A method for constructing a heart-protecting granule fingerprint spectrum based on HPLC and quantitatively detecting components

Abstract

The application discloses a kind of based on HPLC's Baixin granules fingerprint spectrum construction and component quantitative detection method, belong to the technical field of pharmaceutical analysis.The application uses high performance liquid chromatography (HPLC), Baixin granules are made into test solution, with the chromatogram of single medicine decoction piece solution and standard solution as contrast, the chromatographic peak in test solution is attributed and identified, and the HPLC fingerprint spectrum containing 12 common characteristic peaks is obtained, which lays a foundation for the establishment of comprehensive quality control and quality evaluation method of Baixin granules.The method of the application can also be used for quantitative detection of important components in Baixin granules-naringin and salvianolic acid B, with the advantages of high precision, good reproducibility, good stability, high recovery rate, good durability, accurate determination results, providing technical support for high-precision quality monitoring and evaluation of Baixin granules.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of pharmaceutical analysis, in particular to a method for constructing a Baoxin granules fingerprint and quantitatively detecting components based on HPLC. BACKGROUND

[0002] As one of the major diseases that seriously threaten human health, the incidence and mortality of cardiovascular diseases remain high. Baoxin granules, as a new type of traditional Chinese medicine drug (1.1 category), is composed of multiple Chinese medicinal materials such as Radix Astragali, Rhei Radix et Rhizoma with liquor, and Sanguinaria officinalis (S. officinalis) (which has been disclosed in Chinese patent CN113274454A, published on July 8, 2021) and has great potential in the treatment of cardiovascular diseases. Its multi-component synergistic mechanism is expected to become a new breakthrough in clinical treatment. The characteristics of multi-component and multi-target of traditional Chinese medicine prescriptions make their quality significantly affected by factors such as medicinal materials and process. Traditional detection methods have single dimension and cannot meet the stringent quality requirements of new traditional Chinese medicine drugs (1.1 category).

[0003] High performance liquid chromatography (HPLC) fingerprint technology is an analysis method that can comprehensively characterize the complex chemical components of traditional Chinese medicine. By using the characteristic information such as the relative retention time and peak area ratio of the chromatographic peaks, a specific fingerprint is constructed to comprehensively evaluate the overall quality of traditional Chinese medicine prescriptions. This technology has been widely used in the fields of quality control, process optimization, and authenticity identification of traditional Chinese medicine. However, there is no systematic report on the HPLC fingerprint determination method for Baoxin granules, or there are problems such as poor chromatographic separation effect, low characteristic peak coverage, and insufficient method stability. Therefore, it is of great significance to establish a scientific, reliable, and specific HPLC fingerprint determination method for improving the quality control level of Baoxin granules and ensuring its clinical efficacy and safety. SUMMARY

[0004] In view of the problems existing in the prior art, the present application aims to provide a method for constructing a Baoxin granules fingerprint and quantitatively detecting components based on HPLC, which realizes comprehensive analysis of the chemical components of Baoxin granules and precise control of the quality, meets the stringent quality standard requirements of new traditional Chinese medicine drugs (1.1 category), and provides a reliable quality detection basis for the research, production, and clinical application of Baoxin granules.

[0005] To achieve the above-mentioned purpose, the present application provides the following solutions:

[0006] The present application provides a method for determining the HPLC fingerprint of Baoxin granules, comprising the following steps:

[0007] (1) preparing a test sample solution and a standard solution;

[0008] The preparation method of the test sample solution is as follows: taking Baoxin granules, adding a methanol aqueous solution, and then ultrasonic treatment to obtain the test sample solution;

[0009] (2) performing HPLC chromatographic analysis on the test sample solution and the standard solution to obtain the Baoxin Granules HPLC fingerprint spectrum composed of sample common characteristic peaks;

[0010] The HPLC chromatographic analysis conditions are as follows: octadecylsilane-bonded silica gel is used as the filler, acetonitrile is used as the mobile phase A, and 0.1% phosphoric acid solution is used as the mobile phase B; the detection wavelength is 286 nm; and the elution program is as follows:

[0011]

[0012] Further, the preparation method of the test sample solution is as follows: 0.5 g of the Baoxin Granules is accurately weighed, 50 ml of 60% methanol is added, and ultrasonic treatment is performed under the condition of 1100 W and 40 kHz for 15 minutes; after filtration, the test sample solution is obtained.

[0013] Further, the flow rate of the HPLC chromatographic analysis is 0.6 ml per minute, the column temperature is 30 DEG C, and the sample loading amount is 10 ul.

[0014] According to the determination method, the HPLC chromatographic analysis comprises the step of identifying the sample common characteristic peaks in the chromatogram of the test sample solution by taking the chromatogram of the standard solution as a control.

[0015] Further, the standard solution comprises Danshensu solution, Calycosin-7-glucoside solution, Amoroside solution, Glycyrrhizin solution, Naringin solution, Neohesperidin solution, Rosmarinic acid solution, Alkannin solution, Salvianolic acid B solution and Salvianolic acid A solution prepared by using 60% methanol aqueous solution as a solvent.

[0016] Further, the Baoxin Granules HPLC fingerprint spectrum comprises 12 sample common characteristic peaks, wherein, No. 2 peak is Danshensu, No. 3 peak is Calycosin-7-glucoside, No. 4 peak is Amoroside, No. 5 peak is Glycyrrhizin, No. 6 peak is Naringin, No. 7 peak is Neohesperidin, No. 8 peak is Rosmarinic acid, No. 9 peak is Alkannin, No. 10 peak is Salvianolic acid B, and No. 12 peak is Salvianolic acid A.

[0017] The application also provides the application of the Baoxin Granules HPLC fingerprint spectrum determined by the above determination method in the production process quality control, finished product quality evaluation, batch stability analysis and authenticity identification of the Baoxin Granules.

[0018] The Baoxin Granules HPLC fingerprint spectrum constructed by the above determination method can be widely applied to the process optimization in the research and development process of the Baoxin Granules, the quality monitoring in the production process and the quality evaluation work in the preclinical and clinical research stages.

[0019] The application further provides a quality control method of the Baixin granules, which is realized by quantitatively detecting the contents of naringin and salvianolic acid B in the Baixin granules through the above-mentioned determination method.

[0020] Further, the method comprises the following steps:

[0021] (1) The HPLC chromatographic analysis is performed on the sample to be tested by using the above-mentioned determination method, and the peak areas of naringin and salvianolic acid B are determined;

[0022] (2) The naringin standard solution and the salvianolic acid B standard solution with gradient distribution of concentration are prepared, the peak areas of the standard solution are determined by using the above-mentioned determination method, and the standard curve of "concentration-peak area" is constructed;

[0023] (3) The peak areas of naringin and salvianolic acid B determined in step (1) are substituted into the standard curve, and the contents of naringin and salvianolic acid B in the sample to be tested are calculated.

