Analytical and Bioanalytical Chemistry

, Volume 409, Issue 23, pp 5555–5567 | Cite as

Development of a fast and cost-effective gas chromatography–mass spectrometry method for the quantification of short-chain and medium-chain fatty acids in human biofluids

  • Cesare Lotti
  • Josep Rubert
  • Francesca Fava
  • Kieran Tuohy
  • Fulvio Mattivi
  • Urska Vrhovsek
Research Paper


The quantification of short-chain and medium-chain fatty acids is becoming more and more relevant in fecal and plasma samples due to their biological impact, which has been associated with colon rectal cancer and fiber consumption. For these reasons, a fast, cost-effective, and reproducible analytical method is highly required. In this research, a gas chromatography–mass spectrometry method based on full scan and multiple reaction monitoring (MRM) acquisition modes were optimized and validated for the analysis of short-chain and medium-chain fatty acids in three biological samples: human fecal water, fecal fermentation supernatants, and human plasma. Several extraction solvents (acidified water, diethyl ether, dichloromethane, ethyl acetate, and methyl tert-butyl ether (MTBE) were further evaluated, demonstrating that the latter was clearly the most suitable solvent with recoveries from 75.4 to 124.4% and coefficient of variations lower than 20%. The applicability of the GC–MS method was tested, for instance, acetic acid was quantified by using samples of plasma and feces from healthy donors at mean values of 66.9 μM and 24.5 mM, respectively. The optimized protocol could successfully find applications within multi-compartment human studies. In parallel, a second pilot experiment on fecal fermentation supernatants indicated that the proposed protocol is suitable to follow the formation of SCFAs during in vitro fermentation by the human gut microbiota. In summary, the present work provided an improved GC–MS method for precise and accurate quantification of SCFAs and MCFAs in human feces and plasma.


Fecal water Gas chromatography–mass spectrometry Liquid–liquid extraction Nutrition Plasma Short-chain fatty acids 



This study was financially supported by the ADP2016 project, funded by the Autonomous Province of Trento (Italy), and by the Joint Action Biomarkers in Nutrition and Health, Project Food Biomarkers Alliance (FoodBall) of the Joint Programming Initiative a Healthy Diet for a Healthy Life (JPI HDHL) and the respective national funding organization, Ministry of Education, University and Research (MIUR).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

216_2017_493_MOESM1_ESM.pdf (443 kb)
ESM 1 (PDF 442 kb)


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Copyright information

© Springer-Verlag GmbH Germany 2017

Authors and Affiliations

  • Cesare Lotti
    • 1
  • Josep Rubert
    • 1
  • Francesca Fava
    • 1
  • Kieran Tuohy
    • 1
  • Fulvio Mattivi
    • 1
    • 2
  • Urska Vrhovsek
    • 1
  1. 1.Department of Food Quality and NutritionResearch and Innovation Centre, Fondazione Edmund Mach (FEM)San Michele all’AdigeItaly
  2. 2.Center Agriculture Food Environment (CAFE), University of TrentoSan Michele all’AdigeItaly

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