Method for establishing HPLC (High Performance Liquid Chromatography) fingerprint spectrum of lozenge for clearing throat
Through the HPLC fingerprint method, the problem of incomplete ingredient detection in the quality control of laryngeal clearing lozenges was solved, and a fingerprint map for multi-component qualitative and quantitative analysis was established to ensure the uniformity of product quality and the safety of clinical medication.
Patent Information
- Application Number
- CN202510589914.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-08
- Publication Date
- 2025-07-22
AI Technical Summary
In the prior art, the quality control method for clearing throat lozenges is only used as the indicator of astragalus membranaceus, which cannot fully reflect the overall quality of complex components. The existing fingerprint map is not ideal and has poor peak shape, so it is impossible to fully detect chemical components, which affects the effectiveness of quality control.
Using HPLC fingerprinting method, 28 common chromatographic peaks were confirmed by preparing test and reference samples, and a column temperature of 25°C was used, with a detection wavelength of 214 nm. The gradient elution conditions were 0-10min 15%:85%→30%:70%, 25-50min 30%:70%→80%:20%, and 28 common chromatographic peaks were confirmed, and baicalin peaks were selected as reference peaks to establish a fingerprint map for the clear throat lozenges.
A multi-component qualitative and quantitative analysis of the laryngeal clearing lozenges is achieved, which comprehensively reflects product quality and ensures uniform and stable products. It provides a theoretical basis for quality control throughout the process and ensures the safety and effectiveness of clinical medicines.
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Figure CN120352545A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of establishing Chinese medicine fingerprint spectra, and in particular to a method for establishing an HPLC fingerprint spectrum of Qinghouyan Buccal Tablets and the fingerprint spectrum thereof. Background Art
[0002] Qinghouyan Buccal Tablets is a prepared Chinese patent medicine composed of five medicinal materials, namely Scutellaria baicalensis, Scrophularia ningpoensis, Forsythia suspensa, Rehmannia glutinosa, and Ophiopogon japonicus, and has the effects of nourishing yin, clearing the throat, and detoxifying. It is used for acute tonsillitis and angina caused by yin deficiency and lung heat, with symptoms such as sore throat, tonsillar enlargement, cough, hoarseness, dryness and heat in the throat, ear fullness, and earache. The quality of Qinghouyan Buccal Tablets is the premise to ensure the safety and effectiveness of clinical medication. At present, the method used to evaluate the quality of Qinghouyan Buccal Tablets preparations is to control with astragaloside IV as an index. However, Qinghouyan Buccal Tablets are composed of five medicinal materials, with complex components. Only relying on the content measurement of a single component cannot comprehensively describe and evaluate the quality of Qinghouyan Buccal Tablets, and it is difficult to effectively reflect the overall quality of Qinghouyan Buccal Tablets.
[0003] As a comprehensive and quantifiable identification method, Chinese medicine fingerprint spectra have the characteristics of large information volume, strong characteristic, integrity, and ambiguity, which can comprehensively reflect the types and quantities of chemical components contained in Chinese medicine. At present, it has become one of the quality control models that conform to the characteristics of Chinese medicine and is more and more widely used in the quality control of Chinese medicine preparations. However, there are no relevant literature reports on the establishment of fingerprint spectra of Qinghouyan Buccal Tablets at home and abroad.
[0004] The prior art "Study on the HPLC Fingerprint Spectrum of Qinghouyan Mixture" (Jiang Fancheng et al., Evaluation Technology and Methods, Vol. 36, No. 4, 2019) discloses a method for establishing the HPLC fingerprint spectrum of Qinghouyan Mixture. However, this establishment method has the following defects: (1) The separation degree of each characteristic peak is not ideal, and the peak shape is not good, which affects the accurate identification and quantitative analysis of components; (2) The running time is long and some components have no absorption at a wavelength of 270 nm, resulting in these components not being detected, making the fingerprint spectrum incomplete and unable to comprehensively reflect the chemical components of Qinghouyan Mixture, affecting its effectiveness as a quality control tool. Summary of the Invention
[0005] The object of the present invention is to provide a method for establishing an HPLC fingerprint spectrum of Qinghouyan Buccal Tablets that can improve the separation degree and peak shape, has high stability, good accuracy, repeatability, and separation degree, can detect all components at a wavelength of 214 nm, can be used for the production process monitoring and preparation quality control of Qinghouyan Buccal Tablets, provides a certain theoretical basis and support for the establishment of the whole-process quality control system, and ensures the safety and effectiveness of clinical medication.
[0006] To achieve the above object, the present invention adopts the following technical solutions: A method for establishing an HPLC fingerprint of the Qinghouyan Buccal Tablets of the present invention comprises the following steps: (1) Preparation of a test solution and a reference substance mixed solution Preparation of the test solution: Weigh accurately different batches of Qinghouyan Buccal Tablets, place them in volumetric flasks respectively, add an appropriate amount of methanol aqueous solution, ultrasonically treat at room temperature and then cool to room temperature, make up the volume, shake well, and filter. The appropriate amount refers to the amount required to dissolve the Qinghouyan Buccal Tablets.
[0007] Preparation of the reference substance mixed solution: Accurately weigh appropriate amounts of forsythoside A, baicalin, phillyrin, cinnamic acid, baicalein, and wogonin reference substances, accurately weigh them, and prepare a reference substance mixed solution with methanol aqueous solution.
