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 language||English (US)|
|Number of pages||10|
|Journal||Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences|
|State||Published - Aug 15 2016|
Bibliographical noteFunding 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.
© 2016 Elsevier B.V.
Copyright 2016 Elsevier B.V., All rights reserved.
- Carboxylic acid
- Gas chromatography-mass spectrometry
- Metabolic flux
- Polar metabolites
- Tricarboxylic acid cycle