Method for establishing high performance liquid chromatography fingerprint spectrum of big hawthorn pills

Abstract

A method for establishing a large hawthorn pill high performance liquid chromatography fingerprint spectrum relates to the field of medicine, and comprises the following steps: precisely weighing large hawthorn pill medicinal material powder, precisely adding methanol, covering with a plug, weighing, soaking, ultrasonically extracting, taking out, cooling, weighing, complementing weight loss with methanol, uniformly shaking and filtering to obtain a test solution; the method comprises the following steps: accurately weighing a gallic acid reference substance, a 5-hydroxymethylfurfural reference substance and a chlorogenic acid reference substance, adding methanol to prepare mixed reference substance solutions with different mass concentrations, and filtering to obtain the reference substance solutions; and sucking the test solution and the reference solution, injecting the test solution and the reference solution into a high performance liquid chromatograph, measuring and recording a chromatogram, importing detection results of the test solution and the reference solution into fingerprint software, and performing data matching to generate a reference fingerprint, namely the high performance liquid chromatography fingerprint of the big hawthorn pills. According to the invention, the problems of low sensitivity, poor reproducibility and large deviation of measurement results in the existing thin-layer scanning method are solved, and the blank in the field is filled.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, in particular to a method for establishing high performance liquid chromatography (HPLC) fingerprint of Dashan Zha Wan. BACKGROUND

[0002] Dashan Zha Wan is a traditional Chinese medicine preparation, and the quality standard is recorded in Chinese Pharmacopoeia 2025 Edition Volume I. It is composed of three Chinese medicines, i.e. hawthorn, liushenqu (roasted bran) and fried wheat. It has the effect of promoting appetite and digestion, and is used for loss of appetite, indigestion, and abdominal distension caused by food accumulation.

[0003] The content detection of Dashan Zha Wan under the item of Chinese Pharmacopoeia 2025 Edition Volume I is to determine the content of ursolic acid in hawthorn by thin layer scanning method. However, the thin layer scanning method has low sensitivity, poor reproducibility, and large deviation of determination results. Moreover, traditional Chinese medicines and their preparations are complex multi-component systems, and the quality thereof cannot be evaluated only by determining the content of main effective components. A detection method that can provide rich component information should be used.

[0004] Traditional Chinese medicine fingerprint is a comprehensive and quantifiable identification method, which is based on the systematic study of chemical components of traditional Chinese medicines, and is mainly used for evaluating the authenticity, excellence and stability of traditional Chinese medicines and traditional Chinese medicine preparations. At present, the research on the method for establishing HPLC fingerprint of Dashan Zha Wan is not comprehensive. SUMMARY

[0005] In order to solve the problems of low sensitivity, poor reproducibility and large deviation of determination results in the existing method for determining the content of ursolic acid in hawthorn by thin layer scanning method, the present application provides a method for establishing HPLC fingerprint of Dashan Zha Wan, so as to fill the gap in the method for establishing HPLC fingerprint of Dashan Zha Wan.

[0006] The present application establishes the HPLC fingerprint of Dashan Zha Wan (powder) through condition exploration, which can comprehensively reflect the types of chemical components contained in Dashan Zha Wan (powder) and evaluate the overall quality of Dashan Zha Wan (powder).

[0007] The technical solution adopted by the present application to solve the technical problems is as follows:

[0008] The present application provides a method for establishing HPLC fingerprint of Dashan Zha Wan, which specifically comprises the following steps:

[0009] Step one, preparation of test solution;

[0010] Precisely weigh 1.0 g of Dashan Zha Wan medicinal powder, and place it in a conical flask with a plug. Precisely add 25 mL of methanol, cover the plug, weigh, soak, extract for 30 minutes by ultrasonic, take out and cool, make up the weight loss with methanol, shake well, filter, and obtain the test solution.

[0011] Step two, preparation of the reference solution;

[0012] Accurately weigh the gallic acid reference, 5-hydroxymethylfurfural reference, chlorogenic acid reference, add methanol to prepare mixed reference solutions with different mass concentrations, filter to obtain the reference solution;

[0013] Step three, determination method;

[0014] Take 20 μL of the test solution and the reference solution, inject into the high performance liquid chromatograph, determine, record the chromatogram, import the test results of the test solution and the reference solution into the fingerprint software, match the data, generate the control fingerprint, that is, the high performance liquid chromatography fingerprint of Dashan Zha pills.

