Construction Method and Application of Fingerprint of Shiduqing Preparation

Abstract

The present invention relates to a method for constructing and applying a fingerprint of a Shidouqing preparation, which comprises the following steps: extracting the Shidouqing preparation with an extraction solvent to prepare a test solution; performing high performance liquid chromatography (HPLC) detection on the test solution to construct a fingerprint of the Shidouqing preparation; the conditions for the HPLC detection include: mobile phase A is an aqueous phosphoric acid solution, mobile phase B is acetonitrile, and gradient elution is adopted. The method for constructing the fingerprint of the Shidouqing preparation is simple, reliable, highly objective, and has good precision, repeatability, specificity and stability. Further, the characteristic peaks in the fingerprint of the Shidouqing preparation have good resolution, including 16 common peaks, and 4 characteristic peaks such as ferulic acid, astilbin, baicalin, and salvianolic acid B are identified, which can comprehensively reflect the main chemical composition characteristics of the Shidouqing preparation and can provide an overall quality control basis for the quality standard of the Shidouqing preparation.

Description

Technical Field

[0001] This application relates to the technical field of traditional Chinese medicine preparation detection, and particularly to a method for constructing and applying a fingerprint of Shiduqing preparation. Background Art

[0002] Shiduqing preparation is a pure traditional Chinese medicine preparation formulated by selecting a variety of traditional Chinese medicines under the guidance of traditional Chinese medicine theory. It is a mature traditional Chinese medicine formula for skin diseases and a first-line drug for skin allergies that treats both the symptoms and root causes in traditional Chinese medicine. The prescription of Shiduqing preparation includes nine herbs such as Rehmannia glutinosa, Angelica sinensis, Salvia miltiorrhiza, Sophora flavescens, Cryptotympana pustulata, Scutellaria baicalensis, Dictamnus dasycarpus, Smilax glabra, and Glycyrrhiza uralensis, which has the effects of nourishing blood and moistening dryness, resolving dampness and detoxifying, dispelling wind and relieving itching, and is mainly used for those with skin pruritus belonging to the syndrome of blood deficiency and dampness accumulation in the skin. The existing legal quality control methods for Shiduqing preparation are mainly microscopic identification, thin-layer identification, and single content determination method of matrine, which have limitations for the comprehensive quality control of products.

[0003] The traditional Chinese medicine fingerprint is a comprehensive and quantifiable identification method, which is based on the systematic research of the chemical components of traditional Chinese medicine and is mainly used to evaluate the authenticity, superiority, and stability of the quality of traditional Chinese medicine materials and semi-finished traditional Chinese medicine preparations. Traditional Chinese medicines are all multi-component complex systems, so the detection methods that can provide rich identification information and are suitable for them should be used to evaluate their quality. The traditional Chinese medicine fingerprint can comprehensively reflect the types and quantities of chemical components contained in traditional Chinese medicine, and can describe and evaluate the quality of drugs as a whole. Furthermore, it can reflect the quality of traditional Chinese medicine and the overall curative effect reflected by the use of traditional Chinese medicine. As a traditional Chinese medicine preparation, the fingerprint of Shiduqing preparation can reflect its unique chemical composition and pharmacodynamic characteristics, and has wide application value in the quality control of traditional Chinese medicine. Summary of the Invention

[0004] Based on this, the objectives of this application include providing a method for constructing and applying a fingerprint of Shiduqing preparation.

[0005] The technical solution of this application is as follows:

[0006] In one aspect of the present invention, a method for constructing a fingerprint of Shiduqing preparation is provided, including the following steps:

[0007] Use an extraction solvent to extract Shiduqing preparation to prepare a test solution.

[0008] Perform high performance liquid chromatography detection on the test solution to construct a fingerprint of Shiduqing preparation.

[0009] The conditions for the high performance liquid chromatography detection include: mobile phase A is an aqueous phosphoric acid solution, mobile phase B is acetonitrile, and gradient elution is used.

