Method for determining content of flavonoid glycoside in horse chestnut extract

Abstract

The invention belongs to the technical field of chemical detection, and particularly relates to a method for determining the content of flavonoid glycoside in a horse chestnut extract, which adopts high performance liquid chromatography for analysis and detection. Octadecyl silane bonded silica gel is used as a filling agent; an acetonitrile-phosphoric acid aqueous solution is used as a mobile phase A; an acetonitrile-phosphoric acid aqueous solution is used as a mobile phase B; the reference substance solution contains quercetin-3-O-[beta-D-xylosyl (1-> 2)]-beta-D-glucose-3 '-O-beta-D-glucoside, and the content of the quercetin-3-O-[beta-D- The test solution contains the horse chestnut extract. The method disclosed by the invention can be used for effectively controlling and evaluating the quality of the medicine containing the horse chestnut extract, so that the blank in related fields is filled, and a guarantee is provided for stable product quality and medication safety.

Description

Technical Field

[0001] The invention belongs to the technical field of chemical detection, and particularly relates to a method for determining the content of flavonoid glycosides in a horse chestnut extract. Background Art

[0002] Horse chestnut extract has obvious therapeutic effects in treating discomfort in the legs caused by venous dysfunction (chronic venous insufficiency). Horse chestnut extract is mostly imported, and there is no very complete system and standard for quality inspection of horse chestnut extract in China. The European Pharmacopoeia only uses horse chestnut extract to determine the content of total saponins as a quality control indicator. Further determination of the amount of flavonoids in the extract is conducive to comprehensive control of the quality of the extract. Horse chestnut extract contains a variety of flavonol glycosides with quercetin and kaempferol as aglycones. This type of compound has multiple biological activities. Among them, a flavonol glycoside component isolated from horse chestnut extract was determined to have a molecular weight of 758 after comprehensive structural analysis, and the molecular formula is C 32 H 38 O 21 , the chemical name is quercetin-3-O-[β-D-xylosyl (1→2)]-β-D-glucose-3'-O-β-D-glucoside (hereinafter referred to as flavonoid glycoside), CAS number is 250249-92-4. This component can be obtained as a standard product, and its content in horse chestnut extract remains at a relatively stable level of about 0.3~0.4%. By determining this component, the purity and quality of horse chestnut extract in related drugs can be well analyzed. It is urgent to study a determination method for this component to fill the gap in related technologies. Summary of the invention

[0003] In order to solve the problem of imperfect quality detection system of horse chestnut extract in the prior art, the present invention mainly provides a method for determining the content of flavonoid glycosides in horse chestnut extract: A method for determining the content of flavonoid glycosides in a horse chestnut extract is provided, wherein the method adopts high performance liquid chromatography for analysis and detection; octadecylsilane bonded silica gel is used as a filler; acetonitrile-phosphoric acid aqueous solution is used as mobile phase A and mobile phase B; the reference solution contains quercetin-3-O-[β-D-xylosyl (1→2)]-β-D-glucose-3'-O-β-D-glucoside; and the test solution contains a horse chestnut extract. The chromatographic column preferably uses a Waters Xbridge BEH C18, 4.6×100 mm, 2.5 μm or a chromatographic column with equivalent performance.

[0004] Furthermore, the column temperature is 25~40°C.

[0005] Furthermore, the detection wavelength is 250~270nm or 340~360nm.

[0006] Furthermore, the phosphoric acid concentration of the phosphoric acid aqueous solution in the mobile phase A is 0.05-0.15%.

[0007] Further, the elution procedure is: .

[0008] Furthermore, the preparation of the reference solution comprises the following steps: adding a diluent to prepare a reference solution containing 20 µg of quercetin-3-O-[β-D-xylosyl (1→2)]-β-D-glucose-3'-O-β-D-glucoside per 1 mL.

[0009] Furthermore, the preparation of the test solution includes the following steps: taking horse chestnut extract, adding a diluent, ultrasonically mixing, and placing at room temperature; filtering with a microporous filter membrane, and taking the filtrate to obtain a test solution with a horse chestnut extract concentration of 5 mg / mL.

[0010] Furthermore, the diluent is acetonitrile-0.10% phosphoric acid aqueous solution with a volume ratio of 5-30:95-70 or methanol-0.10% phosphoric acid aqueous solution with a volume ratio of 1:9.

[0011] Further, filter with a 0.45 μm microporous filter membrane; discard 1-5 mL of the initial filtrate.

