TY - JOUR
T1 - Extraction, purification, methylation and GC–MS analysis of short-chain carboxylic acids for metabolic flux analysis
AU - Tivendale, Nathan D.
AU - Jewett, Erin M.
AU - Hegeman, Adrian D.
AU - Cohen, Jerry D.
N1 - Publisher Copyright:
© 2016 Elsevier B.V.
PY - 2016/8/15
Y1 - 2016/8/15
N2 - 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 °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.
AB - 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 °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.
KW - Carboxylic acid
KW - Gas chromatography-mass spectrometry
KW - Metabolic flux
KW - Methylation
KW - Polar metabolites
KW - Tricarboxylic acid cycle
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U2 - 10.1016/j.jchromb.2016.05.042
DO - 10.1016/j.jchromb.2016.05.042
M3 - Article
C2 - 27348709
AN - SCOPUS:84975830543
SN - 1570-0232
VL - 1028
SP - 165
EP - 174
JO - Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
JF - Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
ER -