A fingerprint spectrum of a new Xilekang tablet, a construction method and application thereof, and a multi-index component detection method of the Xilekang tablet

Abstract

This invention provides a fingerprint spectrum of Xinlekang tablets, its construction method, and its application, as well as a multi-index component detection method for Xinlekang tablets, relating to the field of pharmaceutical analysis and structural technology. The invention uses chloroform and ammonia as extraction solvents, a C18 column, acetonitrile as mobile phase A, and 0.38–0.42 v / v% phosphoric acid – 0.04–0.06 v / v% triethylamine aqueous solution as mobile phase B. A gradient elution method is employed, with controlled gradient elution program and detection wavelength controlled at 202–206 nm. The test solution and mixed reference solution of Xinlekang tablets are analyzed by high-performance liquid chromatography (HPLC), and the chromatograms of the two solutions are compared and analyzed. The constructed fingerprint spectrum of Xinlekang tablets includes 28 characteristic peaks, including isorhamnetin, rhynchophylline, and reserpine. The abundant chromatographic peaks effectively characterize the quality of Xinlekang tablets, facilitating comprehensive monitoring of drug quality and possessing high application value.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical analysis structure technology, specifically to a fingerprint spectrum of Xinlekang tablets and its construction method and application, and a multi-index component detection method for Xinlekang tablets. Background Technology

[0002] Insomnia is a subjective experience characterized by difficulty falling asleep and / or maintaining sleep, resulting in insufficient sleep quality or quantity to meet normal physiological needs and thus affecting daytime social functioning. It is the most common sleep disorder. Clinically used medications mainly include barbiturates, benzodiazepines, non-benzodiazepines, and traditional Chinese medicine. Compared to Western medicine, traditional Chinese medicine has fewer side effects and can fundamentally solve the problem of insomnia, making it widely accepted by patients.

[0003] Xinlekang tablets have the effects of calming the mind, nourishing the liver, and soothing the nerves. They are suitable for neurasthenia, symptoms such as insomnia, dreaminess, palpitations, and dizziness. It is a compound traditional Chinese medicine tablet composed of three herbs: Uncaria rhynchophylla, Ziziphus jujuba var. spinosa, and Rauvolfia var. sarcodactylis. Uncaria rhynchophylla, the principal herb, has effects such as protecting nerves, lowering blood pressure, protecting the myocardium, anti-cancer, anti-inflammatory, and relieving asthma. Ziziphus jujuba var. spinosa, the secondary herb, has effects such as sedation and hypnosis, anti-anxiety, anti-depression, protecting nerves, protecting the cardiovascular system, protecting the liver, and anti-oxidation. Rauvolfia var. sarcodactylis, another secondary herb, has effects such as lowering blood pressure, anti-arrhythmia, and clearing heat and detoxifying. Xinlekang tablets contain multiple traditional Chinese medicine herbs, making it important to effectively characterize the overall quality of the medicine. To date, there are no reports on using fingerprinting methods for quality control of Xinlekang tablets. Therefore, establishing a fingerprint that can comprehensively reflect the overall quality of Xinlekang tablets is of great significance. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide a fingerprint spectrum of Xinlekang tablets, its construction method and application, and a multi-index component detection method for Xinlekang tablets. The fingerprint spectrum of Xinlekang tablets constructed by this invention has a large number of characteristic peaks, which can comprehensively reflect the overall quality of Xinlekang tablets and is beneficial for comprehensive monitoring of drug quality.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides a method for constructing the fingerprint spectrum of Xinlekang tablets, comprising the following steps:

[0007] The powder of Xinlekang tablets, ammonia and chloroform were mixed and extracted to obtain an extract. The extract was then concentrated and dissolved in methanol to obtain a test solution.

[0008] The test solution and the mixed reference solution were subjected to high performance liquid chromatography (HPLC) to obtain the sample HPLC chromatogram and the reference HPLC chromatogram, respectively. The fingerprint chromatogram of Xinlekang tablets was obtained based on the sample HPLC chromatogram and the reference HPLC chromatogram.

[0009] The reference standards in the mixed control solution include isorhamnetin, rhododendronine, and reserpine;

[0010] The high-performance liquid chromatography (HPLC) detection conditions include: a C18 column; mobile phase A is acetonitrile, and mobile phase B is an aqueous solution of phosphate-triethylamine, wherein the volume fraction of phosphate in mobile phase B is 0.38–0.42%, and the volume fraction of triethylamine is 0.04–0.06%; the elution method is gradient elution, and the gradient elution program includes: 0–15 min, increasing the volume fraction of mobile phase A from 14–16% to 20–22%; 15–30 min, increasing the volume fraction of mobile phase A from 20–22% to 24–26%; 30–65 min, increasing the volume fraction of mobile phase A further... The volume fraction of mobile phase A increases from 24-26% to 39-41%; after 65-70 minutes, the volume fraction of mobile phase A increases from 39-41% to 48-52%; after 70-71 minutes, the volume fraction of mobile phase A increases from 48-52% to 88-92%; after 71-75 minutes, the volume fraction of mobile phase A is 88-92%; after 75-76 minutes, the volume fraction of mobile phase A decreases from 88-92% to 14-16%; after 76-81 minutes, the volume fraction of mobile phase A is 14-16%; the detection wavelength is 202-206 nm.

[0011] The fingerprint spectrum of the Xinlekang tablets contains 28 characteristic peaks, including characteristic peaks of isorhizine, rhizoma lucidae, and reserpine.

[0012] Preferably, the solid-liquid ratio of the powder of the Xinlekang tablet to the ammonia solution is 1g:1.8-2.2mL; and the mass fraction of the ammonia solution is 25-28%.

[0013] The solid-liquid ratio of the powder to chloroform in the Xinlekang tablets is 1g: 24-26mL.

[0014] Preferably, the extraction is reflux extraction, and the extraction time is 28–32 min.

[0015] Preferably, in the mixed reference solution, the concentration of isorhamnetin is 7.6–7.8 μg / mL, the concentration of rhynchophylline is 7.9–8.1 μg / mL, and the concentration of reserpine is 4.7–4.9 μg / mL;

[0016] The solvent in the mixed reference solution includes methanol.

[0017] Preferably, the conditions for high performance liquid chromatography detection further include: a mobile phase flow rate of 0.7–0.9 mL / min, a diode array detector, a column temperature of 28–32 °C, and an injection volume of 9.5–10.5 μL.

