Fingerprint spectrum construction method and fingerprint spectrum of capsule / particle for treating nodules of breast

Abstract

The invention discloses a breast lump removing capsule / granule fingerprint construction method and a fingerprint thereof, and belongs to the field of traditional Chinese medicine quality standard research and application. Respectively injecting the test solution and the single reference solution into a high performance liquid chromatograph, carrying out gradient elution, and carrying out chromatographic analysis to obtain various chromatograms; performing mass spectrometry on the test solution to obtain a mass spectrometry result; taking chromatographic peaks existing in the chromatogram of the test solution as common peaks, and identifying chemical components of the chromatographic peaks in the breast lump removing capsule / particle fingerprint spectrum by combining the chromatogram of the single reference substance solution and the mass spectrum result of the test solution, so as to obtain the breast lump removing capsule / particle fingerprint spectrum. The detection method for the breast lump removing capsule / particle fingerprint spectrum has the advantages of being simple, convenient, stable, high in precision and good in reproducibility, the quality of the breast lump removing capsule / particle can be evaluated more comprehensively and scientifically, and the quality and the curative effect of a product are guaranteed.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine detection, and particularly relates to a fingerprint construction method of Rupi Sanjie capsules / granules and a fingerprint thereof. Background Art

[0002] Rupi Sanjie Capsules and Rupi Sanjie Granules are exclusive products of Shaanxi Bailu Pharmaceutical Co., Ltd. They are compound preparations made from 11 medicinal materials, including Prunella Vulgaris, Chuanxiong, Bombyx Batryticatus, Carapace of Turtle, Bupleurum, Red Peony Root, Rose, Curcuma, Angelica, Corydalis and Oyster. They have the effects of promoting qi and blood circulation, softening and dispersing nodules, etc. They are mainly used for breast hyperplasia caused by qi stagnation and blood stasis, with symptoms such as breast pain, breast lumps, irritability, chest and flank distension, etc. They are often used in the clinical treatment of breast nodules with significant efficacy.

[0003] The quality of traditional Chinese medicine is directly related to its development. In recent years, traditional Chinese medicine has received more and more attention due to its reliable efficacy, minimal side effects, low toxicity and rare drug resistance. However, the quality control of traditional Chinese medicine has become more difficult due to its complex chemical composition and the mutual influence between its components. At present, the quality inspection technology for Rupi Sanjie Capsules / Granules is still insufficient. Most studies only focus on the component analysis of single herbs in the formula. This method ignores the comprehensive consideration of the overall composition of the capsule, and therefore it is difficult to fully reflect its quality status. Summary of the invention

[0004] In order to overcome the shortcomings of the above-mentioned prior art, the purpose of the present invention is to provide a fingerprint construction method of Rupi Sanjie capsules / granules and its fingerprint, so as to solve the technical problem that the existing detection methods cannot fully reflect the quality.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions: The first aspect of the present invention discloses a method for constructing a fingerprint of Rupi Sanjie capsules / granules, comprising the following steps: S1. Prepare the test solution: weigh different batches of Rupi Sanjie capsule powder or Rupi Sanjie granules respectively, add methanol aqueous solution, reflux extraction, and obtain the test solution; S2. Prepare reference substance solution: accurately weigh gallic acid, protocatechuic aldehyde, chlorogenic acid, fraxin, caffeic acid, luteolin, scopoletin, rosmarinic acid, curcumin, curcumenol, benzoylpeonidin, curdione, ellagic acid, protopine, tetrahydropalmatine, p-coumaric acid, benzoic acid and paeoniflorin reference substances, dissolve them, and obtain a single reference substance solution; S3, performing chromatographic analysis on the test solution obtained in S1 and the reference solution in S2 to obtain corresponding chromatograms; S4, importing the chromatogram obtained in S3 into the Chinese medicine chromatographic fingerprint similarity evaluation system, selecting the chromatographic peaks existing in the chromatograms of different batches of Rupi Sanjie Capsules / granules as common peaks, and subjecting the chromatograms of the test solution to data import, multi-point correction and data matching, and performing similarity analysis; S5. Perform high-resolution mass spectrometry analysis on the test solution obtained in S1 to obtain a total ion flow diagram and a mass spectrum result diagram of the chemical components. Import the test data into Xcalibur software, enter the Qual Browser interface, perform data analysis based on the peak conditions of the chemical components, determine the chemical components of each peak in the test sample chromatogram based on the total ion flow diagram and the mass spectrum result diagram of the chemical components in combination with the reference sample chromatogram, and obtain the fingerprint spectrum of Rupi Sanjie Capsules / Granules.

[0006] Preferably, in S1, the methanol aqueous solution is 80% methanol aqueous solution.

[0007] Preferably, in S1, reflux extraction is performed for 30 min.

[0008] Preferably, in S1, the dosage ratio of Rupi Sanjie capsules to methanol aqueous solution is 2.5 g:25 mL, and the dosage ratio of Rupi Sanjie granules to methanol aqueous solution is 3.5 g:25 mL.

