Establishment method of dysphagia pill fingerprint spectrum and established fingerprint spectrum

Abstract

The invention discloses a dysphagia pill fingerprint spectrum establishment method and an established fingerprint spectrum, belongs to the technical field of analysis and detection of traditional Chinese medicine preparations, and provides a dysphagia pill fingerprint spectrum establishment method which comprises the following steps: mixing a dysphagia pill test sample with petroleum ether, performing Soxhlet extraction, mixing medicine residues with a methanol aqueous solution, performing heating reflux, performing column chromatography, and performing freeze drying to obtain a dysphagia pill fingerprint spectrum. Taking filtrate to obtain a test solution; mixing ellagic acid, liquiritin, ammonium glycyrrhizinate and sesamin reference substances with methanol to obtain a mixed reference substance solution; sucking the test solution and the reference solution, injecting into a liquid chromatograph, and collecting a chromatogram; introducing the chromatogram of the test sample into a traditional Chinese medicine chromatographic fingerprint spectrum similarity evaluation system, selecting common chromatographic peaks as common peaks, generating a dysphagia pill contrast fingerprint spectrum, and calculating the relative retention time and the relative peak area of each common peak to obtain the HPLC fingerprint spectrum of the dysphagia pill. The quality of the dysphagia pill can be comprehensively evaluated by using the map, and the quality stability, consistency and controllability of the dysphagia pill are ensured.

Description

Technical Field

[0001] The invention belongs to the technical field of analysis and detection of traditional Chinese medicine preparations, and in particular relates to a method for establishing a fingerprint spectrum of Yege Pills and the fingerprint spectrum obtained by establishing the fingerprint spectrum. Background Art

[0002] The formula of Yege Pills is: walnut kernels, ginkgo, dried persimmon (stem and core removed), fennel, black sesame (fried), sesame oil, jujube (core removed) and licorice. In addition to the above main ingredients, it also contains auxiliary materials such as sucrose and honey, which help to shape the medicine and improve its taste. The main functions of Yege Pills are to nourish the lungs and kidneys, moisten dryness and promote the production of body fluids, and relieve hiccups. It is used for dysphagia, pharyngitis, dysphagia, and dry throat; it can also be used as an auxiliary treatment for atypical hyperplasia of the esophageal mucosal epithelium and esophageal cancer.

[0003] Currently, there is no content test for the effective ingredients of Yege Pills in the current standards, and the "Chinese Pharmacopoeia" has not included the quality standards. The current standards are ministerial standards, which only have a simple microscopic identification. The inspection standards are too backward and cannot reflect the overall quality of the drug. Summary of the invention

[0004] In order to solve the above technical problems, the present invention proposes a method for establishing a fingerprint spectrum of Yege Pills and the fingerprint spectrum obtained by establishing the fingerprint spectrum. The quality of Yege Pills can be evaluated more comprehensively by using the fingerprint spectrum, so as to better ensure the quality stability, consistency and controllability of Yege Pills, thereby ensuring the stable and uniform quality of the preparation.

[0005] To achieve the above object, the present invention provides a method for establishing a fingerprint spectrum of Yege Pills, comprising the following steps:

[0006] (1) Taking a Yege Pill test sample, freezing and crushing to obtain a test sample powder, mixing the test sample powder with petroleum ether, performing Soxhlet extraction, taking a drug residue, mixing the drug residue with a methanol aqueous solution, heating and refluxing, and taking a filtrate to obtain a test sample solution;

[0007] (2) Mixing ellagic acid reference substance, liquiritin reference substance, ammonium glycyrrhizinate reference substance and sesamin reference substance with methanol to obtain a mixed reference substance solution;

[0008] (3) respectively taking the test solution obtained in step (1) and the mixed reference solution obtained in step (2), injecting them into a liquid chromatograph, and collecting 70 min of chromatograms of the test sample and the reference solution;

[0009] (4) The chromatogram of the test sample obtained in step (3) is imported into the "Chinese Herbal Medicine Chromatographic Fingerprint Similarity Evaluation System", and the chromatographic peaks common in the chromatograms of different batches of Yege Pills test samples are selected as common peaks. The median method is used to generate a control fingerprint of Yege Pills, and the relative retention time and relative peak area of ​​each common peak are calculated to obtain the HPLC fingerprint of Yege Pills.

