A method for constructing a reference fingerprint of a xueshuaiwu substance and a fingerprint thereof

Abstract

The application provides a construction method of Xiaoru Wan substance reference fingerprint and the fingerprint, and the construction method comprises the following steps: adopting high performance liquid chromatography combined with high resolution mass spectrometry to analyze a to-be-detected sample, and obtaining a Xiaoru Wan fingerprint. The liquid chromatography-mass spectrometry method adopted by the application is simple in operation, good in stability, high in precision, and good in reproducibility, the constructed fingerprint has rich chemical information, can make various effective components be well separated, and combined with a chromatogram of a reference solution, finally, 10 chemical components are identified, Xiaoru Wan and its pharmaceutical preparations can be effectively and comprehensively detected, the blank of the fingerprint in the quality control of the classic famous prescription Xiaoru Wan is filled, and the Xiaoru Wan and the related preparation research are provided with reference and basis.

Description

Technical Field

[0001] The present invention relates to the technical field of quality analysis and detection of traditional Chinese medicines, and particularly relates to a method for constructing a reference fingerprint of Xiaoru Pills and the fingerprint thereof. Background Art

[0002] The classic traditional Chinese medicine formula Xiaoru Pills originated from "A Hundred Questions for Infants and Children" written by Lu Boshi in the Ming Dynasty. This formula consists of six medicinal materials: Cyperus rotundus, Glycyrrhiza uralensis, Citrus reticulata Blanco, Amomum villosum, and Medicago sativa. The specific prescription and preparation method are as follows: Cyperus rotundus (stir-fried) one tael, Glycyrrhiza uralensis (roasted), and Citrus reticulata Blanco each half tael, Amomum villosum, Medicago sativa (stir-fried) each one tael. Grind the above into fine powder and make pills like millet grains with ice cream. It has the effects of warming the middle-jiao, relieving stuffiness, stopping vomiting, and eliminating milk and food; it is mainly used for vomiting milk or having green stools within 100 days after birth; vomiting from the corners of the mouth; vomiting food and milk; qi stagnation and fright diarrhea; indigestion of retained food.

[0003] At present, the research on Xiaoru Pills is relatively insufficient, lacking in-depth research on quality control, unable to comprehensively reflect the pharmacodynamic characteristics of the combined action of multiple components in traditional Chinese medicines, and also unable to provide sufficient basis for the quality control and evaluation thereof. Summary of the Invention

[0004] In view of the deficiencies of the existing detection methods, the present invention provides a method for constructing a reference fingerprint of Xiaoru Pills and the fingerprint thereof, which can provide a reliable basis for the identification and quality control of Xiaoru Pills.

[0005] The present invention is achieved through the following technical solutions:

[0006] A method for constructing a reference fingerprint of Xiaoru Pills includes the following steps:

[0007] S1. Take the reference of Xiaoru Pills, perform ultrasonic extraction with a methanol solution to obtain a test solution.

[0008] S2. Take reference substances of naringenin, hesperidin, physcion, liquiritin, glycyrrhetinic acid, naringenin, vanillic acid, catechin, liquiritigenin, and neohesperidin, dissolve them separately with a methanol solution to obtain single reference substance solutions.

[0009] S3. Inject the test solution in S1 and each single reference substance solution in S2 into a high performance liquid chromatograph for chromatographic analysis, and perform detection under the same conditions to obtain a test chromatogram and a reference chromatogram.

[0010] S4. Perform high-resolution mass spectrometry analysis on the test solution to obtain a total ion chromatogram and a mass spectrometry result diagram of chemical components.

[0011] S5. Determine the chemical components of each peak in the test chromatogram according to the total ion chromatogram, the mass spectrometry result diagram of chemical components, and the reference chromatogram to obtain the reference fingerprint of Xiaoru Pills.

[0012] Preferably, in S1, the methanol solution is pure methanol.

[0013] Preferably, the liquid chromatography conditions in S3 are as follows: column: Shim-pack VP-ODS column; detector: ultraviolet-visible absorption detector with a detection wavelength of 275 nm.

[0014] Preferably, in S3, the mobile phase for chromatographic analysis is methanol (A) - 0.1% phosphoric acid aqueous solution (B).