[0018] Preferably, the similarity between the liquid chromatography chromatogram of the sample and the liquid chromatography chromatogram of the reference standard is calculated by the software "Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine" and is both >0.95.

[0019] The present invention also provides a fingerprint spectrum of Xinlekang tablets obtained by the fingerprint spectrum construction method described above, which contains 28 characteristic peaks, including characteristic peaks of isorhamnetin, rhynchophylline and reserpine.

[0020] This invention also provides the application of the fingerprint spectrum of Xinlekang tablets described in the above technical solution in the quality control of Xinlekang tablets.

[0021] This invention also provides a method for detecting multiple components of Xinlekang tablets, comprising the following steps:

[0022] The powder of the test tablets, ammonia and chloroform were mixed and extracted to obtain an extract. The extract was then concentrated and dissolved in methanol to obtain the test sample solution.

[0023] The sample solution to be tested was subjected to high performance liquid chromatography to obtain the detection results of multiple indicators of Xinlekang tablets; the multiple indicators include at least two of isorhizine, rhizine and reserpine.

[0024] The high-performance liquid chromatography (HPLC) detection conditions include: a C18 column; mobile phase A is acetonitrile, and mobile phase B is an aqueous solution of phosphate-triethylamine, wherein the volume fraction of phosphate in mobile phase B is 0.38–0.42%, and the volume fraction of triethylamine is 0.04–0.06%; the elution method is gradient elution, and the gradient elution program includes: 0–15 min, increasing the volume fraction of mobile phase A from 14–16% to 20–22%; 15–30 min, increasing the volume fraction of mobile phase A from 20–22% to 24–26%; 30–65 min, increasing the volume fraction of mobile phase A further... The volume fraction of mobile phase A increases from 24-26% to 39-41%; at 65-70 min, the volume fraction of mobile phase A increases from 39-41% to 48-52%; at 70-71 min, the volume fraction of mobile phase A increases from 48-52% to 88-92%; at 71-75 min, the volume fraction of mobile phase A is 88-92%; at 75-76 min, the volume fraction of mobile phase A decreases from 88-92% to 14-16%; at 76-81 min, the volume fraction of mobile phase A is 14-16%; the detection wavelength is 202-206 nm.

[0025] Preferably, the solid-liquid ratio of the powder of the Xinlekang tablet to the ammonia solution is 1g:1.8-2.2mL; and the mass fraction of the ammonia solution is 25-28%.

[0026] The solid-liquid ratio of the powder to chloroform in the Xinlekang tablets is 1g: 24-26mL;

[0027] The extraction was a reflux extraction, and the extraction time was 28–32 min.

[0028] The conditions for high-performance liquid chromatography detection also include: a mobile phase flow rate of 0.7–0.9 mL / min, a diode array detector, a column temperature of 28–32 °C, and an injection volume of 9.5–10.5 μL.

[0029] Xinlekang tablets are a traditional Chinese medicine compound tablet composed of three medicinal herbs—Uncaria rhynchophylla, Ziziphus jujuba var. spinosa, and Rauvolfia purpurea—along with other excipients. The components of these medicinal herbs are numerous and complex, making extraction and liquid chromatography separation challenging. This invention uses ammonia and chloroform as extraction agents, resulting in a greater number of extracted components. This allows for effective characterization of the quality of Xinlekang tablets and facilitates comprehensive quality monitoring of the drug.

[0030] Since fingerprint spectroscopy is not for determining the precise content of a specific component, but rather for fully reflecting the information of the chemical composition, this invention uses a detection wavelength of 202–206 nm, which results in a large number of peaks, more complete information, good absorbance values ​​for each peak, a stable baseline, and avoids the large absorption of near-ultraviolet impurity peaks. This invention uses a C18 column with acetonitrile as mobile phase A and 0.38–0.42 v / v% phosphoric acid – 0.04–0.06 v / v% triethylamine aqueous solution as mobile phase B. A gradient elution method is employed, and the gradient elution program is controlled. The test solution and mixed reference solution of Xinlekang tablets are separately detected by high-performance liquid chromatography (HPLC), and the HPLC chromatograms of the two are compared and analyzed. The obtained fingerprint spectroscopy of Xinlekang tablets contains 28 characteristic peaks, including characteristic peaks of isorhamnetin, rhynchophylline, and reserpine. The fingerprint spectrum constructed by this invention has abundant chromatographic peaks, which is beneficial for comprehensive monitoring of drug quality. It provides an effective means for comprehensively reflecting, monitoring and evaluating the overall quality control of Xinlekang tablets, guides standardized production, and thus better ensures the quality stability, consistency and controllability of Xinlekang tablets. It also improves the overly simplistic current quality standard of Xinlekang tablets and has high application value.

[0031] The high-performance liquid chromatography fingerprinting method established in this invention achieves, for the first time, the quality control of the entire formula of Xinlekang tablets, rather than the identification of a single compound or medicinal material. It can more effectively guide the feeding, strictly standardize production operations, and improve the safety and effectiveness of clinical medication.

[0032] This invention establishes a method for simultaneous detection of multiple components in Xinlekang tablets. This method has the advantage of quantitative monitoring of multiple components in the same chromatographic system. As a quality control method for Xinlekang tablets, it can comprehensively and accurately evaluate the efficacy, safety, and stability of Xinlekang tablets, and control the raw materials of the product more precisely and comprehensively, thus ensuring the quality and efficacy of the product. Moreover, the detection method of this invention has the advantages of being simple, stable, highly precise, and reproducible. Attached Figure Description

[0033] Figure 1 The HPLC chromatogram of the test solution in Example 1;

[0034] Figure 2 This is a superimposed image of the fingerprint spectra of 12 batches of Xinlekang tablets in Example 1;

[0035] Figure 3 This is a similarity evaluation result chart of 12 batches of Xinlekang tablets in Example 1;

[0036] Figure 4 This is the HPLC chromatogram of the test solution lacking Uncaria rhynchophylla in Example 2;

[0037] Figure 5 This is the HPLC chromatogram of the jujube seed-deficient test solution in Example 2;

[0038] Figure 6 The HPLC chromatogram of the Rauvolfia var. rubrum test solution in Example 2 is shown below.

[0039] Figure 7 The HPLC chromatogram of the Uncaria rhynchophylla test solution in Example 3;

[0040] Figure 8 The HPLC chromatogram of the jujube seed test solution in Example 3 is shown below.

