3.5. Analysis of Flavonoids by HPLC and LC–MS

The HPLC and LC–MS analyses of flavonoids were carried out as described before with some modifications [41]. A tissue sample (0.1 g dry weight) was extracted in 1.5 mL of 80% methanol in the water bath for 60 min at 70 °C, centrifuged for 15 min at 4700× g and then filtered through a 0.22-µm pore size filter (Millipore, Billerica, MA, USA) prior to analysis. An HPLC analysis was performed on a Waters 2695 Alliance HPLC system (Waters, Milford, MA, USA) equipped with a photodiode array detector. A C18 column (4.6 mm i.d. × 250 mm) (Waters, Milford, MA, USA) was used with a flow rate of 1 mL·min⁻¹ at 25 °C. Gradient elution was employed using mobile phases of 0.1% trifluoroacetic acid (A) and acetonitrile (B) (Supplementary Table S1). Spectra were measured at a wavelength of 350 nm, and individual flavonoids were identified by comparing the retention time and UV spectra. LC–MS analyses were carried out using an LCQ ion trap mass spectrometer (Finnigan MAT, San Jose, CA, USA) equipped with an ESI source in the positive ion mode. Helium was used as the buffer gas and nitrogen was used as the dry gas (12 L·min⁻¹, 350 °C). The heated metal capillary temperature was 180 °C. The electrospray voltage was at 4.5 kV. The data were analyzed using a DataAnalysis Compass.