Construction method and application of high performance liquid chromatography (HPLC) specific chromatogram of nephritis-relieving granules

Abstract

The invention discloses a construction method and application of an HPLC (High Performance Liquid Chromatography) specific chromatogram of nephritis-relieving granules, and belongs to the technical field of quality control and evaluation of Chinese patent medicines. 16 components such as chlorogenic acid are selected as reference substances, a variety of bioactive components are covered, a specific chromatogram is constructed through high performance liquid chromatography and high resolution mass spectrometry, 35 common peaks are contained, the chemical components and relative contents of different batches of particles are visually displayed, the component difference is accurately presented, and the defects of a traditional method are overcome. 16 characteristic peaks are identified and cover 6 medicinal materials, and an efficient method is provided for quality control. Meanwhile, one or more of the 16 components are used as quality markers, the particle quality is comprehensively evaluated, the limitation of a traditional method is avoided, the 16 components can be detected according to clear requirements, and the result integrity is guaranteed. According to the method, sample pretreatment is simple and convenient, methodology investigation verifies that reproducibility and stability are good, quality standard formulation and industrialization requirements are met, and support is provided for quality control of the nephritis-relieving granules.

Description

Technical Field

[0001] This invention belongs to the field of quality control and evaluation technology of traditional Chinese medicine, specifically relating to a method for constructing HPLC characteristic chromatograms of Shenyanshu granules and its application. Background Technology

[0002] Nephritis Relief Granules, National Medicine Approval Number: Z19991077, is composed of 10 medicinal herbs: Atractylodes lancea, Poria cocos, Imperata cylindrica, Stephania tetrandra, raw ginseng, Polygonatum sibiricum, Cuscuta chinensis, Lycium barbarum, Lonicera japonica, and Taraxacum mongolicum. The formula uses Atractylodes lancea to dry dampness and strengthen the spleen, and ginseng to replenish vital energy as the principal herbs, targeting the core pathogenesis of spleen and kidney deficiency and dampness accumulating in the middle jiao, playing a major role in supporting the body's resistance, eliminating pathogenic factors, and consolidating the foundation. Poria cocos, Polygonatum sibiricum, Lycium barbarum, and Cuscuta chinensis are the assistant herbs, assisting in tonifying the kidneys and replenishing essence, strengthening the spleen and lungs, and replenishing qi and nourishing yin, consolidating the effects of the principal herbs, and preventing the internal generation of dampness and damage to both qi and yin. Stephania tetrandra, Imperata cylindrica, Lonicera japonica, and Taraxacum mongolicum are the adjuvant herbs, also dispelling wind and dampness, clearing heat and detoxifying, cooling blood and stopping bleeding, and treating concurrent symptoms such as damp-heat stagnation and water retention. Poria cocos also serves as the guiding herb, harmonizing the effects of the other herbs, guiding them to their respective meridians, and organically coordinating the overall effects of the formula to enhance efficacy. This formula combines tonifying Qi and Yang with nourishing Yin, strengthening the body's resistance while expelling pathogens. It effectively addresses the pathogenesis of early-stage diabetic nephropathy characterized by spleen and kidney Qi deficiency combined with damp-heat, achieving the combined effects of strengthening the spleen and kidneys, nourishing kidney Yin, and clearing heat and dampness. Clinically, Shenyanshu granules can be used to treat symptoms such as edema, lower back pain, dizziness, and fatigue caused by spleen and kidney Yang deficiency type nephritis.

[0003] With the continuous development of traditional Chinese medicine (TCM), the clinical application of Shenyanshu granules has gradually expanded, and market demand has also increased. However, due to the complexity and diversity of TCM components, quality control has always been a key issue restricting the modernization and internationalization of TCM. For Shenyanshu granules, ensuring the stability and consistency of its quality is directly related to the reliability of clinical efficacy and patient safety. Therefore, establishing scientific, accurate, and effective quality control methods is crucial for ensuring the quality of Shenyanshu granules. Currently, the quality control of TCM mainly uses the content determination of a single component or a few components as a quality control indicator. Although this method can reflect the quality of TCM to a certain extent, it has obvious limitations. TCM is a complex system, and its efficacy is often the result of the synergistic effect of multiple components. The content determination of a single component or a few components cannot comprehensively reflect the overall quality characteristics of TCM, nor can it accurately reflect the intrinsic pharmacodynamic material basis of TCM. Shenyanshu granules contain a variety of bioactive components, which work synergistically to treat kidney diseases. Measuring only the content of one component cannot accurately assess the overall quality of Shenyanshu granules, nor can it effectively predict its clinical efficacy. Furthermore, traditional quality control methods are easily affected by various factors, such as the origin of the medicinal materials, the harvesting season, and the processing techniques. These factors can cause changes in the content of components in traditional Chinese medicine, thereby affecting the accuracy and reliability of quality control results.

[0004] Therefore, traditional quality control methods can no longer meet the needs of modern Chinese medicine quality control. There is an urgent need to establish a more scientific, comprehensive, and accurate quality control method that can reflect the overall chemical characteristics of Shenyanshu granules, so as to improve the scientific nature and systematicness of its quality control. Summary of the Invention

[0005] In view of the problems that traditional Chinese medicine quality control methods only measure one or a few components, which cannot comprehensively and accurately reflect the overall quality of Shenyanshu granules and predict efficacy, and are easily affected by various factors, resulting in unreliable results, this invention only provides a method for constructing HPLC characteristic chromatograms of Shenyanshu granules and its application.

[0006] This invention is achieved through the following technical solution:

[0007] This invention provides a method for constructing the HPLC characteristic chromatogram of Shenyanshu granules, comprising the following steps:

[0008] S1, Prepare a test solution of Shenyanshu granules using different batches of Shenyanshu granules as test samples;

[0009] S2, using chlorogenic acid, 5-hydroxymethylfurfural, neochlorogenic acid, monocaffeoyl tartaric acid, cryptochlorogenic acid, caffeic acid, p-hydroxycinnamic acid, ferulic acid, rutin, luteolin, chicoric acid, isochlorogenic acid B, 3,5-di-O-caffeoylquinic acid, 4,5-di-O-caffeoylquinic acid, isorhamnetin and atractylodes as reference standards, a single reference solution was prepared;

[0010] S3. Under the same conditions, perform high-performance liquid chromatography (HPLC) analysis on the test solution and the single reference solution, and record the corresponding chromatograms. The HPLC mobile phase is acetonitrile-0.1% phosphoric acid aqueous solution, with gradient elution. The elution program is shown in the table below:

[0011]

[0012]

[0013] S4. Export the chromatograms of different batches of test solution and reference solution obtained in S3. Import the chromatograms of the test solution into the Chinese medicine chromatographic fingerprint similarity evaluation system and perform similarity analysis to confirm the reliability of the results.