[0009] Preferably, in S2, the preparation method of the reference substance solution is: accurately weigh each reference substance and mix it with pure methanol solution to prepare a single reference substance solution containing 84 μg of gallic acid, 90 μg of protocatechuic aldehyde, 96 μg of chlorogenic acid, 46 μg of fraxinoside, 95 μg of caffeic acid, 97 μg of luteolin, 74 μg of scopoletin, 85 μg of rosmarinic acid, 33 μg of curcumin, 75 μg of curcumenol, 28 μg of benzoylpeonidin, 75 μg of curdione, 13 μg of ellagic acid, 56 μg of protopine, 57 μg of tetrahydropalmatine, 27 μg of p-coumaric acid, 88 μg of benzoic acid or 99 μg of paeoniflorin per 1 mL.

[0010] Preferably, in S3, the chromatographic conditions are: chromatographic column model Hedera ODS-2-C18, 4.6×250 nm×5 µm; mobile phase is acetonitrile-0.05% phosphoric acid aqueous solution; ultraviolet-visible absorption detector is used, and the detection wavelength is 0~40 min: 260nm; 40~115 min: 240 nm; flow rate: 0.8 mL / min; injection volume: 10 μL.

[0011] Further preferably, in S3, the chromatographic conditions are: gradient elution program: 0-5 min, 2% by volume of acetonitrile; 5-55 min, 2%-25% by volume of acetonitrile; 55-80 min, 25%-40% by volume of acetonitrile; 80-100 min, 40%-75% by volume of acetonitrile; 100-115 min, 75%-2% by volume of acetonitrile.

[0012] 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°C, auxiliary gas temperature 300°C, scanning mode is full scan mode, and the mass-to-charge ratio scanning range m / z is 100-1500.

[0013] Preferably, in S5, according to the mass spectrometry result diagram, the chemical components of each peak in the chromatogram of the test sample are determined, which are: peak 4 is gallic acid, peak 7 is protocatechuic aldehyde, peak 8 is chlorogenic acid, peak 9 is quercetin, peak 10 is caffeic acid, peak 12 is orthoopine, peak 13 is paeoniflorin, peak 14 is p-coumaric acid, peak 15 is tetrahydropalmatine, peak 16 is rosmarinic acid, peak 17 is ellagic acid, peak 19 is benzoic acid, peak 22 is scopoletin, peak 23 is luteolin, peak 24 is benzoylpeoniflorin, peak 27 is curcuminol, peak 28 is curcumin, and peak 29 is curdione, and the fingerprint of Rupi Sanjie Capsules / granules is obtained.

[0014] The second aspect of the present invention discloses the fingerprint of Rupi Sanjie capsules / granules obtained by the above construction method.

[0015] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a method for constructing a fingerprint of Rupi Sanjie capsules / granules. 1) During the preparation of the test solution, reflux extraction is selected for extraction, and the resulting chromatogram components are relatively comprehensive, with good separation, good extraction effect, and more peak information. 2) Gallic acid, protocatechuic aldehyde, chlorogenic acid, quercetin, caffeic acid, luteolin, scopoletin, rosmarinic acid, curcumin, curcumenol, benzoylpeonidin, curdione, ellagic acid, protopine, tetrahydropalmatine, p-coumaric acid, benzoic acid, and paeoniflorin are used as reference substances, and the position and abundance of the reference peaks can be determined to ensure the stability, reproducibility, and specificity of the fingerprint. 3) By systematically analyzing multiple batches of samples, the quality of Rupi Sanjie capsules / granules can be evaluated more comprehensively and scientifically, thereby ensuring the quality and efficacy of the product. 4) The chromatogram was imported into the similarity evaluation system of Chinese medicine chromatographic fingerprint for similarity analysis, which can improve the accuracy and objectivity of the method. 5) The construction of the fingerprint by combining chromatographic analysis with high-resolution mass spectrometry can comprehensively reflect the quality of Rupi Sanjie capsules / granules. The fingerprint of Rupi Sanjie capsules / granules established by this method obtained 29 common peaks, and 18 characteristic peaks were identified, which greatly enriched the spectrum information. The fingerprint obtained was characteristic and could generally reflect the information of the chemical components contained in Rupi Sanjie capsules / granules; and the chromatogram obtained had a stable baseline and good peak shape, which provided a basis for comprehensive detection of the quality of Rupi Sanjie capsules / granules. The stability, repeatability and precision were calculated by relative retention time and relative peak area. The results showed that the RSD values ​​of relative retention time and relative peak area were less than 4.0%, indicating that the method had good reproducibility and reliability. Therefore, this construction method can accurately, clearly and objectively evaluate the quality of Rupi Sanjie capsules / granules, which is of significant importance and practical value for effectively controlling the quality of Rupi Sanjie capsules / granules and ensuring clinical efficacy. ‌

[0016] Furthermore, during the preparation of the test solution, 80% water-methanol solution was selected as the extraction solvent, and the extract chromatogram had the most information, the highest component content, and the best extraction effect.

