title: A targeted metabolomic protocol for short-chain fatty acids and branched-chain amino acids.
abstract: Research in obesity and metabolic disorders that involve intestinal microbiota demands reliable methods for the precise measurement of the short-chain fatty acids (SCFAs) and branched-chain amino acids (BCAAs) concentration. Here, we report a rapid method of simultaneously determining SCFAs and BCAAs in biological samples using propyl chloroformate (PCF) derivatization followed by gas chromatography mass spectrometry (GC-MS) analysis. A one-step derivatization using 100 µL of PCF in a reaction system of water, propanol, and pyridine (v/v/v = 8:3:2) at pH 8 provided the optimal derivatization efficiency. The best extraction efficiency of the derivatized products was achieved by a two-step extraction with hexane. The method exhibited good derivatization efficiency and recovery for a wide range of concentrations with a low limit of detection for each compound. The relative standard deviations (RSDs) of all targeted compounds showed good intra- and inter-day (within 7 days) precision (< 10%), and good stability (< 20%) within 4 days at room temperature (23-25 °C), or 7 days when stored at -20 °C. We applied our method to measure SCFA and BCAA levels in fecal samples from rats administrated with different diet. Both univariate and multivariate statistics analysis of the concentrations of these target metabolites could differentiate three groups with ethanol intervention and different oils in diet. This method was also successfully employed to determine SCFA and BCAA in the feces, plasma and urine from normal humans, providing important baseline information of the concentrations of these metabolites. This novel metabolic profile study has great potential for translational research.
authors.affiliation: Center for Translational Medicine, and Shanghai Key Laboratory of Diabetes Mellitus, Department of Endocrinology and Metabolism, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai 200233, China.
authors.forename: Xiaojiao
authors.initials: X
authors.lastname: Zheng
authors.forename: Yunping
authors.initials: Y
authors.lastname: Qiu
authors.forename: Wei
authors.initials: W
authors.lastname: Zhong
authors.forename: Sarah
authors.initials: S
authors.lastname: Baxter
authors.forename: Mingming
authors.initials: M
authors.lastname: Su
authors.forename: Qiong
authors.initials: Q
authors.lastname: Li
authors.forename: Guoxiang
authors.initials: G
authors.lastname: Xie
authors.forename: Brandon M
authors.initials: BM
authors.lastname: Ore
authors.forename: Shanlei
authors.initials: S
authors.lastname: Qiao
authors.forename: Melanie D
authors.initials: MD
authors.lastname: Spencer
authors.forename: Steven H
authors.initials: SH
authors.lastname: Zeisel
authors.forename: Zhanxiang
authors.initials: Z
authors.lastname: Zhou
authors.forename: Aihua
authors.initials: A
authors.lastname: Zhao
authors.forename: Wei
authors.initials: W
authors.lastname: Jia
keywords: Biological samples; Branched-chain amino acids; Gas chromatography/mass spectrometry; Propyl chloroformate; Short-chain fatty acids; Targeted metabolomics