[0010] Preferably, the freezing temperature in step (1) is -15 to -18°C, and the freezing time is 10 min; the mixing ratio of the test sample powder and petroleum ether in step (1) is 2 g: 60 to 80 mL; the Soxhlet extraction temperature in step (1) is 90°C, and the Soxhlet extraction time is 4 h.

[0011] Preferably, the medicinal residue in step (1) is mixed with 25 mL of methanol aqueous solution, and the volume concentration of the methanol aqueous solution is 70%; the temperature of heating and reflux in step (1) is 80° C., and the heating and reflux time is 30 min.

[0012] Preferably, in step (2), the mixing ratio of ellagic acid reference substance, liquiritin reference substance, ammonium glycyrrhizinate reference substance and sesamin reference substance with methanol is 11 μg: 322 μg: 1065 μg: 210 μg: 10 mL.

[0013] Preferably, in step (3), 10 μL of the test solution obtained in step (1) and 10 μL of the mixed reference solution obtained in step (2) are respectively aspirated and injected into a liquid chromatograph.

[0014] Preferably, the conditions of the liquid chromatography in step (3) are as follows: the chromatographic column is Agilent HC-C18; the specifications of the chromatographic column are 4.6×250 mm, 5 μm; the detection wavelength is 254 nm for 0 to 15 min and 236 nm for 15 to 70 min; the flow rate is 1.0 mL / min; the column temperature is 25° C.; the injection volume is 10 μL; the mobile phase A is acetonitrile, and the mobile phase B is a phosphoric acid solution with a volume concentration of 0.1%, and the gradient elution is 0 to 8 min, A 20%, B 80%; 8 to 14 min, A 20 to 22.5%, B 80 to 77.5%; 14 to 30 min, A 22.5 to 30%, B 77.5 to 70%; 30 to 35 min, A 30 to 60%, B 70 to 40%; 35 to 60 min, A 60%, B40%; 60~61min, A60~100%, B 40~0%.

[0015] Preferably, in step (4), peak No. 6 corresponding to ammonium glycyrrhizinate is used as a reference peak to calculate the relative retention time and relative peak area of ​​each common peak.

[0016] The present invention also provides a fingerprint spectrum of Yege Pills obtained by establishing the method described in any one of the above items.

[0017] Compared with the prior art, the present invention has the following advantages and technical effects:

[0018] The invention discloses a method for establishing a fingerprint spectrum of Yege Pills, and uses liquid chromatography to determine the fingerprint spectrum of Yege Pills with reference to ammonium glycyrrhizinate reference substance. The fingerprint spectrum of Yege Pills established by the method provided by the invention can effectively characterize its quality, is conducive to comprehensive monitoring of product quality, and effectively ensures the quality stability, consistency and controllability of Yege Pills, thereby ensuring safety and effectiveness.

[0019] The present invention establishes a HPLC characteristic spectrum of Yege Pills, calibrates 10 common peaks, and the established fingerprint spectrum has high technical content, good peak shape, and is easy to identify, thus providing a reference for quality control of Yege Pills and improvement of quality standards. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1 It is the liquid chromatogram of the mixed reference solution;

[0022] Figure 2 This is the liquid chromatogram of the Yege Pills test solution;

[0023] Figure 3 This is the control fingerprint of Yege Pills;

[0024] Figure 4 This is the HPLC fingerprint of Yege Pills. DETAILED DESCRIPTION

[0025] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0026] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0027] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0028] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0029] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0030] Example 1

[0031] 1. Instruments and test drugs

[0032] 1. Instrument

[0033] Agilent 1260 high performance liquid chromatograph, Agilent HC-C18 chromatographic column (4.6×250 mm, 5 μm), FA2104N electronic analytical balance, BP211D electronic analytical balance.

[0034] 2. Drug testing

[0035] Yege Pills were provided by the R&D Department of Zhongjing Wanxi Pharmaceutical Co., Ltd., with a total of 15 batches, batch numbers S1 to S15. Methanol and acetonitrile were chromatographically pure, petroleum ether (60 to 90 °C) and phosphoric acid were analytically pure, and water was purified water. The information of the reference substances used is shown in Table 1.