[0041] Figure 9 The HPLC chromatogram of the Rauvolfia test solution in Example 3;

[0042] Figure 10 This is a full-wavelength scan of the diode array detector for the test solution in Example 5;

[0043] Figure 11 The HPLC chromatogram of the test solution in Example 5 using the gradient elution program in Table 5;

[0044] Figure 12 The HPLC chromatogram of the test solution in Example 5 using the gradient elution program shown in Table 6;

[0045] Figure 13 The HPLC chromatogram of the test solution in Example 5 is obtained using the gradient elution program shown in Table 7. Detailed Implementation

[0046] This invention provides a method for constructing the fingerprint spectrum of Xinlekang tablets, comprising the following steps:

[0047] The powder of Xinlekang tablets, ammonia and chloroform were mixed and extracted to obtain an extract. The extract was then concentrated and dissolved in methanol to obtain a test solution.

[0048] The test solution and the mixed reference solution were subjected to high performance liquid chromatography (HPLC) to obtain the sample HPLC chromatogram and the reference HPLC chromatogram, respectively. The fingerprint chromatogram of Xinlekang tablets was obtained based on the sample HPLC chromatogram and the reference HPLC chromatogram.

[0049] The reference standards in the mixed control solution include isorhamnetin, rhododendronine, and reserpine;

[0050] The high-performance liquid chromatography (HPLC) detection conditions include: a C18 column; mobile phase A is acetonitrile, and mobile phase B is an aqueous solution of phosphate-triethylamine, wherein the volume fraction of phosphate in mobile phase B is 0.38–0.42%, and the volume fraction of triethylamine is 0.04–0.06%; the elution method is gradient elution, and the gradient elution program includes: 0–15 min, increasing the volume fraction of mobile phase A from 14–16% to 20–22%; 15–30 min, increasing the volume fraction of mobile phase A from 20–22% to 24–26%; 30–65 min, increasing the volume fraction of mobile phase A further... The volume fraction of mobile phase A increases from 24-26% to 39-41%; after 65-70 minutes, the volume fraction of mobile phase A increases from 39-41% to 48-52%; after 70-71 minutes, the volume fraction of mobile phase A increases from 48-52% to 88-92%; after 71-75 minutes, the volume fraction of mobile phase A is 88-92%; after 75-76 minutes, the volume fraction of mobile phase A decreases from 88-92% to 14-16%; after 76-81 minutes, the volume fraction of mobile phase A is 14-16%; the detection wavelength is 202-206 nm.

[0051] The fingerprint spectrum of the Xinlekang tablets contains 28 characteristic peaks, including characteristic peaks of isorhizine, rhizoma lucidae, and reserpine.

[0052] Unless otherwise specified, the materials and equipment used in this invention are all commercially available products in the field.

[0053] This invention involves mixing the powder of Xinlekang tablets, ammonia, and chloroform to extract the extract, which is then concentrated and dissolved in methanol to obtain a test solution.

[0054] In this invention, the particle size of the powder of the Xinlekang tablets is preferably ≥60 mesh; the powder of the Xinlekang tablets is preferably obtained by grinding the Xinlekang tablets to below 60 mesh after removing the coating.

[0055] In this invention, the mass fraction of the ammonia water is 25-28%, and in specific embodiments it can be 25%, 26%, 27% or 28%; the solid-liquid ratio of the Xinlekang tablet powder to the ammonia water is preferably 1g:1.8-2.2mL, and in specific embodiments it can be 1g:1.8mL, 1g:1.9mL, 1g:2mL, 1g:2.1mL or 1g:2.2mL.

[0056] In this invention, the solid-liquid ratio of the powder of the Xinlekang tablet to chloroform is preferably 1g:24-26mL, and in specific embodiments it can be 1g:24mL, 1g:25mL or 1g:26mL.

[0057] In this invention, the extraction is preferably reflux extraction, and the extraction time is preferably 28 to 32 minutes, which can be 28 minutes, 29 minutes, 30 minutes, 31 minutes or 32 minutes in specific embodiments.

[0058] After extraction, the present invention preferably further includes filtering the obtained extraction system to obtain an extract.

[0059] In this invention, the solid-liquid ratio of the powder of the Xinlekang tablet to methanol is preferably 1g:4.5-5.5mL, and in specific embodiments it can be 1g:4.5mL, 1g:5mL or 1g:5.5mL.

[0060] After dissolution, the present invention preferably further includes filtering the solution obtained by dissolution through a 0.22μm microporous membrane to obtain a test solution.

[0061] After obtaining the test solution, the present invention performs high performance liquid chromatography on the test solution and the mixed reference solution respectively to obtain the sample liquid chromatogram and the reference liquid chromatogram, and obtains the fingerprint spectrum of Xinlekang tablets based on the sample liquid chromatogram and the reference liquid chromatogram.

[0062] In this invention, the reference standards in the mixed reference solution include isorhizine, rhynchophylline, and reserpine. In this invention, the concentration of isorhizine in the mixed reference solution is preferably 7.6–7.8 μg / mL, and in specific embodiments, it can be 7.6 μg / mL, 7.7 μg / mL, or 7.8 μg / mL; the concentration of rhynchophylline is preferably 7.9–8.1 μg / mL, and in specific embodiments, it can be 7.9 μg / mL, 8 μg / mL, or 8.1 μg / mL; the concentration of reserpine is preferably 4.7–4.9 μg / mL, and in specific embodiments, it can be 4.7 μg / mL, 4.8 μg / mL, or 4.9 μg / mL; the solvent in the mixed reference solution preferably includes methanol.

[0063] In this invention, the method for preparing the mixed reference solution preferably includes the following steps: dissolving isorhamnetin reference standard, rhynchophylline reference standard and reserpine reference standard in a solvent, filtering through a 0.22 μm microporous membrane to obtain the mixed reference solution.

