HPLC fingerprint spectrum of Bazheng tablets and construction method and application thereof

Abstract

The invention discloses a Bazheng tablet HPLC (High Performance Liquid Chromatography) fingerprint spectrum as well as a construction method and application thereof, and belongs to the technical field of quality control of traditional Chinese medicine preparations. According to the method, high performance liquid chromatography and mass spectrometry are combined, and geniposidic acid, geniposide, liquiritin and the like are used as reference substances to accurately identify characteristic peaks in a fingerprint spectrum; by designing a gradient elution program, the resolution and accuracy of a chromatogram are improved; a plurality of batches of samples are systematically analyzed, the fingerprint spectrum is successfully constructed, the fingerprint spectrum realizes the characterization of 30 chromatographic common peaks, 10 main chromatographic peaks are clearly and chemically identified, a plurality of medicinal flavors such as liquorice and fructus gardeniae are covered, the chemical component information of the Bazheng tablets is comprehensively and accurately reflected, and the fingerprint spectrum has the advantages of reliability, high sensitivity, high accuracy and the like. Products of different batches and different manufacturers can be rapidly and accurately identified, the preparation quality is comprehensively evaluated, the product quality is ensured, and a powerful guarantee is provided for controlling the quality and ensuring the clinical curative effect.

Description

Technical Field

[0001] The present invention relates to the technical field of quality control of traditional Chinese medicine preparations, and in particular to an eight-positive tablet HPLC fingerprint spectrum and a construction method and application thereof. Background Art

[0002] Bazheng Pian (National Medicine Approval Number: Z20050759) is a traditional Chinese medicine (TCM) formulated with nine herbs: Dianthus radix, Plantago seed (fried), Polygonum multiflorum, Rhubarb, Caulis Atractylodes, Gardenia jasminoides, Licorice root, Juncus effusus, and Talc. Bazheng Pian originates from the Taiping Huimin Heji Bureau prescriptions, a preceptorial prescription from the Song Dynasty. Its strict rules, ingenious formulas, precise use of herbs, and remarkable efficacy have earned it high acclaim among physicians. It has become a classic prescription used by imperial physicians and the general public for the treatment of stranguria (heat stranguria, blood stranguria, stranguria caused by stones, stranguria caused by plaques, and stranguria caused by fatigue). It has significant efficacy against various urinary tract infections and gonorrhea, and can enhance immune function, promoting recovery and spontaneous healing. Therefore, quality control studies are crucial. As a TCM compound preparation, Bazheng Pian contains multiple chemical components, resulting in a complex composition. These components are not independent but rather synergistic in their efficacy, exerting their therapeutic effects. Traditional quality control methods often focus on testing only a single or a few components, making it difficult to fully reflect the overall quality of Bazheng Pian. Therefore, there is an urgent need for a method that can comprehensively and accurately reflect the types and quantities of chemical components in Bazheng tablets to ensure the stability and reliability of its clinical efficacy.

[0003] Fingerprinting is a technology used to characterize and analyze the comprehensive information of chemical components in complex samples such as traditional Chinese medicine. By combining high-performance liquid chromatography (HPLC), gas chromatography (GC) and other technologies with a variety of analytical methods, it can comprehensively analyze the chemical composition of traditional Chinese medicine. Fingerprinting is of great significance in the quality control of traditional Chinese medicine, but there are still many challenges in its actual application. The ingredients of traditional Chinese medicine compound preparations are complex. Some traditional Chinese medicine ingredients are difficult to effectively separate and detect in HPLC due to factors such as polarity and molecular weight, resulting in the absence or overlap of certain important components in the fingerprint, affecting the accuracy and reliability of the fingerprint. In addition, how to choose the appropriate analytical method, how to optimize the chromatographic conditions, and how to ensure the specificity of the fingerprint are also key issues that need to be addressed by fingerprint technology in the quality control of traditional Chinese medicine.