[0014] S5. Perform high-resolution mass spectrometry analysis on the test solution obtained in S1 to obtain the total ion chromatogram; perform data analysis based on the total ion chromatogram and the peak elution of the test chromatogram obtained in S3 to obtain the mass spectrometry results of each chemical component.

[0015] S6. The chromatograms of the test sample and the reference sample obtained in S3, the total ion chromatogram obtained in S5, and the mass spectra of each chemical component are compared to obtain the characteristic chromatogram of Shenyanshu granules composed of common characteristic peaks.

[0016] In S1, different batches of Shenyanshu granules were used as test samples. Methanol solution was added, and the mixture was extracted by ultrasound and filtered to obtain the Shenyanshu granules test sample solution.

[0017] Preferably, the test solution of Shenyanshu granules contains 2.0g of Shenyanshu granules per 25mL of methanol solution, and the ultrasonic extraction conditions are: ultrasonic frequency 40KHz, power 720W, extraction time 20-40min.

[0018] Preferably, the concentration of the methanol solution is 80%.

[0019] Preferably, ultrasonic extraction is performed for 30 minutes.

[0020] Preferably, the filtration is performed using a 0.45μm microporous membrane.

[0021] In S2, the single reference solutions include: chlorogenic acid single reference solution, containing 0.15 mg of chlorogenic acid per 1 mL of methanol solution; 5-hydroxymethylfurfural single reference solution, containing 0.27 mg of 5-hydroxymethylfurfural per 1 mL of methanol solution; neochlorogenic acid single reference solution, containing 0.16 mg of neochlorogenic acid per 1 mL of methanol solution; monocaffeoyl tartaric acid single reference solution, containing 0.27 mg of monocaffeoyl tartaric acid per 1 mL of methanol solution; cryptochlorogenic acid single reference solution, containing 0.18 mg of cryptochlorogenic acid per 1 mL of methanol solution; and caffeic acid single reference solution, containing 0.15 mg of chlorogenic acid per 1 mL of methanol solution. Each mL of methanol solution contains 0.19 mg of caffeic acid; 0.14 mg of p-hydroxycinnamic acid per mL of methanol solution; 0.13 mg of ferulic acid per mL of methanol solution; 0.14 mg of rutin per mL of methanol solution; 0.18 mg of luteolin per mL of methanol solution; 0.26 mg of chicoric acid per mL of methanol solution; and 0.26 mg of isochlorogenic acid B per mL of methanol solution. 0.13 mg; 3,5-di-O-caffeoylquinic acid single reference solution, each 1 mL methanol solution contains 0.15 mg of 3,5-di-O-caffeoylquinic acid; 4,5-di-O-caffeoylquinic acid single reference solution, each 1 mL methanol solution contains 0.28 mg of 4,5-di-O-caffeoylquinic acid; isorhamnetin single reference solution, each 1 mL methanol solution contains 0.17 mg of isorhamnetin; atractylodes single reference solution, each 1 mL methanol solution contains 0.26 mg of atractylodes.

[0022] In S3, the high-performance liquid chromatography detection wavelength is 327 nm; the volumetric flow rate is 1.0 mL / min; the injection volume is 10 μL; and the column temperature is 30 °C.

[0023] S4 specifically involves importing the chromatograms of different batches of Shenyanshu granules test solutions obtained in S3 into the Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System; selecting chromatographic peaks present in all chromatograms of different batches of Shenyanshu granules test solutions as common peaks, generating a reference chromatogram of Shenyanshu granules using the average value calculation method, and calculating the relative retention time and relative 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 Shenyanshu granules test solutions and the common peak patterns; and confirming the reliability of the results based on the similarity result table and the chromatograms of the Shenyanshu granules test solutions.

[0024] In S5, the high-resolution mass spectrometry detection conditions are: electrospray ionization, spray voltage 3500V, sheath gas flow rate 40arb, auxiliary gas flow rate 10arb, capillary temperature 300℃, auxiliary gas temperature 300℃, scanning mode is full scan mode, and mass-to-charge ratio scanning range m / z is 100-1500.

[0025] Specifically, S6 involved comparing the chromatograms of the test solution and the single reference solution of Shenyanshu granules obtained in S3 with the total ion chromatogram and the mass spectrometry results of the chemical components obtained by high-resolution mass spectrometry in S5. The results identified the following peaks in the chromatograms: Peak 2 was 5-hydroxymethylfurfural (retention time 6.942); Peak 4 was neochlorogenic acid (retention time 8.673); Peak 5 was monocaffeoyl tartaric acid (retention time 11.756); Peak 7 was chlorogenic acid (retention time 15.038); Peak 8 was cryptochlorogenic acid (retention time 15.324); Peak 9 was caffeic acid (retention time 19.441); and Peak 10 was p-hydroxycinnamic acid (retention time 27). Peak 969; Peak 11 is ferulic acid, retention time 31.546; Peak 12 is rutin, retention time 33.501; Peak 14 is luteolin, retention time 35.149; Peak 15 is chicoric acid, retention time 36.477; Peak 16 is isochlorogenic acid B, retention time 38.285; Peak 17 is 3,5-di-O-caffeoylquinic acid, retention time 39.688; Peak 18 is 4,5-di-O-caffeoylquinic acid, retention time 43.103; Peak 21 is isorhamnetin, retention time 62.937; Peak 35 is atractylodesin, retention time 82.257; The characteristic spectrum of Shenyanshu granules was obtained.

[0026] Preferably, after obtaining the HPLC characteristic chromatogram of Shenyanshu granules, the HPLC characteristic chromatogram results of Shenyanshu granules are subjected to attribution analysis.

