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3. Extraction
Weigh 5.0 g of the sample into a 100 mL beaker.
Add 20 mL of water and 0.5 mL of 1.0 mol/L phosphoric acid solution.
Perform ultrasonic extraction for 10 minutes.
Adjust the pH to approximately 3.0 using 1.0 mol/L phosphoric acid solution.
Transfer the mixture to a 50 mL volumetric flask, dilute to the mark with water, and mix well.
Transfer the entire sample to a 50 mL plastic centrifuge tube, let stand for 5 minutes, then centrifuge at 8000 rpm for 5 minutes.
Collect 5 mL of the supernatant for purification.
4. Purification – ProElut PLS 500 mg/6 mL (Cat.# 68005)
a. Conditioning: Sequentially add 5 mL of methanol and 5 mL of water.
b. Loading: Add the supernatant to be purified and discard the effluent.
c. Washing: Add 5 mL of 10% aqueous methanol solution and discard the effluent.
d. Elution: Add 5 mL of 40% aqueous methanol solution, collect the eluate, and mix well for HPLC analysis.