Method for establishing fingerprint spectrum of Yangshen Dingzhi granules

Abstract

The present invention relates to a method for establishing a fingerprint spectrum of Yangshen Dingzhi Granules. By examining different sample solution preparations and chromatographic conditions, the method determines the sample solution preparation method and detection conditions for the fingerprint spectrum of the product, identifies six characteristic peaks, including polygalaconitone III and 5-O-methylvisaramide, and effectively reflects the material basis of Yangshen Dingzhi Granules. This method can serve as a basis for quality control of Yangshen Dingzhi Granules and is used to evaluate the authenticity, excellence, and stability of the preparation.

Description

Technical Field

[0001] The present invention relates to a fingerprint spectrum detection method for traditional Chinese medicine preparations, in particular to a method for establishing a fingerprint spectrum of Yangshen Dingzhi granules. Background Art

[0002] A traditional Chinese medicine fingerprint is a chromatogram or spectrum obtained through appropriate processing and analytical methods, which can identify the chemical characteristics of a traditional Chinese medicine. Traditional Chinese medicine fingerprints are primarily used to evaluate the authenticity, quality, and stability of Chinese medicinal materials and semi-finished Chinese medicine preparations. They offer comprehensive and quantifiable results.

[0003] Yangshen Dingzhi Granules are made from Rehmannia root, processed Polygala root, Poria root, wild chrysanthemum, Saposhnikovia root, Cyperus rotundus, red ginseng, and bran-fried Citrus aurantium, which have the effects of nourishing the mind and relieving anxiety.

[0004] Methods of learning features. Summary of the Invention

[0005] To address the technical problems of the prior art, the present invention provides a method for establishing a fingerprint spectrum of Yangshen Dingzhi Granules. This method can comprehensively characterize the chemical characteristics of Yangshen Dingzhi Granules and has the advantages of good stability and high accuracy.

[0006] The technical solution adopted by the present invention to solve the above technical problems is:

[0007] A method for establishing a fingerprint spectrum of Yangshen Dingzhi granules comprises the following steps:

[0008] (1) Prepare the test solution: Take the contents of Yangshen Dingzhi granules, grind them into powder, take 0.5 g of the fine powder, add 50 mL of 70% methanol, ultrasonicate, cool to room temperature, filter, take 30 mL of the filtrate and evaporate to dryness in a water bath, dissolve the residue in methanol, transfer to a 10 mL volumetric flask, dilute to the mark with methanol, shake well, filter, and take the filtrate.

[0009] (2) Preparation of reference solution: Take each reference substance and dissolve it in methanol to make a solution containing 0.1 mg per 1 mL, which will serve as the reference solution;

[0010] (3) Chromatographic conditions and system suitability test: The chromatographic column was filled with octadecyl bonded silica gel; 0.1% phosphoric acid was used as mobile phase A, and acetonitrile was used as mobile phase B;

[0011] The flow rate was 1.0 ml per minute; the column temperature was 30°C; the resolution of naringin and adjacent peaks should be greater than 1.5;

[0012] (4) Take 20 μL of the reference solution and the test solution respectively, inject them into the liquid chromatograph, measure, and record the chromatogram.

[0013] Furthermore, a gradient elution method was adopted, and the elution time and the mobile phase ratio were as follows: 0 min, mobile phase A 90%, mobile phase B 10%; 10 min, mobile phase A 85%, mobile phase B 15%; 50 min, mobile phase A 78%, mobile phase B 22%; 58 min, mobile phase A 70%, mobile phase B 30%; 78 min, mobile phase A 60%, mobile phase B 40%; 98 min, mobile phase A 40%, mobile phase B 60%; 118 min, mobile phase A 40%, mobile phase B 60%; 120 min, mobile phase A 90%, mobile phase B 10%.

[0014] Furthermore, the chromatographic column is a Welch Xtimate@C18 or equivalent chromatographic column with a column length of 2.5 cm, an inner diameter of 4.6 mm, and a particle size of 5 μm.