[0012] Furthermore, the peak area was used for calculation according to the external standard method; the calculation formulas included: response factor = (peak area of ​​reference solution × diluted volume of reference solution) / (sample weight of reference × purity of reference); content = (peak area of ​​main peak in test solution × diluted volume of test solution) / (sample weight of horse chestnut extract × (1-water content of horse chestnut extract) × average value of response factor).

[0013] Take flavonoid glycosides, weigh accurately, and add methanol to prepare a reference stock solution containing approximately 0.2 mg of flavonoid glycosides per 1 mL.

[0014] By adopting the above scheme, the method of the present invention has the following advantages: The method of the present invention can effectively determine the content of quercetin-3-O-[β-D-xylosyl (1→2)]-β-D-glucose-3'-O-β-D-glucoside in horse chestnut extract, and can effectively control and evaluate the quality of drugs containing horse chestnut extract, thus filling the gap in the relevant field and providing guarantee for product quality stability and drug safety.

[0015] By adopting the method of the present invention, the reference solution was sampled 5 times continuously, and the RSD value of the flavonoid glycoside peak area was 0.08% (≤2.0%); the minimum value of the theoretical plate number based on the flavonoid glycoside peak was 33015, which was not less than 20000. The results all met the requirements, indicating that the system applicability of this method was good.

[0016] The flavonoid glycosides of the present invention have a correlation coefficient r of 1.0000 (≥0.999) within a concentration range of 4.13-41.32 μg / mL, a ratio of the Y-axis intercept to the 100% horizontal peak area of ​​0.85% (≤2.0%), and a response factor RSD value of different concentration levels of 0.83% (≤2.0%). The results are in compliance with the regulations, indicating that the flavonoid glycosides have good linearity within a concentration range of 4.13-41.32 μg / mL.

[0017] The blank solvent and the blank auxiliary material of the horse chestnut extract of the present invention have no interference in the determination of the components to be tested; the peak purity index of the flavonoid glycosides of the reference substance and the test solution are both greater than the single point threshold. The results are in compliance with the regulations, indicating that the method has good specificity.

[0018] The detection limit concentration of the flavonoid glycosides of the present invention is 0.2066µg / mL, and the signal-to-noise ratio is 8.1 (≥3); the quantification limit concentration of the flavonoid glycosides is 0.3099µg / mL, the minimum signal-to-noise ratio of the flavonoid glycoside peak in 6 quantitative limit solutions is 10.0 (≥10), the RSD value of the retention time is 0.09% (≤2.0%), and the RSD value of the peak area is 1.96% (≤5.0%). All the values ​​meet the requirements, and the results show that the detection limit and the quantification limit meet the requirements.

[0019] The reference substance, the test solution and the reference substance stock solution of the present invention are placed at room temperature for a period of time. Compared with 0h, the peak area change rate of the reference substance and the test solution at each time point is within the range of 98.0%~102.0%, indicating that the reference substance solution and the test solution are stable within 48h when placed on the injection plate (15°C) and 18h ​​when placed at room temperature, and the reference substance solution is stable within 5 days when placed at 2~8°C. Compared with 0h, the peak area change rate of the reference substance stock solution at each time point is within the range of 98.0%~102.0%, indicating that the reference substance stock solution is stable within 5 days at 2~8°C.