[0017] Furthermore, during the preparation of the test solution, reflux extraction for 30 min was selected, and the obtained chromatogram had better information content with low time and cost.

[0018] Furthermore, during the chromatographic analysis, the mobile phase was acetonitrile-0.05% phosphoric acid, which resulted in a large number of peaks, better peak shape and separation effect. The wavelength was selected as 260 nm for 0-40 min and 240 nm for 40-115 min, and the chromatogram contained the most comprehensive information and had a stable baseline. The column temperature was selected as 35°C, and the separation effect of each component was better.

[0019] Furthermore, during the chromatographic analysis, a gradient elution program was selected as follows: 0-5 min, 2% acetonitrile volume; 5-55 min, 2%-25% acetonitrile volume; 55-80 min, 25%-40% acetonitrile volume; 80-100 min, 40%-75% acetonitrile volume; 100-115 min, 75%-2% acetonitrile volume, which resulted in good separation, high peak height and complete chromatographic information. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The chromatogram obtained in Example 1 during the preparation of the test solution of Rupi Sanjie Capsules of the present invention; Figure 2 The chromatogram obtained in Example 1 during the preparation of the Rupi Sanjie granules test solution of the present invention; Figure 3 The chromatogram and mass spectrum of the gallic acid standard of the present invention; wherein, the chromatogram (A), the mass spectrum (B); Figure 4 The chromatogram and mass spectrum of the protocatechuic aldehyde standard of the present invention; wherein, the chromatogram (A), the mass spectrum (B); Figure 5 The chromatogram and mass spectrum of the chlorogenic acid standard of the present invention; wherein, the chromatogram (A), the mass spectrum (B); Figure 6 The chromatogram and mass spectrum of the standard product of aesculetin of the present invention; wherein, the chromatogram (A), the mass spectrum (B); Figure 7 The chromatogram and mass spectrum of the caffeic acid standard of the present invention; wherein, the chromatogram (A), the mass spectrum (B); Figure 8 The chromatogram and mass spectrum of the original opium alkaloid standard of the present invention; wherein, the chromatogram (A), the mass spectrum (B); Fig. 9 The chromatogram and mass spectrum of the paeoniflorin standard of the present invention; wherein, the chromatogram (A), the mass spectrum (B); Fig.10 The chromatogram and mass spectrum of the p-coumaric acid standard of the present invention; wherein, the chromatogram (A), the mass spectrum (B); Fig.11 The chromatogram and mass spectrum of the tetrahydropalmatine standard of the present invention; wherein, the chromatogram (A), the mass spectrum (B); Fig.12 The chromatogram and mass spectrum of the rosmarinic acid standard of the present invention; wherein, the chromatogram (A), the mass spectrum (B); Fig.13 The chromatogram and mass spectrum of the ellagic acid standard of the present invention; wherein, the chromatogram (A), the mass spectrum (B); Fig.14 The chromatogram and mass spectrum of the benzoic acid standard of the present invention; wherein, the chromatogram (A), the mass spectrum (B); Fig.15 The chromatogram and mass spectrum of the scopoletin standard of the present invention; wherein, the chromatogram (A), the mass spectrum (B); Fig.16 The chromatogram and mass spectrum of the luteolin standard of the present invention; wherein, the chromatogram (A), the mass spectrum (B); Fig.17 The chromatogram and mass spectrum of the benzoylpeonidin standard of the present invention; wherein, the chromatogram (A), the mass spectrum (B); Fig.18 The chromatogram and mass spectrum of the curcuminol standard of the present invention; wherein, the chromatogram (A), the mass spectrum (B); Fig.19 The chromatogram and mass spectrum of the curcumin standard of the present invention; wherein, the chromatogram (A), the mass spectrum (B); Fig. 20 The chromatogram and mass spectrum of the curcuma dione standard of the present invention; wherein, the chromatogram (A), the mass spectrum (B); Fig.21 The fingerprints of 15 batches of Rupi Sanjie Capsules test samples of the present invention; Fig. 22 The fingerprints of 15 batches of Rupi Sanjie granules tested in the present invention; Fig.23 This is a chromatogram obtained by optimizing the extraction method during the preparation of the test solution of the present invention; Fig.24 This is a chromatogram obtained by optimizing the extraction time during the preparation of the test solution of the present invention; Fig.25 The chromatogram obtained by optimizing the mobile phase composition under the chromatographic conditions of the present invention; Fig.26 The chromatogram obtained by optimizing the detection wavelength under the chromatographic conditions of the present invention; Fig. 27 The chromatogram obtained by optimizing the flow rate under the chromatographic conditions of the present invention; Fig.28 The chromatogram obtained by optimizing the elution program under the chromatographic conditions of the present invention. DETAILED DESCRIPTION

[0021] In order to further understand the present invention, the present invention is described below in conjunction with embodiments, which are only further explanations of the features and advantages of the present invention and are not intended to limit the claims of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention.

