Fingerprint spectrum construction method of rupi sanjie capsules / granules and fingerprint spectrum thereof

Abstract

The application discloses a kind of rupi sanjie capsules / granules fingerprint construction method and its fingerprint, belong to the field of traditional Chinese medicine quality standard research and application. The test solution and single control solution are injected into high performance liquid chromatograph, gradient elution, chromatographic analysis is carried out, and each chromatogram is obtained;Mass spectrum analysis is carried out to the test solution, and mass spectrum result is obtained;With the chromatographic peak existing in the chromatogram of test solution as common peak, the chemical components of chromatographic peak in rupi sanjie capsules / granules fingerprint are identified by combining single control solution chromatogram and test solution mass spectrum result, and rupi sanjie capsules / granules fingerprint is obtained.The detection method of rupi sanjie capsules / granules fingerprint provided by the application has the advantages of simple method, stability, high precision and good reproducibility, can more comprehensively and scientifically evaluate the quality of rupi sanjie capsules / granules, so that the quality and curative effect of product are guaranteed.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine detection technology, specifically relating to a method for constructing a fingerprint spectrum of Rupisan capsules / granules and its fingerprint spectrum. Background Technology

[0002] Both Rupisanjie Capsules and Rupisanjie Granules are exclusive products of Shaanxi Bailu Pharmaceutical Co., Ltd. They are compound preparations made from 11 medicinal herbs: Prunella vulgaris, Ligusticum chuanxiong, Bombyx batryticatus, Carapax Trionycis, Bupleurum chinense, Paeonia lactiflora, Rosa rugosa, Curcuma zedoaria, Angelica sinensis, Corydalis yanhusuo, and Ostrea gigas. They have the effects of promoting qi and blood circulation, softening and dispersing nodules, and are mainly used for mammary hyperplasia caused by qi stagnation and blood stasis, with symptoms such as breast pain, breast lumps, irritability, and chest and rib distension. Clinically, they are often used to treat breast nodules with significant efficacy.

[0003] The quality of traditional Chinese medicine (TCM) directly impacts its development. In recent years, TCM has received increasing attention due to its reliable efficacy, minimal side effects, low toxicity, and rare drug resistance. However, the complex chemical composition and interactions between components of TCM increase the difficulty of quality control. Currently, quality testing technologies for Rupisan capsules / granules are insufficient. Most studies focus only on the component analysis of individual herbs in the formula, neglecting the comprehensive consideration of the overall composition of the capsule, thus failing to fully reflect its quality. Summary of the Invention

[0004] In order to overcome the shortcomings of the prior art, the present invention aims to provide a method for constructing a fingerprint spectrum of Rupisan capsules / granules and the fingerprint spectrum thereof, so as to solve the technical problem that the existing detection methods cannot fully reflect the quality.

[0005] To achieve the above objectives, the present invention employs the following technical solution:

[0006] The first aspect of this invention discloses a method for constructing a fingerprint spectrum of Rupisan capsules / granules, comprising the following steps:

[0007] S1. Preparation of test solution: Weigh different batches of Rupisanjie capsule fine powder or Rupisanjie granules, add methanol aqueous solution, reflux extract to obtain test solution;

[0008] S2. Preparation of reference solutions: Accurately weigh gallic acid, protocatechuic aldehyde, chlorogenic acid, fraxin, caffeic acid, luteolin, scopolamine, rosmarinic acid, curcumin, curcumin, benzoyl paeoniflorin, curcumindione, ellagic acid, protopine, fumarate, p-coumaric acid, benzoic acid, and paeoniflorin reference standards, dissolve them, and obtain a single reference solution;

[0009] S3. Perform chromatographic analysis on the test solution obtained in S1 and the reference solution in S2 to obtain the corresponding chromatograms;

[0010] S4. Import the chromatograms obtained in S3 into the chromatographic fingerprint similarity evaluation system for traditional Chinese medicine. Select the chromatographic peaks that are present in the chromatograms of different batches of Rupisan capsules / granules as common peaks. Perform similarity analysis on the chromatograms of the test solution after data import, multi-point correction and data matching.

[0011] S5. Perform high-resolution mass spectrometry analysis on the test solution obtained in S1 to obtain the total ion chromatogram and the mass spectrometry results of the chemical components. Import the detection data into Xcalibur software, enter the Qual Browser interface, and perform data analysis based on the peaks of the chemical components. Based on the total ion chromatogram and the mass spectrometry results of the chemical components, combined with the chromatogram of the reference standard, determine the chemical components of each peak in the chromatogram of the test sample, and obtain the fingerprint spectrum of Rupisan capsules / granules.

[0012] Preferably, in S1, the methanol aqueous solution is an 80% methanol aqueous solution.

[0013] Preferably, in S1, reflux extraction is performed for 30 min.

[0014] Preferably, in S1, the ratio of Rupisanjie capsules to methanol aqueous solution is 2.5 g: 25 mL, and the ratio of Rupisanjie granules to methanol aqueous solution is 3.5 g: 25 mL.

