Method for establishing fingerprint spectrum of asthma-Tai granules by utilizing high performance liquid chromatography and application

Abstract

The invention discloses a method for establishing a fingerprint spectrum of asthma-Tai granules by utilizing high performance liquid chromatography and application, and belongs to the technical field of quality control and management of compound traditional Chinese medicine preparations. The method comprises the following steps: preparing a test solution and a reference solution, carrying out chromatographic analysis through a high performance liquid chromatograph, recording a chromatogram, and carrying out similarity analysis and high-resolution mass spectrometry to finally obtain the fingerprint spectrum of the asthma-Thai granules, the fingerprint spectrum contains 10 common peaks, attribution analysis is carried out, and the source of the asthma-Thai granules is clarified. The method is simple and practicable, has good reproducibility, is accurate and reliable, can confirm multiple components at one time, and improves the detection efficiency. The obtained fingerprint spectrum of the asthma-Thai granules can be used for simultaneously determining 10 components in the asthma-Thai granules, a rapid and efficient technical method is provided for quality control of the asthma-Thai granules, and the method can be used for accurately, clearly and objectively evaluating the quality of the asthma-Thai granules.

Description

Technical Field

[0001] The invention belongs to the technical field of quality control management of compound traditional Chinese medicine preparations, and specifically relates to a method for establishing a fingerprint spectrum of Chuantai granules by utilizing high performance liquid chromatography technology and its application. Background Art

[0002] Chuantai Granule (National Medicine Standard No.: Z20020081) is a traditional Chinese medicine compound preparation that combines the essence of multiple Chinese medicinal materials and shows significant efficacy in the treatment of respiratory diseases. Chuantai Granule is composed of 8 Chinese medicinal materials, including ephedra, scutellaria, bitter almond, coltsfoot, pinellia, wolfberry, cinnamon and licorice. Each medicinal material plays an indispensable role and together constitutes the unique pharmacodynamic system of Chuantai Granule. Ephedra is the main drug to dissipate the cold on the surface, promote lung function and relieve asthma; scutellaria, bitter almond and pinellia are the assistant drugs to clear the heat inside, dissipate the cold on the surface, regulate the spleen and stomach function and enhance the body's disease resistance; while coltsfoot, wolfberry, cinnamon and licorice are the adjuvant drugs to invigorate the kidney, absorb qi, moisten the lung and resolve phlegm, further enhancing the therapeutic effect of Chuantai Granule. For complex diseases such as asthma, chronic bronchitis, and allergic cough, the pathological mechanisms involve multiple physiological processes, including abnormal reactions of the immune system, inflammatory reactions of the respiratory tract, and hyperresponsiveness of the airways. Through multi-target regulation, Chuantai Granules can exert a synergistic therapeutic effect on these pathological mechanisms, thereby achieving the effect of comprehensive treatment.

[0003] However, the efficacy of Chuantai Granules does not come from a single ingredient, but is the result of the interaction and joint action of multiple active ingredients. This complexity makes it difficult for traditional quality control methods, such as single ingredient detection methods, to fully and accurately reflect its overall quality and efficacy. Because many minor ingredients in Chuantai Granules, although they may not be as significant as the main ingredients in content, may play a vital role in the treatment process. These minor ingredients may synergize with the main ingredients to enhance the efficacy of the drug; or by regulating the body's physiological environment, provide favorable conditions for the main ingredients to exert their efficacy. Therefore, ignoring these minor ingredients may lead to deviations in the evaluation of the overall effect of Chuantai Granules.

[0004] With the widespread application of high performance liquid chromatography (HPLC) technology, new ideas and means have been provided for the quality control of traditional Chinese medicine compound preparations. HPLC technology has the advantages of high separation efficiency, fast analysis speed, and high sensitivity, and can achieve the separation and detection of multiple components in complex systems. Therefore, the use of HPLC technology to establish the fingerprint of Chuantai Granules has become a method for accurately, clearly and objectively evaluating the quality of Chuantai Granules. Fingerprint is a chromatogram or spectrum that can mark the chemical characteristics of a substance, with high individual specificity and environmental stability. By comparing the fingerprints of Chuantai Granules from different batches or different sources, the content changes of each component in Chuantai Granules, the interaction relationship between the components, and the overall quality stability can be intuitively understood. This is of great significance and value for controlling the quality of Chuantai Granules and ensuring clinical efficacy. However, there are currently no literature or patent reports on the construction of HPLC fingerprints of Chuantai Granules. Summary of the invention