[0024] Further, the standard solution is prepared by using 60% methanol aqueous solution as a solvent.

[0025] The application discloses the following technical effects:

[0026] 1. Comprehensive nature of the fingerprint spectrum: the HPLC fingerprint spectrum of the Baixin granules constructed by the application through systematic optimization of experimental conditions comprises 12 common characteristic peaks, comprehensively covers the main active ingredients of core medicinal materials such as radix astragali and salvia miltiorrhiza in the Baixin granules, and can completely and accurately present the chemical material basis of the Baixin granules, thereby providing rich and reliable information for quality evaluation.

[0027] 2. Precision of quantitative detection: the application establishes a quantitative analysis method for naringin and salvianolic acid B with key pharmacological activities in the Baixin granules, and exhibits excellent analysis performance through strict methodological verification. In the precision test, the instrument precision RSD value is controlled within 1.5%; in the repeatability test, the similarity of 6 repeated determinations is as high as 0.99 or more; the stability test shows that the test sample solution remains stable within 80 hours, and the RSD value is less than 0.8%; in the sample addition recovery rate test, the recovery rates of naringin and salvianolic acid B are stably 97.2%-98.5% and 96.8%-98.3% respectively; the durability test shows that the method can still maintain stable and reliable detection performance in the face of different instruments, chromatographic columns and experimental condition changes. These excellent indexes ensure the precise control of the quality of the Baixin granules, and meet the high-standard quality requirements of the new Chinese medicine 1.1.

[0028] 3、Technical application value: The determination method and quality control system of the application provide solid technical support for the whole process from research and development to clinical application of Baixin Granules. In the research and development stage, it can help optimize the production process and ensure the stability of drug quality; in the production link, it realizes the precise monitoring of drug quality; in the clinical application, it ensures the consistency and safety of drug efficacy, which has important significance for promoting the development of Baixin Granules as a new Chinese medicine and enhancing the competitiveness of traditional Chinese medicine in the field of cardiovascular disease treatment. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 For the elution gradient of the mobile phase of Example 1;

[0030] Figure 2 For the stacked plot of Example 1 with detection wavelength of 190-230nm; the abscissa is the retention time, and the ordinate is the response signal;

[0031] Figure 3 For the stacked plot of Example 1 with detection wavelength of 240-280nm; the abscissa is the retention time, and the ordinate is the response signal;

[0032] Figure 4 For the stacked plot of Example 1 with detection wavelength of 284-310nm; the abscissa is the retention time, and the ordinate is the response signal;

[0033] Figure 5 For the stacked plot of Example 1 with detection wavelength of 320-360nm; the abscissa is the retention time, and the ordinate is the response signal;

[0034] Figure 6 For the stacked plot of Example 1 with detection wavelength of 370-400nm; the abscissa is the retention time, and the ordinate is the response signal;

[0035] Figure 7 For the stacked plot of the sample extraction method of Example 1;

[0036] Figure 8 For the stacked plot of the sample extraction solvent of Example 1;

[0037] Figure 9 For the comparison chart of the dissolution of the sample by different extraction solvents of Example 1;

[0038] Figure 10 For the stacked plot of the sample extraction time of Example 1;

[0039] Figure 11 For the stacked plot of the sample weight of Example 1;

[0040] Figure 12 For the comparison chart of HPLC atlas of Example 1 with information maximization;

[0041] Figure 13 Fingerprint of Baixin granules for Example 1;

[0042] Figure 14 Fingerprint of Baixin granules for Example 1;

[0043] Figure 15 Specificity of fingerprint of Baixin granules for Example 1;

[0044] Figure 16 Precision of fingerprint of Baixin granules for Example 1;

[0045] Figure 17 Repeatability of fingerprint of Baixin granules for Example 1;

[0046] Figure 18 Intermediate precision of fingerprint of Baixin granules for Example 1;

[0047] Figure 19 Stability of fingerprint of Baixin granules for Example 1;

[0048] Figure 20 Durability test of Baixin granules for Example 1;

[0049] Figure 21 Durability test of Baixin granules for Example 1;

[0050] Figure 22 Durability test of Baixin granules for Example 1;

[0051] Figure 23HPLC stack plot for durability test of Example 1-different brand of column; S1 is ZORBAX SB-C18, S2 is Inertsil ODS-HL, S3 is Ultimate Plus C18, S4 is Inertsustain C18, S5 is ShimNex HEC18-AQ, S6 is Shim-pack GIST C18-AQ;

[0052] Figure 24 Fingerprint of Example 1 control;

[0053] Figure 25 Fingerprint stack plot of Baixin granules sample of Example 1;

[0054] Figure 26 Peak purity investigation plot of Example 2-danshensuan B (left) and naringin (right);

[0055] Figure 27 Specificity investigation plot of Example 2-content determination;

[0056] Figure 28 Linear relationship plot of Example 2-danshensuan B and naringin. DETAILED DESCRIPTION

[0057] The various illustrative embodiments of the present application will now be described in detail, which should be considered to be merely illustrative in nature and not limiting of the application, and which are intended to provide a full and enabling disclosure of certain aspects, features, and embodiments of the present application.

[0058] Example 1

[0059] 1. Optimization of chromatographic conditions

[0060] Preparation of test solution: Take Baixin granules (hereinafter referred to as the product, produced by Zhejiang Kangenbei Pharmaceutical Co., Ltd. according to the following method: accurately take 7g of Astragalus, 5g of Salvia miltiorrhiza, 5g of Ophiopogon, 2g of Poria cocos, 2g of Lantana camara, 1g of Angelica sinensis, 3g of Rehmannia glutinosa, 1g of Citrus aurantium, 1g of Platycodon grandiflorum, 2g of Licorice, 1g of Rhei Radix et Rhizoma, and 1g of Sanguis Draconis. Sanguis Draconis is extracted twice with 70% ethanol, 6 times the amount of ethanol is added each time, and each extraction is performed for 1 hour. The extraction solution is combined, the ethanol is recovered, and the solution is concentrated under reduced pressure to a relative density of 1.15-1.25. The remaining 11 herbs, including Astragalus, are extracted twice with water, 10 times the amount of water is added each time, and each extraction is performed for 1 hour. The extraction solution is filtered, the filtrate is concentrated under reduced pressure to a relative density of 1.15-1.25, 2 times the amount of ethanol is added for precipitation, it is left overnight, filtered, the ethanol is recovered from the filtrate, and the solution is concentrated under reduced pressure to a relative density of 1.15-1.25. The Sanguis Draconis alcohol extraction and the alcohol extraction of the remaining 11 herbs are combined, dried, and granulated. The dried extract is mixed with dextrin at a mass ratio of 1:1, granulated, dried, and made into 1000 grams. The product is packaged and obtained.

