| Description |
An appropriate amount of sample was added to 400 microliters of precooled (4°C) methanol-water (v/v=4:1, containing 0.1% formic acid and 1 mM BHT, containing internal standard). Two steel beads were added, and the mixture was placed in a freezer at -20 °C for 2 min, then was vortexed for 1 min, sonicated in an ice-water bath for 10 min, and centrifuged for 10 min . 300 microliters of supernatant was evaporated. Then, 300 microliters of precooled methanol-water was added to the residue of the supernatant and repeat above step once time. The supernatant was resolubilized with 200 microliters 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. The supernatant was drawn up by a syringe, filtered by using a hydrophilic syringe with a 0.22 micrometers filter, transferred to a brown injection vial, and analyzed by LC-MS. The target metabolites were quantitatively and qualitatively detected by LC-MS. |