krasniqi","sequence":"additional","affiliation":[{"name":"Centre de Recherche Saint-Antoine, Sorbonne Universit\u00e9, INSERM, AP-HP, D\u00e9partement Metomics, H\u00f4pital Saint Antoine, 75000 Paris, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jean-Marc","family":"Lacorte","sequence":"additional","affiliation":[{"name":"H\u00f4pital Piti\u00e9 Salp\u00eatri\u00e8re-Charles Foix, AP-HP, Service de Biochimie Endocrinienne et Oncologique, 75000 Paris, France"},{"name":"Sorbonne Universit\u00e9, Inserm, UMR_S 1166, Research Institute of Cardiovascular Disease, Metabolism and Nutrition, 75000 Paris, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nathalie","family":"Kapel","sequence":"additional","affiliation":[{"name":"H\u00f4pital Piti\u00e9 Salp\u00eatri\u00e8re-Charles Foix, AP-HP, Service de Coprologie Fonctionnelle, 75000 Paris, France"},{"name":"Universit\u00e9 Paris Cit\u00e9, Inserm, UMR_S 1139, 75000 Paris, France"},{"name":"Paris Center for Microbiome Medicine, Federation Hospitalo-Universitaire, 75000 Paris, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Antonin","family":"Lamazi\u00e8re","sequence":"additional","affiliation":[{"name":"Centre de Recherche Saint-Antoine, Sorbonne Universit\u00e9, INSERM, AP-HP, D\u00e9partement Metomics, H\u00f4pital Saint Antoine, 75000 Paris, France"},{"name":"Paris Center for Microbiome Medicine, Federation Hospitalo-Universitaire, 75000 Paris, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Salma","family":"Fourati","sequence":"additional","affiliation":[{"name":"H\u00f4pital Piti\u00e9 Salp\u00eatri\u00e8re-Charles Foix, AP-HP, Service de Biochimie Endocrinienne et Oncologique, 75000 Paris, France"},{"name":"Sorbonne Universit\u00e9, Inserm, UMR_S 1166, Research Institute of Cardiovascular Disease, Metabolism and Nutrition, 75000 Paris, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1891-9253","authenticated-orcid":false,"given":"Thibaut","family":"Eguether","sequence":"additional","affiliation":[{"name":"Centre de Recherche Saint-Antoine, Sorbonne Universit\u00e9, INSERM, AP-HP, D\u00e9partement Metomics, H\u00f4pital Saint Antoine, 75000 Paris, France"},{"name":"Paris Center for Microbiome Medicine, Federation Hospitalo-Universitaire, 75000 Paris, France"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2024,4,19]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1635","DOI":"10.1126\/science.1110591","article-title":"Diversity of the Human Intestinal Microbial Flora","volume":"308","author":"Eckburg","year":"2005","journal-title":"Science"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1564","DOI":"10.1053\/j.gastro.2014.01.058","article-title":"Diet and the Intestinal Microbiome: Associations, Functions, and Implications for Health and Disease","volume":"146","author":"Albenberg","year":"2014","journal-title":"Gastroenterology"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"1107","DOI":"10.1053\/j.gastro.2014.12.036","article-title":"Food, Immunity, and the Microbiome","volume":"148","author":"Tilg","year":"2015","journal-title":"Gastroenterology"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"5573","DOI":"10.1021\/pr300637d","article-title":"Gut Microbial Activity, Implications for Health and Disease: The Potential Role of Metabolite Analysis","volume":"11","author":"Nyangale","year":"2012","journal-title":"J. Proteome Res."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"47","DOI":"10.1021\/acs.jmedchem.7b00244","article-title":"Microbiota\u2013Host Transgenomic Metabolism, Bioactive Molecules from the Inside","volume":"61","author":"Turroni","year":"2018","journal-title":"J. Med. Chem."