[0061] 1.1 Determination of mobile phase ratio

[0062] The stationary phase is octadecylsilane-bonded silica gel (Agilent ZORBAX SB-C18 (4.6 mm x 250 mm, 5 μm)), the mobile phase A is acetonitrile, the mobile phase B is 0.1% phosphoric acid solution, and a specific gradient elution program is designed as shown in Table 1. During the elution process, the flow rate is 1 ml per minute, the column temperature is 30°C, the detection wavelength is 286 nm, and the sample size is 10 μl. The results are shown in Table 1. Figure 1 Elution is performed according to the mobile phase ratio, and the peak shape of different chemical components in the chromatogram is clear and independent, indicating that the above mobile phase ratio and gradient elution program can effectively separate the sample components.

[0063] Table 1 Gradient elution program

[0064]

[0065] 1.2 Determination of column temperature / flow rate

[0066] According to the above-determined mobile phase ratio, the effects of column temperature and flow rate are investigated. When the column temperature is 30°C, the flow rate is set to 0.5-1.0 ml / min (interval 0.1 ml / min); when the flow rate is 0.6 ml / min, the column temperature is set to 25-35°C (interval 2-3°C). The flow rate affects mass transfer and diffusion, and the column temperature changes the viscosity of the mobile phase and the distribution coefficient of the solute, both of which affect the chromatographic behavior of the research object. The results are shown in Table 2.

[0067] Table 2 Column temperature / flow rate investigation separation results

[0068]

[0069] Table 2 shows that the flow rate and column temperature can affect the peak shape and separation of the overall chromatographic peak. Naringin under different chromatographic conditions, the separation degree is up to the standard requirement (> 1.5); Danpolysaccharide B except the flow rate of 1.0 ml / min separation degree does not meet the standard, the rest of the conditions meet the requirements, indicating that it is less affected by the chromatographic conditions.

[0070] After comprehensive consideration, the flow rate of 0.6 ml / min, column temperature of 30℃ as the chromatographic parameters. This selection can guarantee good peak shape and separation, ensure the analysis results of naringin and salvianolic acid B accurate and reliable, provide suitable conditions for Baixin granules fingerprint analysis.

[0071] 1.3 Determination of wavelength

[0072] Precise suction of test sample solution, injection liquid chromatograph, using diode array detector, in 190nm~400nm wavelength under full wavelength scanning. Then extract the chromatogram under different detection wavelength for comparison. Results see Figures 2-6 .

[0073] The results show that in the range of 190nm~250nm and 350nm~400nm, the chromatographic peak information is less, while in the range of 260~340nm, the number of chromatographic peaks is not obvious but the response value is different. Combined with the content of naringin and salvianolic acid B, the maximum absorption wavelength is 284nm and 286nm respectively, after comprehensive consideration, the detection wavelength is temporarily set as 286nm, which is used for the determination of naringin and salvianolic acid B content and fingerprint determination.

[0074] 1.4 Determination of chromatographic conditions

[0075] After the above investigation of the proportion of mobile phase, column temperature and flow rate, and detection wavelength, the following chromatographic conditions and system suitability test conditions are determined:

[0076] With octadecylsilane bonded silica gel as filler (Agilent ZORBAX SB-C18 (4.6mm x 250mm, 5μm)), with acetonitrile as mobile phase A, with 0.1% phosphoric acid solution as mobile phase B, gradient elution according to the record of table 1; the flow rate is 0.6ml per minute; the detection wavelength is 286nm; the column temperature is 30℃, the sample size is 10μl.

[0077] 2, Test sample preparation method investigation

[0078] Determination was carried out according to the chromatographic conditions under the item "1.4 Chromatographic condition determination", by investigating the extraction method, extraction solvent, extraction time and sample weight during the preparation of the test sample, recording the chromatogram, comparing the peak shape, taking the concentration ratio of the peak area of the characteristic peak to the sample weight as the measurement standard, and comprehensively comparing to determine the method more conducive to the extraction of the target component, and the preferred test sample extraction method, thereby providing a reliable sample basis for the subsequent analysis of Baixin Granules.

[0079] 2.1 Extraction method investigation

[0080] Take an appropriate amount of the product, grind it finely, accurately weigh 0.5 g, place it in a conical flask with a stopper, accurately add 80% methanol 50 ml, tightly seal, weigh, investigate two extraction methods of ultrasonic treatment (1100W, 40kHz) and heating reflux (water bath temperature: 80°C), both for 30 minutes, take out, cool down, re-weigh, add 80% methanol to make up for the weight loss, shake well, filter through an organic filter membrane of 0.22 μm, take the filtrate to obtain the test sample solution.

[0081] The results are shown in Figure 7 and Table 3. The chromatographic peak information and response values of the two extraction methods are basically consistent, with a relative average deviation (RAD) value of 0.4%, less than 2.0%. Considering the convenience and feasibility of the operation during the preparation of the test sample solution, ultrasonic treatment is tentatively selected as the extraction method.

[0082] Table 3 Peak area data of extraction method investigation

[0083]

[0084] 2.2 Extraction solvent investigation

[0085] Take an appropriate amount of the product, grind it finely, accurately weigh 0.5 g, place it in a conical flask with a stopper, accurately add 80% methanol 50 ml, tightly seal, weigh, investigate two extraction methods of ultrasonic treatment (1100W, 40kHz) and heating reflux (water bath temperature: 80°C), both for 30 minutes, take out, cool down, re-weigh, add 80% methanol to make up for the weight loss, shake well, filter through an organic filter membrane of 0.22 μm, take the filtrate to obtain the test sample solution.

[0086] The results are shown in Figures 8-9 and Table 4. When methanol is used as the solvent, peaks 1 and 2 show an undesirable peak shape, and the peak shape of peak 1 is also not ideal when 80% ethanol and 60% ethanol are used. From Figure 9From the sample dissolution, when methanol, 80% methanol, 80% ethanol is used as the solvent, the sample appears to be clumped or unevenly dispersed at the bottom, which can lead to uneven sample solution and affect the determination results. In combination with peak shape and sample dissolution, 80% ethanol, 80% methanol, 60% ethanol and methanol are excluded. Table 4 shows that the total peak area of 60% methanol, 30% methanol, 30% ethanol has an RSD value of 0.6% compared to the sample weight and concentration, which is less than 2.0%, indicating that there is no significant difference between different solvents.

[0087] Considering the instability of salvianolic acid B in aqueous solution and the dissolution of the test sample in different solvents, 60% methanol is finally determined as the best extraction solvent.

[0088] Table 4 Peak area data of extraction solvent investigation

[0089]

[0090] 2.3 Extraction time investigation

[0091] Take the product, grind finely, take 0.5g, accurately weigh, place in a conical flask with a plug, accurately add 60% methanol 50ml respectively, tightly plug, weigh, ultrasonic treatment (1100W, 40kHz) for 15 minutes, 30 minutes, 45 minutes, 60 minutes respectively, take out, cool, weigh again, add 60% methanol to make up for the weight loss, shake well, filter through organic membrane 0.22μm, take the filtrate to obtain the test sample solution.