[0022] The present invention provides a method for constructing a fingerprint spectrum of Rupi Sanjie capsules / granules, comprising the following steps: S1. Prepare the test solution: weigh different batches of Rupi Sanjie capsule powder or Rupi Sanjie granules respectively, add methanol aqueous solution, reflux extraction, and obtain the test solution; Wherein, the methanol aqueous solution is 80% methanol aqueous solution, the dosage ratio of Rupi Sanjie capsule to 80% methanol aqueous solution is: 2.5 g:25 mL, and the dosage ratio of Rupi Sanjie granules to 80% methanol aqueous solution is: 3.5 g:25 mL; the preparation method of the Rupi Sanjie capsule / granule test solution is: weigh different batches of Rupi Sanjie capsule / granule fine powder respectively, mix them with 80% methanol solution, reflux extraction for 30 min, shake well, filter, and take the filtrate to pass through a 0.45 µm microporous filter membrane to obtain the Rupi Sanjie capsule / granule test solution.

[0023] S2. Prepare reference solution: Use gallic acid, protocatechuic aldehyde, chlorogenic acid, frasilevin, caffeic acid, luteolin, scopoletin, rosmarinic acid, curcumin, curcumenol, benzoylpeonidin, curdione, ellagic acid, protopine, tetrahydropalmatine, p-coumaric acid, benzoic acid and paeoniflorin as reference substances. Accurately weigh each reference substance and mix them with pure methanol solution to prepare a solution containing 84 μg of gallic acid, 90 μg of protocatechuic aldehyde, 96 μg of chlorogenic acid, 46 μg of frasilevin, 95 μg of caffeic acid, 97 μg of luteolin, 74 μg of scopoletin, 85 μg of rosmarinic acid, 33 μg of curcumin, 75 μg of curcumenol, 28 μg of benzoylpeonidin, 75 μg of curdione μg, ellagic acid 13 μg, orthoopine 56 μg, tetrahydropalmatine 57 μg, p-coumaric acid 27 μg, benzoic acid 88 μg, and paeoniflorin 99 μg as a single reference solution.

[0024] S3. The test solution obtained in S1 and the reference solution obtained in S2 were respectively injected into a high performance liquid chromatograph for chromatographic analysis. The liquid chromatography conditions were as follows: chromatographic column model Hedera ODS-2-C18 (4.6×250 nm×5 µm); the mobile phase was acetonitrile-0.05% phosphoric acid aqueous solution; the column temperature was 35°C; a UV-visible absorption detector was used, and the detection wavelength was 0-40 min: 260 nm; 40-115 min: 240 nm; flow rate: 0.8 mL / min; injection volume: 10 μL; the gradient elution program was as follows: 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%, and the corresponding chromatogram was recorded; S4, importing the chromatogram obtained in S3 into the Chinese medicine chromatographic fingerprint similarity evaluation system, selecting the chromatographic peaks existing in the chromatograms of different batches of Rupi Sanjie Capsules / granules as common peaks, and subjecting the chromatograms of the test solution to data import, multi-point correction and data matching, and performing similarity analysis; S5. Perform high-resolution mass spectrometry analysis on the test solution. The high-resolution mass spectrometry detection conditions are as follows: electrospray ionization, spray voltage 3500 V, sheath gas flow rate 40 arb, auxiliary gas flow rate 10 arb, capillary temperature 300°C, auxiliary gas temperature 300°C, full scan mode, mass-to-charge ratio scanning range m / z 100-1500, obtain total ion flow diagram and mass spectrum result diagram of chemical components, import the detection data into Xcalibur software, and enter Qual Browser interface, data analysis was performed according to the peaks of chemical components, and the chemical components of each peak in the test sample chromatogram were determined according to the total ion flow diagram and the mass spectrometry results of the chemical components combined with the chromatogram of the reference substance, which were: peak 4 was gallic acid, peak 7 was protocatechuic aldehyde, peak 8 was chlorogenic acid, peak 9 was quercetin, peak 10 was caffeic acid, peak 12 was orthoopine, peak 13 was paeoniflorin, peak 14 was p-coumaric acid, peak 15 was tetrahydropalmatine, peak 16 was rosmarinic acid, peak 17 was ellagic acid, peak 19 was benzoic acid, peak 22 was scopoletin, peak 23 was luteolin, peak 24 was benzoylpeoniflorin, peak 27 was curcuminol, peak 28 was curcumin, and peak 29 was curdione.

[0025] The embodiments of the present invention are described in detail below with reference to the examples. If no specific conditions are specified in the examples, the experiments are carried out according to conventional conditions or conditions recommended by the manufacturer. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be obtained commercially.

[0026] 1. Instrument The instruments used in the present invention are shown in Table 1.