[0015] As a preferred embodiment, in step two, 1 mL of the mixed reference solution contains 11.88 μg of gallic acid, 0.191 μg of 5-hydroxymethylfurfural, and 12.5 μg of chlorogenic acid.

[0016] As a preferred embodiment, in step three, the chromatographic conditions determined by the high performance liquid chromatograph are as follows:

[0017] Take octadecylsilane-bonded silica gel as the filler, the injection amount is 20 μL, the detection wavelength is 280 nm, the mobile phase is acetonitrile (A)-0.1% acetic acid aqueous solution (B), and the gradient elution program is as follows: 0-10 min, 5%-8% A; 10-20 min, 8%-8% A; 20-30 min, 8%-13% A; 30-45 min, 13%-16% A; 45-65 min, 16%-27.2% A.

[0018] As a preferred embodiment, in step three, when the high performance liquid chromatograph is determined, an Inertsil ODS-3 chromatographic column is used, the column length is 250 mm, the inner diameter is 4.6 mm, the particle size is 5 μm, the flow rate is 1.0 mL / min, and the column temperature is 35 ℃.

[0019] As a preferred embodiment, in step three, the high performance liquid chromatography fingerprint of Dashan Zha pills has 13 common fingerprint peaks; the 2nd, 3rd and 4th chromatographic peaks in the common fingerprint peaks are respectively indicated as gallic acid, 5-hydroxymethylfurfural and chlorogenic acid.

[0020] The beneficial effects of the present application are:

[0021] The high-performance liquid chromatography fingerprint of hawthorn pills established in this invention contains three common fingerprint peaks, covering the main active ingredients of hawthorn pills. It comprehensively and systematically reflects the overall situation of various active and inactive components in hawthorn pills, exhibiting good stability, precision, and reproducibility. The similarity evaluation results are all higher than 0.98, meeting the technical requirements of fingerprint spectroscopy. The method demonstrates strong consistency and meets the technical requirements of fingerprint spectroscopy.

[0022] This invention effectively solves the problems of low sensitivity, poor reproducibility, and large deviation in the determination of ursolic acid content in hawthorn pills using the thin-layer scanning method. At the same time, it has the advantages of high accuracy in qualitative and quantitative analysis, reliable results, good repeatability, and easy operation. It fills the gap in the method of establishing high-performance liquid chromatography fingerprint of hawthorn pills and is suitable for widespread promotion and use. Attached Figure Description

[0023] Figure 1 This is the HPLC reference fingerprint of the test solution in Example 1.

[0024] Figure 2 The image shows the HPLC chromatogram of hawthorn pills at a wavelength of 254 nm in Example 2.

[0025] Figure 3 The image shows the HPLC chromatogram of hawthorn pills at a wavelength of 280 nm in Example 2.

[0026] Figure 4 This is the HPLC chromatogram under the conditions of gradient elution program 1 in Example 3.

[0027] Figure 5 This is the HPLC chromatogram under the conditions of gradient elution program 2 in Example 3.

[0028] Figure 6 This is the HPLC chromatogram under the conditions of gradient elution program 3 in Example 3.

[0029] Figure 7 This is the HPLC chromatogram under the conditions of gradient elution program 4 in Example 3.

[0030] Figure 8 This is the HPLC chromatogram under the conditions of gradient elution program 5 in Example 3.

[0031] Figure 9 This is the HPLC chromatogram under the conditions of gradient elution program 6 in Example 3.

[0032] Figure 10 The image shows the HPLC chromatogram of the test solution prepared by ultrasound in Example 4 (with 30% methanol as the ultrasonic solvent).

[0033] Figure 11 The image shows the HPLC chromatogram of the test solution prepared by ultrasound in Example 4 (70% methanol was used as the ultrasonic solvent).

[0034] Figure 12 The image shows the HPLC chromatogram of the test solution prepared by ultrasound in Example 4 (100% methanol as the ultrasonic solvent).

[0035] Figure 13 The image shows the HPLC chromatogram after ultrasonic treatment for 30 min in Example 5.

[0036] Figure 14 The image shows the HPLC chromatogram after 1 hour of ultrasonic treatment in Example 5.

[0037] Figure 15 The image shows the HPLC chromatogram after ultrasonic treatment for 1.5 hours in Example 5.

