Method for determining total flavonoids in herba epimedii formula granules and application thereof

Abstract

The invention discloses a method for determining total flavonoids in herba epimedii formula granules and application of the method. The method comprises the following steps: acquiring a characteristic chromatogram of a herba epimedii formula granule test solution; collecting peak areas of chromatographic peaks in the characteristic chromatogram, and summing; the chromatographic peaks comprise a peak 8 and a peak 5; calculating the content of total flavonoids according to a mathematical model Y = aX + b, wherein X is the sum of peak areas, Y is the total flavonoid content, a is larger than or equal to 0.90 and smaller than or equal to 1.20, and b is larger than or equal to 10.0 and smaller than or equal to 11.0. According to the method, the specific chromatogram of the herba epimedii formula granules is qualitatively determined, meanwhile, the content of the total flavonoids in the herba epimedii formula granules can be quantitatively determined by determining the sum of the peak areas of several representative chromatographic peaks in the specific chromatogram, a new method is provided for quality control of the herba epimedii formula granules, and the method is high in efficiency and low in cost.

Description

Technical Field

[0001] This invention belongs to the field of component analysis technology. More specifically, this invention relates to a method for the simultaneous qualitative and quantitative determination of total flavonoids in Epimedium (Epimedium) formulation granules and its application. Background Technology

[0002] Epimedium (Epimedium) granules are granules made from Epimedium (Epimedium) slices through processes such as water extraction, concentration, drying, and granulation. They are used in traditional Chinese medicine clinical prescriptions and their main functions are to tonify kidney yang, strengthen tendons and bones, and dispel wind and dampness. They are used for kidney yang deficiency, impotence and seminal emission, weakness of tendons and bones, rheumatic pain, numbness and contracture.

[0003] The national standard for Epimedium (Epimedium) formula granules is the Chinese National Drug Standard for Formula Granules YBZ-PFKL-2021144. The main quality control indicators include [appearance], [thin-layer chromatography identification], [extractants], [total flavonoid content], and [total flavonol glycoside content]. [Characteristic chromatograms] are precisely determined using high-performance liquid chromatography (HPLC). The chromatograms mainly include flavonoid peaks such as icariin A, icariin B, icariin C, icariin, and cymosin-I, which together characterize the overall features of flavonoid components in Epimedium (Epimedium) formula granules for qualitative identification. The standard also measures [total flavonoid content] using ultraviolet spectrophotometry for quantitative determination of flavonoid components.

[0004] The Chinese national drug standard for formulated granules, YBZ-PFKL-2021144, cannot simultaneously quantify and qualitatively determine the flavonoid components of Epimedium (Epimedium) formulated granules, resulting in low quality testing efficiency and high cost. Summary of the Invention

[0005] Based on this, the purpose of the present invention is to provide a method for simultaneously quantitatively and qualitatively determining the flavonoid components of Epimedium (Epimedium) formulation granules.

[0006] The specific technical solutions for achieving the above-mentioned objectives are as follows.

[0007] A first aspect of the present invention provides a method for determining total flavonoids in Epimedium granules, comprising the following steps:

[0008] (1) Obtain the characteristic spectrum of Epimedium formula granules;

[0009] (2) Collect the peak areas of the chromatographic peaks in the characteristic spectrum and sum them; the chromatographic peaks include peak 8 and peak 5;

[0010] (3) Calculate the total flavonoid content according to the mathematical model Y=aX+b; where X is the sum of the peak areas of the chromatographic peaks mentioned in step (2), Y is the total flavonoid content, 0.90≤a≤1.20, 10.0≤b≤11.0.

[0011] A second aspect of the present invention provides the application of a method for determining total flavonoids in Epimedium granules in the quality control of Epimedium granules.

[0012] The inventors of this invention discovered that among the 12 common peaks in the characteristic chromatogram of Epimedium formula granules, the peak areas of several flavonoid components have a very good linear relationship with the total flavonoid content. Further research revealed that the sum of the peak areas of chromatographic peaks 8 and 5, 8, 5, and 10, 10, and 11, as well as 7, all have a good linear relationship with the total flavonoid content. Therefore, a mathematical model of the sum of peak areas and the total flavonoid content was established. This mathematical model has high accuracy, allowing for the qualitative determination of the characteristic chromatogram of Epimedium formula granules, and also the quantitative determination of the total flavonoid content by measuring the sum of the peak areas of several representative chromatographic peaks in the characteristic chromatogram. This provides a new, efficient, and low-cost method for the quality control of Epimedium formula granules. Attached Figure Description

[0013] Figures 1-9 These are characteristic chromatograms of the Epimedium (Epimedium) formulation granules from batches 001 to 009 in Example 1 of this invention.