[0064] In this invention, the conditions for high-performance liquid chromatography (HPLC) detection include: a C18 column; a mobile phase system comprising mobile phase A and mobile phase B, wherein mobile phase A is acetonitrile and mobile phase B is an aqueous solution of phosphate-triethylamine; the volume fraction of phosphate in mobile phase B is 0.38–0.42%, and in specific embodiments it can be 0.38%, 0.39%, 0.4%, 0.41%, or 0.42%; the volume fraction of triethylamine in mobile phase B is 0.04–0.06%, and in specific embodiments it can be 0.04%, 0.05%, or 0.06%; and the preferred mobile phase flow rate is 0.7–0.9 m / s. The flow rate (L / min) can be 0.7 mL / min, 0.8 mL / min, or 0.9 mL / min in specific embodiments; the detector is preferably a diode array detector with a detection wavelength of 202–206 nm, which can be 202 nm, 203 nm, 204 nm, 205 nm, or 206 nm in specific embodiments; the column temperature is preferably 28–32 °C, which can be 28 °C, 29 °C, 30 °C, 31 °C, or 32 °C in specific embodiments; the injection volume is preferably 9.5–10.5 μL, which can be 9.5 μL, 9.8 μL, 10 μL, 10.2 μL, or 10 μL in specific embodiments.5 μL; the theoretical plate number, calculated based on the Uncaria rhynchophylline peak, should be no less than 20,000; the elution method is gradient elution, and the gradient elution procedure includes: 0–15 min, the volume fraction of mobile phase A is increased from 14–16% to 20–22%, and in a specific embodiment, it can be increased from any one of 14%, 15%, and 16% to any one of 20%, 21%, and 22%; 15–30 min, the volume fraction of mobile phase A is increased from 20–22% to 24–26%, and in a specific embodiment, it can be increased from 15% to 22% to 24–26%. The mobile phase A volume fraction is increased from any one of 20%, 21%, and 22% to any one of 24%, 25%, and 26% over 30-65 minutes; the volume fraction of mobile phase A is increased from 24-26% to 39-41% over 65-70 minutes; in a specific embodiment, the volume fraction of mobile phase A is increased from 39% to 41% to 48-52% over 65-70 minutes. The volume fraction of mobile phase A is increased from any one of 48%, 40%, and 41% to any one of 48%, 49%, 50%, 51%, and 52% over 70-71 minutes; the volume fraction of mobile phase A is increased from 48-52% to 88-92% over 70-71 minutes. In a specific embodiment, it can be increased from any one of 48%, 49%, 50%, 51%, and 52% to any one of 88%, 89%, 90%, 91%, and 92% over 71-75 minutes; the volume fraction of mobile phase A is 88-92%. In a specific embodiment, the volume fraction can be 88%, 89%, 90%, 91%, or 92%; after 75-76 minutes, the volume fraction of mobile phase A decreases from 88-92% to 14-16%, and in a specific embodiment, it can decrease from any one of 88%, 89%, 90%, 91%, and 92% to any one of 14%, 15%, and 16%; after 76-81 minutes, the volume fraction of mobile phase A is 14-16%, and in a specific embodiment, it can be 14%, 15%, or 16%.

[0065] This invention preferably imports the sample liquid chromatography chromatogram into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system to generate a common pattern of characteristic chromatograms, resulting in a fingerprint chromatogram containing 28 characteristic peaks. By comparing the retention times of the fingerprint chromatogram with those of the reference liquid chromatography chromatogram (based on the chromatographic peaks of the reference solution), the fingerprint chromatogram includes characteristic peaks of isorhamnetin, rhynchophylline, and reserpine. In this invention, the sample liquid chromatography chromatogram is preferably calculated using the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" software, and the common peaks with a similarity > 0.95 are considered the fingerprint chromatogram of Xinlekang tablets. In this invention, the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" software is preferably "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System Software (2012A Version)". This invention preferably uses the average method for similarity analysis, with the time window width preferably set to 0.1 min, and identifies a total of 28 common peaks. In an embodiment of the present invention, 12 batches of Xinlekang tablets were specifically analyzed by high performance liquid chromatography, and the obtained chromatograms of the test samples were imported into a similarity evaluation system for chromatographic fingerprinting of traditional Chinese medicine for similarity analysis. The similarity of each batch of Xinlekang tablets was greater than 0.95, and more preferably greater than 0.99.

[0066] In this invention, by comparing the retention time of the fingerprint spectrum with that of the reference liquid chromatography spectrum, it was found that peak 10 in the fingerprint spectrum is isorhamnetin, peak 15 is rhynchophylline, peak 28 is reserpine, peaks 2, 13, 16, 22 and 27 belong to Ziziphus jujuba var. spinosa, peaks 1-12 and 15-27 belong to Uncaria rhynchophylla, and peaks 4, 6, 8, 11, 14, 20, 27 and 28 belong to Rauvolfia purpurea.

[0067] Traditional Chinese medicine (TCM) fingerprinting is an internationally recognized research model and technical platform for controlling the quality of TCM. By analyzing the types and content distribution of effective and ineffective components in TCM, it transforms TCM research methods and quality analysis techniques from analyzing one or a few active ingredients to comprehensively analyzing the entire chemical fingerprint spectrum of TCM, offering universality and cost-effectiveness. TCM fingerprinting reflects the synergistic effects, multi-level complexity, and holistic nature of multiple components in TCM. Xinlekang tablets contain multiple TCM herbs, necessitating overall control of their material composition. Therefore, in addition to "microscopic analysis," some form of "macroscopic analysis" should be used to effectively characterize the overall quality of TCM. This invention, by controlling extraction and HPLC detection conditions, can obtain a fingerprint spectrum with 28 characteristic peaks, effectively characterizing the quality of Xinlekang tablets and facilitating comprehensive quality monitoring of the drug.

[0068] The present invention provides a fingerprint spectrum of Xinlekang tablets obtained by the fingerprint spectrum construction method described above, which contains 28 characteristic peaks, including characteristic peaks of isorhamnetin, rhynchophylline and reserpine.

[0069] This invention also provides the application of the fingerprint spectrum of Xinlekang tablets described in the above technical solution in the quality control of Xinlekang tablets.

[0070] This invention also provides a method for detecting multiple components of Xinlekang tablets, comprising the following steps:

[0071] The powder of the test tablets, ammonia and chloroform were mixed and extracted to obtain an extract. The extract was then concentrated and dissolved in methanol to obtain the test sample solution.

[0072] The sample solution to be tested was subjected to high performance liquid chromatography to obtain the detection results of multiple indicators of Xinlekang tablets; the multiple indicators include at least two of isorhizine, rhizine and reserpine.