[0004] Currently, there is no comprehensive, accurate, and reliable quality control method for Bazheng Tablets. Therefore, developing an HPLC fingerprint that can comprehensively reflect the types and quantities of chemical components in Bazheng Tablets and its construction method are of great significance to ensure the clinical efficacy and safety of Bazheng Tablets. Summary of the Invention

[0005] Given the quality control requirements of Bazheng Tablets, traditional quality control methods rely on the identification or content determination of single compounds, ignoring the interactions and overall effects between components. This leads to problems in existing quality evaluation methods, such as insufficient systematic evaluation and stability evaluation, and inability to fully reflect the material basis of efficacy. There is an urgent need to develop an HPLC fingerprint that can comprehensively reflect the types and quantities of chemical components in Bazheng Tablets to ensure the clinical efficacy and safety of Bazheng Tablets. The present invention aims to provide an HPLC fingerprint of Bazheng Tablets, a method for its construction, and its application, which can be used to comprehensively evaluate and control the quality of Bazheng Tablets, thereby ensuring the stability of product quality and the safety and effectiveness of clinical medication.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] The present invention provides a method for constructing an eight-positive HPLC fingerprint, comprising:

[0008] S1, using different batches of Bazheng tablets as test samples, preparing Bazheng tablets test sample solutions;

[0009] S2, single reference solution was prepared by dissolving geniposide, geniposide, liquiritin, apigenin, myricetin, liquiritigenin, kaempferol, apigenin, emodin, and chrysophanol as reference substances in pure methanol solution;

[0010] S3, under the same conditions, the eight positive tablets test solution and the single reference solution were subjected to high performance liquid chromatography analysis, and the corresponding chromatograms were recorded; the high performance liquid chromatography mobile phase was methanol-water solution, gradient elution, and the elution program was as shown in the following table:

[0011]

[0012] S4, importing the chromatogram of the test solution obtained in S3 into the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012A) and performing similarity analysis to confirm the reliability of the result;

[0013] S5, performing high-resolution mass spectrometry analysis based on the chromatogram of the test solution obtained in S3 to obtain a total ion current diagram; performing data analysis based on the total ion current diagram and the peak conditions of the chromatogram of the test solution obtained in S3 to obtain mass spectrometry results of each chemical component;

[0014] S6, comparing the chromatograms of the test solution and the reference solution obtained in S3, the total ion current obtained in S5, and the mass spectra of each chemical component to obtain an eight-positive HPLC fingerprint consisting of common characteristic peaks.

[0015] In S1, different batches of Bazheng tablets were used as test samples, methanol solution was added, ultrasonic extraction was performed, and filtration was performed to obtain Bazheng tablet test sample solutions.

[0016] Furthermore, the preparation of the Bazheng tablets test solution contains 0.5 g of Bazheng tablets powder in every 20 mL of methanol solution, and the ultrasonic extraction conditions are: ultrasonic power 250 W, extraction 20 to 40 minutes.

[0017] Furthermore, the methanol solution is a pure methanol solution.

[0018] Furthermore, ultrasonic extraction was performed for 30 min.

[0019] Furthermore, the filtration is performed using a 0.45 μm microporous membrane filter.

[0020] In S2, the single reference solution, wherein the geniposide single reference solution contains 87 μg of geniposide per 1 mL of methanol solution; the gardenoside single reference solution contains 86 μg of gardenoside per 1 mL of methanol solution; the liquiritin single reference solution contains 83 μg of liquiritin per 1 mL of methanol solution; the apiosyl liquiritin single reference solution contains 94 μg of apiosyl liquiritin per 1 mL of methanol solution; the myricetin single reference solution contains 84 μg of apiosyl liquiritin per 1 mL of methanol solution; The alcohol solution contains 80μg of myricetin; the single reference solution of glycyrrhizin contains 66μg of glycyrrhizin per 1mL of methanol solution; the single reference solution of kaempferol contains 89μg of kaempferol per 1mL of methanol solution; the single reference solution of apigenin contains 86μg of apigenin per 1mL of methanol solution; the single reference solution of emodin contains 64μg of emodin per 1mL of methanol solution; the single reference solution of chrysophanol contains 65μg of chrysophanol per 1mL of methanol solution.

[0021] In S3, the HPLC detection wavelength is 215-315 nm, the column temperature is 25-30° C., and the flow rate is 0.6-1.0 mL / min.

[0022] Furthermore, the high performance liquid chromatography column is a Hedera ODS-2-C18 (250 mm×4.6 mm, 5 μm) column.

[0023] Furthermore, the HPLC detection wavelength is 254 nm, the column temperature is 30° C., the flow rate is 1.0 mL / min, and the injection volume is 10 μL.