[0015] Furthermore, the detection wavelength is 254 nm.

[0016] Furthermore, the ultrasonic treatment conditions were 250 W, 40 KHz, and 60 minutes.

[0017] Furthermore, the reference substances are polygala tenuifolia root III, 3,6-dierucoylsucrose, 5-O-methylvisalwood alcohol, mongoside, neohesperidin, naringin, rehmannia glutinosa D, polygala tenuifolia root saponin, and ginsenoside Rg1.

[0018] Furthermore, the characteristic spectrum of the test sample should present 14 characteristic peaks, among which the retention times of peaks 4, 5, 6, 7, 9 and 11 are basically consistent with those of the reference substances polygala tenuifolia III, 5-O-methylvisaramide, naringin, 3,6-dierucoylsucrose, neohesperidin and montanadin; the retention times of peaks 6, 7 and 8 should be consistent with those of the reference substances naringin, 3,6-dierucoylsucrose and neohesperidin. The peak corresponding to the retention time of the reference substance naringin peak is designated as S peak, and the relative retention time of each characteristic peak and S peak is calculated, which should be within ±10.0% of the specified value.

[0019] Furthermore, the relative retention time values ​​are: 0.15 (peak 1), 0.19 (peak 2), 0.51 (peak 3), 0.63 (peak 4), 0.94 (peak 5), 1.00 (peak 6, S), 1.04 (peak 7), 1.15 (peak 8), 1.17 (peak 9), 1.32 (peak 10), 1.56 (peak 11), 1.63 (peak 12), 1.69 (peak 13), and 2.17 (peak 14).

[0020] Furthermore, characteristic peaks are matched, and according to the similarity evaluation system of traditional Chinese medicine chromatographic fingerprint, the fingerprint of the test solution and the fingerprint of the reference solution are calculated with similarity not less than 0.90.

[0021] Compared with the existing technology, the beneficial effects of the present invention are:

[0022] The present invention determines the test solution preparation method and detection conditions of the product fingerprint through different test solution preparation and chromatographic conditions, identifies six characteristic peaks such as polygala tenuifolia ketone III and 5-O-methylvisaramide, and better reflects the material basis of Yangshen Dingzhi granules. It can be used as a basis for quality control of Yangshen Dingzhi granules and be used to evaluate the authenticity, excellence and stability of the preparation quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The drawings in the specification, which constitute a part of this application, are used to provide further understanding of this application. The illustrative embodiments of this application and their descriptions are used to explain this application and do not constitute improper limitations on this application.

[0024] Figure 1 This is the chromatogram of Example 1.

[0025] Figure 2 This is the chromatogram of elution condition 1 in Example 2.

[0026] Figure 3 This is the chromatogram of elution condition 2 in Example 2.

[0027] Figure 4 This is the chromatogram of elution condition 3 in Example 2.

[0028] Figure 5 This is the chromatogram in Example 3.

[0029] Figure 6 This is the chromatogram at a wavelength of 203 nm in Example 4.

[0030] Figure 7 This is the chromatogram at a wavelength of 210 nm in Example 4.

[0031] Figure 8 This is the chromatogram at a wavelength of 254 nm in Example 4.

[0032] Figure 9 This is the chromatogram at a wavelength of 283 nm in Example 4.

[0033] Figure 10 This is the chromatogram at a wavelength of 334 nm in Example 4.

[0034] Figure 11 This is a chromatogram for attributing characteristic peaks in Example 5.

[0035] Figure 12 It is the chromatogram under the proposed conditions in Example 5.

[0036] Figure 13 This is the chromatogram when the relative retention time is calculated in Example 5.

[0037] Figure 14 The chromatogram is the characteristic peak limit range in Example 5.

[0038] Figure 15 This is the chromatogram for similarity evaluation in Example 5. DETAILED DESCRIPTION

[0039] In order to enable those skilled in the art to more clearly understand the technical solution of the present disclosure, the technical solution of this patent will be described in detail below in conjunction with specific embodiments. It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of this application.