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"1262","DOI":"10.1126\/science.1223813","article-title":"Host-Gut Microbiota Metabolic Interactions","volume":"336","author":"Nicholson","year":"2012","journal-title":"Science"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"499","DOI":"10.1046\/j.1365-2036.1998.00337.x","article-title":"Review Article: Short Chain Fatty Acids in Health and Disease","volume":"12","author":"Sellin","year":"1998","journal-title":"Aliment. Pharmacol. Ther."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"2325","DOI":"10.1194\/jlr.R036012","article-title":"The Role of Short-Chain Fatty Acids in the Interplay between Diet, Gut Microbiota, and Host Energy Metabolism","volume":"54","author":"Groen","year":"2013","journal-title":"J. Lipid Res."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"9","DOI":"10.1016\/j.ab.2017.03.007","article-title":"Analysis of Short-Chain Fatty Acids in Human Feces: A Scoping Review","volume":"526","author":"Primec","year":"2017","journal-title":"Anal. Biochem."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1031","DOI":"10.1152\/physrev.2001.81.3.1031","article-title":"Short-Chain Fatty Acids and Human Colonic Function: Roles of Resistant Starch and Nonstarch Polysaccharides","volume":"81","author":"Topping","year":"2001","journal-title":"Physiol. Rev."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"123","DOI":"10.1007\/978-1-4615-5967-2_14","article-title":"Butyrate and the Colonocyte. Production, Absorption, Metabolism, and Therapeutic Implications","volume":"427","author":"Lederer","year":"1997","journal-title":"Adv. Exp. Med. Biol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"411","DOI":"10.3920\/BM2020.0057","article-title":"Short Chain Fatty Acids in Human Gut and Metabolic Health","volume":"11","author":"Blaak","year":"2020","journal-title":"Benef. Microbes"},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Al-Qadami, G.H., Secombe, K.R., Subramaniam, C.B., Wardill, H.R., and Bowen, J.M. (2022). Gut Microbiota-Derived Short-Chain Fatty Acids: Impact on Cancer Treatment Response and Toxicities. Microorganisms, 10.","DOI":"10.3390\/microorganisms10102048"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"64","DOI":"10.1186\/s12964-022-00869-5","article-title":"Regulatory Role of Short-Chain Fatty Acids in Inflammatory Bowel Disease","volume":"20","author":"Zhang","year":"2022","journal-title":"Cell Commun. Signal."},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Xiao, L., Liu, Q., Luo, M., and Xiong, L. (2021). Gut Microbiota-Derived Metabolites in Irritable Bowel Syndrome. Front. Cell. Infect. Microbiol., 11.","DOI":"10.3389\/fcimb.2021.729346"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"661493","DOI":"10.3389\/fimmu.2021.661493","article-title":"Serum Short-Chain Fatty Acids and Associations With Inflammation in Newly Diagnosed Patients With Multiple Sclerosis and Healthy Controls","volume":"12","author":"Olsson","year":"2021","journal-title":"Front. Immunol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"5244","DOI":"10.1038\/s41598-021-84881-8","article-title":"Associations of Serum Short-Chain Fatty Acids with Circulating Immune Cells and Serum Biomarkers in Patients with Multiple Sclerosis","volume":"11","author":"Trend","year":"2021","journal-title":"Sci. Rep."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"1275","DOI":"10.1136\/gutjnl-2013-304833","article-title":"A Decrease of the Butyrate-Producing Species Roseburia Hominis and Faecalibacterium Prausnitzii Defines Dysbiosis in Patients with Ulcerative Colitis","volume":"63","author":"Machiels","year":"2014","journal-title":"Gut"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"366","DOI":"10.1093\/carcin\/bgw019","article-title":"Manipulation of the Gut Microbiota Using Resistant Starch Is Associated with Protection against Colitis-Associated Colorectal Cancer in Rats","volume":"37","author":"Hu","year":"2016","journal-title":"Carcinogenesis"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1515","DOI":"10.4161\/15384047.2014.955764","article-title":"Butyrylated Starch Affects Colorectal Cancer Markers Beneficially and Dose-Dependently in Genotoxin-Treated Rats","volume":"15","author":"Toden","year":"2014","journal-title":"Cancer Biol. Ther."