Construction method and application of Tongning tablet fingerprint spectrum based on overall quality evaluation

Abstract

The application discloses a construction method and application of a Tongning tablet fingerprint spectrum based on overall quality evaluation, and belongs to the technical field of quality control of traditional Chinese medicine preparations. The method adopts high performance liquid chromatography and mass spectrometry, takes 5-hydroxymethyl furfural, chlorogenic acid, ferulic acid, calycosin-7-glucoside and calycosin as reference substances, detects different batches of Tongning tablets, and successfully constructs a fingerprint spectrum. The method is simple, high in precision, good in reproducibility, accurate and reliable, can identify at least five components under the same chromatographic conditions at one time, greatly improves the detection efficiency, saves time and cost, can accurately and quickly simultaneously monitor the quality of multiple characteristic components of the Tongning tablet, and provides a strong guarantee for controlling the quality of the Tongning tablet and ensuring the clinical curative effect.

Description

Technical Field

[0001] This invention relates to the field of quality control technology for traditional Chinese medicine preparations, specifically to the construction method and application of the fingerprint spectrum of Tongning tablets based on overall quality evaluation. Background Technology

[0002] Tongning tablets, a commonly used traditional Chinese medicine compound preparation, possess significant effects such as promoting blood circulation, removing blood stasis, reducing swelling, and relieving pain. They are widely used in the clinical treatment of various pain symptoms. The clinical efficacy of traditional Chinese medicine compound preparations is usually the comprehensive result of the synergistic effects of multiple chemical components, such as alkaloids, flavonoids, and organic acids, through multiple targets and pathways. Due to the diversity of Chinese medicinal materials and their complex growth environment, harvesting season, and processing techniques, the chemical basis of Tongning tablets is extremely complex. Therefore, establishing a scientific and rigorous quality evaluation system is crucial for ensuring the stability of the clinical efficacy of Tongning tablets across different batches and for patient safety.

[0003] However, current relevant standards still have significant limitations in the quality control methods for Tongning tablets. Traditional quality evaluation systems mainly rely on microscopic identification, thin-layer chromatography for qualitative analysis, and high-performance liquid chromatography (HPLC) to determine the content of one or two single representative indicator components. This single-indicator component evaluation model is insufficient to comprehensively and objectively reflect the overall quality characteristics and pharmacodynamic material basis of the Tongning tablet compound system. In actual production and supervision, it is highly likely that while the content of some indicator components meets the standards, the overall effective substance group may vary or its proportions may be imbalanced. This quality control blind spot makes it difficult to truly guarantee the uniformity of the preparation's quality.

[0004] Therefore, developing a fingerprint spectral detection method for Tongning tablets based on HPLC technology and establishing a scientific and comprehensive quality evaluation system will provide comprehensive and reliable technical support for improving the quality standards of Tongning tablets, monitoring the production process, and identifying genuine and counterfeit products. Summary of the Invention

[0005] In order to overcome the shortcomings of the prior art, the present invention aims to provide a method for constructing a fingerprint spectrum of Tongning tablets based on overall quality evaluation, so as to solve the technical problem that the existing quality evaluation methods rely only on the identification or content determination of a single compound, resulting in insufficient systematic evaluation and stability evaluation, and failing to fully reflect the pharmacodynamic material basis.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: The first aspect of this invention discloses a method for constructing a fingerprint spectrum of Tongning tablets based on overall quality evaluation, comprising the following steps: S1. Using different batches of Tongning tablets as test samples, prepare Tongning tablet test sample solutions; using 5-hydroxymethylfurfural, chlorogenic acid, ferulic acid, verrucoside glucoside and verrucoside as reference standards, prepare single reference standard solutions. S2. Perform high-performance liquid chromatography analysis on the test solution and reference solution of Tongning tablets, and record the corresponding chromatograms; S3. Perform similarity analysis on the chromatogram of the Tongning tablet test solution obtained in S2 to confirm the reliability of the results; S4. Perform high-resolution mass spectrometry analysis on the test solution obtained in S1 to obtain the total ion chromatogram; perform data analysis based on the total ion chromatogram and the peak elution of the chromatogram of the Tongning tablet test solution obtained in S2 to obtain the mass spectrometry results of each chemical component. S5. Compare the chromatograms of the test solution and reference solution of Tongning tablets obtained in S2, the total ion chromatogram obtained in S4, and the mass spectrometry results of each chemical component to obtain the HPLC fingerprint of Tongning tablets composed of common characteristic peaks.

