6.3.2 Preparation of test solution
6.3.2.1 Mother solution: weigh 0.2 g (accurate to 0.0001g) well-ground sample (6.2) and put it into a 10 mL centrifuge tube. Add 5mL 70% methanol solution (5.4) preheated at 70℃, and after intensively stirring the material by a glass rod, transfer it immediately to 70℃ water bath for 10 min (stir in every 5min). After cooling to ambient temperature, centrifuge the material at a 3500 R / min rotation-speed. 10 min later, transfer the supernate fluid into a 10 mL volumetric flask. Extract the residue by 5mL 70% methanol solution in the same way. Mix the extracting solution to a constant volume of 10 mL, shake up the solution, and then filter it by a 0.45 μm membrane. Prepare the solution for application (24 h preservation time at 4℃).
6.3.2.2 Test solution: pipet 2mL mother solution (6.3.2.1) into a 10 mL volumetric flask, scale the volume by stabilized solution (5.7). Shake up the solution, filter it by 0.45 μm membrane, and prepare the solution for application.
6.3.3 Chromatographic condition
Flow rate of mobile phase: 1mL/min.
Column temperature: 35℃.
Ultraviolet detector: λ=278nm.
Gradient condition: 100% A phase retaining 10 min
↓
From 100% A phase to 68％A phase and 32% B phase in 15 min
↓
68% A phase and 32% B phase retaining in 10 min
↓
100% A phase
6.3.4 Measurement
Carry out the blank operation in steady flow rate and steady column temperature. Suck accurately 10 μL operating fluid in mixed standard series, and inject it into HPLC. Inject 10 μL test solution in the same chromatographic condition, and quantify the test solution by peak area.