[0027] Table 1 Instruments and reagents used in the present invention

[0028] 2. Drugs and reagents The 15 batches of Rupi Sanjie capsules and Rupi Sanjie granules samples used in the present invention were provided by Shaanxi Bailu Pharmaceutical Co., Ltd. (Table 2); reference substances: gallic acid standard (batch number: 110831-201605, purity: 90.08%) purchased from China Food and Drug Administration; rosmarinic acid standard (batch number: C14900895, purity: 97%) purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; tetrahydropalmatine standard (batch number: DSTDY010101, purity: 98%) purchased from Chengdu Lemeitian Pharmaceutical Technology Co., Ltd.; luteolin standard (batch number: AF20030854, purity: 98%), chlorogenic acid standard (batch number: AZ22011851, purity: 98%) were purchased from Chengdu Aifa Biotechnology Co., Ltd.; protopine standard (batch number: 101395, purity: 99.5%), ellagic acid standard (batch number: 200054-2, purity: 98%), curcumin standard (batch number: 200030-1, purity: 99%) were purchased from Jiangsu Yongjian Pharmaceutical Technology Co., Ltd.; protocatechuic aldehyde standard (batch number: 20082402, purity: 99.75%) was purchased from Chengdu Pufeide Biotechnology Co., Ltd.; scopoletin standard (batch number: PS013684, purity: 98%), caffeic acid standard (batch number: PS010522, purity: 98%), curcuminol standard (batch number: PS000319, purity: 98%), esculetin standard (batch number: PS000763, purity: 98%), benzene Formylpaeoniflorin standard (batch number: PS000157, purity: 98%), curcumadione 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 the present invention are shown in Table 3.

[0029] Table 2 Reagents used in the present invention

[0030] Table 3 Reagents used in the present invention

[0031] Example 1 A method for constructing a fingerprint spectrum of Rupi Sanjie capsules / granules, comprising the following steps: S1. Preparation of test solution: accurately weigh 2.5 g of 15 batches of Rupi Sanjie capsules (or accurately weigh 3.5 g of 15 batches of Rupi Sanjie granules), place in a stoppered conical flask, add 25 mL of 80% methanol solution, stopper tightly, reflux extraction for 30 min, shake well, filter, and pass the filtrate through a 0.45 µm microporous filter membrane to obtain the test solution of Rupi Sanjie capsules / granules.

[0032] S2. Preparation of reference solution: accurately weigh each reference substance (gallic acid, protocatechuic aldehyde, chlorogenic acid, frasiletin, caffeic acid, luteolin, scopoletin, rosmarinic acid, curcumin, curcumenol, benzoylpeonidin, curdione, ellagic acid, protopine, tetrahydropalmatine, p-coumaric acid, benzoic acid and paeoniflorin) and place it in a stoppered conical flask, add pure methanol solution to make a solution containing 84 μg of gallic acid, 90 μg of protocatechuic aldehyde, 96 μg of chlorogenic acid, 46 μg of frasiletin, 95 μg of caffeic acid, 97 μg of luteolin, 74 μg of scopoletin, 85 μg of rosmarinic acid, 33 μg of curcumin, 75 μg of curcumenol, 28 μg of benzoylpeonidin, 75 μg of curdione μg, ellagic acid 13 μg, orthoopine 56 μg, tetrahydropalmatine 57 μg, p-coumaric acid 27 μg, benzoic acid 88 μg, and paeoniflorin 99 μg as a single reference solution.

[0033] S3, accurately pipette 10 µL of the test solution obtained in S1 and the reference solution in S2, respectively, and inject them into the HPLC for chromatographic analysis, and record the corresponding chromatograms, such as Figure 1 and Figure 2 shown.

[0034] The liquid chromatography conditions were as follows: chromatographic column: Hedera ODS-2-C18 (4.6×250 nm×5 µm) chromatographic column; detector: UV-visible absorption detector, detection wavelength 0-40 min: 260 nm; 40-115 min: 240 nm; volume 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: Table 4 Elution procedure of Example 1

[0035] S4. Import the chromatogram obtained in S3 into the similarity evaluation system of traditional Chinese medicine chromatographic fingerprint, select the chromatographic peaks existing in the chromatograms of different batches of Rupi Sanjie capsules / granules as common peaks, and perform similarity analysis on the chromatograms of the test solution after data import, multi-point correction and data matching (Tables 5 and 6).

[0036] Table 5 Similarity between samples of each batch of Rupi Sanjie Capsule and the common pattern

[0037] Table 6 Similarity between samples of each batch of Rupi Sanjie Granules and the common pattern

[0038] S5. To determine the chemical components in the fingerprint, the above sample 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°C, auxiliary gas temperature 300°C, full scan mode, mass-to-charge ratio scanning range m / z 100-1500, and the total ion flow diagram and mass spectrum result diagram of the chemical components were obtained ( Figure 3~Figure 20 ). Import the test data into Xcalibur software, enter the Qual Browser interface, and perform data analysis based on the peaks of chemical components.