[0007] Preferably, in S1, different batches of Tongning tablets are taken as test samples, 80% methanol solution is added, ultrasonic extraction is performed, the sample is filtered, the filtrate is concentrated and dried under reduced pressure, 80% methanol solution is added, the sample is dissolved, filtered, and Tongning tablets test sample solution is obtained.

[0008] Preferably, each 20 mL of methanol solution contains 2.0 g of the contents of the Tongning tablets. The extract is extracted by ultrasonication, filtered, and the filtrate is concentrated and dried under reduced pressure. The filtrate is dissolved in 5 mL of methanol solution and filtered through a microporous membrane to obtain the Tongning tablets test solution.

[0009] Preferably, ultrasonic extraction is performed for 30 minutes.

[0010] Preferably, filtration is performed using a 0.45 µm microporous membrane.

[0011] Preferably, in S2, the high-performance liquid chromatography (HPLC) determination conditions are as follows: Shim-pack VP-ODS column; mobile phase A is methanol, mobile phase D is 0.1% phosphoric acid, detection wavelength is 210~310 nm, column temperature is 25~35℃, and flow rate is 0.6~1.0 mL / min.

[0012] Preferably, the mobile phase is methanol-0.1% phosphoric acid, the detection wavelength is 230 nm, the column temperature is 30 °C, and the flow rate is 0.68 mL / min.

[0013] Preferably, after obtaining the HPLC fingerprint of Tongning tablets, the HPLC fingerprint results of Tongning tablets are subjected to attribution analysis.

[0014] In a second aspect, the present invention discloses the application of the HPLC fingerprint of Tongning tablets obtained by the method for constructing a fingerprint spectrum of Tongning tablets based on overall quality evaluation in the detection of the quality of Tongning tablets.

[0015] In a third aspect, the present invention discloses a method for detecting the quality of Tongning tablets. The method utilizes the HPLC fingerprint of Tongning tablets obtained by the method of constructing a fingerprint spectrum of Tongning tablets based on overall quality evaluation to detect the quality of Tongning tablets or their namesake drugs, using one or more of 5-hydroxymethylfurfural, chlorogenic acid, ferulic acid, verrucoside glucoside and verrucoside as quality markers.

[0016] Compared with the prior art, the technical solution of the present invention achieves the following beneficial technical effects: This invention provides a method for constructing a fingerprint chromatogram of Tongning tablets based on overall quality evaluation. Utilizing high-performance liquid chromatography (HPLC) combined with gradient elution and specific chromatographic conditions, this method can accurately separate and detect multiple active ingredients in Tongning tablets, exhibiting high precision, good reproducibility, excellent separation, and high stability. The fingerprint chromatogram constructed using this method ensures consistency and stability between different batches of Tongning tablets, providing a reliable reference standard for quality control. Similarity evaluation with the fingerprint chromatogram allows for rapid and accurate determination of sample quality compliance, ensuring consistency between different batches and contributing to product quality stability and reliability. Compared to traditional quality testing methods, HPLC fingerprinting technology offers higher detection efficiency, enabling the testing of large numbers of samples in a short time and automatically generating test result reports. It provides a comprehensive, objective, and accurate detection and evaluation of Tongning tablet quality, which is of great significance for ensuring clinical efficacy and provides effective assurance for clinical application. The fingerprint spectrum of Tongning tablets constructed based on this method characterized 28 common chromatographic peaks and clearly identified the chemical characteristics of 5 major chromatographic peaks, covering the four prescription herbs: Polygonum multiflorum, Astragalus membranaceus, Angelica sinensis, and Ligusticum chuanxiong. This method comprehensively controls the quality and composition of Tongning tablets. It also makes up for the deficiencies in the content determination of Tongning tablets in the implementation standard, as well as the one-sidedness of existing detection methods that can only reflect the quality of a very small number of drug components. The established fingerprint spectrum determination method is an effective supplement to the quality control method of Tongning tablets, which can reflect the types and quantities of chemical components contained, effectively characterize their quality, and facilitate comprehensive quality control of the product.