},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Song, H.E., Lee, H.Y., Kim, S.J., Back, S.H., and Yoo, H.J. (2019). A Facile Profiling Method of Short Chain Fatty Acids Using Liquid Chromatography-Mass Spectrometry. Metabolites, 9.","DOI":"10.3390\/metabo9090173"},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"McKay, M.J., Castaneda, M., Catania, S., Charles, K.A., Shanahan, E., Clarke, S.J., Engel, A., Varelis, P., and Molloy, M.P. (2023). Quantification of Short-Chain Fatty Acids in Human Stool Samples by LC-MS\/MS Following Derivatization with Aniline Analogues. J. Chromatogr. B Anal. Technol. Biomed. Life Sci., 1217.","DOI":"10.1016\/j.jchromb.2023.123618"},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Liebisch, G., Ecker, J., Roth, S., Schweizer, S., \u00d6ttl, V., Sch\u00f6tt, H.F., Yoon, H., Haller, D., Holler, E., and Burkhardt, R. (2019). Quantification of Fecal Short Chain Fatty Acids by Liquid Chromatography Tandem Mass Spectrometry\u2014Investigation of Pre-Analytic Stability. Biomolecules, 9.","DOI":"10.3390\/biom9040121"},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Eberhart, B.L., Wilson, A.S., O\u2019Keefe, S.J.D., Ramaboli, M.C., and Nesengani, L.T. (2021). A Simplified Method for the Quantitation of Short-Chain Fatty Acids in Human Stool. Anal. Biochem., 612.","DOI":"10.1016\/j.ab.2020.114016"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"121972","DOI":"10.1016\/j.jchromb.2020.121972","article-title":"Development and Validation of a GC-FID Method for the Analysis of Short Chain Fatty Acids in Rat and Human Faeces and in Fermentation Fluids","volume":"1143","author":"Scortichini","year":"2020","journal-title":"J. Chromatogr. B"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"51","DOI":"10.1016\/j.ab.2017.12.001","article-title":"Rapid Profiling Method for Mammalian Feces Short Chain Fatty Acids by GC-MS","volume":"543","author":"Furuhashi","year":"2018","journal-title":"Anal. Biochem."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"73","DOI":"10.1016\/j.jchromb.2018.09.013","article-title":"A Fast and Accurate Way to Determine Short Chain Fatty Acids in Mouse Feces Based on GC\u2013MS","volume":"1099","author":"Han","year":"2018","journal-title":"J. Chromatogr. B Anal. Technol. Biomed. Life Sci."},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Giera, M. (2018). Clinical Metabolomics, Humana Press.","DOI":"10.1007\/978-1-4939-7592-1"},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Ueyama, J., Oda, M., Hirayama, M., Sugitate, K., Sakui, N., Hamada, R., Ito, M., Saito, I., and Ohno, K. (2020). Freeze-Drying Enables Homogeneous and Stable Sample Preparation for Determination of Fecal Short-Chain Fatty Acids. Anal. Biochem., 589.","DOI":"10.1016\/j.ab.2019.113508"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"719","DOI":"10.1016\/j.jchromb.2009.01.039","article-title":"Application of Liquid Chromatography-Mass Spectrometry to Measure Short Chain Fatty Acids in Blood","volume":"877","author":"Bloemen","year":"2009","journal-title":"J. Chromatogr. B Anal. Technol. Biomed. Life Sci."},{"key":"ref_31","unstructured":"(2022). Medical Laboratories\u2014Requirements for Quality and Competence (Standard No. ISO 15189)."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"216","DOI":"10.1016\/j.forsciint.2006.05.021","article-title":"Validation of New Methods","volume":"165","author":"Peters","year":"2007","journal-title":"Forensic Sci. Int."