[0024] S4 specifically includes: importing the chromatograms of different batches of Bazheng tablets test sample solutions obtained in S3 into the Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012A); selecting the chromatographic peaks existing in the chromatograms of different batches of Bazheng tablets test sample solutions as common peaks, generating a reference spectrum of Bazheng tablets using the average value calculation method, and calculating the relative retention time and relative peak area of ​​each common peak; performing similarity analysis after data import, multi-point correction and data matching; obtaining and exporting the similarity result table between the chromatograms of different batches of Bazheng tablets test sample solutions and the common peak pattern; confirming the reliability of the results based on the similarity result table and the chromatograms of the Bazheng tablets test sample solutions.

[0025] In S5, the detection conditions of the high-resolution mass spectrometry are: electrospray ionization, spray voltage of 3500 V, sheath gas flow rate of 40 arb, auxiliary gas flow rate of 10 arb, capillary temperature of 300°C, auxiliary gas temperature of 300°C, scanning mode of full scan mode, and mass-to-charge ratio scanning range of m / z 100-1500.

[0026] S6 is specifically as follows: according to the chromatogram of the test solution of the Bazheng Tablets and the chromatogram of the single reference solution obtained in S3, and combined with the total ion current diagram obtained by high-resolution mass spectrometry in S5 and the mass spectrum of the chemical components, peak 7 gardenia glycoside is used as the reference peak, and peak 5 in the chromatogram is identified as geniposide acid, peak 10 as liquiritin, peak 11 as apigenin, peak 13 as myricetin, peak 15 as glycyrrhizin, peak 21 as kaempferol, peak 22 as apigenin, peak 28 as emodin, and peak 29 as chrysophanol, and the HPLC fingerprint of Bazheng Tablets is obtained.

[0027] Furthermore, after obtaining the HPLC fingerprint of the eight positive tablets, the HPLC fingerprint results of the eight positive tablets were subjected to attribute analysis.

[0028] Furthermore, the chemical components corresponding to the 10 characteristic peaks in the HPLC fingerprint of Bazheng Tablets were attributed and analyzed. Among them, peak 5 geniposide acid is a common peak of fried plantain seeds and gardenia; peak 28 rhein is a common peak of dianthus and rhubarb; peak 15 glycyrrhizin, peak 10 glycyrrhizin, and peak 11 apigenin are derived from licorice; peak 7 gardenoside is derived from gardenia; peak 21 kaempferol and peak 22 apigenin are derived from fried plantain seeds; peak 29 rhein is derived from rhubarb; peak 13 myricetin is derived from Polygonum multiflorum.

[0029] The present invention provides a method for detecting the quality of Bazheng tablets, and utilizes the Bazheng tablets HPLC fingerprint spectrum obtained by the above-mentioned construction method to detect the quality of Bazheng tablets samples.

[0030] Compared with the prior art, the technical solution of the present invention has achieved the following beneficial technical effects:

[0031] The method for constructing an HPLC fingerprint of Bazheng tablets of the present invention adopts ultrasonic extraction with a methanol solution to fully dissolve the chemical components in the Bazheng tablets in the solvent; geniposide, gardenia glycoside, liquiritin and the like are selected as reference substances, which are all important active ingredients in the Bazheng tablets and contribute to the accurate identification of characteristic peaks in the fingerprint; the design of a gradient elution program takes into account the polarity and elution characteristics of different chemical components, thereby improving the resolution and accuracy of the chromatogram; and by systematically analyzing multiple batches of samples, the quality of the Bazheng tablets can be evaluated more comprehensively and scientifically, avoiding the limitations that may be brought about by a single component determination method, thereby providing a scientific and reliable means for quality control of the Bazheng tablets.

[0032] The fingerprint of Bazheng Tablets constructed by the present invention realizes the characterization of 30 common chromatographic peaks and chemically identifies 10 main chromatographic peaks among them, covering 6 medicinal flavors including liquorice, gardenia, rhubarb, plantago seed (fried), herb Dianthus, and Polygonum multiflorum. It can reflect the information of the chemical components contained in Bazheng Tablets in general, ensure the comprehensiveness and accuracy of the fingerprint, and provide the possibility for comprehensive quality control of Bazheng Tablets.

