0.45 μm pvdf syringe filter

Freeze-dried broccoli sample (0.2 g) was extracted with 10 mL methanol/water (60:40, v/v). The supernatant was filtered through a 0.45 μm PVDF syringe filter (VWR Scientific, Seattle, WA), and was transferred into a 2 mL HPLC vial for flavonoid analysis. The UHPLC–PDA–HRMS system consisted of an LTQ Orbitrap XL mass spectrometer with an Accela 1250 binary pump, a PALHTC-Accela1-TMO autosampler, an Accela PDA detector (Thermo Fisher Scientific, San Jose, CA), and a G1316A column compartment (Agilent, Santa Clara, CA)). Separation was carried out on a Synergi Hydro-RP column (250 mm × 2.00 mm, 4 μm. Phenomenex, Torrance, CA) at a flow rate of 0.3 mL/min. The mobile phase consisted of A (0.1% formic acid in water, v/v) and B (0.1% formic acid in acetonitrile, v/v).
The quantification was carried out using molar response factors with rutin as reference standard as described in previous report (Lin et al., 2012 (link)). The concentrations of individual flavonoids based on DW were obtained from the analyses of the freeze-dried samples, and the values were further converted to the concentrations as FW by calculating the moisture losses from FW to DW.