[0027] Preferably, the chemical components corresponding to the 16 characteristic peaks in the characteristic spectrum of Shenyanshu granules were analyzed and assigned. Among them, 5-hydroxymethylfurfural was derived from Imperata cylindrica root; neochlorogenic acid was a common peak of Cuscuta chinensis, Lonicera japonica, Taraxacum mongolicum, and Imperata cylindrica root; monocaffeoyl tartaric acid was derived from Taraxacum mongolicum; chlorogenic acid was a common peak of Cuscuta chinensis, Atractylodes lancea, Lycium barbarum, Lonicera japonica, Taraxacum mongolicum, and Imperata cylindrica root; cryptochlorogenic acid was derived from Imperata cylindrica root; and caffeic acid was a common peak of Lycium barbarum, Taraxacum mongolicum, and Imperata cylindrica root. p-Hydroxycinnamic acid is derived from Imperata cylindrica root; ferulic acid is a common peak of Cuscuta chinensis and Imperata cylindrica root; rutin is a common peak of Cuscuta chinensis, Lycium barbarum, and Taraxacum mongolicum; luteolin is a common peak of Lonicera japonica and Taraxacum mongolicum; chicoric acid is derived from Taraxacum mongolicum; isochlorogenic acid B is a common peak of Cuscuta chinensis and Lonicera japonica; 3,5-di-O-caffeoylquinic acid is derived from Lonicera japonica; 4,5-di-O-caffeoylquinic acid is derived from Lonicera japonica; isorhamnetin is derived from Cuscuta chinensis; atractylodes is derived from Atractylodes lancea.

[0028] This invention provides the HPLC characteristic chromatogram of the nephritis relief granules obtained by the above construction method.

[0029] This invention provides a method for detecting the quality of Shenyanshu granules. The method utilizes the HPLC characteristic chromatogram of Shenyanshu granules to detect the quality of Shenyanshu granule samples or their equivalent drugs. One or more of the following are quality markers: chlorogenic acid, 5-hydroxymethylfurfural, neochlorogenic acid, monocaffeoyl tartaric acid, cryptochlorogenic acid, caffeic acid, p-hydroxycinnamic acid, ferulic acid, rutin, luteolin, chicoric acid, isochlorogenic acid B, 3,5-di-O-caffeoylquinic acid, 4,5-di-O-caffeoylquinic acid, isorhamnetin, and atractylodesin.

[0030] Based on the composition of Shenyanshu granules or similar medicines, the detected components include one or more of the following: chlorogenic acid, 5-hydroxymethylfurfural, neochlorogenic acid, monocaffeoyl tartaric acid, cryptochlorogenic acid, caffeic acid, p-hydroxycinnamic acid, ferulic acid, rutin, luteolin, chicoric acid, isochlorogenic acid B, 3,5-di-O-caffeoylquinic acid, 4,5-di-O-caffeoylquinic acid, isorhamnetin, and atractylodesin.

[0031] The method for detecting the quality of Shenyanshu granules is applied in the construction of quality standards for Shenyanshu granules.

[0032] The method for detecting the quality of Shenyanshu granules is applied in the detection of drugs with the same name and formula as Shenyanshu granules.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] The method for constructing the HPLC characteristic chromatogram of Shenyanshu granules provided by this invention selects 16 components, including chlorogenic acid and 5-hydroxymethylfurfural, as reference standards, covering multiple types of bioactive components in Shenyanshu granules. This method can reflect the chemical composition of the granules from different perspectives, laying the foundation for constructing a characteristic chromatogram that reflects the overall chemical characteristics. Compared with traditional quality control methods that only measure a single or a few components, this method more scientifically and comprehensively reflects the quality characteristics of Shenyanshu granules. The chromatograms of the test sample and reference standards are recorded by high-performance liquid chromatography, and combined with high-resolution mass spectrometry analysis to obtain the total ion chromatogram and the mass spectrometry results of each chemical component, a characteristic chromatogram composed of common characteristic peaks is finally constructed by comparison. This method can accurately present the differences in components between different batches of granules, more scientifically and comprehensively reflecting their quality characteristics, providing rich evidence for quality control, and effectively making up for the shortcomings of traditional methods. The chromatograms of different batches of test sample solutions are imported into a similarity evaluation system for chromatographic fingerprinting of traditional Chinese medicine for similarity analysis. This system assesses quality consistency and stability by calculating the similarity of patterns between batches, enabling it to promptly identify batches with quality abnormalities, ensuring stable and reliable product quality, and providing an effective means for quality control in the production process.

[0035] Furthermore, 16 key characteristic peaks were identified, covering six medicinal herbs: Atractylodes lancea, Imperata cylindrica, Cuscuta chinensis, Lycium barbarum, Lonicera japonica, and Taraxacum mongolicum. This provides a rapid and efficient technical method for quality control of Shenyanshu granules, enabling overall control of the composition and content of Shenyanshu granules.

[0036] The HPLC characteristic chromatogram of Shenyanshu granules established by the method provided in this invention contains a total of 35 common peaks. This characteristic chromatogram contains information on multiple components in Shenyanshu granules, and can intuitively display the chemical composition and relative content of different batches of granules, comprehensively reflecting the overall chemical characteristics of the granules. This provides rich information for quality control and a scientific basis for evaluating clinical efficacy. By monitoring the changes of each component in the characteristic chromatogram, the quality stability of the granules can be understood, its clinical efficacy can be predicted, and the safety and effectiveness of medication for patients can be ensured.

[0037] This invention provides a method for detecting the quality of Shenyanshu granules. It uses one or more of 16 components, including chlorogenic acid and 5-hydroxymethylfurfural, as quality markers, covering different types and efficacy of bioactive substances. This method controls granule quality from multiple perspectives. By selecting multiple components as markers, it can more comprehensively and accurately assess the quality of Shenyanshu granules, avoiding the limitations of traditional methods that rely on only a single or a few components. The method explicitly requires the detection of all 16 components, ensuring that the test results fully represent the chemical composition of Shenyanshu granules. Only when all or most of the key components can be accurately detected can the quality of a batch of granules be considered to meet certain standards. This comprehensive detection requirement helps to identify potential component deficiencies or abnormal changes during the production process, allowing for timely adjustments to the production process and ensuring stable product quality. The method offers rapid and simple sample pretreatment. Methodological evaluation of the constructed characteristic spectral method was conducted, including precision, repeatability, and stability tests, verifying the good reproducibility and stability of the established method. This fully meets the practical needs of quality standard setting and industrial quality control, providing technical support and theoretical reference for the quality control of Shenyanshu granules. Attached Figure Description

[0038] Figure 1 The HPLC characteristic chromatogram of Shenyanshu granules;

[0039] Figure 2 This is a chromatogram obtained by optimizing the extraction method during the preparation of the test sample solution according to the present invention;

[0040] Figure 3 This is a chromatogram obtained by optimizing the extraction solvent during the preparation of the test sample solution according to the present invention;

[0041] Figure 4 This is a chromatogram obtained by optimizing the extraction time during the preparation of the test sample solution according to the present invention;

[0042] Figure 5 This is a chromatogram obtained by optimizing the material-to-liquid ratio during the preparation of the test solution according to the present invention;

[0043] Figure 6 This is a chromatogram obtained by optimizing column temperature under chromatographic conditions according to the present invention;

[0044] Figure 7 To optimize the detection wavelength results under chromatographic conditions in this invention, A is the chromatogram and B is the full wavelength scan (190-800 nm);

[0045] Figure 8 This is a chromatogram obtained by optimizing the flow rate under chromatographic conditions according to the present invention;

[0046] Figure 9This is a chromatogram obtained by optimizing the mobile phase composition under chromatographic conditions according to the present invention;

[0047] Figure 10 This is a chromatogram obtained by optimizing the elution procedure under chromatographic conditions according to the present invention;

[0048] Figure 11 The chromatogram (A), 3D chromatogram (B), and UV chromatogram (C) of chlorogenic acid are shown in this invention.