[0015] Furthermore, in S3, chromatographic analysis employs gradient elution, with the following elution procedure:

[0016]

[0017] Preferably, S3 further includes: exporting the chromatograms of different batches of test solution and reference solution, importing the chromatogram of the test solution into the Chinese medicine chromatographic fingerprint similarity evaluation system and performing similarity analysis to confirm the reliability of the results.

[0018] Furthermore, specifically: the chromatograms of different batches of test sample solutions and reference solutions were imported into the chromatographic fingerprint similarity evaluation system for traditional Chinese medicine; chromatographic peaks present in the chromatograms of different batches of Xiaoru Pills as material references were selected as common peaks; similarity analysis was performed after data import, multi-point correction, and data matching; a similarity result table between the chromatograms of different batches of Xiaoru Pills as material references and the common peak patterns was obtained and exported; the reliability of the results was confirmed based on the similarity result table and the chromatograms of Xiaoru Pills as material references.

[0019] Preferably, in S4, the high-resolution mass spectrometry detection conditions are: electrospray ionization, spray voltage 3500V, sheath gas flow rate 40arb, auxiliary gas flow rate 10arb, capillary temperature 300℃, auxiliary gas temperature 300℃, scanning mode is full scan mode, and mass-to-charge ratio scanning range m / z is 100-1300.

[0020] Preferably, in S5, the chemical components of each peak in the chromatogram of the test sample are as follows: peak 2 is catechin, peak 4 is vanillic acid, peak 5 is emodin methyl ether, peak 7 is glycyrrhizin, peak 9 is hesperidin, peak 13 is glycyrrhizin, peak 19 is naringenin, peak 26 is norihesperidin, peak 30 is citrus reticulatae, and peak 45 is glycyrrhetinic acid.

[0021] The present invention also provides a material-based fingerprint spectrum of the breast-reducing pill obtained by the construction method described above.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] This invention establishes a material reference fingerprint for Xiaoru Pills for the first time. Using high-performance liquid chromatography-high-resolution mass spectrometry (HPLC-MS / MS), the types and quantities of chemical components in Xiaoru Pills were analyzed and confirmed in detail. In particular, key pharmacodynamic components were rapidly, accurately, and scientifically identified and analyzed. This provides a comprehensive overview of the chemical components contained in Xiaoru Pills, reflecting their characteristics and integrity. This is of great significance for the component identification, quality evaluation, and quality standard formulation of Xiaoru Pills. HPLC is a highly efficient and precise analytical technique. Its high separation efficiency, fast analysis speed, high quantitative precision, diverse detector types, and good stability make it an ideal choice for constructing fingerprint profiles. It is not limited by sample volatility or thermal stability; most components in a sample can be analyzed and detected on an HPLC instrument, making it one of the main methods for constructing fingerprint profiles. The HPLC-MS / MS method used in this invention is simple to operate, has good stability, high precision, and good reproducibility.

[0024] Furthermore, this invention optimizes the extraction solvent, extraction method, mobile phase, column temperature, and detection wavelength, ultimately determining that the liquid chromatography uses a Shim-pack VP-ODS (250mm × 4.6mm, 5μm) column with a mobile phase of methanol (A)-0.1% phosphoric acid aqueous solution (B) using gradient elution. The constructed fingerprint spectrum is rich in chemical information, enabling good separation of multiple active ingredients. Combined with the chromatogram of the reference solution, 10 chemical components were ultimately identified, allowing for effective and comprehensive detection of Xiaoru Pill and its pharmaceutical preparations. This fills the gap in fingerprint spectrum technology for the quality control of the classic formula Xiaoru Pill and provides a reference and basis for research on Xiaoru Pill and its related preparations. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 This is a chromatogram obtained during the preparation of the test solution according to the present invention;

[0027] Figure 2 This is a chromatogram obtained during the determination of the extraction solvent in this invention;

[0028] Figure 3 This is a chromatogram obtained during the process of determining the extraction method in this invention;

[0029] Figure 4 This is a chromatogram obtained during the detection wavelength optimization process of this invention;

[0030] Figure 5 This is the chromatogram obtained during the column temperature optimization process of this invention;

[0031] Figure 6 This is a chromatogram obtained during the mobile phase composition optimization process of this invention;

[0032] Figure 7 This is a chromatogram obtained during the elution process optimization process of this invention;

[0033] Figure 8A and Figure 8B These are the positive ion total ion chromatogram and the negative ion total ion chromatogram of the present invention, respectively.