[0033] The application of the HPLC fingerprint of Bazheng Tablets constructed by the present invention can quickly and accurately identify products from different batches and different manufacturers using the HPLC fingerprint of Bazheng Tablets. By comparing the presence or absence of common peaks in the obtained fingerprints, the quality of the preparations can be more comprehensively evaluated, and the quality of the products can be more effectively guaranteed. The method has the advantages of simplicity, high efficiency, good repeatability and stability. It is not only suitable for the quality control of Bazheng Tablets, but also provides a reference and reference for the quality control of other traditional Chinese medicine preparations. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is the HPLC fingerprint of Bazheng tablets;

[0035] Figure 2 This is a chromatogram obtained by optimizing the extraction method during the preparation of the test solution of the present invention;

[0036] Figure 3 This is a chromatogram obtained by optimizing the extraction solvent during the preparation of the test solution of the present invention;

[0037] Figure 4 The chromatogram obtained by optimizing the detection wavelength under the chromatographic conditions of the present invention;

[0038] Figure 5 The chromatogram obtained by optimizing the column temperature in the chromatographic conditions of the present invention;

[0039] Figure 6 The chromatogram obtained by optimizing the flow rate under the chromatographic conditions of the present invention

[0040] Figure 7The chromatogram obtained by optimizing the mobile phase composition under the chromatographic conditions of the present invention;

[0041] Figure 8 The chromatogram obtained by optimizing the elution procedure under the chromatographic conditions of the present invention;

[0042] Figure 9 The chromatogram and mass spectrum of geniposide acid of the present invention are shown in FIG. (A) is the chromatogram and (B) is the mass spectrum;

[0043] Figure 10 The chromatogram and mass spectrum of geniposide of the present invention are shown in Figure 1, (A) is the chromatogram, and (B) is the mass spectrum;

[0044] Figure 11 The chromatogram and mass spectrum of liquiritin of the present invention are shown in Figure 2, (A) is the chromatogram, and (B) is the mass spectrum;

[0045] Figure 12 The chromatogram and mass spectrum of apiolactone liquiritin of the present invention are shown in Figure 1, (A) is the chromatogram, and (B) is the mass spectrum;

[0046] Figure 13 The chromatogram and mass spectrum of myricetin of the present invention are shown in Figure 1, (A) is the chromatogram, and (B) is the mass spectrum;

[0047] Figure 14 The chromatogram and mass spectrum of glycyrrhizin of the present invention are shown in Figures 1 and 2, (A) is the chromatogram, and (B) is the mass spectrum;

[0048] Figure 15 The chromatogram and mass spectrum of kaempferol of the present invention are shown in Figure 5, where (A) is the chromatogram and (B) is the mass spectrum.

[0049] Figure 16 The chromatogram and mass spectrum of apigenin of the present invention are shown in Figure 1, (A) is the chromatogram, and (B) is the mass spectrum;

[0050] Figure 17 The chromatogram and mass spectrum of emodin of the present invention are shown in FIG. (A) is the chromatogram, and (B) is the mass spectrum;

[0051] Figure 18 The chromatogram and mass spectrum of chrysophanol of the present invention are shown in FIG. (A) is the chromatogram, and (B) is the mass spectrum;

[0052] Figure 19 This is the mass spectrum of the eight positive films of the present invention in positive ion mode;

[0053] Figure 20 This is the mass spectrum of the eight positive films of the present invention in negative ion mode;

[0054] Figure 21 This is the fingerprint of the test sample of batch 15 of eight positive films of the present invention. DETAILED DESCRIPTION

[0055] The following examples are used to further illustrate the present invention. However, these examples are only provided for a better understanding of the invention and are not intended to limit the scope or implementation principles of the invention. The embodiments of the present invention are not limited to the following. Unless otherwise specified, the experimental methods used are conventional methods, and the raw materials used are commercially available products.

[0056] The present invention provides a method for constructing an eight-positive HPLC fingerprint, comprising the following steps:

[0057] S1. Prepare Bazheng tablets test solutions using different batches of Bazheng tablets as test samples;

[0058] Remove the coating from the Bazheng tablets, grind into powder, weigh appropriate amounts of powder from different batches, place in a stoppered conical flask, add methanol solution, ultrasonically extract, and filter to obtain the test solution;

[0059] S2, accurately weigh geniposide, geniposide, liquiritin, apigenin, myricetin, liquiritigenin, kaempferol, apigenin, emodin, and chrysophanol, and dissolve them in pure methanol to obtain a single reference solution;

[0060] S3, under the same conditions, the eight positive tablets test solution and the single reference solution were subjected to high performance liquid chromatography analysis, and the corresponding chromatograms were recorded; the high performance liquid chromatography mobile phase was methanol-water solution, gradient elution, and the elution program was as shown in the following table:

[0061] Table 1: Elution program

[0062]

[0063] S4, importing the chromatogram of the test solution obtained in S3 into the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012A) and performing similarity analysis to confirm the reliability of the result;

[0064] S5, performing high-resolution mass spectrometry analysis based on the chromatogram of the test solution obtained in S3 to obtain a total ion current diagram; performing data analysis based on the total ion current diagram and the peak conditions of the chromatogram of the test solution obtained in S3 to obtain mass spectrometry results of each chemical component;

[0065] Perform high-resolution mass spectrometry analysis on the test solution to obtain a total ion current (TIC) and mass spectrometry results of the chemical components. Import the TIC into Xcalibur software and enter the Qual Browser interface to obtain the mass spectrometry results of each chemical component based on the TIC.

[0066] S6, compare the chromatograms of the test solution and the reference solution obtained in S3, the total ion current obtained in S5, and the mass spectra of each chemical component to obtain an eight-positive HPLC fingerprint consisting of common characteristic peaks, see Appendix Figure 1 .

[0067] 2. Optimization process of fingerprint detection:

[0068] (1) Optimization of test solution preparation process

[0069] The present invention conducts experimental investigations on different extraction methods (ultrasound, reflux, and immersion). Figure 2 As shown in the figure, the results showed that the chromatograms obtained by ultrasound and the other two extraction methods were not much different. In comparison, the ultrasound extraction method was simpler, so the ultrasound extraction method was adopted.

[0070] The present invention compares the extraction effects of different extraction solvents (pure ethanol solution, 80% methanol solution, pure methanol solution, aqueous solution), see Appendix Figure 3 As shown, the results showed that when pure methanol solution was used as the extraction solvent, the extract chromatogram had the most information and the highest component content, so pure methanol solution was selected for extraction;

[0071] (2) Optimization of chromatographic conditions:

[0072] The present invention uses a UV-visible absorption detector to investigate the detection wavelength and extracts the chromatograms at 215nm, 245nm, 254nm, 265nm, 280nm and 315nm. Figure 4 As shown in the figure, it was found that when the detection wavelength was 254 nm, the number of chromatographic peaks obtained was large and clear, and the baseline was relatively stable, so 254 nm was selected as the detection wavelength.

[0073] The present invention screens the column temperature (25°C, 30°C, 35°C), see Appendix Figure 5 ,The results showed that the peak shape was the best and the peak height was higher when the column temperature was kept at 30℃, so the column temperature of 30℃ was finally selected;

[0074] The present invention screened the flow rate (0.6mL / min, 0.8mL / min, 1.0mL / min), see the attached Figure 6 ,The results showed that when the flow rate was 1.0mL / min, the peak shape was good and the separation effect of each component was better, so the flow rate of 1.0mL / min was finally selected;

[0075] The present invention compares the elution effects of multiple different elution systems including methanol-water, acetonitrile-water, acetonitrile-0.1% phosphoric acid, and methanol-0.1% phosphoric acid. Figure 7 The results show that when methanol-water is used as the mobile phase, the peak shapes of the chromatographic peaks in the eight positive slices are good, the separation is high, and the baseline is stable, so methanol-water is finally selected as the mobile phase;

[0076] After determining the optimal detection wavelength, column temperature, and flow rate, the present invention screened the best gradient elution program through a large number of experiments. Some elution programs are as follows:

[0077] Table 2: Elution Program 1

[0078]

[0079] Table 3: Elution Program 2

[0080]

[0081] Table 4: Elution Program 3

[0082]

[0083] Table 5: Elution Program 4

[0084]

[0085] Table 6: Elution Program 5

[0086]

[0087]

[0088] Table 7: Elution Program 6

[0089]

[0090] Test results such as Figure 8 As shown, through Figure 8 It can be seen that the chromatographic peak separation obtained by elution program 6 is good, the number of peaks is large, the peak distribution is relatively uniform, and the baseline is stable. Therefore, elution program 6 is selected as the optimal elution program.

[0091] The embodiments of the present invention are described in detail below with reference to the examples. In the examples, if the specific conditions are not specified, the experiments were carried out according to conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used, if the manufacturer is not specified, are all conventional products that can be obtained commercially.

[0092] The instruments and reagents used in the examples are shown in Tables 8 and 9.