[0049] Figure 12 The chromatogram (A), 3D chromatogram (B), and UV chromatogram (C) of 5-hydroxymethylfurfural of this invention are shown below.

[0050] Figure 13 The chromatogram (A), 3D chromatogram (B), and UV chromatogram (C) of the novel chlorogenic acid of this invention are shown.

[0051] Figure 14 The chromatogram (A), 3D chromatogram (B), and UV chromatogram (C) of monocaffeoyl tartaric acid of the present invention are shown.

[0052] Figure 15 The chromatogram (A), 3D chromatogram (B), and UV chromatogram (C) of cryptochlorogenic acid are shown in this invention.

[0053] Figure 16 The chromatogram (A), 3D chromatogram (B), and UV chromatogram (C) of caffeic acid are shown in this invention.

[0054] Figure 17 The chromatogram (A), 3D chromatogram (B), and UV chromatogram (C) of p-hydroxycinnamic acid are shown in this invention.

[0055] Figure 18 The chromatogram (A), 3D chromatogram (B), and UV chromatogram (C) of ferulic acid of the present invention are shown.

[0056] Figure 19 The chromatogram (A), 3D chromatogram (B), and UV chromatogram (C) of rutin in this invention are shown.

[0057] Figure 20 The chromatogram (A), 3D chromatogram (B), and UV chromatogram (C) of luteolin glycoside in this invention are shown.

[0058] Figure 21 The chromatogram (A), 3D chromatogram (B), and UV chromatogram (C) of chicoric acid are shown in this invention.

[0059] Figure 22 The chromatogram (A), 3D chromatogram (B), and UV chromatogram (C) of isochlorogenic acid B are shown in the present invention.

[0060] Figure 23 The chromatogram (A), 3D chromatogram (B), and UV chromatogram (C) of 3,5-di-O-caffeoylquinic acid of this invention are shown.

[0061] Figure 24 The chromatogram (A), 3D chromatogram (B), and UV chromatogram (C) of 4,5-di-O-caffeoylquinic acid of this invention are shown.

[0062] Figure 25 The chromatogram (A), 3D chromatogram (B), and UV chromatogram (C) of isorhamnetin of this invention are shown.

[0063] Figure 26 The chromatogram (A), 3D chromatogram (B), and UV chromatogram (C) of atractylodesin in this invention are shown.

[0064] Figure 27 This is a mass spectrum of the nephritis relief granules of the present invention in positive ion mode;

[0065] Figure 28 This is a mass spectrum of the nephritis relief granules of the present invention in negative ion mode;

[0066] Figure 29 This is the mass spectrum of ginsenoside Rd of the present invention;

[0067] Figure 30 This is a mass spectrum of the oak bark from the present invention;

[0068] Figure 31 This is the mass spectrum of chlorogenic acid / neochlorogenic acid / cryptochlorogenic acid of the present invention;

[0069] Figure 32 This is the mass spectrum of monocaffeoyl tartaric acid of the present invention;

[0070] Figure 33 This is the mass spectrum of coffee acid in this invention;

[0071] Figure 34 This is the mass spectrum of the alkaloids from the bleeding heart tree in this invention;

[0072] Figure 35 This is the mass spectrum of chicoric acid in this invention;

[0073] Figure 36 This is the mass spectrum of hyperoside / isoquercetin of the present invention;

[0074] Figure 37 This is the mass spectrum of rutin in this invention;

[0075] Figure 38 This is the mass spectrum of astragaloside / luteolinoside of the present invention;

[0076] Figure 39 This is the mass spectrum of 3,5-di-O-caffeoylquinic acid / 4,5-di-O-caffeoylquinic acid / isochlorogenic acid A / isochlorogenic acid B / isochlorogenic acid C of the present invention;

[0077] Figure 40 This is the mass spectrum of palmitic acid in this invention;

[0078] Figure 41 This is the mass spectrum of p-hydroxycinnamic acid according to the present invention;

[0079] Figure 42 This is the basal spectrum of the magnolia flower of the present invention;

[0080] Figure 43 This is the mass spectrum of the phenol base of the present invention;

[0081] Figure 44 This is a spectral diagram of Atractylodes lancea from the present invention;

[0082] Figure 45 The characteristic chromatograms of 15 batches of the test sample of the Nephritis Relief Granules of this invention are shown. Detailed Implementation

[0083] 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 test methods used are conventional methods, and all raw materials used are commercially available products.

[0084] This invention provides a method for constructing the HPLC characteristic chromatogram of Shenyanshu granules, comprising the following steps:

[0085] S1, Prepare a test solution of Shenyanshu granules using different batches of Shenyanshu granules as test samples;

[0086] S2, using chlorogenic acid, 5-hydroxymethylfurfural, neochlorogenic acid, monocaffeoyl tartaric acid, cryptochlorogenic acid, caffeic acid, p-hydroxycinnamic acid, ferulic acid, rutin, luteolin, chicoric acid, isochlorogenic acid B, 3,5-di-O-caffeoylquinic acid, 4,5-di-O-caffeoylquinic acid, isorhamnetin and atractylodes as reference standards, a single reference solution was prepared;

[0087] S3. Under the same conditions, perform high-performance liquid chromatography (HPLC) analysis on the test solution and the single reference solution, and record the corresponding chromatograms. The HPLC mobile phase is acetonitrile-0.1% phosphoric acid aqueous solution, with gradient elution. The elution program is shown in the table below:

[0088] Table 1: Elution Procedures for Liquid Chromatography

[0089]

[0090] S4. Export the chromatograms of different batches of test solution and reference solution obtained in S3. Import the chromatograms of the test solution into the Chinese medicine chromatographic fingerprint similarity evaluation system and perform similarity analysis to confirm the reliability of the results.