[0005] In view of the problem that traditional quality control methods are difficult to comprehensively evaluate the overall quality and efficacy of the complex multi-component system of Chuantai Granules, and the existing patents and literature have not reported the technical status of using HPLC to construct a fingerprint of Chuantai Granules, the present invention aims to provide a method and application of establishing a fingerprint of Chuantai Granules using high performance liquid chromatography, through the efficient separation and detection of HPLC, a fingerprint of Chuantai Granules with individual specificity and stability is established, which intuitively reflects the content of each component in Chuantai Granules and the interaction relationship, and provides an accurate, clear and objective evaluation method for controlling its quality and ensuring clinical efficacy.

[0006] The present invention is achieved through the following technical solutions: The present invention provides a method for establishing a fingerprint spectrum of Chuantai granules by using high performance liquid chromatography, comprising the following steps: S1, using different batches of Chuantai granules as test samples to prepare Chuantai granules test sample solutions; S2, prepare a single reference solution with uridine, gallic acid, chlorogenic acid, vitexin, liquiritin, hyperoside, baicalin, melaleuca A glycoside, cinnamic acid and glycyrrhizic acid reference substances; S3, injecting the Chuantai granules test solution and the single reference solution into a high performance liquid chromatograph, respectively, performing chromatographic analysis under the same conditions, and recording the corresponding chromatograms; In S3, the HPLC determination conditions are: Shim-pack VP-ODS chromatographic column; the mobile phase is acetonitrile-0.1% formic acid aqueous solution, and the gradient elution program is as follows: S4, importing the chromatogram of the test solution of Chuantai Granules and the chromatogram of the single reference solution obtained in S3 into the Chinese medicine chromatographic fingerprint similarity evaluation system and performing similarity analysis to confirm the reliability of the results; S5, performing high-resolution mass spectrometry analysis based on the chromatogram of the Chuantai granules test sample solution obtained in S3 to obtain a total ion current diagram; performing data analysis based on the total ion current diagram and the peak conditions of the test sample chromatogram obtained in S3 to obtain a mass spectrometry result diagram of each chemical component; S6, comparing the chromatogram of the test solution of Chuantai Granules and the chromatogram of the single reference solution obtained in S3, the total ion flow diagram obtained in S5, and the mass spectra of each chemical component, to obtain a fingerprint spectrum of Chuantai Granules composed of common characteristic peaks.

[0008] In S1, the contents of different batches of Chuantai granules were taken, methanol solution was added, ultrasonic extraction was performed, filtration was performed, and microporous membrane filtration was performed to obtain Chuantai granule test solution.

[0009] Furthermore, each 20 mL of methanol solution contained 2.0 g of the contents of the Chuantai granules, which were extracted by ultrasonic, filtered, and filtered through a microporous membrane to obtain a Chuantai granule test solution.

[0010] Furthermore, the volume concentration of the methanol solution is 80%.

[0011] Furthermore, ultrasonic extraction was performed for 30 min.

[0012] Furthermore, the solution was filtered twice through a 0.45 µm microfiltration membrane.

[0013] In S2, uridine, gallic acid, chlorogenic acid, vitexin, liquiritin, hyperoside, baicalin, melaleuca alternifolia glycoside A, cinnamic acid and glycyrrhizic acid reference substances were dissolved in methanol solution to prepare a single reference substance solution.

[0014] Furthermore, in S2, the uridine single reference solution contained 0.69 mg of uridine per 1 mL of methanol solution; the gallic acid single reference solution contained 0.62 mg of gallic acid per 1 mL of methanol solution; the chlorogenic acid single reference solution contained 0.63 mg of chlorogenic acid per 1 mL of methanol solution; the vitexin single reference solution contained 0.56 mg of vitexin per 1 mL of methanol solution; the liquiritin single reference solution contained 0.64 mg of liquiritin per 1 mL of methanol solution; the hyperoside single reference solution contained 0.67 mg of hyperoside per 1 mL of methanol solution; the baicalin single reference solution contained 0.89 mg of baicalin per 1 mL of methanol solution; the melaleuca A glycoside single reference solution contained 0.56 mg of melaleuca A glycoside per 1 mL of methanol solution; the cinnamic acid single reference solution contained 0.76 mg of cinnamic acid per 1 mL of methanol solution. mg; in the glycyrrhizic acid single reference solution, each 1 mL of methanol solution contains 0.75 mg of glycyrrhizic acid.

