Determination of Silymarin in Milk Thistle P.E. by HPLC
I. HPLC Conditions:
Column: Inertsil ODS-3 4.6×150mm 5μm
Flow rate: 1.0 mL/min
Detection: 288 nm
Column Temperature: 30 ℃
Injection Volume: 5μl
Mobile phase: A: Metanol
B: Water：phosphoric acid (99.8：0.2, V/V)
T(min) | A(%) | B(%) |
0 | 35 | 65 |
5 | 35 | 65 |
25 | 50 | 50 |
33 | 50 | 50 |
35 | 35 | 65 |
II. Solution Preparation
1. Standard Preparation:
Accurately weigh approximately 5mg of Silvbin Standard into a 25ml volumetric flask. Dissolve and dilute to volume with methanol.
2. Sample Preparation:
Accurately weigh approximately 20mg (assay of Silybin is about 30%) of Milk Thistle P.E. and transfer into a 25ml volumetric flask. Add 20ml methanol and sonicate for 20min. After cool to room temperature and dilute to volume with methanol. Filter through a 0.45μm filter into an HPLC vial or centrifuge to obtain a clear test solution.
III. Calculation
The relative retention times are about 0.68 for silychristin, 0.73 for silydianin, 1.00 for silybin A, 1.05 for silybin B, 1.09 for dehydrosilybin, 1.15 for isosilybin A, and 1.19 for isosilybin B; Separately calculate the percentage of each relevant component of silymarin as silybin (C 25H22O10) against external standard. Calculate the content of silymarin by adding the individual percentages.
