Quantification of Plant Flavonoids by LC-MS/MS

All plants used in this study were grown in Huazhong Agricultural University, Wuhan. Fern and peat moss were collected on campus and identified according to FRPS (http://frps.eflora.cn/). Potato, citrus, palm, populous and bamboo were obtained from college of Horticulture and Forestry Sciences. Arabidopsis, tobacco and crops were grown in greenhouse. Thirteen plant species leaf samples and tuber of potato were collected using liquid nitrogen with two biological replicate sets. The freeze-dried samples were crushed using a mix mill (MM 400, Ratsch) with a zirconia bead for 1 min at 30 Hz, 100 mg dried power were weighted and extracted overnight at 4 °C with 1.0 mL 70% aqueous methanol containing 0.1 mg L⁻¹ lidocaine (internal standard) before analysis using an LC-ESI-MS/MS system^{34 (link)}. Qualification of metabolites was carried out using a scheduled multiple reaction monitoring method^{34 (link)}. The relative signal intensities of flavonoids were standardized by firstly dividing them by the intensities of internal standard and then log 2 transforming them to generate the final data matrix. Flavonoids were quantified based on comparison with standards of apigenin, tricin, apigenin 5-O-glucoside and apigenin 7-O-glucoside.