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Frozen leaves and roots were homogenized i … Frozen leaves and roots were homogenized in 5 µl extraction solvent (methanol:H2O = 4:1) per 1 mg fresh weight of tissues by using a mixer mill (MM300: Retsch GmbH & Co. KG, Haan, Germany) for 3 min at 30 Hz. After centrifugation at 12 000 x g, the cell debris was discarded, and the extracts were centrifuged again. These supernatants were immediately used for the flavonoid analysis.<br />
For flavonoid profiling, a Waters Acquity UPLC system (Waters Co., Massachusetts) fitted with Q-Tof Premier mass spectrometer (Micromass MS Technologies, Manchester, UK) was used. UPLC was carried out on a UPLC BEH C18 column (Φ2.1 mm x 100 mm, Waters) at a flow rate of 0.5 ml/min at 35 °C. An elution gradient with solvent A (0.1% formic acid in H2O) and solvent B (0.1 % formic acid in acetonitrile) and the elution profile—0 min, 100 % A; 20 min, 20 % B—using linear gradients in between the time points was applied. PDA was used for the detection of UV–vis absorption in the range of 210–600 nm. The time-of-flight (TOF) mass analyzer was used for the detection of flavonoid glycosides [M + H]+ and the peak of fragment ions in a positive ion scanning mode with the following setting: desolvation temperature, 180 °C with a nitrogen gas flow of 500 l/h; capillary spray, 3.2 kV; source temperature, 100 °C; and cone voltage was 35 V. rature, 100 °C; and cone voltage was 35 V.
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