[0073] The high-performance liquid chromatography (HPLC) detection conditions include: a C18 column; mobile phase A is acetonitrile, and mobile phase B is an aqueous solution of phosphate-triethylamine, wherein the volume fraction of phosphate in mobile phase B is 0.38–0.42%, and the volume fraction of triethylamine is 0.04–0.06%; the elution method is gradient elution, and the gradient elution program includes: 0–15 min, increasing the volume fraction of mobile phase A from 14–16% to 20–22%; 15–30 min, increasing the volume fraction of mobile phase A from 20–22% to 24–26%; 30–65 min, increasing the volume fraction of mobile phase A further... The volume fraction of mobile phase A increases from 24-26% to 39-41%; at 65-70 min, the volume fraction of mobile phase A increases from 39-41% to 48-52%; at 70-71 min, the volume fraction of mobile phase A increases from 48-52% to 88-92%; at 71-75 min, the volume fraction of mobile phase A is 88-92%; at 75-76 min, the volume fraction of mobile phase A decreases from 88-92% to 14-16%; at 76-81 min, the volume fraction of mobile phase A is 14-16%; the detection wavelength is 202-206 nm.

[0074] In this invention, the preparation method of the sample solution to be tested is the same as the preparation method of the test solution; the conditions for high performance liquid chromatography detection are the same as the conditions for high performance liquid chromatography detection in the fingerprint pattern construction process, and will not be described in detail here.

[0075] In this invention, the multi-index component detection results include qualitative detection results and quantitative detection results. Preferably, the qualitative detection results are obtained using the fingerprint spectrum of Xinlekang tablets described in the above technical solution.

[0076] The present invention does not impose any particular limitation on the method for obtaining the quantitative detection results; any quantitative method well known to those skilled in the art can be used.

[0077] The detection method of the present invention has the advantages of being simple, stable, highly precise, and reproducible.

[0078] To further illustrate the present invention, the fingerprint spectrum construction method, preparation method and application of Xinlekang tablets, as well as the multi-index component detection method, are described in detail below with reference to the embodiments. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0079] The instruments used in the following examples were: an Agilent 1260 high-performance liquid chromatograph (Agilent Technologies, USA); the reagents and medicinal materials used were as follows: isorhamnetin reference standard and rhynchophylline reference standard were purchased from Zhuhai Anzhe Biotechnology Co., Ltd., and reserpine reference standard was purchased from the National Institutes for Food and Drug Control. The batch numbers of Xinlekang tablets (purchased from Taiyuan Pharmaceutical Co., Ltd.) are shown in Table 1. In Example 1, 12 batches of Xinlekang tablets were used; in other examples, the batch number of Xinlekang tablets used was S1.

[0080] Table 112 Batch of Lekang Tablets

[0081] S1 231101 S4 231201 S7 240101 S10 240301 S2 231102 S5 231202 S8 240102 S11 240302 S3 231103 S6 231203 S9 240103 S12 240303

[0082] Example 1

[0083] Construction of fingerprint spectrum of Xinlekang tablets

[0084] Preparation of the test solution: Take 10 tablets of Xinlekang (S1), remove the coating, grind into a fine powder (below 60 mesh), accurately weigh 1.0 g, place in a 100 mL round-bottom flask, add 2 mL of ammonia water (mass concentration 25-28%), add 25 mL of chloroform, heat under reflux for 30 min, filter, concentrate the filtrate to dryness, dissolve the residue in methanol, and dilute to a 5 mL volumetric flask. Filter through a 0.22 μm microporous membrane. The filtrate is the test solution. Prepare the test solutions for the other 11 batches of Xinlekang tablets shown in Table 1 according to the above method.

[0085] Preparation of reference solutions: Take isorhynchine reference standards, rhynchophylline reference standards, and reserpine reference standards, dissolve them in methanol and make up to volume to obtain a mixed reference solution with isorhynchine concentration of 7.70 μg / mL, rhynchophylline concentration of 8.00 μg / mL, and reserpine concentration of 4.78 μg / mL. Filter the solution through a 0.22 μm microporous membrane before HPLC.

[0086] The mixed reference solution and the test solution were separately analyzed by HPLC, and the liquid chromatograms were recorded to obtain the reference liquid chromatogram and the sample liquid chromatogram, respectively. The sample liquid chromatograms were imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)" to analyze the similarity of fingerprint chromatograms of 12 batches of Xinlekang tablets. Using S1 as the reference chromatogram, after shearing the solvent peak in the first 5 minutes, the chromatograms of the 12 batches of samples (S1-S12) were matched. The average method was used to generate the reference chromatogram (R), and the time window width was set to 0.1 min. The results are shown in [Figure Number]. Figures 1-3 And Tables 3-4, Figure 1 The HPLC chromatogram of the test solution (Xinlekang tablets S1) is shown below. Figure 2 The above are the HPLC superimposed chromatograms of the test solutions from 12 batches of Xinlekang tablets. Figure 3 This is a similarity evaluation result chart for 12 batches of Xinlekang tablets.

[0087] The HPLC detection conditions were as follows: a C18 column (250 mm × 4.6 mm, 5 μm) was used, with acetonitrile as mobile phase A and 0.4 v / v% phosphoric acid-0.05 v / v% triethylamine aqueous solution as mobile phase B. Gradient elution was performed at a flow rate of 0.8 mL / min and a column temperature of 30 °C. A diode array detector was used with a detection wavelength of 204 nm and an injection volume of 10 μL. The gradient elution program is shown in Table 2. The theoretical plate number, calculated based on the Uncaria rhynchophylla peak, should not be less than 20,000.

[0088] Table 2 Gradient elution program

[0089] 0 15 85 15 21 79 30 25 75 65 40 60 70 50 50 71 90 10 75 90 10 76 15 85 81 15 85

[0090] Table 312 Similarity Evaluation Results of Xinlekang Tablets Samples

[0091]

[0092]

[0093]

[0094] Table 4. Similarity Evaluation Results of 412 Batches of Xinlekang Tablets

[0095]

[0096] For the calibration of fingerprint peaks, the reference standard Uncaria rhynchophylline (S) was selected, and its retention time was set to 1. The relative retention times of the fingerprint peaks were calculated. Based on the test results of 12 batches of samples ( Figures 1-3(See Tables 3-4). Chromatographic peaks with good stability, strong absorption, and distinct characteristics were selected as common peaks. A total of 28 common fingerprint peaks were identified. The results showed that the relative retention time (RSD) of the common peaks of the test samples was <0.7%. The similarity of the 12 batches of test samples ranged from 0.988 to 0.999, indicating that the 12 batches of test samples had a high degree of similarity.

