A method for establishing HPLC characteristic chromatogram of a medicinal material and decoction piece of Potentilla discolor Bunge, standard decoction and formula granules thereof

Abstract

The application provides a standard decoction, a medicinal slice, a medicinal material and a formula granule of Potentilla discolor and a method for establishing an HPLC characteristic spectrum, and comprises the following steps: S1) extracting Potentilla discolor raw materials by using a solvent to obtain a to-be-tested liquid; and S2) detecting the to-be-tested liquid by using high performance liquid chromatography to obtain an HPLC characteristic spectrum of the Potentilla discolor raw materials; the method for establishing the HPLC characteristic spectrum can effectively separate the chromatographic components of the Potentilla discolor medicinal material, medicinal slice, standard decoction and formula granule, can effectively evaluate the quality of the Potentilla discolor medicinal material, medicinal slice, standard decoction and formula granule, and has important significance for the research on Potentilla discolor.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of analytical detection technology, and in particular to a method for establishing HPLC characteristic chromatogram of standard decoction, medicinal material and its formula granules of Potentilla discolor. BACKGROUND

[0002] Potentilla discolor Bge. is a kind of dry whole plant of Rosaceae Potentilla discolor Bge. It is dug and picked before flowering in summer and autumn, and then the mud and impurities are removed and dried. Potentilla discolor Bge. has the effects of clearing heat and resolving toxicity, stopping diarrhea, stopping bleeding, and is often used for treating damp-heat diarrhea, carbuncle and sore, blood-heat hematochezia, metrorrhagia, eczema and tuberculosis.

[0003] The domestic and foreign literature research shows that the chemical components of Potentilla discolor Bge. mainly include flavonoids, terpenes, steroids, tannins and organic acids. In recent years, no one has reported the characteristic chromatogram of Potentilla discolor Bge. In order to effectively compare and analyze the differences and changes of medicinal material, standard decoction and formula granules, and to overall evaluate and control the quality of Potentilla discolor Bge. medicinal material, standard decoction and formula granules, it is necessary to establish a unified method for determining the characteristic chromatogram of Potentilla discolor Bge. medicinal material, standard decoction and formula granules, which is conducive to the overall evaluation of the scientificity and rationality of the related process of Potentilla discolor Bge., and can overall control the internal quality of Potentilla discolor Bge. medicinal material, standard decoction and formula granules, and ensure the clinical efficacy of Potentilla discolor Bge. formula granules. SUMMARY

[0004] The technical problem solved by the present application is to provide a method for establishing HPLC characteristic chromatogram of Potentilla discolor Bge. medicinal material, medicinal material and its formula granules, which can effectively separate the chromatographic components of Potentilla discolor Bge. medicinal material, medicinal material and its formula granules, and effectively evaluate the quality of Potentilla discolor Bge. medicinal material, medicinal material and its formula granules.

[0005] Therefore, the present application provides a method for establishing HPLC characteristic chromatogram of Potentilla discolor Bge. standard decoction, medicinal material and its formula granules, which comprises the following steps:

[0006] S1) extracting Potentilla discolor Bge. raw materials with a solvent to obtain a test solution;

[0007] S2) detecting the test solution by high performance liquid chromatography to obtain the HPLC characteristic chromatogram of Potentilla discolor Bge. raw materials;

[0008] The conditions of the high performance liquid chromatography are as follows: the chromatographic column is a C18 column, the mobile phase A is acetonitrile, the mobile phase B is 0.2% phosphoric acid solution, 0.2% acetic acid solution or 0.2% formic acid, and gradient elution is used.

[0009] The Potentilla discolor Bge. raw materials are Potentilla discolor Bge. standard decoction, medicinal material and its formula granules.

[0010] Preferably, the method further comprises preparing a reference solution of a control medicinal material and a reference solution of a control substance;

[0011] The preparation of the reference solution of the control medicinal material is specifically: dissolving the control medicinal material of Potentilla discolor Bunge in methanol and obtaining the reference solution of the control medicinal material after ultrasonic treatment;

[0012] The preparation of the reference solution of the control substance is specifically: dissolving isoquercitrin in methanol to obtain the reference solution of the control substance;

[0013] The reference solution of the control substance and the reference solution of the control medicinal material are determined by high performance liquid chromatography to obtain chromatograms of the control substance and the control medicinal material, respectively;

[0014] The components of the HPLC characteristic chromatogram of Potentilla discolor Bunge medicinal material, standard decoction and formula granules are qualitatively determined according to the chromatograms of the control substance and the control medicinal material.

[0015] Preferably, the solvent is methanol, the extraction method is ultrasonic extraction, the extraction time is 15-45 min, and the ratio of the Potentilla discolor Bunge raw material to the solvent is 0.2 g:(20-30) ml.

[0016] Preferably, the gradient elution is specifically:

[0017] 0-15 min, A phase: 10-14%, B phase: 90-86%;

[0018] 15-30 min, A phase: 14-17%, B phase: 86-83%;

[0019] 30-33 min, A phase: 17%, B phase: 17%;

[0020] 33-45 min, A phase: 17-27%, B phase: 83-73%;

[0021] 45-55 min, A phase: 27-45%, B phase: 73-55%;

[0022] 55-60 min, A phase: 45-65%, B phase: 55-35%.

[0023] Preferably, the flow rate of the mobile phase is 0.8-1.2 ml / min, and the detection wavelength is 360 nm.

[0024] Preferably, the column temperature of the chromatographic column is 25-35℃, the column length of the chromatographic column is 250 mm, the inner diameter of the chromatographic column is 4.6 mm, and the particle size is 5 μm.

[0025] Preferably, the similarity of the HPLC characteristic chromatogram of the Herba Potentillae Discolorae, standard decoction and formula granules is evaluated by using the Chinese medicine chromatographic fingerprint similarity evaluation system to obtain the HPLC standard characteristic chromatogram of the Herba Potentillae Discolorae, standard decoction and formula granules, which is composed of 10 characteristic peaks, wherein peak 3 (S): isoquercitrin.

[0026] Preferably, for the standard decoction of the Herba Potentillae Discolorae, the relative retention time of each characteristic peak to the S peak is calculated, and the relative retention time should be within ±10% of the specified value, and the specified value is: 0.33 (peak 1), 0.82 (peak 2), 1.31 (peak 4), 1.34 (peak 5), 1.85 (peak 6), 1.99 (peak 7), 2.07 (peak 8), 2.13 (peak 9), 2.24 (peak 10).

