Gradient conditions for the solvent system were: 0% mobile
phase B for first 8 min, 0–100% B linear gradient from 8 to
13 min, 100% B from 13 to 25 min, 100–0% B linear gradient from
25 to 27 min and 0% B from 27 to 30 min. The flow rate was
1 ml/min. The column temperature was maintained at 40 C in a
column heater chamber. The sample volume was 20 ll. A PDA
detector at 200 nm was used to monitor the eluate.