[0015] Preferably, in S2, the preparation method of the reference solution is as follows: each reference standard is accurately weighed and mixed with pure methanol solution to prepare a single reference solution containing 84 μg gallic acid, 90 μg protocatechuic aldehyde, 96 μg chlorogenic acid, 46 μg fraxin, 95 μg caffeic acid, 97 μg luteolin, 74 μg scopolamine, 85 μg rosmarinic acid, 33 μg curcumin, 75 μg turmeric alcohol, 28 μg benzoylpaeoniflorin, 75 μg turmeric dione, 13 μg ellagic acid, 56 μg protopine, 57 μg coumarin, 27 μg p-coumaric acid, 88 μg benzoic acid, or 99 μg paeoniflorin per 1 mL.

[0016] Preferably, in S3, the chromatographic conditions are as follows: column type Hedera ODS-2-C18, 4.6×250 nm×5 µm; mobile phase is acetonitrile-0.05% phosphoric acid aqueous solution; UV-Vis absorption detector is used, detection wavelength is 0~40 min: 260 nm; 40~115 min: 240 nm; flow rate: 0.8 mL / min; injection volume: 10 μL.

[0017] More preferably, in S3, the chromatographic conditions are as follows: gradient elution program: 0~5 min, acetonitrile volume 2%; 5~55 min, acetonitrile volume 2%~25%; 55~80 min, acetonitrile volume 25%~40%; 80~100 min, acetonitrile volume 40%~75%; 100~115 min, acetonitrile volume 75%~2%.

[0018] Preferably, in S5, the high-resolution mass spectrometry detection conditions are: 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℃, scanning mode is full scan mode, and mass-to-charge ratio scanning range m / z is 100-1500.

[0019] Preferably, in step S5, based on the mass spectrometry results, the chemical components of each peak in the chromatogram of the test sample are determined as follows: peak 4 is gallic acid, peak 7 is protocatechuic aldehyde, peak 8 is chlorogenic acid, peak 9 is fraxin, peak 10 is caffeic acid, peak 12 is protopine, peak 13 is paeoniflorin, peak 14 is p-coumaric acid, peak 15 is fumaric acid, peak 16 is rosmarinic acid, peak 17 is ellagic acid, peak 19 is benzoic acid, peak 22 is scopolamine, peak 23 is luteolin, peak 24 is benzoylpaeoniflorin, peak 27 is curcuminol, peak 28 is curcumin, and peak 29 is curcumindione, thus obtaining the fingerprint chromatogram of Rupisan capsules / granules.

[0020] In a second aspect, the fingerprint spectrum of Rupisan capsules / granules obtained by the above-described construction method is disclosed.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] This invention provides a method for constructing a fingerprint spectrum for Rupisan capsules / granules. 1) During the preparation of the test solution, reflux extraction is selected, resulting in a more comprehensive chromatogram with better separation, better extraction effect, and more peak information. 2) Using gallic acid, protocatechuic aldehyde, chlorogenic acid, fraxin, caffeic acid, luteolin, scopolamine, rosmarinic acid, curcumin, curcumin, benzoylpaeoniflorin, curcumindione, ellagic acid, protopine, fumarate, p-coumaric acid, benzoic acid, and paeoniflorin as reference standards, the position and abundance of reference peaks can be determined, ensuring the stability, reproducibility, and specificity of the fingerprint spectrum. 3) Through systematic analysis of multiple batches of samples, the quality of Rupisan capsules / granules can be evaluated more comprehensively and scientifically, thereby ensuring the quality and efficacy of the product. 4) Importing the chromatogram into a similarity evaluation system for chromatographic fingerprinting of traditional Chinese medicine can improve the accuracy and objectivity of this method. 5) Constructing a fingerprint chromatogram by combining chromatographic analysis with high-resolution mass spectrometry can comprehensively reflect the quality status of Rupisanjie capsules / granules. The fingerprint chromatogram of Rupisanjie capsules / granules established by this method yielded 29 common peaks, and identified 18 characteristic peaks, greatly enriching the chromatographic information. The obtained fingerprint chromatogram has obvious characteristics and can reflect the information of the chemical components contained in Rupisanjie capsules / granules in general. Moreover, the obtained chromatogram has a stable baseline and good peak shape, providing a basis for comprehensive detection of the quality of Rupisanjie capsules / granules. By calculating the stability, repeatability, and precision using relative retention time and relative peak area, the results show that the RSD values ​​of relative retention time and relative peak area are both less than 4.0%, indicating that the method has good reproducibility and reliability. Therefore, this construction method can accurately, clearly, and objectively evaluate the quality of Rupisanjie capsules / granules, which has significant importance and practical value for effectively controlling the quality of Rupisanjie capsules / granules and ensuring clinical efficacy. ‌

[0023] Furthermore, during the preparation of the test solution, 80% water-methanol solution was selected as the extraction solvent, which yielded the extract with the most chromatographic information, the highest component content, and the best extraction effect.

[0024] Furthermore, during the preparation of the test solution, reflux extraction for 30 min yields a better amount of chromatographic information while reducing time and cost.