[0027] Preferably, for the Herba Potentillae Discolorae, the relative retention time of each characteristic peak to the S peak is calculated, and the relative retention time should be within ±10% of the specified value, and the specified value is: 0.33 (peak 1), 0.82 (peak 2), 1.31 (peak 4), 1.35 (peak 5), 1.85 (peak 6), 2.00 (peak 7), 2.08 (peak 8), 2.14 (peak 9), 2.25 (peak 10).

[0028] Preferably, for the formula granules of the Herba Potentillae Discolorae, the relative retention time of each characteristic peak to the S peak is calculated, and the relative retention time should be within ±10% of the specified value, and the specified value is: 0.33 (peak 1), 0.82 (peak 2), 1.31 (peak 4), 1.34 (peak 5), 1.83 (peak 6), 1.97 (peak 7), 2.05 (peak 8), 2.11 (peak 9), 2.21 (peak 10).

[0029] The application provides a method for establishing the HPLC characteristic chromatogram of the Herba Potentillae Discolorae, decoction pieces, standard decoction and formula granules. First, the raw material of the Herba Potentillae Discolorae is extracted by using a solvent to obtain a test solution, and then the test solution is detected by using a high performance liquid chromatography method to obtain the HPLC characteristic chromatogram of the raw material of the Herba Potentillae Discolorae. The method for establishing the HPLC characteristic chromatogram of the Herba Potentillae Discolorae, decoction pieces, standard decoction and formula granules provided by the application uses acetonitrile-0.2% phosphoric acid solution, 0.2% acetic acid solution or 0.2% formic acid as the mobile phase, and isoquercitrin as the reference substance to establish the HPLC characteristic chromatogram of the Herba Potentillae Discolorae, decoction pieces, standard decoction and formula granules, which has the characteristics of good repeatability, high accuracy and good stability, and thus can effectively evaluate the quality of the Herba Potentillae Discolorae, decoction pieces, standard decoction and formula granules. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1Chromatogram of the standard decoction of Potentilla discolor under different flow phases of the present application;

[0031] Figure 2 Chromatogram of the standard decoction of Potentilla discolor under different wavelengths of the present application;

[0032] Figure 3 Chromatogram of the standard decoction of Potentilla discolor under different flow rates of the present application;

[0033] Figure 4 Chromatogram of the standard decoction of Potentilla discolor under different column temperatures of the present application;

[0034] Figure 5 Chromatogram of the standard decoction of Potentilla discolor for delay investigation;

[0035] Figure 6 Chromatogram of the standard decoction of Potentilla discolor under different extraction solvents of the present application;

[0036] Figure 7 Chromatogram of the standard decoction of Potentilla discolor under different extraction methods of the present application;

[0037] Figure 8 Chromatogram of the standard decoction of Potentilla discolor under different extraction times of the present application;

[0038] Figure 9 Chromatogram of the standard decoction of Potentilla discolor under different solvent addition amounts of the present application;

[0039] Figure 10 Characteristic chromatogram peak identification spectrum of the standard decoction of Potentilla discolor of the present application;

[0040] Figure 11 Spectrum of isoquercitrin of the reference substance of the present application;

[0041] Figure 12 Spectrum of isoquercitrin of the standard decoction of Potentilla discolor of the present application;

[0042] Figure 13 Chromatogram of the standard decoction of Potentilla discolor under different instruments of the present application;

[0043] Figure 14 Chromatogram of the standard decoction of Potentilla discolor under different chromatographic columns of the present application;

[0044] Figure 15 Characteristic chromatogram of the standard decoction of Potentilla discolor under different batches of the present application;

[0045] Figure 16 Control chromatogram of the characteristic chromatogram of the standard decoction of Potentilla discolor of the present application;

[0046] Figure 17 Characteristic chromatogram of the medicinal material of Potentilla discolor under different batches of the present application;

[0047] Figure 18 The present application is a contrast characteristic map of the characteristic map of the traditional Chinese medicine of Potentilla discolor Bunge;

[0048] Figure 19 The present application is a chromatogram of the different batches of Potentilla discolor Bunge formula granules;

[0049] Figure 20 The present application is a contrast map of the characteristic map of the Potentilla discolor Bunge formula granules;

[0050] Figure 21 The present application is a contrast map of the characteristic map of the Potentilla discolor Bunge, standard decoction, and granules. DETAILED DESCRIPTION

[0051] In order to further understand the present application, the preferred embodiments of the present application are described below in conjunction with examples, but it should be understood that these descriptions are only for further illustrating the features and advantages of the present application, and are not limitations of the claims of the present application.

[0052] In order to effectively compare and analyze the differences and changes of the Potentilla discolor Bunge medicinal materials, decoction pieces, standard decoction, and formula granules, and overall evaluate the quality of the Potentilla discolor Bunge raw materials, it is of great significance to establish a HPLC characteristic map of the Potentilla discolor Bunge standard decoction, decoction pieces, medicinal materials, and formula granules. Through limiting the related conditions in the high performance liquid chromatography, the HPLC characteristic map of the Potentilla discolor Bunge medicinal materials, decoction pieces, standard decoction, and formula granules established has the characteristics of high accuracy, good stability, and good repeatability, so that the effective separation of the chromatographic components of the Potentilla discolor Bunge medicinal materials, decoction pieces, standard decoction, or formula granules can be realized, and a reliable basis for studying the quality of the Potentilla discolor Bunge is provided. Specifically, the present application discloses a method for establishing a HPLC characteristic map of the Potentilla discolor Bunge standard decoction, decoction pieces, medicinal materials, and formula granules, which comprises the following steps:

[0053] S1) The Potentilla discolor Bunge raw materials are extracted by a solvent to obtain a test solution;

[0054] S2) The test solution is detected by a high performance liquid chromatography to obtain a HPLC characteristic map of the Potentilla discolor Bunge raw materials;

[0055] The conditions of the high performance liquid chromatography are as follows: the chromatographic column is a C18 column, the mobile phase A is acetonitrile, the mobile phase B is a 0.2% phosphoric acid solution, a 0.2% acetic acid solution, or a 0.2% formic acid, and gradient elution is adopted;

[0056] The Potentilla discolor Bunge raw materials are the Potentilla discolor Bunge standard decoction, decoction pieces, medicinal materials, or formula granules.