[0034] Figure 9A and Figure 9B The images show the chromatogram and catechin mass spectrum of the catechin standard of this invention, respectively.

[0035] Figure 10A and Figure 10B The images show the chromatogram and mass spectrum of vanillic acid standard of the present invention, respectively.

[0036] Figure 11A and Figure 11B The images show the chromatogram and mass spectrum of the emodin methyl ether standard of this invention, respectively.

[0037] Figure 12A and Figure 12B The images show the chromatogram and mass spectrum of the glycyrrhizin standard of this invention, respectively.

[0038] Figure 13A and Figure 13B The images show the chromatogram and mass spectrum of the hesperidin standard of this invention, respectively.

[0039] Figure 14A and Figure 14B The images show the chromatogram and chromatogram of the glycyrrhizin standard of this invention, respectively.

[0040] Figure 15A and Figure 15B The images show the chromatogram of the naringin standard of this invention and the mass spectrum of naringin peel, respectively.

[0041] Figure 16A and Figure 16B The images show the chromatogram of the hesperidin standard of this invention and the mass spectrum of hesperidin.

[0042] Figure 17A and Figure 17B The images show the chromatogram and mass spectrum of the hesperidin standard of this invention, respectively.

[0043] Figure 18A and Figure 18BThe images show the chromatogram and mass spectrum of the glycyrrhetinic acid standard of this invention, respectively.

[0044] Figure 19 The fingerprint chromatograms are of 15 batches of Xiaoru Pills tested in this invention. Detailed Implementation

[0045] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0046] It should be noted that the process equipment or apparatus not specifically mentioned in the following embodiments are all conventional equipment or apparatus in the art.

[0047] It should be noted that the terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusion. For example, a process, method, system, product, or apparatus that includes a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to these processes, methods, products, or apparatuses. Furthermore, unless otherwise stated, the numbering of each method step is merely a convenient tool for identifying the method steps, and not intended to limit the order of the method steps or define the scope of the invention. Changes or adjustments to their relative relationships, without substantially altering the technical content, should also be considered within the scope of the invention.

[0048] 1. The method for constructing the material reference fingerprint spectrum of the breast-reducing pills of the present invention includes the following steps:

[0049] S1. Preparation of test solution: Weigh appropriate amounts of different batches of Xiaoru Pill material standard, place them in a stoppered conical flask, extract with methanol solution, filter, and pass the filtrate through a 0.45μm microporous membrane to obtain the test solution;

[0050] S2. Preparation of reference solutions: Accurately weigh the reference standards of hesperidin, hesperidin, emodin methyl ether, glycyrrhizin, glycyrrhetinic acid, naringenin, vanillic acid, catechin, glycyrrhizin, and nobiletin, dissolve them in solvent, and prepare a single reference solution.

[0051] S3. Accurately pipette the test solution from S1 and the reference solution from S2, respectively, and inject them into the high-performance liquid chromatograph. Detect them under the same conditions and record the chromatograms from 0 to 120 min.

[0052] S4. Export the chromatograms of different batches of test solution and reference solution obtained in S3, import the chromatograms of test solution into the Chinese herbal chromatographic fingerprint similarity evaluation system (version 2004A) and perform similarity analysis to confirm the reliability of the results;

[0053] 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. 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 chromatogram of the test sample by combining the mass spectrometry results of the chemical components and the total ion chromatogram with the chromatogram of the reference standard.

[0054] The preparation method of the test solution described in S1 of this invention is as follows: Accurately weigh 5.0 g of the material standard of Xiaoru Pill, place it in a stoppered conical flask, add 25 mL of pure methanol solution, extract by ultrasonication (ultrasonic power 250 W, ultrasonic frequency 40 kHz) for 45 min, filter, and pass the filtrate through a 0.45 μm microporous membrane to obtain the test solution.