[0038] Figure 16 The HPLC chromatogram is for a sample of 1.0 g in Example 6.

[0039] Figure 17 The HPLC chromatogram is for a sample of 2.0 g in Example 6.

[0040] Figure 18 The HPLC chromatogram is for a sample of 3.0 g in Example 6.

[0041] Figure 19 The HPLC chromatogram is for a sample of 4.0 g in Example 6.

[0042] Figure 20 The image shows the matching results of five batches of hawthorn pills (powder) in Example 7.

[0043] Figure 21 This is the HPLC chromatogram of the test solution in Example 7.

[0044] Figure 22 This is the HPLC chromatogram of the reference standard in Example 7. Detailed Implementation

[0045] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0046] Example 1: A method for establishing a high-performance liquid chromatography fingerprint of hawthorn pills

[0047] Step 1: Preparation of the test solution;

[0048] Accurately weigh 1.0g of hawthorn pill powder and place it in a stoppered conical flask. Accurately add 25ml of methanol, stopper the flask, weigh the sample, soak the sample, and extract it by ultrasonication for 30 minutes. Remove the sample, let it cool, weigh it, make up the weight loss with methanol, shake well, and take the filtrate. Filter the filtrate through a 0.22μm microporous membrane to obtain the test solution.

[0049] Step 2: Preparation of the reference solution;

[0050] Accurately weigh gallic acid reference standard, 5-hydroxymethylfurfural reference standard, and chlorogenic acid reference standard, and add methanol to prepare a mixed solution containing 11.88 μg gallic acid, 0.191 μg 5-hydroxymethylfurfural, and 12.5 μg chlorogenic acid per 1 mL. Filter the solution through a 0.22 μm microporous membrane to obtain the reference solution.

[0051] Step 3, Measurement Method;

[0052] Take 20 μL each of the test solution and the reference solution, inject them into the high-performance liquid chromatograph, determine and record the chromatograms. Import the detection results of the test solution and the reference solution into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)" software. After data matching, generate the reference fingerprint chromatogram. Figure 1 ).

[0053] The chromatographic conditions for high-performance liquid chromatography (HPLC) were as follows: octadecylsilane-bonded silica gel was used as the packing material; an Inertsil ODS-3 column was employed with a length of 250 mm, an inner diameter of 4.6 mm, a particle size of 5 μm, a flow rate of 1.0 mL / min, and a column temperature of 35 °C. Acetonitrile was used as mobile phase A, and 0.1% aqueous acetic acid solution was used as mobile phase B for gradient elution. The detection wavelength was 280 nm, and the gradient elution program was as follows: 0–10 min, 5%–8% A; 10–20 min, 8%–8% A; 20–30 min, 8%–13% A; 30–45 min, 13%–16% A; 45–65 min, 16%–27.2% A; and the injection volume was 20 μL.

[0054] Example 2: Investigation of Detection Wavelength

[0055] The fingerprint spectra at detection wavelengths of 254 nm and 280 nm were examined, as shown below. Figure 2 , Figure 3 As shown in the figure, it can be seen from the spectrum that there are more chromatographic peaks and larger peak areas in the spectrum under the wavelength of 280nm, and the peak resolution is better. Therefore, 280nm was selected as the detection wavelength.

[0056] Example 3: Investigation of chromatographic conditions

[0057] The fingerprint spectrum was investigated using acetonitrile (A)-0.1% acetic acid aqueous solution (B) as the mobile phase, under the conditions in Table 1-6, with gradient elution at a flow rate of 1.0 mL / min and a detection wavelength of 280 nm. The results are shown below. Figures 4 to 9 As shown in the figure. The results show that the peak separation is good under the conditions of mobile phase gradient elution 1 in Table 1, which can meet the requirements of fingerprint spectrum. Therefore, the conditions in Table 1, i.e., mobile phase gradient elution 1, are taken as the mobile phase conditions.

