Enzymatic reactions and hairy root extracts were filtered and analyzed on UPLC (Agilent 1290 Infinity II, Santa Clara, CA, United States) with a Eclipse XDB-C18 column (4.6 × 150 mm, 5-μm) maintained at 30 °C. Separation was performed with 0.1% (v/v) formic acid/water (A) and 100% methanol (B) at 1 mL/min flow rate. The linear gradient was 5%–80% B over 16 min, 80%–100% B from 16 to 18 min, 100% B for 2 min, and equilibrated at 5% B for 1 min. The flavonoid compounds were detected at 280 nm with a diode array detector. Flavonoids contents were measured by comparing area of individual peak with standard curves obtained with standard compounds (daidzein and genistein).
The enzymatic products were also detected on LC-MS/MS on an ion-trap TOF mass spectrometer attached to UPLC (Agilent, Santa Clara, CA, United States). Separation was performed on a Eclipse Plus C18 column (2.1 mm × 100 mm, 1.8-μm) using the same solvent system as described above for UPLC. The linear gradient was 5%–60% B over 5 min, 60%–100% B from 5 to 10 min, 100% B for 2 min, and equilibrated at 5% B for 0.1 min. The flow rate was maintained at 0.4 mL/min, and the column temperature was maintained at 30 °C. Flavonoid compounds were detected under negative mode, and the m/z spectra were collected from 500 to 1000 as described previously [44 (link)].
The enzymatic products were also detected on LC-MS/MS on an ion-trap TOF mass spectrometer attached to UPLC (Agilent, Santa Clara, CA, United States). Separation was performed on a Eclipse Plus C18 column (2.1 mm × 100 mm, 1.8-μm) using the same solvent system as described above for UPLC. The linear gradient was 5%–60% B over 5 min, 60%–100% B from 5 to 10 min, 100% B for 2 min, and equilibrated at 5% B for 0.1 min. The flow rate was maintained at 0.4 mL/min, and the column temperature was maintained at 30 °C. Flavonoid compounds were detected under negative mode, and the m/z spectra were collected from 500 to 1000 as described previously [44 (link)].
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