[0015] The HPLC detection wavelength is 210-290 nm, the column temperature is 25-35° C., and the flow rate is 0.6-1.0 mL / min.

[0016] Furthermore, the HPLC detection wavelength was 275 nm, the column temperature was 30°C, the flow rate was 0.6 mL / min, and the injection volume was 20 μL.

[0017] S4 specifically includes: importing the chromatograms of the test solution and reference solution of the Chuantai Granules obtained in S3 into the Chinese medicine chromatographic fingerprint similarity evaluation system; selecting the chromatographic peaks existing in the chromatograms of the test solution of the Chuantai Granules from different batches as common peaks, generating the reference spectrum of the Chuantai Granules by the average value calculation method, and calculating the relative retention time and relative peak area of ​​each common peak; performing similarity analysis after data import, multi-point correction and data matching; obtaining and exporting the similarity result table between the chromatograms of the test solution of the Chuantai Granules from different batches and the common peak pattern; confirming the reliability of the results based on the similarity result table and the chromatogram of the test solution of the Chuantai Granules.

[0018] In S5, the high-resolution mass spectrometry detection conditions are: electrospray ionization, spray voltage 3500 V, sheath gas flow rate 40 arb, auxiliary gas flow rate 10 arb, capillary temperature 300°C, auxiliary gas temperature 300°C, scanning mode is full scan mode, and mass-to-charge ratio scanning range m / z is 100-1500.

[0019] S6 is specifically as follows: according to the chromatogram of the test solution and the single reference solution of Chuantai Granules obtained in S3, combined with the total ion flow diagram obtained by high-resolution mass spectrometry in S5 and the mass spectrum result diagram of the chemical components, a comparison is performed, and peak 27 baicalin is used as the reference peak, and peak 2 in the chromatogram is identified as uridine, peak 5 as gallic acid, peak 13 as chlorogenic acid, peak 18 as vitexin, peak 19 as liquiritin, peak 20 as hyperoside, peak 31 as melaleuca alternifolia A glycoside, peak 33 as cinnamic acid, and peak 35 as glycyrrhizic acid, thereby obtaining the fingerprint of Chuantai Granules.

[0020] In the above method of establishing the fingerprint of Chuantai Granules by using high performance liquid chromatography, after obtaining the fingerprint of Chuantai Granules, the fingerprint results of Chuantai Granules are subjected to attribute analysis.

[0021] Furthermore, the attribution analysis was performed on the 10 common peaks in the fingerprint of Chuantai Granules. The uridine at peak 2 comes from Pinellia ternata; gallic acid at peak 5 and hyperoside at peak 20 come from Coltsfoot Flos; chlorogenic acid at peak 13 comes from Coltsfoot Flos and Lycium barbarum; vitexin at peak 18 comes from Ephedra sinica; glycyrrhizin at peak 19 and glycyrrhizic acid at peak 35 come from Licorice; baicalin at peak 27 and melaleucain A glycoside at peak 31 come from Scutellaria baicalensis; and cinnamic acid at peak 33 comes from Cinnamon Bark.

[0022] The present invention provides a fingerprint spectrum of the Chuantai granules obtained by the above method.

[0023] The present invention provides a method for detecting the quality of Chuantai granules, and utilizes the above-mentioned Chuantai granule fingerprint spectrum to detect the quality of a Chuantai granule sample.

[0024] Compared with the prior art, the present invention has the following beneficial effects: The method for establishing the fingerprint of Chuantai granules by using high performance liquid chromatography provided by the present invention is simple and easy, has good reproducibility, is accurate and reliable, and confirms at least 10 components at one time under the same chromatographic conditions, saving time, saving inspection costs, and improving detection efficiency. Using high performance liquid chromatography, it is possible to separate and detect a variety of components in Chuantai granules, and both the main component and the secondary component can be reflected in its fingerprint, thereby ensuring the comprehensiveness and accuracy of the evaluation; by comparing the chromatogram and the mass spectrum, the chemical components corresponding to each peak in the chromatogram are identified, and a fingerprint of Chuantai granules with clear chemical component information is constructed, which has a high degree of individual specificity and environmental stability, and can not only be used for the quality control of Chuantai granules, but also can provide strong support for its drug efficacy research and clinical application.

