HPLC (High Performance Liquid Chromatography)-based heart-protecting granule fingerprint spectrum construction and component quantitative detection method

Abstract

The invention discloses a heart-protecting granule fingerprint spectrum construction and component quantitative detection method based on HPLC (High Performance Liquid Chromatography), and belongs to the technical field of pharmaceutical analysis. The method comprises the following steps: preparing heart-protecting granules into a test solution by adopting a high performance liquid chromatography (HPLC) technology, affiliating and identifying chromatographic peaks in the test solution by taking chromatograms of a single medicinal material decoction piece solution and a standard solution as a contrast, and constructing an HPLC fingerprint spectrum containing 12 common characteristic peaks. And a foundation is laid for the establishment of a comprehensive quality control and quality evaluation method of the heart-protecting granules. The method disclosed by the invention can also be used for quantitatively detecting important components, namely naringin and salvianolic acid B, in the heart-protecting granules, has the advantages of high precision, good reproducibility, good stability, high recovery rate, good durability and accurate determination result, and provides technical support for high-precision quality monitoring and evaluation of the heart-protecting granules.

Description

Technical Field

[0001] The present invention relates to the technical field of drug analysis, and in particular to a method for constructing a fingerprint of Baoxin granules and quantitatively detecting its components based on HPLC. Background Art

[0002] Cardiovascular disease is one of the major diseases that seriously threatens human health, with high morbidity and mortality rates. Baoxin Granule, a Class 1.1 new Chinese medicine, is made from a combination of multiple Chinese medicinal materials such as astragalus, wine-soaked rhubarb, and scalded leeches (disclosed in Chinese patent CN113274454A, published on July 8, 2021). It has great potential in the treatment of cardiovascular diseases, and its multi-component synergistic mechanism is expected to become a new breakthrough in clinical treatment. The multi-component and multi-target characteristics of traditional Chinese medicine prescriptions make their quality significantly affected by factors such as medicinal materials and processes. Traditional detection methods are single-dimensional and difficult to meet the strict quality requirements of Class 1.1 new Chinese medicines.

[0003] High-performance liquid chromatography (HPLC) fingerprint technology is an analytical method that can comprehensively characterize the complex chemical components of traditional Chinese medicines. By constructing a unique fingerprint spectrum based on characteristic information such as the relative retention time and peak area ratio of chromatographic peaks, it can achieve a comprehensive evaluation of the overall quality of traditional Chinese medicine prescriptions. This technology has been widely used in the fields of traditional Chinese medicine quality control, process optimization, and authenticity identification. However, there has been no systematic report on the HPLC fingerprint determination method for Baoxin Granule, and some problems exist, such as poor chromatographic separation, low coverage of characteristic peaks, and insufficient method stability. Therefore, establishing a scientific, reliable, and exclusive HPLC fingerprint determination method is of great significance for improving the quality control level of Baoxin Granule and ensuring its clinical efficacy and safety. Summary of the Invention

[0004] In response to the problems existing in the above-mentioned prior art, the present invention aims to provide a method for constructing a fingerprint spectrum and quantitatively detecting components of Baoxin Granules based on HPLC, so as to achieve a comprehensive analysis of the chemical components of Baoxin Granules and precise control of their quality, meet the strict quality standard requirements of Class 1.1 new Chinese medicines, and provide a reliable quality detection basis for the research and development, production and clinical application of Baoxin Granules.

[0005] To achieve the above object, the present invention provides the following solutions:

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

[0007] (1) Prepare test solution and standard solution;

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

[0009] (2) performing HPLC chromatography analysis on the test solution and the standard solution to obtain an HPLC fingerprint of Baoxin Granule consisting of common characteristic peaks of the samples;

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

[0011] Time (minutes) Mobile phase A% Mobile phase B% 0～15 2 98 15～25 2→10 98→90 25～35 10→18 90→82 35～50 18 82 50～100 18→28 82→72 100～101 28→90 72→10 101～110 90 10 110～111 <![CDATA[90 → 2]]> 10→98 111～125 2 98 .

[0012] Furthermore, the preparation method of the test solution is: accurately weigh 0.5g of Baoxin granules, add 50ml of 60% methanol, ultrasonically treat for 15 minutes under 1100W and 40kHz conditions, and obtain the test solution after filtering.

[0013] Furthermore, the flow rate of the HPLC chromatography analysis was 0.6 ml per minute, the column temperature was 30° C., and the sample volume was 10 μl.

[0014] 4. The determination method according to claim 1, characterized in that the HPLC chromatographic analysis includes the step of identifying the common characteristic peaks of the samples in the chromatogram of the test solution using the chromatogram of the standard solution as a reference.

[0015] Furthermore, the standard solution includes danshensu solution, calycosin glucoside solution, apigenin isoliquiritin solution, liquiritin solution, naringin solution, neohesperidin solution, rosmarinic acid solution, lithospermic acid solution, salvianolic acid B solution and salvianolic acid A solution prepared with 60% methanol aqueous solution as solvent.

[0016] Furthermore, the HPLC fingerprint of Baoxin Granule contains 12 common characteristic peaks of the samples, among which peak 2 is danshensu, peak 3 is calycosin glucoside, peak 4 is apiosin isoliquiritin, peak 5 is liquiritin, peak 6 is naringin, peak 7 is neohesperidin, peak 8 is rosmarinic acid, peak 9 is lithospermic acid, peak 10 is salvianolic acid B, and peak 12 is salvianolic acid A.

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

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

[0019] The present invention also provides a quality control method for Baoxin Granules, which utilizes the above-mentioned determination method to quantitatively detect the contents of naringin and salvianolic acid B in Baoxin Granules to achieve quality control of Baoxin Granules.

[0020] Further, the following steps are included:

[0021] (1) Using the above-mentioned determination method, the sample to be tested was subjected to HPLC chromatography analysis to determine the peak areas of naringin and salvianolic acid B;

[0022] (2) preparing naringin standard solutions and salvianolic acid B standard solutions with gradient concentration distribution, measuring the peak areas of the standard solutions using the above-mentioned determination method, and constructing a "concentration-peak area" standard curve;

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

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

[0025] The present invention discloses the following technical effects:

[0026] 1. Comprehensiveness of the fingerprint: By systematically optimizing experimental conditions, the present invention constructed the HPLC fingerprint of Baoxin Granule, which contains 12 common characteristic peaks, comprehensively covering the main active ingredients of core medicinal materials such as Astragalus and Salvia miltiorrhiza in Baoxin Granule. It can fully and accurately present the chemical basis of Baoxin Granule, providing rich and reliable information for quality evaluation.

[0027] 2. Quantitative Detection Accuracy: This invention establishes a quantitative analysis method for naringin and salvianolic acid B, key pharmacologically active compounds in Baoxin Granules. This method, rigorously validated, demonstrates excellent analytical performance. In precision testing, the instrument precision RSD value was kept within 1.5%. In repeatability testing, the similarity between six replicates reached over 0.99. Stability testing demonstrated that the test solution remained stable for 80 hours, with an RSD value less than 0.8%. In sample recovery testing, the recoveries of naringin and salvianolic acid B remained stable at 97.2%-98.5% and 96.8%-98.3%, respectively. Durability testing demonstrated that this method maintains stable and reliable detection performance despite variations in instrumentation, chromatographic columns, and experimental conditions. These excellent indicators ensure precise control of Baoxin Granules' quality, meeting the high quality standards of Category 1.1 new Chinese medicines.

