GC-MS analysis

GCMS analysis was performed using an Agilent 7000 triple quadrupole mass spectrometer coupled to an Agilent 7693 GC with an autosampler (Agilent Technologies, Palo Alto, CA).

Two microliters of the sample was injected onto a GC column (30mx0.2mmx0.25um) at a flow rate of 1mL/min. The initial temperature of the oven was set to 70°C kept for 1min followed by a ramp of 5°C/min to 200°C, held for 1 min and increasing to 300°C at 40°C/min with a total run time of 33.5min. The compounds were scanned in EI and spilt less injection mode by selected ion monitoring (SIM) using the following diagnostic ions: succinate, m/z 247; fumarate, m/z 245; alpha ketoglutarate, m/z 288; 2 hydroxyglutaric acid, m/z 247; citrate, m/z 273; glutamate, m/z 246; aspartic acid, m/z 232; asparagine m/z 231; glutamine, m/z 245; and succinate d4, m/z 251. Data acquisition, peak integration and quantitation were done by Mass Hunter software (version B. 07.04). The intra-assay precision was in the range of 1.5-6.0% and the accuracy was between 92-106%. The inter-assay precision and accuracy was in the range of 4.0-9.1% and 92-110% respectively.