A method for constructing a reference fingerprint of a substance of sujinyiwan and the fingerprint thereof

Abstract

The application discloses a kind of Su Ti pills substance benchmark fingerprint and its construction method, belong to the technical field of traditional Chinese medicine analysis.Firstly, preparation test solution and single control solution;Then using Shim-pack VP-ODS (250mm×4.6mm, 5 μm) chromatographic column is carried out chromatographic analysis, mobile phase is acetonitrile (A)-0.1% phosphoric acid aqueous solution (B), gradient elution, constructs Su Ti pills substance benchmark fingerprint;Finally, by similarity analysis and high resolution mass spectrometry analysis, obtain the chemical composition of each peak in test chromatogram.By liquid chromatography-mass spectrometry, it is easy to operate, good stability, high precision and good reproducibility, the chemical information of the constructed fingerprint is rich, can separate a variety of effective components well, combined with control solution chromatogram, finally identify 5 chemical components, can effectively and comprehensively detect Su Ti pills and its pharmaceutical preparations, fill in the blank of fingerprint in Su Ti pills quality control, provide reference and basis for Su Ti pills and its related preparation research.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine analysis technology, specifically relating to a method for constructing a material reference fingerprint spectrum of Suting Pill and its fingerprint spectrum. Background Technology

[0002] The classic formula Su Ting Wan originates from *Yizong Jinjian* (Golden Mirror of Medicine) written by Wu Qian of the Qing Dynasty. This formula consists of two herbs: Perilla seed and Lepidium seed. The prescription and preparation method are as follows: equal parts of roasted Perilla seed and lightly roasted Lepidium seed. Grind them into a fine powder, steam jujube pulp, and form into pills the size of hemp seeds. Its effects are to relieve lung congestion, promote urination, and resolve phlegm. It is indicated for children with phlegm retention in the upper burner, where phlegm accumulates and attacks the lungs, causing wheezing, inability to lie down, facial and bodily edema, a pale red tongue with a thin coating, a deep pulse, and difficulty urinating.

[0003] Classic formulas refer to "prescriptions recorded in ancient Chinese medicine classics that are still widely used today, have definite curative effects, and possess distinct characteristics and advantages." Ancient medical texts contain numerous records and case studies, while modern literature provides extensive clinical and experimental research reports, leading to further refinement and expert consensus in TCM clinical practice. If classic formulas are prepared using modern manufacturing processes to meet modern application requirements, they will differ from the original prescriptions recorded in ancient medical texts. Therefore, it is necessary to establish characteristic chromatograms and fingerprint chromatogram standards to ensure the consistency of efficacy and quality between classic formulas and their original counterparts to the greatest extent possible. TCM fingerprint chromatograms, as a quality control technology and method for TCM, possess multi-component analysis, comprehensiveness, and standardization characteristics. They can comprehensively reflect the overall quality of TCM, fully reflecting the types and quantities of its inherent chemical components, and thus reflecting the quality of the TCM. Currently, research on the component analysis of Su Ting Wan is limited, with most studies focusing on the components of single herbs in the formula. There is a lack of more comprehensive and complete quality control research on Su Ting Wan, which fails to reflect the efficacy characteristics of the combined effects of multiple components in TCM and cannot provide a basis for quality control and evaluation. Summary of the Invention

[0004] In order to overcome the shortcomings of the prior art, the purpose of this invention is to provide a method for constructing a material reference fingerprint spectrum of Suting Pill and its fingerprint spectrum, so as to solve the technical problem that the existing methods cannot comprehensively and fully control the quality of Suting Pill.

[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 material reference fingerprint spectrum of Suting Pill, comprising the following steps:

[0007] S1. Weigh different batches of Suting Pill material standard, extract with methanol solution, filter, and obtain test solution;

[0008] S2. Accurately weigh quercetin-3-O-β-D-glucoside-7-O-β-D-gentiopicroside, quercetin-3-O-β-D-glucopyranoside, rosmarinic acid, luteolin and apigenin reference standards, dissolve them to obtain a single reference standard solution;

[0009] S3. Take the test solution from S1 and the reference solution from S2 for chromatographic analysis to obtain each chromatogram. Select the common peak from the chromatogram of the test solution to construct the material standard fingerprint chromatogram of Suting Pill.