[0058] Table 1. Gradient elution of mobile phase 1

[0059] Time (min) A(％) B(％) 0 5 95 10 8 92 20 8 92 30 13 87 45 16 84 65 27.2 72.8

[0060] Table 2. Gradient elution of mobile phase 2

[0061] Time (min) A(％) B(％) 0 5 95 10 10 90 20 10 90 30 13 87 45 16 84 65 27.2 72.8 70 5 95 75 5 95

[0062] Table 3. Gradient elution of mobile phase 3

[0063]

[0064]

[0065] Table 4. Gradient elution of mobile phase 4

[0066] Time (min) A(％) B(％) 0 5 95 10 9 91 20 9 91 30 13 87 45 16 84 65 27.2 72.8 70 5 95 75 5 95

[0067] Table 5. Mobile phase gradient elution 5

[0068] Time (min) A(％) B(％) 0 5 95 5 6 94 12 12 88 20 15 85 26 19 81 40 33 67 55 55 45 60 75 25 65 90 10 70 5 95 72 5 95

[0069] Table 6. Mobile Phase Gradient Elution

[0070] Time (min) A(％) B(％) 0 5 95 10 9 91 20 10 90 30 13 87 45 16 84 65 27.2 72.8 70 5 95 75 5 95

[0071] Example 4: Investigation of the effect of the test sample solution on solvent treatment

[0072] The HPLC chromatograms of the test sample solution were investigated when it was treated with ultrasound and when the solvents were 30% methanol, 70% methanol, and 100% methanol, respectively. The results are shown below. Figures 10 to 12 As shown, the results indicate that when the ultrasonic treatment is performed at a detection wavelength of 280 nm using 100% methanol as the solvent, the resulting test solution contains a large number of sample peaks with good separation. Therefore, 100% methanol is recommended as the solvent for preparing the test solution.

[0073] Example 5: Investigation of the treatment time of the test sample solution

[0074] The HPLC chromatograms of the test sample after sonication for 30 min, 1 h, and 1.5 h were examined, as shown below. Figures 13 to 15As shown, the results indicate that the test solution treated with sonication for 30 minutes at a detection wavelength of 280 nm can meet the analytical requirements.

[0075] Example 6: Investigation of the dosage of the test sample solution

[0076] HPLC chromatograms were examined for 1.0 g, 2.0 g, 3.0 g, and 4.0 g of the test sample after adding 25 ml of methanol, as shown below. Figures 16 to 19 As shown, the results indicate that adding 25 mL of methanol to 1.0 g of the test sample can fully extract its active ingredients.

[0077] Based on the above comparative analysis, the experiment finally determined the preparation method of the test solution as follows: Under a detection wavelength of 280 nm, take 1.0 g of hawthorn pill powder, accurately weigh it, add 25 mL of methanol, stir well, weigh it, soak it, and ultrasonically extract it for 30 min. The test solution prepared under these conditions can meet the requirements of the analysis.

[0078] Example 7: Similarity evaluation of fingerprint chromatograms of batch hawthorn pills

[0079] Five batches of raw materials for Da Shan Zha Wan were collected, and the batch numbers are shown in Table 7.

[0080] Table 7

[0081] Number Batch number 1 Q202502122 2 Q202505045 3 Q202505046 4 Q202505047 5 Q202505048

[0082] (1) Similarity evaluation;

[0083] Five batches of hawthorn pills (powder) were collected, with two parallel test solutions processed for each batch. The fingerprint analysis was performed using the established fingerprint method described above. The results were imported into the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine (2012 Edition)". After data matching, a control fingerprint was generated (see...). Figure 1 The similarity of the chromatograms of the five batches of test samples was evaluated, and the matching chromatograms are shown in [reference needed]. Figure 20 The similarity results are shown in Table 8. The results indicate that the similarity of the five batches of test samples ranged from 0.993 to 1.000.

[0084] Table 85 shows the similarity evaluation results of the fingerprint spectra of the test samples.