[0025] Furthermore, attribution analysis can further confirm the origin and nature of each peak in the fingerprint spectrum, improve the accuracy and reliability of the fingerprint spectrum, and provide strong support for the in-depth research and application of Chuantai Granules.

[0026] The fingerprint spectrum of the Chuantai granules obtained by the present invention has high individual specificity and stability, and provides an accurate, clear and objective evaluation method for the quality control of the Chuantai granules. The fingerprint spectrum of the Chuantai granules from different batches or different sources can be compared through similarity analysis, so as to timely discover quality differences and ensure the stability and consistency of the product; through the fingerprint spectrum, the content and proportion of each component in the Chuantai granules can be intuitively reflected, so as to timely discover and correct deviations in the production process, which helps to ensure the clinical efficacy and drug safety of the Chuantai granules and improve the satisfaction and trust of patients.

[0027] The method for detecting the quality of Chuantai granules provided by the present invention only measures a single component or a single medicinal material in a drug according to the existing standard, while the present invention uses the fingerprint spectrum of Chuantai granules for detection, successfully characterizes 35 common peaks in the detection of Chuantai granules, and accurately identifies 10 common peaks among them, thereby realizing comprehensive detection of multiple medicinal materials and multiple active ingredients in Chuantai granules at the same time, avoiding errors and limitations that may exist in traditional detection methods, thereby improving the accuracy and reliability of quality detection; by comparing the sample fingerprint spectrum with the standard fingerprint spectrum, it is possible to intuitively judge whether the sample quality meets the requirements, realize standardization and normalization of quality control, help reduce the impact of human factors on quality control, and improve the stability and controllability of the production process; it is possible to accurately evaluate the quality of Chuantai granules, ensure that the content and proportion of its active ingredients meet the standards, help ensure the clinical efficacy and drug safety of Chuantai granules, and reduce the occurrence of poor efficacy or adverse reactions caused by quality problems. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is the HPLC fingerprint of Chuantai granules of the present invention; Figure 2 This is a chromatogram obtained by optimizing the extraction method during the preparation of the test solution of the present invention; Figure 3 This is a chromatogram obtained by optimizing the extraction solvent during the preparation of the test solution of the present invention; Figure 4 This is the full-wavelength 3D scanning image (190nm~800nm) of the test product of the present invention-DAD; Figure 5 The chromatogram obtained by optimizing the detection wavelength in the chromatographic conditions of the present invention; Figure 6 The chromatogram obtained by optimizing the column temperature in the chromatographic conditions of the present invention; Figure 7 The chromatogram obtained by optimizing the flow rate under the chromatographic conditions of the present invention; Figure 8The chromatogram obtained by optimizing the mobile phase composition under the chromatographic conditions of the present invention; Fig. 9 The chromatogram obtained by optimizing the injection amount under the chromatographic conditions of the present invention; Fig.10 A chromatogram obtained by optimizing the elution procedure in the chromatographic conditions of the present invention; Fig.11 is the uridine chromatogram of the present invention; Fig.12 is the gallic acid chromatogram of the present invention; Fig.13 is the chromatogram of chlorogenic acid of the present invention; Fig.14 is the chromatogram of vitexin of the present invention; Fig.15 is the chromatogram of liquiritin of the present invention; Fig.16 is the chromatogram of hyperoside of the present invention; Fig.17 is the chromatogram of baicalin of the present invention; Fig.18 It is the chromatogram of melaleuca polysaccharide A of the present invention; Fig.19 is the chromatogram of cinnamic acid of the present invention; Fig. 20 It is the glycyrrhizic acid chromatogram of the present invention; Fig.21 This is the total ion current diagram of the Chuantai granules of the present invention under the positive ion mode; Fig. 22 This is the total ion current diagram of the Chuantai granules of the present invention under the negative ion mode; Fig.23 This is the mass spectrum of protocatechuic acid of the present invention; Fig.24 This is the mass spectrum of quercetin of the present invention; Fig.25 This is the mass spectrum of liquiritin / isoliquiritin of the present invention; Fig.26 This is the mass spectrum of hyperoside / isoquercetin of the present invention; Fig. 27 is the mass spectrum of scutellariae scutellariae glycoside of the present invention; Fig.28 This is the mass spectrum of Licorice of the present invention; Fig.29 This is the mass spectrum of wogonin / melaleucalyptol A glycoside of the present invention; Fig.30 This is the mass spectrum of Scutellaria baicalensis of the present invention; Fig.31 This is the mass spectrum of glycyrrhizic acid of the present invention; Fig.32 This is the mass spectrum of Licorice flavonoids C of the present invention; Fig.33 This is the mass spectrum of licorice root of the present invention; Fig.34 This is the mass spectrum of the semi-licorice isoflavone B of the present invention; Fig.35 is the mass spectrum of glycyrrhizin of the present invention; Fig.36 This is the mass spectrum of apigenin / apigenin isoliquiritigenin of the present invention; Fig.37 This is the mass spectrum of lycium barbarum acid of the present invention; Fig.38 is the mass spectrum of citric acid of the present invention; Fig.39 is the mass spectrum of chlorogenic acid of the present invention; Fig.40 This is the mass spectrum of amygdalin of the present invention; Fig.41 is the mass spectrum of Scutellaria baicalensis of the present invention; Fig.42 This is the mass spectrum of Vitex quinata of the present invention; Fig.43 This is the mass spectrum of baicalin / norwogonoside of the present invention; Fig.44 is the mass spectrum of rutin of the present invention; Fig.45 These are the fingerprints of 15 batches of Chuantai granules tested in the present invention. DETAILED DESCRIPTION