[0091] S5. Perform high-resolution mass spectrometry analysis on the test solution obtained in S1 to obtain the total ion chromatogram; perform data analysis based on the total ion chromatogram and the peak elution of the test chromatogram obtained in S3 to obtain the mass spectrometry results of each chemical component.

[0092] S6. The chromatograms of the test sample and reference sample obtained in S3, the total ion chromatogram obtained in S5, and the mass spectra of each chemical component were compared to obtain the HPLC characteristic chromatogram of Shenyanshu granules composed of common characteristic peaks. See Appendix. Figure 1 .

[0093] The preferred method for preparing the test solution of Shenyanshu granules in S1 is as follows: Weigh 2.0g of different batches of Shenyanshu granules, place them in a stoppered conical flask, add 25mL of 80% methanol solution, extract by ultrasonication (ultrasonic frequency: 40KHz, power: 720W) for 30min, filter, concentrate by rotary evaporation to 5mL, and filter through a 0.45μm microporous membrane to obtain the test solution of Shenyanshu granules.

[0094] The preferred method for preparing the reference solution in S2 is as follows: Chlorogenic acid, 5-hydroxymethylfurfural, neochlorogenic acid, monocaffeoyl tartaric acid, cryptochlorogenic acid, caffeic acid, p-hydroxycinnamic acid, ferulic acid, rutin, luteolin, chicoric acid, isochlorogenic acid B, 3,5-di-O-caffeoylquinic acid, 4,5-di-O-caffeoylquinic acid, isorhamnetin, and atractylodesin are accurately weighed and dissolved in 80% methanol solution to prepare a solution containing 0.15 mg of chlorogenic acid, 0.27 mg of 5-hydroxymethylfurfural, 0.16 mg of neochlorogenic acid, 0.27 mg of monocaffeoyl tartaric acid, 0.18 mg of cryptochlorogenic acid, 0.19 mg of caffeic acid, 0.14 mg of p-hydroxycinnamic acid, 0.13 mg of ferulic acid, 0.14 mg of rutin, 0.18 mg of luteolin, 0.26 mg of chicoric acid, and 0.13 mg of isochlorogenic acid B per mL. Single reference solutions of 0.15 mg of 3,5-di-O-caffeoylquinic acid, 0.28 mg of 4,5-di-O-caffeoylquinic acid, 0.17 mg of isorhamnetin, and 0.26 mg of atractylodesin.

[0095] The liquid chromatography conditions in S3 are as follows: column: Shim-pack VP-ODS, 250 mm × 4.6 mm, 5 μm column; detector: diode array detector, detection wavelength: 327 nm; flow rate: 1.0 mL / min; injection volume: 10 μL; column temperature: 30 °C.

[0096] 2. Optimization process of feature map detection,

[0097] (1) Optimization of test sample solution preparation

[0098] This invention investigates different extraction methods, including ultrasonic extraction, reflux extraction, maceration extraction, and ultrasonic followed by rotary evaporation concentration, through experiments. (See appendix.) Figure 2 The results showed that the chromatogram obtained by ultrasonic rotary evaporation concentration extraction was more comprehensive and exhibited the best resolution and peak shape symmetry. Therefore, ultrasonic rotary evaporation concentration extraction method was adopted.

[0099] This invention compares the extraction effects of four extraction solvents: pure ethanol solution, pure methanol solution, 80% methanol solution, and aqueous solution. See appendix. Figure 3 The results showed that when pure ethanol and pure methanol were used as extraction solvents, the number of detected components in the chromatogram was small and the peak response values ​​were low, indicating poor extraction efficiency. The extraction efficiency of 80% methanol solution was similar to that of aqueous solution, but when using 80% methanol solution as the extraction solvent, the number of detectable characteristic peaks in the chromatogram was the highest, the peak areas of each major active ingredient were significantly increased, and the baseline was stable. Considering both extraction efficiency and solvent cost, 80% methanol solution was selected for extraction.

[0100] This invention compared the extraction effects of different extraction times of 15 min, 30 min, 45 min, and 60 min. See attached figure. Figure 4 The results showed that when the extraction time was 15 min, the content of each component in the chromatogram was relatively low. When the extraction time was 30 min, 45 min, and 60 min, the number of peaks for each component in the chromatogram was basically the same, and the peak areas of each major active ingredient were also basically the same. Considering the extraction efficiency and energy consumption cost, an ultrasonic extraction time of 30 min was selected.

[0101] This invention investigated different material-to-liquid ratios of 1.0g, 2.0g, and 3.0g. See attached document. Figure 5 Experimental results showed that the peak response values ​​of each component increased with the increase of the solid-liquid ratio. The total peak area under the 3.0g solid-liquid ratio condition was significantly higher than that under the 1.0g solid-liquid ratio condition, but similar to that under the 2.0g solid-liquid ratio condition. Although the 3.0g solid-liquid ratio showed better extraction efficiency, considering the optimal balance between material consumption and extraction efficiency in actual production, and the fact that the 2.0g solid-liquid ratio can still meet basic detection requirements, the optimal solid-liquid ratio condition was finally determined to be 2.0g.

[0102] (2) Optimization of chromatographic conditions:

[0103] This invention screens column temperatures of 25℃, 30℃, and 35℃; see appendix. Figure 6 The results showed that a significant inverted peak appeared at a column temperature of 25℃, and the peak elution was optimal when the column temperature was maintained at 30℃, with better separation of each component. Therefore, a column temperature of 30℃ was ultimately selected.

[0104] This invention employs a diode array detector to investigate the detection wavelength, performing a full wavelength scan of the sample from 190 to 800 nm, and extracting chromatograms at 190 nm, 210 nm, 310 nm, and 327 nm. (See attached diagram) Figure 7 It was found that the chromatogram contained the most comprehensive information and had the most stable baseline when the detection wavelength was 327 nm, so this method was selected as the detection wavelength condition.

[0105] This invention screened different flow rates of 0.6 mL / min, 0.8 mL / min, and 1.0 mL / min, see appendix. Figure 8 The results showed that the peak patterns of each component in the chromatograms were basically consistent under the three flow rate conditions. Although the content of each component was slightly higher at a flow rate of 0.6 mL / min than at a flow rate of 1.0 mL / min, the higher flow rate of 1.0 mL / min shortened the total analysis time, and the resolution of each target component remained high, with peak shape symmetry factors within the ideal range. Based on a comprehensive consideration of analytical efficiency, separation effect, and system stability, a flow rate of 1.0 mL / min was ultimately selected.