[0020] The method of the present invention has high precision and strong repeatability. The system applicability, linearity, range, specificity, detection limit, quantitative limit, solution stability, injection precision, intermediate precision, repeatability, accuracy and durability all meet the requirements of the "Guidelines for Validation of Analytical Methods for Drug Quality Standards" contained in Part IV (General Principle 9101) of the 2015 edition of the Chinese Pharmacopoeia, the "Technical Guidelines for Validation of Analytical Methods for Quality Control of Chemical Drugs" and the "Technical Guidelines for the Standardized Process for Establishing Quality Standards for Chemical Drugs" of CDE, and the "VALIDATION OF ANALYTICAL PROCEDURES: TEXT AND METHODOLOGY" of ICH Q2 (R1), and has the potential to be used as a measurement standard. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1is the blank chromatogram of Example 1; Figure 2 is the spectrum of reference substance 1 of Example 1; Figure 3 is the spectrum of reference substance 2 of Example 1; Figure 4 is the spectrum of the test product 1800339-1-1 of Example 1; Figure 5 is the spectrum of the test product 1800339-2-1 of Example 1; Figure 6 is the spectrum of the test product 1800340-1-1 of Example 1; Figure 7 is the spectrum of the test product 1800340-2-1 of Example 1; Figure 8 is the spectrum of the test product 1800341-1-1 of Example 1; Fig. 9 is the spectrum of the test product 1800341-2-1 of Example 1; Fig.10 is the chromatogram of the reference substance 1 injection of Example 1; Fig.11 It is the chromatogram of the precision reference substance 1-1 of the sample test of the embodiment; Fig.12 It is the chromatogram of the precision reference substance 1-2 of the example sample test; Fig.13 It is the chromatogram of the precision reference substance 1-3 of the example sample test; Fig.14 It is the chromatogram of the precision reference substance 1-4 of the example sample test; Fig.15 It is the chromatogram of the precision reference substance 1-5 of the example sample test; Fig.16 It is the chromatogram of the precision reference substance 1-6 of the example sample test; Fig.17 is the chromatogram of the repeatability test of the sample in the embodiment - test sample 1-1; Fig.18 is the chromatogram of the repeatability test of the sample of the embodiment - test sample 1-2; Fig.19 is the chromatogram of the repeatability test of the sample in the embodiment - test sample 2-1; Fig. 20 is the chromatogram of the repeatability test of the sample of the embodiment - test sample 2-2; Fig.21 is the chromatogram of the repeatability test of the sample in the embodiment - test sample 3-1; Fig. 22is the chromatogram of the repeatability test of the sample in the embodiment - test sample 3-2; Fig.23 is the chromatogram of the repeatability test of the sample in the embodiment - test sample 4-1; Fig.24 is the chromatogram of the repeatability test of the sample in the embodiment - test sample 4-2; Fig.25 is the chromatogram of the repeatability test of the sample in the embodiment - test sample 5-1; Fig.26 is the chromatogram of the repeatability test of the sample in the embodiment - test sample 5-2; Fig. 27 is the chromatogram of the repeatability test of the sample in the embodiment - test sample 6-1; Fig.28 is the chromatogram of the repeatability test of the sample in the embodiment - test sample 6-2; Fig.29 It is the chromatogram of the repeatability test of the sample of the embodiment - the reference substance 1 injection; Fig.30 It is the intermediate precision of the sample test of the embodiment - the chromatogram of the test sample 1-1; Fig.31 It is the intermediate precision of the sample test of the embodiment - the chromatogram of the test sample 2-1; Fig.32 It is the intermediate precision of the sample test of the embodiment - the chromatogram of the test sample 3-1; Fig.33 It is the intermediate precision of the sample test of the embodiment - the chromatogram of the test sample 4-1; Fig.34 It is the intermediate precision of the sample test of the embodiment - the chromatogram of the test sample 5-1; Fig.35 It is the intermediate precision of the sample test of the embodiment - the chromatogram of the test sample 6-1; Fig.36 It is the chromatogram of the intermediate precision test of the sample of the embodiment - the reference substance 1 injection; Fig.37 is a chromatogram of the test accuracy of the sample in the embodiment - low concentration test sample 1-1; Fig.38 is a chromatogram of the example sample test accuracy - low concentration test product 2-1; Fig.39 is a chromatogram of the example sample test accuracy - low concentration test product 3-1; Fig.40 is the chromatogram of the test sample 1-1 in the embodiment sample test accuracy-medium concentration; Fig.41 is the chromatogram of the test sample 2-1 at a medium concentration in the example sample test accuracy; Fig.42is the chromatogram of the test sample 3-1 of the embodiment sample test accuracy-medium concentration; Fig.43 is a chromatogram of the embodiment sample test accuracy-high concentration test sample 1-1; Fig.44 is a chromatogram of the embodiment sample test accuracy-high concentration test sample 2-1; Fig.45 is the chromatogram of the embodiment sample test accuracy - high concentration test sample 3-1; Fig.46 It is a chromatogram of the accuracy of the sample test-reference substance 1 injection. DETAILED DESCRIPTION

[0022] Example 1: Determined by high performance liquid chromatography.

[0023] Chromatographic conditions: octadecylsilane bonded silica gel was used as filler (Waters Xbridge BEH C18, 4.6×100mm, 2.5μm); acetonitrile-phosphoric acid aqueous solution (5:95) (phosphoric acid aqueous solution containing 0.10% phosphoric acid) was used as mobile phase A, and acetonitrile-phosphoric acid aqueous solution (50:50) (phosphoric acid aqueous solution containing 0.10% phosphoric acid) was used as mobile phase B; column temperature was 35°C; detection wavelength was 350nm; flow rate was 1.0mL per minute, and linear gradient elution was performed according to the following table:

[0024] Diluent: acetonitrile-phosphoric acid aqueous solution (10:90) (phosphoric acid aqueous solution contains 0.10% phosphoric acid).