[0014] Figure 10 This is the standard curve of icariin in Example 3 of the present invention.

[0015] Figure 11 This is a graph showing the relationship between the sum of the areas of the four peaks (peaks 8, 5, 10, and 11) and the total flavonoid content in Example 5 of the present invention. Detailed Implementation

[0016] To facilitate understanding of the present invention, a more complete description will be provided below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0017] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used in this invention includes any and all combinations of one or more of the associated listed items.

[0018] In some embodiments of the present invention, a method for determining the total flavonoid content in Epimedium granules is disclosed, comprising the following steps:

[0019] (1) Obtain the characteristic spectrum of Epimedium formula granules;

[0020] (2) Collect the peak areas of the chromatographic peaks in the characteristic spectrum and sum them; the chromatographic peaks include peak 8 and peak 5;

[0021] (3) Calculate the total flavonoid content according to the mathematical model Y=aX+b; where X is the sum of the peak areas of the chromatographic peaks mentioned in step (2), Y is the total flavonoid content, 0.90≤a≤1.20, 10.0≤b≤11.0.

[0022] In one embodiment, the chromatographic peaks in step (2) are peak 8 and peak 5.

[0023] In one implementation, in step (3), 1.10≤a≤1.15 and 10.7≤b≤11.0.

[0024] In one implementation, the mathematical model described in step (3) is y = 1.106X + 10.724.

[0025] In one embodiment, the chromatographic peak in step (2) further includes at least one of peak 10, peak 11 and peak 9.

[0026] In one embodiment, the chromatographic peaks in step (2) are peaks 8, 5, 10 and 11.

[0027] In one implementation, in step (3), 1.00≤a≤1.05, 10.3≤b≤10.5.

[0028] In one implementation, in step (3), 1.01≤a≤1.02, 10.3≤b≤10.4.

[0029] In one implementation, in step (3), the mathematical model is y = 1.0115X + 10.356.

[0030] In one embodiment, the chromatographic peaks in step (2) are peak 8, peak 5 and peak 10.

[0031] In one implementation, in step (3), 1.04≤a≤1.11, 10.4≤b≤10.73;

[0032] In one implementation, in step (3), the mathematical model is y = 1.0444X + 10.407.

[0033] In one embodiment, the chromatographic peaks in step (2) are peaks 8, 5, 10, 11 and 9.

[0034] In one implementation, in step (3), 0.90≤a≤0.96, 10.0≤b≤10.3;

[0035] In one implementation, in step (3), the mathematical model is y = 0.9531X + 10.21.

[0036] In one embodiment, in step (1), high-performance liquid chromatography is used to obtain the characteristic chromatogram of the Epimedium formula granule test solution, and the chromatographic conditions include:

[0037] Chromatographic column: BEH C18; Mobile phase A: acetonitrile; Mobile phase B: 0.08%~0.12% acetic acid solution; Gradient elution: 0→7 min: 15% A→25% mobile phase A; 7 min→15 min: 25% mobile phase A; 15 min→19 min: 25% mobile phase A→35% mobile phase A; 19 min→24 min: 35% mobile phase A→65% mobile phase A; 24 min→26 min: 65% mobile phase A→100% mobile phase A; 26.1 min→28 min: 15% mobile phase A; Flow rate: 0.4±0.1 mL / min; Column temperature: 30±1 ℃; Detection wavelength: 270 ±2 nm; Injection volume: 1±0.1 μL.

[0038] In one embodiment, the test solution is prepared by the following method: take 0.25 g of Epimedium granules, grind them finely, accurately add 50 mL of 70%~75% ethanol, weigh the solution, sonicate for 40 min~50 min, cool, weigh the solution again, replenish the lost weight with 70%~75% ethanol, shake well, take the filtrate, filter, and the test solution is obtained.

[0039] In other embodiments of the present invention, the application of a method for determining total flavonoids in Epimedium granules in the quality control of Epimedium granules is disclosed.

