HPLC (High Performance Liquid Chromatography) fingerprint spectrum of peach-astragalus hematogenic capsule as well as construction and application thereof

Abstract

The invention discloses an HPLC (High Performance Liquid Chromatography) fingerprint spectrum of a peach-astragalus hematopoiesis capsule as well as construction and application thereof, and belongs to the technical field of quality control of traditional Chinese medicine preparations. According to the method, high performance liquid chromatography and mass spectrometry are combined, and formononin, formononetin, calycosin 7-O-beta-D-glucoside, calycosin, ferulic acid, rutin and 5-hydroxymethylfurfural are used as reference substances; and the fingerprint spectrum is successfully constructed by detecting different batches of the peach-astragalus hematopoiesis capsules. The method is simple and easy to implement, high in precision, good in reproducibility, accurate and reliable, at least seven components can be confirmed at a time under the same chromatographic condition, the detection efficiency is greatly improved, and time and cost are saved; quality monitoring can be accurately, rapidly and simultaneously carried out on a plurality of characteristic components of the peach-stilbene hematogenic capsule, and a powerful guarantee is provided for controlling the quality and ensuring the clinical curative effect of the peach-stilbene hematogenic capsule.

Description

Technical Field

[0001] This invention relates to the field of quality control technology for traditional Chinese medicine preparations, specifically to an HPLC fingerprint of Taoqi Shengxue Capsules and its construction and application. Background Technology

[0002] Taoqi Shengxue Capsules (National Drug Approval Number B20020474), a compound traditional Chinese medicine preparation, is composed of ferrous sulfate, astragalus, angelica, and other Chinese medicinal herbs. It has the effect of tonifying qi and nourishing blood, and is clinically used to treat blood deficiency syndrome. This preparation exerts its efficacy through the synergistic effect of multiple components. Its complex chemical composition makes it difficult for traditional single-component quality control methods to fully reflect its overall quality characteristics. Currently, the quality control of Taoqi Shengxue Capsules mainly relies on the quantitative analysis of individual components such as astragaloside A. This method cannot reflect the synergistic effect of multiple components in a compound traditional Chinese medicine, nor can it effectively monitor the impact of production process fluctuations on product quality. Subtle changes in the production process may lead to changes in the content and proportion of various components; single-component testing cannot detect these changes in a timely manner, thus affecting product quality stability and clinical efficacy.

[0003] While high-performance liquid chromatography (HPLC) offers advantages such as excellent separation and accurate quantification, existing methods primarily focus on single-component analysis, lacking comprehensive evaluation of multiple components. Traditional Chinese medicine (TCM) fingerprinting, as a holistic quality control method, can reveal the patterns of quality changes in compound preparations through the overall characterization of their chemical components. Although this technology has been successfully applied in some TCM preparations, research on a specific HPLC fingerprint for Taoqi Shengxue Capsules remains lacking. Particularly in the areas of characteristic peak group screening and quality evaluation standards related to efficacy, a comprehensive system has not yet been established, making it difficult to closely link fingerprint information with the efficacy of the preparation and accurately evaluate the relationship between preparation quality and efficacy.

[0004] Therefore, developing a fingerprint detection method for Taoqi Shengxue Capsules based on HPLC technology and establishing a scientific and comprehensive quality evaluation system are of great significance for improving the quality control level of this preparation and ensuring the stability of clinical efficacy. At the same time, it can provide new technical support for the modernization of quality management of traditional Chinese medicine. Summary of the Invention

[0005] Given the complex composition of Taoqi Shengxue Capsules, existing quality evaluation methods rely solely on the identification or content determination of single compounds, neglecting the interactions between components and the overall effect. This leads to problems such as insufficient systematic evaluation, inadequate stability assessment, and an inability to comprehensively reflect the pharmacodynamic material basis. This invention aims to provide an HPLC fingerprint of Taoqi Shengxue Capsules, its construction, and its application. This fingerprint can be used to comprehensively evaluate and control the quality of Taoqi Shengxue Capsules, thereby ensuring product quality stability and the safety and efficacy of clinical use.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides a method for constructing an HPLC fingerprint of Taoqi Shengxue Capsules, comprising: S1. Using different batches of Taoqi Shengxue Capsules as test samples, test sample solutions were prepared; using gentian glycoside, gentianin, verbascoside 7-O-β-D-glucoside, verbascoside, ferulic acid, rutin, and 5-hydroxymethylfurfural as reference standards, single reference standard solutions were prepared. S2. Under the same conditions, perform high performance liquid chromatography analysis on the test solution and the reference solution of Taoqi Shengxue Capsules, and record the corresponding chromatograms; S3. Import the chromatogram of the test solution obtained in S2 into the Chinese herbal chromatographic fingerprint chromatogram similarity evaluation system (2004 A version) and perform similarity analysis to confirm the reliability of the results; S4. Perform high-resolution mass spectrometry analysis on the test solution obtained in S2 to obtain the total ion chromatogram; perform data analysis on the peak elution of the total ion chromatogram and the test solution obtained in S2 to obtain the mass spectrometry results of each chemical component. S5. The chromatograms of the test solution and the reference solution obtained in S2, the total ion chromatogram obtained in S4, and the mass spectra of each chemical component are compared to obtain the HPLC fingerprint of Taoqi Shengxue Capsules composed of common characteristic peaks.