[0017] This invention provides a method for detecting the quality of Tongning tablets, which can quickly and accurately identify products from different batches and manufacturers. By comparing the presence or absence of common peaks in the obtained fingerprint spectrum, the quality of the preparation can be evaluated more comprehensively, thus more effectively ensuring product quality. It has the advantages of being simple, efficient, repeatable, and stable. It is not only applicable to the quality control of Tongning tablets, but can also provide a reference for the quality control of other traditional Chinese medicine preparations. Attached Figure Description

[0018] Figure 1 This is a chromatogram obtained by optimizing the extraction method during the preparation of the test solution for the Tongning tablets of the present invention; Figure 2 The chromatogram obtained by optimizing the extraction solvent during the preparation of the test solution for the Tongning tablets of the present invention; Figure 3 This is a chromatogram obtained by optimizing the detection wavelength under chromatographic conditions according to the present invention; Figure 4 This is a chromatogram obtained by optimizing column temperature under chromatographic conditions according to the present invention; Figure 5 This is a chromatogram obtained by optimizing the flow rate under chromatographic conditions according to the present invention; Figure 6 This is a chromatogram obtained by optimizing the mobile phase composition under chromatographic conditions according to the present invention; Figure 7 This is a chromatogram obtained by optimizing the elution procedure under chromatographic conditions according to the present invention; Figure 8 This invention provides a test sample for prior art examination. DAD full-wavelength 3D scan map (195 nm~403 nm); Figure 9 The chromatogram (A), UV spectrum (B), and 3D spectrum (C) of 5-hydroxymethylfurfural of this invention are shown. Figure 10 The chromatogram (A), UV spectrum (B), and 3D spectrum (C) of chlorogenic acid are shown in this invention. Figure 11 The chromatogram (A), UV spectrum (B), and 3D spectrum (C) of ferulic acid of the present invention are shown. Figure 12 The chromatogram (A), UV spectrum (B), and 3D spectrum (C) of the present invention are shown. Figure 13 The chromatogram (A), UV spectrum (B), and 3D spectrum (C) of the verbascoside isoflavone of the present invention are shown. Figure 14 This is the total ion chromatogram of the Tongning tablets of the present invention in positive ion mode; Figure 15 This is the total ion flow chromatogram of the Tongning tablets of the present invention in negative ion mode; Figure 16 This is the mass spectrum of isoliquiritigenin of this invention; Figure 17 This is the chlorogenic acid mass spectrum of the present invention; Figure 18 This is the mass spectrum of ferulic acid in this invention; Figure 19 This is the mass spectrum of apigenin glycyrrhizin from the present invention; Figure 20 This is the mass spectrum of glycyrrhizin chalcone of the present invention; Figure 21 This is the mass spectrum of isoglycyrrhizin / glycyrrhizin / Gorgonium chloride of the present invention; Figure 22 This is a mass spectrum of the flower of the *Miscanthus sinensis* according to the present invention; Figure 23 This is the mass spectrum of ginsenoside Ro of the present invention; Figure 24 This is the mass spectrum of rhubarb in this invention; Figure 25 This is the mass spectrum of palmitic acid in this invention; Figure 26 This is the mass spectrum of oleanolic acid in this invention; Figure 27 This is the mass spectrum of the verbascoside isoflavone glucoside of the present invention; Figure 28 This is the mass spectrum of benzoyl aconitine of the present invention; Figure 29 This is the mass spectrum of ligustilide of the present invention; Figure 30 This is the mass spectrum of 5-hydroxymethylfurfural in this invention; Figure 31 This is the mass spectrum of benzoyl neoaconitine of the present invention; Figure 32 This is the mass spectrum of gentian glycosides from the present invention; Figure 33 This is the mass spectrum of benzoyl aconitine of the present invention; Figure 34 This is the glycyrrhizin spectrum of the present invention; Figure 35 This is the mass spectrum of the verbascoside isoflavone of the present invention; Figure 36 This is the mass spectrum of angelica lactone A of the present invention; Figure 37 Fingerprint spectra of different batches of Tongning tablets for testing according to the present invention; Figure 38 This is the HPLC fingerprint of the Tongning tablets of this invention. Detailed Implementation

[0019] The following embodiments are provided to further illustrate the present invention, but these embodiments are only for a better understanding of the invention and are not intended to limit the scope or implementation principles of the invention. The implementation of the present invention is not limited to the following. Unless otherwise specified, all experimental methods used are conventional methods, and all raw materials used are commercially available products. The instruments used in the present invention are shown in Table 1.