[0040] This invention establishes the fingerprint spectrum of Yangshen Dingzhi Granules with reference to General Chapter 9101 "Guiding Principles for Validation of Analytical Methods" and "Technical Requirements for Fingerprint Research of Traditional Chinese Medicine Injections" of the 2020 edition of the "Chinese Pharmacopoeia".

[0041] Yangshen Dingzhi Granules: Rehmannia root, processed Polygala root, Poria root, wild chrysanthemum, Saposhnikovia root, Cyperus rotundus, red ginseng, and stir-fried Citrus aurantium.

[0042] Sample information: Yangshen Dingzhi Granules, production date: 20210912; production batch number: 210912; production unit: nominally "Affiliated Hospital of Shandong University of Traditional Chinese Medicine".

[0043] The reference material information is shown in the following table:

[0044]

[0045]

[0046] With reference to the 2020 edition of the Chinese Pharmacopoeia and literature, a comprehensive analysis of the chemical components of the medicinal materials in Yangshen Dingzhi Granules mainly includes flavonoids, alkaloids, terpenes, glycosides, etc., with complex components. When designing the experiment, the components with ultraviolet absorption in Yangshen Dingzhi Granules were used as fingerprint inspection indicators, and high-performance liquid chromatography was used for inspection. The method screening was based on the characteristic spectra or content determination of the medicinal materials under the prescription composition of the first part of the 2020 edition of the Chinese Pharmacopoeia. The summary and comparison of the determination conditions of the medicinal materials under the prescription composition are as follows:

[0047]

[0048] After comprehensive analysis of the chemical components of the prescription composition and the content determination of the medicinal materials, combined with the material basis of the actual medicinal materials used in the preparation of this product and the determination conditions statistically listed in the above table, the following fingerprint HPLC conditions are tentatively determined for screening the test sample processing. The specific conditions are as follows:

[0049] Chromatographic column: Welch Xtimate@C18 (4.6 mm × 250 mm, 5 μm);

[0050] Mobile phase: Use 0.1% phosphoric acid water as mobile phase A and acetonitrile as mobile phase B, and elute according to the gradient shown in the table below.

[0051]

[0052] Flow rate: 1.0 ml / min;

[0053] Column temperature: 30°C;

[0054] Detection wavelength: tentatively set at 254 nm.

[0055] Example 1 Experimental sample processing and solution preparation

[0056]

[0057]

[0058] Determination method: Accurately measure 20μl of the test solution in the above table and inject them into the liquid chromatograph respectively. According to the tentative fingerprint monitoring conditions, the determination is carried out according to the law. The typical chromatogram results are as follows: Figure 1 shown.

[0059] From the above comparative chromatographic information, it can be seen that when 70% and 100% methanol are selected as the extraction solvents, the number of chromatographic peaks in the transferred sample and the basic baseline separation between the peaks can be achieved, and there is no significant difference in the extraction results of the two solvents; considering the preparation method of the actual extract of this preparation, this experimental study uses 70% methanol as the extraction solvent for the contents of this particle.

[0060] Example 2 Optimization of liquid phase elution conditions for fingerprint

[0061] Considering that the prescription composition of this product has many medicinal flavors and complex ingredients, in order to effectively identify the characteristic fingerprint peaks or common peaks of the material basis of this product as much as possible, it is necessary to optimize the elution mode of the monitored chromatographic conditions so as to separate or elute as many material basis components as possible. The specific experimental plan is designed as follows:

[0062] Preparation of test sample solution: Take about 0.5 g of the fine powder of the sample content (batch number: 2109112), accurately weigh it, place it in a stoppered conical flask, accurately add 50 ml of 70% methanol, ultrasonically treat (250W, 40KHz) for 60 minutes, cool to room temperature, filter, take 30 ml of the filtrate and evaporate to dryness in a water bath, dissolve the residue in methanol, transfer it to a 10 ml volumetric flask, dilute to the scale with methanol, shake well, filter, and take the filtrate.