[0025] Furthermore, during the chromatographic analysis, the mobile phase selected was acetonitrile-0.05% phosphoric acid, which resulted in a large number of peaks with good peak shape and separation. The wavelengths selected were 260 nm for 0–40 min and 240 nm for 40–115 min, providing the most comprehensive information in the chromatogram and ensuring a stable baseline. A column temperature of 35℃ was chosen for optimal separation of the components.

[0026] Furthermore, during the chromatographic analysis, a gradient elution program was selected: 0–5 min, acetonitrile volume 2%; 5–55 min, acetonitrile volume 2%–25%; 55–80 min, acetonitrile volume 25%–40%; 80–100 min, acetonitrile volume 40%–75%; 100–115 min, acetonitrile volume 75%–2%. This resulted in good separation, high peak height, and complete chromatographic information. Attached Figure Description

[0027] Figure 1 This is the chromatogram obtained in Example 1 during the preparation of the test solution of Rupisanjie Capsules according to the present invention;

[0028] Figure 2 This is the chromatogram obtained in Example 1 during the preparation of the test solution of Rupisanjie granules according to the present invention;

[0029] Figure 3 The images show the chromatogram and mass spectrum of the gallic acid standard of the present invention; wherein, chromatogram (A) and mass spectrum (B) are shown.

[0030] Figure 4 The images show the chromatogram and mass spectrum of the protocatechuic aldehyde standard of the present invention; wherein, chromatogram (A) and mass spectrum (B) are shown.

[0031] Figure 5 The images show the chromatogram and mass spectrum of the chlorogenic acid standard of the present invention; wherein, chromatogram (A) and mass spectrum (B) are shown.

[0032] Figure 6 The images show the chromatogram and mass spectrum of the fraxin B standard of the present invention; wherein, chromatogram (A) and mass spectrum (B) are shown.

[0033] Figure 7 The images show the chromatogram and mass spectrum of the caffeic acid standard of the present invention; wherein, chromatogram (A) and mass spectrum (B) are shown.

[0034] Figure 8 The images show the chromatogram and mass spectrum of the original opioid standard of the present invention; wherein, chromatogram (A) and mass spectrum (B) are shown.

[0035] Figure 9 The images show the chromatogram and mass spectrum of the paeoniflorin standard of the present invention; wherein, chromatogram (A) and mass spectrum (B) are shown.

[0036] Figure 10 The images show the chromatogram and mass spectrum of the p-coumaric acid standard of the present invention; wherein, chromatogram (A) and mass spectrum (B) are shown.

[0037] Figure 11 The images show the chromatogram and mass spectrum of the corydaline standard of the present invention; wherein, chromatogram (A) and mass spectrum (B) are shown.

[0038] Figure 12 The chromatogram and mass spectrum of the rosmarinic acid standard of the present invention are shown; wherein, chromatogram (A) and mass spectrum (B) are shown.

[0039] Figure 13 The images show the chromatogram and mass spectrum of the ellagic acid standard of the present invention; wherein, chromatogram (A) and mass spectrum (B) are shown.

[0040] Figure 14 The images show the chromatogram and mass spectrum of the benzoic acid standard of the present invention; wherein, chromatogram (A) and mass spectrum (B) are shown.

[0041] Figure 15 The images show the chromatogram and mass spectrum of the scopolamine standard of the present invention; wherein, chromatogram (A) and mass spectrum (B) are shown.

[0042] Figure 16 The images show the chromatogram and mass spectrum of the luteolin standard of the present invention; wherein, chromatogram (A) and mass spectrum (B) are shown.

[0043] Figure 17 The images show the chromatogram and mass spectrum of the benzoyl paeoniflorin standard of the present invention; wherein, chromatogram (A) and mass spectrum (B) are shown.

[0044] Figure 18 The images show the chromatogram and mass spectrum of the curcuminol standard of the present invention; wherein, chromatogram (A) and mass spectrum (B) are shown.

[0045] Figure 19 The images show the chromatogram and mass spectrum of the curcumin standard of the present invention; wherein, chromatogram (A) and mass spectrum (B) are shown.

[0046] Figure 20 The images show the chromatogram and mass spectrum of the turmeric dione standard of the present invention; wherein, chromatogram (A) and mass spectrum (B) are shown.

[0047] Figure 21 The fingerprint chromatograms of 15 batches of Rupisanjie Capsules tested according to this invention;

[0048] Figure 22 The fingerprint chromatograms of 15 batches of Rupisanjie granules tested according to this invention;

[0049] Figure 23 This is a chromatogram obtained by optimizing the extraction method during the preparation of the test sample solution according to the present invention;

[0050] Figure 24 This is a chromatogram obtained by optimizing the extraction time during the preparation of the test sample solution according to the present invention;

[0051] Figure 25This is a chromatogram obtained by optimizing the mobile phase composition under chromatographic conditions according to the present invention;

[0052] Figure 26 This is a chromatogram obtained by optimizing the detection wavelength under chromatographic conditions according to the present invention;

[0053] Figure 27 This is a chromatogram obtained by optimizing the flow rate under chromatographic conditions according to the present invention;