[0028] 3. Technical Application Value: The assay method and quality control system of this invention provide solid technical support for the entire process of Baoxin Granule, from research and development to clinical application. During the research and development phase, it helps optimize the production process and ensure the stability of drug quality; during the production process, it enables precise monitoring of drug quality; and in clinical application, it ensures the consistency and safety of drug efficacy. This is of great significance for promoting the development of Baoxin Granule as a new Chinese medicine and enhancing the competitiveness of Traditional Chinese Medicine in the treatment of cardiovascular diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The mobile phase elution gradient results of Example 1 are as follows;

[0030] Figure 2 This is a stacked graph of the detection wavelength of 190-230 nm in Example 1; the abscissa is the retention time, and the ordinate is the response signal;

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

[0032] Figure 4 This is a stacked graph of the detection wavelength of 284-310 nm in Example 1; the abscissa is the retention time, and the ordinate is the response signal;

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

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

[0035] Figure 7 This is a stacked diagram of the extraction method of the test sample in Example 1;

[0036] Figure 8 This is a stacked diagram of the solvent extraction of the test sample in Example 1;

[0037] Figure 9 This is a comparison of the dissolution of the test sample by different extraction solvents in Example 1;

[0038] Figure 10 This is a stacked graph of the extraction time of the test sample in Example 1;

[0039] Figure 11 This is a stacked diagram of the sample weights of Example 1;

[0040] Figure 12 To maximize the information of Example 1, the HPLC comparison chart was examined;

[0041] Figure 13 This is the chromatogram of the common peaks in the fingerprint of Baoxin Granule in Example 1;

[0042] Figure 14 This is the fingerprint chromatographic peak identification result diagram of Example 1;

[0043] Figure 15 HPLC stacking diagram for investigation of fingerprint specificity of Example 1;

[0044] Figure 16 HPLC stacking diagram for precision investigation of Example 1;

[0045] Figure 17 HPLC stacking diagram for repeatability inspection of Example 1;

[0046] Figure 18 HPLC stacking diagram for the intermediate precision investigation of Example 1;

[0047] Figure 19 This is a HPLC stacked diagram of the stability study of Example 1;

[0048] Figure 20 This is an HPLC stacking diagram for the durability test of Example 1 - investigation of different flow rates and column temperatures; S1 is a flow rate of 0.5 ml / min, S2 is a flow rate of 0.6 ml / min, S3 is a flow rate of 0.65 ml / min, S4 is a flow rate of 0.7 ml / min, S5 is a column temperature of 28°C, S6 is a column temperature of 30°C, and S7 is a column temperature of 32°C;

[0049] Figure 21 This is an HPLC stacking diagram of the durability test of Example 1 - inspection of instruments from different manufacturers; S1 is Agilent-reference material, S2 is Agilent-test material, S3 is Waters-reference material, and S4 is Waters-test material;

[0050] Figure 22 This is an HPLC stacking diagram of the durability test of Example 1 - investigation of different brands of reagents; S1 is a different brand of reagent 1-blank solvent, S2 is a different brand of reagent 1-reference substance, S3 is a different brand of reagent 1-test product, S4 is a different brand of reagent 2-blank solvent, S5 is a different brand of reagent 2-reference substance, and S6 is a different brand of reagent 2-test product;

[0051] Figure 23This is an HPLC stacking diagram of the durability test of Example 1 - investigation of different brands of chromatographic columns; S1 is ZORBAX SB-C18, S2 is Inertsil ODS-HL, S3 is Ultimate Plus C18, S4 is Inertsustain C18, S5 is ShimNex HEC18-AQ, and S6 is Shim-packG STC18-AQ;

[0052] Figure 24 This is the control fingerprint of Example 1;

[0053] Figure 25 This is a stacked diagram of the fingerprint of the Baoxin Granule sample in Example 1;

[0054] Figure 26 This is a graph showing the peak purity of salvianolic acid B (left) and naringin (right) in Example 2;

[0055] Figure 27 This is a graph showing the specificity of the assay in Example 2;

[0056] Figure 28 This is the linear relationship diagram of salvianolic acid B and naringin in Example 2. DETAILED DESCRIPTION

[0057] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0058] Example 1

[0059] 1. Optimization of chromatographic conditions

[0060] Preparation of test solution: Take Baoxin Granule (hereinafter referred to as this product, produced by Zhejiang Kangenbei Pharmaceutical Co., Ltd., according to the following method: accurately weigh 7g of Astragalus, 5g of Salvia miltiorrhiza, 5g of Ophiopogon japonicus, 2g of Poria, 2g of Zedoaria, 1g of Angelica sinensis, 3g of Rehmannia glutinosa, 1g of Citrus aurantium, 1g of Platycodon grandiflorum, 2g of Radix Glycyrrhizae Preparata, 1g of Rhubarb with wine, and 1g of scalded leech. Leech is extracted twice with 70% ethanol, each time with 6 times the amount of ethanol, each extraction for 1 hour, the extracts are combined, the ethanol is recovered and concentrated under reduced pressure to a relative density of 1.15-1.25. The remaining 11 herbs including Astragalus are extracted with water twice, each time with 10 times the amount of water, each time for 1 hour, the extract is filtered, and the filtrate is filtered. The extract is concentrated under reduced pressure to a relative density of 1.15-1.25, and ethanol is added twice as much as necessary for precipitation. The extract is allowed to stand overnight and filtered. The filtrate is used to recover the ethanol and concentrated under reduced pressure to a relative density of 1.15-1.25. The extract is then combined with a leech alcohol extract paste and dried. The dry extract is added to dextrin in a 1:1 ratio, granulated, dried, and granulated to 1000 g, which is then packaged. (The extract is obtained by grinding an appropriate amount, taking approximately 0.5 g, accurately weighing it, and placing it in a stoppered conical flask. 50 ml of 80% methanol is accurately added, the flask is sealed, and ultrasonic treatment (1100 W, 40 kHz) is performed for 30 minutes. The extract is removed, cooled, shaken, and the filtrate is taken (filtered through a 0.22 μm organic filter membrane).

[0061] 1.1 Determination of mobile phase ratio

[0062] A specific gradient elution program was designed using octadecylsilane bonded silica gel (Agilent ZORBAX SB-C18 (4.6 mm × 250 mm, 5 μm)) as the filler, acetonitrile as the mobile phase A, and 0.1% phosphoric acid solution as the mobile phase B, as shown in Table 1. During the elution process, the flow rate was 1 ml / min; the column temperature was 30°C; the detection wavelength was 286 nm; and the sample volume was 10 μl. Results are shown in Figure 1 When eluted according to the mobile phase ratio, the peaks of different chemical components in the chromatogram were clear and independent, indicating that the above mobile phase ratio and gradient elution procedure can effectively separate the sample components.

[0063] Table 1 Gradient elution program

[0064]

[0065]

[0066] 1.2 Determination of column temperature / flow rate

[0067] Using the mobile phase ratios determined above, the effects of column temperature and flow rate were systematically investigated. At a column temperature of 30°C, the flow rate was set at 0.5–1.0 ml / min (in 0.1 ml / min increments); at a flow rate of 0.6 ml / min, the column temperature was set at 25–35°C (in 2–3°C increments). Flow rate influences mass transfer and diffusion, while column temperature alters mobile phase viscosity and solute partition coefficients, both of which influence the chromatographic behavior of the target. The results are shown in Table 2.

