Extraction, purification, methylation and GC-MS analysis of short-chain carboxylic acids for metabolic flux analysis.
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.
['Tivendale|Nathan D|ND|', 'Jewett|Erin M|EM|', 'Hegeman|Adrian D|AD|', 'Cohen|Jerry D|JD|']
D002264:Carboxylic Acids D002952:Citric Acid Cycle D008401:Gas Chromatography-Mass Spectrometry D019684:Magnoliopsida D064688:Metabolic Flux Analysis D008745:Methylation D010936:Plant Extracts D052616:Solid Phase Extraction
Methylation Carboxylic acid Metabolic flux Gas chromatography-mass spectrometry Polar metabolites Tricarboxylic acid cycle