| MS Description
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BioSource amount:<br />
The harveste … BioSource amount:<br />
The harvested samples were weighed and then each biological sample was put in a 2-ml tube with 5 mm Zirconia beads to be used for metabolite profiling.<br /><br />
For LC-q-TOF-MS analysis:<br />an equivalence of 300 μg FW of the samples was injected.<br /><br />
Sample processing and extraction: <br />The frozen sample in a 2 ml tube was extracted with a concentration of 25 mg FW of tissues per ml extraction medium (methanol/chloroform/water [3:1:1 v/v/v]) containing 10 stable isotope reference compounds using a Retsch mixer mill MM310 (Retsch, Haan, Germany) at a frequency of 30 Hz for 3 min at 4°C. After centrifugation, a 200-μl of the supernatant was used for GC-TOF-MS analysis, while a 100-μl of the supernatant for LC-q-TOF-MS analysis.<br /><br />
Extraction for LC-q-TOF-MS analysis:<br />a 100-μl of the supernatant of each sample in a 1.5 ml tube was used for LC-q-TOF-MS analysis. One hundred-μl of chloroform and one hundred-μl of water containing two reference compounds (0.5 mg/l flavonol-2’-sulfonic acid and 1.0 mg/l ampicilin) at 4°C was added to the tube and then shaken. After 10 min at 4°C, extracts were centrifuged for 3 min at 1,200 x g. The supernatant was transferred and then evaporated to dryness in an SPD2010 SpeedVac® concentrator from ThermoSavant (Thermo electron corporation, Waltham, MA, USA). The extracts were dissolved by 25 μl of 20% aqueous methanol containing 0.5 mg l−1 lidocaine and d-camphor sulfonic acid (final concentration: 1 mg FW per 10-μl sample).<br /><br />
LC-q-TOF-MS conditions: <br />
After filtration of the extracts (Ultrafree-MC, 0.2 μm pore size; Millipore), the sample extracts (3 μl) were analyzed using an LC-MS system equipped with an electrospray ionization (ESI) interface (HPLC, Waters Acquity UPLC system; MS, Waters Q-Tof Premier). The analytical conditions were as follows. HPLC: column, Acquity bridged ethyl hybrid (BEH) C18 (pore size 1.7 μm, length 2.0 × 100 mm, Waters); solvent system, acetonitrile (0.1% formic acid):water (0.1% formic acid); gradient program, 1 : 99 v/v at 0 min, 1 : 99 v/v at 0.1 min, 99.5 : 0.5 at 15.5 min, 99.5 : 0.5 at 17.0 min, 1 : 99 v/v at 17.1 min and 1 : 99 at 20 min; flow rate, 0.3 ml min−1; temperature, 38°C; MS detection: capillary voltage, +3.0 keV; cone voltage, 23 V for positive mode and 35 V for negative mode; source temperature, 120°C; desolvation temperature, 450°C; cone gas flow, 50 l h−1; desolvation gas flow, 800 l/ h; collision energy, 2 V for positive mode and 5 V for negative mode ; detection mode, scan (m/z 100–2000; dwell time 0.45 sec; interscan delay 0.05 sec, centroid). The scans were repeated for 19.5 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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