[0007] In S1, the contents of different batches of Taoqi Shengxue Capsules were taken, methanol solution was added, ultrasonic extraction was performed, and the mixture was filtered. The filtrate was concentrated and dried under reduced pressure, and dissolved in methanol to obtain the test sample Taoqi Shengxue Capsule solution.

[0008] Furthermore, each 15 mL of methanol solution contains 3.0 g of the contents of Taoqi Shengxue Capsules. The contents are extracted by ultrasonication, filtered, and the filtrate is concentrated and dried under reduced pressure. The filtrate is dissolved in 5 mL of methanol solution and filtered through a microporous membrane to obtain the test solution of Taoqi Shengxue Capsules.

[0009] Furthermore, the methanol solution is a pure methanol solution.

[0010] Furthermore, ultrasonic extraction was performed for 30 minutes.

[0011] Furthermore, filtration is achieved using a 0.45 µm microporous membrane.

[0012] In S1, a single reference solution was prepared by dissolving amygdalin, amygdalin, verbascoflavonoid 7-O-β-D-glucoside, verbascoflavonoid, ferulic acid, rutin, and 5-hydroxymethylfurfural as reference standards in methanol.

[0013] Furthermore, in S1, the single reference solution of gentianin contains 56 μg of gentianin per 1 mL of methanol; the single reference solution of gentianin contains 74 μg of gentianin per 1 mL of methanol; the single reference solution of verbascoside 7-O-β-D-glucoside contains 60 μg of verbascoside 7-O-β-D-glucoside per 1 mL of methanol; the single reference solution of verbascoside contains 48 μg of verbascoside per 1 mL of methanol; the single reference solution of ferulic acid contains 46 μg of ferulic acid per 1 mL of methanol; the single reference solution of rutin contains 50 μg of rutin per 1 mL of methanol; and the single reference solution of 5-hydroxymethylfurfural contains 31 μg of 5-hydroxymethylfurfural per 1 mL of methanol.

[0014] In S2, the high-performance liquid chromatography detection wavelength is 210-310 nm, the column temperature is 25-35℃, and the flow rate is 0.6-1.0 mL / min.

[0015] Furthermore, the detection wavelength was 270 nm, the column temperature was 30 °C, the flow rate was 0.6 mL / min, and the injection volume was 10 μL.

[0016] In S2, the high-performance liquid chromatography (HPLC) conditions are as follows: Shim-pack VP-ODS (250 mm × 4.6 mm, 5 μm) column; mobile phase is methanol-0.1% formic acid; gradient elution program is as follows:

[0017] S3 specifically involves: importing the chromatograms of different batches of Taoqi Shengxue Capsules obtained in S2 into the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (Version 2004 A); selecting chromatographic peaks present in all chromatograms of different batches of Taoqi Shengxue Capsules as common peaks, generating a reference chromatogram of Taoqi Shengxue Capsules using the average value calculation method, and calculating the retention time and peak area of ​​each common peak; performing similarity analysis after data import, multi-point correction, and data matching; obtaining and exporting a similarity result table between the chromatograms of different batches of Taoqi Shengxue Capsules and the common peak patterns; and confirming the reliability of the results based on the similarity result table and the chromatograms of the Taoqi Shengxue Capsules.

[0018] In S4, the detection conditions for the high-resolution mass spectrometry are: electrospray ionization, spray voltage of 3500 V, sheath gas flow rate of 40 arb, auxiliary gas flow rate of 10 arb, capillary temperature of 300℃, auxiliary gas temperature of 300℃, scanning mode of full scan mode, and mass-to-charge ratio scanning range of 100-1500 m / z.

[0019] S5 specifically involves comparing the chromatograms of the test solution and the reference solution obtained in S2 with the total ion chromatogram and the mass spectrometry results of the chemical components obtained by high-resolution mass spectrometry in S4. A total of 7 chromatographic peaks were identified, including peak 3 (5-hydroxymethylfurfural, retention time 17.128 min), peak 10 (verrucin 7-O-β-D-glucoside, retention time 42.5542 min), peak 11 (ferulic acid, retention time 43.0801 min), peak 13 (rutin, retention time 47.0642 min), peak 16 (mangosteenin, retention time 50.6792 min), peak 18 (verrucin, retention time 55.1034 min), and peak 19 (mangosteenin, retention time 62.5734 min). The HPLC fingerprint of Taoqi Shengxue Capsules was thus obtained.

[0020] Furthermore, after obtaining the fingerprint spectrum of Taoqi Shengxue Capsules, an attribution analysis was performed on the fingerprint spectrum results of Taoqi Shengxue Capsules.