[0010] S4. Import the chromatograms of different batches of test sample solutions obtained in S3 into the Chinese herbal chromatographic fingerprint similarity evaluation system for similarity analysis to confirm the reliability of the results;

[0011] S5. Perform high-resolution mass spectrometry analysis on the test solution to obtain the total ion chromatogram and the mass spectrometry results of the chemical components. Analyze the data based on the peaks of the chemical components. Determine the chemical components of each peak in the chromatogram of the test sample by combining the total ion chromatogram and the mass spectrometry results of the chemical components with the chromatogram of the reference standard.

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

[0013] More preferably, in S1, ultrasonic extraction is performed for 30 minutes.

[0014] Preferably, in S2, a pure methanol solution is used as the dissolving solvent.

[0015] Preferably, in S2, each 1 mL of a single reference solution contains 30 μg of quercetin-3-O-β-D-glucoside-7-O-β-D-gentiopicroside standard, 30 μg of quercetin-3-O-β-D-glucopyranoside standard, 80 μg of rosmarinic acid standard, 80 μg of luteolin standard, and 70 μg of apigenin standard.

[0016] Preferably, the liquid chromatography conditions in S3 are as follows: column: Shim-pack VP-ODS, 250mm×4.6mm, 5μm; detector: UV-Vis absorption detector, detection wavelength is 254nm.

[0017] More preferably, the flow rate is 0.8 mL / min.

[0018] Preferably, the elution procedure is as follows:

[0019]

[0020] Preferably, S4 specifically involves: importing the chromatograms of different batches of test sample solutions obtained in S3 into the similarity evaluation system for chromatographic fingerprinting of traditional Chinese medicine, selecting the chromatographic peaks present in the chromatograms of different batches of Suting Pill material reference as common peaks; performing similarity analysis after data import, multi-point correction, and data matching; obtaining and exporting the similarity result table between the chromatograms of different batches of Suting Pill material reference and the common peak pattern; and confirming the reliability of the results based on the similarity result table and the chromatograms of Suting Pill material reference.

[0021] Preferably, the high-resolution mass spectrometry detection conditions in S5 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 200-1500.

[0022] Preferably, in S5, the chemical composition of each peak in the fingerprint spectrum is determined as follows: peak 9 is quercetin-3-O-β-D-glucose-7-O-β-D-gentiopicroside, peak 22 is quercetin-3-O-β-D-glucopyranoside, peak 27 is rosmarinic acid, peak 29 is luteolin, and peak 30 is apigenin.

[0023] In a second aspect, the present invention discloses the material reference fingerprint spectrum of Suting Pill obtained by the above method, wherein peak 9 is quercetin-3-O-β-D-glucose-7-O-β-D-gentiopicroside, peak 22 is quercetin-3-O-β-D-glucopyranoside, peak 27 is rosmarinic acid, peak 29 is luteolin, and peak 30 is apigenin.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] This invention provides a method for constructing a material reference fingerprint spectrum for Suting Pill. The method employs liquid chromatography-mass spectrometry (LC-MS) to construct the fingerprint spectrum and analyze and confirm the types and quantities of chemical components contained within. This allows for rapid, accurate, comprehensive, and scientific analysis of key pharmacodynamic components, fully reflecting the integrity and characteristics of Suting Pill. It lays a solid foundation for the formulation and improvement of quality standards for its compound preparations, and is of great significance for the component identification, quality evaluation, and quality standard formulation of Suting Pill. This method is simple to operate, has good stability, high precision, and good reproducibility. The constructed fingerprint spectrum is rich in information, ultimately identifying five common peaks and a standard fingerprint spectrum. It can comprehensively reflect the information of the chemical components contained in Suting Pill, providing a basis for its quality control and pharmacodynamic material basis, and helping to evaluate the overall quality of Suting Pill.

[0026] Furthermore, using an 80% methanol solution for extraction ensures rich chromatographic information, the highest component content, and good separation.

[0027] Furthermore, using ultrasonic extraction for 30 minutes ensures that the chromatogram contains all components and has good separation.