[0055] The reference solution described in S2 of this invention contains, in 1 mL, 50 μg of hesperidin, 60 μg of hesperidin, 20 μg of emodin methyl ether, 90 μg of glycyrrhizin, 80 μg of glycyrrhetinic acid, 100 μg of naringenin, 70 μg of vanillic acid, 30 μg of catechin, 30 μg of glycyrrhizin, and 40 μg of nobiletin.

[0056] The preferred liquid chromatography conditions in S3 of this invention are as follows: Column: Shim-pack VP-ODS (250mm × 4.6mm, 5μm) column; Detector: UV-Vis absorption detector, detection wavelength: 275nm; Mobile phase: methanol (A) - 0.1% phosphoric acid aqueous solution (B), gradient elution, column temperature: 30℃; Flow rate: 0.8mL / min; Injection volume: 20μL; Elution program:

[0057]

[0058] In this invention, S4 specifically involves: importing the chromatograms of different batches of test sample solutions and reference solutions obtained in S3 into the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (Version 2004A); selecting chromatographic peaks present in the chromatograms of different batches of Xiaoru Pills as common peaks, such as... Figure 1 As shown; after data import, data matching and multi-point correction, similarity analysis was performed; a similarity result table between the chromatograms of different batches of Xiaoru Pills and the common peak modes was obtained and exported; based on the similarity result table and the chromatograms of Xiaoru Pills, the reliability of the results was confirmed.

[0059] The high-resolution mass spectrometry detection conditions in S5 of this invention are 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℃, scanning mode is full scan mode, and mass-to-charge ratio scanning range m / z is 100-1300.

[0060] 2. Optimization of fingerprint pattern detection:

[0061] 1) Optimization of the preparation of the test solution for the material reference of the breast milk-reducing pill.

[0062] (1) Optimization of extraction solvent

[0063] Preparation of the test solution: Accurately weigh 5.0 g of the material standard of 15 batches of Xiaoru Pills and place it in a stoppered conical flask. At the same time, weigh 4 portions and add 25 mL of 50% methanol, 70% methanol, pure methanol and 70% ethanol respectively. Extract by ultrasonication for 45 min, filter, and filter the filtrate through a 0.45 μm microporous membrane to obtain the test solution.

[0064] like Figure 2 As shown in the figure, through experimental comparison, it was found that the pure methanol solution extract had the most chromatographic information and the highest component content, so pure methanol solution was chosen for extraction.

[0065] (2) Optimization of extraction method

[0066] Preparation of the test solution: Accurately weigh 5.0 g of the material standard of 15 batches of Xiaoru Pills and place it in a stoppered conical flask. At the same time, weigh 3 portions and perform the following treatments: 1) Add 25 mL of pure methanol solution, heat under reflux for 1 h, evaporate to dryness, and redissolve with 1 mL of methanol; 2) Add 25 mL of pure methanol solution and soak for 24 h; 3) Add 25 mL of pure methanol solution and ultrasonically extract for 45 min; filter, and filter the filtrate through a 0.45 μm microporous membrane to obtain the test solution.

[0067] like Figure 3 As shown in the figure, through experimental comparison, it was found that the chromatogram obtained by adding 25 mL of pure methanol solution and ultrasonic extraction for 45 min had more comprehensive components and better separation. Therefore, the ultrasonic extraction method of 45 min was adopted.

[0068] 2) Optimize chromatographic conditions

[0069] (1) Optimization of detection wavelength

[0070] This invention uses a UV-Vis detector to screen for detection wavelengths (210nm, 254nm, 275nm, 283nm, 300nm, 310nm). The test solution is prepared using the method in S1, and all other chromatographic conditions are the same as in S3 of Example 1. Optimization results are as follows: Figure 4 As shown, the baseline was stable and the separation of each substance was good when the detection wavelength was 275 nm, with rich chromatographic peak information. Therefore, the detection wavelength of 275 nm was selected.

[0071] (2) Flow rate optimization

[0072] This invention screened for flow rates (0.6 mL / min, 0.8 mL / min, 1.0 mL / min). The test solution was prepared using the method in S1, and the remaining chromatographic conditions were the same as in S3 of Example 1. It was found that the chromatogram peaks and resolution were better at a flow rate of 0.8 mL / min, therefore, a flow rate of 0.8 mL / min was selected for subsequent examples.