[0021] Furthermore, the chemical components corresponding to the seven characteristic peaks in the fingerprint spectrum of Taoqi Shengxue Capsules were analyzed. Among them, peak 3 (5-hydroxymethylfurfural), peak 10 (verrucin 7-O-β-D-glucoside), peak 16 (ammonia glycoside), and peak 19 (ammonia glycoside) were derived from Astragalus membranaceus; peak 11 (ferulic acid) and peak 18 (verrucin) were derived from Angelica sinensis; and peak 13 (rutin) was derived from Lycium barbarum. This invention provides an HPLC fingerprint of Taoqi Shengxue Capsules obtained by the above-described construction method.

[0022] The method for detecting the quality of Taoqi Shengxue Capsules is applied in the construction of quality standards for Taoqi Shengxue Capsules.

[0023] The method for detecting the quality of Taoqi Shengxue Capsules is applied to the detection of drugs with the same name and formula as Taoqi Shengxue Capsules.

[0024] This invention provides a method for detecting the quality of Taoqi Shengxue Capsules. The method utilizes the HPLC fingerprint of Taoqi Shengxue Capsules to detect the quality of Taoqi Shengxue Capsules or similar drug samples. The quality markers are one or more of the following: styracin, styracin, isoflavone 7-O-β-D-glucoside, isoflavone, ferulic acid, rutin, and 5-hydroxymethylfurfural. The detected components include one or more of the following: mongholic acid, mongholic acid, verbascoflavonoid 7-O-β-D-glucoside, verbascoflavonoid, ferulic acid, rutin, and 5-hydroxymethylfurfural.

[0025] Compared with the prior art, the technical solution of the present invention achieves the following beneficial technical effects: The present invention provides a method for constructing an HPLC fingerprint of Taoqi Shengxue Capsules. Utilizing high-performance liquid chromatography (HPLC) combined with gradient elution and specific chromatographic conditions, this method can accurately separate and detect multiple active ingredients in Taoqi Shengxue Capsules, exhibiting high precision, good reproducibility, excellent separation, and high stability. The fingerprint constructed using this method ensures consistency and stability between different batches of Taoqi Shengxue Capsules, providing a reliable reference standard for quality control. Similarity evaluation with the fingerprint spectrum allows for rapid and accurate determination of sample quality compliance, ensuring consistency between different batches and contributing to product quality stability and reliability. Compared to traditional quality testing methods, HPLC fingerprinting technology offers higher detection efficiency, enabling the testing of large numbers of samples in a short time and automatically generating test result reports. It provides a comprehensive, objective, and accurate detection and evaluation of the quality of Taoqi Shengxue Capsules, which is of great significance for ensuring clinical efficacy and provides effective assurance for clinical application.

[0026] The fingerprint spectrum of Taoqi Shengxue Capsules constructed in this invention characterizes 19 common chromatographic peaks and provides clear chemical identification for 7 of the main chromatographic peaks, covering the three prescription herbs: Astragalus membranaceus, Angelica sinensis, and Lycium barbarum. This allows for comprehensive control of the quality and composition of Taoqi Shengxue Capsules. This invention overcomes the shortcomings in the content determination of Taoqi Shengxue Capsules in the implementation standards, and addresses the limitations of existing detection methods that only reflect the quality of a very small number of drug components. The established fingerprint spectrum determination method is an effective supplement to the quality control methods of Taoqi Shengxue Capsules, reflecting the types and quantities of chemical components contained within, effectively characterizing their quality, and facilitating comprehensive quality control of the product.

[0027] This invention provides a quality testing method for Taoqi Shengxue Capsules, which can quickly and accurately identify products from different batches and manufacturers. By comparing the presence or absence of common peaks in the obtained fingerprint spectra, the quality of the preparation can be comprehensively evaluated, thus more effectively ensuring product quality. It has the advantages of being simple, efficient, repeatable, and stable. It is not only applicable to the quality control of Taoqi Shengxue Capsules, but can also provide a reference for the quality control of other traditional Chinese medicine preparations. Attached Figure Description

