7890b 7000d system

The cell samples were added with 1 mL extract of chloroform: methanol (2:1 v/v) and vortexed for 1 min. Then, 0.5 mL of phosphate-buffered saline (PBS) was added to the samples and mixed thoroughly to remove the supernatant. Nitrogen was used to dry the samples, mixed with 2 mL of acidified methanol, and incubated at 80 °C for 2 h. After methyl esterification, the samples were extracted with hexane (1 mL of hexane). To quantify medium- and long-chain fatty acids, Supelco 37-component FAME (fatty acid methyl ester) mix (Sigma-Aldrich) was used to construct a calibration curve for the concentration range of 0.5–1000 mg/L. The extracted sample was analyzed by GC–MS operating in a single ion monitoring mode using an Agilent 7890B-7000D system with an Agilent DB-WAX capillary GC column. The initial temperature was 50 °C, maintained at this temperature for 3 min, increased to 220 °C at 10 °C/min, and maintained at 220 °C for 20 min. A quality control sample was used to test and evaluate the stability and repeatability of the system. The temperature was set to 280 °C for the injection port and 230 °C for the ion source. The electron bombardment ionization source, selected ion monitor scanning mode, and electron energy were 70 eV. Mass data were analyzed using ChemStation (Agilent) to determine the concentration of each compound.