[0010] In one embodiment, the program for the gradient elution includes:

[0011] From 0 to 15 minutes, the volume percentage of mobile phase B increases from 5% to 20%;

[0012] From 15 minutes to 25 minutes, the volume percentage of mobile phase B increases from 20% to 21%;

[0013] From 25 minutes to 35 minutes, the volume percentage of mobile phase B increases from 21% to 28%;

[0014] From 35 minutes to 40 minutes, the volume percentage of mobile phase B increases from 28% to 30%;

[0015] From 40 minutes to 50 minutes, the volume percentage of mobile phase B increases from 30% to 48%;

[0016] From 50 minutes to 60 minutes, the volume percentage of mobile phase B increases from 48% to 55%;

[0017] From 60 minutes to 65 minutes, the volume percentage of mobile phase B is maintained at 55%.

[0018] In one embodiment, in the phosphoric acid aqueous solution, the volume percentage content of phosphoric acid is 0.05% - 0.2%.

[0019] In one embodiment, the high performance liquid chromatography detection satisfies at least one of the following conditions:

[0020] (1) The detection wavelength is 200 nm - 300 nm;

[0021] (2) The flow rate is 0.5 mL / min - 2 mL / min;

[0022] (3) The column temperature is 25°C - 35°C;

[0023] (4) The injection volume is 1 μL - 10 μL.

[0024] In one embodiment, the extraction step satisfies at least one of the following conditions:

[0025] (1) The extraction method is ultrasonic extraction or reflux extraction;

[0026] (2) The extraction time is 15 min - 60 min;

[0027] (3) The extraction solvent includes methanol or methanol aqueous solution; Optionally, in the methanol aqueous solution, the volume percentage of methanol is 0.1% - 100% methanol aqueous solution.

[0028] In one embodiment, the method for constructing the fingerprint of the Shidutang preparation further includes the following steps:

[0029] Dissolve the reference substance using a dissolution reagent to prepare a mixed reference substance solution;

[0030] Perform high performance liquid chromatography detection on the mixed reference substance solution to obtain a reference chromatogram;

[0031] Identify the common peaks of the fingerprint chromatogram of the Shiduqing preparation based on the reference chromatogram;

[0032] Among them, the reference substance includes one or more of ferulic acid, astilbin, baicalin, salvianolic acid B, matrine, oxymatrine, tanshinone IIA, liquiritin, ammonium glycyrrhizinate, acteoside, and rehmannioside D.

[0033] In one embodiment, the Shiduqing preparation includes Shiduqing tablets, Shiduqing capsules, or Shiduqing granules.

[0034] In one embodiment, the dissolution reagent includes methanol or an aqueous methanol solution; optionally, in the aqueous methanol solution, the volume percentage of methanol is 0.1% - 100%.

[0035] In one embodiment, the fingerprint chromatogram of the Shiduqing preparation contains 16 common peaks. Among them, the 4 identified characteristic peaks include: peak 6 is ferulic acid, peak 7 is astilbin, peak 10 is baicalin, and peak 11 is salvianolic acid B.

[0036] Another aspect of the present invention provides the application of the method for constructing the fingerprint chromatogram of the Shiduqing preparation as described above in the quality control of the Shiduqing preparation.

[0037] Compared with the prior art, the present application has the following beneficial effects:

[0038] The present application detects and analyzes the Shiduqing preparation under specific high performance liquid chromatography conditions to construct a fingerprint chromatogram of the Shiduqing preparation containing multiple characteristic peaks. The method for constructing the fingerprint chromatogram of the Shiduqing preparation is simple, reliable, highly objective, and has good precision, repeatability, specificity, and stability.