[0073] (3) Optimization of column temperature

[0074] This invention screened column temperatures (28℃, 30℃, 35℃) for chromatographic conditions. The test solution was prepared using the method in S1, and the remaining chromatographic conditions were the same as in S3 of Example 1. The optimization results are as follows: Figure 5 As shown, column temperature has little effect on the peak elution of the chromatogram, and a column temperature of 30℃ was maintained.

[0075] (4) Optimization of mobile phase composition

[0076] This invention compares the elution effects of four different elution systems—methanol-water, acetonitrile-water, methanol-0.1% phosphoric acid aqueous solution, 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 6 As shown, it was found that when methanol-0.1% phosphoric acid aqueous solution was used as the mobile phase, the separation effect of each component in Xiaoru Pill was better, the baseline was stable, and the chromatographic information was complete. Therefore, methanol-0.1% phosphoric acid aqueous solution was finally selected as the mobile phase.

[0077] (5) Optimization of the elution process

[0078] After determining the optimal mobile phase composition, the present invention screened the optimal gradient elution procedure 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. Partial elution procedures are as follows:

[0079] Table 1 Elution Procedure 1

[0080]

[0081] Table 2 Elution Procedure 2

[0082]

[0083] Table 3 Elution Procedure 3

[0084]

[0085] Table 4 Elution Procedure 4

[0086]

[0087] Table 5 Elution Procedure 5

[0088]

[0089] Table 6 Elution Procedure 6

[0090]

[0091] Table 7 Elution Procedure 7

[0092]

[0093]

[0094] Table 8 Elution Procedure

[0095]

[0096] Test results as follows Figure 7 As shown. (Through) Figure 7 It can be seen that elution program 8 has good resolution, high peak height, stable baseline, and complete chromatographic information. Therefore, elution program 8 is selected as the optimal elution program.

[0097] The present invention will be described in detail below using specific embodiments as examples.

[0098] 1. Instruments

[0099] The instruments used in the embodiments of this invention are shown in Table 9.

[0100] Table 9. Instruments and reagents used in this invention

[0101]

[0102] 2. Medicines and reagents

[0103] Material standards for Xiaoru Pills: Prepared in the laboratory; glycyrrhizin (batch number: AFBI0102, purity: 98%), glycyrrhizin (batch number: AFBH3102, purity: 98%), glycyrrhetinic acid (batch number: AF21021652, purity: 98%), naringenin (batch number: AFCB2758, purity: 98%), all purchased from Chengdu Aifa Biotechnology Co., Ltd.; hesperidin (batch number: YJ0509, purity: 98%), and hesperidin (batch number: 200134-3, purity: 96%). The following reagents were used: hesperidin (batch number: YJ0275, purity: 98%), purchased from Jiangsu Yongjian Pharmaceutical Technology Co., Ltd.; vanillic acid (batch number: PS010559, purity: 98%), purchased from Chengdu Pusi Biotechnology Co., Ltd.; catechin (batch number: 110877-201604, purity: 99.2%), purchased from the China National Institutes for Food and Drug Control; and emodin methyl ether (batch number: DHJM20200419, purity: 98%), purchased from Nanjing Chunqiu Bioengineering Co., Ltd. The reagents used are shown in Table 10.

[0104] Table 10 Reagents used in this invention

[0105]

[0106] Example 1

[0107] A method for constructing a material reference fingerprint spectrum for a breast-reducing pill and the fingerprint spectrum thereof, comprising the following steps:

[0108] S1. Preparation of the test solution:

[0109] Accurately weigh 5.0 g of the Xiehuang Powder material standard from 15 batches and place it in a stoppered conical flask. Add 25 mL of pure methanol solution, extract by ultrasonication for 45 min, filter, and pass the filtrate through a 0.45 μm microporous membrane to obtain the test solution.

[0110] S2. Preparation of the reference solution:

[0111] Accurately weigh each reference standard and place it in a stoppered conical flask. Add pure methanol solution to prepare a single reference standard solution containing 50 μg hesperidin, 60 μg hesperidin, 20 μg emodin methyl ether, 90 μg glycyrrhizin, 80 μg glycyrrhetinic acid, 100 μg naringenin, 70 μg vanillic acid, 30 μg catechin, 30 μg glycyrrhizin, and 40 μg nobiletin per 1 mL.

