| Description |
An appropriate amount of sample was added to 400 μL of precooled (4°C) methanol-water (v/v=4:1, containing 0.1% formic acid and 1 mM BHT, containing internal standard succinate-2,2,3,3-d4). Two steel beads were added, and the mixture was placed in a freezer at -20 °C for 2 min, placed into a grinder for grinding (60 Hz,2 min), vortexed for 1 min, sonicated in an ice-water bath for 10 min, and centrifuged for 10 min (4 °C,12,000 rpm). After 10 min (4 °C,12,000 rpm),300 μL of supernatant was evaporated. Then,300 μL of precooled (4 °C) methanol-water (v/v=4:1, containing 0.1% formic acid,1 mM BHT, containing internal standard succinic acid-2,2,3,3-d4) was added to the residue of the supernatant; vortexing was performed for 1 min, sonication was carried out in an ice-water bath for 10 min, and the supernatant was centrifuged for 10 min (4 °C,12000 rpm), vortexed for 1 min, sonicated for 10 min, and centrifuged for 10 min (4 °C,12,000 rpm). Then,300 μL of supernatant was evaporated; the supernatant was resolubilized with 200 uL of 95% aqueous acetonitrile (containing L-2-chlorophenylalanine), vortexed for 30 seconds, sonicated for 5 min in an ice-water bath, and centrifuged for 5 min (4 °C,13000 pm). The supernatant was drawn up by a syringe, filtered by using a hydrophilic syringe with a 0.22-μm filter, transferred to a brown injection vial, and analyzed by LC-MS. The target metabolites were quantitatively and qualitatively detected by LC-MS. |