[0092] The results are shown in Figure 10 and Table 5. Comparing the chromatographic peaks of 15 minutes, 30 minutes, 45 minutes and 60 minutes, the peak shape is good, the RSD value of the total peak area to the sample weight and concentration is 0.4%, which is less than 2.0%, indicating that there is no significant difference in different extraction times. Considering factors such as energy saving, the extraction time is determined to be 15 minutes.

[0093] Table 5 Peak area data of extraction time investigation

[0094]

[0095] 2.4 Sample weight investigation

[0096] Take the product, grind finely, take 0.25g, 0.5g, 1.0g, 2.0g, accurately weigh, place in a conical flask with a plug, accurately add 60% methanol 50ml respectively, tightly plug, weigh, ultrasonic treatment (1100W, 40kHz) for 15 minutes, take out, cool, weigh again, add 60% methanol to make up for the weight loss, shake well, filter through organic membrane 0.22μm, take the filtrate to obtain the test sample solution.

[0097] The results are shown inFigure 11 and Table 6, the peak shapes of different sample weights were compared and the concentration ratio was calculated, with an RSD value of 1.0%, less than 2.0%. Since the overall chromatographic peak response value was low when the sample weight was 0.25 g, and it was difficult to filter when the sample weight was 1.0 g and 2.0 g. After comprehensive consideration, the sample weight of 0.5 g was selected to ensure accuracy and avoid filtration problems.

[0098] Table 6 Peak area data for sample weight investigation

[0099]

[0100] 2.5 Preliminary determination of test sample preparation method

[0101] Based on the above investigation results of extraction method, extraction solvent, extraction time and sample weight, the test sample preparation method was preliminarily determined as follows:

[0102] Take the product, grind finely, take 0.5 g, accurately weigh, put in a conical flask with a plug, accurately add 50 ml of 60% methanol, tightly plug, weigh, ultrasonic treatment (1100 W, 40 kHz) for 15 minutes, cool, weigh again, make up the weight loss with 60% methanol, shake well, filter through an organic filter membrane of 0.22 μm, take the filtrate, and obtain.

[0103] 3. Information maximization investigation

[0104] To investigate whether the main components contained in the test sample are reflected in the atlas and whether the principle of maximum effective information is met, sample preparation and determination were carried out according to the method determined in "1.4 Chromatographic condition determination" and "2.5 Preliminary determination of test sample preparation method". Under the proposed gradient elution program, the elution time was extended to 300 minutes (mobile phase ratio: acetonitrile-0.1% phosphoric acid (28:72)) after 100 minutes. According to this operation procedure, blank solution and test sample solution were injected in turn, and the chromatogram was recorded at the same time.

[0105] The results are shown in Figure 12 Under the conditions of mobile phase ratio acetonitrile-0.1% phosphoric acid (28:72) and last gradient extension 2 times, the test sample chromatogram had no obvious chromatographic peak after about 120 minutes. This result shows that the main component information of the sample has been basically obtained, thereby basically meeting the information maximization principle.

[0106] 4. Peak assignment and chromatographic peak identification

[0107] 4.1 Drug taste peak assignment and chromatographic peak identification

[0108] Take the prescribed amount of Radix Astragali, Radix Salviae Miltiorrhizae, Radix Rehmanniae, Radix Ophiopogonis, Fructus Aurantii, Herba Lycopi, Poria, Rhei Radix et Rhizoma, Angelica sinensis, Platycodon grandiflorum, and Glycyrrhiza uralensis, accurately weigh, place in a conical flask with a stopper, add 50 ml of water, heat and decoct for 30 minutes, filter, evaporate the filtrate to dryness, then from "add 60% methanol 50 ml", prepare single herb piece solution (among which, the solution of Limax marginatus is prepared: take the prescribed amount of Limax marginatus, accurately weigh, place in a conical flask with a stopper, add 70% ethanol 50 ml, reflux extract for 30 minutes, filter, evaporate the filtrate to dryness, from "add 60% methanol 50 ml", prepare in the same way as the preparation method of the test sample) according to the preparation method of the test sample. At the same time, prepare the corresponding negative solution of each medicine to determine the attribution of each chromatographic peak in Baixin Granules.

[0109] Prepare the standard solution: accurately weigh Danshensu, Calycosin-7-Glucoside, Isoliquiritigenin, Liquiritin, Naringin, Neo-Hesperidin, Rosmarinic Acid, Shikonin, Salvianolic Acid B, and Salvianolic Acid A reference substances, respectively, and place them in a volumetric flask. Add an appropriate amount of 60% methanol solution, ultrasonically treat to completely dissolve, cool, dilute to the mark with 60% methanol solution, shake well, and prepare each standard solution containing 5 μg of Danshensu, 5 μg of Calycosin-7-Glucoside, 5 μg of Isoliquiritigenin, 5 μg of Liquiritin, 25 μg of Naringin, 15 μg of Neo-Hesperidin, 5 μg of Rosmarinic Acid, 5 μg of Shikonin, 50 μg of Salvianolic Acid B, and 10 μg of Salvianolic Acid A in 1 ml.

[0110] Inject the test sample solution and each standard control solution for detection, compare the chromatographic results of the test sample solution with those of each standard control solution, and identify the chromatographic peaks. The results are shown in Table 7 and Table 8. Figure 13 、 Figure 14 、Table 7 and Table 8.

[0111] Table 7 Retention time of chromatographic peaks in single herb piece characteristic chromatogram

[0112]

[0113] Table 8 Attribution of peaks of each medicine and identification results of chromatographic peaks

[0114]

[0115] 4.2 Selection of reference peaks

[0116] The elution time of naringin in the chromatogram is 61.874 minutes, which is in the middle of the entire chromatogram. It can serve as an intermediate reference point, effectively correlating and comparing components at different time points. Its peak shape is good and its response value is moderate, which can reduce errors caused by fluctuations in experimental conditions, thus making the experimental results more accurate and reliable. The elution time of salvianolic acid B in the chromatogram is 84.903 minutes, which is relatively late. It can provide an effective reference for subsequent component analysis. Its peak shape is good and its response value is moderate, which can accurately reflect the component information in later stages to aid in qualitative and quantitative analysis.

[0117] Taking all factors into consideration, the peaks of the two substances are located in the middle and late stages, respectively, covering different time periods, and are close to other peaks, which facilitates the comparison of the relationship between components. At the same time, the two substances do not react with the sample, ensuring the authenticity and reliability of the results, and have a certain degree of purity and good stability, which can reduce the interference of impurities and ensure the consistency of chromatographic behavior. Therefore, salvianolic acid B and naringin were selected as reference substances.