[0039] Furthermore, the separation degree of each characteristic peak in the fingerprint chromatogram of the Shiduqing preparation is good, containing 16 common peaks, and 4 characteristic peaks such as ferulic acid, astilbin, baicalin, and salvianolic acid B are identified, which can comprehensively reflect the main chemical component characteristics of the Shiduqing preparation; in addition, the present application also examines the medicinal material attribution of each characteristic peak, and the detection results of different batches of Shiduqing tablets have a high similarity. The generated reference fingerprint chromatogram can be used for the overall similarity evaluation of the material basis of Shiduqing tablets, and can provide an overall quality control basis for the quality standard of the Shiduqing preparation. Description of the Drawings

[0040] To more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present application. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0041] Figure 1 For the HPLC superimposed fingerprint and the reference fingerprint R of Shiduqing tablets in Example 1.

[0042] Figure 2 For the high performance liquid chromatography detection chart of the mixed reference solution in Example 1.

[0043] Figure 3 For the high performance liquid chromatography detection chart of the attribution of the characteristic peaks of the fingerprint of Shiduqing tablets in Example 1 to the medicinal materials. Among them, A: Shiduqing tablets; B: Rehmannia glutinosa; C: Scutellaria baicalensis; D: Salvia miltiorrhiza; E: Glycyrrhiza uralensis; F: Angelica sinensis; G: Sophora flavescens; H: Cryptotympana pustulata; I: Smilax glabra.

[0044] Figure 4 For the high performance liquid chromatography detection chart of Shiduqing tablets, the mixed reference solution and the blank test solution in Example 1. Among them, A: Shiduqing tablets; B: mixed reference solution; C: blank solvent.

[0045] Figure 5 For the high performance liquid chromatography detection chart of the intermediate precision of the instrument for the fingerprint of Shiduqing tablets in Example 1. Among them, A: liquid phase equipment J-20-C-008; B: liquid phase equipment J-20-C-093.

[0046] Figure 6 For the high performance liquid chromatography detection chart of the intermediate precision of the chromatographic column for the fingerprint of Shiduqing tablets in Example 1. A: chromatographic column JZ-SY-C002; B: chromatographic column QC-C18-24-0401; C: chromatographic column QC-C18-22-0604. Specific Embodiments

[0047] The following further elaborates on the present application in conjunction with specific embodiments. The present application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosed content of the present application more thorough and comprehensive.

[0048] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which the present application belongs. The terms used in the description of the present application in this specification are only for the purpose of describing specific embodiments and are not intended to limit the present application.

[0049] Unless otherwise specified or there is a contradiction, the terms or phrases used in this document have the following meanings:

[0050] In this document, "one or more" refers to any one, any two, or any two or more of the listed items.

[0051] In this document, terms such as "further", "even further", "especially", etc. are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0052] In this application, when it comes to numerical ranges, unless otherwise specified, the above numerical ranges are considered continuous and include the minimum and maximum values of the range, as well as every value between such minimum and maximum values. Further, when the range refers to integers, it includes every integer between the minimum and maximum values of the range. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be combined. In other words, unless otherwise specified, all ranges disclosed in this document should be understood to include any and all sub-ranges subsumed therein.

[0053] This application only specifically discloses some numerical ranges. However, any lower limit can be combined with any upper limit to form a range not explicitly recited; and any lower limit can be combined with other lower limits to form a range not explicitly recited, and similarly any upper limit can be combined with any other upper limit to form a range not explicitly recited. In addition, each individually disclosed point or single numerical value itself can be used as a lower limit or upper limit and combined with any other point or single numerical value or combined with other lower limits or upper limits to form a range not explicitly recited. The use of numerical ranges expressed by endpoints includes all numbers within the range and any range within the range. For example, 1 to 5 includes 1, 1.1, 1.3, 1.5, 2, 2.75, 3, 3.80, 4, and 5, etc.

[0054] In this application, for the percentage content involved, unless otherwise specified, it refers to the mass percentage for solid-liquid mixtures and solid-solid mixtures, and the volume percentage for liquid-liquid mixtures.

[0055] In this application, for the percentage concentration involved, unless otherwise specified, it refers to the final concentration. The final concentration refers to the proportion of the added component in the system after adding the component.