[0040] In the following embodiments of the present invention, the reagents used include: Epimedium reference material (batch number: 121632-202103, source: National Institutes for Food and Drug Control, content: approximately 0.5g); methanol and acetonitrile (Zhangshu Kehai Chemical Co., Ltd.) were chromatographically pure, other reagents were analytically pure, and water was purified water. The instruments used included: Vanquish Core high-performance liquid chromatograph 10493 (Thermo Fisher Scientific (China) Co., Ltd.), GL2204C electronic balance 104120 (Shanghai Youke Instrument Co., Ltd.), KM7200DE ultrasonic cleaner 10408 (Kunshan Meimei Ultrasonic Instrument Co., Ltd.), UV-6100 ultraviolet-visible spectrophotometer 10424 (Shanghai Yuanxi Instrument Co., Ltd.), BEH C18 column (2.1mm×150mm, 1.7μm), HH-S6 digital display constant temperature water bath 10405 (Changzhou Guoyu Instrument Manufacturing Co., Ltd.), and electric heating mantles 10422 and 10423 (Tianjin Test Instrument Co., Ltd.).

[0041] In the following embodiments of the present invention, a total of 18 batches of Epimedium (Epimedium) formula granule samples were used, of which 9 batches (batch numbers 001~009) were used for modeling and 9 batches (batch numbers 010~018) were used for verification. Each batch of samples was made from Epimedium (Epimedium) slices. The details are shown in Table 1.

[0042] Table 1

[0043]

[0044]

[0045] The present invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0046] Example 1: Determination of the characteristic chromatogram of Epimedium (Epimedium) formulation granules

[0047] Includes the following steps:

[0048] 1. Preparation of Epimedium (Epimedium) formulation granule samples

[0049] Take 5000 g of Epimedium (Epimedium) slices, extract twice with water. For the first extraction, add 10 times the amount of water and decoct for 1 hour. For the second extraction, add 8 times the amount of water and decoct for 1 hour. Combine the decoctions, filter, and concentrate the filtrate to a clear extract with a relative density of 1.05~1.09 (80℃). Dry, pulverize, sieve, add appropriate amount of excipients, mix well, granulate, and prepare 1000 g of the extract. A total of 18 batches were prepared, as shown in Table 1 (except for the batches of slices, all others were the same).

[0050] 2. Preparation of the test solution

[0051] Nine batches (batch numbers 001-009) of Epimedium (Epimedium) formula granules were taken separately. 1.25 g of each Epimedium (Epimedium) formula granule was weighed, and 50 mL of 75% ethanol was precisely added. The mixture was placed in a stoppered conical flask, weighed, and ultrasonically treated (power 250W, frequency 40 kHz) for 45 minutes. After cooling, the weight was measured again. The lost weight was replenished with 75% ethanol, and the mixture was shaken well. The filtrate was filtered through a microporous membrane to obtain the test solution. A total of nine test solutions were prepared.

[0052] 3. Preparation of reference solution

[0053] Take 1 g of Epimedium reference material, place it in a stoppered conical flask, add 25 mL of 75% ethanol, heat under reflux for 45 minutes, cool, shake well, filter, and take the filtrate as the reference solution.

[0054] Take an appropriate amount of icariin reference standard, accurately weigh it, and add methanol to prepare a solution containing 0.1 mg per 1 mL, which will be used as the reference solution.

[0055] 4. Determination of the characteristic chromatogram of Epimedium (Epimedium) formulation granules

[0056] HPLC chromatographic conditions include:

[0057] Column: BEH C18 (2.1mm × 150mm, 1.7μm)

[0058] Mobile phase A: acetonitrile; Mobile phase B: 0.1% acetic acid solution

[0059] Gradient elution: 0→7 min: 15% A→25% mobile phase A;

[0060] 7 min → 15 min: 25% mobile phase A;

[0061] 15 min → 19 min: 25% mobile phase A → 35% mobile phase A;

[0062] 19 min → 24 min: 35% mobile phase A → 65% mobile phase A;

[0063] 24 min → 26 min: 65% mobile phase A → 100% mobile phase A;

[0064] 26.1 min → 28 min: 15% mobile phase A

[0065] Flow rate: 0.4 mL / min

[0066] Column temperature: 30 ℃

[0067] Detection wavelength: 270 nm

[0068] Injection volume: 1 μL.

[0069] 1 μL each of the reference solution of the reference medicinal material, the reference standard solution, and the test solution were precisely pipetted into the liquid chromatograph and analyzed. Characteristic chromatograms of nine batches of Epimedium (Epimedium) formula granules were obtained, as shown below. Figures 1-9 As shown.

[0070] 5. Collect the common peak areas in the feature spectrum.

[0071] The peak areas of 12 common peaks in the characteristic chromatograms of 9 batches of Epimedium (Epimedium) formula granules were collected, as shown in Table 2.