[0008] (2) Establishment of the fingerprint Perform injection analysis on the test solution and the reference substance mixed solution respectively by HPLC to obtain the superimposed HPLC fingerprint of the test solution and the HPLC fingerprint of the reference substance mixed solution, and confirm 28 common chromatographic peaks according to the superimposed HPLC fingerprint of the test solution; Import the fingerprint data of the test solution into the Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints (2012 Edition), use one of the test solution fingerprints as the reference fingerprint, set the time window width to 0.1 min, perform multi-point calibration and data matching, and generate the HPLC fingerprint of the Qinghouyan Buccal Tablets by the mean method; Compare the HPLC fingerprint of the reference substance mixed solution with the HPLC fingerprint of the Qinghouyan Buccal Tablets, and 6 of the common chromatographic peaks are identified, confirming forsythoside A at peak No. 8, baicalin at peak No. 13, phillyrin at peak No. 16, cinnamic acid at peak No. 22, baicalein at peak No. 24, and wogonin at peak No. 26, and use the baicalin peak as the reference peak for similarity analysis.
[0009] Preferably, in step (1), accurately weigh 0.2 - 1.0 g of different batches of Qinghouyan Buccal Tablets, place them in 50 mL volumetric flasks respectively, add an appropriate amount of methanol aqueous solution with a volume percentage of 30 - 90%, ultrasonically treat at room temperature for 30 - 60 min; Add a methanol aqueous solution with a volume percentage of 30 - 90% to prepare a reference substance mixed solution containing 20 μg of forsythoside A, 150 μg of baicalin, 3 μg of phillyrin, 0.7 μg of cinnamic acid, 10 μg of baicalein, and 4 μg of wogonin per 1 mL. Most preferably, accurately weigh 0.5 g of different batches of Qinghouyan Buccal Tablets and select a methanol aqueous solution with a volume percentage of 50%.
[0010] Preferably, in step (2), the HPLC chromatographic conditions are as follows: an Agilent ZORBAX SB-C18 chromatographic column with a column specification of 250 mm × 4.6 mm and 5 μm is used; the column temperature is 25°C; the detection wavelength is 214 nm; acetonitrile is used as mobile phase A, and a 0.01 mol / L potassium dihydrogen phosphate solution with a pH of 3.3 is used as mobile phase B for gradient elution. In the prior art, gradient elution is mostly carried out using acetonitrile-0.1% phosphoric acid, acetonitrile-3% acetic acid, acetonitrile-0.01 mol / L potassium dihydrogen phosphate, etc. for the separation and content determination of single herbs in Qinghouyan lozenges. The inventor found through investigating the elution effects of these three systems that in the chromatograms eluted by the acetonitrile-0.2% phosphoric acid and acetonitrile-3% acetic acid systems, the number of chromatographic peaks is small and not sufficient to comprehensively represent the material basis of Qinghouyan lozenges. However, when using acetonitrile-0.01 mol / L potassium dihydrogen phosphate as the elution solvent, the chromatographic peaks obtained under this condition are significantly better than those of other elution systems.
[0011] Preferably, the gradient elution program is as follows: from 0 to 10 min, the ratio of mobile phase A to mobile phase B is 15%:85% → 15%:85%; from 10 to 25 min, the ratio of mobile phase A to mobile phase B is 15%:85% → 30%:70%; from 25 to 50 min, the ratio of mobile phase A to mobile phase B is 30%:70% → 80%:20%.
[0012] Preferably, in step (2), the 28 common chromatographic peaks are: peak 1 with a relative retention time of 0.146, peak 2 with a relative retention time of 0.170, peak 3 with a relative retention time of 0.241, peak 4 with a relative retention time of 0.418, peak 5 with a relative retention time of 0.668, peak 6 with a relative retention time of 0.716, peak 7 with a relative retention time of 0.744, peak 8 with a relative retention time of 757, peak 9 with a relative retention time of 0.798, peak 10 with a relative retention time of 0.835, peak 11 with a relative retention time of 0.931, peak 12 with a relative retention time of 0.970, peak 13 with a relative retention time of 1.000, peak 14 with a relative retention time of 1.075, peak 15 with a relative retention time of 1.094, peak 16 with a relative retention time of 1.126, peak 17 with a relative retention time of 1.141, peak 18 with a relative retention time of 1.160, peak 19 with a relative retention time of 1.180, peak 20 with a relative retention time of 1.200, peak 21 with a relative retention time of 1.283, peak 22 with a relative retention time of 1.301, peak 23 with a relative retention time of 1.416, peak 24 with a relative retention time of 1.431, peak 25 with a relative retention time of 1.511, peak 26 with a relative retention time of 1.629, peak 27 with a relative retention time of 1.649, and peak 28 with a relative retention time of 1.670.
[0013] A fingerprint of Qinghouyan Buccal Tablets obtained by the above method for establishing the HPLC fingerprint of Qinghouyan Buccal Tablets.
