*4.5. Purification and Identification of the Biotransformation Product*

One hundred milliliters of the reaction mixture (1 mg/mL of 8-OHDe, 125 μg/mL of DgAS, 300 mM of sucrose, 50 mM of phosphate buffer, pH 7) was carried out at 40 ◦C for 30 min. At the end of the reaction, equal volume of methanol was added into the reaction mixture to stop the reaction. Two hundred milliliters of the reaction mixture with 50% of methanol was applied in a preparative YL9100 HPLC system (YoungLin, Gyeonggi-do, Korea). The stationary phase was the Inertsil ODS 3 column (10 mM, 20 i.d. × 250 mM, GL Sciences, Eindhoven, The Netherlands), and the mobile phase was the same as those in the UPLC system, but with a flow rate of 15 mL/min. The detection condition was 254 nm, and the sample volume was 10 mL for one injection. The product from each run was collected, concentrated under vacuum, and lyophilized with a freeze dryer. From the 100 mL of reaction, 90 mg of the major compound was purified. The chemical structure of the major compound was determined with mass and nmR spectral analysis. The mass spectral analysis was performed on a Finnigan LCQ Duo mass spectrometer (ThermoQuest Corp., San Jose, CA, USA) with ESI. 1H- and

13C-NMR, DEPT, HSQC, HMBC, COSY, and NOESY spectra were recorded on a Bruker AV-700 nmR spectrometer (Bruker Corp., Billerica, MA, USA) at ambient temperature. Standard pulse sequences and parameters were used for the nmR experiments, and all chemical shifts were reported in parts per million (ppm, δ).