[0028] Furthermore, the selection of a detection wavelength of 254 nm ensures a stable detection baseline, good separation of various substances, and abundant chromatographic peak information.

[0029] Furthermore, a flow rate of 0.8 mL / min was selected to ensure good chromatographic peak elution and resolution.

[0030] Furthermore, selecting acetonitrile-0.1% phosphoric acid aqueous solution as the mobile phase and the elution procedure described above can ensure good separation of each component in Suting Pill, with high peaks, stable baselines, and complete chromatographic information. Attached Figure Description

[0031] Figure 1 This is a chromatogram obtained during the detection wavelength optimization process of this invention;

[0032] Figure 2 This is a chromatogram obtained during the flow rate optimization process of this invention;

[0033] Figure 3 This is the chromatogram obtained during the column temperature optimization process of this invention;

[0034] Figure 4 This is a chromatogram obtained during the mobile phase composition optimization process of this invention;

[0035] Figure 5 This is a chromatogram obtained during the elution process optimization process of this invention;

[0036] Figure 6 The chromatogram of 30 common peaks of the Suting Pill material standard of the present invention;

[0037] Figure 7 The chromatogram (A) of the quercetin-3-O-β-D-glucose-7-O-β-D-gentiopicroside standard and the mass spectrum (B) of quercetin-3-O-β-D-glucose-7-O-β-D-gentiopicroside are shown below.

[0038] Figure 8 The chromatogram (A) and mass spectrum (B) of the quercetin-3-O-β-D-glucopyranoside standard of the present invention are shown.

[0039] Figure 9The chromatogram (A) and mass spectrum (B) of the rosmarinic acid standard of the present invention are shown.

[0040] Figure 10 The chromatogram (A) of the luteolin standard and the mass spectrum (B) of the luteolin are shown in the figure.

[0041] Figure 11 The chromatogram (A) and celery spore mass spectrum (B) of the apigenin standard of the present invention are shown below.

[0042] Figure 12 The fingerprint chromatograms are of 15 batches of Suting Pills tested according to the present invention. Detailed Implementation

[0043] To enable those skilled in the art to understand the features and effects of the present invention, the following descriptions and definitions are only general descriptions of the terms and expressions mentioned in the specification and claims. Unless otherwise specified, all technical and scientific terms used herein have the ordinary meaning understood by those skilled in the art regarding the present invention, and in the event of any conflict, the definitions in this specification shall prevail.

[0044] The theories or mechanisms described and disclosed herein, whether right or wrong, should not in any way limit the scope of the invention, that is, the contents of the invention can be implemented without being limited by any particular theory or mechanism.

[0045] In this document, all features defined by numerical ranges or percentage ranges, such as numerical values, quantities, contents, and concentrations, are for the sake of brevity and convenience only. Accordingly, descriptions of numerical ranges or percentage ranges should be considered as covering and specifically disclosing all possible sub-ranges and individual numerical values ​​(including integers and fractions) within those ranges.

[0046] In this article, unless otherwise specified, “contains,” “includes,” “containing,” “has,” or similar terms cover the meanings of “composed of” and “mainly composed of,” for example, “A contains a” covers the meanings of “A contains a and others” and “A contains only a.”

[0047] For the sake of brevity, not all possible combinations of the technical features in each implementation scheme or embodiment are described herein. Therefore, as long as there is no contradiction in the combination of these technical features, the technical features in each implementation scheme or embodiment can be combined arbitrarily, and all possible combinations should be considered within the scope of this specification.

[0048] Some of the instruments used in this paper are shown in Table 1. Unless otherwise specified, conventional instruments and equipment in this field were used.

[0049] Table 1. Instruments used in this invention

[0050]

[0051] Experimental methods not specified in this article are generally performed under standard conditions or as recommended by the manufacturer.