[0039] S6. According to the total ion flow chart and the mass spectrometry results of the chemical components combined with the chromatogram of the reference substance, the chemical components of each peak in the chromatogram of the test sample were determined, and the peak No. 4 in the fingerprint was determined to be gallic acid with a retention time of 15.478 min, peak No. 7 was protocatechuic aldehyde with a retention time of 31.093 min, peak No. 8 was chlorogenic acid with a retention time of 37.166 min, peak No. 9 was quercetin with a retention time of 38.405 min, peak No. 10 was caffeic acid with a retention time of 39.640 min, peak No. 12 was protoopine with a retention time of 45.393 min, peak No. 13 was paeoniflorin with a retention time of 46.115 min, peak No. 14 was p-coumaric acid with a retention time of 47.912 min, peak No. 15 was tetrahydropalmatine with a retention time of 49.433 min, peak No. 16 was rosmarinic acid with a retention time of 50.706 min, peak No. 17 was ellagic acid with a retention time of 52.813 min. min, peak 19 is benzoic acid with a retention time of 55.945 min, peak 22 is scopoletin with a retention time of 62.134 min, peak 23 is luteolin with a retention time of 72.101 min, peak 24 is benzoylpaeoniflorin with a retention time of 77.149 min, peak 27 is curcumenol with a retention time of 97.336 min, peak 28 is curcumin with a retention time of 98.611 min, and peak 29 is curdion with a retention time of 101.662 min. The fingerprints of 15 batches of Rupi Sanjie Capsules are as follows: Fig.21 As shown in the figure, the fingerprints of 15 batches of Rupi Sanjie granules were obtained. Fig. 22 shown.

[0040] S7. Methodological Review 1. Precision experiment The test solution obtained in S1 was taken and injected in parallel 6 times according to the chromatographic conditions in S3, with an injection volume of 10 μL. Gallic acid, protocatechuic aldehyde, chlorogenic acid, fraxinoyl alcohol, caffeic acid, orthoopine, paeoniflorin, p-coumaric acid, tetrahydropalmatine, rosmarinic acid, ellagic acid, benzoic acid, scopoletin, luteolin, benzoylpeoniflorin, curcuminol, curcumin, and curdione 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 precision of the instrument was good.

[0041] 2. Stability test The test solution obtained in S1 was taken and analyzed at 0 h, 2 h, 4 h, 8 h, 12 h and 24 h according to the chromatographic conditions in S3, with an injection volume of 10 μL. Gallic acid, protocatechuic aldehyde, chlorogenic acid, fraxinoyl alcohol, caffeic acid, orthoopine, paeoniflorin, p-coumaric acid, tetrahydropalmatine, rosmarinic acid, ellagic acid, benzoic acid, scopoletin, luteolin, benzoylpeoniflorin, curcuminol, curcumin and curdione 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 values ​​were calculated. The results showed that the RSDs were 0.35%~0.69% and 0.93%~3.31%, respectively, and the RSDs were all less than 4%, indicating that the test solution of Rupi Sanjie Capsules / Granules had good stability within 24 h.

[0042] 3. Repeatability Experiment Six portions of Rupi Sanjie capsule / granule test solution were taken and analyzed according to the chromatographic conditions in S3. Gallic acid, protocatechuic aldehyde, chlorogenic acid, fraxinoside, caffeic acid, orthoopine, paeoniflorin, p-coumaric acid, tetrahydropalmatine, rosmarinic acid, ellagic acid, benzoic acid, scopoletin, luteolin, benzoylpeoniflorin, curcuminol, curcumin and curdione 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 values ​​were calculated. The results showed that the RSDs were 0.28%-0.71% and 0.66%-3.09%, respectively. The RSDs were all less than 4%, and the method had good repeatability.

[0043] Example 2 The purpose of this example is to investigate the extraction effects of different extraction solvents (methanol, ethanol, acetonitrile, water, 0.05% phosphoric acid water) during the preparation of the test solution, and the other conditions are the same as those in Example 1. The results show that methanol extraction has the best effect.

[0044] Next, the extraction effects of different methanol extraction concentrations (30% methanol solution, 50% methanol solution, 80% methanol solution, pure methanol solution) were compared, and the other conditions were the same as in Example 1. The results showed that when 80% methanol solution was used as the extraction solvent, the extract chromatogram had the most information and the highest component content.

[0045] Example 3 The purpose of this example is to investigate the effects of different extraction methods (ultrasound, reflux, immersion) on the detection of Rupi Sanjie capsule / granule test solution during the preparation of the test solution. The chromatographic conditions are the same as those in Example 1, and the preparation conditions of the other test solutions are the same as those in Example 1. The test results are shown in Fig.23 As shown, it can be seen that the chromatogram obtained by reflux extraction has more comprehensive components, better separation, better extraction effect, and more peak information.