[0036] Table 1 Reference material information

[0037] Reference batch number source Ellagic acid 111959-201903 China Food and Drug Inspection Institute Licorice glycoside 111610-202209 China Food and Drug Inspection Institute Ammonium glycyrrhizinate 110731-202122 China Food and Drug Inspection Institute Sesamin 110836-201706 China Food and Drug Inspection Institute

[0038] 2. Methods and Results

[0039] 1. Chromatographic conditions

[0040] The chromatographic column was Agilent HC-C18 (4.6×250mm, 5μm), and the detection wavelength was: 0~15min, 254nm; 15~70min, 236nm. The flow rate was 1.0mL / min; the column temperature was 25℃; the injection volume was 10μL. The mobile phase A was acetonitrile, and the mobile phase B was a 0.1% volume concentration phosphoric acid solution. The gradient elution method was as shown in Table 2 below (the following % refers to volume percentage).

[0041] Table 2 Gradient elution method

[0042] Time (min) Mobile phase A (%) Mobile phase B (%) 0～8 20 80 8～14 20～22.5 80～77.5 14～30 22.5～30 77.5～70 30～35 30～60 70～40 35～60 60 40 60～61 60～100 40～0

[0043] Accurately pipette 10 μL of the test solution and the reference solution respectively, inject into the high performance liquid chromatograph, measure, and record the chromatogram.

[0044] 2. Solution preparation

[0045] Preparation of test solution: Cut the Yege Pills sample into pieces, freeze at -15℃ for 10 min, and then grind to obtain the test powder. Accurately weigh 2 g, place in a Soxhlet extractor, add 70 mL of petroleum ether (60-90℃), heat and reflux at 90℃ for 4 h, discard the petroleum ether liquid, wait until the solvent on the residue evaporates completely, transfer to a conical flask, add 25 mL of 70% methanol aqueous solution by volume, heat and reflux at 80℃ for 30 min, filter, and take the filtrate to obtain the test solution.

[0046] Preparation of mixed reference solution: Take ellagic acid, liquiritin, ammonium glycyrrhizinate, and sesamin reference substances, accurately weigh them, add methanol to prepare a mixed reference stock solution containing 11 μg ellagic acid, 322 μg liquiritin, 1065 μg ammonium glycyrrhizinate, and 210 μg sesamin per 1 mL, dilute the stock solution 10 times, and use it as the mixed reference solution.

[0047] 3. Methodological investigation of multi-index component content determination

[0048] (1) Linear relationship

[0049] The mixed reference substance stock solution was diluted separately to obtain mixed reference substance solutions of a series of concentrations, which were injected sequentially. The reference substance concentration was taken as the abscissa X and the corresponding peak area was taken as the ordinate Y. Linear regression analysis was performed on each component and the linear regression equation was calculated. The results are shown in Table 3 below.

[0050] Table 3 Linear regression equation

[0051] Element Regression equation <![CDATA[R 2 ]]> Ellagic acid Y=8.495x-0.302 0.9995 Licorice glycoside Y=17.886x+2.2643 0.9994 Ammonium glycyrrhizinate Y=60.912x+1.8114 1.000 Sesamin Y=21.579x+0.6879 1.000

[0052] (2) Precision test

[0053] Take the same reference solution, repeat the injection 6 times, measure the peak area, calculate the RSD, and the data are shown in Table 4 below.

[0054] Table 4 Precision data

[0055] Serial number Ellagic acid Licorice glycoside Ammonium glycyrrhizinate Sesamin 1 85.7 185.7 608.7 216.0 2 85.3 187.7 609.6 216.2 3 85.0 186.2 608.4 215.8 4 86.3 187.3 609.3 216.2 5 86.8 188.2 608.1 215.9 6 86.1 186.5 609.1 216.2 Mean 85.9 186.9 608.8 216.0 RSD(%) 0.78 0.52 0.10 0.10

[0056] The results showed that the RSD of the peak area of ​​each component was less than 2.0%, indicating that the instrument had good precision.

[0057] (3) Stability test

[0058] Take the same test solution and inject it at 0, 4, 8, 12, 18, and 24 hours respectively, and calculate the RSD. The data are shown in Table 5 below.