[0014] Therefore, the present invention has the following beneficial effects: It provides a method for establishing the fingerprint of Qinghouyan Buccal Tablets, and qualitatively and quantitatively analyzes multiple effective components of multiple medicinal materials, which can comprehensively reflect the quality information of Qinghouyan Buccal Tablets, ensure the uniform and stable quality of the product. According to the change trend and degree of the relative peak area of the reference substance, determine the key process control links, formulate corresponding key point quality control standards, which can be used for the production process monitoring of Qinghouyan Buccal Tablets, provide a certain theoretical basis and support for the establishment of the whole-process quality control system, and ensure the safety and effectiveness of clinical medication. Description of the Drawings
[0015] Figure 1 It is the superimposed HPLC fingerprint of the test solution in Example 1.
[0016] Figure 2 It is the HPLC fingerprint of the reference substance mixed solution in Example 1.
[0017] Figure 3 It is the HPLC fingerprint of the Qinghouyan Buccal Tablets obtained in Example 1.
[0018] Figure 4 It is the superimposed HPLC fingerprint of the blank excipient sample solution, Qinghouyan Buccal Tablets solution, each reference substance solution and the mixed reference substance solution in the specificity test.
[0019] Figure 5 It is the superimposed HPLC fingerprint of 6 test samples of Qinghouyan Buccal Tablets in the repeatability test.
[0020] Figure 6 It is the superimposed HPLC fingerprint of 6 test samples of Qinghouyan Buccal Tablets in the intermediate precision test.
[0021] Figure 7 It is the superimposed HPLC fingerprint of 7 test samples of Qinghouyan Buccal Tablets in the stability test.
[0022] Figure 8 It is the superimposed HPLC chromatogram of the extract of each single herb.
[0023] Figure 9 It is the HPLC chromatogram of the test solution of Qinghouyan Buccal Tablets and each negative sample solution.
[0024] Figure 10 It is the comparison chart of the HPLC fingerprint of the Qinghouyan Buccal Tablets obtained in Comparative Example 1 and Comparative Example 2 and the HPLC fingerprint of the Qinghouyan Buccal Tablets obtained in Example 1.
[0025] Figure 11 It is the comparison chart of the HPLC fingerprint of the Qinghouyan Buccal Tablets obtained in Comparative Example 3 and Comparative Example 4 and the HPLC fingerprint of the Qinghouyan Buccal Tablets obtained in Example 1.
[0026] Figure 12 It is the comparison chart of the HPLC fingerprint of the Qinghouyan Buccal Tablets obtained in Comparative Example 5 and Comparative Example 6 and the HPLC fingerprint of the Qinghouyan Buccal Tablets obtained in Example 1. Detailed implementation manners
[0027] The present invention will be further described below in conjunction with the accompanying drawings and detailed implementation manners.
[0028] The instruments and reagents used in the following examples are as follows: Instruments: Electronic balance (XSE205DU, Mettler-Toledo); pH meter (SevenCompact S210, Mettler-Toledo); High performance liquid chromatograph (Agilent 1260, Agilent Technologies, DAD detector); Ultrasonic cleaner (YM-1001LUS, Yumeng).
[0029] Test drugs: forsythoside A (batch number: 110810 - 202209, content 96.4%, National Institutes for Food and Drug Control); baicalin (batch number: 110715 - 202223, content 97.2%, National Institutes for Food and Drug Control); phillyrin (batch number: 110821 - 202318, content 95.8%, National Institutes for Food and Drug Control); cinnamic acid (batch number: 110786 - 202305, content 99.8%, National Institutes for Food and Drug Control); baicalein (batch number: 111595 - 202309, content 98.6%, National Institutes for Food and Drug Control); wogonin (batch number: H - 220300NU1, content 97.71%, CFW); Qinghouyan Buccal Tablets (batch numbers: 230201, 230202, 230203, 231001, 231002, 231003, Zhejiang Conba Pharmaceutical Co., Ltd.). Reagents: chromatographic methanol and acetonitrile were purchased from Merck, USA; phosphoric acid was purchased from Scharlur; potassium dihydrogen phosphate was purchased from Sinopharm Chemical Reagent Co., Ltd.; the water used was purified water (self - made).
[0030] Example 1 (1) Preparation of test solution and reference substance mixed solution Preparation of test solution: Accurately weigh 0.5 g of 6 batches of Qinghouyan Buccal Tablets respectively, place them in 50 - mL volumetric flasks, add an appropriate amount of 50% (v / v) methanol - water solution, ultrasonically treat (frequency 45 kHz) at room temperature for 30 min, then cool to room temperature, make up the volume, shake well, and filter through a 0.45 - μm organic microporous membrane. Preparation of reference substance mixed solution: Accurately take appropriate amounts of reference substances of forsythoside A, baicalin, phillyrin, cinnamic acid, baicalein, and wogonin, accurately weigh them, and add a 50% (v / v) methanol - water solution to prepare a reference substance mixed solution containing 20 μg of forsythoside A, 150 μg of baicalin, 3 μg of phillyrin, 0.7 μg of cinnamic acid, 10 μg of baicalein, and 4 μg of wogonin per 1 mL.