[0057] In step S1), the raw material of Potentilla discolor Bunge is first extracted by solvent to obtain the test solution; in this step, the solvent is preferably selected from methanol, and the peak shape of the characteristic peak is moderate at this time. The extraction method is ultrasonic extraction or reflux extraction. Since ultrasonic extraction is fast and simple, the extraction method is preferably ultrasonic extraction. The extraction time is 15-45 min, more specifically 15 min, 30 min or 45 min. In the present application, the extraction time is preferably 30 min, and the peak shape of the chromatogram is moderate. The ratio of the raw material of Potentilla discolor Bunge to the solvent is 0.2 g:(20-30) ml; more specifically 0.2 g:25 ml, and the peak shape of each chromatographic peak of the chromatogram is moderate at this time.

[0058] In step S2), the test solution is detected by high performance liquid chromatography to obtain the HPLC characteristic spectrum of the raw material of Potentilla discolor Bunge; this step also includes preparing a reference solution of a control medicinal material and a reference solution of a control substance;

[0059] The preparation of the reference solution of the control medicinal material is specifically as follows: the control medicinal material of Potentilla discolor Bunge is dissolved in methanol, and a reference solution of the control medicinal material is obtained after ultrasonic treatment; wherein the ratio of the control medicinal material of Potentilla discolor Bunge to the methanol is 0.5 g:50 ml, the ultrasonic treatment time is 30 min, the power is 600 W, and the frequency is 40 kHz.

[0060] The preparation of the reference solution of the control substance is specifically as follows: isoquercitrin is dissolved in methanol to obtain a reference solution of the control substance; wherein the ratio of isoquercitrin to methanol is 80 μg:1 ml.

[0061] The reference solution of the control substance and the reference solution of the control medicinal material are then determined by high performance liquid chromatography to obtain the chromatograms of the control substance and the control medicinal material, respectively.

[0062] The components of the HPLC characteristic spectrum of Potentilla discolor Bunge medicinal material, decoction pieces, standard decoction and formula granules are qualitatively determined according to the chromatograms of the control substance and the control medicinal material.

[0063] In the process of high performance liquid chromatography detection in the present application, the gradient elution is specifically as follows:

[0064] 0-15 min, A phase: 10-14%, B phase: 90-86%;

[0065] 15-30 min, A phase: 14-17%, B phase: 86-83%;

[0066] 30-33 min, A phase: 17%, B phase: 17%;

[0067] 33-45 min, A phase: 17-27%, B phase: 83-73%;

[0068] 45~55min, A phase: 27~45%, B phase, 73~55%;

[0069] 55~60min, A phase: 45~65%, B phase, 55~35%.

[0070] Wherein, the mobile phase A is acetonitrile, the mobile phase B is 0.2% phosphoric acid solution, 0.2% acetic acid solution or 0.2% formic acid, the phosphoric acid of the different mobile phases is relatively stable, therefore, the mobile phase is selected from acetonitrile-0.2% phosphoric acid solution. The flow rate of the mobile phase is 0.8-1.2ml / min, more specifically, the flow rate of the mobile phase is 0.8ml / min, 1.0ml / min or 1.2ml / min, different flow rates can effectively separate the chromatographic peaks, and in the application, 1.2ml / min is preferably used. The detection wavelength is 360nm, the chromatographic peak information is large at this wavelength, and the chromatogram limit is more stable.

[0071] The chromatographic column is C18, the column length is 250mm, the inner diameter is 4.6mm, and the particle size is 5μm; the column temperature is 25-35℃, more specifically 30℃, under which conditions each chromatographic peak can be effectively separated.

[0072] Under the detection of the above high performance liquid chromatography, the HPLC characteristic spectrum of the P. chinensis raw material is obtained. Specifically, when the P. chinensis raw material is P. chinensis medicinal material, the HPLC characteristic spectrum of the P. chinensis medicinal material can be obtained; when the P. chinensis raw material is P. chinensis decoction piece, the HPLC characteristic spectrum of the P. chinensis decoction piece can be obtained; when the P. chinensis raw material is P. chinensis standard decoction, the HPLC characteristic spectrum of the P. chinensis standard decoction can be obtained; and when the P. chinensis raw material is P. chinensis formula granule, the HPLC characteristic spectrum of the P. chinensis formula granule can be obtained.

[0073] The application first establishes a characteristic spectrum method for simultaneously determining P. chinensis medicinal material, P. chinensis decoction piece and P. chinensis formula granule by high performance liquid chromatography; the chromatographic conditions are as follows: octadecylsilane bonded silica gel is used as the filler; acetonitrile is used as the mobile phase A, and 0.2% phosphoric acid solution is used as the mobile phase B, gradient elution is carried out according to the provisions in Table 20; the flow rate is 1.2ml / min; the column temperature is 30℃; the detection wavelength is 360nm; and the theoretical plate number calculated according to the isoquercitrin peak should not be less than 5000.

[0074] Table 20 Gradient elution data table of P. chinensis raw material

[0075] Time (min) Mobile phase A (%) Mobile phase B (%) 0～15 10→14 90→86 15～30 14→17 86→83 30～33 17 83 33～45 17→27 83→73 45～55 27→45 73→55 55～60 45→65 55→35

[0076] The method can effectively separate the components of the Potentilla discolor chromatographic peak, and successfully identify isoquercitrin; the traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2012 version) is used to synthesize the characteristic chromatograms of Potentilla discolor and Potentilla discolor formula granules into a control chromatogram; the control chromatogram of the characteristic chromatograms of Potentilla discolor medicinal materials, standard decoction, and Potentilla discolor granules is established; as shown in Figure 16 , Figure 18 and Figure 20 .

[0077] For the final regulation of Potentilla discolor medicinal materials: the test sample chromatogram should present 10 characteristic peaks, and should correspond to the retention time of the 10 characteristic peaks in the reference chromatogram of the control medicinal materials, among which peak 3 should correspond to the retention time of the isoquercitrin reference peak. The peak corresponding to the isoquercitrin reference peak is the S peak, and the relative retention time of each characteristic peak to the S peak is calculated, which should be within ±10% of the specified value. The specified value is: 0.33 (peak 1), 0.82 (peak 2), 1.31 (peak 4), 1.35 (peak 5), 1.85 (peak 6), 2.00 (peak 7), 2.08 (peak 8), 2.14 (peak 9), 2.25 (peak 10).

[0078] The 16 batches of Potentilla discolor medicinal materials were synthesized using the traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2012 version), and the control characteristic chromatogram of the characteristic chromatogram of Potentilla discolor medicinal materials was established, as shown in Figure 18 .