[0025] Preparation of reference solution: Take an appropriate amount of flavonoid glycosides (from Shanghai Traditional Chinese Medicine Standardization Research Center, batch: 05-2028-201808, content: 98.9%), accurately weigh, and add methanol to make a reference stock solution containing about 0.2 mg flavonoid glycosides per 1 mL. Accurately measure an appropriate amount of the reference stock solution, add a diluent to make a reference solution containing about 20 μg flavonoid glycosides per 1 mL, shake well, and obtain.

[0026] Prepare the test solution: take about 100 mg of horse chestnut extract, accurately weigh it, put it in a 20 mL volumetric flask, add diluent, ultrasonically treat it for about 20 minutes, let it cool to room temperature, dilute it to the scale with diluent, shake it well, filter it with a 0.45 μm microporous filter membrane, and take the filtrate to obtain the test solution.

[0027] Determination method: Accurately measure 20µl of reference solution and test solution respectively and inject them into liquid chromatograph. The chromatogram includes Figures 1 to 10 ; Calculate by peak area using the external standard method to obtain the result.

[0028] The content of horse chestnut extracts from different batches was determined twice for each batch, and the results were averaged. The results are shown in Table 1 below.

[0029] Table 1: Assay results:

[0030] It can be seen from the above table and the corresponding spectra that the minimum value of flavonoid glycoside content detected in different batches of horse chestnut extracts is 0.31%, and the average value is 0.35%.

[0031] Example sample test: Injection precision: Diluent: acetonitrile-phosphoric acid aqueous solution (10:90) (phosphoric acid aqueous solution contains 0.10% phosphoric acid).

[0032] Reference solution: Take an appropriate amount of flavonoid glycosides, weigh accurately, and add methanol to make a reference stock solution containing about 0.2 mg of flavonoid glycosides per 1 mL. Accurately measure an appropriate amount of reference stock solution, add diluent to make a reference solution containing about 20 μg of flavonoid glycosides per 1 mL, shake well, and you have it.

[0033] Accurately measure 20 μl of the reference solution and inject it into the liquid chromatograph. Inject the sample 6 times continuously and calculate the RSD of the retention time and peak area respectively. The chromatogram is as follows: Figures 11 to 16 , calculate the RSD value. Acceptable standard: The reference solution was injected 6 times in a row, and the RSD value of the flavonoid glycoside retention time and peak area should not be greater than 2.0%. The results are shown in Table 2.

[0034] Table 2: Injection precision results:

[0035] As can be seen from the table above, the reference solution was injected 6 times in a row, and the RSD values ​​of the retention time and peak area of ​​flavonoid glycosides were 0.06% and 0.05% (≤2.0%) respectively. The results were in line with the regulations, indicating that the injection precision of this method was good.

[0036] Repeatability: Diluent: acetonitrile-phosphoric acid aqueous solution (10:90) (phosphoric acid aqueous solution contains 0.10% phosphoric acid).

[0037] Reference solution: Take an appropriate amount of flavonoid glycosides, weigh accurately, and add methanol to make a reference stock solution containing about 0.2 mg of flavonoid glycosides per 1 mL. Accurately measure an appropriate amount of reference stock solution, add diluent to make a reference solution containing about 20 μg of flavonoid glycosides per 1 mL, shake well, and the solution is ready. Prepare 2 portions in parallel, and mark them as reference solution 1 and reference solution 2, respectively.

[0038] Test solution: Take about 100 mg of horse chestnut extract, weigh accurately, place in a 20 mL volumetric flask, add an appropriate amount of diluent, ultrasonically treat for about 20 minutes, let it cool to room temperature, dilute to scale with diluent, shake well, filter with a 0.45 μm microporous membrane, and take the filtrate. Prepare 6 copies in parallel.

[0039] Accurately measure 20 μl of each of the above solutions and inject them into the liquid chromatograph respectively. Inject the reference solution 1 continuously for 5 times, the reference solution 2 continuously for 2 times, and the test solution continuously for 2 times. The chromatogram is as follows: Figures 17 to 29 , the external standard method was used to calculate the flavonoid glycoside content of the 6 test solutions, and the RSD value was calculated. Acceptable standard: The RSD of the flavonoid glycoside content of the 6 test solutions should not be greater than 3.0%. The results are shown in Table 3.