[0031] (2) Establishment of fingerprint Use HPLC to inject and analyze the test solution and the reference substance mixed solution respectively to obtain the superimposed HPLC fingerprint of the test solution (as shown in Figure 1 ) and the HPLC fingerprint of the reference substance mixed solution (as shown in Figure 2As shown in [figure], and based on the superimposed HPLC fingerprint of the test solution, 28 common chromatographic peaks were confirmed; the fingerprint data of the test solution were imported into the Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicines (2012 version). Using the fingerprint of the test solution with batch number 230201 as the reference fingerprint, the time window width was set to 0.1 min. After multi-point calibration and data matching, the HPLC fingerprint of Qinghouyan Buccal Tablets was generated by the mean method (as shown in Figure 3As shown in the figure); the HPLC fingerprint of the reference substance mixed solution was compared with the HPLC fingerprint of Qinghouyan Buccal Tablets. Among them, 6 common chromatographic peaks were identified, and it was confirmed that peak 8 was forsythoside A, peak 13 was baicalin, peak 16 was phillyrin, peak 22 was cinnamic acid, peak 24 was baicalein, and peak 26 was wogonin. The baicalin peak was used as the reference peak for similarity analysis; the HPLC chromatographic conditions were as follows: an Agilent ZORBAX SB-C18 chromatographic column was used, with a column specification of 250 mm × 4.6 mm and 5 μm; the column temperature was 25 °C; the detection wavelength was 214 nm; acetonitrile was used as mobile phase A, and a 0.01 mol / L potassium dihydrogen phosphate solution with pH 3.3 was used as mobile phase B for gradient elution. The gradient elution program was as follows: 0 - 10 min, mobile phase A:mobile phase B was 15%:85% → 15%:85%; 10 - 25 min, mobile phase A:mobile phase B was 15%:85% → 30%:70%; 25 - 50 min, mobile phase A:mobile phase B was 30%:70% → 80%:20%; the 28 common chromatographic peaks were respectively: peak 1 with a relative retention time of 0.146, peak 2 with a relative retention time of 0.170, peak 3 with a relative retention time of 0.241, peak 4 with a relative retention time of 0.418, peak 5 with a relative retention time of 0.668, peak 6 with a relative retention time of 0.716, peak 7 with a relative retention time of 0.744, peak 8 with a relative retention time of 757, peak 9 with a relative retention time of 0.798, peak 10 with a relative retention time of 0.835, peak 11 with a relative retention time of 0.931, peak 12 with a relative retention time of 0.970, peak 13 with a relative retention time of 1.000, peak 14 with a relative retention time of 1.075, peak 15 with a relative retention time of 1.094, peak 16 with a relative retention time of 1.126, peak 17 with a relative retention time of 1.141, peak 18 with a relative retention time of 1.160, peak 19 with a relative retention time of 1.180, peak 20 with a relative retention time of 1.200, peak 21 with a relative retention time of 1.283, peak 22 with a relative retention time of 1.301, peak 23 with a relative retention time of 1.416, peak 24 with a relative retention time of 1.431, peak 25 with a relative retention time of 1.511, peak 26 with a relative retention time of 1.629, peak 27 with a relative retention time of 1.649, and peak 28 with a relative retention time of 1.670.
[0032] It can be seen that the resolution of peak 13 (baicalin peak) is good, the peak area is large, and the retention time is moderate. Therefore, the baicalin peak was selected as the reference peak, and the similarity analysis was carried out on the HPLC fingerprints of 6 batches of Qinghouyan Buccal Tablets and the HPLC fingerprint of Qinghouyan Buccal Tablets.
[0033] 1. Methodology investigation of fingerprint 1.1 Specificity test Take the blank excipient sample of Qinghouyan Buccal Tablets and Qinghouyan Buccal Tablets (batch number 230202), and prepare the blank excipient sample solution and Qinghouyan Buccal Tablets solution of Qinghouyan Buccal Tablets according to the preparation method of the test solution in Example 1; prepare each reference substance solution and reference substance mixed solution according to the preparation method of the reference substance mixed solution in Example 1, and inject samples respectively according to the chromatographic conditions in Example 1 to obtain the superimposed HPLC fingerprints of the blank excipient sample solution of Qinghouyan Buccal Tablets, Qinghouyan Buccal Tablets solution, each reference substance solution and reference substance mixed solution (as Figure 4 shown). The results show that the blank excipient sample solution of Qinghouyan Buccal Tablets has no interference on the fingerprint detection of Qinghouyan Buccal Tablets, and the method is feasible.
[0034] By locating with each reference substance solution, it is confirmed that the peak at 18.615 min is forsythoside A (peak No. 8), the peak at 4.578 min is baicalin (peak No. 13), the peak at 27.670 min is phillyrin (peak No. 16), the peak at 31.971 min is cinnamic acid (peak No. 22), the peak at 35.174 min is baicalein (peak No. 24), and the peak at 40.034 min is wogonin (peak No. 26). Taking baicalin as the reference peak (S), the relative retention times are 0.757, 1.000, 1.126, 1.301, 1.431, 1.629 respectively. A total of 28 common peaks are determined in the HPLC fingerprint with baicalin as the reference peak.