[0063] Monitoring chromatographic conditions

[0064] Chromatographic column: octadecyl bonded silica gel as filler (Welch Xtimate@C18; 4.6 mm × 250 mm, 5 μm);

[0065] Mobile phase A: 0.1% phosphoric acid solution

[0066] Mobile phase B: acetonitrile

[0067] Flow rate: 1.0ml / min

[0068] Column temperature: 30°C

[0069] Detection wavelength: 254nm

[0070] Design the following gradient elution conditions and conduct the experiment: Elution condition ①

[0071] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 97 3 20 97 3 30 85 15 60 78 22 80 70 30 90 60 40 100 40 60 118 40 60 120 97 3

[0072] Elution conditions②

[0073]

[0074]

[0075] Elution conditions ③

[0076] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 90 10 10 85 15 50 78 22 58 70 30 78 60 40 98 40 60 118 40 60 120 90 10

[0077] Determination method: Accurately measure 20 μl of the above test solution and inject it into the liquid chromatograph according to the above elution conditions ① to ③ respectively, and record the typical chromatogram. The results are as follows Figure 2 、 3 , as shown in 4.

[0078] From the above experiments, it can be seen that, compared with the above elution conditions, when following elution condition ③, the fingerprint spectrum of the test solution of the test sample shows that the separation of each peak is better, and the peak response is better than the other two elution conditions, and the analysis time is moderate. Therefore, this study tentatively uses the mobile phase system elution condition ③ as the basic fingerprint determination condition for this particulate matter.

[0079] Example 3 Location of the main material basis of Yangshen Dingzhi granules

[0080] Take an appropriate amount of the reference substance in the following table and prepare the positioning solution concentration as shown in the following table:

[0081] Table Research Reference Solution Information

[0082]

[0083] Determination method: Take 20 μl of the above reference solution and inject it into the liquid chromatograph respectively. Determine according to the conditions optimized in Example 2 and record the chromatogram. Figure 5 shown.

[0084] The results of the aforementioned reference solution positioning indicate that rehmannia glutinosa D exhibits a relatively low response under these detection conditions, hindering the identification of the characteristic peaks of the product's particle fingerprint. To more effectively characterize the product's properties, this study primarily examined substances with a strong response at specific wavelengths as characteristic peaks of the fingerprint. Therefore, further optimization of the specific wavelength is necessary.

[0085] Example 4 Optimization of the UV Absorption Wavelength of the Fingerprint

[0086] In order to ensure the effective traceability and monitoring of the particulate material basis of this product, the optimal characteristic ultraviolet absorption wavelength of the particulate material basis of Yangshen Dingzhi was explored; this study combined the detection conditions under the corresponding medicinal materials in the first volume of the Chinese Pharmacopoeia 2020 edition. The ultraviolet absorption of the drug basis of this product is mainly concentrated near 203nm, 210nm, 254nm, 283nm, and 334nm wavelengths; to ensure the comprehensiveness of the material basis detection, feature positioning is used to examine the characteristics of the chromatograms at different wavelengths to confirm the required wavelengths for fingerprint monitoring in this study. The test solution of Example 1 and the reference solution of Example 2 were respectively injected into the liquid chromatograph, and the chromatograms with detection wavelengths of 203nm, 210nm, 254nm, 283nm, and 334nm were mainly recorded. The experimental results are as follows Figure 6-10 .

[0087] From the above experiments, it can be seen that when the fingerprint is detected at wavelengths of 203nm, 210nm, 254nm, 283nm, and 334nm, the characteristic indicators of the drugs in the prescription of this product, such as polygalaenoside, polygalaenoside III, 3,6-dierucoylsucrose, 5-O-methylvisamyl alcohol, montanol, neohesperidin, naringin, ginsenoside Rg1, etc., all respond; compared with the condition of 254nm wavelength, the ultraviolet characteristics of each material basis in the sample respond better; therefore, it is confirmed that the ultraviolet monitoring wavelength of the conditions of this study is 254nm.