[0028] Figure 1 This is a chromatogram obtained by optimizing the extraction method during the preparation of the test solution for Taoqi Shengxue Capsules of the present invention; Figure 2 The chromatogram obtained by optimizing the extraction solvent during the preparation of the test solution for the Taoqi Shengxue Capsule of the present invention; Figure 3 This is a chromatogram obtained by optimizing the detection wavelength under chromatographic conditions according to the present invention; Figure 4This is a chromatogram obtained by optimizing column temperature under chromatographic conditions according to the present invention; Figure 5 This is a chromatogram obtained by optimizing the flow rate under chromatographic conditions according to the present invention; Figure 6 This is a chromatogram obtained by optimizing the mobile phase composition under chromatographic conditions according to the present invention; Figure 7 This is a chromatogram obtained by optimizing the elution procedure under chromatographic conditions according to the present invention; Figure 8 This is a 3D scan image (190nm~400nm) of the test sample-DAD full wavelength during the prior art examination of this invention; Figure 9 The chromatogram (A), UV spectrum (B), and 3D spectrum (C) of 5-hydroxymethylfurfural of this invention are shown. Figure 10 The chromatogram (A), UV spectrum (B), and 3D spectrum (C) of ferulic acid of the present invention are shown. Figure 11 The chromatogram (A), UV spectrum (B), and 3D spectrum (C) of the gentian glycosides of this invention are shown. Figure 12 The chromatogram (A), UV spectrum (B), and 3D spectrum (C) of the gentianin of this invention are shown. Figure 13 The chromatogram (A), UV spectrum (B), and 3D spectrum (C) of the verbascoside isoflavone of the present invention are shown. Figure 14 The chromatogram (A), UV spectrum (B), and 3D spectrum (C) of rutin in this invention are shown. Figure 15 The chromatograms (A), UV chromatograms (B), and 3D chromatograms (C) of the verbascoside 7-O-β-D-glucoside of the present invention are shown. Figure 16 This is the total ion chromatogram in positive ion mode for the Taoqi Shengxue Capsules of the present invention; Figure 17 This is the total ion chromatogram of the Taoqi Shengxue Capsules of the present invention in negative ion mode; Figure 18 This is the mass spectrum of 5-hydroxymethylfurfural in this invention; Figure 19 This is the mass spectrum of ferulic acid in this invention; Figure 20 This is the mass spectrum of atractylodes lactone I of the present invention; Figure 21 This is the mass spectrum of atractylodes lactone II of the present invention; Figure 22 This is the mass spectrum of atractylone in this invention; Figure 23 This is the mass spectrum of rutin in this invention; Figure 24This is the mass spectrum of chlorogenic acid / cryptochlorogenic acid / neochlorogenic acid of the present invention; Figure 25 This is the mass spectrum of gentian glycosides from the present invention; Figure 26 This is a mass spectrum of the flower of the *Miscanthus sinensis* according to the present invention; Figure 27 This is the mass spectrum of the verbascoside isoflavone of the present invention; Figure 28 This is the mass spectrum of the 7-O-β-D-glucoside of verbascoside in this invention; Figure 29 This is the gallic acid mass spectrum of the present invention; Figure 30 This is the isochlorogenic acid mass spectrum of the present invention; Figure 31 Fingerprint chromatograms of different batches of Taoqi Shengxue Capsules for testing according to this invention; Figure 32 This is the HPLC fingerprint of the test solution of Taoqi Shengxue Capsules of the present invention. Detailed Implementation

[0029] The following embodiments are provided to further illustrate the present invention, but these embodiments are only for a better understanding of the invention and are not intended to limit the scope or implementation principles of the invention. The implementation of the present invention is not limited to the following. Unless otherwise specified, all experimental methods used are conventional methods, and all raw materials used are commercially available products. The instruments used in the present invention are shown in the table below.

[0030]

[0031] The different batches of Taoqi Shengxue Capsules used in this invention were all purchased from the market. The Taoqi Shengxue Capsules were produced by Shaanxi Haoqijun Pharmaceutical Co., Ltd. The reagents used are shown in the table below.

[0032]

[0033] Reference standards: Aristolochic acid (batch number: PS000671, purity: 98%), aristolochic acid (batch number: PS000674, purity: 98%), and verbascoside (batch number: PS000684, purity: 98%) were all purchased from Chengdu Pusi Biotechnology Co., Ltd.; ferulic acid (batch number: AF21021753, purity: 98%) was purchased from Chengdu Efa Biotechnology Co., Ltd.; rutin (batch number: 100080-200306, purity: 91.7%) was purchased from the National Institutes for Food and Drug Control; 5-hydroxymethylfurfural (batch number: C15467552, purity: 97%) was purchased from Shanghai Maclean Biochemical Technology Co., Ltd.; and verbascoside 7-O-β-D-glucoside (batch number: 21022604, purity: 98%) was purchased from Chengdu Pufeide Biotechnology Co., Ltd.

[0034] This invention provides an HPLC fingerprint of Taoqi Shengxue Capsules, the specific construction method of which is as follows: S1. Using different batches of Taoqi Shengxue Capsules as test samples, test sample solutions were prepared; using gentian glycoside, gentianin, verbascoside 7-O-β-D-glucoside, verbascoside, ferulic acid, rutin, and 5-hydroxymethylfurfural as reference standards, single reference standard solutions were prepared. S2. Under the same conditions, perform high performance liquid chromatography analysis on the test solution and the reference solution of Taoqi Shengxue Capsules, and record the corresponding chromatograms; S3. Import the chromatogram of the test solution obtained in S2 into the Chinese herbal chromatographic fingerprint chromatogram similarity evaluation system (2004 A version) and perform similarity analysis to confirm the reliability of the results; S4. Perform high-resolution mass spectrometry analysis on the test solution obtained in S2 to obtain the total ion chromatogram; perform data analysis on the peak elution of the total ion chromatogram and the test solution obtained in S2 to obtain the mass spectrometry results of each chemical component. S5. The chromatograms of the test solution and the reference solution obtained in S2, the total ion chromatogram obtained in S4, and the mass spectra of each chemical component are compared to obtain the HPLC fingerprint of Taoqi Shengxue Capsules composed of common characteristic peaks.

