Simultaneous quantification of straight-chain and branched-chain short chain fatty acids by gas chromatography mass spectrometry

J Chromatogr B Analyt Technol Biomed Life Sci. 2018 Aug 15:1092:359-367. doi: 10.1016/j.jchromb.2018.06.028. Epub 2018 Jun 18.


Biomedical research in areas such as metabolic disorders, neuromodulatory, and immunomodulatory conditions involves lipid metabolism and demands a reliable and inexpensive method for quantification of short chain fatty acids (SCFAs). We report a GC-MS method for analysis of all straight-chain and branched-chain SCFAs using pentafluorobenzyl bromide (PFBBr) as derivatization reagent. We optimized the derivatization and GC-MS conditions using a mixture containing all eight SCFA standards, i.e., five straight-chain and three branched-chain SCFAs. The optimal derivatization conditions were derivatization time 90 min, temperature 60 °C, pH 7, and (CH3)2CO:H2O ratio 2:1 (v:v). Comparing the performance of different GC column configurations, a 30 m DB-225ms hyphenated with a 30 m DB-5ms column in tandem showed the best separation of SCFAs. Using the optimized experiment conditions, we simultaneously detected all SCFAs with much improved detection limit, 0.244-0.977 μM. We further applied the developed method to measure the SCFAs in mouse feces and all SCFAs were successfully quantified. The recovery rates of the eight SCFAs ranged from 55.7% to 97.9%.

Keywords: GC–MS; Hyphenated GC column; Metabolomics; PFBBr; Short chain fatty acids.

MeSH terms

  • Animals
  • Fatty Acids, Volatile / analysis*
  • Fatty Acids, Volatile / chemistry
  • Fatty Acids, Volatile / metabolism
  • Feces / chemistry
  • Gas Chromatography-Mass Spectrometry / methods*
  • Limit of Detection
  • Linear Models
  • Male
  • Metabolomics / methods*
  • Mice
  • Mice, Inbred C57BL
  • Reproducibility of Results


  • Fatty Acids, Volatile