[0020] Table 1 Experimental Apparatus

[0021] The different batches of Tongning tablets used in this invention were all produced by Shaanxi Ouke Pharmaceutical Co., Ltd., and the reagents used are shown in Table 2.

[0022] Table 2 Experimental Reagents

[0023] Reference standards: 5-hydroxymethylfurfural reference standard (batch number: 111626-202417, purity: 98%), chlorogenic acid (batch number: 110753-202520, purity: 97.9%), ferulic acid (batch number: 110773-202316, purity: 99.3%), verbascoside (batch number: 111920-202308, purity: 96.9%), and verbascoside reference standard (batch number: 112117-202402, purity: 99.6%), purchased from the National Institutes for Food and Drug Control.

[0024] This invention provides a method for constructing a fingerprint spectrum of Tongning tablets based on overall quality evaluation, comprising the following steps: S1. Using different batches of Tongning tablets as test samples, prepare Tongning tablet test sample solutions; using 5-hydroxymethylfurfural, chlorogenic acid, ferulic acid, verrucoside glucoside and verrucoside as reference standards, prepare single reference standard solutions. The preparation method of the test solution for Tongning tablets is as follows: Weigh 2.0 g of the contents of different batches of Tongning tablets, place them in a stoppered conical flask, add 20 mL of 80% methanol solution, extract by ultrasonication for 30 min, filter, concentrate and dry the filtrate under reduced pressure, add 5 mL of 80% methanol solution, dissolve, filter, and filter the filtrate through a 0.45 µm microporous membrane to obtain the test solution for Tongning tablets; The preparation method of the single reference solution is as follows: Accurately weigh 5-hydroxymethylfurfural, chlorogenic acid, ferulic acid, vernix caseosa glucoside and vernix caseosa, dissolve them in pure methanol solution to prepare a solution containing 0.89 mg of 5-hydroxymethylfurfural, 0.24 mg of chlorogenic acid, 0.39 mg of ferulic acid, 0.29 mg of vernix caseosa glucoside and 0.19 mg of vernix caseosa in 1 mL; S2. Under the same conditions, perform high performance liquid chromatography analysis on the test solution and reference solution of Tongning tablets, and record the corresponding chromatograms; The high-performance liquid chromatography (HPLC) conditions were as follows: column: Shim-pack VP-ODS (250 mm × 4.6 mm, 5 μm) column; detector: diode array detector; detection wavelength: 230 nm; flow rate: 0.8 mL / min; injection volume: 10 μL; column temperature: 30℃; mobile phase: methanol (A) - 0.1% phosphoric acid aqueous solution (D), gradient elution, and the elution program is shown in Table 3. Table 3 Elution Procedure 5

[0025] S3. Import the chromatogram of the test solution obtained in S2 into the Chinese herbal chromatographic fingerprint chromatogram similarity evaluation system (2004 A version) and perform similarity analysis to confirm the reliability of the results; S4. Perform high-resolution mass spectrometry analysis on the Tongning tablet test solution obtained in S1 to obtain the total ion chromatogram; perform data analysis on the peak elution of the total ion chromatogram and the test solution obtained in S2 to obtain the mass spectrometry results of each chemical component. S5. Compare the chromatograms of the test solution and the reference solution obtained in S2, the total ion chromatogram obtained in S4, and the mass spectra of each chemical component to obtain the HPLC fingerprint of Tongning tablets composed of common characteristic peaks.