[0035] The preferred method for preparing the test solution of Taoqi Shengxue Capsules in S1 is as follows: Weigh 3.0 g of the contents of different batches of Taoqi Shengxue Capsules, place them in a stoppered conical flask, add 15 mL of pure methanol solution, extract by ultrasonication for 30 min, filter, concentrate and dry the filtrate under reduced pressure, add 5 mL of pure methanol solution, dissolve, filter, and filter the filtrate through a 0.45 µm microporous membrane to obtain the test solution of Taoqi Shengxue Capsules.

[0036] The preferred method for preparing the standard solution in S1 is as follows: accurately weigh mangiferin, mangiferin, verbascoflavonoid 7-O-β-D-glucoside, verbascoflavonoid, ferulic acid, rutin, and 5-hydroxymethylfurfural, and dissolve them in pure methanol to prepare a single reference solution containing 56 μg of mangiferin, 74 μg of mangiferin, 60 μg of verbascoflavonoid 7-O-β-D-glucoside, 48 μg of verbascoflavonoid, 46 μg of ferulic acid, 50 μg of rutin, and 31 μg of 5-hydroxymethylfurfural per 1 mL.

[0037] The liquid chromatography conditions in S2 were as follows: column: Shim-pack VP-ODS (250 mm × 4.6 mm, 5 μm) column; detector: diode array detector; detection wavelength: 270 nm; flow rate: 0.6 mL / min; injection volume: 10 μL; column temperature: 30℃; mobile phase: methanol (A) - 0.1% formic acid aqueous solution (D), gradient elution, and the elution program is shown in Table 1. Table 1: Elution Procedures for Liquid Chromatography

[0038] This invention provides an optimized process for the HPLC fingerprint detection of the above-mentioned Taoqi Shengxue Capsules: (1) Optimization of test sample solution preparation This embodiment investigates different extraction methods (ultrasound, reflux, impregnation, and ultrasonic followed by vacuum concentration and drying) through experiments. See the appendix for details. Figure 1 , by appendix Figure 1 Data shows that the ultrasonic-assisted vacuum concentration and drying extraction method can more comprehensively and efficiently extract the active ingredients from the preparation through vacuum concentration and drying. Therefore, the ultrasonic-assisted vacuum concentration and drying extraction method was adopted. This example compares the extraction effects of different extraction solvents (pure ethanol solution, 80% methanol solution, pure methanol solution, and water). See the appendix for details. Figure 2 , by appendix Figure 2 The data shows that when pure methanol solution is used as the extraction solvent, the extract has the most chromatographic information and the highest component content. Therefore, pure methanol solution is selected for extraction. (2) Optimization of chromatographic conditions This embodiment uses a diode array detector to investigate the detection wavelength, extracting chromatograms at 210 nm, 245 nm, 254 nm, 270 nm, 290 nm, and 310 nm. See the appendix for details. Figure 3 The chromatogram contains the most comprehensive information and has a stable baseline when the detection wavelength is 270 nm; therefore, this method was selected as the detection wavelength condition. This embodiment screened column temperatures (25℃, 30℃, 35℃), see appendix for details. Figure 4 The optimal column temperature of 30℃ resulted in the best peak elution and separation of components; therefore, 30℃ was ultimately selected as the column temperature. This embodiment screened different flow rates (0.6 mL / min, 0.8 mL / min, and 1.0 mL / min), as detailed in the appendix. Figure 5The optimal flow rate of 0.6 mL / min resulted in the best peak elution and separation of components; therefore, 0.6 mL / min was ultimately selected as the optimal flow rate. After determining the optimal detection wavelength, column temperature, and flow rate, this invention screened mobile phases (methanol-water, methanol-0.1% formic acid, acetonitrile-0.1% formic acid, methanol-0.1% phosphoric acid, acetonitrile-0.1% phosphoric acid, and acetonitrile-water) through extensive experiments. See the appendix for details. Figure 6 Ultimately, methanol-0.1% formic acid was chosen as the mobile phase, and the elution program was optimized. Part of the elution program is as follows: Table 2: Elution Procedure 1

[0039] Table 3: Elution Procedure 2

[0040] Table 4: Elution Procedure 3

[0041] Table 5: Washing Procedure 4

[0042] Table 6: Washing Procedure 5

[0043] Table 7: Elution Procedure 6

[0044] Test results as follows Figure 7 As shown, elution program 4 exhibits good resolution, high peak height, stable baseline, and complete chromatographic information. Therefore, elution program 4 is selected as the optimal elution program. The invention is further illustrated below with specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0045] Example 1 Based on the above-mentioned optimized conditions, this embodiment provides a method for constructing the HPLC fingerprint of Taoqi Shengxue Capsules.