[0054] Figure 28 This is a chromatogram obtained by optimizing the elution procedure under chromatographic conditions according to the present invention. Detailed Implementation

[0055] To further understand the present invention, the following description, in conjunction with embodiments, is provided. These descriptions are merely illustrative of the features and advantages of the invention and are not intended to limit the scope of the claims. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0056] This invention provides a method for constructing the fingerprint spectrum of Rupisan capsules / granules, comprising the following steps:

[0057] S1. Preparation of test solution: Weigh different batches of Rupisanjie capsule fine powder or Rupisanjie granules, add methanol aqueous solution, reflux extract to obtain test solution;

[0058] The methanol-water solution is an 80% methanol-water solution. The ratio of Rupisanjie capsules to 80% methanol-water solution is 2.5 g: 25 mL, and the ratio of Rupisanjie granules to 80% methanol-water solution is 3.5 g: 25 mL. The preparation method of the Rupisanjie capsule / granule test solution is as follows: weigh different batches of Rupisanjie capsule / granule fine powder, mix with 80% methanol solution, reflux for 30 min, shake well, filter, and filter the filtrate through a 0.45 µm microporous membrane to obtain the Rupisanjie capsule / granule test solution.

[0059] S2. Preparation of reference solutions: Using gallic acid, protocatechuic aldehyde, chlorogenic acid, fraxin, caffeic acid, luteolin, scopolamine, rosmarinic acid, curcumin, curcuminol, benzoylpeonidin, curcumidine, ellagic acid, protopine, fumarate, p-coumaric acid, benzoic acid, and paeoniflorin as reference standards, accurately weigh each reference standard and mix them with pure methanol solution to prepare a solution containing 84 μg gallic acid, 90 μg protocatechuic aldehyde, 96 μg chlorogenic acid, fraxin 46 μg fraxin, 95 μg caffeic acid, 97 μg luteolin, scopolamine 74 μg, rosmarinic acid 85 μg, curcumin 33 μg, curcuminol 75 μg, benzoylpeonidin 28 μg, and curcumidine 75 μg per mL. A single reference solution containing μg of ellagic acid, 13 μg of protopine, 56 μg of coumarin, 57 μg of p-coumaric acid, 27 μg of benzoic acid, 88 μg of paeoniflorin, and 99 μg of paeoniflorin.

[0060] S3. Inject the test solution obtained in S1 and the reference solution obtained in S2 into a high-performance liquid chromatograph (HPLC) for chromatographic analysis. The HPLC conditions are as follows: column type: Hedera ODS-2-C18 (4.6×250 nm×5 µm); mobile phase: acetonitrile-0.05% phosphoric acid aqueous solution; column temperature: 35℃; UV-Vis detector: detection wavelength: 0~40 min: 260 nm; 40~115 min: 240 nm; flow rate: 0.8 mL / min; injection volume: 10 μL; gradient elution program: 0~5 min, acetonitrile volume 2%; 5~55 min, acetonitrile volume 2%~25%; 55~80 min, acetonitrile volume 25%~40%; 80~100 min, acetonitrile volume 40%~75%; 100~115 min, acetonitrile volume 75%~2%. Record the corresponding chromatograms.

[0061] S4. Import the chromatograms obtained in S3 into the chromatographic fingerprint similarity evaluation system for traditional Chinese medicine. Select the chromatographic peaks that are present in the chromatograms of different batches of Rupisan capsules / granules as common peaks. Perform similarity analysis on the chromatograms of the test solution after data import, multi-point correction and data matching.

[0062] S5. Perform high-resolution mass spectrometry analysis on the test solution. The high-resolution mass spectrometry detection conditions are: 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, mass-to-charge ratio scan range m / z 100-1500. Obtain the total ion chromatogram and the mass spectrometry results of the chemical composition. Import the detection data into Xcalibur software and enter Qual... The browser interface analyzes the data based on the peaks of the chemical components. The chemical components of each peak in the chromatogram of the test sample are determined by combining the total ion chromatogram, the mass spectrometry results of the chemical components, and the chromatogram of the reference standard. These components are: Peak 4 is gallic acid, Peak 7 is protocatechuic aldehyde, Peak 8 is chlorogenic acid, Peak 9 is fraxin, Peak 10 is caffeic acid, Peak 12 is protopine, Peak 13 is paeoniflorin, Peak 14 is p-coumaric acid, Peak 15 is fumaric acid, Peak 16 is rosmarinic acid, Peak 17 is ellagic acid, Peak 19 is benzoic acid, Peak 22 is scopolamine, Peak 23 is luteolin, Peak 24 is benzoylpaeoniflorin, Peak 27 is curcumin, Peak 28 is curcumin, and Peak 29 is curcumindione.

[0063] 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.

[0064] 1. Instruments

[0065] The instruments used in this invention are shown in Table 1.