[0079] For the final regulation of Potentilla discolor standard decoction: the test sample chromatogram should present 10 characteristic peaks, and should correspond to the retention time of the 10 characteristic peaks in the reference chromatogram of the control medicinal materials, among which peak 3 should correspond to the retention time of the corresponding reference peak. The peak corresponding to the isoquercitrin reference peak is the S peak, and the relative retention time of each characteristic peak to the S peak is calculated, which should be within ±10% of the specified value. The specified value is: 0.33 (peak 1), 0.82 (peak 2), 1.31 (peak 4), 1.34 (peak 5), 1.85 (peak 6), 1.99 (peak 7), 2.07 (peak 8), 2.13 (peak 9), 2.24 (peak 10).

[0080] The 16 batches of Potentilla discolor standard decoction were synthesized using the traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2012 version), and the control chromatogram of the characteristic chromatogram of Potentilla discolor standard decoction was established, as shown in Figure 16 .

[0081] For the final regulation of the Potentilla discolor formula granules, 10 characteristic peaks should be presented in the test sample chromatogram, and should correspond to the retention time of 10 characteristic peaks in the reference chromatogram of the control medicinal material, wherein peak 3 should correspond to the retention time of the isoquercitrin control substance reference peak. The peak corresponding to the isoquercitrin control substance reference peak is the S peak, and the relative retention time of each characteristic peak to the S peak is calculated, which should be within ±10% of the specified value. The specified value is: 0.33 (peak 1), 0.82 (peak 2), 1.31 (peak 4), 1.34 (peak 5), 1.83 (peak 6), 1.97 (peak 7), 2.05 (peak 8), 2.11 (peak 9), and 2.21 (peak 10).

[0082] The three batches of Potentilla discolor formula granules were synthesized by using the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 version), and the control chromatogram of the characteristic chromatogram of the Potentilla discolor formula granules was established, as shown in Figure 20

[0083] The present application first establishes a characteristic chromatogram method for simultaneously determining Potentilla discolor medicinal materials, slices, standard decoctions and formula granules by using high performance liquid chromatography. The test sample preparation method of the method has the advantages of simple preparation and wide selection range, and the method can achieve effective separation under the conditions of 70% methanol, methanol, 50% ethanol as the extraction solvent and ultrasonic extraction and reflux extraction as the extraction method.

[0084] The present application first establishes a characteristic chromatogram method for simultaneously determining Potentilla discolor medicinal materials, slices, standard decoctions and formula granules by using high performance liquid chromatography, and successfully identifies isoquercitrin, wherein isoquercitrin is first identified.

[0085] The present application has the characteristics of strong operability, convenient analysis and stability, and uses high performance liquid chromatographic fingerprint technology to control the quality of Potentilla discolor medicinal materials, slices, standard decoctions and formula granules, so that the product quality is effectively controlled.

[0086] The present application first establishes a characteristic chromatogram method for simultaneously determining Potentilla discolor medicinal materials, slices, standard decoctions and formula granules by using high performance liquid chromatography, and successfully identifies isoquercitrin, wherein isoquercitrin is first identified. Figure 21

[0087] The present application can analyze the differences and changes of Potentilla discolor medicinal materials, slices, standard decoctions and formula granules, which is beneficial to the overall evaluation of the scientificity and rationality of the related process of Potentilla discolor, and can better control the internal quality of Potentilla discolor medicinal materials, slices, standard decoctions and formula granules, and ensure the clinical efficacy of Potentilla discolor formula granules.

[0088] ​​For a further understanding of the present application, the HPLC characteristic chromatogram establishment method of the medicinal material, decoction piece, standard decoction and formula granule of Potentilla discolor provided by the present application is described in detail below in combination with examples, and the protection scope of the present application is not limited by the following examples.

[0089] Example 1

[0090] 1 Standard Decoction of Potentilla discolor

[0091] The HPLC characteristic chromatogram of the standard decoction of Potentilla discolor is established, which can be used as the overall quality control method of the standard decoction of Potentilla discolor.

[0092] 1.1 Materials, reagents and instruments

[0093] High performance liquid chromatograph: Agilent inifity 1260 type high performance liquid chromatograph, Waters 2695 type high performance liquid chromatograph, Shimadzu LC-20 type high performance liquid chromatograph;

[0094] Electronic balance: ME204E / 02, MS205DM, XP26 (Mettler-Toledo Instruments Co., Ltd.);

[0095] Ultrapure water machine: cell type 1810A (Shanghai Moore Scientific Instruments Co., Ltd.);

[0096] Ultrasonic cleaner: KQ600-DB type (600W, 40KHz; Kunshan Ultrasonic Instrument Co., Ltd.);

[0097] Chromatographic column: Waters C18 (4.6mm×250mm, 5μm);

[0098] Acetonitrile (Sigma Aldrich, chromatographically pure); formic acid (chromatographically pure); water is ultrapure water, and other reagents are analytically pure;

[0099] Isoquercitrin reference substance (China Food and Drug Inspection Research Institute, batch number: 111809-2022055, purity: 96.3%);

[0100] Potentilla discolor reference medicinal material (China Food and Drug Inspection Research Institute, batch number: 121162-201704);

[0101] Standard decoction of Potentilla discolor (prepared by Sichuan New Green Medicine Science and Technology Development Co., Ltd.).

[0102] 1.2 Chromatographic conditions and system suitability test

[0103] The column was packed with octadecylsilane-bonded silica gel (250 mm column length, 4.6 mm inner diameter, 5 μm particle size); acetonitrile was used as mobile phase A, and 0.2% phosphoric acid solution was used as mobile phase B, with gradient elution performed according to the specifications in Table 1; the flow rate was 1.2 ml / min; the column temperature was 30 °C; the detection wavelength was 360 nm; the theoretical plate number, calculated based on the isoquercitrin peak, should not be less than 5000.

[0104] Table 1 Gradient elution data of standard decoction of Agrimonia pilosa

[0105]

[0106]

[0107] 1.3 Preparation of the reference solution

[0108] Take 0.5g of Agrimonia pilosa reference material, place it in a stoppered conical flask, add 50ml of methanol, sonicate (600W power, 40kHz frequency) for 30 minutes, cool, filter, and take the filtrate as the reference solution for the reference material.

[0109] Take an appropriate amount of isoquercitrin standard and add methanol to prepare a solution containing 80 μg per 1 ml, which will be used as the reference solution.

[0110] 1.4 Preparation of the test solution

[0111] Take 0.2g of this product, place it in a stoppered conical flask, add 25ml of methanol, seal tightly, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and collect the filtrate to obtain the product.

