Establishment of HPLC fingerprint of Yiqihuoxue Zhongyao granules and its application

Abstract

This invention relates to a method for constructing and applying an HPLC fingerprint of a traditional Chinese medicine granule for invigorating qi and promoting blood circulation, belonging to the field of biomedical technology. This invention provides a method for establishing an HPLC fingerprint of the traditional Chinese medicine granule Qishen Jianxin Granules and its application. The application includes using HPLC fingerprinting with isoflavone glucoside, glycyrrhizin, and ammonium glycyrrhizate as references to achieve quality control of the Qishen Jianxin Granules, and quantifying key components by determining the content of astragaloside A. This invention provides a traditional Chinese medicine granule with definite efficacy, strict quality control, and stable controllability, and its quality control method. It achieves overall characteristic control through HPLC fingerprinting and quantifies key components through the determination of astragaloside A content, providing dual assurance of preparation quality and efficacy. It is highly practical and has good industrialization and promotion value.

Description

Technical Field

[0001] This invention relates to a method for constructing and applying HPLC fingerprints of traditional Chinese medicine granules that invigorate qi and promote blood circulation, belonging to the field of biomedical technology. Background Technology

[0002] Qishen Jianxin Granules are granules prepared from extracts of Astragalus membranaceus, Sophora flavescens, and honey-processed Glycyrrhiza uralensis. This product is an in-house preparation of Zhongshan Hospital affiliated with Fudan University and has a 30-year history of clinical application. Clinically, it is used to treat viral myocarditis and related premature beats and arrhythmias, possessing the effects of invigorating qi and blood circulation, strengthening the body's foundation, and detoxifying. Myocarditis is clinically common and prone to protracted illness; invigorating qi and blood circulation, strengthening the body's foundation, and detoxifying are commonly used and effective treatments in traditional Chinese medicine. Traditional Chinese medicine granules are widely used due to their convenience and stable efficacy. However, current quality control methods often rely on single-component quantification, which is insufficient to comprehensively reflect the overall quality of the preparation, and batch-to-batch consistency is difficult to control precisely. Fingerprint spectroscopy, as a core technology for the overall quality evaluation of traditional Chinese medicine, can achieve "holistic identification" through multiple characteristic peaks. However, existing methods suffer from insufficient specificity, complex gradient procedures, and poor reproducibility. Therefore, there is an urgent need to establish a fingerprint spectroscopy construction method with strong specificity, good reproducibility, and simple operation to meet the quality control requirements of traditional Chinese medicine granules, which is of great significance for improving the quality controllability and clinical stability of traditional Chinese medicine preparations. Summary of the Invention

[0003] The purpose of this invention is to solve the technical problems of quality control for the traditional Chinese medicine granule "Qi Shen Jian Xin Granules". This invention provides a method for establishing the fingerprint spectrum of "Qi Shen Jian Xin Granules" and the fingerprint spectrum thereof, overcoming the shortcomings of existing product quality control technologies, making its quality control more comprehensive and scientific, and achieving the purpose of controlling product quality and monitoring the safe production of pharmaceuticals. It solves the problems of incompleteness and poor reproducibility of existing quality control methods, providing a traditional Chinese medicine granule with definite efficacy, strict quality control, and stable controllability, and its quality control method.

[0004] To address the aforementioned problems, the present invention provides an application of HPLC fingerprinting for a traditional Chinese medicine granule, namely, Qishen Jianxin Granules. This application includes using HPLC fingerprinting with isoflavone glucoside, glycyrrhizin, and ammonium glycyrrhizate as references to achieve quality control of Qishen Jianxin Granules, and quantifying key components by determining the content of astragaloside A.

[0005] Preferably, the astragaloside A content is not less than 1 mg per bag of Qishen Jianxin Granules, calculated as astragaloside A.

[0006] This invention provides a method for establishing an HPLC fingerprint of the traditional Chinese medicine granule Qishen Jianxin Granules, comprising the following steps:

[0007] Step 1, Preparation of reference solution: Take appropriate amounts of verbenafil glucoside reference standard, glycyrrhizin reference standard and glycyrrhizic acid ammonium reference standard, accurately weigh them, add 60% methanol to prepare a solution containing 20 μg of verbenafil glucoside, 0.1 mg of glycyrrhizin and 0.3 mg of glycyrrhizic acid ammonium per ml.