[0035] 1.2 Repeatability test Take the same batch of Qinghouyan Buccal Tablets (batch number 230202), parallel 6 portions, numbered S2 - S7, and prepare 6 parallel test solutions of Qinghouyan Buccal Tablets according to the preparation method of the test solution in Example 1. Inject samples respectively according to the chromatographic conditions in Example 1 to obtain 6 HPLC fingerprints of Qinghouyan Buccal Tablets test solutions (as Figure 5 shown). Import the HPLC fingerprint data of the 6 Qinghouyan Buccal Tablets test solutions into the Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints (2012 version) for similarity analysis with the HPLC fingerprint of Qinghouyan Buccal Tablets. The analysis results are shown in Table 1.
[0036] Table 1 Similarity of fingerprints in the repeatability test of Qinghouyan Buccal Tablets It can be seen from Table 1 that the RSD of the similarity of the fingerprints of the 6 Qinghouyan Buccal Tablets solutions is 0, indicating that the method has good repeatability.
[0037] 1.3 Intermediate precision test At different times, with different instruments, and by different analysts, Qinghouyan Buccal Tablets (batch number 230202) were taken, and 6 samples of Qinghouyan Buccal Tablets test solutions were prepared in parallel according to the preparation method of the test solution in Example 1, and were separately injected and determined according to the chromatographic conditions in Example 1, obtaining 6 HPLC fingerprint spectra of Qinghouyan Buccal Tablets test samples (as Figure 6 shown). The data of the 6 HPLC fingerprint spectra of Qinghouyan Buccal Tablets test samples were imported into the Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints (2012 version) for similarity analysis with the HPLC fingerprint spectrum of Qinghouyan Buccal Tablets, and the RSD was calculated together with the 6 parallel Qinghouyan Buccal Tablet solutions in the repeatability test. The calculation results are shown in Table 2.
[0038] Table 2 Fingerprint spectrum similarity of intermediate precision test for Qinghouyan Buccal Tablets As can be seen from Table 2, the RSD of the fingerprint spectrum similarity of 12 Qinghouyan Buccal Tablet solutions is 0, indicating that the precision of this method is good.
[0039] 1.4 Stability test Take the same batch of Qinghouyan Buccal Tablets (batch number 230202), prepare the Qinghouyan Buccal Tablet solution according to the preparation method of the test solution in Example 1, and inject and analyze according to the chromatographic conditions in Example 1. The injection times are 0 hour, 4 hours, 8 hours, 12 hours, 24 hours, 48 hours, and 72 hours respectively, obtaining 7 HPLC fingerprint spectra of Qinghouyan Buccal Tablets test samples (as Figure 7 shown). The data of the 7 HPLC fingerprint spectra of Qinghouyan Buccal Tablets test samples were imported into the Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints (2012 version) for similarity analysis with the HPLC fingerprint spectrum of Qinghouyan Buccal Tablets. The analysis results are shown in Table 3.
[0040] Table 3 Fingerprint spectrum similarity of stability test for Qinghouyan Buccal Tablet solution According to Table 3, the RSD of the fingerprint spectrum similarity of 7 Qinghouyan Buccal Tablets test samples is 0, indicating that the solution has good stability within 72 hours.
[0041] 1.5 Analysis of the correlation between the finished product of Qinghouyan Buccal Tablets and each single herb and the attribution of common peaks Prepared according to the process of Qinghouyan Buccal Tablets, the extraction solutions of each single herb of Scutellaria baicalensis, Scrophularia ningpoensis, Forsythia suspensa, Rehmannia glutinosa, and Ophiopogon japonicus in Qinghouyan Buccal Tablets were separately extracted. The extraction solutions of each single herb were separately injected and analyzed by HPLC (the chromatographic conditions are the same as in Example 1), obtaining the HPLC chromatograms of the extraction solutions of each single herb (as Figure 8As shown in the figure); the HPLC fingerprint of Qinghouyan Buccal Tablets and the HPLC chromatograms of the extracts of each single herb were compared and analyzed by a DAD detector, and the retention times of the chromatographic peaks were compared. Finally, the attribution peaks of the common chromatographic peaks in the fingerprint of Qinghouyan Buccal Tablets on the herb chromatograms were confirmed. The results are shown in Table 4.
[0042] Table 4 Analysis of the Attribution of the Common Peaks in the Fingerprint of the Finished Product of Qinghouyan Buccal Tablets From the analysis of the above results, it can be seen that among the 28 common peaks in the fingerprint of Qinghouyan Buccal Tablets, peaks 6, 7, 9, 10, 11, 13, 14, 15, 17, 18, 19, 20, 21, 23, 24, 26, 27, and 28 come from Scutellaria baicalensis Georgi herbs; peaks 12, 20, and 22 come from Scrophularia ningpoensis Hemsl. herbs; peaks 1, 2, 5, 6, 7, 8, 10, 11, 14, 16, 21, and 25 come from Forsythia suspensa (Thunb.) Vahl herbs; peaks 2, 3, 9, 10, and 18 come from Rehmannia glutinosa Libosch. herbs; peaks 2 and 3 come from Ophiopogon japonicus (Thunb.) Ker-Gawl. herbs; peak 4 has no herb attribution, and it is possible that new components were generated during the decocting process. Thus, it can be seen that the fingerprint of Qinghouyan Buccal Tablets can basically represent the material basis of Qinghouyan Buccal Tablets.