[0112] 1.5 Determination Method

[0113] Accurately pipette 10 μl each of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0114] 1.6 Chromatographic conditions and system suitability test

[0115] 1.6.1 Selection of mobile phase

[0116] Based on the experimental conditions outlined above, the separation effects of three different mobile phases were investigated: acetonitrile-0.2% acetic acid, acetonitrile-0.2% phosphoric acid, and acetonitrile-0.2% formic acid. The results are as follows: Figure 1 As shown, Figure 1 The chromatograms of standard decoctions of *Potentilla chinensis* under different mobile phases are shown below. Figure 1 It can be seen that the differences between the chromatographic peaks are not significant under different mobile phase conditions, but the properties of phosphoric acid are relatively stable. Therefore, acetonitrile-0.2% phosphoric acid solution is used as the mobile phase for the determination of the characteristic chromatogram of Agrimonia pilosa standard decoction.

[0117] 1.6.2 Wavelength selection

[0118] Based on the above experimental conditions, the full wavelength scanning of the test solution was carried out by using a diode array detector, and the chromatograms of the test solution at 300 nm, 320 nm, 340 nm, 360 nm, and 380 nm were extracted, respectively, as shown in Figure 2 Figure 2 The chromatograms of the standard decoction of Potentilla discolor at different wavelengths were known from Figure 2 It can be seen that when the detection wavelength is 360 nm, the amount of chromatographic peak information is larger, and the baseline of the chromatogram is more stable, so the detection wavelength is determined as 360 nm.

[0119] 1.6.3 Flow rate investigation

[0120] Based on the above experimental conditions, the flow rates of 0.8 ml / min, 1.0 ml / min, and 1.2 ml / min were investigated, respectively, as shown in Figure 3 Table 2, and Table 3, Figure 3 The chromatograms of the standard decoction of Potentilla discolor at different flow rates were shown in

[0121] Table 2: Flow rate investigation-retention time characteristic peak data table

[0122]

[0123] Table 3: Flow rate investigation-relative retention time characteristic peak data table

[0124]

[0125] The results showed that different flow rates could effectively separate each chromatographic peak, and the temporary flow rate was 1.2 ml / min.

[0126] 1.6.4 Column temperature investigation

[0127] Based on the above experimental conditions, the column temperatures of 25℃, 30℃, and 35℃ were investigated, respectively, as shown in Figure 4 Table 4, and Table 5, Figure 4 The chromatograms of the standard decoction at different column temperatures were shown in

[0128] Table 4: Column temperature investigation-retention time characteristic peak data table

[0129]

[0130] Table 5: Column temperature investigation-relative retention time characteristic peak data table

[0131]

[0132] ​The results show that each chromatographic peak can be effectively separated under the condition of column temperature of 30°C, and the temporary column temperature is 30°C.

[0133] 1.6.5 Delayed Test

[0134] Based on the above proposed experimental conditions, the delayed test was carried out. The results are shown in Figure 5 Figure 5 The chromatogram of the standard decoction of Potentilla discolor for delayed test; it can be seen from Figure 5 that there is basically no chromatographic peak after 60 minutes of the sample, so the sample detection time is set to 60 minutes.

[0135] 1.6.6 System Suitability Test Results

[0136] Octadecylsilane-bonded silica gel is used as the filler (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile is used as the mobile phase A, and 0.2% phosphoric acid solution is used as the mobile phase B, which is gradient eluted according to the following table 6; the flow rate is 1.2 ml per minute; the column temperature is 30°C; the detection wavelength is 360 nm; the theoretical plate number should not be less than 5000 calculated by the peak of isoquercitrin;

[0137] Table 6 Data table of gradient elution of Potentilla discolor standard decoction

[0138] Time (min) Mobile phase A (%) Mobile phase B (%) 0～15 10→14 90→86 15～30 14→17 86→83 30～33 17 17 33～45 17→27 83→73 45～55 27→45 73→55 55～60 45→65 55→35

[0139] 1.7 Preparation of Test Solution

[0140] 1.7.1 Extraction Solvent Investigation

[0141] Take 0.2 g of the product, put it in a conical flask with a plug, and investigate the test product extraction solvents of methanol, 30% methanol, 70% methanol, ethanol, 50% ethanol, and water, each 25 ml, tightly plug, ultrasonic treatment (power 600 W, frequency 40 kHz) for 30 minutes, cool down, shake well, filter, and take the filtrate, i.e. the result is shown in Figure 6 Figure 6 The chromatogram of Potentilla discolor standard decoction under different extraction solvents, it can be seen from Figure 6 that when the extraction solvent is methanol, the peak shape of each characteristic peak is moderate, and the extraction solvent is temporarily set to methanol.

[0142] 1.7.2 Extraction Method Investigation

[0143] Take 0.2 g of the product, put it in a conical flask with a plug, add methanol 25 ml, tightly plug, and investigate the test product extraction methods of reflux and ultrasonic, respectively, with an extraction time of 30 minutes, cool down, shake well, filter, and take the filtrate, i.e. the result is shown in Figure 7 Figure 7 The chromatogram of Potentilla discolor standard decoction under different extraction methods, it can be seen from Figure 7 ​​​As shown in the table, there is little difference in the effect of reflux and ultrasonic extraction on the test sample, but the ultrasonic method is fast and simple, so the test sample extraction method is determined to be ultrasonic extraction.

[0144] 1.7.3 Extraction time investigation

[0145] Take 0.2 g of the product, place it in a conical flask with a plug, add 25 ml of methanol, tightly plug, and ultrasonically treat (power 600 W, frequency 40 kHz) for 15 minutes, 30 minutes, and 45 minutes, respectively, to investigate the test sample extraction time. After cooling, shake well, filter, and take the filtrate, you get it, as shown in the table below. Figure 8 As shown in the table, Figure 8 The chromatograms of the standard decoction of Potentilla discolor under different extraction times are shown in the table below. Figure 8 As can be seen from the table,

[0146] 1.7.4 Investigation of solvent addition amount

[0147] Take 0.2 g of the product, place it in a conical flask with a plug, add 10 ml, 25 ml, and 50 ml of methanol, tightly plug, and ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes. After cooling, shake well, filter, and take the filtrate, you get it, as shown in the table below. Figure 9 As shown in the table, Figure 9 The chromatograms of the standard decoction of Potentilla discolor under different solvent addition amounts are shown in the table below. Figure 9 As can be seen from the table,

[0148] Based on the above, the preparation method of the characteristic chromatogram of the test sample solution of the standard decoction of Potentilla discolor is determined as follows: take 0.2 g of the product, place it in a conical flask with a plug, add 25 ml of methanol, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and take the filtrate, you get it.