[0118] 5. Methodological Validation

[0119] 5.1 Specificity Test

[0120] Following the chromatographic conditions and preparation method determined in "1.4 Determination of Chromatographic Conditions" and "2.5 Preliminary Determination of Test Sample Preparation Method," precisely pipettes of 60% methanol solution (blank solvent) (S3), excipient solution (S4), reference solution (S2), and test sample solution (S1) were injected into the liquid chromatograph, and the chromatograms were recorded. Results are shown below. Figure 15 The blank solvent and excipients showed no interference at the chromatographic peak positions, indicating that the method has good specificity.

[0121] 5.2 Precision Test

[0122] Following the chromatographic conditions and sample preparation method determined in "1.4 Determination of Chromatographic Conditions" and "2.5 Preliminary Determination of Sample Preparation Method," a sample solution (1 part) was prepared and injected six times consecutively. The chromatogram was recorded. Results are shown below. Figure 16 Tables 9 and 10 show the obtained chromatograms. The similarity was calculated according to the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine" (2012 edition). All similarity results were above 0.98, with an RSD of 0.00%. Using naringin as the reference peak S1 and salvianolic acid B as the reference peak S2, the relative retention times of some chromatographic peaks and the reference peaks were calculated. The relative retention times of peaks 1, 2, 3, 4, and 5 with peak S1, and the relative retention times of peaks 7, 8, 9, 11, and 12 with peak S2, all had RSD values ​​less than 2.0%, indicating good instrument precision.

[0123] Table 9. Similarity of Instrument Precision Examination

[0124]

[0125] Table 10 Instrument precision test - relative retention time results

[0126]

[0127] 5.3 Repeatability Test

[0128] Following the chromatographic conditions and preparation method determined in "1.4 Determination of Chromatographic Conditions" and "2.5 Preliminary Determination of Test Sample Preparation Method," test sample solutions (6 portions) were prepared, and chromatograms were recorded. Results are shown below. Figure 17 Tables 11 and 12 show the obtained chromatograms. Similarity was calculated using the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine" (2012 edition). All similarity results were above 0.98, with an RSD of 0.00%. The relative retention times of the chromatographic peaks with reference peaks S1 (naringin) and S2 (tanshinone B) were calculated. The relative retention times of peaks 1, 2, 3, 4, and 5 with peak S1, and the relative retention times of peaks 7, 8, 9, 11, and 12 with peak S2, all had RSD values ​​less than 2.0%, indicating good repeatability.

[0129] Table 11 Repeatability Test - Similarity Calculation Results

[0130]

[0131] Table 12 Repeatability Test - Relative Retention Time Results

[0132]

[0133] 5.4 Intermediate Precision Test

[0134] Different determination times, different high-performance liquid chromatographs, and different experimental personnel (B) were selected. Test solutions (6 parallel replicates) were prepared according to section "8.3.5 Preliminary Determination of Test Sample Preparation Method," and the samples were injected and analyzed. Chromatograms were recorded. Results are shown in […]. Figure 18, Table 13 and Table 14, the obtained chromatograms were calculated for similarity according to "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (2012 edition), and the similarity results were all above 0.98, and the RSD value was 0.04%. Combined with the data of "5.3 repeatability test", the relative standard deviation (RSD) value of the intermediate precision calculated by personnel A and personnel B was 0.07%. Taking naringin as the reference peak S1 and salvianolic acid B as the reference peak S2, the relative retention time of the chromatographic peaks and the reference peaks was calculated. The relative retention time of peaks 1, 2, 3, 4, 5 was calculated with S1 peak, and the relative retention time of peaks 7, 8, 9, 11, 12 was calculated with S2 peak. The results showed that the similarity of personnel A was 0.984, and the RSD value was 0.00%, and the operation repeatability was good; the similarity of personnel B fluctuated between 0.982 and 0.983, and the RSD value was 0.04%, and the RSD value of the similarity of the two was 0.07%, and the overall situation was good. In terms of relative retention time, except that the RSD value of peak 1 was greater than 2%, the RSD values of the other peaks were less than 2%, the data consistency of personnel A was strong, and only peak 1 of personnel B was different from A and had good stability. In summary, although personnel A and B had differences, the overall intermediate precision in the experiment was good.

[0135] Table 13 Similarity of intermediate precision test

[0136]

[0137] Table 14 Results of intermediate precision test- relative retention time

[0138]

[0139] 5.5 Stability test

[0140] Take the test solution ① under "5.3 repeatability test", and place it for 0, 2, 4, 6, 8, 10, 12, 18, 25, 32, 42, 65 and 80 hours, then inject and determine according to the proposed chromatographic conditions, and record the chromatogram. The results are shown in Figure 19 , Table 15 and Table 16, the obtained chromatograms were calculated for similarity according to "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (2012 edition), and the similarity results were all above 0.98, and the RSD value was 0.00%. Taking naringin as the reference peak S1 and salvianolic acid B as the reference peak S2. The relative retention time of peaks 1, 2, 3, 4, 5 was calculated with S1 peak, and the relative retention time of peaks 7, 8, 9, 11, 12 was calculated with S2 peak, and the RSD value was less than 2.0%, indicating that the test solution was stable after being placed for 80 hours.

[0141] Table 15 Stability test-similarity calculation results

[0142]

[0143] Table 16 Stability Study - Relative Retention Time Results

[0144]

[0145] 5.6 Durability Test

[0146] The original chromatographic conditions established in this experiment were: column temperature 30℃, flow rate 0.6 ml / min, Agilent instrument, acetonitrile (Wokai)-phosphoric acid (Tianjin Damao) reagent, and ZORBAX SB-C18 column. Using the chromatograms obtained under these original conditions as a baseline, the robustness of these chromatographic conditions was investigated under different column temperatures, flow rates, instrument manufacturers, reagent brands, and column conditions. Robustness was assessed by calculating the similarity of the obtained chromatograms based on common peaks.

[0147] The results of the flow rate and column temperature durability test are shown in Figure 20 See Table 17. The chromatographic similarity under the original chromatographic conditions was 0.984. The results were investigated at column temperatures of 28℃ and 32℃, and flow rates of 0.5 ml / min, 0.65 ml / min, and 0.7 ml / min. The calculated RAD values ​​under each condition compared to the original conditions were all less than 2%. This indicates that the method has good tolerance to small changes in column temperature and flow rate, demonstrating good robustness.

[0148] The results of the instrument durability test are as follows: Figure 21 See Table 18. When using a Waters instrument, the RAD value was 0.11% compared to the spectrum obtained with the Agilent instrument. This result indicates that instruments from different manufacturers have little impact on this method, demonstrating good instrument versatility.

[0149] The results of the reagent durability study are shown below. Figure 22 See Table 18. When using reagent brand 2, the RAD value of its chromatogram was 0.06% compared with the original conditions (using reagents from Sinopharm Group and Tianjin Damao). This indicates that different brands of reagents have minimal impact on this method, reflecting the good adaptability of this method to reagents. This characteristic has important guiding significance for practical reagent selection.