[0026] This invention provides an optimized process for constructing the fingerprint spectrum of Tongning tablets based on overall quality evaluation, including the following steps: (1) Optimization of test sample solution preparation Experiments were conducted to investigate different extraction methods (ultrasound, reflux, impregnation, and ultrasonic followed by vacuum concentration and drying). See details in the attached document. Figure 1 ,Depend on Figure 1 Data shows that the ultrasonic-assisted vacuum concentration and drying extraction method can extract the active ingredients in the preparation more comprehensively and efficiently. Therefore, the ultrasonic-assisted vacuum concentration and drying extraction method is adopted. The extraction effects of different extraction solvents (pure ethanol solution, 80% methanol solution, pure methanol solution, and water) were compared. See details in [link to relevant documentation]. Figure 2 ,Depend on Figure 2 The data shows that when 80% methanol solution is used as the extraction solvent, the extract chromatogram contains the most information and the component content is the highest. Therefore, 80% methanol solution is selected for extraction. (2) Optimization of chromatographic conditions A diode array detector was used to investigate the detection wavelength, and chromatograms were extracted at 210 nm, 230 nm, 245 nm, 254 nm, 280 nm, and 310 nm. See details [link to relevant documentation]. Figure 3 The chromatogram contains the most comprehensive information and has a stable baseline when the detection wavelength is 230 nm, so this method is selected as the detection wavelength condition. The selection was based on column temperature (25℃, 30℃, 35℃), see appendix for details. Figure 4 The column temperature was 30℃, which resulted in the best peak elution and good separation of the components. Therefore, the column temperature of 30℃ was finally selected. This embodiment screens for flow rates (0.6 mL / min, 0.8 mL / min, 1.0 mL / min), see details below. Figure 5The peak elution was optimal at a flow rate of 0.8 mL / min, and the separation of each component was better. Therefore, the final flow rate was selected as 0.8 mL / min. After determining the optimal detection wavelength, column temperature, and flow rate, this invention screened mobile phases (methanol-water, methanol-0.1% formic acid, acetonitrile-0.1% formic acid, methanol-0.1% phosphoric acid, acetonitrile-0.1% phosphoric acid, and acetonitrile-water) through extensive experiments. See the appendix for details. Figure 6 Finally, methanol-0.1% phosphoric acid was selected as the mobile phase, and the elution program was optimized. Some of the elution programs are shown in Tables 3 to 8: Table 4 Elution Procedure 1

[0027] Table 5 Washing Procedure 2

[0028] Table 6 Elution Procedure 3

[0029] Table 7 Elution Procedure 4

[0030] Table 8 Elution Procedure 6

[0031] Test results as follows Figure 7 As shown, elution program 5 exhibits good resolution, high peak height, stable baseline, and complete chromatographic information. Therefore, elution program 5 is selected as the optimal elution program.

[0032] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0033] Example 1 Based on the above-mentioned optimization conditions, this embodiment provides a method for constructing a fingerprint spectrum of Tongning tablets based on overall quality evaluation.

[0034] S1. Preparation of the test solution Weigh 2.0 g of the contents of 15 batches of Tongning tablets and place them in a stoppered conical flask. Add 20 mL of 80% methanol solution, extract by sonication for 30 min, filter, concentrate and dry the filtrate under reduced pressure, add 5 mL of 80% methanol solution to dissolve, filter, and filter the filtrate through a 0.45 µm microporous membrane to obtain the Tongning tablet test solution.

[0035] S2. Preparation of reference solution Accurately weigh 5-hydroxymethylfurfural, chlorogenic acid, ferulic acid, verrucoside glucoside, and verrucoside, and dissolve them in pure methanol solution to prepare a single reference solution containing 0.89 mg of 5-hydroxymethylfurfural, 0.24 mg of chlorogenic acid, 0.39 mg of ferulic acid, 0.29 mg of verrucoside glucoside, and 0.19 mg of verrucoside per 1 mL.