[0106] This invention compares the elution performance of several different elution systems—methanol-0.1% phosphoric acid, acetonitrile-0.1% phosphoric acid, methanol-0.1% formic acid, acetonitrile-0.1% formic acid, methanol-water, and acetonitrile-water—under the same gradient. (See appendix.) Figure 9 The results showed that compared to the methanol system, the acetonitrile system had more detectable characteristic peaks and larger peak areas for each major active ingredient in its chromatogram. The acetonitrile-water elution system exhibited significant peak overlap and low separation efficiency. While the acetonitrile-0.1% formic acid elution system showed some separation ability, its peak shape was not as ideal as that of the acetonitrile-0.1% phosphoric acid elution system, and its baseline stability was also slightly worse. Using acetonitrile-0.1% phosphoric acid as the mobile phase resulted in better separation of the components in the Shenyanshu granules; therefore, acetonitrile-0.1% phosphoric acid was ultimately selected as the mobile phase. After determining the optimal mobile phase composition, this invention screened the best gradient elution program through numerous experiments. Some of the elution programs are as follows:

[0107] Table 2: Elution Procedure 1

[0108]

[0109] Table 3: Elution Procedure 2

[0110]

[0111] Table 4: Elution Procedure 3

[0112]

[0113] Table 5: Elution Procedure 4

[0114]

[0115] Table 6: Elution Procedure 5

[0116]

[0117] Table 7: Elution Procedure 6

[0118]

[0119] Test results as follows Figure 10 As shown. (Through) Figure 10 It can be seen that elution program 6 has good resolution, high peak height, stable baseline, and complete chromatographic information. Therefore, elution program 6 is selected as the optimal elution program.

[0120] The embodiments of the present invention will be described in detail below with reference to the examples. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0121] The instruments and reagents used in the embodiments are as follows: The instruments used in this invention are shown in Table 8; the different batches of nephritis relief granules used in this invention were all purchased from Shaanxi Jianmin Pharmaceutical Co., Ltd., and the reagents used are shown in Table 9.

[0122] Table 8: Instruments and reagents used in this invention

[0123]

[0124] Table 9: Reagents used in this invention

[0125]

[0126] Reference standards: chlorogenic acid (batch number: 110753-202119, purity 96.3%), neochlorogenic acid (batch number: 112110-202401, purity 99.2%), ferulic acid (batch number: 110773-202316, purity 99.3%), isorhamnetin (batch number: 110860-202513, purity 98.0%), 5-hydroxymethylfurfural (batch number: 110753-202316, purity 99.3%), 5-hydroxymethylfurfural (batch number: 110860-202513, purity 98.0%), 5-hydroxymethylfurfural (batch number: 110753-202119, purity 96.3%), neochlorogenic acid (batch number: 112110-202401, purity 99.2%), 5-hydroxymethylfurfural (batch number: 110753-202119, purity 96.3 ... Product No.: 111626-202417, Purity: 99.8%), Monocaffeoyl tartaric acid (Batch No.: 112087-202101, Purity: 98.3%), Cryptochlorogenic acid (Batch No.: 112111-202401, Purity: 96.7%), Caffeic acid (Batch No.: 110885-201703, Purity: 99.7%), Atractylodesin (Batch No.: 111924- 202207, purity 99.7%), p-hydroxycinnamic acid (batch number: 112037-202102, purity 99.7%), chicoric acid (batch number: 111752-202105, purity 98.3%), rutin (batch number: 100080-202513, purity 94.2%), luteolin (batch number: 111720-202312, purity 98%). 1%) was purchased from the National Institutes for Food and Drug Control; isochlorogenic acid B (batch number: 104028-240901, purity 98.5%), 3,5-di-O-caffeoylquinic acid (batch number: 103963, purity 98%), and 4,5-di-O-caffeoylquinic acid (batch number: 104311-230601, purity 99%) were all purchased from Jiangsu Yongjian Pharmaceutical Technology Co., Ltd.

[0127] Example 1

[0128] This embodiment provides a method for constructing the HPLC characteristic chromatogram of Shenyanshu granules, including the following steps:

[0129] S1. Preparation of the test solution for Shenyanshu granules

[0130] Weigh 2.0g of different batches of Shenyanshu granules and place them in a stoppered conical flask. Add 25mL of 80% methanol solution, extract by sonication for 30min, filter, concentrate by rotary evaporation to 5mL, and filter through a 0.45μm microporous membrane to obtain the Shenyanshu granules test solution.

[0131] S2. Preparation of reference solution

[0132] Accurately weigh chlorogenic acid, 5-hydroxymethylfurfural, neochlorogenic acid, monocaffeoyl tartaric acid, cryptochlorogenic acid, caffeic acid, p-hydroxycinnamic acid, ferulic acid, rutin, luteolin, chicoric acid, isochlorogenic acid B, 3,5-di-O-caffeoylquinic acid, 4,5-di-O-caffeoylquinic acid, isorhamnetin, and atractylodesin reference standards, and dissolve them in 80% methanol solution to prepare a solution containing 0.15 mg of chlorogenic acid, 0.27 mg of 5-hydroxymethylfurfural, 0.16 mg of neochlorogenic acid, 0.27 mg of monocaffeoyl tartaric acid, 0.18 mg of cryptochlorogenic acid, 0.19 mg of caffeic acid, 0.14 mg of p-hydroxycinnamic acid, 0.13 mg of ferulic acid, 0.14 mg of rutin, 0.18 mg of luteolin, 0.26 mg of chicoric acid, and 0.13 mg of isochlorogenic acid B per mL. Single reference solutions of 0.15 mg of 3,5-di-O-caffeoylquinic acid, 0.28 mg of 4,5-di-O-caffeoylquinic acid, 0.17 mg of isorhamnetin, and 0.26 mg of atractylodesin.

[0133] S3. Inject the test solution and the reference solution into the high-performance liquid chromatograph (HPLC) for chromatographic analysis. Detection is performed under the same conditions, and the corresponding chromatograms are recorded. The HPLC conditions are as follows: Column: Shim-pack VP-ODS (250 mm × 4.6 mm, 5 μm) column; Detector: Diode array detector, detection wavelength: 327 nm; Flow rate: 1.0 mL / min; Injection volume: 10 μL; Column temperature: 30 ℃; Mobile phase: Acetonitrile (A) - 0.1% phosphoric acid aqueous solution (B), gradient elution, elution program as shown in Table 1.