[0008] Step 2, Preparation of the test solution: Take about 1g of the test sample, accurately weigh it, place it in a stoppered conical flask, accurately add 10ml of 60% methanol, stopper tightly, weigh it, sonicate at 250W, 40kHz for 10 minutes, cool it, weigh it again, replenish the lost weight with 60% methanol, shake well, let it stand, take the supernatant, filter it, and take the filtrate to obtain the test solution;

[0009] Step 3, Determination: Accurately pipette 10 μL each of the reference solution and the test solution into the liquid chromatograph, determine and record the chromatograms, and compare the chromatograms.

[0010] Preferably, the detection conditions for the high-performance liquid chromatograph in step 3 above are as follows:

[0011] Chromatographic column: Agilent ZORBAX SB-C18 with octadecylsilane-bonded silica gel as the packing material, 25 cm in length, 4.6 mm in inner diameter, and 5 μm in particle size; flow rate: 1 ml / min; column temperature: 25 °C; detection wavelength: 230 nm.

[0012] Mobile phase: Acetonitrile was used as mobile phase A, and 0.05% formic acid solution was used as mobile phase B;

[0013] Perform gradient elution as specified in the table below; the theoretical plate number, calculated based on the glycyrrhizin peak, should be no less than 5000.

[0014] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0-50 3→55 97→45 50-52 55→70 45→30 52-57 70 30 57-59 70→3 30→97 59-65 3 97

[0015] .

[0016] Preferably, in step 3 above, the HPLC fingerprint chromatogram of the test sample should show chromatographic peaks with the same retention time as the reference peak; according to the similarity evaluation system for chromatographic fingerprint chromatograms of traditional Chinese medicine, the similarity between the fingerprint chromatogram of the test sample and the reference fingerprint chromatogram should not be less than 0.90.

[0017] Preferably, the chromatographic peaks with the same retention time include three peaks: verbascoside, glycyrrhizin, and ammonium glycyrrhizate; the HPLC fingerprint of Astragalus and Ginseng Heart-Strengthening Granules includes three peaks: verbascoside, glycyrrhizin, and ammonium glycyrrhizate.

[0018] This invention provides an application of the HPLC fingerprint of a traditional Chinese medicine granule, Qishen Jianxin Granules. The application further includes the determination of astragaloside A content, the determination comprising the following steps:

[0019] Step 4: Preparation of reference solution: Take an appropriate amount of astragaloside A reference standard, accurately weigh it, and add methanol to prepare a solution containing 0.4 mg per ml.

[0020] Step 5: Preparation of the test solution: Take the Astragalus and Ginseng Heart-Strengthening Granules from the "Containment Weight Difference" section, grind them into a fine powder, weigh approximately 5g, place them in a stoppered conical flask, accurately add 50ml of 70% methanol, seal tightly, weigh, heat under reflux for 1 hour, cool, weigh again, replenish the lost weight with 70% methanol, shake well, filter, accurately measure 25ml of the filtrate, evaporate to dryness, add 25ml of water to dissolve the residue, extract 4 times with water-saturated n-butanol, 25ml each time, combine the n-butanol solutions, wash 3 times with ammonia solution, 30ml each time, discard the washings, evaporate the n-butanol solution to dryness, add methanol to dissolve the residue, transfer to a 5ml volumetric flask, add methanol to the mark, shake well, filter, and take the filtrate to obtain the test solution.

[0021] Step 6, Determination: Accurately pipette 5 μl, 10 μl, or 20 μl of the reference solution and 10–20 μl of the test solution into the liquid chromatograph, respectively, and determine the result by calculating the logarithmic equation using the external standard two-point method; each bag of Qishen Jianxin Granules contains not less than 1 mg of astragalus, calculated as astragaloside A.