[0093] Table 8: Instruments and reagents used in the present invention

[0094]

[0095] Drugs and reagents: The different batches of Bazheng tablets samples used in the present invention were purchased from the market and produced by Shaanxi Momede Qixuehe Pharmaceutical Co., Ltd.

[0096] Table 9: Reagents used in the present invention

[0097]

[0098] Reference substances: apigenin reference substance (batch number: 111901-202205, purity: 98.4%), gardenia glycoside reference substance (batch number: 110749-202320, purity: 98.1%), liquiritin reference substance (batch number: 111610-201908, purity: 95.0%), emodin reference substance (batch number: 110756-202414, purity: 98.5%), myricetin reference substance (batch number: 111860-202204, purity: 91.9%), chrysophanol reference substance (batch number: 110 796-202423, purity: 99.8%), and geniposide reference substance (batch number: 111828-202406, purity: 97.6%) were purchased from China Food and Drug Inspection Institute; apigenin reference substance (batch number: PS011457, purity: 98%) was purchased from Chengdu Pusi Biotechnology Co., Ltd.; glycyrrhizin reference substance (batch number: ABFH3012, purity: 98%) and kaempferol reference substance (batch number: AF20100214, purity: 98%) were purchased from Chengdu Aifa Biotechnology Co., Ltd.

[0099] Example 1

[0100] An eight-positive HPLC fingerprint and its construction method and application, comprising the following steps:

[0101] S1. Preparation of test solution

[0102] Weigh different batches of Bazheng tablets respectively, remove the coating, grind into powder, accurately weigh 0.5 g of powder, place it in a stoppered conical flask, add 20 mL of pure methanol solution, ultrasonically extract (ultrasonic power 250 W, frequency 40 kHz) for 30 min, filter, and take the filtrate to pass through a 0.45 μm microporous filter membrane to obtain the test solution.

[0103] S2. Preparation of reference solution

[0104] Accurately weigh each reference substance, place it in a stoppered conical flask, and add pure methanol solution to prepare a single reference substance solution containing 87 μg of geniposide, 86 μg of geniposide, 83 μg of liquiritin, 94 μg of apigenin, 80 μg of myricetin, 66 μg of liquiritigenin, 89 μg of kaempferol, 86 μg of apigenin, 64 μg of emodin, and 65 μg of chrysophanol per 1 mL.

[0105] S3. Inject the test solution and reference solution obtained in S1 and S2 above into a high-performance liquid chromatograph and analyze under the same conditions. Record the chromatogram of the test solution. The liquid chromatography conditions are as follows: Column: Hedera ODS-2-C18 (250 mm × 4.6 mm, 5 μm); Detector: UV-visible absorption detector; Detection wavelength: 254 nm; Volume flow rate: 1.0 mL / min; Injection volume: 10 μL; Mobile phase: methanol (A)-water solution (B), gradient elution. The elution program is shown in Table 1.

[0106] S4. Import the chromatograms of the different batches of Bazheng tablets test sample solutions obtained in S3 into the Chinese medicine chromatographic fingerprint similarity evaluation system (2012A version); select the chromatographic peaks that exist in the chromatograms of the different batches of Bazheng tablets test sample solutions as common peaks, use the average value calculation method to generate a reference spectrum of Bazheng tablets, and calculate the relative retention time and relative peak area of ​​each common peak; after data import, multi-point correction and data matching, perform similarity analysis; obtain and export the similarity result table between the chromatograms of the different batches of Bazheng tablets test sample solutions and the common peak pattern; confirm the reliability of the results based on the similarity result table and the chromatograms of the Bazheng tablets test sample solutions.

[0107] S5. According to the chromatogram of the test solution of the Bazheng Tablets and the chromatogram of the single reference solution obtained in S3, and combined with the total ion current diagram obtained by high-resolution mass spectrometry and the mass spectrum result diagram of the chemical components, comparison is performed, and with gardenia glycoside (peak 7) as the reference peak, peak 5 in the chromatogram is identified as geniposide acid, peak 10 as liquiritin, peak 11 as apigenin, peak 13 as myricetin, peak 15 as glycyrrhizin, peak 21 as kaempferol, peak 22 as apigenin, peak 28 as emodin, and peak 29 as chrysophanol, and the fingerprint of Bazheng Tablets is obtained.

