HPLC Analysis of Soybean Leaf Flavonoids

HPLC analysis was conducted following the protocol described in Dowd et al., 2018 and Riddick et al., 2018, on a Shimadzu LC-20 HPLC system (LC-20AT quaternary pump, DGU-20A5 degasser, SIL-20A HT autosampler, and a SPD M20A photodiode array detector, running under Shimadzu LCSolutions version 1.22 chromatography software, Columbia, MD, USA)^{13 (link),14 (link)}. The column used was an Inertsil ODS-3 reverse phase C-18 column (5 µ, 250 ×4.6 mm from Varian, Palo Alto, CA). For phenolic compound analysis, the initial conditions were 20% methanol with 0.25% trifluroacetic acid and 80% water with 0.25% trifluroacetic acid, at a flow rate of 1 mL min⁻¹. The column was held at the initial conditions for 2 minutes, then developed to 100% methanol with 0.25% trifluroacetic acid in a linear gradient over 55 minutes. Five-point standard curves for the evaluation of the concentration of the identified flavonoids in soybean leaves were prepared with commercially purchased kaempferol, genistein, and daidzein for the determination of extinction coefficients at 280 and 340 nm.