[0066] Table 1. Instruments and reagents used in this invention

[0067]

[0068] 2. Medicines and Reagents

[0069] The 15 batches of Rupisanjie capsules and granules used in this invention were all provided by Shaanxi Bailu Pharmaceutical Co., Ltd. (Table 2); the reference standards were: gallic acid standard (batch number: 110831-201605, purity: 90.08%) purchased from the National Institutes for Food and Drug Control; rosmarinic acid standard (batch number: C14900895, purity: 97%) purchased from Shanghai Maclean Biochemical Technology Co., Ltd.; and corydaline standard (batch number: DSTDY010101, purity: 98%). Purchased from Chengdu Lemeitian Pharmaceutical Technology Co., Ltd.; Luteolin standard (batch number: AF20030854, purity: 98%) and chlorogenic acid standard (batch number: AZ22011851, purity: 98%) were purchased from Chengdu Efa Biotechnology Co., Ltd.; Protopine standard (batch number: 101395, purity: 99.5%), ellagic acid standard (batch number: 200054-2, purity: 98%), and curcumin standard (batch number: 200030-1, purity: 99%) were all purchased from [unclear - likely a company name]. Jiangsu Yongjian Pharmaceutical Technology Co., Ltd.; Protocatechuic aldehyde standard (batch number: 20082402, purity: 99.75%), purchased from Chengdu Pufeide Biotechnology Co., Ltd.; Scopolamine standard (batch number: PS013684, purity: 98%), caffeic acid standard (batch number: PS010522, purity: 98%), turmeric alcohol standard (batch number: PS000319, purity: 98%), fraxetin standard (batch number: PS000763, purity: 98%), benzene Formoylpaeoniflorin standard (batch number: PS000157, purity: 98%), curcuminidine standard (batch number: PS011300, purity: 98%), p-coumaric acid standard (batch number: PS020451, purity: 98%), benzoic acid standard (batch number: PS012647, purity: 98.35%), and paeoniflorin standard (batch number: 2300223, purity: 98%) were all purchased from Chengdu Pusi Biotechnology Co., Ltd.; the reagents used in this invention are shown in Table 3.

[0070] Table 2 Reagents used in this invention

[0071]

[0072] Table 3 Reagents used in this invention

[0073]

[0074] Example 1

[0075] A method for constructing the fingerprint spectrum of Rupisan capsules / granules includes the following steps:

[0076] S1. Preparation of the test solution: Accurately weigh 2.5 g of 15 batches of Rupisanjie capsules (or accurately weigh 3.5 g of 15 batches of Rupisanjie granules), place them in a stoppered conical flask, add 25 mL of 80% methanol solution, stopper tightly, reflux for 30 min, shake well, filter, and filter the filtrate through a 0.45 µm microporous membrane to obtain the Rupisanjie capsule / granule test solution.

[0077] S2. Preparation of reference solutions: Accurately weigh each reference standard (gallic acid, protocatechuic aldehyde, chlorogenic acid, fraxin, caffeic acid, luteolin, scopolamine, rosmarinic acid, curcumin, turmeric alcohol, benzoylpeonidin, turmeric dione, ellagic acid, protopine, fumarate, p-coumaric acid, benzoic acid, and paeoniflorin) and place them in a stoppered conical flask. Add pure methanol solution to prepare a solution containing 84 μg gallic acid, 90 μg protocatechuic aldehyde, 96 μg chlorogenic acid, fraxin aldehyde, 46 μg caffeic acid, 95 μg luteolin, 97 μg scopolamine, 74 μg rosmarinic acid, 85 μg curcumin, 33 μg turmeric alcohol, 75 μg benzoylpeonidin, 28 μg turmeric dione, and 75 μg turmeric dione per mL. A single reference solution containing μg of ellagic acid, 13 μg of protopine, 56 μg of coumarin, 57 μg of p-coumaric acid, 27 μg of benzoic acid, 88 μg of paeoniflorin, and 99 μg of paeoniflorin.

[0078] S3. Accurately pipette 10 µL each of the test solution obtained in S1 and the reference solution in S2, and inject them separately into the high-performance liquid chromatograph (HPLC) for chromatographic analysis. Record the corresponding chromatograms, such as... Figure 1 and Figure 2 As shown.

[0079] The liquid chromatography conditions were as follows: column: Hedera ODS-2-C18 (4.6 × 250 nm × 5 µm) column; detector: UV-Vis absorption detector, detection wavelength: 0–40 min: 260 nm; 40–115 min: 240 nm; flow rate: 0.8 mL / min; injection volume: 10 μL; mobile phase: acetonitrile-0.05% phosphoric acid aqueous solution, gradient elution, the elution program is shown in Table 4.

[0080] Table 4 Elution Procedure for Example 1

[0081]

[0082] S4. Import the chromatograms obtained in S3 into the chromatographic fingerprint similarity evaluation system for traditional Chinese medicine. Select the chromatographic peaks that are present in the chromatograms of different batches of Rupisan capsules / granules as common peaks. Perform similarity analysis on the chromatograms of the test solution after data import, multi-point correction and data matching (Tables 5 and 6).

[0083] Table 5. Similarity between samples from different batches of Rupisanjie Capsules and the common pattern.