[0085] S1 S2 S3 S4 S5 Reference Fingerprint S1 1.000 1.000 0.997 0.996 0.994 0.998 S2 1.000 1.000 0.997 0.996 0.994 0.998 S3 0.997 0.997 1.000 0.999 0.998 0.999 S4 0.996 0.996 0.999 1.000 0.998 0.999 S5 0.994 0.994 0.998 0.998 1.000 0.998 S6 0.993 0.994 0.997 0.998 1.000 0.998 S7 0.995 0.996 0.994 0.996 0.995 0.998 S8 0.995 0.996 0.995 0.997 0.996 0.999 S9 0.995 0.996 0.997 0.998 0.997 0.999 S10 0.996 0.996 0.997 0.997 0.997 0.999 Reference Fingerprint 0.998 0.998 0.999 0.999 0.998 1.000 S6 S7 S8 S9 S10 Reference Fingerprint S1 0.993 0.995 0.995 0.995 0.996 0.998 S2 0.994 0.996 0.996 0.996 0.996 0.998 S3 0.997 0.994 0.995 0.997 0.997 0.999 S4 0.998 0.996 0.997 0.998 0.997 0.999 S5 1.000 0.995 0.996 0.997 0.997 0.998 S6 1.000 0.996 0.996 0.998 0.997 0.998 S7 0.996 1.000 1.000 0.999 0.999 0.998 S8 0.996 1.000 1.000 0.999 0.999 0.999 S9 0.998 0.999 0.999 1.000 1.000 0.999 S10 0.997 0.999 0.999 1.000 1.000 0.999 Reference Fingerprint 0.998 0.995 0.999 0.999 0.999 1.000

[0086] Chlorogenic acid was selected as the reference, and its retention time was set to 1. The relative retention times of the fingerprint peaks were calculated. The relative retention times should be relatively constant. Based on the detection results of 5 batches of samples, chromatographic peaks with good stability, strong absorption, and obvious characteristics were selected as common peaks. A total of 13 common fingerprint peaks were identified through matching. Figure 21Peak 2 is gallic acid, peak 3 is 5-hydroxymethylfurfural, and peak 4 is chlorogenic acid. Figure 22 ).

[0087] (2) Stability test;

[0088] Hawthorn pills (powder) were taken as samples and prepared into test solutions according to the test solution preparation method. Under the above chromatographic conditions, the samples were injected at 0, 6, and 12 hours, and fingerprint spectra were recorded. Chlorogenic acid was used as a reference peak, and the relative retention time and relative peak area ratios of each common peak were calculated, along with the RSD values. The results showed that the relative retention time RSD of the 13 specified common peaks was <2% (Table 9), and the relative peak area RSD was <3% (Table 10). The data files were imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)" for similarity evaluation. The results showed that the similarity of the fingerprint spectra of each test sample was greater than 0.98 (Table 11), indicating that the samples had good stability within 12 hours and met the requirements of fingerprint chromatographic research techniques.

[0089] Table 9. Results of relative retention time for stability.

[0090]

[0091] Table 10 Results of relative peak area for stability

[0092]

[0093]

[0094] Table 11 Stability Similarity Evaluation Results

[0095] S1 S2 S3 Reference Fingerprint S1 1.000 0.998 0.999 0.999 S2 0.998 1.000 0.999 1.000 S3 0.999 0.999 1.000 1.000 Reference Fingerprint 0.999 1.000 1.000 1.000

[0096] (3) Precision test;

[0097] One sample solution of hawthorn pills (powder) was injected six times under the chromatographic conditions described above. Using chlorogenic acid as a reference peak, the relative retention time and relative peak area ratios of each common peak were calculated, and the RSD values ​​were also calculated. The results showed that the RSD of the relative retention time of the 13 specified common peaks was <2% (Table 12), and the RSD of the relative peak area was <3% (Table 13). The data file was imported into the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine (2012 Edition)" for similarity evaluation. The results showed that the similarity of the fingerprint chromatograms of the test samples was all greater than 0.98 (Table 14), indicating good instrument precision and meeting the requirements of fingerprint chromatographic research techniques.

[0098] Table 12 Results of Precision Relative Retention Time

[0099]

[0100]

[0101] Table 13 Precision Relative Peak Area Results

[0102]

[0103] Table 14 Precision Similarity Evaluation Results

[0104] S1 S2 S3 S4 S5 S6 Reference Fingerprint S1 1.000 0.995 0.998 0.997 0.998 0.998 0.998 S2 0.995 1.000 0.998 0.998 0.998 0.996 0.998 S3 0.998 0.998 1.000 0.999 0.999 0.999 1.000 S4 0.997 0.998 0.999 1.000 1.000 1.000 1.000 S5 0.998 0.998 0.999 1.000 1.000 0.999 1.000 S6 0.998 0.996 0.999 1.000 0.999 1.000 0.999 Reference Fingerprint 0.998 0.998 1.000 1.000 1.000 0.999 1.000

[0105] (4) Repeatability test;

[0106] Five samples of hawthorn pills (powder) were injected separately under the chromatographic conditions described above. Using chlorogenic acid as a reference peak, the relative retention time and relative peak area ratios of each common peak were calculated, and the RSD values ​​were also calculated. The results showed that the RSD of the relative retention time of the 13 specified common peaks was <2% (Table 15), and the RSD of the relative peak area was <3% (Table 16). The data files were imported into the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine (2012 Edition)" for similarity evaluation. The results showed that the similarity of the fingerprint chromatograms of each test sample was greater than 0.98 (Table 17), indicating good repeatability and meeting the requirements of fingerprint chromatographic research techniques.