[0040] Table 3: Repeatability results:

[0041] As can be seen from the table above, the RSD of the flavonoid glycoside content of the 6 test solutions is 0.13% (≤3.0%). The results meet the requirements, indicating that the method has good repeatability.

[0042] Intermediate precision: According to the experimental operation under the repeatability item, different analysts use different instruments on different dates to measure the same batch of samples. Compare with the repeatability results and calculate the RSD value of the flavonoid glycoside content of the 12 test solutions. Acceptable standard: The RSD value of the flavonoid glycoside content of the 12 test solutions should not exceed 6.0%. The chromatogram is as follows Figures 30 to 36 , the results are shown in Table 4.

[0043] Table 4: Intermediate precision results:

[0044] As can be seen from the table above, the RSD value of the flavonoid glycoside content of the 12 test solutions is 0.66% (≤6.0%). The results meet the requirements, indicating that the intermediate precision of this method is good.

[0045] Accuracy: Diluent: acetonitrile-phosphoric acid aqueous solution (10:90) (phosphoric acid aqueous solution contains 0.10% phosphoric acid).

[0046] Reference solution: Take an appropriate amount of flavonoid glycosides, weigh accurately, and add methanol to make a reference stock solution containing about 0.2 mg of flavonoid glycosides per 1 mL. Accurately measure an appropriate amount of reference stock solution, add diluent to make a reference solution containing about 20 μg of flavonoid glycosides per 1 mL, shake well, and the solution is ready. Prepare 2 portions in parallel, and mark them as reference solution 1 and reference solution 2, respectively.

[0047] Recovery test solution: Take about 50 mg of horse chestnut extract, accurately weigh it, and place it in 9 20 mL volumetric flasks. Accurately measure 3 portions of reference stock solution (0.5 mL, 1.0 mL, and 1.5 mL), respectively, and place them in the above 9 20 mL volumetric flasks. Add an appropriate amount of diluent, ultrasonically treat for about 20 minutes, bring to room temperature, dilute to the scale with diluent, and shake well to obtain low (50%), medium (100%), and high (150%) concentration levels of recovery test solutions.

[0048] Accurately measure 20 μl of each of the above solutions and inject them into the liquid chromatograph respectively. The reference solution 1 is injected 5 times continuously, the reference solution 2 is injected 2 times continuously, and the recovery rate test solution is injected 2 times continuously. The chromatogram is recorded. The test solution is shown in Figures 37 to 46 , Acceptable criteria: The average recovery of flavonoid glycosides at low, medium and high concentration levels should be in the range of 92%~105%, and the RSD value should be no more than 3.0%. The results are shown in Table 5.

[0049] Table 5: Sample recovery results:

[0050] As can be seen from the table above, the average recoveries of flavonoid glycosides at low, medium and high concentration levels were 98.47%, 98.67% and 97.99% (within the range of 92% to 105%), and the RSD values ​​were 1.52%, 0.05% and 0.53% (≤3.0%), respectively. The results were in line with the regulations, indicating that the accuracy of this method was good.

[0051] Durability: Diluent: acetonitrile-phosphoric acid aqueous solution (10:90) (phosphoric acid aqueous solution contains 0.10% phosphoric acid).

[0052] Reference solution: Take an appropriate amount of flavonoid glycosides, weigh accurately, and add methanol to make a reference stock solution containing about 0.2 mg of flavonoid glycosides per 1 mL. Accurately measure an appropriate amount of reference stock solution, add diluent to make a reference solution containing about 20 μg of flavonoid glycosides per 1 mL, shake well, and the solution is ready. Prepare 2 portions in parallel, and mark them as reference solution 1 and reference solution 2, respectively.

[0053] Test solution: Take about 100 mg of horse chestnut extract, accurately weigh it, put it in a 20 mL volumetric flask, add an appropriate amount of diluent, ultrasonically treat it for about 20 minutes, let it cool to room temperature, dilute it to the scale with diluent, shake it well, filter it with a 0.45 μm microporous filter membrane, and take the filtrate to obtain the test solution.

[0054] The changes in the chromatographic behavior of the instrument when the column temperature ±2°C, wavelength ±2nm, flow rate ±0.1mL / min, phosphoric acid concentration in the mobile phase ±0.01%, initial proportion of the mobile phase ±1%, and the use of chromatographic columns of the same brand but different batches and different serial numbers in the same batch were investigated, and the repeatability results under the original conditions were compared to investigate the durability of the method. The chromatographic parameters are shown in Table 6.