[0043] 2. Methodological Investigation on the Determination of 6 Active Ingredients in Qinghouyan Buccal Tablets 2.1 Specificity Test Negative control samples lacking Scutellaria baicalensis Georgi, Forsythia suspensa (Thunb.) Vahl, and Scrophularia ningpoensis Hemsl. were prepared according to the prescription and operated according to the steps of "1.1 Specificity Test" to obtain the HPLC chromatograms of the test solution of Qinghouyan Buccal Tablets and each negative sample solution (as Figure 9 shown), and it can be seen that each negative sample has no interference in the determination of the contents of forsythoside A, baicalin, phillyrin, cinnamic acid, baicalein, and wogonin.
[0044] 2.2 Linearity and Range Appropriately weighed reference substances of forsythoside A, baicalin, phillyrin, cinnamic acid, baicalein, and wogonin were precisely weighed and diluted to different mass concentrations, and determined according to the chromatographic conditions in Example 1. Linear regression was performed with the reference substance mass concentration as the abscissa (X) and the peak area as the ordinate (Y). The results are shown in Table 5.
[0045] Table 5 Results of Linearity and Range According to Table 5, it can be seen that the linear relationships of each component are good within their respective linear ranges.
[0046] 2.3 Accuracy Test Take 0.25 g of Qinghouyan Buccal Tablets (batch number 230202) respectively, weigh accurately, place in a 50 mL volumetric flask, add appropriate amounts of forsythoside A, baicalin, phillyrin, cinnamic acid, baicalein, and wogonin reference substance solutions respectively, prepare 6 portions in parallel according to the preparation method of the test solution in Example 1, and inject samples for determination according to the chromatographic conditions in Example 1 respectively, record the chromatograms, calculate the recovery rate by the external standard method, and the calculation results are shown in Table 6.
[0047] Table 6 Spiked Recovery Rates of Each Component As can be seen from Table 6, the recovery rates of each component are all within 85% - 110% (n = 6), indicating that this method has good accuracy.
[0048] 2.4 Repeatability Test Carry out the repeatability test according to the method of "1.2 Repeatability Test" in Example 1, calculate the content by the external standard method, and the calculation results are shown in Table 7.
[0049] Table 7 Results of Content Determination in Repeatability Test (mg / g) As can be seen from Table 7, the average contents of forsythoside A, baicalin, phillyrin, cinnamic acid, baicalein, and wogonin are 2.006 mg / g, 15.223 mg / g, 0.294 mg / g, 0.066 mg / g, 0.843 mg / g, and 0.418 mg / g respectively, and the RSDs are 0.36%, 0.25%, 2.61%, 2.86%, 0.71%, and 0.40% (n = 6). It shows that this method has good repeatability.
[0050] 2.5 Intermediate Precision Test At different times, with different instruments and different analysts, take Qinghouyan Buccal Tablets (batch number 230202), prepare 6 portions in parallel according to the preparation method of the test solution in Example 1, and inject samples for determination according to the chromatographic conditions in Example 1 respectively, record the chromatograms, and calculate the RSD together with the 6 portions of test solution of Qinghouyan Buccal Tablets in "2.4 Repeatability Test", a total of 12 portions of test solution, and the calculation results are shown in Table 8.
[0051] Table 8 Results of Content Determination in Intermediate Precision Test (μg / g) As can be seen from Table 8, the RSDs of forsythoside A, baicalin, phillyrin, cinnamic acid, baicalein, and wogonin are 0.56%, 1.06%, 2.40%, 5.44%, 0.88%, and 2.66% (n = 12), indicating that this method has good precision.
[0052] 2.6 Solution Stability Test Take Qinghouyan Buccal Tablets (batch number 230202), prepare the test solution of Qinghouyan Buccal Tablets according to the preparation method of the test solution in Example 1. At the same time, prepare the reference substance solutions of forsythoside A (20 μg / mL), baicalin (150 μg / mL), phillyrin (3 μg / mL), cinnamic acid (0.7 μg / mL), baicalein (10 μg / mL), and wogonin (4 μg / mL) respectively. Inject the test solution of Qinghouyan Buccal Tablets and each reference substance solution into the chromatograph at 0 hour, 4 hours, 8 hours, 12 hours, 24 hours, and 48 hours according to the chromatographic conditions in Example 1, record the chromatogram and calculate the peak area. The calculation results are shown in Table 9 and Table 10.
[0053] Table 9 Results of the Stability Test of the Reference Substance Solution Table 10 Results of the Stability Test of the Test Solution of Qinghouyan Buccal Tablets It can be seen from Table 9 that within 36 hours, the RSDs of the peak areas of the main peaks of forsythoside A, baicalin, phillyrin, cinnamic acid, baicalein, and wogonin in the chromatogram of the reference substance solution are 0.55%, 0.39%, 0.78%, 2.45%, 2.16%, and 0.36% respectively, and the RSDs of the peak areas of the main peaks of forsythoside A, baicalin, phillyrin, cinnamic acid, baicalein, and wogonin in the chromatogram of the test solution are 0.84%, 0.16%, 3.42%, 9.35%, 2.39%, and 2.89% respectively, indicating that the reference substance solution and the test solution are stable within 48 hours and both meet the determination requirements.
