A method for establishing a fingerprint spectrum of Yege Pills and the fingerprint spectrum obtained

Abstract

The present invention discloses a method for establishing a fingerprint spectrum of Yege Pills and the fingerprint spectrum obtained by establishing the fingerprint spectrum, belonging to the technical field of analysis and detection of traditional Chinese medicine preparations. The method for establishing a fingerprint spectrum of Yege Pills is provided, comprising: mixing a Yege Pill test sample with petroleum ether, performing Soxhlet extraction, mixing the drug residue with a methanol-water solution, heating and refluxing, and taking the filtrate to obtain a test solution; mixing ellagic acid, liquiritin, ammonium glycyrrhizate, and sesamin reference substances with methanol to obtain a mixed reference solution; aspirating the test solution and the reference solution, injecting them into a liquid chromatograph, and collecting a chromatogram; importing the test sample chromatogram into a "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System", selecting common chromatographic peaks as common peaks, generating a Yege Pill reference fingerprint spectrum, calculating the relative retention time and relative peak area of ​​each common peak, and obtaining an HPLC fingerprint spectrum of Yege Pills. The spectrum can be used to more comprehensively evaluate the quality of Yege Pills, ensuring the quality stability, consistency, and controllability of Yege Pills.

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 the establishment. Background Art

[0002] The formula for Yege Pills consists of walnut kernels, ginkgo nuts, dried persimmons (stem and pit removed), fennel, roasted black sesame seeds, sesame oil, pitted jujubes, and licorice. Besides these key ingredients, it also includes auxiliary ingredients like sucrose and honey, which help to enhance the pill's form and taste. Yege Pills primarily nourish the lungs and kidneys, moisten dryness, promote salivation, and relieve hiccups. It is used to treat dysphagia, pharyngitis, dysphagia, and dry throat. It can also be used as an adjunctive treatment for atypical hyperplasia of the esophageal mucosal epithelium and esophageal cancer.

[0003] Currently, there is no content test for the active ingredients of Yege Pills in the current standards, and the "Chinese Pharmacopoeia" does not include 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 resulting fingerprint spectrum. The spectrum can be used to more comprehensively evaluate the quality of Yege Pills, thereby better ensuring 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) Take a Yege Pill test sample, freeze it, and crush it to obtain a test sample powder. The test sample powder is mixed with petroleum ether, and Soxhlet extraction is performed. The drug residue is collected, and the drug residue is mixed with a methanol aqueous solution, heated under reflux, and the filtrate is collected to obtain a test sample solution;

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

[0008] (3) The test solution obtained in step (1) and the mixed reference solution obtained in step (2) were respectively aspirated and injected into a liquid chromatograph, and a 70-min chromatogram of the test sample and the reference solution was collected;

[0009] (4) The chromatogram of the test sample obtained in step (3) was imported into the "Similarity Evaluation System of Chromatographic Fingerprints of Traditional Chinese Medicines", and the common chromatographic peaks in the chromatograms of the test samples of different batches of Yege Pills were selected as common peaks. The median method was used to generate the control fingerprint of Yege Pills, and the relative retention time and relative peak area of ​​each common peak were 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 minutes; the mixing ratio of the test sample powder and petroleum ether in step (1) is 2g:60 to 80mL; the Soxhlet extraction temperature in step (1) is 90°C, and the Soxhlet extraction time is 4 hours.

[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 the heating reflux in step (1) is 80° C., and the heating reflux time is 30 minutes.

[0012] Preferably, in step (2), the mixing ratio of ellagic acid reference substance, liquiritin reference substance, ammonium glycyrrhizate reference substance and sesamin reference substance to 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 the 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 0-15 min and 236 nm for 15-70 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%, with a gradient elution of 0-8 min, A 20%, B 80%; 8-14 min, A 20-22.5%, B 80-77.5%; 14-30 min, A 22.5-30%, B 77.5-70%; 30-35 min, A 30-60%, B 70-40%; 35-60 min, A 60%, B40%; 60~61min, A60~100%, B 40~0%.