[0022] Preferably, the detection conditions of the high performance liquid chromatograph in step 6 are as follows: chromatographic column: octadecylsilane-bonded silica gel as the packing material; mobile phase: acetonitrile-water 32:68 as the mobile phase; detection by evaporative light scattering detector; the theoretical plate number calculated based on the astragaloside A peak should not be less than 4000.

[0023] This invention provides a method for establishing an HPLC fingerprint of Astragalus and Ginseng Heart-Strengthening Granules. The method uses the similarity evaluation of chromatographic fingerprints of traditional Chinese medicine. The HPLC fingerprint of Astragalus and Ginseng Heart-Strengthening Granules established in step 3 above is compared with the fingerprint of a reference substance. If the similarity is ≥0.9, the product is considered qualified.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] This invention enables comprehensive quality control of traditional Chinese medicine (TCM) preparations, better aligning with the characteristics of TCM compound formulas. Utilizing HPLC fingerprinting technology, it comprehensively reflects the overall information of multiple chemical components in the preparation, shifting from "single-component control" to "holistic characteristic control," thus more scientifically and realistically reflecting the multi-component, multi-target, and synergistic effects of TCM compound formulas. This invention possesses strong specificity, effectively identifying authenticity and quality. The method provided is stable, reliable, and reproducible; it is simple to operate, highly efficient, and widely applicable; and it achieves a high level of quality control, meeting modern TCM standards.

[0026] This invention provides a Chinese herbal medicine granule with definite efficacy, strict quality control, and stable and controllable quality, as well as its quality control method. It achieves overall characteristic control through HPLC fingerprinting and quantifies key components through astragaloside A content determination, thus providing dual protection for the quality and efficacy of the preparation. It is highly practical and has good industrialization and application value. Attached Figure Description

[0027] Figure 1 This is a comparative fingerprint spectrum for the present invention. Detailed Implementation

[0028] To make the present invention more apparent and understandable, preferred embodiments are described in detail below:

[0029] Example

[0030] I. Obtaining fingerprint patterns:

[0031] 1.1 Determine the fingerprint analysis conditions

[0032] According to the determination method of high performance liquid chromatography (General Chapter 0512, Part IV, Chinese Pharmacopoeia 2025 Edition), the acetonitrile-phosphoric acid, acetonitrile-acetic acid, and acetonitrile-formic acid systems, as well as different formic acid ratios, column temperatures, and chromatographic columns, were investigated. The gradient elution program was optimized, and the fingerprint analysis conditions were determined by combining the chromatographic results at different wavelengths during full-wavelength scanning. Simultaneously, the methanol concentration in the extraction solvent, the sample amount, and the ultrasonic time were investigated to determine the preparation method of the sample solution. All conditions used should ensure good precision, repeatability, and stability. Different brands of chromatographic columns showed slight differences in the separation of certain characteristic peaks; ultimately, an Agilent ZORBAX SB-C18 (25cm × 4.6mm, 5μm) column was selected.

[0033] 1.2 Chromatographic conditions and system suitability test:

[0034] The column was packed with octadecylsilane-bonded silica gel (Agilent ZORBAX SB-C18, 25 cm column length, 4.6 mm inner diameter, 5 μm particle size); acetonitrile was used as mobile phase A, and 0.05% formic acid solution was used as mobile phase B, with gradient elution performed according to the specifications in the table below; the flow rate was 1 ml / min; the column temperature was 25 °C; and the detection wavelength was 230 nm. The theoretical plate number, calculated based on the glycyrrhizin peak, should be no less than 5000.

[0035] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0-50 3→55 97→45 50-52 55→70 45→30 52-57 70 30 57-59 70→3 30→97 59-65 3 97

[0036] Preparation of reference solution: Take appropriate amounts of verbenafil glucoside reference standard, glycyrrhizin reference standard and glycyrrhizic acid ammonium reference standard, accurately weigh them, add 60% methanol to prepare a solution containing 20 μg of verbenafil glucoside, 0.1 mg of glycyrrhizin and 0.3 mg of glycyrrhizic acid ammonium per ml.