[0054] 2.7 Determination of the Contents of Index Components Take 6 batches of Qinghouyan Buccal Tablets, prepare the test solution of Qinghouyan Buccal Tablets according to the preparation method of the test solution in Example 1, inject the samples according to the chromatographic conditions in Example 1 respectively, record the peak areas of each index component, and calculate by the external standard method. The content results of each index component in different batches of Qinghouyan Buccal Tablets are shown in Table 11.
[0055] Table 11 Determination Results of the Contents of 6 Components in 6 Batches of Qinghouyan Buccal Tablets (mg / g, n = 2) Batch number Phillyrin A Baicalin Phillyrin Cinnamic acid Baicalein Wogonin 230201 2.037 15.537 0.269 0.063 0.857 0.432 230202 1.989 15.428 0.284 0.066 0.842 0.441 230203 0.637 16.397 0.149 0.054 1.020 0.520 231001 2.404 17.450 0.297 0.062 0.870 0.376 231002 2.196 17.386 0.319 0.063 0.809 0.360 231003 2.005 17.786 0.345 0.061 0.874 0.406 The above test results show that the method for establishing the fingerprint of Qinghouyan Buccal Tablets of the present invention can comprehensively analyze the overall components of Qinghouyan Buccal Tablets by establishing the fingerprint of Qinghouyan Buccal Tablets, can basically represent and reflect the active ingredients and material basis of Qinghouyan Buccal Tablets, has certain discriminative significance for the quality control of different technological processes of the preparation, provides a certain theoretical basis and support for the establishment of the whole-process quality control system, can comprehensively reflect the quality information of Qinghouyan Buccal Tablets, and ensure the uniform and stable quality of the products.
[0056] Comparative Example 1 Compared with Example 1, the difference in Comparative Example 1 is that the detection wavelength in the HPLC chromatographic conditions is 200 nm, and the rest is exactly the same as in Example 1.
[0057] Comparative Example 2 Compared with Example 1, the difference in Comparative Example 2 is that the detection wavelength in the HPLC chromatographic conditions is 270 nm, and the rest is exactly the same as in Example 1.
[0058] The comparison diagrams of the HPLC fingerprints of the Qinghouyan Buccal Tablets obtained in Comparative Example 1 and Comparative Example 2 with the HPLC fingerprint of the Qinghouyan Buccal Tablets obtained in Example 1 are as Figure 10 shown. It can be seen from this that in the HPLC fingerprint of the Qinghouyan Buccal Tablets obtained in Comparative Example 1, the number of chromatographic peaks is significantly less than that in Example 1; while in the HPLC fingerprint of the Qinghouyan Buccal Tablets obtained in Comparative Example 2, although the number of chromatographic peaks has no obvious difference from that in Example 1, the baseline fluctuates greatly.
[0059] Comparative Example 3 Compared with Example 1, the difference in Comparative Example 3 is that in the HPLC chromatographic conditions, acetonitrile is used as mobile phase A, and pH 3.3, 0.1% phosphoric acid is used as mobile phase B for gradient elution, and the rest is exactly the same as in Example 1.
[0060] Comparative Example 4 Compared with Example 1, the difference in Comparative Example 4 is that in the HPLC chromatographic conditions, acetonitrile is used as mobile phase A, and pH 3.3, 3% glacial acetic acid solution is used as mobile phase B for gradient elution, and the rest is exactly the same as in Example 1.
[0061] The comparison diagrams of the HPLC fingerprints of the Qinghouyan Buccal Tablets obtained in Comparative Example 3 and Comparative Example 4 with the HPLC fingerprint of the Qinghouyan Buccal Tablets obtained in Example 1 are as Figure 11 shown. It can be seen from this that in the HPLC fingerprint of the Qinghouyan Buccal Tablets obtained in Comparative Example 3, the front edge of the phillyrin peak is wrapped with other component peaks and is not completely separated, affecting the quantitative analysis of phillyrin; while no peak appears in the HPLC fingerprint of the Qinghouyan Buccal Tablets obtained in Comparative Example 4; in the HPLC fingerprint of the Qinghouyan Buccal Tablets obtained in Example 1, the resolution and peak shape of each characteristic peak are good.
[0062] Comparative Example 5 Comparative Example 5 is different from Example 1 in that in the HPLC chromatographic conditions, the pH of mobile phase B is 3.5, and the rest are exactly the same as those in Example 1.
[0063] Comparative Example 6 Comparative Example 6 is different from Example 1 in that in the HPLC chromatographic conditions, the pH of mobile phase B is 3.0, and the rest are exactly the same as those in Example 1.
[0064] The comparison charts of the HPLC fingerprint spectra of the Qinghouyan buccal tablets obtained in Comparative Example 5 and Comparative Example 6 with the HPLC fingerprint spectrum of the Qinghouyan buccal tablet obtained in Example 1 are as Figure 12 shown. It can be seen therefrom that in the HPLC fingerprint spectrum of the Qinghouyan buccal tablet obtained in Comparative Example 5, the resolution between phillyrin and the adjacent peak is not completely separated; in the HPLC fingerprint spectrum of the Qinghouyan buccal tablet obtained in Comparative Example 6, the forsythoside A peak and the adjacent peak are not completely separated, and the phillyrin peak overlaps with the adjacent peak; in the HPLC fingerprint spectrum of the Qinghouyan buccal tablet obtained in Example 1, the characteristic peaks are basically separated.