[0015] Preferably, in step (4), peak No. 6 corresponding to ammonium glycyrrhizate 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 of Yege Pills established by any of the establishment methods.

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

[0018] The present invention discloses a method for establishing a fingerprint of Yege Pills. Liquid chromatography is used to determine the fingerprint using an ammonium glycyrrhizate reference substance. The fingerprint of Yege Pills established using the method provided herein can effectively characterize their quality, facilitate comprehensive product quality monitoring, and effectively ensure the quality stability, consistency, and controllability of Yege Pills, thereby ensuring their safety and effectiveness.

[0019] The present invention establishes an HPLC characteristic spectrum of Yege Pills and calibrates 10 common peaks. The established fingerprint spectrum has high technical content, good peak shape, and is easy to identify, 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 following briefly introduces the drawings required for use in the embodiments. 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 any 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 rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

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

[0027] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice 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 any conflict with any incorporated document, the contents 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 described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be 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 reagents

[0032] 1. Instruments

[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., totaling 15 batches, numbered S1 to S15. Methanol and acetonitrile were chromatographically grade, petroleum ether (60-90°C) and phosphoric acid were analytically grade, and water was purified. Information on the reference substances used is shown in Table 1.

[0036] Table 1 Reference substance information

[0037] Reference substances batch number source Ellagic acid 111959-201903 China Food and Drug Inspection Institute Liquiritin 111610-202209 China Food and Drug Inspection Institute Ammonium glycyrrhizate 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 an Agilent HC-C18 (4.6×250 mm, 5 μm). The detection wavelengths were: 254 nm for 0–15 min and 236 nm for 15–70 min. The flow rate was 1.0 mL / min, the column temperature was 25°C, and the injection volume was 10 μL. Mobile phase A was acetonitrile, and mobile phase B was 0.1% by volume phosphoric acid. Gradient elution was employed. The gradient elution method is shown in Table 2 (% 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 reference solution respectively, inject them into the high performance liquid chromatograph, perform the measurement, and record the chromatogram.

[0044] 2. Solution preparation

[0045] Preparation of test solution: Cut the Yege Pill sample into pieces, freeze it at -15℃ for 10 min, and then grind it to obtain the test powder. Accurately weigh 2 g and place it 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 is completely evaporated, transfer it 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: accurately weigh ellagic acid, liquiritin, ammonium glycyrrhizate, and sesamin reference substances, add methanol to prepare a mixed reference stock solution containing 11 μg ellagic acid, 322 μg liquiritin, 1065 μg ammonium glycyrrhizate, and 210 μg sesamin per 1 mL. Dilute the stock solution 10-fold to prepare 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 used as the abscissa X and the corresponding peak area as the ordinate Y. Linear regression analysis was performed on each component to calculate the linear regression equation. 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 Liquiritin Y=17.886x+2.2643 0.9994 Ammonium glycyrrhizate Y=60.912x+1.8114 1.000 Sesamin Y=21.579x+0.6879 1.000

[0052] (2) Precision experiment

[0053] The same reference solution was injected 6 times, the peak area was measured, and the RSD was calculated. The data are shown in Table 4 below.

[0054] Table 4 Precision data

[0055] Serial number Ellagic acid Liquiritin Ammonium glycyrrhizate 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] The same test solution was injected at 0, 4, 8, 12, 18, and 24 hours, and the RSD was calculated. The data are shown in Table 5 below.

[0059] Table 5 Stability data

[0060] Time (h) Ellagic acid Liquiritin Ammonium glycyrrhizate 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 Liquiritin Ammonium glycyrrhizate 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 RSDs of the peak areas of the four components were all less than 2.0%, indicating that the 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 Liquiritin sample recovery data

[0072]

[0073] Table 9 Ammonium glycyrrhizate 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 Random determination results of 4 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 glycyrrhizate, and peak No. 8 was sesamin. Taking peak No. 6 ammonium glycyrrhizate 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 the 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 Tables 12 and 13, the relative retention time RSDs of the common peaks were less than 5%, and the relative peak area RSDs were less than 5%, indicating that the determined chromatographic conditions had good precision.