[0037] Preparation of the test solution: Weigh about 1g of this product accurately, place it in a stoppered conical flask, add 10ml of 60% methanol accurately, stopper tightly, weigh, sonicate (250W, 40kHz) for 10 minutes, cool, weigh again, replenish the lost weight with 60% methanol, shake well, let stand, take the supernatant, filter, and take the filtrate to obtain the test solution.

[0038] Determination method: Accurately pipette 10 μL each of the reference solution and the test solution into the liquid chromatograph, measure and record the chromatogram to obtain the result.

[0039] The fingerprint chromatogram of the test sample should show chromatographic peaks with the same retention times as the reference peaks. Calculated using the similarity evaluation system for chromatographic fingerprints of traditional Chinese medicine, the similarity between the fingerprint chromatogram of the test sample and the reference fingerprint chromatogram should not be less than 0.90. The reference fingerprint chromatogram is as follows: Figure 1 Peak 1 is citriol isoflavone glucoside, peak 2 is glycyrrhizin, and peak 3 is ammonium glycyrrhizate.

[0040] II. Content Determination

[0041] In the "Qi Shen Jian Xin Granules" formula, Astragalus membranaceus is used in the largest quantity. Astragaloside A is the main component of Astragalus membranaceus. Modern pharmacological studies have shown that Astragaloside A inhibits myocardial fibrosis by regulating mitochondrial autophagy. Therefore, this experiment selected Astragaloside A as an indicator component and measured its content to control the quality of the preparation and ensure clinical efficacy.

[0042] Referring to the methods for determining the content of astragaloside A in Astragalus membranaceus raw materials, Astragalus membranaceus granules, and other compound preparations containing Astragalus membranaceus, a method for determining the content of Astragaloside A in Qishen Jianxin Granules was developed. The extraction method (ammonia hydrolysis and ultrasonication followed by reflux) and washing method (ammonia solution and sodium hydroxide solution) were investigated. The method showed good specificity, linearity, precision, and accuracy. Based on the content results from different batches, the limit for the content of astragaloside A was determined to be: each bag of this product contains astragaloside A (C... 41 H 68 O 14 The total dose shall not be less than 1 mg.

[0043] Chromatographic conditions and system suitability test

[0044] Octadecylsilane-bonded silica gel was used as the filler; acetonitrile-water (32:68) was used as the mobile phase; and evaporative light scattering detector was used for detection. The theoretical plate number, calculated based on the astragaloside A peak, should be no less than 4000.

[0045] Preparation of reference solution

[0046] Take an appropriate amount of astragaloside A reference standard, accurately weigh it, and add methanol to prepare a solution containing 0.4 mg per 1 ml.

[0047] Preparation of test solution

[0048] Take the product from the "Variation in Content" section, grind it into a fine powder, accurately weigh about 5g, place it in a stoppered conical flask, accurately add 50ml of 70% methanol, seal tightly, weigh, heat under reflux for 1 hour, cool, weigh again, replenish the lost weight with 70% methanol, shake well, filter, accurately measure 25ml of the subsequent filtrate, evaporate to dryness, dissolve the residue in 25ml of water, extract 4 times with water-saturated n-butanol, 25ml each time, combine the n-butanol extracts, wash 3 times with ammonia solution, 30ml each time, discard the washings, evaporate the n-butanol extract to dryness, dissolve the residue in methanol, transfer to a 5ml volumetric flask, add methanol to the mark, shake well, filter, and collect the subsequent filtrate to obtain the final product.

[0049] Determination method

[0050] Accurately pipette 5 μl (or 10 μl) and 20 μl of the reference solution, and 10–20 μl of the test solution, respectively, into the liquid chromatograph, determine the result, and calculate using the logarithmic equation of the external standard two-point method.

[0051] Each sachet of this product contains astragalus root extract as astragaloside A (C). 41 H 68 O 14 The total dose shall not be less than 1 mg.