[0046] S1. Preparation of the test solution: Weigh 3.0 g of the contents of different batches of Taoqi Shengxue Capsules and place them in a stoppered conical flask. Add 15 mL of pure methanol solution, extract by sonication for 30 min, filter, concentrate and dry the filtrate under reduced pressure, add 5 mL of pure methanol solution to dissolve, filter, and filter the filtrate through a 0.45 µm microporous membrane to obtain the test solution of Taoqi Shengxue Capsules.

[0047] S2. Preparation of the reference solution: Accurately weigh out styracin, styracin, verbascoflavonoid 7-O-β-D-glucoside, verbascoflavonoid, ferulic acid, rutin, and 5-hydroxymethylfurfural, and dissolve them in pure methanol solution. Each 1 mL contains a single reference solution containing 56 μg of styracin, 74 μg of styracin, 60 μg of verbascoflavonoid 7-O-β-D-glucoside, 48 μg of verbascoflavonoid, 46 μg of ferulic acid, 50 μg of rutin, and 31 μg of 5-hydroxymethylfurfural.

[0048] S3. Chromatographic analysis: The test solution in S1 and the reference solution in S2 were injected separately into a high-performance liquid chromatograph (HPLC) for chromatographic analysis. Detection was performed under the same conditions using an UltiMate 3000 HPLC system. The HPLC conditions were as follows: column: Shim-pack VP-ODS (250 mm × 4.6 mm, 5 μm); detector: diode array detector, detection wavelength: 270 nm; flow rate: 0.6 mL / min; injection volume: 10 μL; column temperature: 30℃; mobile phase: methanol (A) - 0.1% formic acid aqueous solution (D), gradient elution, using the preferred elution program parameters 4 from Example 1. Chromatograms of different single reference solutions are shown below. Figures 9-15 As shown.

[0049] S4. High-performance liquid chromatography-mass spectrometry analysis Based on the chromatograms of the Taoqi Shengxue Capsule test solution and the single reference solution obtained in S3, high-resolution mass spectrometry (HMS) analysis was performed on the Taoqi Shengxue Capsule test solution. The HMS detection conditions were: electrospray ionization, spray voltage 3500 V, sheath gas flow rate 40 arb, auxiliary gas flow rate 10 arb, capillary temperature 300℃, auxiliary gas temperature 300℃, full scan mode, and mass-to-charge ratio scan range m / z 100-1500. The total ion chromatogram was obtained. Figure 16 and Figure 17 The total ion chromatogram was imported into Xcalibur software, and data analysis was performed based on the peak elution of the test solution obtained in S3 to obtain the mass spectrometry results of each chemical component. Figures 18-30High-resolution mass spectrometry analysis of the material basis of Taoqi Shengxue Capsules revealed seven chemical components. Combined with the HPLC-MS component analysis results of extracts from six herbs in the formulation (ferrous sulfate, astragalus, angelica, walnut kernel, wolfberry, and stir-fried atractylodes macrocephala) in the previous stage of this invention, the seven chemical components were further analyzed to identify components from three herbs. The results showed that 5-hydroxymethylfurfural, verbascoside 7-O-β-D-glucoside, gentianin, and gentianin were derived from astragalus; ferulic acid and verbascoside were derived from angelica; and rutin was derived from wolfberry.

[0050] S5, Reliability Verification The chromatograms of different batches of Taoqi Shengxue Capsule test solutions obtained in S3 were imported into the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (Version 2004 A); chromatograms of different batches of Taoqi Shengxue Capsule test solutions were selected ( Figure 31 Chromatographic peaks present in all samples were taken as common peaks. A reference chromatogram of Taoqi Shengxue Capsules was generated using the average value calculation method. The retention time and peak area of ​​each common peak were calculated. After data import, multi-point correction, and data matching, similarity analysis was performed. A similarity result table between the chromatograms of different batches of Taoqi Shengxue Capsules test solutions and the common peak patterns was obtained and exported. Based on the similarity results, the reliability of the results was confirmed for the chromatograms of Taoqi Shengxue Capsules test solutions.