[0029] In order to further understand the present invention, the present invention is described below in conjunction with embodiments. These descriptions are only to further explain the features and advantages of the present invention and are not intended to limit the claims of the present invention.

[0030] It should be noted that the terms "first", "second", etc. in the specification and claims of the present invention and the above-mentioned drawings are used to distinguish similar objects, and are not necessarily used to describe a specific order or sequence. It should be understood that the data used in this way can be interchanged where appropriate, so that the embodiments of the present invention described herein can be implemented in an order other than those illustrated or described herein. In addition, the terms "including" and "having" and any variations thereof are intended to cover non-exclusive inclusions, for example, a process, method, system, product or device that includes a series of steps or units is not necessarily limited to those steps or units that are clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products or devices.

[0031] The present invention provides a method for establishing a fingerprint spectrum of Chuantai granules by using high performance liquid chromatography, comprising the following steps: S1, weigh different batches of Chuantai granules, add 80% methanol solution, and perform ultrasonic extraction to obtain Chuantai granule test solution; S2, take uridine, gallic acid, chlorogenic acid, vitexin, liquiritin, hyperoside, baicalin, melaleuca A glycoside, cinnamic acid, and glycyrrhizic acid, dissolve in methanol to obtain a single reference solution; S3, injecting the Chuantai granules test solution and the single reference solution into a high performance liquid chromatograph for chromatographic analysis, performing detection under the same conditions, and recording the corresponding chromatograms; S4, performing high-resolution mass spectrometry analysis on the test solution to obtain a total ion current diagram, importing the total ion current diagram into Xcalibur software, performing data analysis based on the peaks of the chromatogram of the test solution, and obtaining a mass spectrum result diagram of each chemical component; a total of 23 chemical components were analyzed through high-resolution mass spectrometry analysis of the Chuantai granule material benchmark, and combined with the HPLC-MS component analysis results of 8 medicinal material extracts such as ephedra, scutellaria, and bitter almond in the preparation prescription in the early stage of the present invention, the 23 chemical components were attributed and analyzed. The results showed that protocatechuic acid and vitexin were derived from ephedra; baicalin, baicalin, melaleuca A glycoside, wogonin, baicalein, baicalin, norwogonin glycoside were derived from scutellaria; quercetin, amygdalin, and rutin were derived from bitter almonds; quercetin, chlorogenic acid, and rutin were derived from coltsfoot flowers; citric acid and chlorogenic acid were derived from pinellia tuber; quercetin, lycium acid, and rutin were derived from wolfberry; licorice glycoside, isoliquiritigenin, glycyrrhizic acid, glycyrrhizin C, glabridin, semi-licorice isoflavone B, glycyrrhizin, apigenin, apigenin isoliquiritigenin were derived from licorice; S5, import the chromatograms of the test solution and reference solution of Chuantai Granules from different batches into the Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 A Version); select the chromatographic peaks existing in the chromatograms of the test solution of Chuantai Granules from different batches as common peaks, generate the reference spectrum of Chuantai Granules by the average value calculation method, and calculate the relative retention time and relative peak area of ​​each common peak; perform similarity analysis after data import, multi-point correction and data matching; obtain and export the similarity result table between the chromatograms of the test solution of Chuantai Granules from different batches and the common peak pattern; confirm the reliability of the result based on the similarity result table and the chromatogram of the test solution of Chuantai Granules.