[0108] At the same time, the present invention uses the automatically generated reference spectrum R to generate a common chromatographic peak pattern. Analysis and calculation show that the common chromatographic peaks of 15 batches of Bazheng tablets test solutions have relatively good similarity, indicating that the fingerprint spectrum of Bazheng tablets established by this method can well detect the quality of Bazheng tablets and different batches of Bazheng tablets. The results are shown in Table 10.

[0109] Table 10: Similarity between each batch of samples and the common chromatographic peak pattern

[0110]

[0111] Example 2

[0112] Methodological research on fingerprint detection methods:

[0113] (1) Precision study

[0114] The test solution prepared by the method of Example 1 was taken and analyzed according to the detection method of Example 1. The sample was injected in parallel 6 times with an injection volume of 10 μL. Gardenia jasminoides was used as the reference peak. The peak area and retention time were analyzed and the RSD value was calculated. The results showed that the RSD of the retention time was less than 1.457%, and the RSD of the peak area was less than 2.668%, indicating that the parallel injection precision of the equipment was good. The results are shown in Table 11.

[0115] Table 11: Peak area, retention time and corresponding RSD values ​​of characteristic peaks of each batch of eight positive film samples

[0116]

[0117]

[0118] (2) Stability study

[0119] The test solution prepared by the method of Example 1 was taken and analyzed according to the detection method of Example 1. The sample was injected and analyzed at different times of 0, 2, 6, 12, 18, and 24 hours, with an injection volume of 10 μL. Gardenia glycoside was used as the reference peak. The peak area and retention time of the common peak of the HPLC fingerprint of the sample were analyzed and the RSD value was calculated. The results showed that the RSD of the retention time was less than 1.539%, and the RSD of the peak area was less than 2.729%, indicating that the chromatographic peak of the test solution of Bazhengpian was almost unchanged within 24 hours, and the stability was good. The results are shown in Table 12.

[0120] Table 12: Peak area, retention time and corresponding RSD values ​​of characteristic peaks of eight positive samples at different times

[0121]

[0122]

[0123] (3) Reproducible studies

[0124] Six batches of sample solutions were prepared according to the test solution method in Example 1. Referring to the chromatographic conditions of Example 1, the injection volume was 10 μL, and Gardenia jasminoides was used as the reference peak. The peak area and retention time of the common peaks in the HPLC fingerprint of the samples were analyzed and the RSD values ​​were calculated. The results showed that the RSD of the retention time was less than 1.653%, and the RSD of the peak area was less than 2.823%, indicating that the sample chromatographic peaks had good reproducibility and the method had good repeatability. The results are shown in Table 13.

[0125] Table 13: Peak area, retention time and corresponding RSD values ​​of characteristic peaks of each batch of eight positive film samples

[0126]

[0127] The above experimental results demonstrate that the fingerprint spectrum construction method for the Eight-Positive Tablet provided by the present invention has good stability, high precision, and good repeatability. It can comprehensively and objectively evaluate the quality of the Eight-Positive Tablet, providing quality assurance for clinical efficacy. The above examples are merely exemplary embodiments of the present invention and are not intended to limit the scope of the present invention, which is defined by the claims.

[0128] The above content is merely an example and explanation of the concept of the present invention. Any modifications or additions made to the described specific embodiments or replacements made by technicians in this technical field in a similar manner shall fall within the scope of protection of the present invention as long as they do not deviate from the concept of the invention or exceed the scope defined by the claims.

Claims

1. A method for constructing an eight-positive HPLC fingerprint, characterized in that: include: S1, using different batches of Bazheng tablets as test samples to prepare Bazheng tablets test sample solutions; S2, single reference solution was prepared by dissolving geniposide, geniposide, liquiritin, apigenin, myricetin, liquiritigenin, kaempferol, apigenin, emodin, and chrysophanol as reference substances in pure methanol solution; S3, under the same conditions, the eight positive tablets test solution and the single reference solution were subjected to high performance liquid chromatography analysis, and the corresponding chromatograms were recorded; the high performance liquid chromatography mobile phase was methanol-water solution, gradient elution, and the elution program was as shown in the following table: S4, importing the chromatogram of the test solution obtained in S3 into the traditional Chinese medicine fingerprint similarity evaluation system and performing similarity analysis to confirm the reliability of the result; S5, performing high-resolution mass spectrometry analysis based on the chromatogram of the test solution obtained in S3 to obtain a total ion current graph; Data analysis was performed based on the total ion current graph and the peaks in the chromatogram of the test solution obtained in S3 to obtain the mass spectrometry results of each chemical component; S6, comparing the chromatograms of the test solution and the reference solution obtained in S3, the total ion current obtained in S5, and the mass spectra of each chemical component to obtain an eight-positive HPLC fingerprint consisting of common characteristic peaks.