[0089] (2) Stability test

[0090] The same test solution (S1) was injected at 0, 4, 8, 12, 18, and 24 h, and the retention time and peak area were measured respectively. Taking peak 6 ammonium glycyrrhizate 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 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] As shown in Tables 14 and 15, for the same batch of test solutions, the relative retention time RSDs of the common peaks were less than 5%, and the relative peak area RSDs were less than 5%, when measured at 0, 4, 8, 12, 18, and 24 hours. This indicates that the test results were stable when the test solutions were measured at different times within 24 hours according to the established fingerprint conditions.

[0097] (3) Repeatability test

[0098] Take the same test solution (S1) and repeat the injection 6 times. The retention time and peak area are measured respectively. Taking peak No. 6 ammonium glycyrrhizate as the reference peak, the relative retention time and relative peak area of ​​each common peak and the reference peak are calculated. 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] As shown in Tables 16 and 17, the relative retention time RSDs of the common peaks were less than 5%, and the relative peak area RSDs were less than 5%, indicating that the results were reproducible under the determined chromatographic conditions.

[0105] 6. Similarity evaluation

[0106] Take 15 batches of Yege Pills samples, prepare the test solution according to the test solution preparation method, inject the sample 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 imported into the Chinese medicine chromatographic fingerprint similarity evaluation system, and the common chromatographic peaks in each batch of Yege Pills were selected. The median method was used to generate the control fingerprint (R) (such as Figure 3 ), the control fingerprint contains 10 common peaks. The control fingerprint is marked with peaks 1 to 10 according to the order of peak appearance, and peak 6 is used as the reference peak. 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 similarity evaluation system of Chinese medicine chromatographic fingerprint was used to evaluate the fingerprints of 15 batches of Yege Pills. The similarities were all greater than , and the results are shown in Table 18.

[0111] Table 1815 Batch of Yege Pills Similarity

[0112]

[0113]

[0114] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for establishing a fingerprint of Yege Pills, characterized in that: The following steps are involved: (1) Take the Yege Pills test sample, freeze it, and crush it to obtain the test sample powder. The test sample powder is mixed with petroleum ether, and Soxhlet extraction is performed. The drug residue is collected, and the drug residue is mixed with methanol aqueous solution, heated to reflux, and the filtrate is collected to obtain the test sample solution; (2) Mix ellagic acid reference substance, liquiritin reference substance, ammonium glycyrrhizate reference substance, and sesamin reference substance with methanol to obtain a mixed reference solution; (3) The test solution obtained in step (1) and the mixed reference solution obtained in step (2) were respectively aspirated and injected into a liquid chromatograph, and the chromatograms of the test sample and the reference solution were collected over a period of 70 minutes; (4) The chromatogram of the test sample obtained in step (3) was imported into the "Chinese Herbal Medicine Chromatographic Fingerprint Similarity Evaluation System", and the chromatographic peaks common in the chromatograms of the test samples of different batches of Yege Pills were selected as common peaks. The control fingerprint of Yege Pills was generated using the median method, and the relative retention time and relative peak area of ​​each common peak were calculated to obtain the HPLC fingerprint of Yege Pills; The liquid chromatography conditions 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 from 0 to 15 min and 236 nm from 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, the mobile phase B is a phosphoric acid solution with a volume concentration of 0.1%, and the gradient elution is from 0 to 8 min, A 20%, B 80%; 8~14min, A 20~22.5%, B 80~77.5%; 14~30min, A 22.5~30%, B 77.5~70%; 30~35min, A 30~60%, B 70~40%; 35~60min, A 60%, B 40%; 60~61min, A 60~100%, B 40~0%.

2. The method according to claim 1, wherein: The freezing temperature in step (1) is -15~-18°C, and the freezing time is 10 minutes; the mixing ratio of the test sample powder and petroleum ether in step (1) is 2g:60~80mL; 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, wherein: 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 the heating reflux in step (1) is 80° C., and the heating reflux time is 30 min.

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

5. The method according to claim 1, wherein: 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) were respectively aspirated and injected into the liquid chromatograph.

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

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