An optimized analytical method for cellular targeted quantification of primary metabolites in tricarboxylic acid cycle and glycolysis using gas chromatography-tandem mass spectrometry and its application in three kinds of hepatic cell lines

In this study, we established a repeatable gas chromatography-tandem mass spectrometry (GC-MS/MS) method with selected reaction monitoring (SRM) mode for simultaneous quantification of several primary metabolites in these two pathways, including glucose, 3-phosphoglycerate, phosphoenolpyruvate (PEP), pyruvate, lactate, citrate, cis-aconitate, isocitrate, α-ketoglutarate, succinate, fumarate and malate. There are many solvents to extract the metabolites in these two pathways, however, which one is more effective still remains unclear. Sample pretreatment was optimized for solvent types and volumes to advance the extraction efficiency of metabolites. 500 μL of 75% methanol-methyl tert-butyl ether (MTBE) was finally selected for the extraction of targeted metabolites in cells due to its highest extraction efficiency. Activated carbon as an effective adsorbent was successfully applied to the removal of endogenous targeted metabolites in cells for getting the analyte-free surrogate matrices. A series of methodological studies verified the validity of this optimized approach which was applied to quantify and compare the targeted metabolites in three common hepatic cells. The developed GC-MS/MS method provided a better way to determine the metabolites of energy metabolism in cellular metabolomics, facilitating the application of targeted quantification metabolomics to precisely discover the metabolic alterations.Graphical abstract
Source: Journal of Pharmaceutical and Biomedical Analysis - Category: Drugs & Pharmacology Source Type: research