[0097] Example 2

[0098] Negative control experiment

[0099] Preparation of the test solution for Uncaria rhynchophylla: Weigh 7g of Ziziphus jujuba seeds, soak them in 120mL of water for 24h, then decoct twice, the first time for 6h and the second time for 2h. Combine the decoctions and concentrate to dryness. Add 0.04g of total alkaloids of Rauvolfia, add 2mL of ammonia water (mass concentration 25-28%), add 25mL of chloroform, heat under reflux for 30min, filter, concentrate the filtrate to dryness, dissolve the residue in methanol, and make up to 5mL in a volumetric flask. Filter the filtrate through a 0.22μm microporous membrane to obtain the test solution for Uncaria rhynchophylla.

[0100] Preparation of the test solution for jujube seed deficiency: Weigh 14g of Uncaria rhynchophylla, soak in 120mL of water for 24h, then decoct twice, the first time for 6h and the second time for 2h. Combine the decoctions and concentrate to dryness. Add 0.04g of total alkaloids of Rauvolfia, add 2mL of ammonia water (mass concentration 25-28%), add 25mL of chloroform, heat under reflux for 30min, filter, concentrate the filtrate to dryness, dissolve the residue in methanol, and make up to 5mL in a volumetric flask. Filter the filtrate through a 0.22μm microporous membrane to obtain the test solution for jujube seed deficiency.

[0101] Preparation of Rauvolfia elegans test solution: Weigh 14g of Uncaria rhynchophylla and 7g of Ziziphus jujuba var. spinosa, add 120mL of water and soak for 24h, then decoct twice, the first time for 6h and the second time for 2h. Combine the decoctions and concentrate to dryness, add 2mL of ammonia water (mass concentration 25-28%), add 25mL of chloroform, heat under reflux for 30min, filter, concentrate the filtrate to dryness, dissolve the residue in methanol, and make up to 5mL in a volumetric flask. Filter the filtrate through a 0.22μm microporous membrane to obtain the Rauvolfia elegans test solution.

[0102] According to the HPLC detection conditions in Example 1, each negative test solution was subjected to HPLC detection.

[0103] Figure 4 The HPLC chromatogram of the test solution lacking Uncaria rhynchophylla is shown. Figure 5 The HPLC chromatogram of the test solution for jujube seed deficiency is shown. Figure 6 The HPLC chromatogram of the Rauvolfia var. rubrum test solution shows that there is no interference from any negative test sample, and the resolution of each component peak is greater than 1.5.

[0104] Example 3

[0105] Identification and attribution of common peaks

[0106] Preparation of Uncaria rhynchophylla test solution: Weigh 14g of Uncaria rhynchophylla, soak in 120mL of water for 24h, then decoct twice, the first time for 6h and the second time for 2h. Combine the decoctions and concentrate to dryness. Add 2mL of ammonia water (mass concentration 25-28%), add 25mL of chloroform, heat under reflux for 30min, filter, concentrate the filtrate to dryness, dissolve the residue in methanol, and make up to 5mL in a volumetric flask. Filter the filtrate through a 0.22μm microporous membrane to obtain the Uncaria rhynchophylla test solution.

[0107] Preparation of jujube seed test solution: Weigh 7g of jujube seeds, soak in 120mL of water for 24h, then decoct twice, the first time for 6h and the second time for 2h. Combine the decoctions and concentrate to dryness. Add 2mL of ammonia water (mass concentration 25-28%), add 25mL of chloroform, heat under reflux for 30min, filter, concentrate the filtrate to dryness, dissolve the residue in methanol, and make up to 5mL in a volumetric flask. Filter the filtrate through a 0.22μm microporous membrane to obtain the jujube seed test solution.

[0108] Preparation of Rauvolfia test solution: Weigh 0.04 g of total alkaloids from Rauvolfia, add 120 mL of water and soak for 24 h, then decoct twice, the first time for 6 h and the second time for 2 h. Combine the decoctions and concentrate to dryness, add 2 mL of ammonia water (mass concentration 25-28%), add 25 mL of chloroform, heat under reflux for 30 min, filter, concentrate the filtrate to dryness, dissolve the residue in methanol, and make up to 5 mL in a volumetric flask. Filter the filtrate through a 0.22 μm microporous membrane to obtain the Rauvolfia test solution.

[0109] According to the HPLC detection conditions in Example 1, the test solutions of each single medicinal material were subjected to HPLC detection.

[0110] Figure 7 The HPLC chromatogram of the Uncaria rhynchophylla test solution is shown. Figure 8 The HPLC chromatogram of the jujube seed sample solution is shown. Figure 9 HPLC chromatogram of the Rauvolfia test solution. Figures 1-2 and Figures 7-9 Comparison yielded the fingerprint spectrum of Xinlekang tablets, identifying three components: isorhizine (peak 10), rhizine (peak 15), and reserpine (peak 28). Figure 1 In comparison, peaks 2, 13, 16, 22, and 27 belong to Ziziphus jujuba var. spinosa; peaks 1–12 and 15–27 belong to Uncaria rhynchophylla; and peaks 4, 6, 8, 11, 14, 20, 27, and 28 belong to Rauvolfia purpurea.

[0111] This invention established HPLC fingerprint chromatograms of 12 batches of Xinlekang tablets, with a similarity greater than 0.988. Through systematic component identification and attribution of single medicinal materials with common peaks in different batches of the drug, it comprehensively reflects the chemical information contained in Xinlekang tablets and the current status of each component, which can more comprehensively reflect the quality of Xinlekang tablets and provide a reference for the quality control of Xinlekang tablets.

[0112] Example 4

[0113] Investigation of the preparation method of the test solution

[0114] The HPLC detection conditions were the same as in Example 1.

[0115] (1) Examination of extraction methods

[0116] Common extraction methods include reflux extraction, maceration extraction, ultrasonic extraction, and Soxhlet extraction. Because Soxhlet extraction is complicated, reflux extraction, maceration extraction, and ultrasonic extraction were investigated.

[0117] Reflux extraction: the method for preparing the test solution in Example 1.

[0118] Immersion extraction: The heating reflux extraction for 30 min in the preparation process of the test sample solution in Example 1 was replaced with immersion extraction at room temperature for 12 h.

[0119] Ultrasonic extraction: The heating reflux extraction for 30 min in the preparation process of the test solution in Example 1 was replaced by ultrasonic extraction for 30 min at room temperature, power 220 W, and frequency 40 kHz.