[0068] Table 2 Column temperature / flow rate separation results

[0069] Chromatographic conditions Separation of naringin front and back peaks Separation of front and back peaks of salvianolic acid B 0.5ml / min-30℃ 19.60 / 4.32 11.17 / 2.24 0.6ml / min-30℃ 18.73 / 4.30 12.10 / 2.05 0.7ml / min-30℃ 18.03 / 4.54 13.05 / 2.24 0.8ml / min-30℃ 16.60 / 4.57 13.09 / 2.24 1.0ml / min-30℃ 16.05 / 10.71 12.98 / 1.01 0.6ml / min-25℃ 19.11 / 1.86 12.61 / 2.26 0.6ml / min-28℃ 18.25 / 4.26 12.99 / 2.23 0.6ml / min-30℃ 18.73 / 4.30 12.10 / 2.05 0.6ml / min-32℃ 19.86 / 4.77 11.44 / 2.10 0.6ml / min-35℃ 19.94 / 5.02 13.19 / 2.01

[0070] Table 2 shows that flow rate and column temperature affect overall chromatographic peak shape and resolution. Naringin achieved resolution that met the standard (>1.5) under all chromatographic conditions. Salvianolic acid B met the standard resolution under all conditions, except at a flow rate of 1.0 ml / min, indicating that it is less affected by chromatographic conditions.

[0071] After comprehensive consideration, a flow rate of 0.6 ml / min and a column temperature of 30°C were selected as chromatographic parameters. This selection ensures good peak shape and resolution, ensuring accurate and reliable analysis of naringin and salvianolic acid B, and providing optimal conditions for Baoxin Granule fingerprint analysis.

[0072] 1.3 Determination of detection wavelength

[0073] Accurately draw the test solution and inject it into the liquid chromatograph. Use a diode array detector to perform a full wavelength scan at a wavelength of 190nm to 400nm. Then extract the chromatograms at different detection wavelengths for comparison. Figure 2-Figure 6 .

[0074] The results showed that there were fewer chromatographic peaks within the 190nm-250nm and 350nm-400nm wavelength ranges, while the number of chromatographic peaks in the 260nm-340nm range did not differ significantly, but the response values ​​did differ. Considering that the maximum absorption wavelengths for naringin and salvianolic acid B are 284nm and 286nm, respectively, a detection wavelength of 286nm was tentatively selected for the determination of naringin and salvianolic acid B, as well as for fingerprint analysis.

[0075] 1.4 Determination of chromatographic conditions

[0076] After the above investigations on the mobile phase ratio, column temperature and flow rate, and detection wavelength, the following chromatographic conditions and system suitability test conditions were preliminarily determined:

[0077] Octadecylsilane bonded silica gel was used as the filler (Agilent ZORBAX SB-C18 (4.6 mm × 250 mm, 5 μm)), acetonitrile was used as the mobile phase A, and 0.1% phosphoric acid solution was used as the mobile phase B. Gradient elution was performed according to the description in Table 1; the flow rate was 0.6 ml per minute; the detection wavelength was 286 nm; the column temperature was 30°C, and the sample volume was 10 μl.

[0078] 2. Investigation of test sample preparation methods

[0079] The determination was carried out according to the chromatographic conditions under "1.4 Determination of Chromatographic Conditions". By examining factors such as the extraction method, extraction solvent, extraction time and sample weight during the preparation of the test sample, the chromatograms were recorded, the peak shapes were compared, and the ratio of the peak area of ​​the characteristic peak to the concentration of the sample weight was used as the measurement standard. A comprehensive comparison was made to determine the method that was more conducive to the extraction of the target component, and the extraction method of the test sample was optimized, thereby providing a reliable sample basis for the subsequent analysis of Baoxin Granules.

[0080] 2.1 Investigation of extraction methods

[0081] Take an appropriate amount of this product, grind it into powder, take 0.5g, weigh it accurately, put it in a stoppered conical flask, accurately add 50ml of 80% methanol, stopper it, weigh it, and examine two extraction methods: ultrasonic treatment (1100W, 40kHz) and heating reflux (water bath temperature: 80℃). Both extractions were conducted for 30 minutes. Take it out, let it cool, weigh it again, add 80% methanol to make up for the lost weight, shake it evenly, filter it through a 0.22μm organic filter membrane, take the filtrate, and obtain the test solution.

[0082] See the results Figure 7 and Table 3. The chromatographic peak information content and response values ​​of the two extraction methods were 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 solution, ultrasonic treatment was tentatively proposed as the extraction method.

[0083] Table 3 Peak area data of extraction method

[0084]

[0085] 2.2 Extraction solvent investigation

[0086] Take an appropriate amount of this product, grind it into powder, take 0.5g, weigh it accurately, put it into a stoppered conical flask, and accurately add 50ml each of 30% methanol, 60% methanol, 80% methanol, methanol, 30% ethanol, 60% ethanol, and 80% ethanol, stopper it tightly, weigh it, and treat it ultrasonically (1100W, 40kHz) for 30 minutes. Take it out, let it cool, weigh it again, add the corresponding reagent to make up for the lost weight, shake it well, filter it through a 0.22μm organic filter membrane, take the filtrate to obtain the test solution.

[0087] See the results Figure 8-Figure 9 and Table 4. When methanol is used as the solvent, peaks 1 and 2 show unsatisfactory peak shapes, and the peak shape of peak 1 in 80% ethanol and 60% ethanol is also unsatisfactory. Figure 9 Regarding sample dissolution, when using methanol, 80% methanol, and 80% ethanol as solvents, the sample exhibited clumping or uneven dispersion at the bottom, potentially leading to an uneven sample solution and affecting the measurement results. Based on the peak shape and sample dissolution, 80% ethanol, 80% methanol, 60% ethanol, and methanol were excluded. Table 4 shows that the RSD value of the total peak area to sample weight and concentration ratio for 60% methanol, 30% methanol, and 30% ethanol was 0.6%, less than 2.0%, indicating no significant differences between the different solvents.

[0088] Taking into account the instability of salvianolic acid B in aqueous solution and the solubility of the test sample in different solvents, 60% methanol was finally determined to be the optimal extraction solvent.

[0089] Table 4 Extraction solvent investigation peak area data

[0090]

[0091] 2.3 Extraction time investigation

[0092] Take an appropriate amount of this product, grind it into powder, take 0.5g, weigh it accurately, put it into a stoppered conical flask, accurately add 50ml of 60% methanol respectively, stopper it, weigh it, and treat it ultrasonically (1100W, 40kHz) for 15 minutes, 30 minutes, 45 minutes, and 60 minutes respectively. Take it out, let it cool, weigh it again, add 60% methanol to make up for the lost weight, shake it well, filter it through a 0.22μm organic filter membrane, take the filtrate to obtain the test solution.

[0093] See the results Figure 10 and Table 5. Comparison of the chromatographic peaks at 15, 30, 45, and 60 minutes reveals good peak shape. The RSD of the total peak area to sample weight and concentration ratio is 0.4%, less than 2.0%, indicating no significant differences between extraction times. Taking into account factors such as energy conservation, the extraction time was determined to be 15 minutes.