[0084]

[0085] Table 6. Similarity between batches of Rupisanjie granules and the common pattern

[0086]

[0087] S5. To determine the chemical composition in the fingerprint spectrum, the above-mentioned test solution was subjected to high-resolution mass spectrometry analysis. The high-resolution mass spectrometry 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 and the mass spectrometry results of the chemical composition were obtained. Figures 3-20 Import the detection data into Xcalibur software, enter the Qual Browser interface, and perform data analysis based on the peak values ​​of the chemical components.

[0088] S6. Based on the total ion chromatogram and the mass spectrometry results of the chemical components, combined with the chromatogram of the reference standard, determine the chemical components of each peak in the chromatogram of the test sample. The fingerprint chromatogram shows the following peaks: Peak 4 is gallic acid (retention time 15.478 min), Peak 7 is protocatechuic aldehyde (retention time 31.093 min), Peak 8 is chlorogenic acid (retention time 37.166 min), Peak 9 is fraxin (retention time 38.405 min), Peak 10 is caffeic acid (retention time 39.640 min), Peak 12 is protopine (retention time 45.393 min), Peak 13 is paeoniflorin (retention time 46.115 min), Peak 14 is p-coumaric acid (retention time 47.912 min), Peak 15 is fumarate (retention time 49.433 min), Peak 16 is rosmarinic acid (retention time 50.706 min), and Peak 17 is ellagic acid (retention time 52.813 min). Peak 19 is benzoic acid, retention time 55.945 min; peak 22 is scopolamine, retention time 62.134 min; peak 23 is luteolin, retention time 72.101 min; peak 24 is benzoylpaeoniflorin, retention time 77.149 min; peak 27 is turmeric alcohol, retention time 97.336 min; peak 28 is curcumin, retention time 98.611 min; peak 29 is turmeric dione, retention time 101.662 min. The fingerprint chromatograms of the 15 batches of Rupisanjie capsules obtained are as follows: Figure 21 As shown, the fingerprint spectra of the 15 batches of Rupisanjie Granules test samples are as follows: Figure 22As shown.

[0089] S7. Methodological Investigation

[0090] 1. Precision Experiment

[0091] The test solution obtained in S1 was injected in parallel 6 times under the chromatographic conditions in S3, with an injection volume of 10 μL. Gallic acid, protocatechuic aldehyde, chlorogenic acid, fraxin, caffeic acid, protopine, paeoniflorin, p-coumaric acid, fumaric acid, ellagic acid, benzoic acid, scopolamine, luteolin, benzoylpaeoniflorin, curcumin, curcumin, and curcumindione were used as reference peaks. The relative retention time and relative peak area were analyzed and the RSD value was calculated. The results showed that the RSDs were 0.18%~0.33% and 0.84%~2.42%, respectively. The RSDs were all less than 3%, indicating that the instrument had good precision.

[0092] 2. Stability test

[0093] The test solution obtained in S1 was analyzed by injection at 0 h, 2 h, 4 h, 8 h, 12 h and 24 h according to the chromatographic conditions in S3. The injection volume was 10 μL. Gallic acid, protocatechuic aldehyde, chlorogenic acid, fraxin, caffeic acid, protopine, paeoniflorin, p-coumaric acid, fumaric acid, rosmarinic acid, ellagic acid, benzoic acid, scopolamine, luteolin, benzoylpaeoniflorin, curcumin, curcumin and curcumindione were used as reference peaks. The relative retention time and relative peak area of ​​the common peaks in the HPLC fingerprint of the sample were analyzed and the RSD value was calculated. The results showed that the RSD was 0.35%~0.69% and 0.93%~3.31%, respectively. The RSD was less than 4%, indicating that the test solution of Rupisan capsules / granules had good stability within 24 h.

[0094] 3. Repeatability experiment

[0095] Six samples of Rupisan capsules / granules were taken and analyzed according to the chromatographic conditions in S3. Gallic acid, protocatechuic aldehyde, chlorogenic acid, fraxin, caffeic acid, protopine, paeoniflorin, p-coumaric acid, fumaric acid, ellagic acid, benzoic acid, scopolamine, luteolin, benzoylpaeoniflorin, curcumin, and curcuminone were used as reference peaks. The relative retention time and relative peak area of ​​the common peaks in the HPLC fingerprint of the samples were analyzed and the RSD value was calculated. The results showed that the RSDs were 0.28%~0.71% and 0.66%~3.09%, respectively, and the RSDs were all less than 4%, indicating that the method had good repeatability.

[0096] Example 2

[0097] The purpose of this embodiment is to examine the extraction efficiency of different extraction solvents (methanol, ethanol, acetonitrile, water, and 0.05% phosphoric acid solution) during the preparation of the test solution, with all other conditions being the same as in Example 1. The results show that methanol extraction has the best effect.

[0098] Next, the extraction effects of different methanol concentrations (30% methanol solution, 50% methanol solution, 80% methanol solution, and pure methanol solution) were compared, with all other conditions remaining the same as in Example 1. The results showed that when 80% methanol solution was used as the extraction solvent, the extract produced the most chromatographic information and had the highest component content.