[0056] The temperature parameters in this application, unless otherwise specified, allow for both constant temperature treatment and treatment within a certain temperature range. The constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. Fluctuations within ranges such as ±5°C, ±2°C, ±1°C, ±0.5°C, ±0.4°C, ±0.3°C, ±0.2°C, ±0.1°C are allowed. Normal temperature or room temperature in this application refers to the situation where no temperature control operation is applied, generally referring to 4°C to 35°C, preferably 20 ± 5°C.

[0057] In this application, among the technical features described in an open-ended manner, it includes both a closed technical solution composed of the listed features and an open technical solution containing the listed features.

[0058] Traditional Chinese medicine fingerprint is a comprehensive and quantifiable identification method. It is based on the systematic research of the chemical components of traditional Chinese medicine and is mainly used to evaluate the authenticity, superiority, and stability of the quality of traditional Chinese medicine materials and semi-finished traditional Chinese medicine preparations. Traditional Chinese medicines are all multi-component complex systems. Therefore, to evaluate their quality, a detection method that is suitable for them and can provide rich identification information should be used. The traditional Chinese medicine fingerprint can comprehensively reflect the types and quantities of chemical components contained in traditional Chinese medicine, and can provide an overall description and evaluation of the drug quality. Furthermore, it can reflect the quality of traditional Chinese medicine and the overall curative effect reflected by traditional Chinese medicine in clinical use. As a traditional Chinese medicine preparation, the fingerprint of Shiduqing preparation can reflect its unique chemical composition and pharmacodynamic characteristics, and has wide application value in the quality control of traditional Chinese medicine.

[0059] Based on this, in one aspect of the present invention, a method for constructing a fingerprint of Shiduqing preparation is provided, including the following steps:

[0060] Extract the Shiduqing preparation with an extraction solvent to prepare a test solution;

[0061] Perform high performance liquid chromatography detection on the test solution to construct a fingerprint of the Shiduqing preparation;

[0062] The conditions for the high performance liquid chromatography detection include: mobile phase A is an aqueous phosphoric acid solution, mobile phase B is acetonitrile, and gradient elution is used.

[0063] In some examples, the gradient elution program includes:

[0064] 0 - 15 min, the volume percentage of mobile phase B increases from 5% to 20%;

[0065] 15 min - 25 min, the volume percentage of mobile phase B increases from 20% to 21%;

[0066] 25 min - 35 min, the volume percentage of mobile phase B increases from 21% to 28%;

[0067] From 35 min to 40 min, the volume percentage of mobile phase B increases from 28% to 30%.

[0068] From 40 min to 50 min, the volume percentage of mobile phase B increases from 30% to 48%.

[0069] From 50 min to 60 min, the volume percentage of mobile phase B increases from 48% to 55%.

[0070] From 60 min to 65 min, the volume percentage of mobile phase B remains at 55%.

[0071] In some examples, in the phosphoric acid aqueous solution, the volume percentage of phosphoric acid is 0.05% - 0.2%. It can be understood that the volume percentage of phosphoric acid in the phosphoric acid aqueous solution includes but is not limited to 0.05%, 0.1%, 0.15%, 0.2%.

[0072] In some examples, the conditions for high performance liquid chromatography detection include: the detection wavelength is 200 nm - 300 nm. It can be understood that the detection wavelength of the high performance liquid chromatography includes but is not limited to 200 nm, 210 nm, 220 nm, 230 nm, 240 nm, 250 nm, 260 nm, 270 nm, 280 nm, 290 nm, 300 nm.

[0073] In some examples, the conditions for high performance liquid chromatography detection include: the flow rate is 0.5 mL / min - 2 mL / min. It can be understood that the flow rate for high performance liquid chromatography detection includes but is not limited to 0.5 mL / min, 0.6 mL / min, 0.7 mL / min, 0.8 mL / min, 0.9 mL / min, 1 mL / min, 1.1 mL / min, 1.2 mL / min, 1.3 mL / min, 1.4 mL / min, 1.5 mL / min, 1.6 mL / min, 1.7 mL / min, 1.8 mL / min, 1.9 mL / min, 2 mL / min.