[0094] Table 5 Peak area data of extraction time

[0095]

[0096] 2.4 Sample weighing inspection

[0097] Take an appropriate amount of this product, grind it into powder, take 0.25g, 0.5g, 1.0g, and 2.0g, weigh them accurately, place them in a stoppered conical flask, accurately add 50ml of 60% methanol respectively, stopper it, weigh it, and treat it ultrasonically (1100W, 40kHz) for 15 minutes. Take it out, let it cool, weigh it again, add 60% methanol to make up for the lost weight, shake it well, filter it through an organic membrane with a thickness of 0.22μm, take the filtrate to obtain the test solution.

[0098] See the results Figure 11 Comparing the peak shapes of the chromatographic samples at different sample weights and calculating the concentration ratios, as shown in Table 6, revealed an RSD of 1.0%, less than 2.0%. Due to the low overall peak response at a sample weight of 0.25 g, filtration was more difficult at sample weights of 1.0 g and 2.0 g. Taking all factors into consideration, selecting a sample weight of 0.5 g ensured accuracy while avoiding filtration challenges.

[0099] Table 6 Peak area data of sample weight inspection

[0100]

[0101] 2.5 Preliminary determination of test sample preparation method

[0102] Based on the above investigation results on the extraction method, extraction solvent, extraction time and sample weight, the test sample preparation method was preliminarily determined as follows:

[0103] Take an appropriate amount of this product, grind it into powder, take 0.5g, weigh it accurately, put it into a stoppered conical flask, accurately add 50ml of 60% methanol, stopper it tightly, weigh it, ultrasonically treat it (1100W, 40kHz) for 15 minutes, let it cool, weigh it again, make up the lost weight with 60% methanol, shake it well, filter it through a 0.22μm organic filter membrane, and take the filtrate to obtain it.

[0104] 3. Information maximization investigation

[0105] To determine whether the main components of the test sample are reflected in the chromatogram and whether the principle of maximizing effective information is met, sample preparation and analysis were carried out according to the methods specified in "1.4 Determination of Chromatographic Conditions" and "2.5 Preliminary Determination of Test Sample Preparation Method." Following the proposed gradient elution procedure, the elution time was extended after 100 minutes to 300 minutes (the mobile phase ratio was acetonitrile-0.1% phosphoric acid (28:72)). Following this procedure, blank solution and test sample solution were injected sequentially, and the chromatograms were recorded simultaneously.

[0106] See the results Figure 12Under the conditions of a mobile phase ratio of acetonitrile and 0.1% phosphoric acid (28:72) and a final gradient duration twice as long, the sample chromatogram showed no distinct peaks after approximately 120 minutes. This result indicates that the primary components of the sample have been largely obtained, thus essentially meeting the principle of maximum information.

[0107] 4. Peak attribution and chromatographic peak identification

[0108] 4.1 Medicinal flavor peak attribution and chromatographic peak identification

[0109] According to the amount of the prescription, weigh the appropriate amount of Astragalus, Salvia miltiorrhiza, Rehmannia glutinosa, Radix Ophiopogonis, Fructus Aurantii Immaturus, Herba Lycopodii, Poria, Rhubarb (wine-processed), Angelica sinensis, Platycodon grandiflorum, and Radix Glycyrrhizae (roasted liquor) in a stoppered conical flask, add 50 ml of water to each, heat and boil for 30 minutes, filter, evaporate the filtrate to dryness, and then prepare single-ingredient solutions in the same manner as the test sample starting from "add 50 ml of 60% methanol" (for the preparation of scalded leech: weigh the scalded leech slices in the prescription according to the amount of the prescription, weigh the appropriate amount, place in a stoppered conical flask, add 50 ml of 70% ethanol, reflux extract for 30 minutes, filter, evaporate the filtrate to dryness, and prepare in the same manner as the test sample starting from "add 50 ml of 60% methanol"). At the same time, prepare the negative solution corresponding to each medicinal ingredient to determine the attribution of each chromatographic peak in Baoxin Granules.

[0110] Preparation of standard solutions: Accurately weigh danshensu, calycosin glucoside, apiosin isoliquiritin, liquiritin, naringin, neohesperidin, rosmarinic acid, lithospermic acid, salvianolic acid B, and salvianolic acid A reference substances into a volumetric flask. Add an appropriate amount of 60% methanol in water and sonicate to dissolve completely. Allow to cool, then dilute to the mark with 60% methanol in water and shake well to prepare 1 ml of each standard solution containing 5 μg danshensu, 5 μg calycosin glucoside, 5 μg apiosin isoliquiritin, 5 μg liquiritin, 25 μg naringin, 15 μg neohesperidin, 5 μg rosmarinic acid, 5 μg lithospermic acid, 50 μg salvianolic acid B, and 10 μg salvianolic acid A.

[0111] The test solution and each standard reference solution were injected together for testing, and the chromatographic peaks were identified by comparing the chromatographic results of the test solution with those of the standard reference solution. Figure 13 、 Figure 14 , Table 7 and Table 8.

[0112] Table 7 Retention time of chromatographic peaks in characteristic spectra of single herbal medicine pieces

[0113]

[0114]

[0115] Table 8: Attribution of each medicinal flavor peak and chromatographic peak identification results

[0116]

[0117]

[0118] 4.2 Reference peak selection

[0119] The chromatographic peak of naringin, with an elution time of 61.874 minutes, is located in the middle of the chromatogram, serving as an intermediate reference point, effectively correlating and comparing components from different time periods. Its good peak shape and moderate response reduce errors caused by fluctuations in experimental conditions, thereby making experimental results more accurate and reliable. The chromatographic peak of salvianolic acid B, with a relatively late elution time of 84.903 minutes, provides an effective reference for subsequent component analysis. Its good peak shape and moderate response accurately reflect component information, aiding qualitative and quantitative analysis.

[0120] Taking all factors into consideration, the two peaks are respectively in the middle and late stages, 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 do not react with the sample to ensure the authenticity and reliability of the results, and have a certain 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.

[0121] 5. Methodological Validation

[0122] 5.1 Specificity test

[0123] According to the chromatographic conditions and test sample preparation method determined in "1.4 Determination of Chromatographic Conditions" and "2.5 Preliminary Determination of Test Sample Preparation Method", accurately aspirate 60% methanol solution (blank solvent) (S3), excipient solution (S4), reference solution (S2), and test sample solution (S1), inject them into the liquid chromatograph, and record the chromatogram. Figure 15 , blank solvent and negative excipients had no interference at the chromatographic peak position, indicating that the method had good specificity.

[0124] 5.2 Precision test

[0125] Prepare the test solution (1 portion) according to the chromatographic conditions and test sample preparation method determined in "1.4 Determination of Chromatographic Conditions" and "2.5 Preliminary Determination of Test Sample Preparation Method", inject 6 injections continuously, and record the chromatogram. Figure 16, Tables 9 and 10. The similarities of the obtained chromatograms were calculated according to the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine" (2012 Edition). The similarity results were all above 0.98, with RSD values ​​of 0.00%. Using naringin as reference peak S1 and salvianolic acid B as reference peak S2, the relative retention times of some chromatographic peaks were calculated relative to the reference peaks. The relative retention times of Peaks 1, 2, 3, 4, and 5 relative to Peak S1, and the relative retention times of Peaks 7, 8, 9, 11, and 12 relative to Peak S2, all had RSD values ​​less than 2.0%, indicating good instrument precision.