[0134] S4. Import the chromatograms of the 15 batches of Shenyanshu granules test solutions obtained in S3 into the Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System; select the chromatographic peaks present in the chromatograms of different batches of Shenyanshu granules test solutions as common peaks, generate the reference chromatogram of Shenyanshu granules using the average value calculation method, and calculate the relative retention time and relative peak area of ​​each common peak; perform similarity analysis after data import, multi-point correction and data matching; obtain and export the similarity result table between the chromatograms of different batches of Shenyanshu granules test solutions and the common peak patterns; confirm the reliability of the results based on the similarity result table and the chromatograms of Shenyanshu granules test solutions.

[0135] S5. Perform high-resolution mass spectrometry analysis on the test solution to obtain the total ion chromatogram. Import the total ion chromatogram into Xcalibur software and perform data analysis based on the peak elution of the chromatogram of the test solution to obtain the mass spectrometry results of each chemical component.

[0136] The high-resolution mass spectrometry detection conditions were as follows: electrospray ionization, spray voltage 3500V, sheath gas flow rate 40arb, auxiliary gas flow rate 10arb, capillary temperature 300℃, auxiliary gas temperature 300℃, full scan mode, and mass-to-charge ratio scan range m / z of 100-1500.

[0137] S6. Based on the chromatograms of the test solution of Shenyanshu granules and the single reference solution obtained in S3 (see Appendix). Figures 11-26 ), and combined with the total ion chromatogram obtained by high-resolution mass spectrometry and the mass spectra of chemical components (see Appendix) Figure 27 ~Attached Figure 44 By comparing the chromatograms, the following peaks were identified: peak 2 (5-hydroxymethylfurfural), peak 4 (neochlorogenic acid), peak 5 (monocaffeoyl tartaric acid), peak 7 (chlorogenic acid), peak 8 (cryptochlorogenic acid), peak 9 (caffeic acid), peak 10 (p-hydroxycinnamic acid), peak 11 (ferulic acid), peak 12 (rutin), peak 14 (luteolin), peak 15 (chicoric acid), peak 16 (isochlorogenic acid B), peak 17 (3,5-di-O-caffeoylquinic acid), peak 18 (4,5-di-O-caffeoylquinic acid), peak 21 (isorhamnoside), and peak 35 (atractylaminophen). The HPLC characteristic chromatogram of Shenyanshu granules was obtained. (See attached image.) Figure 45 .

[0138] 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 15 batches of Shenyanshu granules test samples have relatively good similarity, indicating that the characteristic chromatogram of Shenyanshu granules established by this method can effectively detect the quality of Shenyanshu granules and 15 batches of Shenyanshu granules. The results are shown in Table 10.

[0139] Table 10: Similarity between each batch of samples and the common chromatographic peak pattern

[0140]

[0141]

[0142] Example 2: Methodological study of the feature map detection method:

[0143] (1) Precision study

[0144] 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. The peak area and retention time were analyzed and the RSD value was calculated. The results showed that the RSD of the retention time was less than 1.54% and the RSD of the peak area was less than 1.67%, indicating that the parallel injection precision of the device was good.

[0145] Table 11: Peak area and retention time of common characteristic peaks for each batch of Shenyanshu granules samples, and RSD values ​​calculated.

[0146]

[0147]

[0148] (2) Stability Study

[0149] 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 times (0, 2, 6, 12, 18, and 24 hours) with an injection volume of 10 μL. The peak area and retention time of the common characteristic peaks in the HPLC chromatograms of the samples were analyzed, and the RSD values ​​were calculated. The results showed that the RSD of the retention time was less than 1.38%, and the RSD of the peak area was less than 2.39%, indicating that the chromatographic peaks of the Shenyanshu granules test solution showed almost no change within 24 hours, demonstrating good stability.

[0150] Table 12: Peak area and retention time of common characteristic peaks in Shenyanshu granules samples at different time points, and RSD values ​​were calculated.

[0151]

[0152] (3) Reproducibility studies

[0153] Six batches of sample solutions were prepared according to the test solution method in Example 1. Under the chromatographic conditions of Example 1, the injection volume was 10 μL. The peak area and retention time of the common characteristic peaks in the HPLC characteristic chromatograms of the samples were analyzed and the RSD value was calculated. The results showed that the RSD of the retention time was less than 1.87% and the RSD of the peak area was less than 1.96%, indicating that the sample chromatographic peaks had good reproducibility and the method had good repeatability.

[0154] Table 13: Peak area and retention time of common characteristic peaks in each batch of Shenyanshu granules samples, and RSD value calculated.

[0155]

[0156] The above experimental results show that the characteristic spectrum construction method of Shenyanshu granules provided by the present invention has the characteristics of good stability, high precision and good repeatability, and can comprehensively and objectively evaluate the quality of Shenyanshu granules, providing quality assurance for clinical efficacy.

[0157] 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 characteristic chromatogram of Shenyanshu granules, characterized in that, Includes the following steps: S1, Prepare a test solution of Shenyanshu granules using different batches of Shenyanshu granules as test samples; S2, using chlorogenic acid, 5-hydroxymethylfurfural, neochlorogenic acid, monocaffeoyl tartaric acid, cryptochlorogenic acid, caffeic acid, p-hydroxycinnamic acid, ferulic acid, rutin, luteolin, chicoric acid, isochlorogenic acid B, 3,5-di-O-caffeoylquinic acid, 4,5-di-O-caffeoylquinic acid, isorhamnetin and atractylodes as reference standards, a single reference solution was prepared; S3. Under the same conditions, perform high-performance liquid chromatography (HPLC) analysis on the test solution and the single reference solution, and record the corresponding chromatograms. The HPLC mobile phase is acetonitrile-0.1% phosphoric acid aqueous solution, with gradient elution. The elution program is shown in the table below: S4. Export the chromatograms of different batches of test solution and reference solution obtained in S3. Import the chromatograms of the test solution into the Chinese medicine chromatographic fingerprint similarity evaluation system and perform similarity analysis to confirm the reliability of the results. S5, perform high-resolution mass spectrometry analysis on the test solution obtained in S1 to obtain the total ion chromatogram; Data analysis was performed based on the total ion chromatogram and the peak elution of the test sample obtained in S3 to obtain the mass spectrometry results of each chemical component; S6. The chromatograms of the test sample and the reference sample obtained in S3, the total ion chromatogram obtained in S5, and the mass spectra of each chemical component are compared to obtain the HPLC characteristic chromatogram of Shenyanshu granules composed of common characteristic peaks.