2. The method for constructing an eight-positive HPLC fingerprint according to claim 1, characterized in that: In S1, different batches of Bazheng tablets were used as test samples, methanol solution was added, ultrasonic extraction was performed, and filtration was performed to obtain Bazheng tablet test sample solutions.

3. The method for constructing an eight-positive HPLC fingerprint according to claim 1, characterized in that: In S2, the single reference solution, wherein the geniposide single reference solution contains 87 μg of geniposide per 1 mL of methanol solution; the gardenoside single reference solution contains 86 μg of gardenoside per 1 mL of methanol solution; the liquiritin single reference solution contains 83 μg of liquiritin per 1 mL of methanol solution; the apiosyl liquiritin single reference solution contains 94 μg of apiosyl liquiritin per 1 mL of methanol solution; the myricetin single reference solution contains 84 μg of apiosyl liquiritin per 1 mL of methanol solution; The alcohol solution contains 80μg of myricetin; the single reference solution of glycyrrhizin contains 66μg of glycyrrhizin per 1mL of methanol solution; the single reference solution of kaempferol contains 89μg of kaempferol per 1mL of methanol solution; the single reference solution of apigenin contains 86μg of apigenin per 1mL of methanol solution; the single reference solution of emodin contains 64μg of emodin per 1mL of methanol solution; the single reference solution of chrysophanol contains 65μg of chrysophanol per 1mL of methanol solution.

4. The method for constructing an eight-positive HPLC fingerprint according to claim 1, characterized in that: In S3, the HPLC detection wavelength is 215-315 nm, the column temperature is 25-30° C., and the flow rate is 0.6-1.0 mL / min.

5. The method for constructing an eight-positive HPLC fingerprint according to claim 1, characterized in that: S4 is specifically as follows: importing the chromatograms of the different batches of Bazheng tablets test solution obtained in S3 into the Chinese medicine chromatographic fingerprint similarity evaluation system; selecting the chromatographic peaks existing in the chromatograms of the different batches of Bazheng tablets test solution as common peaks, generating a control spectrum of Bazheng tablets by the average value calculation method, and calculating the relative retention time and relative peak area of ​​each common peak; performing similarity analysis after data import, multi-point correction and data matching; obtaining and exporting the similarity result table between the chromatograms of the different batches of Bazheng tablets test solution and the common peak pattern; confirming the reliability of the results based on the similarity result table and the chromatogram of the Bazheng tablets test solution.

6. The method for constructing an eight-positive HPLC fingerprint according to claim 1, characterized in that: In S5, the detection conditions of the high-resolution mass spectrometry are: electrospray ionization, spray voltage of 3500 V, sheath gas flow rate of 40 arb, auxiliary gas flow rate of 10 arb, capillary temperature of 300°C, auxiliary gas temperature of 300°C, scanning mode of full scan mode, and mass-to-charge ratio scanning range of m / z 100-1500.

7. The method for constructing an eight-positive HPLC fingerprint according to claim 1, characterized in that: S6 is specifically as follows: according to the chromatogram of the test solution of the Bazheng Tablets and the chromatogram of the single reference solution obtained in S3, and combined with the total ion current diagram obtained by high-resolution mass spectrometry in S5 and the mass spectrum of the chemical components, peak 7 gardenia glycoside is used as the reference peak, and peak 5 in the chromatogram is identified as geniposide acid, peak 10 as liquiritin, peak 11 as apigenin, peak 13 as myricetin, peak 15 as glycyrrhizin, peak 21 as kaempferol, peak 22 as apigenin, peak 28 as emodin, and peak 29 as chrysophanol, and the HPLC fingerprint of Bazheng Tablets is obtained.

8. The method for constructing an eight-positive HPLC fingerprint according to claim 1, characterized in that: After obtaining the HPLC fingerprint of Bazheng tablets, the HPLC fingerprint results of Bazheng tablets were subjected to attribute analysis.

9. Eight positive HPLC fingerprints obtained according to the construction method according to any one of claims 1 to 8.

10. A method for detecting the quality of eight-positive film, characterized in that: The quality of the Bazheng tablets sample is detected using the Bazheng tablets HPLC fingerprint spectrum described in claim 9.

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