[0126] Table 9 Similarity of instrument precision inspection

[0127] Peak Similarity Precision 1 (S1) 0.984 Precision 2 (S2) 0.984 Precision 3 (S3) 0.984 Precision 4 (S4) 0.984 Precision 5 (S5) 0.984 Precision 6 (S6) 0.984 RSD (%) 0.00

[0128] Table 10 Instrument precision investigation - relative retention time results

[0129]

[0130]

[0131] 5.3 Repeatability test

[0132] According to the chromatographic conditions and test sample preparation method determined in "1.4 Determination of chromatographic conditions" and "2.5 Preliminary determination of test sample preparation method", prepare the test sample solution (6 portions) and record the chromatogram. Figure 17 , Table 11, and Table 12. The similarities of the obtained spectra were calculated according to the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine" (2012 Edition). The similarity results were all above 0.98, with RSD values ​​of 0.00%. Using naringin as reference peak S1 and salvianolic acid B as reference peak S2, the relative retention times of the chromatographic peaks were calculated relative to the reference peaks. The relative retention times of Peaks 1, 2, 3, 4, and 5 relative to Peak S1, and the relative retention times of Peaks 7, 8, 9, 11, and 12 relative to Peak S2, all had RSD values ​​less than 2.0%, indicating good reproducibility.

[0133] Table 11 Repeatability inspection-similarity calculation results

[0134] Peak Similarity Repeatability 1 (S1) 0.984 Repeatability 2 (S2) 0.984 Repeatability 3 (S3) 0.984 Repeatability 4 (S4) 0.984 Repeatability 5 (S5) 0.984 Repeatability 6 (S6) 0.984 RSD (%) 0.00

[0135] Table 12 Repeatability study - relative retention time results

[0136] Peak 1 2 3 4 5 7 8 9 11 12 Repeatability 1 (S1) 0.22 0.43 0.75 0.77 0.79 0.82 0.85 0.90 1.08 1.09 Repeatability 2 (S2) 0.22 0.42 0.75 0.77 0.79 0.82 0.85 0.90 1.08 1.09 Repeatability 3 (S3) 0.22 0.42 0.75 0.77 0.79 0.82 0.85 0.90 1.08 1.09 Repeatability 4 (S4) 0.22 0.43 0.75 0.77 0.79 0.82 0.85 0.90 1.08 1.09 Repeatability 5 (S5) 0.22 0.43 0.75 0.77 0.79 0.82 0.85 0.90 1.08 1.09 Repeatability 6 (S6) 0.22 0.43 0.75 0.77 0.79 0.82 0.85 0.90 1.08 1.09 RSD (%) 0.00 1.21 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

[0137] 5.4 Intermediate precision test

[0138] Select different measurement time, different HPLC instrument, different experimenter (B), prepare the test sample solution (6 parallels) according to "8.3.5 Preliminary determination of test sample preparation method", inject the sample for measurement, and record the chromatogram. Figure 18 , Tables 13 and 14. The similarities of the resulting spectra were calculated according to the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine" (2012 Edition). The similarity results were all above 0.98, with an RSD of 0.04%. Combined with the data from "5.3 Repeatability Test," the intermediate precision relative standard deviation (RSD) obtained by personnel A and B was 0.07%. Using naringin as reference peak S1 and salvianolic acid B as reference peak S2, the relative retention times of the chromatographic peaks were calculated relative to the reference peaks. The relative retention times of Peaks 1, 2, 3, 4, and 5 were calculated relative to Peak S1, and the relative retention times of Peaks 7, 8, 9, 11, and 12 were calculated relative to Peak S2. The results showed that the similarity across multiple measurements by person A was 0.984, with an RSD of 0.00%, indicating good repeatability. The similarity for person B fluctuated between 0.982 and 0.983, with an RSD of 0.04%. The RSD for the similarity between the two groups was 0.07%, indicating good overall performance. Regarding relative retention times, except for Peak 1, where the RSD value was greater than 2%, all other peaks were less than 2%, indicating strong consistency for person A. For person B, only Peak 1 differed from Person A, and the data showed good stability. In summary, despite some differences between persons A and B, the overall experimental precision was good.

[0139] Table 13 Similarity of intermediate precision inspection

[0140]

[0141] Table 14 Intermediate precision investigation - relative retention time results

[0142]

[0143] 5.5 Stability test

[0144] 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 the sample and measure according to the proposed chromatographic conditions and record the chromatogram. Figure 19, Table 15 and Table 16. The similarities of the obtained spectra were calculated according to the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine" (2012 Edition). The similarity results were all above 0.98, with RSD values ​​of 0.00%. Naringin was used as reference peak S1, and salvianolic acid B was used as reference peak S2. The relative retention times of peaks 1, 2, 3, 4, and 5 relative to peak S1, and the relative retention times of peaks 7, 8, 9, 11, and 12 relative to peak S2, all had RSD values ​​less than 2.0%, indicating that the test solution had good stability after 80 hours of storage.

[0145] Table 15 Stability investigation-similarity calculation results

[0146]

[0147]

[0148] Table 16 Stability Study - Relative Retention Time Results

[0149] Peak 1 2 3 4 5 7 8 9 11 12 Stability 0h 0.22 0.43 0.75 0.77 0.79 0.82 0.85 0.90 1.08 1.09 Stability 2h 0.22 0.43 0.75 0.77 0.79 0.82 0.85 0.90 1.08 1.09 Stability 4h 0.22 0.42 0.75 0.77 0.79 0.82 0.85 0.90 1.08 1.09 Stability 6h 0.22 0.42 0.75 0.77 0.79 0.82 0.85 0.90 1.08 1.09 Stability 8h 0.22 0.42 0.75 0.77 0.79 0.82 0.85 0.90 1.08 1.09 Stability 10h 0.22 0.42 0.75 0.77 0.79 0.82 0.85 0.90 1.08 1.09 Stability 12h 0.22 0.42 0.75 0.77 0.79 0.82 0.85 0.90 1.08 1.09 Stability 18h 0.22 0.43 0.75 0.77 0.79 0.82 0.85 0.90 1.08 1.09 Stability 25h 0.22 0.43 0.75 0.77 0.79 0.82 0.85 0.90 1.08 1.09 Stability 32h 0.22 0.43 0.75 0.78 0.79 0.82 0.85 0.90 1.08 1.09 Stability 42h 0.22 0.43 0.75 0.77 0.79 0.82 0.85 0.90 1.08 1.09 Stability 65h 0.22 0.43 0.75 0.78 0.79 0.82 0.85 0.90 1.08 1.09 Stability 80h 0.22 0.42 0.75 0.78 0.79 0.82 0.85 0.90 1.08 1.09 RSD (%) 0.00 1.22 0.00 0.57 0.00 0.00 0.00 0.00 0.00 0.00

[0150] 5.6 Durability test

[0151] The original chromatographic conditions established for this experiment were: column temperature 30°C, flow rate 0.6 ml / min, Agilent instrumentation, reagents acetonitrile (Wokai)-phosphoric acid (Tianjin Damao), and a ZORBAX SB-C18 column. Chromatograms obtained under these original conditions served as baseline results. The robustness of these chromatographic conditions was then investigated under different column temperatures, flow rates, instrumentation from different manufacturers, reagent brands, and column conditions. Robustness was assessed by calculating similarity based on shared peaks in the resulting chromatograms.