[0149] 1.8 Methodology investigation

[0150] 1.8.1 Chromatographic peak identification

[0151] Preparation of test sample solution: Prepare the test sample solution of the standard decoction of Potentilla discolor according to the experimental conditions proposed above.

[0152] Preparation of reference solution: Take 0.5 g of Potentilla discolor control drug, place it in a conical flask with a plug, add 50 ml of methanol, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, cool, filter, and take the filtrate as the reference solution of the control drug.

[0153] Take an appropriate amount of isoquercitrin reference substance, add methanol to prepare a solution containing 80 μg per 1 ml as the reference solution of the control substance.

[0154] Preparation of negative control solution: According to the experimental conditions as above, the negative control solution of standard decoction of P. heterophylla was prepared.

[0155] The characteristic peaks of the standard decoction of P. heterophylla were located as shown in Figure 10-12 Figure 10 The characteristic peak identification spectrum of the standard decoction of P. heterophylla is shown in Figure 11 The spectrum of quercitrin as a control is shown in Figure 12 The spectrum of quercitrin in the standard decoction of P. heterophylla is shown in. It can be seen from the figure that peak 3 is quercitrin. In the following methodology investigation, 10 characteristic peaks in the sample were investigated.

[0156] 1.8.2 Precision test

[0157] The test sample solution of the standard decoction of P. heterophylla was taken, and 10 μl was continuously injected for 6 times according to the proposed experimental method. The retention time and peak area of each characteristic peak were calculated; as shown in Tables 7 and 8.

[0158] Table 7 Precision investigation-retention time data table

[0159]

[0160] Table 8 Precision investigation-peak area data table

[0161]

[0162]

[0163] The results showed that the RSD of the retention time of each characteristic peak was 0.01% to 0.06%, and the RSD of the peak area of the characteristic peak was 0.62% to 1.40%, indicating that the precision of the instrument was good.

[0164] 1.8.3 Reproducibility investigation

[0165] Six portions of the standard decoction of P. heterophylla were accurately weighed, prepared and determined according to the proposed experimental method, and the relative retention time and relative peak area of each characteristic peak were calculated; as shown in Table 9.

[0166] Table 9 Reproducibility investigation-relative retention time characteristic peak data table

[0167]

[0168] The results showed that the RSD of the relative retention time of the 6 samples was 0.00% to 0.08%, indicating that the method had good reproducibility.

[0169] 1.8.4 Intermediate precision investigation

[0170] 1.8.4.1 Investigation of different instruments​

[0171] On the basis of the above experimental conditions, the standard decoction of Potentilla discolor was precisely weighed to prepare the test solution, which was determined on Agilent (1260) and Shimadzu high performance liquid chromatography, and the relative retention time was calculated, as shown in Table 10. Figure 13 Figure 13 Table 10. Data table of relative retention time characteristic peaks of instrument durability investigation

[0172] Table 10. Data table of relative retention time characteristic peaks of instrument durability investigation

[0173]

[0174] The results showed that when the test samples were detected by the above two instruments, the RSD of the relative retention time of each characteristic peak was 0.21% to 4.56%, indicating that the instrument durability was good.

[0175] 1.8.4.2 Investigation of different personnel and time

[0176] On the basis of the above experimental conditions, two samples of the standard decoction of Potentilla discolor were precisely weighed by different personnel (A, B) at different times (T1, T2) to prepare test samples, which were determined and the relative retention time and relative peak area were calculated, as shown in Tables 11-12.

[0177] Table 11. Data table of relative retention time characteristic peaks of personnel and time investigation

[0178]

[0179] Table 12. Data table of relative peak area of personnel and time investigation

[0180]

[0181] The results showed that when the same sample was determined by different personnel at different times, the RSD of the relative retention time of each characteristic peak was 0.00% to 0.06%, indicating that the method was stable.

[0182] 1.8.5 Durability investigation

[0183] 1.8.5.1 Investigation of column durability

[0184] On the basis of the above experimental conditions, the standard decoction of Potentilla discolor was precisely weighed to prepare the test solution, which was determined on Agilent (1260) and Shimadzu high performance liquid chromatography, and the relative retention time was calculated, as shown in Table 10. Figure 14 ​Table 13 Chromatographic column durability investigation - relative retention time data table Figure 14 Chromatograms of the standard decoction of Potentilla discolor under different chromatographic columns;

[0185] Table 13 Chromatographic column durability investigation - relative retention time data table

[0186]

[0187] The results show that, by using the above three chromatographic columns to detect the sample, the RSD of the relative retention time of the characteristic peaks is 0.69% to 6.30%, indicating that the chromatographic column durability is good.

[0188] 1.8.5.2 Stability Investigation

[0189] On the basis of the above proposed experimental conditions, the same test solution was taken, and the retention time was measured at 0 h, 4 h, 8 h, 12 h, 16 h, and 24 h, as shown in Tables 14 and 15.

[0190] Table 14 Stability Investigation - Retention Time Characteristic Peak Data Table

[0191]

[0192] Table 15 Stability Investigation - Peak Area Characteristic Peak Data Table

[0193]

[0194] The results show that the RSD of the retention time of the characteristic peaks is 0.02% to 0.13%, and the RSD of the peak area of the characteristic peaks is 0.24% to 2.77%. The sample solution is stable within 24 hours.

[0195] In summary, the RSD of the relative retention time and peak area of each characteristic peak meets the requirements in the above investigations, and the method is good. The above 10 characteristic peaks are included in the subsequent investigation.

[0196] 1.9 Determination of Characteristic Peaks and Establishment of Control Chromatogram

[0197] The proposed method was used to determine the characteristic chromatograms of 16 batches of samples, and the relative retention time and relative peak area were calculated, as shown in Tables 16 and 17. Figure 15 Figure 15 Table 16 Relative Retention Time Characteristic Peak Data Table of Standard Decoction of Potentilla discolor

[0198] Table 16 Relative Retention Time Characteristic Peak Data Table of Standard Decoction of Potentilla discolor

[0199]

[0200] ​

[0201] Table 17. Relative peak area data table of standard decoction of Herba Pyrrosiae

[0202]

[0203] According to the principle of stable relative retention time and detectable and relatively high peak of each batch sample, a total of 10 peaks with better durability were selected as characteristic peaks. According to the results of methodological investigation and 16 batches of standard decoction verification, the theoretical plate number was tentatively determined to be not less than 5000 according to the calculation of isoquercitrin peak.