[0150] The results of the column robustness test are shown in Figure 23and Table 18. Partial chromatographic columns (such as Inertsustain C18, etc.) are not involved in the calculation because they are not suitable for the conditions. For chromatographic columns such as Inertsil ODS-HL, Ultimate Plus C18, etc., the obtained chromatograms have a RAD value less than 2% compared with the original conditions (using a ZORBAX SB-C18 chromatographic column). This indicates that the method has good universality among suitable chromatographic columns, showing good robustness and providing more flexibility for the selection of chromatographic columns.

[0151] In summary, through the investigation of flow rate, column temperature, instrument, reagent, and the robustness of the chromatographic column, the present chromatographic method shows good robustness, adaptability to different conditions, and universality in the selection of related equipment and reagents under various condition changes, which provides a strong basis for the stability and reliability of the method in practical application.

[0152] Table 17 Robustness test - column temperature / flow rate similarity calculation results

[0153]

[0154] Table 18 Robustness test - different instrument manufacturers / different brand reagents / different brand chromatographic columns similarity calculation results

[0155]

[0156] 6. Establishment of fingerprint spectrum

[0157] Fifteen batches of Baoxin Granules (batch numbers are shown in Table 19) were prepared and determined according to the chromatographic conditions and test sample preparation method determined in “1.4 Chromatographic condition determination” and “2.5 Test sample preparation method preliminary determination”. After the chromatogram was integrated, it was imported into the “Traditional Chinese Medicine Chromatographic Fingerprint Spectrum Similarity Evaluation System” (2012 edition), and the superimposed chromatogram was established by using the median method, multi-point calibration, and automatic matching. The control chromatogram (R) is shown in Figure 24 , and the fingerprint spectrum stacking chart of Baoxin Granule samples is shown in Figure 25 .

[0158] The similarity of 12 common peaks of 15 batches of Baoxin Granules and the control fingerprint spectrum was calculated, and the results are shown in Table 19. According to the similarity calculated by the fingerprint spectrum, the similarity of S2-S15 samples and the control fingerprint spectrum was 0.982, 0.985, 0.978, 0.990, 0.990, 0.991, respectively, indicating that the chemical composition of the 15 batches of samples was similar, and the quality was stable and uniform.

[0159] According to the above results, the fingerprint standard is determined as follows: the test sample fingerprint should present the same chromatographic peaks as the reference material in terms of retention time, and there should be 12 corresponding common peaks. The Mark peak matching is used, and the similarity of the test sample fingerprint and the control fingerprint should not be less than 0.90 according to the similarity evaluation system of traditional Chinese medicine chromatographic fingerprint.

[0160] Table 19 Similarity calculation results of 15 batches of Baoxin granules

[0161]

[0162] Example 2 Quantitative detection of salvianolic acid B and naringin based on the fingerprint construction method

[0163] 1. Purity of salvianolic acid B and naringin peaks

[0164] Preparation and determination were performed according to the chromatographic conditions and test sample preparation method determined in Example 1, and the results are shown in Table 2. Figure 26 Naringin and salvianolic acid B in the test sample were detected at a wavelength of 286 nm, and the UV purity values were both greater than the specified 995, indicating that naringin and salvianolic acid B had high purity and less impurity interference under the detection conditions, meeting the purity requirements for subsequent experiments.

[0165] 2. Methodology verification

[0166] Preparation of the reference solution: Take the naringin reference and salvianolic acid B reference, accurately weigh, and prepare a mixed solution containing 25 μg of naringin and 50 μg of salvianolic acid B per 1 ml of 60% methanol, and obtain it.

[0167] Preparation of the test sample solution: Take the product, mix well, grind finely, accurately weigh 0.5 g, accurately weigh, place in a conical bottle with a plug, accurately add 50 ml of 60% methanol, tightly plug, weigh, ultrasonic treatment (1100 W, 40 kHz) for 15 minutes, cool, weigh again, make up the weight loss with 60% methanol, shake well, filter, and take the filtrate, and obtain it.

[0168] Determination method: accurately take 10 μl of the reference solution and the test sample solution, inject into the liquid chromatograph, and determine, and obtain it.

[0169] 2.1 Investigation of specificity

[0170] Since salvianolic acid B is contained in Danshen and naringin is contained in Zhike, the negative sample of Baoxin granules without Danshen and the negative sample of Baoxin granules without Zhike are prepared, and the excipients and negative sample solutions are prepared according to the test sample preparation method determined in Example 1. Then, the blank solvent, excipients, reference, negative sample, and test sample solutions are accurately taken for determination.

[0171] The results are shown in Table 3. Figure 27The chromatogram of the negative sample without Salvia miltiorrhiza had no chromatographic peak at the retention time corresponding to Danshensuan B, and the chromatogram of the negative sample without Aurantii Fructus had no chromatographic peak at the retention time corresponding to Naringin. The blank solvent and excipients had no interference. Therefore, the determination of Danshensuan B and Naringin in the product by the method has specificity.

[0172] 2.2 Linear relationship investigation

[0173] Preparation of reference solution: Take Naringin and Danshensuan B reference substances in appropriate amounts, accurately weigh, and add 60% methanol to prepare a reference stock solution containing 250 μg of Naringin and 500 μg of Danshensuan B per 1 ml. Accurately take the reference stock solution in appropriate amounts, and dilute to a series of different concentrations. The concentration of Danshensuan B is 5.107 μg, 10.21 μg, 51.07 μg, 255.4 μg, and 510.7 μg per 1 ml. The concentration of Naringin is 2.339 μg, 4.679 μg, 23.39 μg, 117.0 μg, and 233.9 μg per 1 ml.

[0174] Determine according to the chromatographic conditions determined in Example 1, record the peak area of the chromatographic peak, and draw the standard curve with the concentration of the reference substance (μg / ml) as the abscissa and the peak area (mAU) as the ordinate.

[0175] For Danshensuan B, the linear relationship investigation results are shown in Table 19 and Table 20. The regression equation is y = 21.2259x + 4.1668, and the correlation coefficient is 1.0000. The concentration of Danshensuan B is linear within the range of 5.107 μg / ml-510.7 μg / ml. Figure 28

[0176] For Naringin, the linear relationship investigation results are shown in Table 21 and Table 22. The regression equation is y = 28.8702x + 7.2030, and the correlation coefficient is 1.0000. The concentration of Naringin is linear within the range of 2.339 μg / ml-233.9 μg / ml. Figure 28

[0177] Table 20 Linear relationship investigation

[0178]

[0179] 2.3 Precision test

[0180] Take the reference solution No. 3 under the item “2.2 Linear relationship investigation”, determine according to the chromatographic conditions determined in Example 1, and continuously inject 5 times to calculate the RSD values of the retention time and peak area of Danshensuan B and Naringin. The results are shown in Table 21. The RSD values of the retention time and peak area of Danshensuan B and Naringin are all less than 2.0%, indicating that the proposed analysis method has good injection precision and meets the verification precision requirements. ​​

[0181] Table 21 Precision test results (n=5)

[0182]

[0183] 2.4 Reproducibility test

[0184] The tester (A) took an appropriate amount of the product, finely ground, and accurately weighed 0.5 g (a total of 6 times), prepared the test sample and determined. The results are shown in Table 22. The RSD values of naringin and salvianolic acid B were less than 2.0%, indicating that the method was reproducible.

