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Extraction for LC-q-TOF-MS to detect lipid … Extraction for LC-q-TOF-MS to detect lipids<br />
Each frozen samplewas milled using mixer mill MM301 (Retsch) at a frequency of 20 Hz for 2 min at 4°C. After that, frozen powder was extracted with 20 fold volume of extraction solvent (chloroform/methanol/waer[50 : 100 : 31.45, v/v])containing 0.25mM of 1,2-dioctanoyl-sn-glycero-3-phosphocholine (SIGMA). Samples were vigorously mixed using a vortex mixture. 52.5 μl of water and 52.5 μl of chloroform were added to 200 μl of extract and then vigorously mixed for 5 min at room temperature. After standing for 15 min on ice, the samples were centrifuged at 1,000 ×g at 5°C for 5 min. The supernatant (85 μl) was transferred to a 2 ml tube with insert. Each extract was evaporated to dryness by SPD2010 SpeedVac® concentrator (Thermo Fisher Scientific). The residue was dissolved in 162μl of ethanol, and centrifuged at 10,000×g at 45°C for 15 min. Two hundred microliter of the supernatant was transferred to a glass tube for lipid analysis.<br /><br />
LC-q-TOF-MS conditions to detect lipids<br />
Sample extracts (1 μl) were analyzed using an LC-MS system equipped with an electrospray ionization (ESI) interface (HPLC, Waters Acquity UPLC system; MS, Waters Xevo G2 Qtof). Two-solvent (A and B) system was used for separation of each metabolite. Compositions of these solvents were as follows: solvent A, acetonitrile: water:1 M ammonium acetate:formic acid = (158 g:800g:10 ml:1 ml); solvent B, acetonitrile:2-propanol:water:1 M ammonium acetate:formic acid = (79 g:711 g:10 ml:1 ml). The analytical conditions were as follows. HPLC: column, Acquity UPLC HSS T3 (pore size 1.8 μm, 1.0 i.d × 50 mm long, Waters); gradient program, 35% B at 0 min, 70% B at 3 min, 85% B at 7 min, 90% B at 10 min, 90% B at 12 min and 35% B at 12.5 min; flow rate, 0.15 ml/min; temperature, 55°C; MS detection: capillary voltage, +3.0 kV; cone voltage, 20 V for positive mode and 40 V for negative mode; source temperature, 120°C; desolvation temperature, 450°C; cone gas flow, 50 l/h; desolvation gas flow, 450 l/h; collision energy, 6 V; detection mode, scan (m/z 100–2000; scan time, 0. 5 sec; centroid). The scans were repeated for 15 min in a single run. The data were recorded using MassLynx version 4.1 software (Waters). ng MassLynx version 4.1 software (Waters).
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