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"577","DOI":"10.4155\/bio-2017-0226","article-title":"Proposal for Risk-Based Scientific Approach on Full and Partial Validation for General Changes in Bioanalytical Method","volume":"10","author":"Mochizuki","year":"2018","journal-title":"Bioanalysis"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"300","DOI":"10.1016\/j.ejps.2010.12.008","article-title":"Workshop\/Conference Report on EMA Draft Guideline on Validation of Bioanalytical Methods","volume":"42","author":"Blume","year":"2011","journal-title":"Eur. J. Pharm. Sci."},{"key":"ref_35","unstructured":"FDA, and ICH (2024, April 09). Bioanalytical Method Validation, Available online: https:\/\/www.fda.gov\/media\/128343\/download."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"1405","DOI":"10.1080\/19490976.2020.1759489","article-title":"Freeze-Dried Fecal Samples Are Biologically Active after Long-Lasting Storage and Suited to Fecal Microbiota Transplantation in a Preclinical Murine Model of Clostridioides Difficile Infection","volume":"11","author":"Reygner","year":"2020","journal-title":"Gut Microbes"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"818","DOI":"10.1007\/s11306-013-0500-6","article-title":"A Targeted Metabolomic Protocol for Short-Chain Fatty Acids and Branched-Chain Amino Acids","volume":"9","author":"Zheng","year":"2013","journal-title":"Metabolomics"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"53","DOI":"10.2174\/1874091X01004010053","article-title":"The Impact of the Level of the Intestinal Short Chain Fatty Acids in Inflammatory Bowel Disease Patients Versus Healthy Subjects","volume":"4","author":"Abdulamir","year":"2010","journal-title":"Open Biochem. J."},{"key":"ref_39","doi-asserted-by":"crossref","unstructured":"Lemay, J.-A., Yamamoto, M., Kroezen, Z., Shanmuganathan, M., Ly, R., Hart, L., Pai, N., and Britz-McKibbin, P. (2021). Lyophilized Fecal Short-Chain Fatty Acid and Electrolyte Determination by Capillary Electrophoresis with Indirect UV Detection for Assessment of Pediatric Inflammatory Bowel Disease. J. Pharm. Biomed. Anal., 192.","DOI":"10.1016\/j.jpba.2020.113658"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"2016","DOI":"10.1097\/MIB.0000000000000836","article-title":"Emerging Biomarkers for the Diagnosis and Monitoring of Inflammatory Bowel Diseases","volume":"22","author":"Poullis","year":"2016","journal-title":"Inflamm. Bowel Dis."},{"key":"ref_41","doi-asserted-by":"crossref","unstructured":"Kaczmarczyk, O., D\u0105bek-Drobny, A., Wo\u017aniakiewicz, M., Pa\u015bko, P., Dobrowolska-Iwanek, J., Wo\u017aniakiewicz, A., Pi\u0105tek-Guziewicz, A., Zagrodzki, P., Mach, T., and Zwoli\u0144ska-Wcis\u0142o, M. (2021). Fecal Levels of Lactic, Succinic and Short-Chain Fatty Acids in Patients with Ulcerative Colitis and Crohn Disease: A Pilot Study. J. Clin. Med., 10.","DOI":"10.3390\/jcm10204701"},{"key":"ref_42","first-page":"43","article-title":"Association between Fecal Levels of Short-Chain Fatty Acids and Serum Pro- and Anti-Inflammatory Cytokines in Patients with Inflammatory Bowel Disease","volume":"62","author":"Kaczmarczyk","year":"2022","journal-title":"Folia Med. Cracov."}],"container-title":["Microorganisms"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-2607\/12\/4\/828\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T14:31:11Z","timestamp":1760106671000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-2607\/12\/4\/828"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,4,19]]},"references-count":42,"journal-issue":{"issue":"4","published-online":{"date-parts":[[2024,4]]}},"alternative-id":["microorganisms12040828"],"URL":"https:\/\/doi.org\/10.3390\/microorganisms12040828","relation":{},"ISSN":["2076-2607"],"issn-type":[{"value":"2076-2607","type":"electronic"}],"subject":[],"published":{"date-parts":[[2024,4,19]]}}}