[0099] Example 3

[0100] The purpose of this embodiment is to investigate the effect of different extraction methods (ultrasound, reflux, and maceration) on the detection of Rupisan capsules / granules during the preparation of the test solution. The chromatographic conditions were the same as in Example 1, and the preparation conditions for all other test solutions were the same as in Example 1. The detection results are as follows: Figure 23 As shown, the chromatogram obtained by reflux extraction has a more comprehensive composition, better separation, better extraction effect, and more peak information.

[0101] Example 4

[0102] The purpose of this embodiment is to investigate the effect of different extraction times (30 min, 60 min, 90 min) on the detection of Rupisanjie capsules / granules during the preparation of the test solution. The chromatographic conditions were the same as in Example 1, and the preparation conditions for the remaining test solutions were also the same as in Example 1. The detection results are as follows: Figure 24 As shown, the chromatograms obtained from reflux extraction for 30 min and 90 min yielded better chromatogram information, with little difference between them. Given that 30 min is more time- and cost-effective, 30 min was chosen as the extraction time.

[0103] Example 5

[0104] The purpose of this embodiment is to investigate the effect of different mobile phases (acetonitrile-0.1% phosphoric acid, methanol-water, acetonitrile-water, acetonitrile-0.05% phosphoric acid, acetonitrile-0.05% formic acid) on the detection of Rupisanjie capsules / granules in the chromatographic analysis process. The test solution was prepared using the method in Example 1, and all other chromatographic conditions were the same as in Example 1. The detection results are as follows: Figure 25 As shown, the mobile phase of acetonitrile-0.05% phosphoric acid has a larger number of peaks and better peak shape and separation effect.

[0105] Example 6

[0106] The purpose of this embodiment is to investigate the effect of different wavelengths (210 nm, 240 nm, 254 nm, 260 nm, 310 nm) on the detection of Rupisanjie capsules / granules in chromatographic analysis. A UV-Vis absorption detector was used to investigate the detection wavelength. The test solution was prepared using the method in Example 1, and all other chromatographic conditions were the same as in Example 1. The detection results are as follows: Figure 26 As shown, the chromatogram contains the most comprehensive information and has a stable baseline when the wavelength is 0~40 min: 260 nm; 40~115 min: 240 nm. Therefore, this method is selected as the detection wavelength condition.

[0107] Example 7

[0108] The purpose of this embodiment is to investigate the effect of different column temperatures (25℃, 30℃, 35℃) on the detection of Rupisan capsules / granules in the chromatographic analysis process. The test solution was prepared using the method of Example 1, and all other chromatographic conditions were the same as in Example 1. The detection results are as follows: Figure 27 As shown in the figure, the separation effect of each component is better when the column temperature is 35℃, so this column temperature of 35℃ is selected.

[0109] Example 8

[0110] The purpose of this embodiment is to investigate the effect of different elution programs on the detection of Rupisanjie capsules / granules in chromatographic analysis. The test solution was prepared using the method in Example 1, and elution programs were set for each solution. All other chromatographic conditions were the same as in Example 1. Some elution programs are shown in Tables 4 and 7-10.

[0111] Table 7 Elution Procedure 1

[0112]

[0113] Table 8 Washing Procedure 2

[0114]

[0115] Table 9 Elution Procedure 3

[0116]

[0117] Table 10 Elution Procedure 4

[0118]

[0119] Test results as follows Figure 28 As shown, it can be seen that the separation is good, the peak height is high, and the chromatographic information is complete when the elution program of Example 1 is selected. Therefore, the elution program of Example 1 is selected as the optimal elution program.

[0120] The above experimental results show that the method for constructing a high-performance liquid chromatography combined with high-resolution mass spectrometry fingerprint of Rupisan capsules / granules provided by the present invention has the characteristics of good stability, high precision, and good repeatability. It can comprehensively and objectively evaluate the quality of Rupisan capsules / granules and provide quality assurance for clinical efficacy.

[0121] This invention, through screening extraction solvents, extraction methods, mobile phases, column temperatures, and flow rates, uses octadecylsilane-bonded silica gel as the packing material and acetonitrile and phosphoric acid aqueous solution as the mobile phase. Employing specific elution conditions, it significantly improves the separation of multiple active ingredients while shortening the detection time. This results in a fingerprint spectrum containing more characteristic peaks, greatly enriching the spectral information. High-resolution mass spectrometry is used to jointly identify 18 chemical components, enabling effective and comprehensive detection of Rupisanjie capsules / granules. This invention establishes for the first time a fingerprint spectrum quality evaluation method for Rupisanjie capsules / granules. Using this method, the types and quantities of the main chemical components contained in the traditional Chinese medicine Rupisanjie capsules / granules are comprehensively detected, providing a rapid, convenient, and accurate objective and comprehensive quality assessment. This method has the advantages of simplicity, stability, high precision, and good reproducibility.

[0122] The above embodiments are merely exemplary embodiments of the present invention and are not intended to limit the present invention. The scope of protection of the present invention is defined by the claims. Those skilled in the art can make various modifications or equivalent substitutions to the present invention within its spirit and scope of protection, and such modifications or equivalent substitutions should also be considered to fall within the scope of protection of the present invention.