[0072] Table 2

[0073]

[0074] Note: S in Table 2 represents a common peak.

[0075] Example 2: Methodological validation of the method for determining the characteristic chromatogram of Epimedium (Epimedium) formulation granules.

[0076] 1. Repeatability test

[0077] Six portions of Epimedium (batch number 006) formula granules were taken, and test solutions were prepared according to the method in step 2 of Example 1. Chromatographic analysis was performed under the conditions in step 4 of Example 1, and the chromatograms were recorded. The "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 version)" was used for evaluation. Using icariin as the reference peak (S), the relative retention time, relative peak area, and corresponding RSD of the remaining 11 common peaks were calculated. The results showed that the similarity of the fingerprint chromatograms of the six samples was greater than 0.90, and the RSDs of the relative retention time and relative peak area of ​​the 11 common peaks were 0.10%~0.50% and 0.26%~1.26%, respectively, indicating good method repeatability.

[0078] 2. Precision test

[0079] Epimedium (batch number 006) formulation granules were used to prepare a test solution according to step 2 of Example 1. The solution was injected six times consecutively under the chromatographic conditions described in step 4 of Example 1, and the chromatograms were recorded. The "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)" was used for evaluation. Using icariin as the reference peak (S), the relative retention time, relative peak area, and corresponding RSD of the remaining 11 common peaks were calculated. The results showed that the similarity of the fingerprint chromatograms corresponding to the six injections was greater than 0.90. The RSDs of the relative retention time and relative peak area of ​​the 11 common peaks were 0.14%~0.43% and 0.17%~0.84%, respectively, indicating good method precision.

[0080] 3. Stability test

[0081] Epimedium (batch number 006) formulation granules were used to prepare a test solution according to step 2 of Example 1. The solution was analyzed at 0, 2, 4, 6, 8, 12, and 24 h under the chromatographic conditions described in step 4 of Example 1, and the chromatograms were recorded. The "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 version)" was used for evaluation. Using icariin as the reference peak (S), the relative retention time, relative peak area, and corresponding RSD of the remaining 11 common peaks were calculated. The results showed that the similarity of the fingerprint chromatograms at each time point was greater than 0.90. The RSDs of the relative retention time and relative peak area of ​​the 11 common peaks were 0.35%–0.63% and 0.42%–1.59%, respectively, indicating that the test solution had good stability within 24 h.

[0082] Example 3: Determination of total flavonoid content in Epimedium (Epimedium) formulation granules

[0083] 1. Preparation of reference solution

[0084] Take an appropriate amount of icariin reference standard, accurately weigh it, and add dilute ethanol (50%) to prepare a solution containing 0.1 mg per mL, which is the reference standard solution.

[0085] 2. Preparation of standard curve

[0086] Accurately measure 0.2 mL, 0.5 mL, 1.0 mL, 1.5 mL, 3.0 mL, and 6.0 mL of the reference solution into separate 10 mL volumetric flasks. Add dilute ethanol (50%) to the mark, and mix well. Using dilute ethanol (50%) as a blank, measure the absorbance at 270 nm using ultraviolet-visible spectrophotometry (General Chapter 0401, Chinese Pharmacopoeia 2020 Edition). Plot a standard curve with absorbance on the ordinate and concentration on the abscissa. Figure 10 As shown. Regression equation: Y = 0.0003 + 0.0133 * Abs, R 2 =0.9999, indicating that the concentration of icariin has a good linear relationship in the range of 0.0020 mg / mL to 0.0608 mg / mL.

[0087] 3. Preparation of the test solution

[0088] Nine batches (batch numbers 001-009) of Epimedium (Epimedium) formula granules were taken. Approximately 0.65 g of Epimedium (Epimedium) formula granules equivalent to Epimedium slices were weighed, heated to dryness in a water bath, and 50 mL of dilute ethanol was precisely added. The mixture was placed in a stoppered conical flask, weighed, and sonicated (250 W, 40 kHz) for 30 minutes. After cooling, the mixture was weighed again. The lost weight was replenished with 50% dilute ethanol, shaken well, and filtered. 1 mL of the filtrate was precisely pipetted into a 50 mL volumetric flask, and 50% dilute ethanol was added to the mark. The mixture was shaken well to obtain the test solution. A total of nine test solutions were prepared.