[0046] Example 4 The purpose of this example is to investigate the effect of different extraction times (30 min, 60 min, 90 min) on the detection of Rupi Sanjie capsule / granule test solution during the preparation of the test solution. The chromatographic conditions are the same as those in Example 1, and the preparation conditions of the other test solutions are the same as those in Example 1. The test results are as follows Fig.24 As shown in the figure, it can be seen that the chromatograms obtained by reflux extraction for 30 min and 90 min have better information content and the difference is not large. Considering that 30 min saves more time and cost. Therefore, 30 min is selected as the extraction time condition.

[0047] Example 5 The purpose of this example 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 Rupi Sanjie capsule / granule test solution during chromatographic analysis. The test solution was prepared by the method of Example 1, and the other chromatographic conditions were the same as those of Example 1. The test results are shown in Fig.25 As shown, it can be seen that when the mobile phase is acetonitrile-0.05% phosphoric acid, the number of peaks is larger, and the peak shape and separation effect are better.

[0048] Example 6 The purpose of this example is to investigate the effect of different wavelengths (210 nm, 240 nm, 254 nm, 260 nm, 310 nm) on the detection of Rupi Sanjie capsule / granule test solution during chromatographic analysis. The detection wavelength was investigated using a UV-visible absorption detector. The test solution was prepared using the method of Example 1, and the other chromatographic conditions were the same as those of Example 1. The test results are shown in Fig.26 As shown, it can be seen that when the wavelength is 0~40 min: 260 nm; 40~115 min: 240 nm, the chromatogram contains the most comprehensive information and the baseline is stable, so this method is selected as the detection wavelength condition.

[0049] Example 7 The purpose of this example is to investigate the effect of different column temperatures (25°C, 30°C, 35°C) on the detection of Rupi Sanjie capsule / granule test solution during chromatographic analysis. The test solution was prepared by the method of Example 1, and the other chromatographic conditions were the same as those of Example 1. The test results are shown in Fig. 27 As shown, it can be seen that the separation effect of each component is better when the column temperature is 35℃, so the column temperature is selected as 35℃.

[0050] Example 8 The purpose of this example is to investigate the influence of different elution procedures on the detection of Rupi Sanjie Capsule / Granule test solution during chromatographic analysis. The test solution was prepared by the method of Example 1, and the elution procedures were set respectively. The other chromatographic conditions were the same as those in Example 1. Some elution procedures are shown in Table 4, Table 7 to Table 10: Table 7 Elution procedure 1

[0051] Table 8 Elution procedure 2

[0052] Table 9 Elution procedure 3

[0053] Table 10 Elution program 4

[0054] Test results such as Fig.28 As shown, it can be seen that when the elution program of Example 1 is selected, the separation is good, the peak height is high, and the chromatographic information is complete. Therefore, the elution program of Example 1 is selected as the optimal elution program.

[0055] The above experimental results indicate that the method for constructing a fingerprint of Rupi Sanjie capsules / granules by high performance liquid chromatography combined with high resolution mass spectrometry 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 Rupi Sanjie capsules / granules, providing quality assurance for clinical efficacy.

[0056] The present invention screens the extraction solvent, extraction method, mobile phase, column temperature, flow rate and other conditions, uses octadecylsilane bonded silica gel as a filler, acetonitrile and phosphoric acid aqueous solution as mobile phases, and adopts specific elution conditions. While shortening the detection time, the separation effect of multiple effective ingredients is significantly improved, so that more characteristic peaks are included in the fingerprint spectrum, which greatly enriches the spectrum information, and 18 chemical components are jointly identified by high-resolution mass spectrometry, which can effectively and comprehensively detect Rupi Sanjie Capsules / Granules. The present invention establishes a fingerprint spectrum quality evaluation method for Rupi Sanjie Capsules / Granules for the first time, and uses the method of the present invention to comprehensively detect the types and quantities of the main chemical components contained in the Chinese patent medicine Rupi Sanjie Capsules / Granules, and objectively and comprehensively evaluates its quality in a fast, convenient and accurate manner. The method has the advantages of simplicity, stability, high precision and good reproducibility.

[0057] The above embodiments are only exemplary embodiments of the present invention and are not intended to limit the present invention. The protection scope of the present invention is defined by the claims. Those skilled in the art may make various modifications or equivalent substitutions to the present invention within the essence and protection scope of the present invention, and such modifications or equivalent substitutions shall also be deemed to fall within the protection scope of the present invention.

