Sample preparation methodology for mouse heart metabolomics using comprehensive two-dimensional gas chromatography...

The investigation of naturally volatile and derivatized metabolites in mammalian tissues by comprehensive two-dimensional (2D) gas chromatography coupled with time-of-flight mass spectrometry (GC × GC-TOFMS) can provide the data for a comprehensive analysis of the pathophysiology of disease processes. When relative quantification is needed for hypothesis testing, the preparation of sample tissue must provide clear evidence that a quantitative relationship exists between the final detected signal and the amount of metabolite in the tissue. Herein, we report the optimization of a metabolite extraction method for mouse heart tissue for GC × GC-TOFMS analysis. A recursive extraction experiment was initially performed to measure the extraction efficiency of representative target metabolites (sugars, tricarboxylic acid cycle metabolites, amino acids, lipid and signaling molecules) in the aqueous fraction of a three-phase extraction system involving tissue, methanol:water, and chloroform. Some metabolites suffered from incomplete extraction with a single extraction of ≈ 40 mg in 600 μl organic and 400 μl aqueous phases, possibly caused by saturation effects. Subsequent experiments, calibrating resulting metabolite signal to the mass of heart tissue extracted, demonstrated that doubling the solvent volumes and a lower tissue mass was needed to provide accurate relative quantification of the derivatized mouse heart metabolome. We demonstrate quantitative extraction of metabolites from ≈ 20 mg of heart tissue using 1200 μl organic phase (chloroform) and 800 μl aqueous phase (methanol:water in equal parts by volume).