Chan James Chun Yip, Kioh Dorinda Yan Qin, Yap Gaik Chin, Lee Bee Wah, Chan Eric Chun Yong
Department of Pharmacy, National University of Singapore, 18 Science Drive 4, 117543, Singapore.
Department of Paediatrics, National University of Singapore, Yong Loo Lin School of Medicine, 1E Kent Ridge Road, NUHS Tower Block, Level 12, 119228, Singapore.
J Pharm Biomed Anal. 2017 May 10;138:43-53. doi: 10.1016/j.jpba.2017.01.044. Epub 2017 Jan 28.
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 via C- and C-aniline respectively, facilitated the accurate quantitation of C-aniline derivatized endogenous SCFAs based on calibration of exogenously C-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.
已开发并验证了一种用于定量测定人类婴儿粪便中肠道微生物衍生短链脂肪酸（SCFA）的新型液相色谱串联质谱（LCMSMS）方法。在15分钟的分析时间内实现了12种SCFA（乙酸、丁酸、己酸、2,2-二甲基丁酸、2-乙基丁酸、异丁酸、异戊酸、2-甲基丁酸、4-甲基戊酸、丙酸、新戊酸和戊酸）的基线色谱分离。通过分别对内源性和加标的粪便中的SCFA进行C-和C-苯胺顺序衍生化，基于外源性C-衍生化SCFA的校准，促进了C-苯胺衍生化内源性SCFA的准确定量。在LCMSMS分析之前对衍生化试剂进行优化淬灭，进一步将由于未淬灭的苯胺与LCMS系统中存在的残留乙酸在线衍生化而产生的混淆色谱峰降低到可忽略不计的水平。以前的SCFA分析中未涉及常见LCMS改性剂残留乙酸在SCFA分析中的影响。首次在107份健康婴儿粪便样本中检测并定量了总共9种SCFA（乙酸、丁酸、己酸、异丁酸、异戊酸、2-甲基丁酸、4-甲基戊酸、丙酸和戊酸）。婴儿粪便中SCFA的丰度和多样性从3周起随时间变化，并在12个月末趋于稳定。这反过来反映了婴儿产生SCFA的肠道微生物群群落的成熟。总之，这种新方法适用于未来研究SCFA在儿科健康和疾病中的生物学作用。