[0152] The results of flow rate and column temperature durability test are shown in Figure 20 and Table 17. The chromatographic similarity under the original chromatographic conditions was 0.984. Column temperatures of 28°C and 32°C, and flow rates of 0.5 ml / min, 0.65 ml / min, and 0.7 ml / min were tested. The calculated RAD values ​​for each condition compared to the original conditions were less than 2%. This demonstrates that this method tolerates small changes in column temperature and flow rate well, demonstrating robustness.

[0153] The results of the instrument durability inspection are shown in Figure 21 and Table 18. When the Waters instrument was used, the RAD value was 0.11% compared to the original Agilent instrument. This result indicates that the influence of different manufacturers' instruments on this method is minimal, and this method demonstrates good instrument versatility.

[0154] The results of the reagent durability test are shown in Figure 22 and Table 18. When using Brand 2 reagent, the RAD value of the spectrum compared to the original conditions (using Sinopharm and Tianjin Damao reagents) was 0.06%. This indicates that different brands of reagents have minimal impact on this method, reflecting the method's good adaptability to reagents, a characteristic that is of great guiding significance for actual reagent selection.

[0155] The results of the column durability test are shown in Figure 23 and Table 18. Some columns, such as the Inertsustain C18, were not included in the calculations due to their incompatibility with these conditions. For columns such as the Inertsil ODS-HL and Ultimate Plus C18, the resulting chromatograms exhibited RAD values ​​less than 2% compared to the original conditions (using a ZORBAX SB-C18 column). This demonstrates the method's good versatility across applicable columns, demonstrating robustness and providing greater flexibility in column selection.

[0156] In summary, through the investigation of flow rate and column temperature, instruments, reagents and durability of chromatographic columns, it can be seen that this chromatographic method shows good durability under various changes in conditions, adaptability to different conditions and versatility in the selection of relevant equipment and reagents, which provides a strong basis for the stability and reliability of this method in practical applications.

[0157] Table 17 Durability Study - Column Temperature / Flow Rate Similarity Calculation Results

[0158] Chromatographic conditions Similarity RAD% original condition 0.984 — 0.5ml / min 0.985 0.05 0.65ml / min 0.986 0.10 0.7ml / min 0.985 0.05 28℃ 0.985 0.05 32℃ 0.994 0.51

[0159] Table 18 Durability test - similarity calculation results of instruments from different manufacturers / reagents from different brands / chromatographic columns from different brands

[0160]

[0161] 6. Establishment of fingerprint

[0162] Fifteen batches of Baoxin Granules (batch numbers shown in Table 19) were prepared and measured according to the chromatographic conditions and test sample preparation methods specified in “1.4 Determination of Chromatographic Conditions” and “2.5 Preliminary Determination of Test Sample Preparation Methods”. The chromatograms were integrated and imported into the “Chinese Herbal Chromatographic Fingerprint Similarity Evaluation System” (2012 Edition). The median method was used and multi-point calibration was performed to automatically match and establish an overlay map. A reference map (R) was also established. Figure 24 , Baoxin Granule sample fingerprint stacking diagram is shown in Figure 25 .

[0163] The similarity between the fingerprints of 15 batches of Baoxin Granule and the control was calculated based on the 12 common peaks, and the results are shown in Table 19. The similarities calculated between the fingerprints of samples S2-S15 and the control were 0.982, 0.985, 0.978, 0.990, 0.990, and 0.991, respectively, indicating that the chemical composition of the 15 batches of samples is similar and their quality is stable and uniform.

[0164] Based on the above results, the fingerprint standard was determined as follows: the test sample fingerprint should present chromatographic peaks with the same retention time as the reference chromatographic peaks, and there should be 12 corresponding common peaks. Mark peak matching was used, and according to the Chinese medicine chromatographic fingerprint similarity evaluation system, the similarity between the test sample fingerprint and the reference fingerprint should not be less than 0.90.

[0165] Table 1915 Similarity calculation results of Baoxin granules

[0166]

[0167]

[0168] Example 2 Quantitative detection of salvianolic acid B and naringin based on fingerprint construction method

[0169] 1. Purity of salvianolic acid B and naringin peaks

[0170] The chromatographic conditions and sample preparation method determined in Example 1 were used for preparation and determination. The results are shown in Figure 26 When the UV purity values ​​of naringin and salvianolic acid B in the test samples were detected at a wavelength of 286 nm, they were both greater than the specified 995, indicating that the purity of naringin and salvianolic acid B was high under these detection conditions, with little interference from impurities, and they met the purity requirements of subsequent experiments.

[0171] 2. Methodological Validation

[0172] Preparation of reference solution: Take appropriate amount of naringin reference substance and salvianolic acid B reference substance, accurately weigh them, and add 60% methanol to make a mixed solution containing 25 μg of naringin and 50 μg of salvianolic acid B per 1 ml.

[0173] Preparation of test solution: Take the product, mix it, grind it into powder, take 0.5g, accurately weigh it, put it into a stoppered conical flask, accurately add 50ml of 60% methanol, stopper it, weigh it, ultrasonically treat it (1100W, 40kHz) for 15 minutes, let it cool, weigh it again, make up the lost weight with 60% methanol, shake it well, filter it, and take the filtrate to obtain it.

[0174] Determination method: Accurately aspirate 10 μl of reference solution and test solution, inject into liquid chromatograph, and determine.

[0175] 2.1 Specificity Investigation

[0176] Because Danshen contains salvianolic acid B and Fructus Aurantii contains naringin, negative samples of Baoxin Granule lacking Danshen and Fructus Aurantii were prepared. Then, excipient and negative sample solutions were prepared according to the test sample preparation method determined in Example 1. Blank solvent, excipients, reference substance, negative sample, and test solution were then accurately aspirated for analysis.

[0177] See the results Figure 27 The chromatogram of the negative sample of Salvia miltiorrhiza has no chromatographic peak at the corresponding retention time of salvianolic acid B, and the chromatogram of the negative sample of Citrus aurantium has no chromatographic peak at the corresponding retention time of naringin, and there is no interference from the blank solvent and excipients. Therefore, this method is specific for determining the contents of salvianolic acid B and naringin in this product.

[0178] 2.2 Linear relationship investigation

[0179] Preparation of Reference Solution: Accurately weigh appropriate amounts of naringin and salvianolic acid B reference substances, then add 60% methanol to prepare reference stock solutions containing 250 μg of naringin and 500 μg of salvianolic acid B per 1 ml. Accurately measure appropriate amounts of each reference stock solution and dilute them into a series of solutions with different concentrations: salvianolic acid B concentrations of 5.107 μg, 10.21 μg, 51.07 μg, 255.4 μg, and 510.7 μg per ml; and naringin concentrations of 2.339 μg, 4.679 μg, 23.39 μg, 117.0 μg, and 233.9 μg per ml.

[0180] The chromatographic conditions determined in Example 1 were used for determination, and the peak areas of the chromatographic peaks were recorded. Standard curves were drawn with the concentration of the reference substance (μg / ml) as the abscissa and the peak area (mAU) as the ordinate.