[0112] S3. Accurately pipette 20 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 and record the chromatograms from 0 to 120 min.

[0113] 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: 275 nm; Mobile phase: methanol (A) - 0.1% phosphoric acid aqueous solution (B), gradient elution, the elution program is shown in Table 11.

[0114] Table 11 Elution Procedure

[0115]

[0116] S4. Chromatograms of different batches of test solution obtained in S3 ( Figure 19 The chromatograms of the reference solution and the standard solution were imported into the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (Version 2004A). Chromatographic peaks present in the chromatograms of different batches of Xiaoru Pill reference materials were selected as common peaks. After data import, multi-point correction, and data matching, similarity analysis was performed. A similarity result table between the chromatograms of different batches of Xiaoru Pill reference materials and the common peak patterns was obtained and exported. The reliability of the results was confirmed based on the similarity result table and the chromatograms of Xiaoru Pill reference materials. The results show that the fingerprint chromatogram method for Xiaoru Pill reference materials constructed in this invention has good similarity among the common peaks, indicating that the fingerprint chromatogram established by this method can effectively detect the quality of Xiaoru Pill reference materials. Detailed fingerprint chromatogram similarity data are shown in Table 12.

[0117] Table 12 Similarity between each batch of samples and the common pattern

[0118]

[0119] S5. To determine the chemical components in the fingerprint spectrum, the test solution was subjected to high-resolution mass spectrometry analysis to obtain the total ion chromatogram and the mass spectrometry results of 10 chemical components. Figure 8A , Figure 8B , Figure 9A , Figure 9B , Figure 10A , Figure 10B , Figure 11A , Figure 11B , Figure 12A , Figure 12B , Figure 13A , Figure 13B , Figure 14A , Figure 14B , Figure 15A , Figure 15B , Figure 16A , Figure 16B , Figure 17A , Figure 17B , Figure 18A , Figure 18BImport the detection data into Xcalibur software, enter the Qual Browser interface, perform data analysis based on the peaks of 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.

[0120] S6. Based on the high-resolution mass spectrometry results and the reference chromatogram, peak 2 in the fingerprint chromatogram was determined to be catechin, peak 4 to be vanillic acid, peak 5 to be emodin methyl ether, peak 7 to be glycyrrhizin, peak 9 to be hesperidin, peak 13 to be glycyrrhizin, peak 19 to be naringenin, peak 26 to be norihesperidin, peak 30 to be citrus tomentosa, and peak 45 to be glycyrrhetinic acid.

[0121] Example 2: Methodological study on the construction of a material reference fingerprint spectrum for a breast-reducing pill:

[0122] S1, Stability Study

[0123] 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. The glycyrrhizin peak (peak 7) was used as the reference peak. 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 showed that the RSD of the relative retention time was within 0.01% to 1.74%, and the RSD of the relative peak area was less than 2.80%. The results indicate that the Xiaoru Pill test solution has good stability within 24h.

[0124] S2, Precision Experiment

[0125] The test solution prepared by the method in Example 1 was taken and measured 6 times consecutively according to the detection method in Example 1. The glycyrrhizin peak 7 was used as the reference peak. 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 showed that the RSD of the relative retention time was less than 0.80% and the RSD of the relative peak area was less than 3.00%, indicating that the method has good precision.

[0126] S3, Repeatable Experiment

[0127] Six test solutions prepared by the method in Example 1 were taken and analyzed according to the detection method in Example 1. The glycyrrhizin peak (peak 7) was used as the reference peak. 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 showed that the RSD of the relative retention time was less than 0.23% and the RSD of the relative peak area was less than 2.99%. The results showed that the chromatographic peaks of the samples were reproducible, indicating that the method had good repeatability.

[0128] Based on the above experimental results, the method for constructing the high-performance liquid chromatography combined with high-resolution mass spectrometry fingerprint of Xiaoru Pills provided by this invention has the characteristics of good repeatability, high precision, and excellent stability. This method can comprehensively and objectively evaluate the quality of Xiaoru Pills, providing a reliable quality assurance for its clinical efficacy.