[0059] Table 5 Stability data

[0060] Time (h) Ellagic acid Licorice glycoside Ammonium glycyrrhizinate Sesamin 0 113.2 175.5 455.3 180.4 4 112.3 174.8 454.9 180.6 8 113.9 174.9 453.1 180.2 12 112.2 177.7 447.4 177.6 18 111.0 175.1 453.8 180.1 24 111.9 171.7 449.6 180.4 Mean 112.4 174.9 452.3 179.9 RSD(%) 0.92 1.1 0.71 0.64

[0061] The results showed that the peak area RSDs of the four components were all less than 2.0%, indicating that the test solution had good stability within 24 hours.

[0062] (4) Repeatability test

[0063] Take 6 portions of the test solution, inject them, and calculate the peak area RSD. The results are shown in Table 6 below.

[0064] Table 6 Repeatability data

[0065] Serial number Ellagic acid Licorice glycoside Ammonium glycyrrhizinate Sesamin 1 136.0 181.4 604.3 214.2 2 136.7 183.8 604.6 214.2 3 137.1 181.6 604.2 214.4 4 136.2 182.3 603.3 214.3 5 137.3 179.0 601.8 214.7 6 136.1 179.7 601.3 214.4 Mean 136.5 181.3 603.2 214.4 RSD(%) 0.40 0.96 0.24 0.09

[0066] The results showed that the RSD of the peak areas of the four components were all less than 2.0%, indicating that this method had good repeatability.

[0067] (5) Sample recovery test

[0068] Take 6 portions of the test solution, add the mixed reference solution respectively, measure, and calculate the sample recovery rate. The sample recovery rate results are shown in Tables 7, 8, 9 and 10 below.

[0069] Table 7 Ellagic acid sample recovery data

[0070]

[0071] Table 8 Liquorice glycoside recovery data

[0072]

[0073] Table 9 Ammonium glycyrrhizinate sample recovery data

[0074]

[0075] Table 10 Sesamin sample recovery data

[0076]

[0077] 4. Content determination of 4 random batches of samples

[0078] Table 11 The test results of 4 random batches of Yege Pills samples

[0079]

[0080] 5. Construction of the characteristic spectrum of Yege Pills

[0081] (1) Precision test

[0082] The same test solution (S1) was taken and injected 6 times continuously to determine the retention time and peak area respectively. Among them, peak No. 2 was ellagic acid, peak No. 3 was liquiritin, peak No. 6 was ammonium glycyrrhizinate, and peak No. 8 was sesamin. Taking peak No. 6 ammonium glycyrrhizinate as the reference peak, the relative retention time and relative peak area of ​​each common peak and the reference peak were calculated. The relative retention time results are shown in Table 12, and the relative peak area results are shown in Table 13.

[0083] Table 12 Relative retention time of each common peak

[0084]

[0085]

[0086] Table 13 Relative peak areas of common peaks

[0087] Peak 1 2 3 4 5 6 average value RSD(%) 1 0.325 0.339 0.343 0.333 0.339 0.343 0.337 2.062 2 0.213 0.236 0.243 0.238 0.242 0.240 0.236 4.727 3 0.316 0.317 0.323 0.314 0.329 0.316 0.319 1.849 4 0.237 0.237 0.239 0.232 0.240 0.232 0.236 1.419 5 0.153 0.156 0.157 0.155 0.161 0.158 0.157 1.822 6(S) 1.000 1.000 1.000 1.000 1.000 1.000 1.000 0.000 7 0.118 0.120 0.120 0.119 0.120 0.117 0.119 1.008 8 0.321 0.327 0.336 0.326 0.338 0.323 0.329 2.081 9 0.154 0.154 0.159 0.152 0.157 0.149 0.154 2.375 10 1.510 1.556 1.571 1.520 1.570 1.511 1.540 1.883

[0088] As shown in Table 12 and Table 13, the relative retention time RSD of each common peak is less than 5%, and the relative peak area RSD is less than 5%, indicating that the determined chromatographic conditions have good precision.

[0089] (2) Stability test

[0090] The same test solution (S1) was taken and injected at 0, 4, 8, 12, 18 and 24 h, and the retention time and peak area were measured respectively. Peak 6 ammonium glycyrrhizinate was used as the reference peak, and the relative retention time and relative peak area of ​​each common peak and the reference peak were calculated. The relative retention time results are shown in Table 14, and the relative peak area results are shown in Table 15.

