title: An isotope-labeled chemical derivatization method for the quantitation of short-chain fatty acids in human feces by liquid chromatography-tandem mass spectrometry.
abstract: Short-chain fatty acids (SCFAs) are produced by anaerobic gut microbiota in the large bowel. Qualitative and quantitative measurements of SCFAs in the intestinal tract and the fecal samples are important to understand the complex interplay between diet, gut microbiota and host metabolism homeostasis. To develop a new LC-MS/MS method for sensitive and reliable analysis of SCFAs in human fecal samples, 3-nitrophenylhydrazine (3NPH) was employed for pre-analytical derivatization to convert ten C2-C6 SCFAs to their 3-nitrophenylhydrazones under a single set of optimized reaction conditions and without the need of reaction quenching. The derivatives showed excellent in-solution chemical stability. They were separated on a reversed-phase C18 column and quantitated by negative-ion electrospray ionization - multiple-reaction monitoring (MRM)/MS. To achieve accurate quantitation, the stable isotope-labeled versions of the derivatives were synthesized in a single reaction vessel from (13)C6-3NPH, and were used as internal standard to compensate for the matrix effects in ESI. Method validation showed on-column limits of detection and quantitation over the range from low to high femtomoles for the ten SCFAs, and the intra-day and inter-day precision for determination of nine of the ten SCFAs in human fecal samples was ≤8.8% (n=6). The quantitation accuracy ranged from 93.1% to 108.4% (CVs≤4.6%, n=6). This method was used to determine the SCFA concentrations and compositions in six human fecal samples. One of the six samples, which was collected from a clinically diagnosed type 2 diabetes patient showed a significantly high molar ratio of branch-chain SCFAs to straight-chain SCFAs than the others. In summary, this work provides a new LC-MS/MS method for precise and accurate quantitation of SCFAs in human feces.
authors.affiliation: University of Victoria - Genome BC Proteomics Centre, University of Victoria, Vancouver Island Technology Park, 3101-4464 Markham Street, Victoria, BC V8Z 7X8, Canada.
authors.forename: Jun
authors.initials: J
authors.lastname: Han
authors.affiliation: University of Victoria - Genome BC Proteomics Centre, University of Victoria, Vancouver Island Technology Park, 3101-4464 Markham Street, Victoria, BC V8Z 7X8, Canada.
authors.forename: Karen
authors.initials: K
authors.lastname: Lin
authors.affiliation: University of Victoria - Genome BC Proteomics Centre, University of Victoria, Vancouver Island Technology Park, 3101-4464 Markham Street, Victoria, BC V8Z 7X8, Canada.
authors.forename: Carita
authors.initials: C
authors.lastname: Sequeira
authors.affiliation: University of Victoria - Genome BC Proteomics Centre, University of Victoria, Vancouver Island Technology Park, 3101-4464 Markham Street, Victoria, BC V8Z 7X8, Canada; Department of Biochemistry and Microbiology, University of Victoria, Petch Building Room 207, 3800 Finnerty Road, Victoria, BC V8P 5C2, Canada. Electronic address: christoph@proteincentre.com.
authors.forename: Christoph H
authors.initials: CH
authors.lastname: Borchers
mesh_terms: D002138:Calibration; D002853:Chromatography, Liquid / Q000379:methods*; D005232:Fatty Acids, Volatile / Q000032:analysis*; D005243:Feces / Q000737:chemistry*; D006801:Humans; D007553:Isotope Labeling; D057230:Limit of Detection; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry
chemical_list: D005232:Fatty Acids, Volatile
keywords: 3-Nitrophenylhydrazine; Chemical derivatization; Isotope labeling; Short-chain fatty acid; Ultrahigh performance liquid chromatography–multiple-reaction monitoring mass spectrometry