Claims

1. A method for constructing a fingerprint of Rupi Sanjie capsules / granules, characterized in that: The following steps are involved: S1. Prepare the test solution: weigh different batches of Rupi Sanjie capsule powder or Rupi Sanjie granules respectively, add methanol aqueous solution, reflux extraction, and obtain the test solution; S2. Prepare reference substance solution: accurately weigh gallic acid, protocatechuic aldehyde, chlorogenic acid, fraxin, caffeic acid, luteolin, scopoletin, rosmarinic acid, curcumin, curcumenol, benzoylpeonidin, curdione, ellagic acid, protopine, tetrahydropalmatine, p-coumaric acid, benzoic acid and paeoniflorin reference substances, dissolve them, and obtain a single reference substance solution; S3, performing chromatographic analysis on the test solution obtained in S1 and the reference solution in S2 to obtain corresponding chromatograms; S4, importing the chromatogram obtained in S3 into the Chinese medicine chromatographic fingerprint similarity evaluation system, selecting the chromatographic peaks existing in the chromatograms of different batches of Rupi Sanjie Capsules / granules as common peaks, and subjecting the chromatograms of the test solution to data import, multi-point correction and data matching, and performing similarity analysis; S5. Perform high-resolution mass spectrometry analysis on the test solution obtained in S1 to obtain a total ion flow diagram and a mass spectrum result diagram of the chemical components. Import the test data into Xcalibur software, enter the Qual Browser interface, perform data analysis based on the peak conditions of the chemical components, determine the chemical components of each peak in the test sample chromatogram based on the total ion flow diagram and the mass spectrum result diagram of the chemical components in combination with the reference sample chromatogram, and obtain the fingerprint spectrum of Rupi Sanjie Capsules / Granules.

2. The method for constructing the fingerprint of Rupi Sanjie capsules / granules according to claim 1, characterized in that: In S1, the methanol aqueous solution is 80% methanol aqueous solution.

3. The method for constructing the fingerprint of Rupi Sanjie capsules / granules according to claim 1, characterized in that: In S1, reflux extraction was performed for 30 min.

4. The method for constructing the fingerprint of Rupi Sanjie capsules / granules according to claim 1, characterized in that: In S1, the dosage ratio of Rupi Sanjie capsules to methanol aqueous solution is 2.5 g:25 mL, and the dosage ratio of Rupi Sanjie granules to methanol aqueous solution is 3.5 g:25 mL.

5. The method for constructing the fingerprint of Rupi Sanjie capsules / granules according to claim 1, characterized in that: In S2, the preparation method of the reference solution is as follows: each reference substance is accurately weighed and mixed with pure methanol solution to prepare a single reference solution containing 84 μg of gallic acid, 90 μg of protocatechuic aldehyde, 96 μg of chlorogenic acid, 46 μg of quercetin, 95 μg of caffeic acid, 97 μg of luteolin, 74 μg of scopoletin, 85 μg of rosmarinic acid, 33 μg of curcumin, 75 μg of curcumenol, 28 μg of benzoylpeonidin, 75 μg of curdione, 13 μg of ellagic acid, 56 μg of protopine, 57 μg of coumarin, 27 μg of p-coumaric acid, 88 μg of benzoic acid or 99 μg of paeoniflorin per 1 mL.

6. The method for constructing the fingerprint of Rupi Sanjie capsules / granules according to claim 1, characterized in that: In S3, the chromatographic conditions were as follows: chromatographic column model Hedera ODS-2-C18, 4.6×250 nm×5 µm; mobile phase was acetonitrile-0.05% phosphoric acid aqueous solution; UV-visible absorption detector was used, and the detection wavelength was 0-40 min: 260 nm; 40-115 min: 240 nm; Flow rate: 0.8 mL / min; injection volume: 10 μL.

7. The method for constructing the fingerprint of Rupi Sanjie capsules / granules according to claim 6, characterized in that: In S3, the chromatographic conditions are as follows: gradient elution program: 0-5 min, 2% volume of acetonitrile; 5-55 min, 2%-25% volume of acetonitrile; 55-80 min, 25%-40% volume of acetonitrile; 80-100 min, 40%-75% volume of acetonitrile; 100-115 min, 75%-2% volume of acetonitrile.

8. The method for constructing the fingerprint of Rupi Sanjie capsules / granules according to claim 1, characterized in that: In S5, 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°C, auxiliary gas temperature 300°C, scanning mode was full scan mode, and the mass-to-charge ratio scanning range m / z was 100-1500.

9. The method for constructing the fingerprint of Rupi Sanjie capsules / granules according to claim 1, characterized in that: In S5, according to the mass spectrometry result diagram, the chemical components of each peak in the chromatogram of the test sample were determined, which were: peak 4 was gallic acid, peak 7 was protocatechuic aldehyde, peak 8 was chlorogenic acid, peak 9 was quercetin, peak 10 was caffeic acid, peak 12 was orthoopine, peak 13 was paeoniflorin, peak 14 was p-coumaric acid, peak 15 was tetrahydropalmatine, peak 16 was rosmarinic acid, peak 17 was ellagic acid, peak 19 was benzoic acid, peak 22 was scopoletin, peak 23 was luteolin, peak 24 was benzoylpeoniflorin, peak 27 was curcuminol, peak 28 was curcumin, and peak 29 was curdione, and the fingerprint of Rupi Sanjie Capsules / Granules was obtained.

10. The fingerprint of Rupi Sanjie capsules / granules obtained by the construction method according to any one of claims 1 to 9.

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