title: Extraction, purification, methylation and GC-MS analysis of short-chain carboxylic acids for metabolic flux analysis.
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 12-15h 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.
authors.affiliation: Department of Horticultural Science and Microbial and Plant Genomics Institute, University of Minnesota, St. Paul, MN, 55108, USA. Electronic address: ntivenda@umn.edu.
authors.forename: Nathan D
authors.initials: ND
authors.lastname: Tivendale
authors.affiliation: Department of Horticultural Science and Microbial and Plant Genomics Institute, University of Minnesota, St. Paul, MN, 55108, USA; Department of Plant Biology, University of Minnesota, St. Paul, MN, 55108, USA.
authors.forename: Erin M
authors.initials: EM
authors.lastname: Jewett
authors.affiliation: Department of Horticultural Science and Microbial and Plant Genomics Institute, University of Minnesota, St. Paul, MN, 55108, USA; Department of Plant Biology, University of Minnesota, St. Paul, MN, 55108, USA.
authors.forename: Adrian D
authors.initials: AD
authors.lastname: Hegeman
authors.affiliation: Department of Horticultural Science and Microbial and Plant Genomics Institute, University of Minnesota, St. Paul, MN, 55108, USA.
authors.forename: Jerry D
authors.initials: JD
authors.lastname: Cohen
mesh_terms: D002264:Carboxylic Acids / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D002952:Citric Acid Cycle; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D019684:Magnoliopsida / Q000737:chemistry / Q000378:metabolism*; D064688:Metabolic Flux Analysis / Q000379:methods; D008745:Methylation; D010936:Plant Extracts / Q000737:chemistry / Q000378:metabolism; D052616:Solid Phase Extraction / Q000379:methods
chemical_list: D002264:Carboxylic Acids; D010936:Plant Extracts
keywords: Carboxylic acid; Gas chromatography-mass spectrometry; Metabolic flux; Methylation; Polar metabolites; Tricarboxylic acid cycle