[0074] In some examples, the conditions for high performance liquid chromatography detection include: the column temperature is 25°C - 35°C. It can be understood that the column temperature for high performance liquid chromatography detection includes but is not limited to 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C.

[0075] In some examples, the conditions for high performance liquid chromatography detection include: the injection volume is 1 μL - 10 μL. It can be understood that the injection volume for high performance liquid chromatography detection includes but is not limited to 1 μL, 3 μL, 5 μL, 7 μL, 9 μL, 10 μL.

[0076] In some of these examples, the conditions for the extraction step include: the extraction method is ultrasonic extraction or reflux extraction.

[0077] In some of these examples, the conditions for the extraction step include: the extraction time is 15 min - 60 min. It can be understood that the extraction time includes but is not limited to 15 min, 20 min, 30 min, 40 min, 45 min, 50 min, 60 min.

[0078] In some of these examples, the conditions for the extraction step include: the extraction solvent includes methanol or an aqueous methanol solution. Optionally, in the aqueous methanol solution, the volume percentage of methanol is 0.1% - 100%.

[0079] In some of these examples, the method for constructing the fingerprint of the Shiduting preparation further includes the following steps:

[0080] Dissolve the reference substance using a dissolution reagent to prepare a mixed reference substance solution;

[0081] Perform high performance liquid chromatography detection on the mixed reference substance solution to obtain a reference chromatogram;

[0082] Identify the common peaks of the chromatographic peaks of the fingerprint of the Shiduting preparation according to the reference chromatogram;

[0083] Among them, the reference substance includes one or more of ferulic acid, astilbin, baicalin, salvianolic acid B, matrine, oxymatrine, tanshinone IIA, liquiritin, ammonium glycyrrhizinate, acteoside, and rehmannioside D.

[0084] In some of these examples, the Shiduting preparation includes Shiduting tablets, Shiduting capsules, or Shiduting granules. Further, the Shiduting preparation is Shiduting tablets.

[0085] In one of the embodiments, the dissolution reagent includes methanol or an aqueous methanol solution; optionally, in the aqueous methanol solution, the volume percentage of methanol is 0.1% - 100%.

[0086] In some of these examples, the fingerprint of the Shiduting preparation contains 16 common peaks, among which, the 4 identified characteristic peaks include: peak 6 is ferulic acid, peak 7 is astilbin, peak 10 is baicalin, and peak 11 is salvianolic acid B.

[0087] Another aspect of the present invention provides the application of the method for constructing the fingerprint of the Shiduting preparation as described above in the quality control of the Shiduting preparation.

[0088] The following is a further illustration in conjunction with specific embodiments. For the raw materials involved in the following specific embodiments, unless otherwise specified, they can all be sourced commercially; for the instruments used, unless otherwise specified, they can all be sourced commercially; for the processes involved, unless otherwise specified, they are all conventional choices of those skilled in the art.

[0089] Example 1

[0090] I. Materials and Methods

[0091] Some of the instruments and materials used in the example are as follows:

[0092] Testing instruments: electronic analytical balance, high performance liquid chromatography, ultrasonic cleaner.

[0093] Test samples: 19 batches of Shiduqing tablets used in the example are from Guangzhou Nuojin Pharmaceutical Co., Ltd., with batch numbers 325B004, 3259007, 3254005, 3254003, 3259001, 4252003, 4252002, 3257006, 325A010, 3257009, 325A010, 3257009, 325A004, 325B005, 3259008, 4251010, 325C002, 4252005, 325A003.

[0094] The preparation method of the test sample solution is as follows: accurately weigh the Shiduqing tablets, grind them finely, take 2 g, add 40 mL of methanol, and ultrasonicate for 45 min. Cool to room temperature, weigh again, make up the lost mass with methanol, shake well, filter through a 0.45 μm microporous membrane, and collect the subsequent filtrate to obtain the solution.

[0095] Reference substances include ferulic acid, astilbin, baicalin, salvianolic acid B, matrine, oxymatrine, tanshinone IIA, liquiritin, ammonium glycyrrhizinate, acteoside, and rehmannioside D.

