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The frozen samples were first freeze-dried for 24 h. After freeze-drying, the samples were crushed using a beads beater in MeOH. 800 µL MeOH and 800 mg beads were added to the samples and the homogenizer (Bullet blender, Next advance) was ran for 3 min and further sonication was applied to samples for 10 min. Then, samples were shaken by hand for 3 min and vortexed. After maintaining the sample at -20 °C for 1 h, the samples were vortexed for 10 s and centrifuged for 5 min (4 °C and 28,000 g). Finally, 400 µL of the supernatants were filtered using the syringeless filter having 0.2 µm pore size (Mini-prep, Whatman) and were injected to LC-MS analysis.

After sample injection, the gradient system was operated as follows: 5% mobile phase B from 0 to 2 min; a linear increase to 35% to 99% (mobile phase B) from 2 to 13.5 min; maintain 99% (mobile phase B) from 13.5 to 15 min; a linear decrease to 5% (mobile phase B) from 15 to 15.5 min; and maintaining 5% B (mobile phase B) from 15.5 to 20 min. The flow rate was 400 μl/min and column temperature was 40 °C. The injection volume of samples was 10uL. Electrospray ionization was used in the positive ion mode.

Electrospray ionization was used in the positive ion mode, and the ions were scanned in the range of 50–2500 m/z (MS). LC−QTOF-MS was operated as follows: ion source gas pressure, 50 psi; curtain gas pressure, 25 psi; source temperature, 500 °C; ion spray voltage, 5500 V; declustering potential voltage, 80 V; and collision energy voltage, 15 V.