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Flavonol analyses were performed in quintu … Flavonol analyses were performed in quintuplicate. Frozen leaves were homogenized in 5 μL of extraction solvent (methanol: CH3COOH:H2O = 9:1:10, 0.02 mM naringenin-7-O-glucoside) per milligram of fresh weight of tissue in a mixer mill (MM300; Retsch) for 5 min at 30 Hz. After centrifugation at 12,000g, the supernatants were immediately used for flavonoid analysis.
For flavonol profiling, a Waters Acquity UPLC system (Waters) fitted with a Q-TOF Premier mass spectrometer (Micromass MS Technologies) was used. UPLC was performed on a UPLC phenyl C18 column (Φ2.1 mm × 100 mm; Waters) at a flow rate of 0.5 mL/min at 35°C. Compounds were separated with a linear elution gradient with solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in acetonitrile) from 0 min, 100% A to 10 min, 40% B. PDA was used for the detection of UV-visible absorption in the range of 210 to 500 nm. The TOF mass analyzer was used for the detection of flavonol glycosides [M + H]+ and the peak of fragment ions in a positive ion scanning mode with the following setting: desolvation temperature, 400°C with a nitrogen gas flow of 600 L/h; capillary spray, 3.0 kV; source temperature, 150°C; and cone voltage of 10 V for flavonoid glycosides [M + H]+ or 30 V for fragment ions. osides [M + H]+ or 30 V for fragment ions.
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