[0204] Finally, it is stipulated that 10 characteristic peaks should be present in the test sample chromatogram, and should correspond to the retention time of the 10 characteristic peaks in the reference material chromatogram of the control medicinal material, among which peak 3 should correspond to the retention time of the corresponding control reference peak, and the peak corresponding to the isoquercitrin control reference peak is the S peak. The relative retention time of each characteristic peak to the S peak should be within ±10% of the specified value. The specified value is: 0.33 (peak 1), 0.82 (peak 2), 1.31 (peak 4), 1.34 (peak 5), 1.85 (peak 6), 1.99 (peak 7), 2.07 (peak 8), 2.13 (peak 9), 2.24 (peak 10).

[0205] The similarity evaluation system for traditional Chinese medicine chromatographic fingerprint (2012 version) was used to synthesize 16 batches of standard decoction of Herba Pyrrosiae, and the control chromatogram of the characteristic chromatogram of the standard decoction of Herba Pyrrosiae was established, as shown in Figure 16 Peak 3 (S): isoquercitrin; column: Waters C18 4.6mm x 250mm, 5μm

[0206] Example 2 Herba Pyrrosiae

[0207] Based on the results of the investigation of the characteristic chromatogram of the standard decoction of Herba Pyrrosiae, the high performance liquid chromatographic characteristic chromatogram of Herba Pyrrosiae was established, which can be used as a method for the overall quality control of Herba Pyrrosiae.

[0208] 2.1 Materials, reagents and instruments

[0209] 2.1.1 Experimental instruments and materials

[0210] High performance liquid chromatograph: Agilent 1260 type high performance liquid chromatograph, Shimadzu-20D type high performance liquid chromatograph;

[0211] Electronic balance: ME204E / 02, MS205DU, XP26 (Mettler-Toledo Instruments Co., Ltd.);

[0212] Ultra-pure water machine: cell type 1810A (Shanghai Moore Scientific Instruments Co., Ltd.);

[0213] Ultrasonic cleaner: KQ600-DB type (600W, 40KHz; Kunshan Ultrasonic Instrument Co., Ltd.);

[0214] Chromatographic column: Agilent C18 4.6mmx250mm, 5μm; Waters C18 4.6mmx250mm, 5μm; Kromasil C18 4.6mmx250mm, 5μm.

[0215] 2.1.2 Reagents and reagents

[0216] Acetonitrile (Sigma Aldrich, chromatographically pure); formic acid (chromatographically pure); water is ultrapure water, and other reagents are analytical pure;

[0217] Isoquercitrin reference substance (China Institute for Drug Control, batch number: 111809-2022055, purity: 96.3%);

[0218] Reference material of Potentilla discolor Bunge (China Institute for Drug Control, batch number: 121162-201704).

[0219] 2.2 Chromatographic conditions and system suitability test

[0220] The same as 1.2 Chromatographic conditions and system suitability test.

[0221] 2.3 Preparation of reference solution

[0222] The same as 1.3 Preparation of reference solution.

[0223] 2.4 Preparation of test solution

[0224] Take the powder (pass through No. 3 sieve) 1g, add methanol 25ml to dissolve, ultrasonic treatment for 30 minutes, filter, take the filtrate, as the test solution.

[0225] 2.5 Determination method

[0226] The same as 1.5 Chromatographic conditions and system suitability test.

[0227] 2.6 Determination of characteristic peaks and establishment of reference chromatogram

[0228] The prepared method was used to determine the characteristic chromatogram of 16 batches of samples, and the relative retention time and relative peak area were calculated, as shown in Figure 17 , Table 18, Figure 17 The characteristic chromatogram of Potentilla discolor Bunge of different batches; S1-S15 are XXLS2023076344-XXLS202307648 respectively; S16 is 010613-2307001;

[0229] Table 18 Relative retention time data table of Herba Pyrolae characteristic chromatogram

[0230]

[0231]

[0232] According to the principle of relative retention time stability and each batch sample can be detected and the peak is relatively high, a total of 10 peaks with good repeatability were selected as characteristic peaks. The relative retention time RSD of 10 characteristic peaks of 16 batches of Herba Pyrolae was less than 1.0%.

[0233] Finally, it is stipulated that 10 characteristic peaks should be present in the test sample chromatogram, and should correspond to the retention time of the 10 characteristic peaks in the reference chromatogram of the control drug, among which peak 3 should correspond to the retention time of the isoquercitrin reference peak; the peak corresponding to the isoquercitrin reference peak is S peak, the relative retention time of each characteristic peak to S peak is calculated, which should be within ±10% of the specified value; the specified value is: 0.33 (peak 1), 0.82 (peak 2), 1.31 (peak 4), 1.35 (peak 5), 1.85 (peak 6), 2.00 (peak 7), 2.08 (peak 8), 2.14 (peak 9), 2.25 (peak 10).

[0234] The 16 batches of Herba Pyrolae were synthesized by using Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 version), and the control characteristic chromatogram of Herba Pyrolae characteristic chromatogram was established, as shown in Figure 18 Peak 3 (S): isoquercitrin, chromatographic column: Waters C18 4.6mm×250mm, 5μm.

[0235] Example 3 Herba Pyrolae formula granules

[0236] A high performance liquid characteristic chromatogram of Herba Pyrolae formula granules was established, which can be used as a whole quality control method for Herba Pyrolae formula granules.

[0237] 3.1 Materials, reagents and instruments

[0238] The same as 1.1. Materials, reagents and instruments.

[0239] Herba Pyrolae formula granules (prepared by Sichuan Xinglve Pharmaceutical Science and Technology Development Co., Ltd.).

[0240] 3.2 Chromatographic conditions and system suitability test

[0241] The same as 1.2 Chromatographic conditions and system suitability test.

[0242] 3.3 Preparation of reference solution

[0243] The same as 1.3 Preparation of reference solution.

[0244] 3.4 Preparation of Test Solution

[0245] Take 0.2 g of the product, grind finely, and place in a conical flask with a stopper. Add 25 ml of methanol, and ultrasonically treat for 30 minutes. Allow to cool, shake well, and filter. Take the filtrate, and you have obtained it.

[0246] 3.5 Assay Method

[0247] The same as 1.5 Chromatographic Conditions and System Suitability Test.

[0248] 3.6 Determination of Characteristic Peaks and Establishment of Reference Chromatogram

[0249] The proposed method was used to determine the characteristic chromatograms of 3 batches of samples, and the relative retention times and relative peak areas were calculated. As shown in Table 19, Figure 19 , Table 19 shows the chromatograms of different batches of Baihuacaoganli Granules; Figure 19

[0250] Table 19 Relative Retention Time Characteristic Peak Data Table of Baihuacaoganli Granules

[0251]

[0252]

[0253] According to the principles of stable relative retention time and detectable peaks in each batch of samples, a total of 10 peaks with relatively high peaks were selected as characteristic peaks, including peak 1, peak 2, peak 3, peak 4, peak 5, peak 6, peak 7, peak 8, peak 9, and peak 10. According to the results of the methodological investigation and the verification results of 3 batches of granules, the theoretical plate number was tentatively determined to be not less than 5000 based on the calculation of isoquercitrin peak.

