GC-MS Analysis of Tumor Metabolites

For gas chromatography-tandem mass spectrometry (GC-MS), subcutaneous tumor fragments weighing 5–15 mg were homogenized using an electronic tissue disruptor (Qiagen) in ice-cold 80:20 methanol:water (vol:vol) followed by with three freeze-thaw cycles in liquid nitrogen. The supernatant was collected after a 10 min centrifugation at 13,000xg at 4°C then lyophilized. To analyze isotope enrichment in the plasma, whole blood was chilled on ice then centrifuged for 1 minute at 13,000xg at 4°C to separate the plasma. Aliquots of 20–40 μl of plasma were added to 80:20 methanol:water to extract the metabolites, then lyophilized using a SpeedVac (Thermo), and re-suspended in 40 μl anhydrous pyridine. This solution was added to pre-prepared GC-MS autoinjector vials containing 80 μl N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (MTBSTFA) to derivatize polar metabolites. The samples were incubated at 70°C for 1 hour, then aliquots of 1 μl were injected for analysis. Samples were analyzed using either an Agilent 6890 or an Agilent 7890 gas chromatograph coupled to an Agilent 5973N or 5975C Mass Selective Detector, respectively. The observed distributions of mass isotopologues were corrected for natural abundance^{39 (link)}.