[0107] Table 15 Results of Repeatability Relative Retention Time

[0108]

[0109] Table 16 Repeatability Relative Peak Area Results

[0110]

[0111] Table 17 Results of Repeatability Similarity Evaluation

[0112] S1 S2 S3 S4 S5 Reference Fingerprint S1 1.000 0.998 0.997 0.997 0.998 0.998 S2 0.998 1.000 0.999 0.999 1.000 1.000 S3 0.997 0.999 1.000 1.000 0.999 0.999 S4 0.997 0.999 1.000 1.000 1.000 1.000 S5 0.998 1.000 0.999 1.000 1.000 1.000 Reference Fingerprint 0.998 1.000 0.999 1.000 1.000 1.000

[0113] This invention discloses a method for establishing a high-performance liquid chromatography fingerprint of hawthorn pills. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The product of this invention has been described through preferred embodiments, and those skilled in the art can clearly modify or appropriately change and combine the product described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

Claims

1. A method for establishing a high-performance liquid chromatography fingerprint of hawthorn pills, characterized in that, Includes the following steps: Step 1: Preparation of the test solution; Accurately weigh 1.0g of hawthorn pill powder, place it in a stoppered conical flask, accurately add 25mL of methanol, stopper the flask, weigh the sample, soak it, extract it by sonication for 30 minutes, remove it, cool it, weigh it, make up the weight loss with methanol, shake well and filter to obtain the test solution. Step 2: Preparation of the reference solution; Accurately weigh gallic acid reference standard, 5-hydroxymethylfurfural reference standard, and chlorogenic acid reference standard, add methanol to prepare mixed reference standard solutions of different mass concentrations, and obtain the reference standard solution after filtration; Step 3, Measurement Method; Take 20 μL each of the test solution and the reference solution and inject them into the high-performance liquid chromatograph. Measure and record the chromatograms. Import the detection results of the test solution and the reference solution into the fingerprinting software. After data matching, generate the reference fingerprint spectrum, which is the high-performance liquid chromatogram of Da Shan Zha Wan (a traditional Chinese medicine).

2. The method for establishing a high-performance liquid chromatography fingerprint of hawthorn pills according to claim 1, characterized in that, In step two, 1 mL of the mixed reference solution contains 11.88 μg of gallic acid, 0.191 μg of 5-hydroxymethylfurfural, and 12.5 μg of chlorogenic acid.

3. The method for establishing a high-performance liquid chromatography fingerprint of hawthorn pills according to claim 1, characterized in that, In step three, the chromatographic conditions determined by the high-performance liquid chromatograph are as follows: Octadecylsilane-bonded silica gel was used as the packing material, with an injection volume of 20 μL and a detection wavelength of 280 nm. The mobile phase was acetonitrile (A)-0.1% aqueous acetic acid (B), and the gradient elution program was as follows: 0–10 min, 5%–8% A; 10–20 min, 8%–8% A; 20–30 min, 8%–13% A; 30–45 min, 13%–16% A; 45–65 min, 16%–27.2% A.

4. The method for establishing a high-performance liquid chromatography fingerprint of hawthorn pills according to claim 1, characterized in that, In step three, the high-performance liquid chromatography (HPLC) uses an Inertsil ODS-3 column with a length of 250 mm, an inner diameter of 4.6 mm, a particle size of 5 μm, a flow rate of 1.0 mL / min, and a column temperature of 35 °C.

5. The method for establishing a high-performance liquid chromatography fingerprint of hawthorn pills according to claim 1, characterized in that, In step three, the high-performance liquid chromatography fingerprint of the hawthorn pill has 13 common fingerprint peaks; among the common fingerprint peaks, peaks 2, 3, and 4 are identified as gallic acid, 5-hydroxymethylfurfural, and chlorogenic acid, respectively.

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