[0052] In this article, the material standard used for Suting Pills was prepared by taking equal amounts of roasted perilla seeds and roasted lepidium seeds, crushing them, sieving them, and adding them to jujube paste to make pills. The quercetin-3-O-β-D-glucoside standard (batch number: AZBJ1013, purity: 98%), quercetin-3-O-β-D-glucopyranoside standard (batch number: AZ21080601, purity: 98%), luteolin standard (batch number: AF20030854, purity: 98%), and apigenin standard (batch number: AF21021152, purity: 98%) used in the examples were all purchased from Chengdu Efa Biotechnology Co., Ltd.; the rosmarinic acid standard (batch number: C14900895, purity: 97%) was purchased from Shanghai Maclean Biochemical Technology Co., Ltd. The reagents used in this paper are shown in Table 2. Unless otherwise specified, all raw materials used were commercially available products of standard specifications in this field.

[0053] Table 2. Reagents used in this invention

[0054]

[0055]

[0056] 1. The method for constructing a material reference fingerprint spectrum of Suting Pill according to the present invention includes the following steps:

[0057] S1. Preparation of test solution: Weigh 1.0 g of Su Ting Wan material standard from different batches, place it in a stoppered conical flask, extract with 25 mL of methanol solution, filter, and pass the filtrate through a 0.45 μm microporous membrane to obtain the test solution.

[0058] S2. Preparation of reference solution: Accurately weigh quercetin-3-O-β-D-glucose-7-O-β-D-gentiopicroside, quercetin-3-O-β-D-glucopyranoside, rosmarinic acid, luteolin and apigenin reference standards, dissolve them in solvent to prepare a single reference solution;

[0059] The solvent is selected from at least one of 50% methanol, 70% methanol, 80% methanol and pure methanol; each 1 mL of single reference solution contains 30 μg of quercetin-3-O-β-D-gluco-7-O-β-D-gentiopicroside standard, 30 μg of quercetin-3-O-β-D-glucopyranoside standard, 80 μg of rosmarinic acid standard, 80 μg of luteolin standard and 70 μg of apigenin standard;

[0060] S3. Accurately pipette 10 μL each of the test solution in S1 and the reference solution in S2, and inject them into the high performance liquid chromatograph for chromatographic analysis. Perform the detection under the same conditions, record the chromatograms from 0 to 75 min, select the common peaks from the characteristic chromatograms of the test solution, and construct the material standard fingerprint chromatogram of Suting Pill.

[0061] 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 herbal chromatographic fingerprint similarity evaluation system (version 2004A). After data import, multi-point correction and data matching, and similarity analysis, confirm the reliability of the results.

[0062] S5. Perform high-resolution mass spectrometry analysis on the test solution to obtain the total ion chromatogram and the mass spectrometry results of the five chemical components. Import the detection data into Xcalibur software, enter the Qual Browser interface, perform data analysis based on the peaks of the chemical components, and determine the chemical components of each peak in the fingerprint spectrum based on the total ion chromatogram and the mass spectrometry results of the chemical components combined with the chromatogram of the reference standard.

[0063] The high-resolution mass spectrometry detection conditions were as follows: electrospray ionization (ESI), 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 200-1500.

[0064] 2. The method for constructing the above-mentioned material reference fingerprint spectrum of Suting Pill is optimized:

[0065] S1. Optimization of the preparation of the test solution for the material reference of Suting Pill.

[0066] (1) Optimization of extraction solvent

[0067] This invention screened extraction solvents (50% methanol, 70% methanol, 80% methanol, and pure methanol) using methanol solutions of different proportions. It was found that when the extraction solvent was 80% methanol, the extract had rich chromatographic information, the highest component content, and good separation. Therefore, 80% methanol solution was selected for extraction.

[0068] (2) Optimization of extraction method

[0069] This invention screened different extraction methods (ultrasound, reflux, immersion) and different extraction times (30 min, 45 min, 60 min), and found that the chromatogram obtained by ultrasound extraction for 30 min had more comprehensive components and better separation. Therefore, the ultrasound extraction method for 30 min was adopted.

[0070] S2. Optimize chromatographic conditions.

[0071] (1) Optimization of detection wavelength

[0072] This invention uses a UV-Vis detector to screen detection wavelengths (254nm, 265nm, 280nm, 310nm, 330nm). The test solution is prepared using the method in S1 of Example 1, and all other chromatographic conditions are the same as in S3 of Example 1. Optimization results are as follows: Figure 1 As shown, the baseline was stable and the separation of each substance was good when the detection wavelength was 254 nm, with rich chromatographic peak information. Therefore, the detection wavelength of 254 nm was selected.

