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

•Previously-published methylation methods are adapted for short-chain organic acids.•The most suitable methods are applied to purified plant extracts.•These methods may be used in metabolic flux analysis of these types of compounds.•These methods offer significant advantages over current derivatizat...

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Published in:Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Analytical technologies in the biomedical and life sciences, 2016-08, Vol.1028, p.165-174
Main Authors: Tivendale, Nathan D., Jewett, Erin M., Hegeman, Adrian D., Cohen, Jerry D.
Format: Article
Language:English
Subjects:
Carboxylic acid
Carboxylic Acids - analysis
Carboxylic Acids - isolation & purification
Carboxylic Acids - metabolism
Citric Acid Cycle
Gas chromatography-mass spectrometry
Gas Chromatography-Mass Spectrometry - methods
Magnoliopsida - chemistry
Magnoliopsida - metabolism
Metabolic flux
Metabolic Flux Analysis - methods
Methylation
Plant Extracts - chemistry
Plant Extracts - metabolism
Polar metabolites
Solid Phase Extraction - methods
Tricarboxylic acid cycle
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Summary:•Previously-published methylation methods are adapted for short-chain organic acids.•The most suitable methods are applied to purified plant extracts.•These methods may be used in metabolic flux analysis of these types of compounds.•These methods offer significant advantages over current derivatization strategies. 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 12–15h 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.
ISSN:1570-0232
1873-376X
DOI:10.1016/j.jchromb.2016.05.042