| MS Description
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<Sample processing and extraction>&l … <Sample processing and extraction><br />
The lyophilized sample in a 2 ml tube was frozen and then homogenized with a 5 mm of zirconia bead by a Mixer Mill (Retsch, Haan, Germany) at 20 Hz for 1 min. Five mg dry weight (DW) of the lyophilized samples were weighed for GC-MS and LC-MS analyses, while 25 mg DW of the samples for CE-MS analysis.<br /><br />
<Extraction for LC-MS>
Five mg DW per 150 μl of extraction medium (methanol/water [2:5
v/v] with reference compounds [0.5 mg l-1 flavonol-2'-sulfonic acid and 1.0 mg l-1 ampicilin])
each sample was used for the extraction of plant material using a Retsch mixer mill MM310
at a frequency of 20 Hz for 5 min at 4°C. After centrifugation for 10 min at 15,000 × g, the
supernatant was transferred into a 2 ml tube. Thirty volumes of methanol were added to the
tube and then extracted again using the mixer mill at a frequency of 20 Hz for 5 min at 4°C.
After centrifugation for 10 min at 15,000 × g, the resulting supernatant was transferred into
the tube. Two hundred-μl aliquot of the extracts was filtered using an Oasis® HLB -μelusion
plate (30 μm, Waters Co., Massachusetts, USA). The extracts were evaporated to dryness in an
SPD2010 SpeedVac® concentrator from ThermoSavant (Thermo electron corporation, Waltham,
MA, USA). The extracts were dissolved by 160 μl of 20% aqueous methanol containing 0.5 mg l-1
lidocaine and d-camphor sulfonic acid.
<LC-TOF/MS conditions>
After filtration of the extracts (Ultrafree-MC, 0.2 μm pore size; Millipore), the sample extracts (5
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 μ m,
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 fiow, 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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