[0073] (2) Flow rate optimization

[0074] This invention screened for flow rates (0.8 mL / min, 0.9 mL / min, 1.0 mL / min). The test solution was prepared using the method in S1 of Example 1, and all other chromatographic conditions were the same as in S3 of Specific Embodiment 1. The optimization results are as follows: Figure 2 As shown, the chromatogram peaks and resolution were found to be better when the flow rate was 0.8 mL / min. Therefore, the flow rate of 0.8 mL / min was selected for subsequent Example 1.

[0075] (3) Optimization of column temperature

[0076] This invention screens column temperatures (28℃, 30℃, 35℃) in chromatographic conditions. The test solution is prepared using the method in S1 of Example 1, and the remaining chromatographic conditions are the same as in S3 of Example 1. The optimization results are as follows: Figure 3 As shown, column temperature has little effect on the peak elution of the chromatogram, and a column temperature of 30℃ was maintained.

[0077] (4) Optimization of mobile phase composition

[0078] This invention compares the elution effects of four different elution systems—methanol-water, acetonitrile-0.1% formic acid aqueous solution, acetonitrile-water, and acetonitrile-0.1% phosphoric acid aqueous solution—under different gradients. The test solution was prepared using the method in S1 of Example 1, and all other chromatographic conditions were the same as in S3 of Example 1. Optimization results are as follows: Figure 4As shown, it was found that when acetonitrile-0.1% phosphoric acid aqueous solution was used as the mobile phase, the separation effect of each component in Suting Pill was better, the baseline was stable, and the chromatographic information was complete. Therefore, acetonitrile-0.1% phosphoric acid aqueous solution was finally selected as the mobile phase.

[0079] (5) Optimization of the elution process

[0080] After determining the optimal mobile phase composition, the present invention screened the optimal gradient elution program through numerous experiments. The test solution was prepared using the method in S1 of Example 1, and all other chromatographic conditions were the same as in S3 of Example 1. Some elution programs are shown in Tables 3 to 8:

[0081] Table 3 Elution Procedure 1

[0082]

[0083] Table 4 Elution Procedure 2

[0084]

[0085] Table 5 Elution Procedure 3

[0086]

[0087]

[0088] Table 6 Elution Procedure 4

[0089]

[0090] Table 7 Elution Procedure 5

[0091]

[0092] Table 8 Elution Procedure 6

[0093]

[0094] Test results as follows Figure 5 As shown. (Through) Figure 5 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.

[0095] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0096] Example 1

[0097] A method for constructing a material reference fingerprint spectrum of Su Ting Wan includes the following steps:

[0098] S1. Preparation of Suting Pill Material Standard Test Solution: Accurately weigh 1.0 g of Suting Pill material standard from 15 batches, place them in a stoppered conical flask, add 25 mL of 80% methanol solution, and extract by ultrasonication (ultrasonic power 250 W, ultrasonic frequency 40 kHz) for 30 min. Filter, and pass the filtrate through a 0.45 μm microporous membrane to obtain the test solution.

[0099] S2. Preparation of reference solutions: Accurately weigh quercetin-3-O-β-D-glucoside-7-O-β-D-gentiopicroside, quercetin-3-O-β-D-glucopyranoside, rosmarinic acid, luteolin, and apigenin reference standards, place them in stoppered conical flasks, add pure methanol solution to prepare single reference solutions containing 30 μg of quercetin-3-O-β-D-glucoside-7-O-β-D-gentiopicroside standard, 30 μg of quercetin-3-O-β-D-glucopyranoside standard, 80 μg of rosmarinic acid standard, 80 μg of luteolin standard, and 70 μg of apigenin standard per 1 mL.

[0100] S3. Accurately pipette 10 μL each of the test solution in S1 and the reference solution in S2, and inject them into the high performance liquid chromatograph for chromatographic analysis. Perform the detection under the same conditions, record the chromatograms from 0 to 75 min, select the common peaks from the characteristic chromatograms of the test solution, and construct the material standard fingerprint chromatogram of Suting Pill.