[0089] 4. Determination of total flavonoid content

[0090] Using dilute ethanol as a blank solution, the absorbance of each test solution was measured at 270 nm. The total flavonoid concentration in the Epimedium (Epimedium) formula granule sample was calculated by substituting the values ​​into the regression equation of step 2, and the content was further calculated. The results are shown in Table 3.

[0091] Table 3

[0092]

[0093] Example 4: Methodological validation of the method for determining the total flavonoid content of Epimedium (Epimedium) formulation granules.

[0094] 1. Precision test

[0095] The absorbance of Epimedium (Epimedium) formula granules (batch number 006) was measured at 270 nm using dilute ethanol (50%) as a blank solution. The measurement was repeated 6 times. The RSD of the 6 absorbance values ​​was 0.52%, indicating that the method has good precision.

[0096] 2. Stability test

[0097] The test solution of Epimedium (batch number 006) formula granules was taken and the absorbance at 270 nm was measured at 0, 2, 4, 6 and 8 h using dilute ethanol (50%) as blank solution. The RSD of the five absorbance values ​​was 0.94%, indicating that the test solution was relatively stable within 8 h.

[0098] 3. Repeatability test

[0099] Take 6 portions of Epimedium (batch number 006) formulation granules and prepare 6 test solutions according to step 3 of Example 3. Use dilute ethanol (50%) as a blank solution and measure the absorbance at 270 nm. The RSD value of the absorbance values ​​of the 6 test solutions is 1.29%, indicating that the method has good repeatability.

[0100] 4. Spiking recovery test

[0101] Six portions of Epimedium (batch number 006, total flavonoid content 90.38 mg / g) formulation granules were weighed out, and approximately 20 mg of icariin was added. Six test solutions were prepared according to step 3 of Example 3. The absorbance was measured at 270 nm using a 50% dilute ethanol blank solution. The total flavonoid content of the six test solutions was obtained through a standard curve. The average recovery rate was calculated to be 98.25%, with an RSD of 1.58%, indicating good recovery.

[0102] Example 5: Linear Relationship Analysis of Common Peaks and Total Flavonoid Content in Characteristic Spectra of Epimedium (Epimedium) Formula Granules

[0103] 1. Standardized conversion of measurement indicators

[0104] Based on the characteristic spectrum of Epimedium (Epimedium) formula granules or the relationship between the corresponding medicinal materials in the total flavonoid determination and the test solution, the peak areas of 12 peaks in 9 batches of test solutions were converted into the peak areas corresponding to each gram of Epimedium slices, and the total flavonoid content in 9 batches of test solutions was converted into the total flavonoid content corresponding to each gram of Epimedium slices. The results are shown in Table 4.

[0105] Table 4

[0106]

[0107] Using Excel software, a linear relationship analysis was performed on the common peak area and total flavonoid content in Table 4, and the results are shown in Table 5.

[0108] Table 5

[0109]

[0110] As shown in Table 5, all 12 chromatographic peaks were positively correlated with the total flavonoid content. The order of correlation strength was as follows: peak 8 (r=0.9662) > peak 5 (r=0.9349) > peak 10 (r=0.8229) > peak 11 (r=0.7101) > peak 9 (r=0.6332) > peak 7 (r=0.3412) > peak 2 (r=0.2907) > peak 12 (r=0.2447) > peak 2. Peak 3 (r=0.1732) > Peak 4 (r=0.1349) > Peak 6 (r=0.049) > Peak 1 (r=0.0316). The peak areas of these 12 peaks were successively added and combined according to the strength of their correlation, forming 11 sets of common peak combinations (see Table 6). The linear relationship between the 11 common peak combinations and the total flavonoid content was analyzed using EXCEL software. The linear correlation equations are shown in Table 7.

[0111] Table 6

[0112]

[0113]

[0114] Table 7

[0115]

[0116] As shown in Table 7, among the 11 common peak combinations, the third combination showed the best correlation with the total flavonoid content. Therefore, the optimal mathematical model was the linear equation y = 1.0115x + 10.356, which corresponds to the sum of the peak areas (X) of peaks 8, 5, 10, and 11 and the total flavonoid content (Y). The linear relationship is as follows: Figure 11 As shown.

[0117] Example 6: Verification of the accuracy of prediction results from the optimal mathematical model

[0118] Nine batches of Epimedium (Epimedium) formula granules (see batch numbers 010~018 in Table 1) were taken respectively, and characteristic spectra were obtained according to the method of Example 1. The sum of the peak areas of the four common peaks (peak 5, peak 8, peak 10, peak 11) of the nine batches of samples was obtained and substituted into the linear equation y = 1.0115x + 10.356 to obtain the calculated value of total flavonoid content (see Table 9).