[0055] Table 6: Parameters of chromatographic condition changes:

[0056] Accurately measure 20 μl of the above reference solution and test solution, and inject them into the liquid chromatograph respectively. The reference solution 1 is continuously injected 5 times, the reference solution 2 is continuously injected 2 times, and each portion of the test solution is continuously injected 2 times. The durability results are shown in Tables 7 to 13. Acceptable standard: Under each durability condition, the system suitability meets the requirements. The measured test sample content is not significantly different from the original condition (RSD value should be no more than 5%).

[0057] Table 7: Durability results (column temperature):

[0058] Table 8: Durability results (detection wavelength):

[0059] Table 9: Durability Results (Flow Rate):

[0060] Table 10: Durability results (phosphoric acid concentration):

[0061] Table 11: Robustness results (initial ratio of mobile phase):

[0062] Table 12: Durability results (column):

[0063] Table 13: Theoretical plate number of flavonoid glycosides under various conditions of durability experiment:

[0064] Note: "*" Theoretical plate numbers are all based on the first reference sample under each change condition. As can be seen from the table above, under the conditions of column temperature, wavelength, flow rate, phosphoric acid concentration in the mobile phase, initial proportion of the mobile phase, chromatographic columns of the same brand but different batches and different serial numbers of the same batch, the theoretical plate numbers of flavonoid glycosides are all greater than 20,000; compared with the repeatability results under the original conditions, there is no significant difference between the test sample content and the content measured under the original conditions (RSD value is not greater than 5%). The results are in compliance with the regulations, indicating that this method has good durability.

Claims

1. A method for determining the content of flavonoid glycosides in horse chestnut extract, characterized in that: High performance liquid chromatography was used for analysis and detection; octadecylsilane bonded silica gel was used as filler; acetonitrile-phosphoric acid aqueous solution was used as mobile phase A and mobile phase B; the reference solution contained quercetin-3-O-[β-D-xylosyl (1→2)]-β-D-glucose-3'-O-β-D-glucoside; and the test solution contained horse chestnut extract.

2. The method for determining the content of flavonoid glycosides in horse chestnut extract according to claim 1, characterized in that: The column temperature is 25~40℃.

3. The method for determining the content of flavonoid glycosides in horse chestnut extract according to claim 1, characterized in that: The detection wavelength is 250~270nm or 340~360nm.

4. The method for determining the content of flavonoid glycosides in horse chestnut extract according to claim 1, characterized in that: The phosphoric acid concentration of the phosphoric acid aqueous solution in mobile phase A is 0.05-0.15%.

5. The method for determining the content of flavonoid glycosides in horse chestnut extract according to claim 1 or 2, characterized in that: The elution procedure is: 。 6. The method for determining the content of flavonoid glycosides in horse chestnut extract according to claim 1, characterized in that: Preparation of the reference solution The method comprises the following steps: adding a diluent to prepare a reference solution containing 20 µg of quercetin-3-O-[β-D-xylosyl (1→2)]-β-D-glucose-3'-O-β-D-glucoside per 1 mL.

7. The method for determining the content of flavonoid glycosides in horse chestnut extract according to claim 1, characterized in that: The preparation of the test solution comprises the following steps: taking a horse chestnut extract, adding a diluent, ultrasonically mixing, and cooling to room temperature; filtering with a microporous filter membrane, and taking a filtrate to obtain a test solution with a concentration of 5 mg / mL of horse chestnut extract.

8. The method for determining the content of flavonoid glycosides in horse chestnut extract according to claim 6 or 7, characterized in that: The diluent is acetonitrile-0.10% phosphoric acid aqueous solution with a volume ratio of 5-30:95-70 or methanol-0.10% phosphoric acid aqueous solution with a volume ratio of 1:

9.

9. The method for determining the content of flavonoid glycosides in horse chestnut extract according to claim 7, characterized in that: Filter with a 0.45 μm microporous membrane; discard 1-5 mL of the initial filtrate.

10. The method for determining the content of flavonoid glycosides in horse chestnut extract according to claim 1, characterized in that: Calculated by peak area using the external standard method; the calculation formula includes: response factor = (peak area of ​​reference solution × diluted volume of reference solution) / (sample weight of reference × purity of reference); content = (peak area of ​​main peak in test solution × diluted volume of test solution) / (sample weight of horse chestnut extract × (1-water content of horse chestnut extract) × average value of response factor).

Images