Analysis of metabolites from the tricarboxylic acid cycle for yeast and bacteria samples using gas chromatography mass spectrometry

We here explain step by step the implementation of gas chromatography coupled with tandem mass spectrometry for the quantitative analysis of intracellular metabolites from the tricarboxylic acid (TCA) cycle such as citrate, isocitrate, alpha-ketoglutarate, succinate, malate, and fumarate. Isotope dilution is used to correct for potential metabolite losses during sample processing, matrix effects, incomplete derivatization, and liner contamination. All measurements are performed in selected reaction monitoring (SRM) mode. Standards and samples are first diluted with a fixed volume of a mixture of fully 13C-labeled internal standards and then derivatized to give trimethylsilyl-methoxylamine derivatives prior GC-MS/MS analysis