[0052] III. Use of this invention:

[0053] This invention provides a method for constructing and applying an HPLC fingerprint of a traditional Chinese medicine granule for invigorating qi and promoting blood circulation. The method achieves overall characteristic control through HPLC fingerprinting and quantifies key components through astragaloside A content determination, thus providing dual assurance for the quality and efficacy of the preparation. The specific steps are as follows:

[0054] Step 1: Obtain and compare fingerprint patterns;

[0055] Step 1.1 Preparation of reference solution: Take appropriate amounts of verbenafil glucoside reference standard, glycyrrhizin reference standard and glycyrrhizic acid ammonium reference standard, accurately weigh them, add 60% methanol to prepare a solution containing 20 μg of verbenafil glucoside, 0.1 mg of glycyrrhizin and 0.3 mg of glycyrrhizic acid ammonium per ml.

[0056] Step 1.2, Preparation of the test solution: Take about 1g of the test sample, accurately weigh it, place it in a stoppered conical flask, accurately add 10ml of 60% methanol, stopper tightly, weigh it, sonicate (250W, 40kHz) for 10 minutes, cool it, weigh it again, replenish the lost weight with 60% methanol, shake well, let it stand, take the supernatant, filter it, and take the filtrate to obtain the test solution;

[0057] Step 1.3, Determination: Accurately pipette 10 μL each of the reference solution and the test solution into the liquid chromatograph, determine and record the chromatograms, and compare the chromatograms.

[0058] Step 2, Astragaloside A (C 41 H 68 O 14 Content determination:

[0059] Step 2.1 Preparation of reference solution: Take an appropriate amount of astragaloside A reference standard, accurately weigh it, and add methanol to prepare a solution containing 0.4 mg per ml.

[0060] Step 2.2, Preparation of the test solution: Take the test sample from the content difference section, grind it finely, take about 5g, accurately weigh it, place it in a stoppered conical flask, accurately add 50ml of 70% methanol, stopper tightly, weigh it, heat under reflux for 1 hour, cool, weigh it again, replenish the lost weight with 70% methanol, shake well, filter, accurately measure 25ml of the subsequent filtrate, evaporate to dryness, add 25ml of water to dissolve the residue, extract 4 times with water-saturated n-butanol, 25ml each time, combine the n-butanol solutions, wash 3 times with ammonia solution, 30ml each time, discard the washings, evaporate the n-butanol solution to dryness, add methanol to dissolve the residue, transfer it to a 5ml volumetric flask, add methanol to the mark, shake well, filter, and take the subsequent filtrate to obtain the test solution.

[0061] Step 2.3, Determination: Accurately pipette 5 μl (or 10 μl) and 20 μl of the reference solution and 10–20 μl of the test solution into the liquid chromatograph, respectively, and determine the result by calculating the logarithmic equation using the external standard two-point method.

[0062] It must be guaranteed that each bag of this product contains astragalus root extract as astragaloside A (C). 41 H 68 O 14 The total dose shall not be less than 1 mg.

[0063] "Astragalus and Ginseng Heart-Strengthening Granules"

[0064]

Functions and Indications

[0065]

Dosage and Administration

[0066]

Specifications

[0067] [Storage] Sealed.

[0068] [Validity Period] Tentatively 24 months.

[0069] Currently, traditional Chinese medicine compound preparations used for myocarditis and improving cardiac function are mostly composed of multiple herbs such as Astragalus membranaceus and Glycyrrhiza uralensis, resulting in complex compositions and unclear pharmacodynamic material bases. Existing quality control methods generally suffer from the following defects: 1. They only measure the content of a single component, failing to reflect the overall chemical information of the compound and thus unable to comprehensively control the quality of the preparation; 2. They lack detection methods capable of overall identification and evaluation of batch-to-batch consistency, easily leading to problems where different batches of products have similar appearance and taste but significant differences in internal quality; 3. They lack highly specific characteristic chromatograms or fingerprint spectra, making it difficult to effectively distinguish genuine from counterfeit products and identify differences in raw material sources and preparation processes; 4. Existing methods have poor reproducibility and weak specificity, failing to meet the quality control requirements of modern, standardized, and large-scale production of traditional Chinese medicine. To overcome the shortcomings of the above-mentioned existing technologies, ensure stable, uniform, and controllable preparation quality, and guarantee reliable clinical efficacy, this invention provides a highly specific, reproducible, and comprehensive HPLC fingerprint quality control method capable of evaluating the overall quality of the preparation.