[0254] The relative retention times of each characteristic peak are stable and within the range of the average value ± 10%, so the relative retention time of each peak is tentatively set to be within the range of ± 10%.

[0255] The final specification is that the test sample chromatogram should present 10 characteristic peaks, which should correspond to the retention times of the 10 characteristic peaks in the reference chromatogram of the control medicinal material, and peak 3 should correspond to the retention time of the isoquercitrin reference peak. The peak corresponding to the isoquercitrin reference peak is the S peak, and the relative retention times of each characteristic peak and the S peak should be within the range of ± 10% of the specified value. The specified value is 0.33 (peak 1), 0.82 (peak 2), 1.31 (peak 4), 1.34 (peak 5), 1.83 (peak 6), 1.97 (peak 7), 2.05 (peak 8), 2.11 (peak 9), and 2.21 (peak 10).

[0256] ​The three batches of P. chinensis formula granules were synthesized by using the similarity evaluation system of traditional Chinese medicine chromatographic fingerprint (2012 version), and a control chromatogram of P. chinensis formula granule characteristic chromatogram was established, as shown in Figure 20 . Figure 20 The control chromatogram of P. chinensis formula granule characteristic chromatogram is shown in

[0257] The above examples are only used to help understand the method of the present application and its core idea. It should be noted that for those skilled in the art, without departing from the principles of the present application, the present application can be improved and modified, and these improvements and modifications also fall within the protection scope of the claims of the present application.

[0258] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present application. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to the embodiments shown herein, but will conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for establishing HPLC characteristic chromatogram of standard decoction, pieces, medicinal materials and formula granules of Pyrola rotundifolia, comprising the following steps: S1) extracting Pyrola rotundifolia raw materials with a solvent to obtain a test solution; the solvent is methanol, and the extraction method is ultrasonic extraction; S2) detecting the test solution by high performance liquid chromatography to obtain the HPLC characteristic chromatogram of Pyrola rotundifolia raw materials; The conditions of the high performance liquid chromatography are as follows: the chromatographic column is C18 column, the mobile phase A is acetonitrile, the mobile phase B is 0.2% phosphoric acid solution, 0.2% acetic acid solution or 0.2% formic acid, and gradient elution; the detection wavelength of the mobile phase is 360 nm; The Pyrola rotundifolia raw materials are standard decoction, pieces, medicinal materials or formula granules of Pyrola rotundifolia; The gradient elution is specifically as follows: 0-15 min, A phase: 10-14%, B phase: 90-86%; 15-30 min, A phase: 14-17%, B phase: 86-83%; 30-33 min, A phase: 17%, B phase: 83%; 33-45 min, A phase: 17-27%, B phase: 83-73%; 45-55 min, A phase: 27-45%, B phase: 73-55%; 55-60 min, A phase: 45-65%, B phase: 55-35%.

2. The establishment method according to claim 1, characterized by, It also includes preparation of reference solution of control medicinal materials and reference solution of control; The preparation of the reference solution of control medicinal materials is specifically as follows: dissolving Pyrola rotundifolia control medicinal materials with methanol and obtaining the reference solution of control medicinal materials after ultrasonic treatment; The preparation of the reference solution of control is specifically as follows: dissolving isoquercitrin with methanol to obtain the reference solution of control; The reference solution of control and the reference solution of control medicinal materials are detected by high performance liquid chromatography to obtain the chromatograms of the reference solution of control and the reference solution of control medicinal materials, respectively; The components of the HPLC characteristic chromatogram of Pyrola rotundifolia medicinal materials, standard decoction and formula granules are qualitatively determined according to the chromatograms of the reference solution of control and the reference solution of control medicinal materials.

3. The establishment method according to claim 1 or 2, characterized by, The extraction time is 15-45 min; the ratio of the Pyrola rotundifolia raw materials to the solvent is 0.2 g: (20-30) ml.

4. The establishment method according to claim 1 or 2, characterized by, The flow rate of the mobile phase is 0.8-1.2 ml / min.

5. The establishment method according to claim 1 or 2, characterized by, The column temperature of the chromatographic column is 25-35℃, the column length of the chromatographic column is 250 mm, the inner diameter of the chromatographic column is 4.6 mm, and the particle size is 5 μm.

6. The establishment method according to claim 1 or 2, characterized by, The similarity of the HPLC characteristic chromatogram of Pyrola rotundifolia medicinal materials, standard decoction and formula granules is evaluated by using traditional Chinese medicine chromatographic fingerprint similarity evaluation system to obtain the HPLC standard characteristic chromatogram of Pyrola rotundifolia medicinal materials, standard decoction and formula granules composed of 10 characteristic peaks, wherein peak 3 is isoquercitrin.

7. The establishment method according to claim 6, characterized by, For the standard decoction of Pyrola rotundifolia, the relative retention time of each characteristic peak to the S peak of isoquercitrin reference peak is calculated, and the relative retention time should be within ±10% of the specified value, and the specified value is as follows: 0.33 (peak 1), 0.82 (peak 2), 1.31 (peak 4), 1.34 (peak 5), 1.85 (peak 6), 1.99 (peak 7), 2.07 (peak 8), 2.13 (peak 9), 2.24 (peak 10).

8. The establishment method according to claim 6, characterized by, For the herb of Potentilla discolor, taking the reference peak of isoquercitrin as S peak, the relative retention time of each characteristic peak to S peak should be within ±10% of the specified value, which is: 0.33 (peak 1), 0.82 (peak 2), 1.31 (peak 4), 1.35 (peak 5), 1.85 (peak 6), 2.00 (peak 7), 2.08 (peak 8), 2.14 (peak 9), 2.25 (peak 10).

9. The establishment method according to claim 6, wherein, For the formula granules of Potentilla discolor, taking the reference peak of isoquercitrin as S peak, the relative retention time of each characteristic peak to S peak should be within ±10% of the specified value, which is: 0.33 (peak 1), 0.82 (peak 2), 1.31 (peak 4), 1.34 (peak 5), 1.83 (peak 6), 1.97 (peak 7), 2.05 (peak 8), 2.11 (peak 9), 2.21 (peak 10).

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