2. The method for constructing the HPLC characteristic chromatogram of Shenyanshu granules according to claim 1, characterized in that, In step S1, different batches of Shenyanshu granules were used as test samples. Methanol solution was added, and the mixture was extracted using ultrasound and filtered to obtain a Shenyanshu granules test sample solution. In step S2, the single reference solution contained the following: chlorogenic acid single reference solution, containing 0.15 mg of chlorogenic acid per 1 mL of methanol solution; 5-hydroxymethylfurfural single reference solution, containing 0.27 mg of 5-hydroxymethylfurfural per 1 mL of methanol solution; neochlorogenic acid single reference solution, containing 0.16 mg of neochlorogenic acid per 1 mL of methanol solution; monocaffeoyl tartaric acid single reference solution, containing 0.27 mg of monocaffeoyl tartaric acid per 1 mL of methanol solution; cryptochlorogenic acid single reference solution, containing [missing information - likely a specific ingredient or value] per 1 mL of methanol solution. Cryptochlorogenic acid 0.18 mg; caffeic acid single reference solution, each 1 mL methanol solution contains 0.19 mg caffeic acid; p-hydroxycinnamic acid single reference solution, each 1 mL methanol solution contains 0.14 mg p-hydroxycinnamic acid; ferulic acid single reference solution, each 1 mL methanol solution contains 0.13 mg ferulic acid; rutin single reference solution, each 1 mL methanol solution contains 0.14 mg rutin; luteolin single reference solution, each 1 mL methanol solution contains 0.18 mg luteolin; chicoric acid single reference solution, each 1 mL methanol solution contains 0.26 mg chicoric acid; isochlorogenic acid B single reference solution, each 1 mL methanol solution contains isochlorogenic acid B. 0.13 mg; 3,5-di-O-caffeoylquinic acid single reference solution, each 1 mL methanol solution contains 0.15 mg of 3,5-di-O-caffeoylquinic acid; 4,5-di-O-caffeoylquinic acid single reference solution, each 1 mL methanol solution contains 0.28 mg of 4,5-di-O-caffeoylquinic acid; isorhamnetin single reference solution, each 1 mL methanol solution contains 0.17 mg of isorhamnetin; atractylodes single reference solution, each 1 mL methanol solution contains 0.26 mg of atractylodes.

3. The method for constructing the HPLC characteristic chromatogram of Shenyanshu granules according to claim 1, characterized in that, In S3, the high-performance liquid chromatography detection wavelength is 327 nm; the volumetric flow rate is 1.0 mL / min; the injection volume is 10 μL; and the column temperature is 30 °C.

4. The method for constructing the HPLC characteristic chromatogram of Shenyanshu granules according to claim 1, characterized in that, S4 specifically involves importing the chromatograms of different batches of Shenyanshu granules test solutions obtained in S3 into the Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System; selecting chromatographic peaks present in all chromatograms of different batches of Shenyanshu granules test solutions as common peaks, generating a reference chromatogram of Shenyanshu granules using the average value calculation method, and calculating the relative retention time and relative 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 Shenyanshu granules test solutions and the common peak patterns; and confirming the reliability of the results based on the similarity result table and the chromatograms of the Shenyanshu granules test solutions.

5. The method for constructing the HPLC characteristic chromatogram of Shenyanshu granules according to claim 1, characterized in that, In S5, the high-resolution mass spectrometry detection conditions are: electrospray ionization, spray voltage 3500V, sheath gas flow rate 40arb, auxiliary gas flow rate 10arb, capillary temperature 300℃, auxiliary gas temperature 300℃, scanning mode is full scan mode, and mass-to-charge ratio scanning range m / z is 100-1500.

6. The method for constructing the HPLC characteristic chromatogram of Shenyanshu granules according to claim 1, characterized in that, Specifically, S6 involved comparing the chromatograms of the test solution and the single reference solution of Shenyanshu granules obtained in S3 with the total ion chromatogram and the mass spectrometry results of the chemical components obtained by high-resolution mass spectrometry in S5. The results identified the following peaks in the chromatograms: Peak 2 was 5-hydroxymethylfurfural (retention time 6.942); Peak 4 was neochlorogenic acid (retention time 8.673); Peak 5 was monocaffeoyl tartaric acid (retention time 11.756); Peak 7 was chlorogenic acid (retention time 15.038); Peak 8 was cryptochlorogenic acid (retention time 15.324); Peak 9 was caffeic acid (retention time 19.441); and Peak 10 was p-hydroxycinnamic acid (retention time 27.96). Peaks 9 and 11 are ferulic acid, with a retention time of 31.546; peak 12 is rutin, with a retention time of 33.501; peak 14 is luteolin, with a retention time of 35.149; peak 15 is chicoric acid, with a retention time of 36.477; peak 16 is isochlorogenic acid B, with a retention time of 38.285; peak 17 is 3,5-di-O-caffeoylquinic acid, with a retention time of 39.688; peak 18 is 4,5-di-O-caffeoylquinic acid, with a retention time of 43.103; peak 21 is isorhamnetin, with a retention time of 62.937; peak 35 is atractylodesin, with a retention time of 82.

257. The HPLC characteristic chromatogram of Shenyanshu granules was obtained.

7. The method for constructing the HPLC characteristic chromatogram of the nephritis-relieving granules according to claim 1, characterized in that, After obtaining the HPLC characteristic chromatogram of Shenyanshu granules, the HPLC characteristic chromatogram results of Shenyanshu granules were subjected to attribute analysis.

8. The HPLC characteristic chromatogram of Shenyanshu granules obtained by the construction method according to any one of claims 1 to 7.

9. A method for detecting the quality of Shenyanshu granules, characterized in that, The quality of Shenyanshu granules or its equivalent drugs was determined by using the HPLC characteristic chromatogram of Shenyanshu granules as described in claim 8, with one or more of the following as quality markers: chlorogenic acid, 5-hydroxymethylfurfural, neochlorogenic acid, monocaffeoyl tartaric acid, cryptochlorogenic acid, caffeic acid, p-hydroxycinnamic acid, ferulic acid, rutin, luteolin, chicoric acid, isochlorogenic acid B, 3,5-di-O-caffeoylquinic acid, 4,5-di-O-caffeoylquinic acid, isorhamnetin, and atractylodesin.

10. The method for detecting the quality of Shenyanshu granules according to claim 9, characterized in that, The following components were detected in the material basis of Shenyanshu granules or its equivalent formula: chlorogenic acid, 5-hydroxymethylfurfural, neochlorogenic acid, monocaffeoyl tartaric acid, cryptochlorogenic acid, caffeic acid, p-hydroxycinnamic acid, ferulic acid, rutin, luteolin, chicoric acid, isochlorogenic acid B, 3,5-di-O-caffeoylquinic acid, 4,5-di-O-caffeoylquinic acid, isorhamnetin, and atractylodesin.

Images