[0065] The above-described embodiments are only a preferred solution of the present invention, and do not impose any form of limitation on the present invention. There are other variants and modifications without exceeding the technical solutions recorded in the claims.
Claims
1. Method for establishing HPLC fingerprint of Qinghouyan Buccal Tablets, characterized in that It includes the following steps: (1) Prepare the test solution and the reference substance mixed solution Prepare the test solution: Accurately weigh different batches of Qinghouyan Buccal Tablets, place them in volumetric flasks respectively, add an appropriate amount of methanol aqueous solution, ultrasonically treat at room temperature and then cool to room temperature, make up the volume, shake well, and filter; Prepare the reference substance mixed solution: Accurately take appropriate amounts of forsythoside A, baicalin, phillyrin, cinnamic acid, baicalein, and wogonin reference substances, accurately weigh them, and prepare a reference substance mixed solution with methanol aqueous solution; (2) Establish the fingerprint Inject the test solution and the reference substance mixed solution for analysis by HPLC respectively to obtain the superimposed HPLC fingerprint of the test solution and the HPLC fingerprint of the reference substance mixed solution, and confirm 28 common chromatographic peaks according to the superimposed HPLC fingerprint of the test solution; Import the fingerprint data of the test solution into the Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints (2012 Edition), take the fingerprint of one of the test solutions as the reference fingerprint, set the time window width to 0.1 min, perform multi-point calibration and data matching, and generate the HPLC fingerprint of Qinghouyan Buccal Tablets by the mean method; Compare the HPLC fingerprint of the reference substance mixed solution with the HPLC fingerprint of Qinghouyan Buccal Tablets, and 6 common chromatographic peaks are identified, confirming forsythoside A at peak No. 8, baicalin at peak No. 13, phillyrin at peak No. 16, cinnamic acid at peak No. 22, baicalein at peak No. 24, and wogonin at peak No.
26. Take the baicalin peak as the reference peak for similarity analysis.
2. The method for establishing the HPLC fingerprint of the Qinghouyan Buccal Tablets according to claim 1, wherein In step (1), accurately weigh 0.2 - 1.0 g of different batches of Qinghouyan Buccal Tablets, place them in 50 mL volumetric flasks respectively, add an appropriate amount of methanol aqueous solution with a volume percentage of 30 - 90%, ultrasonically treat at room temperature for 30 - 60 min; Add methanol aqueous solution with a volume percentage of 30 - 90% to prepare a reference substance mixed solution containing 20 μg of forsythoside A, 150 μg of baicalin, 3 μg of phillyrin, 0.7 μg of cinnamic acid, 10 μg of baicalein, and 4 μg of wogonin per 1 mL.
3. The method for establishing the HPLC fingerprint of the Qinghouyan Buccal Tablets according to claim 1, wherein, In step (2), the HPLC chromatographic conditions are as follows: The chromatographic column uses an Agilent ZORBAX SB-C18 chromatographic column with a column specification of 250 mm × 4.6 mm, 5 μm; The column temperature is 25°C; The detection wavelength is 214 nm; Acetonitrile is used as mobile phase A, and a 0.01 mol / L potassium dihydrogen phosphate solution with pH 3.3 is used as mobile phase B for gradient elution.
4. The method for establishing the HPLC fingerprint of the Qinghouyan Buccal Tablets according to claim 3, characterized in that, The gradient elution program is as follows: 0 - 10 min, mobile phase A:mobile phase B is 15%:85% → 15%:85%; 10 - 25 min, mobile phase A:mobile phase B is 15%:85% → 30%:70%; 25 - 50 min, mobile phase A:mobile phase B is 30%:70% → 80%:20%.
5. The method for establishing the HPLC fingerprint of Qinghouyan Buccal Tablets according to claim 1, characterized in that, In step (2), the 28 common chromatographic peaks are respectively: peak 1 with a relative retention time of 0.146, peak 2 with a relative retention time of 0.170, peak 3 with a relative retention time of 0.241, peak 4 with a relative retention time of 0.418, peak 5 with a relative retention time of 0.668, peak 6 with a relative retention time of 0.716, peak 7 with a relative retention time of 0.744, peak 8 with a relative retention time of 757, peak 9 with a relative retention time of 0.798, peak 10 with a relative retention time of 0.835, peak 11 with a relative retention time of 0.931, peak 12 with a relative retention time of 0.970, peak 13 with a relative retention time of 1.000, peak 14 with a relative retention time of 1.075, peak 15 with a relative retention time of 1.094, peak 16 with a relative retention time of 1.126, peak 17 with a relative retention time of 1.141, peak 18 with a relative retention time of 1.160, peak 19 with a relative retention time of 1.180, peak 20 with a relative retention time of 1.200, peak 21 with a relative retention time of 1.283, peak 22 with a relative retention time of 1.301, peak 23 with a relative retention time of 1.416, peak 24 with a relative retention time of 1.431, peak 25 with a relative retention time of 1.511, peak 26 with a relative retention time of 1.629, peak 27 with a relative retention time of 1.649, and peak 28 with a relative retention time of 1.
670.
6. A fingerprint of Qinghouyan Buccal Tablets obtained by the method for establishing the HPLC fingerprint of Qinghouyan Buccal Tablets described in claim 1.