[0032] S6. Based on the chromatogram of the test solution of Chuantai Granules and the chromatogram of the single reference solution, combined with the total ion flow obtained by high-resolution mass spectrometry and the mass spectrum results of the chemical components, baicalin (peak 27) was used as the reference peak, and peak 2 in the chromatogram was identified as uridine, peak 5 as gallic acid, peak 13 as chlorogenic acid, peak 18 as vitexin, peak 19 as liquiritin, peak 20 as hyperoside, peak 31 as melaleuca A glycoside, peak 33 as cinnamic acid, and peak 35 as glycyrrhizic acid, and the fingerprint of Chuantai Granules was obtained, see Appendix. Figure 1 .

[0033] Among them, the preparation method of the test solution of Chuantai granules in S1 is preferably: weigh 2.0 g of Chuantai granules from 15 batches respectively, place them in a stoppered conical flask, add 20 mL of 80% methanol solution, ultrasonically extract for 30 min, filter, spin dry, add 5 mL of water to reconstitute, and pass the solution through a 0.45 µm microporous filter membrane twice to obtain the test solution of Chuantai granules.

[0034] The method for preparing the standard solution in S2 is preferably as follows: uridine, gallic acid, chlorogenic acid, vitexin, liquiritin, hyperoside, baicalin, melaleuca A glycoside, cinnamic acid, and glycyrrhizic acid are accurately weighed and dissolved in pure methanol solution to form a single reference solution containing 0.69 mg uridine, 0.62 mg gallic acid, 0.63 mg chlorogenic acid, 0.56 mg vitexin, 0.64 mg liquiritin, 0.67 mg hyperoside, 0.89 mg baicalin, 0.56 mg melaleuca A glycoside, 0.76 mg cinnamic acid, and 0.75 mg glycyrrhizic acid per 1 mL.

[0035] The liquid chromatography conditions in S3 are as follows: chromatographic column: Shim-pack VP-ODS (250 mm×4.6 mm, 5 μm) chromatographic column; detector: diode array detector, detection wavelength is 275 nm; volume flow rate: 0.6 mL / min; injection volume: 10 μL; mobile phase: acetonitrile (A)-0.1% phosphoric acid aqueous solution (B), gradient elution, the elution program is shown in Table 1: Table 1: HPLC elution program

[0036] The present invention provides an optimization process for establishing a fingerprint spectrum of Chuantai granules using high performance liquid chromatography: (1) Optimization of test solution preparation Different extraction methods (ultrasound, reflux, maceration, filtration after ultrasound, and reconstitution of the filtrate by spinning it dry with 5 mL of water) were experimentally investigated. The results are shown in the attached Figure 2 As shown in the figure, after analysis, it was determined that the final extraction method was ultrasound followed by filtration. The chromatogram components of the filtrate after spin drying and re-dissolving with 5 mL of water were relatively comprehensive and had good separation, so ultrasound followed by filtration and re-dissolving with 5 mL of water after spin drying were selected as the method for further operation; this method efficiently released the target components through ultrasonic extraction, combined with filtration to remove impurities, and further purified by spin drying and concentration and re-dissolving. The final chromatogram components were comprehensive and had excellent separation, indicating that it had high extraction efficiency, less impurity interference, simple operation and controllable solvent dosage. The present invention compares the extraction effects of different extraction solvents (50% methanol solution, 80% methanol solution, 90% methanol solution, pure methanol solution, water, ethanol, acetonitrile), and the results are shown in the attached Figure 3 As shown, the results showed that when 80% methanol solution was used as the extraction solvent, the extract chromatogram had the most information and the highest component content, indicating that it was significantly better than other solvents in terms of extraction efficiency and component coverage of the target components; compared with other solvents, 80% methanol solution has both polar and non-polar properties, which can more comprehensively dissolve and extract a variety of target substances while reducing interference from impurities, and is highly efficient, universal and practical.

[0037] This paper compares the extraction effects of different extraction times (30 min, 45 min, 1 h), and the results are shown in the attached Figure 4 As shown in the figure, the results showed that the chromatogram obtained when ultrasonic extraction was performed for 30 min had the highest component content, the best peak shape, and high separation, so 30 min was selected as the ultrasonic extraction time.