[0070] The advantages of this invention are:

[0071] 1. This invention enables overall quality control of traditional Chinese medicine preparations, which is more in line with the characteristics of traditional Chinese medicine compound prescriptions. This invention uses HPLC fingerprinting technology, which can comprehensively reflect the overall information of multiple chemical components in the preparation, realizing the transformation from "single component control" to "overall characteristic control", and more scientifically and realistically reflecting the essence of multi-component, multi-target, and synergistic effects of traditional Chinese medicine compound prescriptions.

[0072] 2. This invention has strong specificity and can effectively distinguish between genuine and counterfeit products and between superior and inferior products. This invention uses isoflavone glucoside, glycyrrhizin, and ammonium glycyrrhizate as references to establish specific, stable, and repeatable characteristic peaks, which can quickly distinguish between genuine, counterfeit, and inferior products, and solve the problems of traditional methods being difficult to qualitatively identify and easily confused.

[0073] 3. The method provided by this invention is stable, reliable, and reproducible. This invention optimizes chromatographic conditions, gradient elution procedures, and extraction methods. Methodological validation shows that the precision, repeatability, stability, and robustness are all good.

[0074] 4. This invention is simple to operate, has high detection efficiency, and is highly applicable; the test sample of this invention is extracted by ultrasound, which is simple and time-saving; the chromatographic conditions are mild and versatile, requiring no complicated pretreatment, making it suitable for daily rapid testing in pharmaceutical factories and drug testing institutes, and can be used for quality monitoring of raw materials, intermediates, and finished products throughout the entire process.

[0075] 5. This invention has a high level of quality control and meets the requirements of modern Chinese medicine standards. The method of this invention fully complies with the high performance liquid chromatography guidelines in the Chinese Pharmacopoeia. It is technologically advanced, has clear indicators, and allows for data traceability, which significantly improves the quality control level of preparations and provides strong assurance for the safety, efficacy, and quality control of drugs.

[0076] 6. This invention achieves overall characteristic control through HPLC fingerprinting and quantifies key components through astragaloside A content determination, thus providing dual assurance for the quality and efficacy of the preparation. It forms a complete technical solution with outstanding innovation and strong practicality, and has good industrialization and protection value.

[0077] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any form or substance. It should be noted that those skilled in the art can make various improvements and additions without departing from the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention. Any modifications, alterations, and equivalent changes made by those skilled in the art based on the above-disclosed technical content without departing from the spirit and scope of the present invention are equivalent embodiments of the present invention. Furthermore, any modifications, alterations, and evolutions made to the above embodiments based on the essential technology of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. The application of an HPLC fingerprint of a traditional Chinese medicine granule, Qishen Jianxin Granules, characterized in that, The application includes quality control of Astragalus and Ginseng Heart-Strengthening Granules by using HPLC fingerprinting with isoflavone glucoside, glycyrrhizin, and ammonium glycyrrhizate as references, and quantitative determination of key components by measuring the content of astragaloside A.

2. The application according to claim 1, characterized in that, The astragaloside A content is no less than 1 mg per bag of Qishen Jianxin Granules, calculated as astragaloside A.

3. A method for establishing an HPLC fingerprint of the traditional Chinese medicine granule Qishen Jianxin Granules, characterized in that, Includes the following steps: Step 1, Preparation of reference solution: Take appropriate amounts of verbenafil glucoside reference standard, glycyrrhizin reference standard and glycyrrhizic acid ammonium reference standard, accurately weigh them, add 60% methanol to prepare a solution containing 20 μg of verbenafil glucoside, 0.1 mg of glycyrrhizin and 0.3 mg of glycyrrhizic acid ammonium per ml. Step 2, Preparation of the test solution: Take about 1g of the test sample, accurately weigh it, place it in a stoppered conical flask, accurately add 10ml of 60% methanol, stopper tightly, weigh it, sonicate at 250W, 40kHz for 10 minutes, cool it, weigh it again, replenish the lost weight with 60% methanol, shake well, let it stand, take the supernatant, filter it, and take the filtrate to obtain the test solution; Step 3, Determination: Accurately pipette 10 μL each of the reference solution and the test solution into the liquid chromatograph, determine and record the chromatograms, and compare the chromatograms.