[0101] The liquid chromatography conditions were as follows: column: Shim-pack VP-ODS (250 mm × 4.6 mm, 5 μm) column; detector: UV-Vis absorption detector, detection wavelength: 254 nm; column temperature: 30 ℃; flow rate: 0.8 mL / min; mobile phase: acetonitrile (A) - 0.1% phosphoric acid aqueous solution (B), gradient elution, and the elution program is shown in Table 8.

[0102] S4. Import the different batches of test sample solutions obtained in S3 into the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (Version 2004A); select the chromatographic peaks present in the chromatograms of different batches of Suting Pill material reference as common peaks, such as... Figure 6 As shown; after data import, multi-point correction and data matching, similarity analysis was performed; a similarity result table between the material reference chromatograms of different batches of Suting Pill and the common peak mode was obtained and exported; based on the similarity result table and the material reference chromatogram of Suting Pill, the reliability of the results was confirmed.

[0103] The results show that the fingerprint spectrum method for Suting Pill material reference constructed in this invention has good similarity among the common peaks, indicating that the fingerprint spectrum established by this method can effectively detect the quality of Suting Pill material reference. Detailed fingerprint spectrum similarity data are shown in Table 9.

[0104] Table 9. Similarity between each batch of samples and the shared pattern

[0105]

[0106] S5. To determine the chemical components in the fingerprint spectrum, the test solution was subjected to high-resolution mass spectrometry (HMS) analysis. The HMS detection conditions were: electrospray ionization (ESI), 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 200-1500. The total ion chromatogram and mass spectra of five chemical components were obtained. The detection data were imported into Xcalibur software, and the Qual Browser interface was used for data analysis based on the peaks of the chemical components. The analysis was then combined with the total ion chromatogram and the mass spectra of the chemical components, along with the chromatogram of the reference standard. Figures 7-11 The chemical composition of each peak in the fingerprint spectrum was determined.

[0107] like Figure 12 As shown, based on the high-resolution mass spectrometry results and the reference chromatogram, peak 9 in the fingerprint chromatogram was identified as quercetin-3-O-β-D-glucose-7-O-β-D-gentiopicroside (retention time 23.909 min), peak 22 as quercetin-3-O-β-D-glucopyranoside (retention time 40.254 min), peak 27 as rosmarinic acid (retention time 48.216 min), peak 29 as luteolin (retention time 56.613 min), and peak 30 as apigenin (retention time 63.302 min).

[0108] Example 2

[0109] Methodological study on the construction method of the above-mentioned Su Ting Wan material reference fingerprint spectrum:

[0110] S1, Stability Study

[0111] The test solution prepared by the method in Example 1 was analyzed at 0h, 2h, 4h, 8h, 12h, and 24h according to the detection method in Example 1. Quercetin-3-O-β-D-glucose-7-O-β-D-gentiopicroside, quercetin-3-O-β-D-glucopyranoside, rosmarinic acid, luteolin, and apigenin were used as reference peaks. The peak area and retention time of the common peaks in the HPLC fingerprint of the samples were analyzed, and the RSD value was calculated. The results are shown in Table 10. The RSD of the relative retention time was less than 0.15%, and the RSD of the relative peak area was less than 2.80%, indicating that the Suting Pill test solution has good stability within 24h.

[0112] Table 10 Peak area and retention time in stability study

[0113]

[0114] S2, Precision Experiment

[0115] The test solution prepared by the method in Example 1 was analyzed six times consecutively according to the detection method in Example 1. Quercetin-3-O-β-D-glucose-7-O-β-D-gentiopicroside, quercetin-3-O-β-D-glucopyranoside, rosmarinic acid, luteolin, and apigenin were used as reference peaks. The peak area and retention time of the common peaks in the HPLC fingerprint of the sample were analyzed, and the RSD value was calculated. The results are shown in Table 11. The RSD of the relative retention time was less than 0.20%, and the RSD of the relative peak area was less than 2.90%, indicating that the method has good precision.

