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Extraction for IT-MS<br />
Each Samp … Extraction for IT-MS<br />
Each Sample (50 mg) was extracted with 750 μl of chloroform/MeOH(1:1, v/v) containing 1.25μM
1,2-dioctanoyl-sn-glycero-3-phosphocholine (SIGMA) followed by centrifugation at 10,000g at 4°C for 5 min.
The supernatant was transferred to a 2ml tube, and the extraction procedure was repeated again.
The combined supernatant was evaporated to dryness by SPD2010 SpeedVac® concentrator.
The residue was dissolved in 750μl of ethanol, and centrifuged at 10,000g at 4°C for 5 min.
Six hundred microlitter of the supernatant was transferred to a glass tube for polar-lipid analysis.<br /><br />
LC-IT-TOF-MS conditions<br />
Extracts (0.5μl, ca. 33.3μg of each sample) was analyzed by LC-MS with ESI interface (LC, Shimadzu LC-20AD system; MS, Shimadzu LCMS-IT-TOF) operated by Shimadzu LCMSsolution software (version 3.60). Two-solvent system was used for separation of each metabolite. The analytical conditions were as follows. Column, Shim-pack XR-ODS (2.0 mm I.D., 50 mm long); solvent A, water (1% 1M ammonium formate and 0.1% formic acid); solvent B, acetonitrile/isopropyl alcohol (40:60, v/v. 1% 1M ammonium formate and 0.1% formic acid);gradient program, 40% B at 0 min, 75% B at 3 min, 95% B at 10 min, 100% B at 19 min, 100% B at 27 min, 40% B at 27.01 min (total run time, 30 min); flow rate, 0.3 ml/min; column temperature, 55°C; MS interface voltage, 4.50 kV, nebulizer gas, 1.50 L/min; CDL temperature 200.0℃, heat block temperature, 200°C; detection mode, scan (m/z 150 ̃1600, positive); scan time, 0.25 sec; ion accumulation time, 20 msec. 0.25 sec; ion accumulation time, 20 msec.
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