[0119] Nine batches of Epimedium (Epimedium) formula granules (batch numbers 010~018) were taken respectively, and the total flavonoid content was measured according to the method in Example 3 (see Table 8).

[0120] Table 8

[0121]

[0122] As shown in Table 8, the calculated and measured values ​​of the total flavonoid content of the nine batches of Epimedium (Epimedium) formula granules deviated by less than ±3, indicating that the prediction results of the optimal mathematical model y = 1.0115x + 10.356 are relatively accurate and can be used to directly calculate the total flavonoid content of Epimedium (Epimedium) formula granules.

[0123] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0124] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A method for determining total flavonoids in Epimedium granules, characterized in that, Includes the following steps: (1) Obtain the characteristic spectrum of Epimedium formula granules; (2) Collect the peak areas of the chromatographic peaks in the characteristic spectrum and sum them; the chromatographic peaks include peak 8 and peak 5; (3) Calculate the total flavonoid content according to the mathematical model Y=aX+b; where X is the sum of the peak areas of the chromatographic peaks mentioned in step (2), Y is the total flavonoid content, 0.90≤a≤1.20, 10.0≤b≤11.

0.

2. The method for determining total flavonoids in Epimedium granules according to claim 1, characterized in that, The chromatographic peaks mentioned in step (2) are peak 8 and peak 5; And / or, in step (3), 1.10≤a≤1.15, 10.7≤b≤11.

0.

3. The method for determining total flavonoids in Epimedium granules according to claim 2, characterized in that, The mathematical model mentioned in step (3) is y = 1.106X + 10.

724.

4. The method for determining total flavonoids in Epimedium granules according to claim 1, characterized in that, The chromatographic peaks mentioned in step (2) also include at least one of peak 10, peak 11 and peak 9.

5. The method for determining total flavonoids in Epimedium granules according to claim 4, characterized in that, The chromatographic peaks mentioned in step (2) are peaks 8, 5, 10 and 11; And / or, in step (3), 1.00≤a≤1.05, 10.3≤b≤10.5; preferably 1.01≤a≤1.02, 10.3≤b≤10.

4.

6. The method for determining total flavonoids in Epimedium granules according to claim 5, characterized in that, In step (3), the mathematical model is y = 1.0115X + 10.

356.

7. The method for determining total flavonoids in Epimedium granules according to claim 4, characterized in that, The chromatographic peaks mentioned in step (2) are peak 8, peak 5 and peak 10; in step (3), 1.04≤a≤1.11, 10.4≤b≤10.73; Preferably, the mathematical model is y = 1.0444X + 10.

407.

8. The method for determining total flavonoids in Epimedium granules according to claim 4, characterized in that, The chromatographic peaks mentioned in step (2) are peak 8, peak 5, peak 10, peak 11 and peak 9; in step (3), 0.90≤a≤0.96, 10.0≤b≤10.3; Preferably, in step (3), the mathematical model is y = 0.9531X + 10.

21.

9. The method for determining total flavonoids in Epimedium granules according to any one of claims 1 to 8, characterized in that, In step (1), high performance liquid chromatography (HPLC) was used to obtain the characteristic chromatogram of the Epimedium formula granule test solution. The chromatographic conditions included: Chromatographic column: BEH C18; Mobile phase A: acetonitrile; Mobile phase B: 0.08%~0.12% acetic acid solution; Gradient elution: 0→7min: 15%A→25% mobile phase A; 7min→15min: 25% mobile phase A; 15min→19min: 25% mobile phase A→35% mobile phase A; 19min→24min: 35% mobile phase A→65% mobile phase A; 24min→26min: 65% mobile phase A→100% mobile phase A; 26.1min→28min: 15% mobile phase A; Flow rate: 0.4±0.1 mL / min; Column temperature: 30±1 ℃; Detection wavelength: 270 ±2nm; Injection volume: 1±0.1 μL; And / or, the test solution is prepared by the following method: take 0.25 g of Epimedium (Epimedium) formulation granules, grind them finely, accurately add 50 mL of 70%~75% ethanol, weigh them, sonicate for 40 min~50 min, cool, weigh them again, replenish the lost weight with 70%~75% ethanol, shake well, take the filtrate, filter, and the test solution is obtained.

10. The application of the method for determining total flavonoids in Epimedium granules according to any one of claims 1 to 9 in the quality control of Epimedium granules.

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