[0120] HPLC analysis of each test solution showed that the chromatographic area of ​​reflux extraction was significantly larger than that of maceration extraction and ultrasonic extraction. Therefore, reflux extraction was selected.

[0121] (2) Investigation of extraction solvent

[0122] The extraction effects of methanol, ethanol, chloroform, and dichloromethane were investigated. In the preparation of the test solution in Example 1, chloroform was replaced with ethanol, chloroform, and dichloromethane, respectively. HPLC analysis of each test solution showed that the chloroform extract had the most chromatographic peaks, while the methanol and ethanol extracts had significantly fewer peaks than the chloroform extract, and the dichloromethane extract had fewer peaks than the trichloromethane extract. Therefore, chloroform, which had the most extraction peaks, was selected as the extraction solvent.

[0123] (3) Examination of extraction time

[0124] The test solution was prepared according to the method described in Example 1, with extraction times of 20 min, 30 min, and 40 min. HPLC analysis of each test solution showed that the extraction effects were similar across the three methods, but the total peak area was largest when refluxed for 30 min. Therefore, a reflux time of 30 min was selected.

[0125] Example 5

[0126] Investigation of the detection method for the test sample

[0127] (1) Investigation of detection wavelength

[0128] The test solution prepared in Example 1 was scanned across the entire wavelength range using a diode array detector, and the spectra were analyzed. The results are shown in the figure. Figure 10 It can be seen that the HPLC chromatogram at a wavelength of 204 nm has the most chromatographic peaks and the best separation effect. Therefore, 204 nm was selected as the determination wavelength for fingerprint chromatogram.

[0129] (2) Investigation of the composition of the mobile phase

[0130] The test solution was analyzed by HPLC according to the method of Example 1, with the only difference being that the mobile phase B was 0.2 v / v% phosphoric acid aqueous solution and 0.4 v / v% phosphoric acid aqueous solution, respectively. The results showed that the separation of each component in the chromatogram of the test solution was better when using the acetonitrile-0.4 v / v% phosphoric acid aqueous solution elution system.

[0131] The effect of the amount of triethylamine added in mobile phase B (0.05 v / v%, 0.06 v / v%, and 0.07 v / v%) on peak shape and separation efficiency was further investigated. The results showed that the elution effect was best when 0.05 v / v% triethylamine was added. Therefore, acetonitrile was selected as mobile phase A, and 0.4 v / v% phosphoric acid-0.05 v / v% triethylamine-water solution was selected as mobile phase B for gradient elution.

[0132] (3) Examination of gradient elution procedure

[0133] The test solution was analyzed by HPLC according to the method of Example 1, the only difference being that the gradient elution programs are shown in Tables 5-7, and the HPLC chromatograms of the test solution are shown in Tables 5-7. Figure 11 (Table 5) Figure 12 (Table 6) and Figure 13 (Table 7)

[0134] Table 5 Gradient elution program

[0135] 0 20 80 20 25 75 55 40 60 70 70 30 71 90 10 75 90 10 80 20 80 90 20 80

[0136] Table 6 Gradient elution program

[0137] 0 15 85 30 25 75 65 40 60 80 70 30 81 90 10 85 90 10 86 15 85 91 15 85

[0138] Table 7 Gradient elution program

[0139] 0 20 80 10 25 75 20 25 75 25 40 60 35 40 60 50 70 30 51 90 10 55 90 10 60 20 80 65 20 80

[0140] By comparison Figure 1 and Figures 11-13 It can be seen that, compared with the gradient elution programs in Tables 5-7, the gradient elution program shown in Table 2 results in more chromatographic peaks and better separation of each peak.

[0141] Example 6

[0142] Methodological investigation

[0143] Stability Test: Test solutions were prepared according to the method in Example 1. HPLC analysis was performed at 0h, 2h, 6h, 10h, 24h, and 48h after preparation (detection conditions were the same as in Example 1). Fingerprint chromatograms were recorded, and the RSD values ​​of the relative peak area and relative retention time of each common peak were calculated. Results showed that the RSD of the relative retention time of common peaks with a single peak area greater than 5% of the total peak area was less than 1%, and the RSD of the relative peak area was less than 3%. Visual observation of the overall appearance of the chromatographic fingerprint chromatograms of the five test samples showed no significant changes. The data files were imported into the "Similarity Evaluation System for Chromatographic Fingerprint Spectra of Traditional Chinese Medicine (2012 Edition)" for similarity evaluation. Results showed that the similarity of the fingerprint chromatograms of each test sample was greater than 0.98, indicating that the samples had good stability within 48h, meeting the requirements of fingerprint chromatographic research techniques.

[0144] Precision test: 15 μL of the same test sample solution was accurately pipetted and measured five times consecutively, recording the chromatograms. Using rhizome alkaloid as a reference peak, the relative retention time and peak area ratio of each characteristic peak were calculated, and their RSD values ​​were also calculated. The results showed that the RSD of the relative retention time of common peaks with a single peak area greater than 5% of the total peak area was less than 1%, and the RSD of the relative peak area was less than 3%. Visual observation of the overall appearance of the five test sample chromatographic fingerprints showed no significant changes. The data files were imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)" for similarity evaluation. The results showed that the similarity of the fingerprint chromatograms of each test sample was greater than 0.99, indicating good instrument precision and meeting the requirements of fingerprint chromatographic research techniques.

[0145] Reproducibility Test: Following the method in Example 1, five parallel solutions of the same batch of test sample powder were prepared and analyzed by HPLC, and the chromatograms were recorded. Using rhynchophylline as a reference peak, the relative retention time and peak area ratio of each characteristic peak were calculated, and their RSD values ​​were also calculated. The results showed that the RSD of the relative retention time of common peaks with a single peak area greater than 5% of the total peak area was less than 1%, and the RSD of the relative peak area was less than 3%. The overall appearance of the chromatographic fingerprints of the five test samples showed no significant changes. The data files were imported into the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine (2012 Edition)" for similarity evaluation. The results showed that the similarity of the fingerprints of each test sample was greater than 0.98, indicating good repeatability of the method and meeting the requirements of fingerprint research techniques.