[0091] Table 14 Relative retention time of each common peak

[0092] Peak 1 2 3 4 5 6 average value RSD(%) 1 0.205 0.206 0.205 0.206 0.204 0.206 0.205 0.357 2 0.310 0.311 0.310 0.310 0.310 0.313 0.311 0.404 3 0.325 0.326 0.325 0.325 0.325 0.327 0.325 0.283 4 0.592 0.592 0.592 0.592 0.591 0.594 0.592 0.157 5 0.660 0.657 0.656 0.655 0.655 0.657 0.656 0.314 6(S) 1.000 1.000 1.000 1.000 1.000 1.000 1.000 0.000 7 1.013 1.014 1.014 1.014 1.014 1.014 1.014 0.046 8 1.199 1.200 1.200 1.199 1.200 1.200 1.200 0.017 9 1.279 1.280 1.280 1.279 1.280 1.279 1.280 0.027 10 1.316 1.317 1.317 1.316 1.317 1.316 1.316 0.026

[0093] Table 15 Relative peak areas of the common peaks

[0094]

[0095]

[0096] It can be seen from Table 14 and Table 15 that for the same batch of test solutions, when measured at 0, 4, 8, 12, 18, and 24 hours, the relative retention time RSD of each common peak is less than 5%, and the relative peak area RSD is less than 5%. This indicates that the test solution is measured at different times within 24 hours according to the determined fingerprint spectrum establishment conditions, and the measurement results are stable.

[0097] (3) Repeatability test

[0098] Take the same test solution (S1) and repeat the injection 6 times, and measure the retention time and peak area respectively. Take peak No. 6 ammonium glycyrrhizinate as the reference peak, and calculate the relative retention time and relative peak area of ​​each common peak and the reference peak. The relative retention time results are shown in Table 16, and the relative peak area results are shown in Table 17.

[0099] Table 16 Relative retention time of each common peak

[0100] Peak 1 2 3 4 5 6 average value RSD(%) 1 0.203 0.199 0.204 0.203 0.207 0.205 0.204 1.220 2 0.307 0.304 0.308 0.307 0.311 0.310 0.308 0.766 3 0.323 0.321 0.323 0.323 0.326 0.325 0.323 0.591 4 0.590 0.589 0.590 0.590 0.592 0.592 0.591 0.250 5 0.649 0.648 0.651 0.654 0.652 0.653 0.651 0.366 6(S) 1.000 1.000 1.000 1.000 1.000 1.000 1.000 0.000 7 1.013 1.014 1.014 1.014 1.014 1.014 1.014 0.031 8 1.197 1.197 1.198 1.198 1.199 1.199 1.198 0.079 9 1.275 1.277 1.277 1.278 1.279 1.279 1.277 0.102 10 1.312 1.313 1.314 1.315 1.315 1.315 1.314 0.097

[0101] Table 17 Relative peak areas of the common peaks

[0102]

[0103]

[0104] It can be seen from Table 16 and Table 17 that the relative retention time RSD of each common peak is less than 5%, and the relative peak area RSD is less than 5%, indicating that the results have good repeatability when the determined chromatographic conditions are used.

[0105] 6. Similarity evaluation

[0106] Take 15 batches of Yege Pills samples, prepare the test solution according to the test solution preparation method, inject and measure according to the chromatographic conditions, and record the chromatogram (such as Figure 1 and Figure 2 ) The chromatograms of 15 batches of Yege Pills were introduced into the Chinese medicine chromatographic fingerprint similarity evaluation system, and the common chromatographic peaks in each batch of Yege Pills were selected, and the reference fingerprint (R) was generated by the median method (such as Figure 3 ), the reference fingerprint contains 10 common peaks. The reference fingerprint is calibrated with peaks 1 to 10 according to the order of peak appearance, peak 6 is used as the reference peak, and the relative peak areas and relative retention times of the remaining 9 common peaks are calculated to obtain the HPLC fingerprint of Yege Pills (such as Figure 4 ).

