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 12-15 h extraction at -80 degrees C. The extracts are then subject to reduction (to stabilize S-keto acids), purified by strong anion exchange solid phase extraction and methylated with methanolic HCI. This method could also be readily adapted to quantify many other short-chain carboxylic acids. (C) 2016 Elsevier B.V. All rights reserved.