4. The method for establishing an HPLC fingerprint of the traditional Chinese medicine granule Qishen Jianxin Granules according to claim 3, characterized in that, The detection conditions for the high-performance liquid chromatograph in step 3 are as follows: Chromatographic column: Agilent ZORBAX SB-C18 with octadecylsilane-bonded silica gel as the packing material, 25 cm in length, 4.6 mm in inner diameter, and 5 μm in particle size; flow rate: 1 ml / min; column temperature: 25 °C; detection wavelength: 230 nm. Mobile phase: Acetonitrile was used as mobile phase A, and 0.05% formic acid solution was used as mobile phase B; Perform gradient elution as specified in the table below; the theoretical plate number, calculated based on the glycyrrhizin peak, should be no less than 5000. 。 5. The method for establishing an HPLC fingerprint of the traditional Chinese medicine granule Qishen Jianxin Granules according to claim 3, characterized in that, In step 3, the HPLC fingerprint chromatogram of the test sample should show chromatographic peaks with the same retention time as the reference peak; according to the similarity evaluation system for chromatographic fingerprint chromatograms of traditional Chinese medicine, the similarity between the fingerprint chromatogram of the test sample and the reference fingerprint chromatogram should not be less than 0.

90.

6. The method for establishing an HPLC fingerprint of the traditional Chinese medicine granule Qishen Jianxin Granules according to claim 5, characterized in that, The chromatographic peaks with the same retention time include three peaks: verbascoside, glycyrrhizin, and ammonium glycyrrhizate; the HPLC fingerprint of Astragalus and Ginseng Heart-Strengthening Granules includes three peaks: verbascoside, glycyrrhizin, and ammonium glycyrrhizate.

7. The application according to claim 1, characterized in that: The application also includes the determination of astragaloside A content, the determination comprising the following steps: Step 4: Preparation of reference solution: Take an appropriate amount of astragaloside A reference standard, accurately weigh it, and add methanol to prepare a solution containing 0.4 mg per ml. Step 5: Preparation of the test solution: Take the Astragalus and Ginseng Heart-Strengthening Granules from the "Containment Weight Difference" section, grind them into a fine powder, weigh approximately 5g, place them in a stoppered conical flask, accurately add 50ml of 70% methanol, seal tightly, weigh, heat under reflux for 1 hour, cool, weigh again, replenish the lost weight with 70% methanol, shake well, filter, accurately measure 25ml of the filtrate, evaporate to dryness, add 25ml of water to dissolve the residue, extract 4 times with water-saturated n-butanol, 25ml each time, combine the n-butanol solutions, wash 3 times with ammonia solution, 30ml each time, discard the washings, evaporate the n-butanol solution to dryness, add methanol to dissolve the residue, transfer to a 5ml volumetric flask, add methanol to the mark, shake well, filter, and take the filtrate to obtain the test solution. Step 6, Determination: Accurately pipette 5 μl, 10 μl, or 20 μl of the reference solution and 10–20 μl of the test solution into the liquid chromatograph and determine the result. Calculate the result using the logarithmic equation with external standard two-point method. Each bag of Qishen Jianxin Granules contains not less than 1 mg of astragalus, calculated as astragaloside A.

8. The application according to claim 7, characterized in that, The detection conditions for the high performance liquid chromatograph in step 6 are as follows: chromatographic column: octadecylsilane-bonded silica gel as the packing material; mobile phase: acetonitrile-water 32:68 as the mobile phase; detection by evaporative light scattering detector; the theoretical plate number calculated based on the astragaloside A peak should not be less than 4000.

9. The application of a method for establishing an HPLC fingerprint of Astragalus and Ginseng Heart-Strengthening Granules, characterized in that: The HPLC fingerprint of Astragalus and Ginseng Heart-Strengthening Granules established in step 3 of claim 3 was evaluated for similarity with the fingerprint of the reference substance. If the similarity is ≥0.9, the product is qualified.

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