A novel LCMSMS method for quantitative measurement of short-chain fatty acids in human stool derivatized with12C- and13C-labelled aniline.
A novel liquid chromatography tandem mass spectrometry (LCMSMS) method for the quantitative measurement of gut microbial-derived short-chain fatty acids (SCFAs) in human infant stool has been developed and validated. Baseline chromatographic resolution was achieved for 12 SCFAs (acetic, butyric, caproic, 2,2-dimethylbutyric, 2-ethylbutyric, isobutyric, isovaleric, 2-methylbutyric, 4-methylvaleric, propionic, pivalic and valeric acids) within an analysis time of 15min. A novel sequential derivatization of endogenous and spiked SCFAs in stool via12C- and13C-aniline respectively, facilitated the accurate quantitation of12C-aniline derivatized endogenous SCFAs based on calibration of exogenously13C-derivatized SCFAs. Optimized quenching of derivatization agents prior to LCMSMS analysis further reduced to negligible levels the confounding chromatographic peak due to in-line derivatization of unquenched aniline with residual acetic acid present within the LCMS system. The effect of residual acetic acid, a common LCMS modifier, in analysis of SCFAs has not been addressed in previous SCFA assays. For the first time, a total of 9 SCFAs (acetic, butyric, caproic, isobutyric, isovaleric, 2-methylbutyric, 4-methylvaleric, propionic and valeric acids) were detected and quantitated in 107 healthy infant stool samples. The abundance and diversity of SCFAs in infant stool vary temporally from 3 weeks onwards and stabilize towards the end of 12 months. This in turn reflects the maturation of infant SCFA-producing gut microbiota community. In summary, this novel method is applicable to future studies that investigate the biological roles of SCFAs in paediatric health and diseases.
Department of Pharmacy, National University of Singapore, 18 Science Drive 4, 117543, Singapore.
James Chun Yip
JC
Chan
Department of Pharmacy, National University of Singapore, 18 Science Drive 4, 117543, Singapore.
Dorinda Yan Qin
DY
Kioh
Department of Paediatrics, National University of Singapore, Yong Loo Lin School of Medicine, 1E Kent Ridge Road, NUHS Tower Block, Level 12, 119228, Singapore.
Gaik Chin
GC
Yap
Department of Paediatrics, National University of Singapore, Yong Loo Lin School of Medicine, 1E Kent Ridge Road, NUHS Tower Block, Level 12, 119228, Singapore. Electronic address: paeleebw@nus.edu.sg.
Bee Wah
BW
Lee
Department of Pharmacy, National University of Singapore, 18 Science Drive 4, 117543, Singapore; Singapore Institute for Clinical Sciences (SICS), Brenner Centre for Molecular Medicine, 30 Medical Drive, 117609, Singapore. Electronic address: eric.chan@nus.edu.sg.
Eric Chun Yong
EC
Chan
D019342:Acetic Acid / Q000737:chemistry; D000814:Aniline Compounds / Q000737:chemistry*; D002247:Carbon Isotopes / Q000737:chemistry*; D002853:Chromatography, Liquid / Q000379:methods*; D005232:Fatty Acids, Volatile / Q000737:chemistry*; D005243:Feces / Q000737:chemistry*; D006801:Humans; D010421:Pentanoic Acids / Q000737:chemistry; D053719:Tandem Mass Spectrometry / Q000379:methods*
D000814:Aniline Compounds; D002247:Carbon Isotopes; D005232:Fatty Acids, Volatile; D010421:Pentanoic Acids; C038780:n-pentanoic acid; D019342:Acetic Acid; C023650:aniline
(13)C-Aniline derivatization; Butyric acid; Human gut microbiome; Human gut microbiota; Human infant stool; Short-chain fatty acids