[0116] Table 11 Peak area and retention time in precision studies

[0117]

[0118] S3, Repeatability Experiment

[0119] Six test solutions prepared according to the method in Example 1 were taken and analyzed according to the detection method in Example 1. Quercetin-3-O-β-D-glucose-7-O-β-D-gentiopicroside, quercetin-3-O-β-D-glucopyranoside, rosmarinic acid, luteolin, and apigenin were used as reference peaks. The peak area and retention time of the common peaks in the HPLC fingerprint of the samples were analyzed, and the RSD value was calculated. The results are shown in Table 12. The RSD of the relative retention time was less than 0.15%, and the RSD of the relative peak area was less than 2.70%. The results show that the chromatographic peaks of the samples are reproducible, indicating that the method has good repeatability.

[0120] Table 12 Peak area and retention time in repeatability studies

[0121]

[0122]

[0123] The above experimental results show that the method for constructing the material reference fingerprint spectrum of Suting Pill provided by the present invention is simple to operate, has good stability, high precision, and good reproducibility. The constructed fingerprint spectrum is rich in chemical information and can reflect the information of the chemical components contained in Suting Pill in an overall manner, providing a basis for its quality control and pharmacodynamic material basis. It helps to evaluate the quality of Suting Pill as a whole and provides quality assurance for clinical efficacy.

[0124] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. A method for constructing a material reference fingerprint spectrum of Su Ting Wan, characterized in that, Includes the following steps: S1. Weigh different batches of Suting Pill material standard, extract with methanol solution, filter, and obtain test solution; S2. Accurately weigh quercetin-3-O-β-D-glucoside-7-O-β-D-gentiopicroside, quercetin-3-O-β-D-glucopyranoside, rosmarinic acid, luteolin and apigenin reference standards, dissolve them to obtain a single reference standard solution; S3. Take the test solution from S1 and the reference solution from S2 for chromatographic analysis to obtain each chromatogram. Select the common peak from the chromatogram of the test solution to construct the material standard fingerprint chromatogram of Suting Pill. The chromatographic conditions were as follows: column: Shim-pack VP-ODS, 250 mm × 4.6 mm, 5 μm; Detector: UV-Vis absorption detector, detection wavelength 254 nm, flow rate 0.8 mL / min, elution program as follows: S4. Import the chromatograms of different batches of test sample solutions obtained in S3 into the Chinese medicine chromatographic fingerprint similarity evaluation system for similarity analysis to confirm the reliability of the results; S5. Perform high-resolution mass spectrometry analysis on the test solution to obtain the total ion chromatogram and the mass spectrometry results of the chemical components. Analyze the data based on the peaks of the chemical components. Determine the chemical components of each peak in the chromatogram of the test sample by combining the total ion chromatogram and the mass spectrometry results of the chemical components with the chromatogram of the reference standard.

2. The method for constructing a material reference fingerprint spectrum of Suting Pill according to claim 1, characterized in that, In S1, the methanol solution is an 80% methanol solution.

3. The method for constructing a material reference fingerprint spectrum of Su Ting Wan according to claim 2, characterized in that, In S1, ultrasonic extraction was performed for 30 minutes.

4. The method for constructing a material reference fingerprint spectrum of Su Ting Wan according to claim 1, characterized in that, S4 specifically involves: importing the chromatograms of different batches of test sample solutions obtained in S3 into the similarity evaluation system for chromatographic fingerprinting of traditional Chinese medicine; selecting chromatographic peaks present in the chromatograms of different batches of Sutingwan material reference as common peaks; 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 Sutingwan material reference and the common peak pattern; and confirming the reliability of the results based on the similarity result table and the chromatograms of Sutingwan material reference.

5. The method for constructing a material reference fingerprint spectrum of Su Ting Wan according to claim 1, characterized in that, The high-resolution mass spectrometry detection conditions in S5 are as follows: 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 200-1500.

6. The method for constructing a material reference fingerprint spectrum of Suting Pill according to claim 1, characterized in that, In S5, the chemical composition of each peak in the fingerprint spectrum was determined as follows: peak 9 is quercetin-3-O-β-D-glucose-7-O-β-D-gentiopicroside, peak 22 is quercetin-3-O-β-D-glucopyranoside, peak 27 is rosmarinic acid, peak 29 is luteolin, and peak 30 is apigenin.

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