[0051] S6, fingerprint spectrum of Taoqi Shengxue Capsules Based on the chromatograms of the test solution and the single reference solution of Taoqi Shengxue Capsules obtained in S3, and by comparing them with the total ion chromatogram and the mass spectrometry results of the chemical components obtained by high-resolution mass spectrometry, a total of 7 chromatographic peaks were identified. Peak 3 was 5-hydroxymethylfurfural, retention time 17.128 min; peak 10 was verbascoside 7-O-β-D-glucoside, retention time 42.5542 min; peak 11 was ferulic acid, retention time 43.0801 min; peak 13 was rutin, retention time 47.0642 min; peak 16 was gentianoside, retention time 50.6792 min; peak 18 was verbascoside, retention time 55.1034 min; and peak 19 was gentianoside, retention time 62.5734 min. The fingerprint chromatogram of Taoqi Shengxue Capsules was constructed using the method described above. (See Appendix) Figure 32As shown. By comparing the high-performance liquid chromatograms of the sample with those of seven reference standards, including vernix caseosa, and combining the HPLC-MS component analysis results of the extracts of six medicinal materials (ferrous sulfate, Astragalus membranaceus, Angelica sinensis, walnut kernel, Lycium barbarum, and stir-fried Atractylodes macrocephala) in the formulation of this invention, the chemical components corresponding to the seven characteristic peaks in the fingerprint spectrum of Taoqi Shengxue Capsules were assigned and analyzed. Among them, peak 3 (5-hydroxymethylfurfural), peak 10 (vernix caseosa 7-O-β-D-glucoside), peak 16 (amongoronin), and peak 19 (amongoronin) are derived from Astragalus membranaceus; peak 11 (ferulic acid) and peak 18 (vernix caseosa flavonoids) are derived from Angelica sinensis; and peak 13 (rutin) is derived from Lycium barbarum.

[0052] Example 2: Methodological Study of Fingerprint Detection Method (1) Similarity study Meanwhile, this invention uses an automatically generated reference chromatogram R to generate a common chromatographic peak mode. Analysis and calculation show that the common chromatographic peaks of different batches of Taoqi Shengxue Capsules have relatively good similarity, indicating that the fingerprint chromatogram of Taoqi Shengxue Capsules established by this method can effectively detect the quality of Taoqi Shengxue Capsules. The results are shown in Table 8.

[0053] Table 8: Similarity between each batch of Taoqi Shengxue Capsules samples and common chromatographic peak patterns

[0054] (2) Precision study The test solution prepared by the method in Example 1 was analyzed according to the detection method in Example 1. Six parallel injections were performed with an injection volume of 10 μL each. The peak area and retention time of the common characteristic peaks were analyzed, and the RSD values ​​were calculated, as shown in Table 9. The data in Table 9 show that the RSD of the retention time was less than 0.643%, and the RSD of the peak area was less than 1.408%, indicating that the parallel injection precision of this device is good.

[0055] Table 9: Peak area and retention time of common characteristic peaks for each batch of Taoqi Shengxue Capsules, with calculated RSD values.

[0056] (3) Stability study The test solution prepared by the method in Example 1 was analyzed according to the detection method in Example 1. Injections were performed at different time points (0, 2, 6, 12, 18, and 24 h) with an injection volume of 10 μL. The peak area and retention time of the common characteristic peaks in the HPLC fingerprint of the samples were analyzed, and the RSD values ​​were calculated. See Table 10 for details. As shown in Table 10, the RSD of the retention time was less than 0.536%, and the RSD of the peak area was less than 1.848%, indicating that the chromatographic peaks of the Taoqi Shengxue Capsule test solution showed almost no change within 24 h, demonstrating good stability.

[0057] Table 10: Peak area and retention time of common characteristic peaks in Taoqi Shengxue Capsule samples at different times, and RSD values ​​were calculated.

[0058] (4) Reproducibility studies Six batches of test solutions were prepared according to the method for preparing the test solution in Example 1. Under the same chromatographic conditions as in Example 1, the injection volume was 10 μL. The peak area and retention time of the common characteristic peaks in the HPLC fingerprint of the samples were analyzed, and the RSD values ​​were calculated. See Table 11 for details. As shown in Table 11, the results indicate that the RSD of the retention time was less than 0.723%, and the RSD of the peak area was less than 1.964%, indicating good reproducibility of the chromatographic peaks and good repeatability of the method.

[0059] Table 11: Peak area and retention time of common characteristic peaks in each batch of Taoqi Shengxue Capsules samples, and RSD values ​​calculated.

[0060] The above experimental results demonstrate that the fingerprinting method for Taoqi Shengxue Capsules provided by this invention exhibits good stability, high precision, and good repeatability. It can comprehensively and objectively evaluate the quality of Taoqi Shengxue Capsules, providing quality assurance for clinical efficacy. The above embodiments are merely exemplary embodiments of this invention and are not intended to limit the invention. The scope of protection of this invention is defined by the claims.

[0061] The above description is merely an example and illustration of the concept of the present invention. Any modifications or additions made by those skilled in the art to the specific embodiments described, or any substitutions made in a similar manner, as long as they do not deviate from the inventive concept or exceed the scope defined by the claims, shall fall within the protection scope of the present invention.