[0096] The preparation method of the mixed reference substance solution is as follows: take appropriate amounts of ferulic acid reference substance, astilbin reference substance, baicalin reference substance, and salvianolic acid B reference substance, accurately weigh them, and make a mixed solution containing 100 μg of ferulic acid, 100 μg of astilbin, 100 μg of baicalin, and 100 μg of salvianolic acid B in 1 mL of methanol to obtain the solution.

[0097] Medicinal materials include: rehmannia root, scutellaria root, salvia root, licorice root, angelica root, sophora root, cicada slough, and glabrous greenbrier rhizome;

[0098] The preparation method of the medicinal material solution is as follows: According to the proportion of the standard prescription, take 2.2 g of Rehmannia glutinosa, 0.4 g of Scutellaria baicalensis, 1.0 g of Salvia miltiorrhiza, 0.7 g of Glycyrrhiza uralensis, 1.7 g of Angelica sinensis, 1.7 g of Sophora flavescens, 0.7 g of Cryptotympana pustulata, and 0.4 g of Smilax glabra respectively, add 40 mL of methanol to each, and ultrasonicate for 45 min. Cool to room temperature, weigh again, make up the lost mass with methanol, shake well, filter through a 0.45 μm microporous membrane, and collect the subsequent filtrate, which is the obtained solution.

[0099] The chromatographic conditions are shown in Table 1 below:

[0100] Table 1

[0101]

[0102] II. Establishment of fingerprint and identification of common peaks

[0103] Prepare the test solution for 19 batches of Shiduqing tablets according to the above-mentioned preparation method of the test solution. Inject the test solution, the medicinal material solution, and the mixed reference substance solution for injection analysis under the above-mentioned chromatographic conditions respectively. The results are as shown in Figure 1 and Figure 2 By analyzing the fingerprint of the existing 19 batches of Shiduqing tablets, find their common characteristic peaks. Import the chromatograms of 19 batches of Shiduqing tablets into the "Similarity Evaluation System for Traditional Chinese Medicine Fingerprints (2012 Edition)", use the chromatogram of sample S11 as the reference chromatogram, automatically match and generate the superimposed fingerprint in the form of all-spectrum peaks, and generate the reference fingerprint R by the average method. Identify the common peaks by comparing with the chromatogram of the mixed reference substance solution.

[0104] The results show that through the fingerprint analysis of Shiduqing tablets and their negative samples, 16 common peaks are matched in the chromatograms of 19 batches of Shiduqing tablets. By comparing with the chromatogram of the mixed reference substance solution, 4 characteristic peaks are identified, namely ferulic acid (peak No. 6), astilbin (peak No. 7), baicalin (peak No. 10), and salvianolic acid B (peak No. 11) in sequence.

[0105] III. Similarity analysis of fingerprint

[0106] Through chromatogram comparison, determine 16 characteristic peaks with good resolution and stable peak emergence in each batch of fingerprint. And use the "Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints (2012 Edition)" to generate the reference fingerprint according to these 16 peaks and conduct similarity analysis. The results are shown in Table 2.

[0107] Table 2 Similarity evaluation results of 19 batches of Shiduqing tablets

[0108]

[0109] The results showed that the similarity of the batch with the lowest similarity was 0.998, and the rest were all above 0.999. This indicates that the similarities of the Shiduqing tablets in each batch are relatively high, and the generated reference fingerprint can be used for the overall similarity evaluation of the material basis of Shiduqing tablets.

[0110] IV. Attribution of Single Herbs to Each Characteristic Peak

[0111] By analyzing the fingerprint of Shiduqing tablets and each single herb under the above chromatographic conditions respectively, the attribution of each characteristic peak of Shiduqing tablets to herbs was investigated. The results are as Figure 3 shown in Table 3.