[0036] S3, Chromatographic Analysis The test solution of Tongning tablets in S1 and the reference solution in S2 were injected separately into a high-performance liquid chromatograph (HPLC) for chromatographic analysis. Detection was performed under the same conditions using a Vanquish HPLC system. The HPLC conditions were as follows: column: Shim-pack VP-ODS (250 mm × 4.6 mm, 5 μm); detector: diode array detector, detection wavelength: 230 nm; flow rate: 0.8 mL / min; injection volume: 10 μL; column temperature: 30℃; mobile phase: methanol (A) - 0.1% phosphoric acid aqueous solution (D), gradient elution, using the elution program parameters shown in Table 3. The chromatograms of the different single reference solutions are shown below. Figures 8-13 As shown, the chromatogram of the test solution of Tongning tablets is as follows. Figure 37 As shown.

[0037] S4. Reliability Verification The chromatograms of different batches of Tongning tablets obtained in S3 were imported into the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (Version 2004 A); chromatograms of different batches of Tongning tablets were selected ( Figure 37 Chromatographic peaks present in all samples were taken as common peaks. A reference chromatogram of Tongning tablets was generated using the average value calculation method. The retention time and peak area of ​​each common peak were calculated. After data import, multi-point correction and data matching, similarity analysis was performed. A similarity result table between the chromatograms of different batches of Tongning tablet test solutions and the common peak patterns was obtained and exported. The reliability of the results was confirmed based on the similarity results and the chromatograms of the Tongning tablet test solutions.

[0038] S5. High-performance liquid chromatography-mass spectrometry analysis Based on the chromatogram of the Tongning tablets test solution obtained in S3, high-resolution mass spectrometry (HMS) analysis was performed on the Tongning tablets test solution. The HMS detection conditions were: electrospray ionization, spray voltage 3500 V, sheath gas flow rate 40 arb, auxiliary gas flow rate 10 arb, capillary temperature 300℃, auxiliary gas temperature 300℃, full scan mode, and mass-to-charge ratio scan range m / z 100-1500. The total ion chromatogram was obtained. Figure 14 and Figure 15The total ion chromatogram was imported into Xcalibur software, and data analysis was performed based on the peak elution of the test solution obtained in S3 to obtain the mass spectrometry results of each chemical component. Figures 16-36 ).

[0039] S6, fingerprint spectrum of Tongning tablets Based on the chromatograms of the test solution and the single reference solution of Tongning tablets obtained in S3, and by comparing them with the total ion chromatogram and the mass spectrometry results of the chemical components obtained by high-resolution mass spectrometry, a total of 5 chromatographic peaks were identified. Among them, peak 2 is 5-hydroxymethylfurfural, with a retention time of 12.459 min; peak 5 is chlorogenic acid, with a retention time of 26.365 min; peak 7 is ferulic acid, with a retention time of 39.604 min; peak 8 is romaine isoflavone glucoside, with a retention time of 40.362 min; and peak 13 is romaine isoflavone, with a retention time of 58.015 min. The fingerprint spectrum of Tongning tablets was constructed using the method described above. Figure 38 ).

[0040] By comparing the high-performance liquid chromatograms (HPLC) of five reference standards, including 5-hydroxymethylfurfural, and combining the HPLC-MS component analysis results of extracts from nine medicinal herbs in the formulation (Astragalus membranaceus, Ginseng, Atractylodes macrocephala, Angelica sinensis, Polygonum multiflorum, Ligusticum chuanxiong, processed Aconitum carmichaelii, processed Aconitum kusnezoffii, and Glycyrrhiza uralensis) in the previous invention, the chemical components corresponding to the five characteristic peaks in the fingerprint spectrum of Tongning tablets were assigned. Among them, peak 2 (5-hydroxymethylfurfural) originated from Polygonum multiflorum and Astragalus membranaceus; peak 5 (chlorogenic acid) originated from Angelica sinensis; peak 7 (ferulic acid) originated from Angelica sinensis and Ligusticum chuanxiong; and peaks 8 (verruca flavonoid glucoside) and 13 (verruca flavonoids) originated from Astragalus membranaceus.

[0041] Example 2 This embodiment conducts methodological research based on the above construction method.

[0042] (1) Similarity study In this embodiment, an automatically generated reference chromatogram R is used to generate a common chromatographic peak pattern. The analysis and calculation show that the common chromatographic peaks of different batches of Tongning tablets have relatively good similarity, indicating that the fingerprint chromatogram of Tongning tablets established by this method can effectively detect the quality of Tongning tablets (Table 9).