[0088] Based on the results of the above studies, the determination conditions of the fingerprint spectrum of Yangshen Dingzhi Granules are summarized as follows:

[0089] Chromatographic column: Use octadecyl bonded silica gel as the filler (such as: Welch Xtimate@C18; 4.6mm×250mm, 5μm or equivalent column efficiency); Mobile phase: 0.1% phosphoric acid water as mobile phase A, acetonitrile as mobile phase B, and gradient elution according to the table below.

[0090] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 90 10 10 85 15 50 78 22 58 70 30 78 60 40 98 40 60 118 40 60 120 90 10

[0091] Flow rate: 1.0ml / min

[0092] Column temperature: 30°C;

[0093] Injection volume: 20 μl;

[0094] Detection wavelength: 254nm

[0095] Test solution: Grind 0.5 g of the sample content into fine powder, accurately weigh, and place in a stoppered conical flask. Accurately add 50 ml of 70% methanol, ultrasonically treat (250 W, 40 kHz) for 60 minutes, cool to room temperature, filter, and take 30 ml of the filtrate to dryness in a water bath. Dissolve the residue in methanol and transfer to a 10 ml volumetric flask. Add methanol to dilute to the scale, shake well, filter, and take the filtrate.

[0096] Determination method: Accurately measure 20μl of the test solution, inject it into the liquid chromatograph, determine it according to the method, and record the fingerprint chromatogram.

[0097] Example 5 Attribution of characteristic peaks

[0098] The known chemical components of Yangshen Dingzhi Granules include rehmannoside D, polygalactone III, 3,6-dierucoylsucrose, 5-O-methylvisalwood alcohol, montanol, neohesperidin, naringin, and ginsenoside Rg1. These components were located and identified in the chromatogram of the test solution. Reference solution: Accurately weigh appropriate amounts of each of rehmannoside D, polygalactone III, 3,6-dierucoylsucrose, 5-O-methylvisalwood alcohol, montanol, neohesperidin, naringin, and ginsenoside Rg1. Dissolve these in methanol to create solutions containing approximately 0.1 mg of each component per 1 ml. This serves as the reference solution.

[0099] Determination method: According to the chromatographic conditions determined in Example 4 above, the sample was injected and measured, and the peaks in the chromatogram of the test solution (batch number: 210912) were compared to attribute the characteristic peaks. The results are shown in Figure 11 .

[0100] The characteristic peaks in the chromatogram of the test solution were identified and seven known components were marked. Among them, 5-O-methylvisaramide, polygalaconone III, 3,6-dierucoylsucrose, naringin, neohesperidin, and montanin were relatively obvious and could be considered as characteristic peaks. Ginsenoside Rg1 and rehmannia glutinosa were detected weakly and were not considered as characteristic peaks.

[0101] Among the known components detected in the test solution, the peaks of "5-O-methylvisaramide, polygala tenuifolia Ⅲ, 3,6-dierucoylsucrose, naringin, neohesperidin, and montmorillonin" are more obvious and are located in the middle position of the fingerprint characteristic spectrum. The peaks of 3,6-dierucoylsucrose, naringin, and neohesperidin can better reflect the separation effect of the spectrum, so they are used as references.

[0102] Preparation of reference solution: Take appropriate amount of 3,6-dierucoylsucrose, naringin, and neohesperidin reference substances, weigh accurately, and dissolve them in methanol to prepare a solution containing approximately 0.1 mg of each per 1 ml, which serves as the reference solution.

[0103] Take an appropriate amount of the contents of one batch of Yangshen Dingzhi granules for the test, and prepare 5 test solutions according to the same method as the test solution. Under the proposed chromatographic conditions, accurately measure 20μl and inject them into the liquid chromatograph for determination according to the law, and record the chromatogram. The results are as follows: Figure 12 .