[0185] Table 22 Reproducibility results (n=6)

[0186]

[0187] 2.5 Intermediate precision test

[0188] Select different determination times, different high-performance liquid chromatographs, and different testers (B), take an appropriate amount of the product, finely ground, and accurately weigh about 0.5 g (a total of 6 times), prepare the test sample and determine. The results are shown in Table 23. The average content of naringin and salvianolic acid B determined by operators A and B was 2.211 mg / g and 4.925 mg / g, respectively, and the RSD values were less than 4.0%, indicating that the method had good intermediate precision.

[0189] Table 23 Naringin / salvianolic acid B-intermediate precision test results

[0190]

[0191] 2.6 Stability test

[0192] Take the test sample solution under item "2.4 Reproducibility test" No. 1, and determine by sampling at 0, 2, 4, 6, 8, 10, 12, 18, 25, 32, 42, 65, and 80 hours after preparation. Calculate the RSD values of the peak areas of naringin and salvianolic acid B. The results are shown in Table 24. The RSD values of naringin and salvianolic acid B were less than 2.0%, indicating that the test sample solution had good stability within 80 hours.

[0193] Table 24 Stability results (n=13)

[0194]

[0195] 2.7 Accuracy test

[0196] Take the product, grind, take 0.25g, accurately weigh (parallel 9), respectively, in the conical flask with plug. The 9 samples were divided into 3 gradients, according to the mass ratio 1:0.5, 1:1, 1:1.5, respectively, add appropriate amount of mixed control solution (the concentration of naringin and salvianolic acid B is determined according to the mean value of "9.6.3.2 repeatability test"), prepare the sample and determine. Record the peak area and calculate the recovery. The results are shown in Table 25 and Table 26, the recovery of naringin is 96.3%~98.2%, the average recovery is 98%, which meets the requirements of pharmacopoeia (92%~105%), indicating that the method for determining the content of naringin has good accuracy; the recovery of salvianolic acid B is 96.9%~98.7%, the average recovery is 98%, which meets the requirements of pharmacopoeia (92%~105%); indicating that the method for determining the content of salvianolic acid B has good accuracy.

[0197] Table 25 Naringin-accuracy test results (n=9)

[0198]

[0199] Table 26 Salvianolic acid B-accuracy test results (n=9)

[0200]

[0201] 2.8 Range test

[0202] The applicability of the established method for determining the content of naringin and salvianolic acid B of the product under different sample weight concentrations was investigated to provide basis for evaluating the accuracy and reliability of the method. The product was taken in appropriate amount, ground, and prepared into sample at 80%, 100%, and 120% sample weight concentration levels, and determined. The results are shown in Table 27, the relative standard deviation (RSD) of the content determination results of naringin and salvianolic acid B at different concentrations are all less than 2.0%, indicating that the method has good accuracy and repeatability within the specified range.

[0203] Table 27 Range test results

[0204]

[0205] 2.9 Durability test

[0206] The test method is the same as that of Example 1 "5.6 durability test". The original chromatographic conditions established in this test are as follows: column temperature 30℃, flow rate 0.6ml / min, using Agilent instrument, reagent is acetonitrile (Wokai)-phosphoric acid (Tianjin Damao), chromatographic column is ZORBAX SB-C18. The content of naringin is 2.180mg / g, and the content of salvianolic acid B is 5.003mg / g.

[0207] The results are shown in Table 28 and Table 29. The RAD values of the contents of salvianolic acid B and naringin determined under each micro-change condition are less than 4.0%, and do not cause significant fluctuations in the determination results, ensuring the consistency and reliability of the determination results, and proving the durability of the column temperature at 28-32℃, the flow rate at 0.5-0.7ml / min, and the use of different brands of chromatographic columns, different instruments, and different brands of reagents, fully showing the stability and accuracy of the method, and providing strong data support for chromatographic analysis practice.

[0208] Table 28 Naringin Durability Test Results

[0209]

[0210] Table 29 Salvianolic Acid B Durability Test Results

[0211]

[0212] 3. Determination of quantitative detection method

[0213] The methodological validation data of the method, such as system applicability, linear relationship, precision, stability, repeatability, and accuracy, are all good, meeting the requirements for content determination, and can be used for the content determination of Baixin Granules. The final determination method is as follows:

[0214] Preparation of reference solution: Take appropriate amounts of naringin and salvianolic acid B reference substances, accurately weigh, and prepare a mixed solution containing 25μg of naringin and 50μg of salvianolic acid B per 1ml in 60% methanol.

[0215] Preparation of test sample solution: Take an appropriate amount of the product, grind it finely, take about 0.5g, accurately weigh, place it in a conical flask with a plug, accurately add 60% methanol 50ml, tightly plug, weigh, ultrasonic treatment (1100W, 40kHz) for 15 minutes, take out, cool, weigh again, add 60% methanol to make up for the weight loss, shake well, filter, and take the filtrate, which is the test sample solution.

[0216] Determination method: accurately pipette 10μl of the reference solution and the test sample solution respectively, inject into the liquid chromatograph, and determine.

[0217] 4. Sample detection

[0218] The sample preparation and determination of 6 batches of Baoxin Granules were carried out according to the requirements in item 4, Determination of quantitative detection method. The results are shown in Table 30. The content of naringin in 6 batches of samples ranged from 1.711 mg / g to 2.703 mg / g, with an average of 2.071 mg / g; the content of salvianolic acid B ranged from 4.924 mg / g to 6.977 mg / g, with an average of 5.918 mg / g. These data reflect the differences in the contents of the two indicator components in different batches of Baoxin Granules, providing a direct basis for the evaluation of product quality. At the same time, the contents of naringin and salvianolic acid B fluctuate to some extent among different batches, which may be related to the quality of raw materials, production process and other factors. Further in-depth research can be carried out to optimize the production process and improve the stability of product quality.

[0219] Table 30 Sample test results

[0220]

[0221] 5. Comparison of quality standards

[0222] 5.1 Instruments and reagents

[0223] Three batches of clinical samples with batch numbers 210601, 221001 and 240401 were used, which were produced by Zhejiang Kangenbeier Pharmaceutical Co., Ltd.

