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Freeze-dried powder of plant material was … Freeze-dried powder of plant material was extracted with 75% methanol containing 25 µM of 7-hydroxy-5-methylflavone as an internal standard. After homogenization using a Mixer Mill MM 300 (Quiagen) at 25 Hz for 2 min twice, homogenates were centrifuged (17,400 g, 5 min, 4 ºC). The supernatant was filtered through 0.2 µm PTFE membrane (Millipore). Hydrophobic compounds in the filtrate were removed by absorbing to C18 silica column (MonoSpin C18, GL Science, Tokyo, Japan).
20 µl of methanol solution was applied to a TSK-gel column ODS-100V (4.6 x 250 mm, 5 µm; TOSOH). Water (HPLC grade; solvent A) and acetonitrile (HPLC grade; solvent B) were used as the mobile phase with 0.1% v/v formic acid added to both solvents. The gradient program was as follows: 3% B (0 min), 97% B (90 min), 97% B (100 min), 3% B (100.1), and 3% B (107 min). The flow rate was set to 0.25 ml/min (0-100 min) and 0.5 ml/min (100.1-107 min). The flow rate was set to 0.5 ml/min, and the column oven temperature was set at 40 ºC. Compounds are detected in ESI-positive mode in the range m/z 100-1500. Multistage MSn analyses were carried out using collision induced dissociation (CID) in a linear ion trap detector at a normalized collision energy of 35.0% and an isolation width of 4.0 (m/z), and monitored by both ion trap detector and FT-ICR detector at 25,000 (at m/z 400) mass resolution. The ESI setting was as follows: spray voltage 4.0 kV and capillary temperature 300 ºC. Nitrogen sheath gas and auxiliary gas were set at 40 and 15 arbitrary units, respectively. To monitor HPLC elution, a photodiode array detector was used in the wavelength range 200–650 nm.
Mass scan events were set as follows (referred as method 5): full mass scan with FT-ICR in resolution 12,500, MS2 scans for the most intense 5 ions of full mass scan with IT, and MS3 scans for the most 2 ions of MS2 scan with IT. A dynamic exclusion setting was applied as follows: repeat count, 3; repeat duration, 30 sec; exclusion list size, 500; margin, 10 ppm, and exclusion duration, 20.
The data were acquired by Xcalibur software version 2.07 (Thermo Fisher Scientific). e version 2.07 (Thermo Fisher Scientific).
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