[0043] Table 9. Similarity between each batch of Tongning tablets and the common chromatographic peak pattern

[0044] (2) Precision study The test solution of the Tongning tablets prepared by the method in Example 1 was analyzed according to the detection method in Example 1. Six parallel injections were performed with an injection volume of 10 μL each. The peak area and retention time of the common characteristic peaks were analyzed, and the RSD values ​​were calculated (see Table 10). As shown in Table 10, the RSD of the retention time was less than 0.048%, and the RSD of the peak area was less than 2.674%, indicating that the parallel injection precision of this device is good.

[0045] Table 10. Peak area and retention time of common characteristic peaks for each batch of Tongning tablets, with calculated RSD values.

[0046] (3) Stability study The test solution prepared by the method in Example 1 was analyzed according to the detection method in Example 1. Injections were performed at different time points (0, 2, 6, 12, 18, and 24 h) with an injection volume of 10 μL. The peak areas and retention times of the common characteristic peaks in the HPLC fingerprint of the samples were analyzed, and the RSD values ​​were calculated (see Table 11). As shown in Table 11, the RSD of the retention time was less than 0.031%, and the RSD of the peak area was less than 2.935%, indicating that the chromatographic peaks of the Tongning tablet test solution showed almost no change within 24 h, demonstrating good stability.

[0047] Table 11. Peak areas and retention times of common characteristic peaks in Tongning tablet samples at different times, and RSD values ​​were calculated.

[0048] (4) Reproducibility studies Six batches of test solutions were prepared according to the method for preparing the test solution in Example 1. Under the same chromatographic conditions as in Example 1, the injection volume was 10 μL. The peak area and retention time of the common characteristic peaks in the HPLC fingerprint of the samples were analyzed, and the RSD values ​​were calculated (see Table 12). As shown in Table 12, the RSD of the retention time was less than 0.094%, and the RSD of the peak area was less than 1.443%, indicating good reproducibility of the chromatographic peaks and good repeatability of the method.

[0049] Table 12. Peak areas and retention times of common characteristic peaks in each batch of Tongning tablets, with calculated RSD values.

[0050] The above experimental results show that the fingerprint spectrum construction method for Tongning tablets provided by the present invention has the characteristics of good stability, high precision and good repeatability, and can comprehensively and objectively evaluate the quality of Tongning tablets, providing quality assurance for clinical efficacy.

[0051] The above description is merely an example and illustration of the concept of the present invention. Any modifications or additions made by those skilled in the art to the specific embodiments described, or any substitutions made in a similar manner, as long as they do not deviate from the inventive concept or exceed the scope defined by the claims, shall fall within the protection scope of the present invention.

Claims

1. A method for constructing a fingerprint spectrum of Tongning tablets based on overall quality evaluation, characterized in that, Includes the following steps: S1. Using different batches of Tongning tablets as test samples, prepare Tongning tablet test sample solutions; using 5-hydroxymethylfurfural, chlorogenic acid, ferulic acid, verrucoside glucoside and verrucoside as reference standards, prepare single reference standard solutions. S2. Perform high-performance liquid chromatography analysis on the test solution and reference solution of Tongning tablets, and record the corresponding chromatograms; S3. Perform similarity analysis on the chromatogram of the Tongning tablet test solution obtained in S2 to confirm the reliability of the results; S4. Perform high-resolution mass spectrometry analysis on the test solution obtained in S1 to obtain the total ion chromatogram; Data analysis was performed based on the total ion chromatogram and the peak elution of the Tongning tablet test solution obtained in S2 to obtain the mass spectrometry results of each chemical component; S5. Compare the chromatograms of the test solution and reference solution of Tongning tablets obtained in S2, the total ion chromatogram obtained in S4, and the mass spectrometry results of each chemical component to obtain the HPLC fingerprint of Tongning tablets composed of common characteristic peaks.

2. The method for constructing the fingerprint spectrum of Tongning tablets based on overall quality evaluation according to claim 1, characterized in that, In S1, different batches of Tongning tablets were taken as test samples, 80% methanol solution was added, ultrasonic extraction was performed, the sample was filtered, the filtrate was concentrated and dried under reduced pressure, 80% methanol solution was added, the sample was dissolved, filtered, and Tongning tablets test sample solution was obtained.