[0112] Table 3 Attribution of Characteristic Peaks in the Fingerprint of 19 Batches of Shiduqing Tablets to Herbs

[0113]

[0114] V. Methodology Validation

[0115] 5.1 Specificity Investigation

[0116] Precisely pipette 5 μL each of the test solution of Shiduqing tablets, the mixed reference substance solution, and the blank solvent (0 - 100% methanol aqueous solution), inject them into the liquid chromatograph, and determine according to the chromatographic conditions described in Table 1 to check whether there is interference from the extraction solvent. The results are as Figure 4 shown.

[0117] The results showed that the blank solvent had basically no absorption under the chromatographic conditions and had no interference on the chromatographic analysis of the test sample, indicating that the established method has good specificity.

[0118] 5.2 Repeatability Investigation

[0119] Take 6 portions of the same batch of Shiduqing tablet samples, weigh them, and prepare the test solutions, and perform injection analysis under the optimal chromatographic conditions. Taking the 10th peak as the reference peak, calculate the RSD values of the relative peak areas and relative retention times of each common peak. The results are shown in Table 4 and Table 5.

[0120] Table 4 Relative Peak Areas in the Repeatability Investigation of the Fingerprint of Shiduqing Tablets

[0121]

[0122] Table 5 Relative Retention Times in the Repeatability Investigation of the Fingerprint of Shiduqing Tablets

[0123]

[0124] The results showed that the RSDs of the relative peak areas of each common peak were all less than 3.48% (n = 6), and the RSDs of the relative retention times were all less than 0.03% (n = 6), indicating that the method has good repeatability.

[0125] 5.3 Precision Investigation

[0126] Take the test sample of Shiduqing Tablets, inject it continuously for 6 times, and determine it under the optimal chromatographic conditions. Taking the 10th peak as the reference peak, calculate the RSD values of the relative peak areas of each common peak. The results are shown in Table 6 and Table 7.

[0127] Table 6 Relative Peak Areas in the Precision Investigation of the Fingerprint of Shiduqing Tablets

[0128]

[0129] Table 7 Relative Retention Times in the Precision Investigation of the Fingerprint of Shiduqing Tablets

[0130]

[0131] The results show that the RSDs of the relative peak areas of each common peak are all less than 2.99% (n = 6), and the RSDs of the relative retention times are all less than 0.05% (n = 6), indicating good instrument precision.

[0132] 5.4 Stability Investigation

[0133] The test samples of Shiduqing Tablets were determined under the optimal chromatographic conditions at 0, 6h, 12h, 18h, 24h, 30h, and 36h after preparation respectively. Taking the 10th peak as the reference peak, calculate the RSD values of the peak areas. The results are shown in Table 8 and Table 9.

[0134] Table 8 Relative Peak Areas in the Stability Investigation of the Fingerprint of Shiduqing Tablets

[0135]

[0136] Table 9 Relative Retention Times in the Stability Investigation of the Fingerprint of Shiduqing Tablets

[0137]

[0138] The results show that the RSDs of the relative peak areas of each common peak are all less than 3.58% (n = 7), and the RSDs of the relative retention times are all less than 0.10% (n = 7), indicating good stability of this method.

[0139] 5.5 Intermediate Precision Investigation

[0140] 5.5.1 Intermediate Precision Investigation of Liquid Phase

[0141] Currently, the main liquid phase in the laboratory is Thermo Fisher U3000 (equipment numbers: J-20-C-093, J-20-C-008). Prepare 1 test sample of Shiduqing Tablets and determine its fingerprint on different liquid chromatographs, and use the Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints (2012 Edition) for similarity analysis. The results are shown in Table 10 and Figure 5 .

[0142] Table 10 Similarity Evaluation of the Intermediate Precision of the Fingerprint of Shiduqing Tablets by Instrument

[0143]

[0144] The results showed that the similarity of the fingerprint of Shiduqing tablets measured by liquid chromatography with different numbers reached 0.999 or above, indicating good intermediate precision of the liquid chromatography.