[0038] (2) Optimization of chromatographic conditions The present invention uses a diode array detector to investigate the detection wavelength and performs a full wavelength scan of 190-800 nm on the sample. Figure 5 As shown, the results show that when the detection wavelength condition is 275 nm, the chromatogram contains the most comprehensive information and the baseline is stable, so this method is selected as the detection wavelength condition; The present invention screens the column temperature (25°C, 30°C, 35°C), see Appendix Figure 6 As shown in the results, it was found that when the column temperature was maintained at 30°C, the peak elution was optimal and the separation effect of each component was better, so the column temperature of 30°C was finally selected.

[0039] The present invention screened the flow rate (0.6 mL / min, 0.8 mL / min, 1.0 mL / min), see Appendix Figure 7 As shown in the figure, the results show that when the flow rate is maintained at 0.6 mL / min, the peak elution is the best and the separation effect of each component is better, so the flow rate is finally selected as 0.6 mL / min; The present invention screened the injection volume (10 μL, 15 μL, 20 μL), see Appendix Fig. 9 As shown in the figure, the results show that when the injection volume is 20 μL, the peak is the best and the separation effect of each component is good, so the injection volume is finally selected as 20 μL; The present invention compares the elution effects of multiple different elution systems such as acetonitrile-0.1% acetic acid, methanol-0.1% acetic acid, acetonitrile-0.1% formic acid, methanol-0.1% formic acid, acetonitrile-water, methanol-water, methanol-0.1% phosphoric acid, acetonitrile-0.1% phosphoric acid under different gradients, see Appendix Figure 8 As shown, the results showed that when acetonitrile-0.1% formic acid was used as the mobile phase, the separation effect of the various components in the Chuantai granules was better, so acetonitrile-0.1% formic acid was finally selected as the mobile phase.

[0040] After determining the optimal mobile phase composition, the present invention screened the best gradient elution program through a large number of experiments, and some elution programs are as follows: Table 2: Elution Program 1

[0041] Table 3: Elution Program 2

[0042] Table 4: Elution Program 3

[0043] Table 5: Elution Program 4

[0044] Table 6: Elution Program 5

[0045] Table 7: Elution Program 6

[0046] Test results such as Fig.10 As shown. Fig.10 It can be seen that elution program 6 has good separation, high peak height, stable baseline, and complete chromatographic information. Therefore, elution program 6 is selected as the optimal elution program.

[0047] The embodiments of the present invention are described in detail below with reference to the examples. If no specific conditions are specified in the examples, the experiments are carried out according to conventional conditions or conditions recommended by the manufacturer. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be obtained commercially.

[0048] The instruments and reagents used in the embodiments are as follows: Experimental equipment: The instruments used in the present invention are shown in Table 8.

[0049] Table 8: Instruments and reagents used in the present invention

[0050] Drugs and reagents: Different batches of Chuantai granules samples used in the present invention were purchased from the market and produced by Shaanxi Kanghui Pharmaceutical Co., Ltd. (batch numbers: 201201, 210903, 210904, 210907, 211212, 211213, 211215, 220102, 220103, 220104, 220107, 221203, 221205, 221207, 221209).

[0051] The reagents used in the present invention are shown in Table 9.

[0052] Table 9: Reagents used in the present invention

[0053] Reference substances: uridine reference substance (batch number: 100635-201902, purity: 99.9%), gallic acid reference substance (batch number: 110831-202407, purity: 90.8%), chlorogenic acid reference substance (batch number: 110753-202119, purity: 96.3%), vitexin reference substance (batch number: 111687-202306, purity: 99.5%), liquiritin reference substance (batch number: 111610-202209, purity: 95.2%), Hypericum perforatum The glycoside reference substance (batch number: 111521-202310: 94.7%), baicalin (batch number: 110715-202223: 97.2%), and cinnamic acid reference substance (batch number: 110786-202305, purity: 99.8%) were purchased from China Food and Drug Inspection Institute; the melaleuca alternifolia glycoside reference substance (batch number: PS011092: 98%) and glycyrrhizic acid reference substance (batch number: 20070203, purity: 98.27%) were purchased from Chengdu Pufeide Biotechnology Co., Ltd.