[0181] For salvianolic acid B, the linear relationship results are shown in Figure 28 and Table 20. Its regression equation is: y = 21.2259x + 4.1668; correlation coefficient: 1.0000. The concentration of salvianolic acid B showed good linearity in the range of 5.107 μg / ml to 510.7 μg / ml.

[0182] For naringin, the linear relationship results are shown in Figure 28 and Table 22. Regression equation: y = 28.8702x + 7.2030; correlation coefficient: 1.0000. Naringin concentrations showed good linearity within the range of 2.339 μg / ml to 233.9 μg / ml.

[0183] Table 20 Linear relationship investigation

[0184]

[0185]

[0186] 2.3 Precision test

[0187] The reference sample solution No. 3 under "2.2 Linear Relationship Investigation" was injected five times continuously according to the chromatographic conditions specified in Example 1. The RSDs of the retention times and peak areas of salvianolic acid B and naringin were calculated. The results are shown in Table 21. The RSDs of the retention times and peak areas of salvianolic acid B and naringin were all less than 2.0%, indicating that the proposed analytical method has good injection precision and meets the validation precision requirements.

[0188] Table 21 Precision test results (n=5)

[0189] Number of injections Retention time of salvianolic acid B (min) Salvianolic acid B peak area Naringin retention time (min) Naringin peak area 1 85.191 1138.976 62.085 790.304 2 85.228 1139.660 62.087 789.107 3 85.184 1140.529 62.099 790.758 4 85.098 1137.522 62.049 790.304 5 85.109 1138.630 62.036 790.523 RSD (%) 0.1 0.1 0.1 0.1

[0190] 2.4 Repeatability test

[0191] Experimenter (A) took an appropriate amount of this product, ground it into a fine powder, and accurately weighed 0.5 g (6 portions) to prepare the test sample and assay it. The results are shown in Table 22. The RSD values ​​for naringin and salvianolic acid B were both less than 2.0%, indicating good reproducibility of this method.

[0192] Table 22 Repeatability results (n=6)

[0193] Serial number Naringin content (mg / g) Salvianolic acid B content (mg / g) 1 2.189 5.031 2 2.183 4.995 3 2.183 5.005 4 2.177 5.003 5 2.171 4.986 6 2.174 4.997 mean 2.180 5.003 RSD (%) 0.4 0.4

[0194] 2.5 Intermediate precision test

[0195] Using different measurement times, different HPLC instruments, and different experimenters (B), appropriate amounts of the product were ground finely. Approximately 0.5 g of each sample (6 portions) was accurately weighed and prepared as test samples. The results are shown in Table 23. The average naringin content in the samples measured by operators A and B was 2.211 mg / g, and the average salvianolic acid B content was 4.925 mg / g. The RSD values ​​for both samples were less than 4.0%, indicating good intermediate precision.

[0196] Table 23 Naringin / salvianolic acid B-intermediate precision test results

[0197]

[0198]

[0199] 2.6 Stability test

[0200] Samples of the test solution No. 1 under "2.4 Repeatability Test" were injected and tested at 0, 2, 4, 6, 8, 10, 12, 18, 25, 32, 42, 65, and 80 hours after preparation. The RSDs of the peak areas for naringin and salvianolic acid B were calculated. The results are shown in Table 24. The RSDs for the peak areas for naringin and salvianolic acid B were all less than 2.0%, indicating good stability of the test solution over an 80-hour period.

[0201] Table 24 Stability results (n=13)

[0202] Time (hours) Naringin peak area Salvianolic acid B peak area 0 646.557 1082.515 2 644.846 1079.571 4 645.210 1077.082 6 645.777 1077.197 8 644.847 1076.491 10 643.952 1075.411 12 643.940 1074.285 18 645.331 1080.248 25 643.708 1077.276 32 645.570 1080.413 42 647.065 1081.282 65 647.472 1082.178 80 649.103 1080.753 RSD (%) 0.3 0.3

[0203] 2.7 Accuracy test

[0204] Grind an appropriate amount of this product, then accurately weigh 0.25g (9 replicates) and place each into a stoppered conical flask. Divide these 9 samples into three gradients, adding appropriate amounts of a mixed reference solution (the concentrations of salvianolic acid B and naringin are determined based on the mean values ​​in "9.6.3.2 Repeatability Test") at mass ratios of 1:0.5, 1:1, and 1:1.5, respectively. Prepare the test sample and perform the assay. Record the peak areas and calculate the recovery. The results are shown in Tables 25 and 26. The recovery of naringin ranged from 96.3% to 98.2%, with an average recovery of 98%, meeting the pharmacopoeial requirements (92% to 105%), demonstrating that this method has good accuracy for determining naringin content. The recovery of salvianolic acid B ranged from 96.9% to 98.7%, with an average recovery of 98%, meeting the pharmacopoeial requirements (92% to 105%), demonstrating that this method has good accuracy for determining salvianolic acid B content.

[0205] Table 25 Naringin-Accuracy Test Results (n=9)

[0206]

[0207]

[0208] Table 26 Salvianolic acid B-accuracy test results (n=9)

[0209]

[0210] 2.8 Range Test

[0211] The suitability of the established method for the determination of naringin and salvianolic acid B at different sample concentrations was investigated to provide a basis for evaluating the accuracy and reliability of the method. An appropriate amount of the product was ground into a fine powder. Samples were prepared and assayed at three sample concentrations: 80%, 100%, and 120%. The results are shown in Table 27. The relative standard deviations (RSDs) of the naringin and salvianolic acid B determinations at different concentrations were all less than 2.0%, demonstrating that the method has good accuracy and repeatability within the specified range.

[0212] Table 27 Range test results

[0213] concentration Naringin content (mg / g) Salvianolic acid B content (mg / g) 80％ 2.144 4.930 100％ 2.139 4.917 120％ 2.124 4.872 RSD (%) 0.5 0.7

[0214] 2.9 Durability test

[0215] The test method was the same as in "5.6 Durability Test" of Example 1. The original chromatographic conditions established for this test were: column temperature 30°C, flow rate 0.6 ml / min, Agilent instrumentation, acetonitrile (Wokai)-phosphoric acid (Tianjin Damao) reagents, and a ZORBAX SB-C18 column. The naringin content was 2.180 mg / g, and the salvianolic acid B content was 5.003 mg / g.

[0216] The results are shown in Tables 28 and 29. The RAD values ​​of the salvianolic acid B and naringin contents measured under various slight change conditions were all less than 4.0%, which did not cause significant fluctuations in the measurement results, ensuring the consistency and reliability of the measurement results. It also proved the durability of the method when the column temperature was 28℃~32℃, the flow rate was 0.5ml / min~0.7ml / min, and when different brands of chromatographic columns, different instruments, and different brands of reagents were used, fully demonstrating the stability and accuracy of the method, providing strong data support for chromatographic analysis practice.

[0217] Table 28 Naringin-Durability Test Results

[0218]

[0219] Table 29 Salvianolic acid B-durability test results

[0220]

[0221] 3. Determination of quantitative detection method

[0222] The method's system suitability, linearity, precision, stability, repeatability, accuracy and other methodological validation data are all good and meet the requirements for content determination. It can be used for content determination of Baoxin Granule preparations. The final method is as follows:

[0223] Preparation of reference solution: Take appropriate amount of naringin and salvianolic acid B reference substances respectively, weigh accurately, add 60% methanol to make a mixed solution containing 25 μg of naringin and 50 μg of salvianolic acid B per 1 ml.