3. The method for constructing the fingerprint spectrum of Tongning tablets based on overall quality evaluation according to claim 1, characterized in that, In S1, the single reference solution of 5-hydroxymethylfurfural contained 0.89 mg of 5-hydroxymethylfurfural per 1 mL of methanol solution; the single reference solution of chlorogenic acid contained 0.24 mg of chlorogenic acid per 1 mL of methanol solution; the single reference solution of ferulic acid contained 0.39 mg of ferulic acid per 1 mL of methanol solution; the single reference solution of verbascoside glucoside contained 0.29 mg of verbascoside glucoside per 1 mL of methanol solution; and the single reference solution of verbascoside contained 0.19 mg of verbascoside per 1 mL of methanol solution.

4. The method for constructing the fingerprint spectrum of Tongning tablets based on overall quality evaluation according to claim 1, characterized in that, In S2, the high-performance liquid chromatography (HPLC) conditions are as follows: Shim-pack VP-ODS column; mobile phase A is methanol, mobile phase D is 0.1% phosphoric acid, detection wavelength is 210–310 nm, column temperature is 25–35 °C, flow rate is 0.6–1.0 mL / min, and gradient elution program is as follows: 。 5. The method for constructing the fingerprint spectrum of Tongning tablets based on overall quality evaluation according to claim 1, characterized in that, S3 specifically involves: importing the chromatograms of different batches of Tongning tablets test solutions obtained in S2 into the Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System; selecting chromatographic peaks present in all batches of Tongning tablets test solutions as common peaks, generating a reference chromatogram of Tongning tablets using the average value calculation method, and calculating the retention time and peak area of ​​each common peak; performing similarity analysis after data import, multi-point correction, and data matching; obtaining and exporting a similarity result table between the chromatograms of different batches of Tongning tablets test solutions and the common peak patterns; and confirming the reliability of the results based on the similarity result table and the chromatograms of the Tongning tablets test solutions.

6. The method for constructing the fingerprint spectrum of Tongning tablets based on overall quality evaluation according to claim 1, characterized in that, In S4, the detection conditions for 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℃, auxiliary gas temperature of 300℃, scanning mode of full scan mode, and mass-to-charge ratio scanning range of 100-1500 m / z.

7. The method for constructing the fingerprint spectrum of Tongning tablets based on overall quality evaluation according to claim 1, characterized in that, S5 specifically involves comparing the chromatograms of the test solution and the reference solution obtained in S2 with the total ion chromatogram and the mass spectrometry results of the chemical components obtained by high-resolution mass spectrometry in S4. A total of 5 chromatographic peaks were identified. Among them, peak 2 is 5-hydroxymethylfurfural with a retention time of 12.459 min; peak 5 is chlorogenic acid with a retention time of 26.365 min; peak 7 is ferulic acid with a retention time of 39.604 min; peak 8 is verbascoside glucoside with a retention time of 40.362 min; and peak 13 is verbascoside with a retention time of 58.015 min. The HPLC fingerprint of Tongning tablets was thus obtained.

8. The application of the HPLC fingerprint of Tongning tablets obtained by the method for constructing the fingerprint spectrum of Tongning tablets based on overall quality evaluation as described in any one of claims 1 to 7 in the detection of the quality of Tongning tablets.

9. A method for testing the quality of Tongning tablets, characterized in that, The HPLC fingerprint of Tongning tablets obtained by the method for constructing the fingerprint spectrum of Tongning tablets based on overall quality evaluation as described in any one of claims 1 to 7 is used to detect the quality of Tongning tablets or its equivalent drugs, with one or more of 5-hydroxymethylfurfural, chlorogenic acid, ferulic acid, verrucoside glucoside and verrucoside as quality markers.

10. A method for detecting the quality of Tongning tablets according to claim 9, characterized in that, The material basis of Tongning tablets or its namesake drugs includes the following detected components: 5-hydroxymethylfurfural, chlorogenic acid, ferulic acid, verrucoside glucoside and verrucoside.

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