[0104] The above fingerprint test data were imported into the traditional Chinese medicine fingerprint similarity software. After multi-point correction peak matching of the traditional Chinese medicine fingerprint similarity software, the fingerprint spectra of 5 samples were compared. The results showed that the repeatability of the fingerprint spectra was good.

[0105] The chromatograms of 5 test sample solutions from one batch of Yangshen Dingzhi granules were analyzed and compared, and the common peaks were determined by calculation using the "Chinese Medicine Fingerprint Similarity Software".

[0106] Results: The chromatograms of 5 test solutions of 1 batch of Yangshen Dingzhi Granules were separated into more than 20 peaks (such as Figure 13 ), of which there are 14 characteristic peaks, and the characteristic peak area accounts for more than 90% of the total peak area. Taking naringin as the reference peak, the relative retention time is calculated as follows:

[0107]

[0108] According to the above chromatogram peak table, the relative retention time is calculated and the characteristic peaks are calibrated. The results are as follows:

[0109]

[0110]

[0111] Based on the above experimental results, the characteristic spectrum of Yangshen Dingzhi Granules was determined: 14 characteristic peaks should be presented in the characteristic spectrum of the test sample, and the relative retention times of each characteristic peak and the naringin peak are as follows: 0.15 (peak 1), 0.19 (peak 2), 0.51 (peak 3), 0.63 (peak 4), 0.94 (peak 5), 1.00 (peak 6, S), 1.04 (peak 7), 1.15 (peak 8), 1.17 (peak 9), 1.32 (peak 10), 1.56 (peak 11), 1.63 (peak 12), 1.69 (peak 13), 2.17 (peak 14); among which Peak 4: Polygala tenuifolia III, Peak 5: 5-O-methylvisaramide, Peak 6 (S): naringin, Peak 7: 3,6-dierucoylsucrose, Peak 9: neohesperidin, Peak 11: montanadin.

[0112] Characteristic peak limit range:

[0113] The characteristic spectrum of the test sample should show 14 characteristic peaks (such as Figure 14 The retention time of peaks 4, 5, 6, 7, 9 and 11 are basically consistent with those of the reference substances polygalaconitone III, 5-O-methylvisaramide, naringin, 3,6-dierucoylsucrose, neohesperidin and montanin; the retention time of peaks 6, 7 and 8 should be consistent with the retention time of the reference substances of naringin, 3,6-dierucoylsucrose and neohesperidin. The peak corresponding to the retention time of the reference substance naringin is designated as S peak. The relative retention time of each characteristic peak and S peak is calculated, and it should be within ±10.0% of the specified value.

[0114] Similarity evaluation

[0115] The fingerprint test data of one batch of Yangshen Dingzhi Granules (210912) and the characteristic reference substance were imported into the Chinese medicine fingerprint similarity software. The control fingerprint of the Yangshen Dingzhi Granules fingerprint was obtained by the Chinese medicine fingerprint similarity software. The fingerprint test data of each batch were calculated by the MARK peak (characteristic peak) similarity. The results are shown in Figure 15 .

[0116]

[0117] Results: The similarity between the fingerprint of Yangshen Dingzhi Granules and the MARK peak (characteristic peak) was not less than 0.90.

[0118] The experiment determined the test solution preparation method and detection conditions of the product's fingerprint through different test solution preparation and chromatographic conditions, and identified six characteristic peaks including Polygala tenuifolia III and 5-O-methylvisamol, which better reflected the material basis of Yangshen Dingzhi Granules and can be used as a basis for quality control of Yangshen Dingzhi Granules to evaluate the authenticity, excellence and stability of the preparation quality.

[0119] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or make equivalent replacements for parts thereof. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention. Although the above describes the specific embodiments of the present invention in conjunction with the accompanying drawings, it is not intended to limit the protection scope of the present invention. Those skilled in the art should understand that, based on the technical solution of the present invention, various modifications or variations that can be made by those skilled in the art without creative work are still within the protection scope of the present invention.

