The AoBTHP was analyzed with high-performance liquid chromatography (HPLC) using a Waters Alliance 2690/2695 Separations Module (Waters Corporation, Milford, MA, USA) coupled to a Waters 2996 photodiode array detector (UV/DAD) (Waters Corporation, MA). The used column was a Purospher® Star RP-18 end-capped, particle size 5 μm, LiChroCART® 250 × 4 mm, connected to a guard column with the same stationary phase.
Two mobile phases were employed: water + 0.1% formic acid (solvent A) and acetonitrile (solvent B). The flow rate was 0.6 mL/min, and the following gradient solvent was used: from 0 to 13 min → 95:5 (A:B) to 83:17 (A:B), from 13 to 45 min → 83:17 (A:B) to 67:33 (A:B), from 45 to 46 min → 67:33 (A:B) to 60:40 (A:B), from 46 to 50 min ® 60:40 (A:B) to 25:75 (A:B). This was followed by washing and reconditioning of the column. The flow rate of the mobile phase was 0.6 mL/min, and the temperature of the column thermostat was 25 °C. The samples of AoB and the standards were solubilized in methanol (10 mg/mL) and filtered through a polytetrafluoroethylene (PTFE) syringe filter (0.2 μm), before the analysis.
The chromatogram was monitored with Maxplot (240–650 nm), and data were collected and analyzed using Waters Millennium®32 Chromatography Manager (Waters Corporation, MA). The injection volume was 10 μL.
Two mobile phases were employed: water + 0.1% formic acid (solvent A) and acetonitrile (solvent B). The flow rate was 0.6 mL/min, and the following gradient solvent was used: from 0 to 13 min → 95:5 (A:B) to 83:17 (A:B), from 13 to 45 min → 83:17 (A:B) to 67:33 (A:B), from 45 to 46 min → 67:33 (A:B) to 60:40 (A:B), from 46 to 50 min ® 60:40 (A:B) to 25:75 (A:B). This was followed by washing and reconditioning of the column. The flow rate of the mobile phase was 0.6 mL/min, and the temperature of the column thermostat was 25 °C. The samples of AoB and the standards were solubilized in methanol (10 mg/mL) and filtered through a polytetrafluoroethylene (PTFE) syringe filter (0.2 μm), before the analysis.
The chromatogram was monitored with Maxplot (240–650 nm), and data were collected and analyzed using Waters Millennium®32 Chromatography Manager (Waters Corporation, MA). The injection volume was 10 μL.