Extraction, purification, methylation and GC–MS analysis of short-chain carboxylic acids for metabolic flux analysis

Nathan D. Tivendale, Erin M. Jewett, Adrian D. Hegeman, Jerry D. Cohen

Research output: Contribution to journalArticlepeer-review

1 Scopus citations

Abstract

Dynamic metabolic flux analysis requires efficient and effective methods for extraction, purification and analysis of a plethora of naturally-occurring compounds. One area of metabolism that would be highly informative to study using metabolic flux analysis is the tricarboxylic acid (TCA) cycle, which consists of short-chain carboxylic acids. Here, we describe a newly-developed method for extraction, purification, derivatization and analysis of short-chain carboxylic acids involved in the TCA cycle. The method consists of snap-freezing the plant material, followed by maceration and a 12–15 h extraction at −80 °C. The extracts are then subject to reduction (to stabilize β-keto acids), purified by strong anion exchange solid phase extraction and methylated with methanolic HCl. This method could also be readily adapted to quantify many other short-chain carboxylic acids.

Original languageEnglish (US)
Pages (from-to)165-174
Number of pages10
JournalJournal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
Volume1028
DOIs
StatePublished - Aug 15 2016

Bibliographical note

Funding Information:
This research was funded by the National Science Foundation ( IOS-1238812 ). We further acknowledge supplemental support from the Gordon and Margaret Bailey Endowment for Environmental Horticulture and the Minnesota Agricultural Experiment Station.

Publisher Copyright:
© 2016 Elsevier B.V.

Copyright:
Copyright 2016 Elsevier B.V., All rights reserved.

Keywords

  • Carboxylic acid
  • Gas chromatography-mass spectrometry
  • Metabolic flux
  • Methylation
  • Polar metabolites
  • Tricarboxylic acid cycle

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