[0146] Traditional Chinese medicine (TCM) fingerprinting is an internationally recognized research model and technical platform for controlling the quality of TCM. By analyzing the types and content distribution of effective and ineffective components in TCM, it transforms TCM research methods and quality analysis techniques from analyzing one or a few active ingredients to comprehensively analyzing the entire chemical fingerprint spectrum of TCM, offering universality and cost-effectiveness. TCM fingerprinting reflects the synergistic effects, multi-level complexity, and holistic nature of multiple components in TCM. Xinlekang tablets contain multiple TCM herbs, necessitating overall control of their material composition. In addition to "microscopic analysis," some form of "macroscopic analysis" should be used to effectively characterize the overall quality of TCM. This invention, by controlling extraction and HPLC detection conditions, can obtain a fingerprint spectrum with 28 characteristic peaks, effectively characterizing the quality of Xinlekang tablets and facilitating comprehensive monitoring of drug quality. Furthermore, the method of this invention has the advantages of simplicity, stability, high precision, and good reproducibility.

[0147] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for constructing a fingerprint spectrum of Xinlekang tablets, comprising the following steps: The powder of Xinlekang tablets, ammonia and chloroform were mixed and extracted to obtain an extract. The extract was then concentrated and dissolved in methanol to obtain a test solution. The test solution and the mixed reference solution were subjected to high performance liquid chromatography (HPLC) to obtain the sample HPLC chromatogram and the reference HPLC chromatogram, respectively. The fingerprint chromatogram of Xinlekang tablets was obtained based on the sample HPLC chromatogram and the reference HPLC chromatogram. The reference standards in the mixed control solution include isorhamnetin, rhododendronine, and reserpine; The high-performance liquid chromatography (HPLC) detection conditions include: a C18 column; mobile phase A is acetonitrile, and mobile phase B is an aqueous solution of phosphate-triethylamine, wherein the volume fraction of phosphate in mobile phase B is 0.38-0.42%, and the volume fraction of triethylamine is 0.04-0.06%; the elution method is gradient elution, and the gradient elution program includes: 0-15 min, increasing the volume fraction of mobile phase A from 14-16% to 20-22%; 15-30 min, increasing the volume fraction of mobile phase A from 20-22% to 24-26%; 30-65 min, increasing the volume fraction of mobile phase A further... The volume fraction of mobile phase A increases from 24-26% to 39-41%; after 65-70 min, the volume fraction of mobile phase A increases from 39-41% to 48-52%; after 70-71 min, the volume fraction of mobile phase A increases from 48-52% to 88-92%; after 71-75 min, the volume fraction of mobile phase A is 88-92%; after 75-76 min, the volume fraction of mobile phase A decreases from 88-92% to 14-16%; after 76-81 min, the volume fraction of mobile phase A is 14-16%; the detection wavelength is 202-206 nm. The fingerprint spectrum of the Xinlekang tablets contains 28 characteristic peaks, including characteristic peaks of isorhizine, rhizoma lucidae, and reserpine.

2. The fingerprint map construction method according to claim 1, characterized in that, The solid-liquid ratio of the powder to ammonia in the Xinlekang tablets is 1g:1.8~2.2mL; the mass fraction of the ammonia is 25~28%. The solid-liquid ratio of the powder in the Xinlekang tablets to chloroform is 1g:24~26mL.

3. The fingerprint mapping method according to claim 1 or 2, characterized in that, The extraction was performed by reflux extraction, and the extraction time was 28-32 minutes.

4. The fingerprint map construction method according to claim 1, characterized in that, In the mixed reference solution, the concentration of isorhynchine was 7.6-7.8 μg / mL, the concentration of rhynchophylline was 7.9-8.1 μg / mL, and the concentration of reserpine was 4.7-4.9 μg / mL; The solvent in the mixed reference solution includes methanol.

5. The fingerprint map construction method according to claim 1, characterized in that, The conditions for high-performance liquid chromatography detection also include: a mobile phase flow rate of 0.7~0.9 mL / min, a diode array detector, a column temperature of 28~32℃, and an injection volume of 9.5~10.5 μL.

6. The fingerprint map construction method according to claim 1, characterized in that, The liquid chromatography chromatograms of the samples and the reference samples were calculated using the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine" software, and the similarity was >0.

95.

7. The application of the fingerprint spectrum construction method of Xinlekang tablets according to any one of claims 1 to 6 in the quality control of Xinlekang tablets.

8. A method for detecting multiple components of Xinlekang tablets, comprising the following steps: The powder of the test tablets, ammonia and chloroform were mixed and extracted to obtain an extract. The extract was then concentrated and dissolved in methanol to obtain the test sample solution. The sample solution to be tested was subjected to high performance liquid chromatography to obtain the detection results of multiple index components of Xinlekang tablets; the multiple index components include isorhamnetin, rhynchophylline and reserpine. The high-performance liquid chromatography (HPLC) detection conditions include: a C18 column; mobile phase A is acetonitrile, and mobile phase B is an aqueous solution of phosphate-triethylamine, wherein the volume fraction of phosphate in mobile phase B is 0.38-0.42%, and the volume fraction of triethylamine is 0.04-0.06%; the elution method is gradient elution, and the gradient elution program includes: 0-15 min, increasing the volume fraction of mobile phase A from 14-16% to 20-22%; 15-30 min, increasing the volume fraction of mobile phase A from 20-22% to 24-26%; 30-65 min, increasing the volume fraction of mobile phase A further... The volume fraction of mobile phase A increases from 24-26% to 39-41%; at 65-70 min, the volume fraction of mobile phase A increases from 39-41% to 48-52%; at 70-71 min, the volume fraction of mobile phase A increases from 48-52% to 88-92%; at 71-75 min, the volume fraction of mobile phase A is 88-92%; at 75-76 min, the volume fraction of mobile phase A decreases from 88-92% to 14-16%; at 76-81 min, the volume fraction of mobile phase A is 14-16%; the detection wavelength is 202-206 nm.

9. The multi-index component detection method according to claim 8, characterized in that, The solid-liquid ratio of the powder to ammonia in the Xinlekang tablets is 1g:1.8~2.2mL; the mass fraction of the ammonia is 25~28%. The solid-liquid ratio of the powder in the Xinlekang tablets to chloroform is 1g: 24~26mL; The extraction was a reflux extraction, and the extraction time was 28-32 minutes. The conditions for high-performance liquid chromatography detection also include: a mobile phase flow rate of 0.7~0.9 mL / min, a diode array detector, a column temperature of 28~32℃, and an injection volume of 9.5~10.5 μL.

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