HPLC analysis

Chromatographic analysis was performed on a Waters 2960 HPLC system (Milford, MA, USA) with a quaternary pump, an automatic injector, a 996 photodiode array detector, and a Waters Empower software for peak identification. A gradient mobile phase system of acetonitriol (eluent A) and water (eluent B) was applied as follows: 36% A and 64% B (0  min), 36% A and 64% B (10  min), 50% A and 50% B (20  min), 65% A and 35% B (35  min), 75% A and 25% B (40 min), 100% A (45 min), 100% A (50  min), 36% A and 64% B (53  min), and 36% A and 64% B (60  min). The flow rate was set at 1  ml/min. The sample volume injected was set at 10 μl, and the detection wavelength was set at 202 nm.