Claims

1. A method for constructing a fingerprint spectrum of Rupisan capsules / granules, characterized in that, Includes the following steps: S1. Preparation of test solution: Weigh different batches of Rupisanjie capsule fine powder or Rupisanjie granules, add 80% methanol aqueous solution, reflux and extract to obtain test solution; S2. Preparation of reference solutions: Accurately weigh gallic acid, protocatechuic aldehyde, chlorogenic acid, fraxin, caffeic acid, luteolin, scopolamine, rosmarinic acid, curcumin, curcumin, benzoyl paeoniflorin, curcumindione, ellagic acid, protopine, fumarate, p-coumaric acid, benzoic acid, and paeoniflorin reference standards, dissolve them, and obtain a single reference solution; S3. Chromatographic analysis was performed on the test solution obtained in S1 and the reference solution obtained in S2. The chromatographic conditions were as follows: column type Hedera ODS-2-C18, 4.6×250 nm×5 µm; mobile phase was acetonitrile-0.05% phosphoric acid aqueous solution; UV-Vis absorption detector was used, and the detection wavelength was 260 nm from 0 to 40 min and 240 nm from 40 to 115 min. Flow rate: 0.8 mL / min; injection volume: 10 μL; gradient elution program: 0–5 min, acetonitrile volume 2%; 5–55 min, acetonitrile volume 2%–25%; 55–80 min, acetonitrile volume 25%–40%; 80–100 min, acetonitrile volume 40%–75%; 100–115 min, acetonitrile volume 75%–2%, to obtain the corresponding chromatograms; S4. Import the chromatograms obtained in S3 into the chromatographic fingerprint similarity evaluation system for traditional Chinese medicine. Select the chromatographic peaks that are present in the chromatograms of different batches of Rupisan capsules / granules as common peaks. Perform similarity analysis on the chromatograms of the test solution after data import, multi-point correction and data matching. S5. Perform high-resolution mass spectrometry analysis on the test solution obtained in S1 to obtain the total ion chromatogram and the mass spectrometry results of the chemical components. Import the detection data into Xcalibur software, enter the Qual Browser interface, and perform data analysis based on the peaks of the chemical components. Based on the total ion chromatogram and the mass spectrometry results of the chemical components, combined with the chromatogram of the reference standard, determine the chemical components of each peak in the chromatogram of the test sample, and obtain the fingerprint spectrum of Rupisan capsules / granules.

2. The method for constructing the fingerprint spectrum of a Rupisan capsule / granule according to claim 1, characterized in that, In S1, reflux extraction was performed for 30 min.

3. The method for constructing the fingerprint spectrum of a Rupisan capsule / granule according to claim 1, characterized in that, In S1, the ratio of Rupisanjie capsules to methanol aqueous solution is 2.5 g: 25 mL, and the ratio of Rupisanjie granules to methanol aqueous solution is 3.5 g: 25 mL.

4. The method for constructing the fingerprint spectrum of a Rupisan capsule / granule according to claim 1, characterized in that, In S2, the preparation method of the reference solution is as follows: each reference standard is accurately weighed and mixed with pure methanol solution to prepare a single reference solution containing 84 μg gallic acid, 90 μg protocatechuic aldehyde, 96 μg chlorogenic acid, 46 μg fraxin, 95 μg caffeic acid, 97 μg luteolin, 74 μg scopolamine, 85 μg rosmarinic acid, 33 μg curcumin, 75 μg turmeric alcohol, 28 μg benzoylpaeoniflorin, 75 μg turmeric dione, 13 μg ellagic acid, 56 μg protopine, 57 μg coumarin, 27 μg p-coumaric acid, 88 μg benzoic acid, or 99 μg paeoniflorin per 1 mL.

5. The method for constructing the fingerprint spectrum of a Rupisan capsule / granule according to claim 1, characterized in that, In S5, the high-resolution mass spectrometry detection conditions are: 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℃, scanning mode is full scan mode, and mass-to-charge ratio scanning range m / z is 100-1500.

6. The method for constructing the fingerprint spectrum of a Rupisan capsule / granule according to claim 1, characterized in that, In S5, based on the mass spectrometry results, the chemical components of each peak in the chromatogram of the test sample were determined as follows: peak 4 is gallic acid, peak 7 is protocatechuic aldehyde, peak 8 is chlorogenic acid, peak 9 is fraxin, peak 10 is caffeic acid, peak 12 is protopine, peak 13 is paeoniflorin, peak 14 is p-coumaric acid, peak 15 is fumaric acid, peak 16 is rosmarinic acid, peak 17 is ellagic acid, peak 19 is benzoic acid, peak 22 is scopolamine, peak 23 is luteolin, peak 24 is benzoylpaeoniflorin, peak 27 is curcuminol, peak 28 is curcumin, and peak 29 is curcumindione. The fingerprint chromatogram of Rupisan capsules / granules was obtained.

7. The fingerprint spectrum of Rupisan capsules / granules obtained by the construction method according to any one of claims 1 to 6.

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