Claims

1. A method for constructing the HPLC fingerprint of Taohong Qisheng Capsules, characterized in that, Comprise the following steps: S1, with different batches of peach qi blood capsules as test preparation preparation peach qi blood capsules test solution; With mutamycin, formononetin, calycosin 7-O-beta-D-glucoside, calycosin, ferulic acid, rutin, 5-hydroxymethyl furfural as control, preparation of single control solution; S2, under the same conditions, peach qi blood capsules test solution and control solution were analyzed by high performance liquid chromatography, and the corresponding chromatogram was recorded; S3, the test solution chromatogram obtained in S2 was introduced into the traditional Chinese medicine chromatographic fingerprint similarity evaluation system and similarity analysis was carried out, and the reliability of the results was confirmed; S4, the high resolution mass spectrum of peach qi blood capsules test solution was analyzed, and the total ion flow chart was obtained; According to the total ion flow chart and the peak condition of the peach qi blood capsules test solution obtained in S2, data analysis was carried out, and the mass spectrum result chart of each chemical component was obtained; S5, the test solution chromatogram and control solution chromatogram obtained in S2, the total ion flow chart obtained in S4 and the mass spectrum chart of each chemical component were compared, and the HPLC fingerprint of peach qi blood capsules composed of common characteristic peaks was obtained.

2. The method for constructing the HPLC fingerprint of the Taoshengxue Capsules according to claim 1, characterized in that, In S1, different batches of peach qi blood capsules were taken as test preparation, methanol solution was added, ultrasonic extraction was carried out, filtration was carried out, the filtrate was concentrated under reduced pressure and dried, methanol solution was added, dissolved, microporous membrane filtration was carried out, and test solution of peach qi blood capsules was obtained.

3. The method for constructing the HPLC fingerprint of the Taoshengxue capsule according to claim 1, characterized in that, In S2, the high performance liquid chromatography determination conditions were as follows: Shim-pack VP-ODS chromatographic column; The mobile phase was methanol-0.1% formic acid, the detection wavelength was 210-310 nm, the column temperature was 25-35 ℃, the flow rate was 0.6-1.0 mL / min, and the gradient elution program was as follows: 。 4. The method for constructing the HPLC fingerprint of the Taoshengxue capsule according to claim 1, characterized in that, S3 is as follows: the chromatogram of different batches of peach qi blood capsules test solution obtained in S2 is introduced into the traditional Chinese medicine chromatographic fingerprint similarity evaluation system; The chromatographic peaks existing in the chromatogram of different batches of peach qi blood capsules test solution are selected as common peaks, the control chromatogram of peach qi blood capsules is generated by using average value calculation method, the retention time and peak area of each common peak are calculated; After data import, multi-point correction and data matching, similarity analysis is carried out; The similarity result table between the chromatogram of different batches of peach qi blood capsules test solution and the common peak mode is obtained and exported; According to the similarity result table and the chromatogram of peach qi blood capsules test solution, the reliability of the results is confirmed.

5. The method for constructing the HPLC fingerprint of the Taoshengxue capsule according to claim 1, characterized in that, In S4, the detection conditions of high resolution mass spectrometry are as follows: electrospray ionization, spray voltage is 3500 V, sheath gas flow rate is 40 arb, auxiliary gas flow rate is 10 arb, capillary temperature is 300 ℃, auxiliary gas temperature is 300 ℃, scanning mode is full scanning mode, mass to charge ratio scanning range m / z is 100-1500.

6. The method for constructing the HPLC fingerprint of the Taoshengxue capsule according to claim 1, characterized in that, S5 is specifically: according to the chromatogram of the test sample solution and the chromatogram of the control sample solution obtained in S2, combined with the total ion current diagram obtained by high resolution mass spectrometry in S4 and the mass spectrum result diagram of the chemical components, 7 chromatographic peaks are identified, wherein peak No. 3 is 5-hydroxymethyl furfural, the retention time is 17.128 min; peak No. 10 is calycosin-7-O-β-D-glucoside, the retention time is 42.5542 min; peak No. 11 is ferulic acid, the retention time is 43.0801 min; peak No. 13 is rutin, the retention time is 47.0642 min; peak No. 16 is astragalin, the retention time is 50.6792 min; peak No. 18 is calycosin, the retention time is 55.1034 min; peak No. 19 is formononetin, the retention time is 62.5734 min, and the HPLC fingerprint of the Taoshen Shengxue capsule is obtained.

7. The method according to claim 1, characterized in that, After obtaining the fingerprint of the Taoshen Shengxue capsule, the attribution analysis of the fingerprint result of the Taoshen Shengxue capsule is performed.

8. The Taoshen Shengxue capsule HPLC fingerprint obtained by the construction method of any one of claims 1-7.

9. A method for detecting the quality of Taoqishengxue capsules, characterized in that, Using the Taoshen Shengxue capsule HPLC fingerprint of claim 8, the quality of the Taoshen Shengxue capsule or its homonymic and same prescription medicine is detected, and one or more of astragalin, formononetin, calycosin-7-O-β-D-glucoside, calycosin, ferulic acid, rutin and 5-hydroxymethyl furfural is used as a quality marker.

10. The method for detecting the quality of the Taoqishengxue capsule according to claim 9, characterized in that, The quality basis of the Taoshen Shengxue capsule or its homonymic and same prescription medicine includes the following detected components: astragalin, formononetin, calycosin-7-O-β-D-glucoside, calycosin, ferulic acid, 5-hydroxymethyl furfural and rutin.

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