[0107] The relative retention times and peak areas of the 10 peaks in the HPLC fingerprint are as follows:

[0108] The relative retention times of peaks 1 to 10 are: peak 1 0.208; peak 2 0.306; peak 3 0.322; peak 4 0.589; peak 5 0.650; peak 6 1.000; peak 7 1.015; peak 8 1.197; peak 9 1.276; peak 10 1.313;

[0109] The relative peak areas of peaks 1 to 10 are: peak 1 0.335; peak 2 0.208; peak 3 0.319; peak 4 0.238; peak 5 0.137; peak 6 1.000; peak 7 0.171; peak 8 0.329; peak 9 0.155; peak 10 1.538.

[0110] The “Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System” was used to evaluate the similarity of 15 batches of Yege Pills fingerprints, and the similarities were all greater than , the results are shown in Table 18.

[0111] Table 1815 batches of Yege Pills similarity

[0112]

[0113]

[0114] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for establishing a fingerprint spectrum of Yege Pills, characterized in that: The following steps are involved: (1) Taking a Yege Pill test sample, freezing and crushing to obtain a test sample powder, mixing the test sample powder with petroleum ether, performing Soxhlet extraction, taking a drug residue, mixing the drug residue with a methanol aqueous solution, heating and refluxing, and taking a filtrate to obtain a test sample solution; (2) Mixing ellagic acid reference substance, liquiritin reference substance, ammonium glycyrrhizinate reference substance and sesamin reference substance with methanol to obtain a mixed reference substance solution; (3) respectively taking the test solution obtained in step (1) and the mixed reference solution obtained in step (2), injecting them into a liquid chromatograph, and collecting 70 min of chromatograms of the test sample and the reference solution; (4) The chromatogram of the test sample obtained in step (3) is imported into the "Chinese Herbal Medicine Chromatographic Fingerprint Similarity Evaluation System", and the chromatographic peaks common in the chromatograms of different batches of Yege Pills test samples are selected as common peaks. The median method is used to generate a control fingerprint of Yege Pills, and the relative retention time and relative peak area of ​​each common peak are calculated to obtain the HPLC fingerprint of Yege Pills.

2. The method according to claim 1, characterized in that: The freezing temperature in step (1) is -15 to -18°C, and the freezing time is 10 minutes; the mixing ratio of the test sample powder and petroleum ether in step (1) is 2 g: 60 to 80 mL; the Soxhlet extraction temperature in step (1) is 90°C, and the Soxhlet extraction time is 4 hours.

3. The method according to claim 1, characterized in that: The medicinal residue in step (1) is mixed with 25 mL of methanol aqueous solution, wherein the volume concentration of the methanol aqueous solution is 70%; the temperature of heating and reflux in step (1) is 80° C., and the time of heating and reflux is 30 min.

4. The method according to claim 1, characterized in that: In step (2), the mixing ratio of ellagic acid reference substance, liquiritin reference substance, ammonium glycyrrhizinate reference substance and sesamin reference substance with methanol is 11 μg: 322 μg: 1065 μg: 210 μg: 10 mL.

5. The method according to claim 1, characterized in that: In step (3), 10 μL of the test solution obtained in step (1) and 10 μL of the mixed reference solution obtained in step (2) are respectively aspirated and injected into the liquid chromatograph.

6. The method according to claim 1, characterized in that: The conditions of the liquid chromatography in step (3) are as follows: the chromatographic column is Agilent HC-C18; the specifications of the chromatographic column are 4.6×250 mm, 5 μm; the detection wavelength is 254 nm for 0 to 15 min and 236 nm for 15 to 70 min; the flow rate is 1.0 mL / min; the column temperature is 25° C.; the injection volume is 10 μL; the mobile phase A is acetonitrile, and the mobile phase B is a phosphoric acid solution with a volume concentration of 0.1%, and the gradient elution is 0 to 8 min, A 20%, B 80%; 8 to 14 min, A 20 to 22.5%, B 80 to 77.5%; 14 to 30 min, A 22.5 to 30%, B 77.5 to 70%; 30 to 35 min, A 30 to 60%, B 70 to 40%; 35 to 60 min, A 60%, B 40%; 60~61min, A60~100%, B 40~0%.

7. The method according to claim 1, characterized in that: In step (4), peak No. 6 corresponding to ammonium glycyrrhizinate is used as a reference peak to calculate the relative retention time and relative peak area of ​​each common peak.

8. The fingerprint of Yege Pills established by the method as described in any one of claims 1 to 7.

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