, Volume 9, Issue 4, pp 818–827 | Cite as

A targeted metabolomic protocol for short-chain fatty acids and branched-chain amino acids

  • Xiaojiao Zheng
  • Yunping Qiu
  • Wei Zhong
  • Sarah Baxter
  • Mingming Su
  • Qiong Li
  • Guoxiang Xie
  • Brandon M. Ore
  • Shanlei Qiao
  • Melanie D. Spencer
  • Steven H. Zeisel
  • Zhanxiang Zhou
  • Aihua ZhaoEmail author
  • Wei JiaEmail author
Original Article


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 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 statistical analysis of the concentrations of these targeted 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.


Propyl chloroformate Short-chain fatty acids Branched-chain amino acids Gas chromatography–mass spectrometry Biological samples Targeted metabolomics 



Short-chain fatty acids


Branched-chain amino acids


Propyl chloroformate


Gas chromatography–mass spectrometry


Relative standard deviations


Capillary electrophoresis


High performance liquid chromatography


Gas chromatography


Headspace solid-phase microextraction






Internal standard


Retention time


Partial least squares regression


Limit of detection


Medium chain fatty acids



This work was financially supported by the NIH Grants 1R01AA020212-01 and 3P30DK056350-10.

Supplementary material

11306_2013_500_MOESM1_ESM.docx (596 kb)
Supplementary material 1 (DOCX 595 kb)


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Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Xiaojiao Zheng
    • 1
    • 2
  • Yunping Qiu
    • 3
  • Wei Zhong
    • 3
  • Sarah Baxter
    • 4
  • Mingming Su
    • 4
  • Qiong Li
    • 3
  • Guoxiang Xie
    • 3
  • Brandon M. Ore
    • 4
  • Shanlei Qiao
    • 3
  • Melanie D. Spencer
    • 5
  • Steven H. Zeisel
    • 5
  • Zhanxiang Zhou
    • 3
  • Aihua Zhao
    • 1
    Email author
  • Wei Jia
    • 1
    • 3
    Email author
  1. 1.Center for Translational Medicine, and Shanghai Key Laboratory of Diabetes Mellitus, Department of Endocrinology and MetabolismShanghai Jiao Tong University Affiliated Sixth People’s HospitalShanghaiChina
  2. 2.School of PharmacyShanghai Jiao Tong UniversityShanghaiChina
  3. 3.Center for Translational Biomedical Research, University of North Carolina at GreensboroKannapolisUSA
  4. 4.David H. Murdock Research InstituteKannapolisUSA
  5. 5.UNC Nutrition Research Institute, University of North Carolina at Chapel HillKannapolisUSA

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