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

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Vol. 1028; pp. 165 - 174
• Main Authors: Tivendale, Nathan D., Jewett, Erin M., Hegeman, Adrian D., Cohen, Jerry D.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 15.08.2016
• Subjects: anion exchange; Carboxylic acid; carboxylic acids; Carboxylic Acids - analysis; Carboxylic Acids - isolation & purification; Carboxylic Acids - metabolism; Citric Acid Cycle; derivatization; Gas chromatography-mass spectrometry; Gas Chromatography-Mass Spectrometry - methods; hydrochloric acid; maceration; Magnoliopsida - chemistry; Magnoliopsida - metabolism; Metabolic flux; metabolic flux analysis; Metabolic Flux Analysis - methods; metabolism; Methylation; Plant Extracts - chemistry; Plant Extracts - metabolism; Polar metabolites; solid phase extraction; Solid Phase Extraction - methods; Tricarboxylic acid cycle; Tricarboxylic acid cycle; Metabolic flux; Gas chromatography-mass spectrometry; Polar metabolites; Methylation; Carboxylic acid
• ISSN: 1570-0232; 1873-376X
• DOI: 10.1016/j.jchromb.2016.05.042

•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.