[0129] This invention optimizes extraction conditions such as solvent, extraction method, mobile phase, column temperature, and flow rate, using octadecylsilane-bonded silica gel as the packing material, methanol and phosphoric acid-containing aqueous solution as mobile phases A and B, and employing specific elution conditions. This series of optimizations significantly improves the separation of multiple active ingredients while shortening the detection time, resulting in a fingerprint spectrum containing more characteristic peaks and enriching the spectral information. Through combined high-resolution mass spectrometry analysis, 10 chemical components were successfully identified, enabling this method to effectively and comprehensively detect Xiaoru Pills and their pharmaceutical preparations. This invention established HPLC fingerprints for 15 different batches of Xiaoru Pills, identifying 45 common chromatographic peaks. The peak groups in the chromatograms of the 15 batches of Xiaoru Pills were basically consistent, establishing an HPLC fingerprint spectrum for Xiaoru Pills. This spectrum can comprehensively reflect the information of the chemical components contained in Xiaoru Pills, demonstrating the characteristics and integrity of Xiaoru Pills, providing a reference for its pharmacodynamic material basis and quality control, and contributing to the overall quality evaluation of Xiaoru Pills.

[0130] This invention establishes for the first time a fingerprint spectrum quality evaluation method for Xiaoru Pills. This method comprehensively detects the types and quantities of the main chemical components contained in the classic formula Xiaoru Pills, providing a rapid, convenient, and accurate objective and comprehensive assessment of its quality. This method has advantages such as simplicity, stability, high precision, and good reproducibility.

[0131] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them. Although the foregoing embodiments have been described in detail, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein, and such modifications or substitutions will not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for constructing a material reference fingerprint spectrum for a breast-reducing pill, characterized in that, Includes the following steps: S1. Eliminate the milk pellet material standard and use pure methanol for ultrasonic extraction to obtain the test solution; S2. Take the reference standards of hesperidin, hesperidin, emodin methyl ether, glycyrrhizin, glycyrrhetinic acid, naringenin, vanillic acid, catechin, glycyrrhizin and nobiletin, and dissolve them in methanol solution to obtain single reference standard solutions. S3. Inject the test solution from S1 and each single reference solution from S2 into a high-performance liquid chromatograph for chromatographic analysis. Detection is performed under the same conditions to obtain the chromatograms of the test sample and the reference solutions. The liquid chromatography conditions are: column: Shim-pack VP-ODS column; Detector: UV-Vis absorption detector, detection wavelength 275 nm; mobile phase: methanol-0.1% phosphoric acid aqueous solution; chromatographic analysis used gradient elution, the elution program is as follows: S4. Perform high-resolution mass spectrometry analysis on the test solution to obtain the total ion chromatogram and the mass spectrometry results of the chemical composition; S5. 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 material reference fingerprint spectrum of Xiaoru Pill.

2. The method for constructing a material reference fingerprint spectrum for a breast-reducing pill according to claim 1, characterized in that, S3 also includes: exporting the chromatograms of different batches of test solution and reference solution, importing the chromatograms of the test solution into the Chinese medicine chromatographic fingerprint similarity evaluation system and performing similarity analysis to confirm the reliability of the results.

3. The method for constructing a material reference fingerprint spectrum for a breast-reducing pill according to claim 2, characterized in that, Specifically, the process involves: importing the chromatograms of different batches of test solutions and reference solutions into a similarity evaluation system for chromatographic fingerprinting of traditional Chinese medicine; selecting chromatographic peaks present in the chromatograms of different batches of Xiaoru Pills 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 Xiaoru Pills and the common peak patterns; and confirming the reliability of the results based on the similarity result table and the chromatograms of Xiaoru Pills.

4. The method for constructing a material reference fingerprint spectrum for a breast-reducing pill according to claim 1, characterized in that, In S4, 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 100-1300.

5. The method for constructing a material reference fingerprint spectrum for a breast-reducing pill according to claim 1, characterized in that, In S5, the chemical components of each peak in the chromatogram of the test sample are as follows: peak 2 is catechin, peak 4 is vanillic acid, peak 5 is emodin methyl ether, peak 7 is glycyrrhizin, peak 9 is hesperidin, peak 13 is glycyrrhizin, peak 19 is naringenin, peak 26 is norihesperidin, peak 30 is citrus reticulatae, and peak 45 is glycyrrhetinic acid.

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