Db 17ht column

GC-MS analyses were performed using an Agilent 6890 N gas chromatograph (Santa Clara, CA, USA), equipped with a DB-17ht column (30 m × 0.25 mm i.d. ×0.15 μm film, J & W Scientific) and a retention gap (deactivated fused silica, 5 m × 0.53 mm i.d.), and coupled to an Agilent 5973 MSD single quadruple mass spectrometer. The autosampler storage tray was maintained below 5 °C with a cooler system. The derivatized macrophage extract (1 μL) was injected using Agilent 7683 autosampler in splitless mode. The injector temperature was 230 °C and carrier gas (helium) flow was 0.8 mL/min. The transfer line was 280 °C and the MS source temperature was 230 °C. The column temperature was set at 70 °C for 0.1 min, raised to 225 °C at 5 °C/min, and then 310 °C at 55 °C/min and held there for 4 min. After a five minute solvent delay, mass spectra were acquired using electron ionization (EI) with a selected-ion-monitoring (SIM) mode as in Table S2. Metabolic intermediates of glycolysis, TCA, aspartate-argininosuccinate shunt, GABA shunt and urea cycle were included in this study (Table S1).

All lipids were derivatized to trimethylsilyl ethers using 1:1 (vol: vol) Bis(trimethylsilyl)trifluoroacetamide: pyridine and heating at 70 °C for 1 h before analysis on an Agilent 7890B Series GC. Lipids were separated on a 60 m Agilent DB17HT column (60 m x 0.25 mm i.d. x 0.1 μm film thickness) with helium as the carrier gas at constant flow of 1.1 mL/min and programed as follows: 100 °C for 2 min; then 8 °C/min to 250 °C and held for 10 min; then 3 °C/min to 330 oC and held for 17 min. In all, 2 μL of each sample was injected in splitless mode at 250 °C. The GC was coupled to a 5977 A Series MSD with the ion source at 230 °C and operated at 70 eV in EI mode scanning from 50-850 Da in 0.5 s. Lipids were analyzed using Agilent MassHunter Qualitative Analysis (B.06.00) and identified based on retention time and spectra by comparison to previously confirmed laboratory standards, published spectra^{31 (link),63 (link),64 (link)}, and spectra deposited in the American Oil Chemists’ Society (AOCS) Lipid Library (http://lipidlibrary.aocs.org/index.cfm) or the National Institute of Standards and Technology (NIST) databases.