[0145] 5.5.2 Intermediate Precision of Chromatographic Column

[0146] Prepare 1 portion of the test sample of Shiduqing tablets, and use three ODS C18 (4.6×250mm, 5μm) chromatographic columns (department numbers: JZ-SY-C002, QC-C18-24-0401, QC-C18-22-0604) to measure the fingerprint, and use the "Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints (2012 Edition)" for similarity analysis. The results are shown in Table 11 and Figure 6 .

[0147] Table 11 Similarity Evaluation of the Intermediate Precision of the Fingerprint of Shiduqing Tablets by Instrument

[0148]

[0149] The results showed that the similarity of the fingerprint of Shiduqing tablets measured by liquid chromatography with different numbers reached 0.999 or above, indicating good intermediate precision of the instrument.

[0150] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope described in this specification.

[0151] The above-described embodiments only represent several implementation manners of the present application. Their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the patent application. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present application, several modifications and improvements can still be made, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims, and the description can be used to explain the content of the claims.

Claims

1. A method for constructing a fingerprint of Shiduqing Tablets, characterized in that: The following steps are involved: The wet-du-qing tablets are extracted using an extraction solvent to prepare a test solution; the extraction solvent is methanol or a methanol-water solution; The test solution was subjected to high performance liquid chromatography to construct a fingerprint of Shiduqing Tablets, wherein the fingerprint of Shiduqing Tablets contained 16 common peaks, and the identified 4 characteristic peaks included: peak 6 was ferulic acid, peak 7 was astilbin, peak 10 was baicalin, and peak 11 was salvianolic acid B; The conditions for the high performance liquid chromatography detection include: the chromatographic column is an octadecyl bonded silica gel column; the detection wavelength is 220 nm; the mobile phase A is a phosphoric acid aqueous solution, the mobile phase B is acetonitrile, and gradient elution is used; the procedure of the gradient elution includes: 0-15min, the volume percentage of the mobile phase B increased from 5% to 20%; 15min-25min, the volume percentage of the mobile phase B increased from 20% to 21%; 25min-35min, the volume percentage of the mobile phase B increased from 21% to 28%; 35min-40min, the volume percentage of the mobile phase B increased from 28% to 30%; 40min-50min, the volume percentage of the mobile phase B increased from 30% to 48%; 50min-60min, the volume percentage of the mobile phase B increased from 48% to 55%; From 60 min to 65 min, the volume percentage of the mobile phase B was maintained at 55%.

2. The method for constructing the fingerprint of Shiduqing Tablets according to claim 1, characterized in that: In the phosphoric acid aqueous solution, the volume percentage of phosphoric acid is 0.05%-0.2%.

3. The method for constructing the fingerprint of Shiduqing Tablets according to claim 1, characterized in that: The high performance liquid chromatography detection satisfies at least one of the following conditions: (1) Flow rate: 0.5 mL / min-2 mL / min; (2) Column temperature is 25℃-35℃; (3) The injection volume is .

4. The method for constructing the fingerprint of Shiduqing Tablets according to claim 1, characterized in that: The extraction step satisfies at least one of the following conditions: (1) The extraction method is ultrasonic extraction or reflux extraction; (2) The extraction time is 15min-60min.

5. The method for constructing the fingerprint of Shiduqing Tablets according to claim 1, characterized in that: The method for constructing the fingerprint spectrum of the Shiduqing tablets also includes the following steps: Dissolving the reference substance using a dissolving reagent to prepare a reference substance mixed solution; Performing high performance liquid chromatography on the reference substance mixed solution to obtain a reference spectrum; According to the reference spectrum, the chromatographic peaks of the fingerprint spectrum of the Shiduqing tablets are identified as common peaks; Wherein, the reference substances include ferulic acid, astilbin, baicalin and salvianolic acid B.

6. The method for constructing the fingerprint of Shiduqing Tablets according to claim 5, characterized in that: The dissolving agent includes methanol or a methanol aqueous solution.

7. Application of the method for constructing the fingerprint of Shiduqing tablets according to any one of claims 1 to 6 in the quality control of Shiduqing tablets.

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