Claims

1. A method for establishing a fingerprint spectrum of Yangshen Dingzhi granules, characterized in that: The steps include: (1) Prepare the test solution: Take the contents of Yangshen Dingzhi granules, grind them into powder, take 0.5 g of the fine powder, add 50 mL of 70% methanol, ultrasonically treat, cool to room temperature, filter, take 30 mL of the filtrate and evaporate to dryness in a water bath, add methanol to dissolve the residue and transfer it to a 10 mL volumetric flask, add methanol to dilute to the mark, shake well, filter, and take the filtrate. (2) Preparation of reference solution: Take the reference substance and dissolve it in methanol to make a solution containing 0.1 mg per 1 mL, which is used as the reference solution; (3) Chromatographic conditions and system suitability test: The chromatographic column is filled with octadecyl bonded silica gel; 0.1% phosphoric acid was used as mobile phase A and acetonitrile was used as mobile phase B; The flow rate was 1.0 ml per minute; the column temperature was 30°C; the resolution of naringin and adjacent peaks should be greater than 1.5; (4) Take 20µL of the reference solution and the test solution respectively, inject them into the liquid chromatograph, measure, and record the chromatogram; Gradient elution was used, with the elution time and mobile phase ratio being: 0 min, mobile phase A 90%, mobile phase B 10%; 10 min, mobile phase A 85%, mobile phase B 15%; 50 min, mobile phase A 78%, mobile phase B 22%; 58 min, mobile phase A 70%, mobile phase B 30%; 78 min, mobile phase A 60%, mobile phase B 40%; 98 min, mobile phase A 40%, mobile phase B 60%; 118 min, mobile phase A 40%, mobile phase B 60%; 120 min, mobile phase A 90%, mobile phase B 10%; The reference substances are polygala tenuifolia root Ⅲ, 3,6-dierucoylsucrose, 5-O-methylvisalwood alcohol, mongoside, neohesperidin, naringin, rehmannia root glycoside D, polygala tenuifolia root saponin and ginsenoside Rg1.

2. The establishment method according to claim 1, characterized in that The Yangshen Dingzhi granules are composed of the following components: prepared rehmannia root, processed polygala root, poria root, siler, cyperus rotundus, red ginseng, wild chrysanthemum, and stir-fried aurantium with bran.

3. The establishment method according to claim 1, characterized in that The chromatographic column was Welch Xtimate@C18 with a length of 2.5 cm, an inner diameter of 4.6 mm, and a particle size of 5 μm.

4. The establishment method according to claim 1, characterized in that The detection wavelength is 254 nm.

5. The establishment method according to claim 1, characterized in that: The ultrasonic treatment conditions were 250 W, 40 KHz, and 60 min.

6. The establishment method according to claim 1, characterized in that: The characteristic spectrum of the test sample should present 14 characteristic peaks, among which the retention times of peaks 4, 5, 6, 7, 9 and 11 are basically consistent with those of the reference substances polygalaconitone III, 5-O-methylvisaramide, naringin, 3,6-dierucoylsucrose, neohesperidin and montanadin; the retention times of peaks 6, 7 and 8 should be consistent with those of the reference substances naringin, 3,6-dierucoylsucrose and neohesperidin. The peak corresponding to the retention time of the reference substance naringin peak is designated as S peak. The relative retention time of each characteristic peak and S peak is calculated, which should be within ±10.0% of the specified value.

7. The establishment method according to claim 1, characterized in that: The relative retention time values ​​are: 0.15-peak 1, 0.19-peak 2, 0.51-peak 3, 0.63-peak 4, 0.94-peak 5, 1.00-peak 6, S, 1.04-peak 7, 1.15-peak 8, 1.17-peak 9, 1.32-peak 10, 1.56-peak 11, 1.63-peak 12, 1.69-peak 13, 2.17-peak 14.

8. The establishment method according to claim 1, characterized in that: Characteristic peak matching: According to the similarity evaluation system of traditional Chinese medicine chromatographic fingerprint, the fingerprint of the test solution and the fingerprint of the reference solution are calculated with similarity not less than 0.90.

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