[0224] Preparation of test solution: Take an appropriate amount of this product, grind it into powder, take about 0.5g, accurately weigh it, put it into a stoppered conical flask, accurately add 50ml of 60% methanol, stopper it tightly, weigh it, and treat it ultrasonically (1100W, 40kHz) for 15 minutes. Take it out, let it cool, weigh it again, add 60% methanol to make up for the lost weight, shake it well, filter it, and take the filtrate to obtain it.

[0225] Determination method: Accurately aspirate 10 μl of reference solution and test solution respectively, inject into liquid chromatograph and determine.

[0226] 4. Sample testing

[0227] According to the requirements under "4. Determination of Quantitative Detection Method," test solutions were prepared and measured for six batches of Baoxin Granule samples. The results are shown in Table 30. The naringin content in the six samples ranged from 1.711 mg / g to 2.703 mg / g, with an average of 2.071 mg / g; the salvianolic acid B content 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 content of these two index components in different batches of Baoxin Granule, providing a direct basis for evaluating product quality. Furthermore, the content of naringin and salvianolic acid B fluctuated between batches, which may be related to various factors such as raw material quality and production process. Further in-depth research is needed to optimize the production process and improve the stability of product quality.

[0228] Table 30 Sample test results

[0229]

[0230] 5. Comparative study of quality standards

[0231] 5.1 Instruments and reagents

[0232] Three batches of clinical samples were used, with batch numbers 210601, 221001, and 240401, produced by Zhejiang Kangenbei Pharmaceutical Co., Ltd.

[0233] 5.2 Comparative study test

[0234] According to the current quality standards and the determination method determined in this example, three batches of Baoxin Granule clinical samples were measured, and the determination results of the salvianolic acid B content of each batch of samples under two standards were recorded.

[0235] ① Current quality standard content determination method: Determine with reference to high performance liquid chromatography (General Rule 0512).

[0236] Chromatographic conditions and system suitability test: octadecylsilane bonded silica gel was used as the filler; methanol-acetonitrile-formic acid-water (25:10:1:64) was used as the mobile phase; the detection wavelength was 286 nm; and the theoretical plate number was greater than 5000.

[0237] Preparation of reference solution: Take an appropriate amount of salvianolic acid B reference substance, accurately weigh it, and add 75% methanol to make a solution containing 40 μg per 1 ml.

[0238] Preparation of test solution: Take the product, grind it into powder, take about 0.5g, weigh it accurately, put it into a stoppered conical flask, add 50ml of 75% methanol, weigh it, ultrasonicate (300W, 40kHz) for 10min, take it out, let it cool, weigh it again, make up the lost weight with 75% methanol, shake it well, filter it, and take the filtrate to obtain it.

[0239] Determination method: Accurately aspirate 10 μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0240] ② The determination method determined in this example: see "4. Determination of quantitative detection method" for details.

[0241] The results are shown in Table 31. The salvianolic acid B content of Baoxin Granule samples with batch numbers 210601, 221001, and 240401 under the current quality standard was higher than that under the new quality standard, with a relative average deviation (RAD) ranging from 1.7% to 2.8%, a relatively small difference. This indicates that, despite variations in salvianolic acid B content when the quality standard is raised, the content remains relatively stable, which has positive implications for the quality controllability of Baoxin Granule. Preliminary inferences suggest that changes in its content have no significant negative impact on safety and efficacy, but further research is needed to accurately assess its clinical significance. Furthermore, the salvianolic acid B content of different batches of Baoxin Granule under both the current and new quality standards fluctuated, suggesting that attention should be paid to batch-to-batch consistency and process optimization during production.

[0242] In summary, there is no significant difference in the content of salvianolic acid B under the current and new quality standards. In the future, we need to continue to pay attention to the quality consistency between batches and enterprises and optimize measures to ensure the stable quality of Baoxin Granules.

[0243] Table 31 Summary of comparative study on salvianolic acid B between current standard and new standard

[0244]

[0245] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for determining the HPLC fingerprint of Baoxin granules, characterized in that: The steps include: (1) Prepare test solution and standard solution; The preparation method of the test solution is as follows: taking Baoxin granules, adding methanol aqueous solution, and then ultrasonically treating to obtain the test solution; (2) performing HPLC chromatography analysis on the test solution and the standard solution to obtain an HPLC fingerprint of Baoxin Granule consisting of common characteristic peaks of the samples; The HPLC chromatographic analysis conditions are as follows: octadecylsilane bonded silica gel as filler, acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B; detection wavelength of 286 nm; elution procedure: 。 2. The measuring method according to claim 1, wherein The preparation method of the test solution is as follows: accurately weigh 0.5 g of Baoxin granules, add 50 ml of 60% methanol, ultrasonically treat for 15 minutes under 1100 W and 40 kHz conditions, and filter to obtain the test solution.

3. The measuring method according to claim 1, wherein The flow rate of the HPLC chromatography analysis was 0.6 ml per minute, the column temperature was 30° C., and the sample volume was 10 μl.

4. The measuring method according to claim 1, wherein The HPLC chromatographic analysis includes the steps of identifying the common characteristic peaks of the samples in the chromatogram of the test solution using the chromatogram of the standard solution as a control.

5. The measuring method according to claim 1, wherein The standard solution includes danshensu solution, calycosin glucoside solution, apigenin isoliquiritin solution, liquiritin solution, naringin solution, neohesperidin solution, rosmarinic acid solution, lithospermic acid solution, salvianolic acid B solution and salvianolic acid A solution prepared with 60% methanol aqueous solution as solvent.

6. The measuring method according to claim 1, wherein The HPLC fingerprint of the Baoxin Granule contains 12 common characteristic peaks of the samples, among which peak 2 is danshensu, peak 3 is calycosin isoflavone glucoside, peak 4 is apigenin isoliquiritin, peak 5 is liquiritin, peak 6 is naringin, peak 7 is neohesperidin, peak 8 is rosmarinic acid, peak 9 is lithospermic acid, peak 10 is salvianolic acid B, and peak 12 is salvianolic acid A.

7. Application of the HPLC fingerprint of Baoxin Granule determined by the determination method according to any one of claims 1 to 6 in the quality control of the Baoxin Granule production process, the quality evaluation of the finished product, the batch stability analysis and the authenticity identification.

8. A quality control method for Baoxin granules, characterized in that: The quality control method is to achieve quality control of Baoxin Granules by quantitatively detecting the content of naringin and salvianolic acid B in Baoxin Granules using the determination method described in any one of claims 1 to 6.

9. The quality control method according to claim 8, characterized in that: The steps include: (1) Using the determination method according to any one of claims 1 to 6, the sample to be tested is subjected to HPLC chromatography analysis to determine the peak areas of naringin and salvianolic acid B; (2) preparing a naringin standard solution and a salvianolic acid B standard solution with a concentration gradient distribution, measuring the peak area of ​​the standard solution using the determination method described in any one of claims 1 to 6, and constructing a "concentration-peak area" standard curve; (3) Substituting the peak areas of naringin and salvianolic acid B measured in step (1) into the standard curve, the contents of naringin and salvianolic acid B in the sample to be tested are calculated.

10. The quality control method according to claim 9, characterized in that: The standard solution was prepared using 60% methanol aqueous solution as solvent.

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