[0224] 5.2 Comparative study test

[0225] According to the current quality standards and the determination method determined in this example, the contents of salvianolic acid B in three batches of Baoxin Granules clinical samples were determined under two standards, and the results of each batch of samples were recorded.

[0226] ① Current quality standard content determination method: determined by reference to high performance liquid chromatography (general rule 0512).

[0227] Chromatographic conditions and system suitability test: octadecylsilane-bonded silica gel as the filler; methanol-acetonitrile-formic acid-water (25:10:1:64) as the mobile phase; detection wavelength of 286 nm; theoretical plate number greater than 5000.

[0228] Preparation of reference solution: take salvianolic acid B reference substance, accurately weigh, add 75% methanol to make a solution containing 40 μg per 1 ml, and get it.

[0229] Preparation of test sample solution: take the product, grind finely, take about 0.5 g, accurately weigh, put into a conical flask with plug, add 75% methanol 50 ml, weigh, ultrasonic (300 W, 40 kHz) for 10 min, take out, cool, weigh again, make up the weight loss with 75% methanol, shake well, filter, take the filtrate, and get it.

[0230] Determination method: 10 μl of the control solution and the sample solution were precisely pipetted, injected into the liquid chromatograph, and determined, and then the determination was completed.

[0231] 2. The determination method determined in this example is described in detail in "4. Determination of the quantitative detection method".

[0232] The results are shown in Table 31. The contents of salvianolic acid B in the samples of batch numbers 210601, 221001 and 240401 of Baoxin granules were higher than those under the current quality standard, the relative average deviation (RAD) was 1.7%~2.8%, and the difference was small. This indicates that when the quality standard is upgraded, the content of salvianolic acid B is relatively stable although there is a difference, which is of positive significance for the controllability of the quality of Baoxin granules. It is preliminarily inferred that the content change has no significant negative impact on safety and effectiveness, but accurate evaluation of clinical significance still needs more research. In addition, the contents of salvianolic acid B in different batches of Baoxin granules under the current and new quality standards fluctuate, suggesting that the consistency of quality between batches should be paid attention to in production and the production process should be optimized.

[0233] In summary, the difference in the content of salvianolic acid B under the current and new quality standards is not significant, and subsequent attention should be paid to the consistency of quality between batches and between enterprises and optimization measures should be taken to ensure the stable quality of Baoxin granules.

[0234] Table 31 Summary of the content of salvianolic acid B under the current and new standards

[0235]

[0236] The above-described examples only describe the preferred modes of the present application and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements to the technical solutions of the present application made by those of ordinary skill in the art should fall within the scope of protection determined by the claims of the present application.

Claims

1. A method for determining the HPLC fingerprint of Baixin granules, characterized in that, It comprises the following steps: (1) preparing a test sample solution and a standard solution; The preparation method of the test sample solution is as follows: 0.5 g of Baixin Granules is precisely weighed, 50 ml of 60% methanol is added, and ultrasonic treatment is performed under the condition of 1100 W and 40 kHz; after filtration, the test sample solution is obtained; The standard solution comprises Danshensu solution, Calycosin-7-Glucoside solution, Astragali Glucosido Vitexin solution, Glycyrrhizin solution, Naringin solution, Neohesperidin solution, Rosmarinic Acid solution, Alkannin solution, Salvianolic Acid B solution and Salvianolic Acid A solution prepared by using 60% methanol aqueous solution as a solvent; (2) performing HPLC chromatographic analysis on the test sample solution and the standard solution to obtain a Baixin Granules HPLC fingerprint spectrum composed of sample common characteristic peaks; The HPLC chromatographic analysis conditions are as follows: octadecylsilane-bonded silica gel is used as a filler, acetonitrile is used as mobile phase A, 0.1% phosphoric acid solution is used as mobile phase B, the detection wavelength is 286 nm, the flow rate is 0.5-0.7 ml per minute, the column temperature is 28-32°C, and the elution program is as follows: ； The Baixin Granules is made of the following raw materials: Radix Astragali 7 g, Radix Salviae Miltiorrhizae 5 g, Radix Ophiopogonis 5 g, Poria Cocos 2 g, Lycopi Herba 2 g, Radix Angelicae Sinensis 1 g, Radix Rehmanniae 3 g, Fructus Aurantii 1 g, Platycodonis Radix 1 g, Glycyrrhizae 2 g, Rhei Radix et Rhizoma 1 g, and Aquilariae Resinatum 1 g.

2. The assay method according to claim 1, characterized by The preparation method of the test sample solution is as follows: 0.5 g of Baixin Granules is precisely weighed, 50 ml of 60% methanol is added, and ultrasonic treatment is performed under the condition of 1100 W and 40 kHz for 15 minutes; after filtration, the test sample solution is obtained.

3. The assay method according to claim 1, characterized by, The flow rate of the HPLC chromatographic analysis is 0.6 ml per minute, the column temperature is 30°C, and the sample loading amount is 10 μl.

4. The assay method according to claim 1, characterized by, The HPLC chromatographic analysis comprises the step of identifying sample common characteristic peaks in the chromatogram of the test sample solution by taking the chromatogram of the standard solution as a control.

5. The assay method according to claim 1, characterized by, The Baixin Granules HPLC fingerprint spectrum contains 12 sample common characteristic peaks, wherein, No. 2 peak is Danshensu, No. 3 peak is Calycosin-7-Glucoside, No. 4 peak is Astragali Glucosido Vitexin, No. 5 peak is Glycyrrhizin, No. 6 peak is Naringin, No. 7 peak is Neohesperidin, No. 8 peak is Rosmarinic Acid, No. 9 peak is Alkannin, No. 10 peak is Salvianolic Acid B, and No. 12 peak is Salvianolic Acid A.

6. The application of the Baixin Granules HPLC fingerprint spectrum determined by the determination method of any one of claims 1-5 in production process quality control, finished product quality evaluation, batch stability analysis and authenticity identification of the Baixin Granules.

7. A method for quality control of a heart protecting granule, characterized by, The quality control method is to realize the quality control of the Baixin Granules by quantitatively detecting the contents of Naringin and Salvianolic Acid B in the Baixin Granules by using the determination method of any one of claims 1-5.

8. The quality control method according to claim 7, characterized by, It comprises the following steps: (1) performing HPLC chromatographic analysis on the test sample by using the determination method of any one of claims 1-5 to determine the peak areas of Naringin and Salvianolic Acid B; (2) preparing naringin standard solution and salvianolic acid B standard solution with gradient distribution of concentration, using the determination method of any one of claims 1-6 to determine the peak area of the standard solution, and constructing a standard curve of "concentration-peak area"; (3) substituting the peak area of naringin and salvianolic acid B determined in step (1) into the standard curve, and calculating the content of naringin and salvianolic acid B in the sample to be determined.

9. The quality control method according to claim 8, characterized in that, The standard solution is prepared by using 60% methanol aqueous solution as a solvent.

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