[0054] Example 1 This embodiment provides a method for establishing a fingerprint spectrum of Chuantai granules using high performance liquid chromatography, comprising the following steps: S1, using different batches of Chuantai granules as test samples to prepare Chuantai granules test sample solutions; Weigh 2.0 g of Chuantai granules from 15 batches respectively, place them in a stoppered conical flask, add 20 mL of 80% methanol solution, extract by ultrasonic for 30 min, filter, spin dry, add 5 mL of water to reconstitute, and pass the solution through a 0.45 µm microporous filter membrane twice to obtain the Chuantai granule test solution.

[0055] S2, prepare a single reference solution with uridine, gallic acid, chlorogenic acid, vitexin, liquiritin, hyperoside, baicalin, melaleuca alternifolia glycoside A, cinnamic acid and glycyrrhizic acid reference substances.

[0056] Accurately weigh uridine, gallic acid, chlorogenic acid, vitexin, liquiritin, hyperoside, baicalin, melaleuca A glycoside, cinnamic acid, and glycyrrhizic acid and dissolve them in methanol to form a single reference solution containing 0.69 mg uridine, 0.62 mg gallic acid, 0.63 mg chlorogenic acid, 0.56 mg vitexin, 0.64 mg liquiritin, 0.67 mg hyperoside, 0.89 mg baicalin, 0.56 mg melaleuca A glycoside, 0.76 mg cinnamic acid, and 0.75 mg glycyrrhizic acid per 1 mL.

[0057] S3, inject the Chuantai granules test solution and the single reference solution into the high performance liquid chromatograph, perform chromatographic analysis under the same conditions, and record the corresponding chromatograms (see Appendix Fig.11 ~Attached Fig. 20 ); The liquid chromatography conditions were as follows: chromatographic column: Shim-pack VP-ODS (250 mm×4.6 mm, 5 μm) column; detector: diode array detector, detection wavelength was 275 nm; volume flow rate: 0.6 mL / min; injection volume: 20 μL; mobile phase: acetonitrile (A)-0.1% phosphoric acid aqueous solution (B), gradient elution, and the elution program is shown in Table 1.

[0058] S4, importing the chromatograms of different batches of test sample solutions and the chromatogram of a single reference sample solution obtained in S3 into the Chinese medicine chromatographic fingerprint similarity evaluation system and performing similarity analysis to confirm the reliability of the results.

[0059] The chromatograms of 15 batches of Chuantai granules test solution and reference solution were imported into the Chinese medicine chromatographic fingerprint similarity evaluation system (2012 A version); the chromatographic peaks existing in the chromatograms of different batches of Chuantai granules test solution were selected as common peaks, and the reference spectrum of Chuantai granules was generated by the average value calculation method, and the relative retention time and relative peak area of ​​each common peak were calculated; similarity analysis was performed after data import, multi-point correction and data matching; the similarity result table between the chromatograms of different batches of Chuantai granules test solution and the common peak pattern was obtained and exported; the reliability of the result was confirmed based on the similarity result table and the chromatogram of Chuantai granules test solution.

[0060] S5, high-resolution mass spectrometry analysis is performed based on the chromatograms of different batches of test sample solutions obtained in S3 to obtain a total ion current diagram ( Figure 21-22 ); According to the total ion flow chart and the peak of the test sample chromatogram obtained in S3, data analysis was performed to obtain the mass spectrum result chart of each chemical component ( Figure 23-Figure 44 ).

[0061] The high-resolution mass spectrometry detection conditions were as follows: electrospray ionization, spray voltage 3500 V, sheath gas flow rate 40 arb, auxiliary gas flow rate 10 arb, capillary temperature 300°C, auxiliary gas temperature 300°C, full scan mode, and mass-to-charge ratio scanning range m / z 100-1500.

[0062] The test solution is subjected to high-resolution mass spectrometry analysis to obtain a total ion current diagram, which is then imported into Xcalibur software, and data analysis is performed based on the peaks in the chromatogram of the test solution to obtain mass spectrometry results of each chemical component; S6, comparing the chromatograms of the test sample and the reference sample obtained in S3, the total ion flow diagram obtained in S4, and the mass spectra of each chemical component, to obtain a fingerprint of the Chuantai granules consisting of common characteristic peaks.

[0063] Using baicalin (peak 27) as the reference peak, peak 2 in the chromatogram was identified as uridine, peak 5 as gallic acid, peak 13 as chlorogenic acid, peak 18 as vitexin, peak 19 as liquiritin, peak 20 as hyperoside, peak 31 as melaleuca A glycoside, peak 33 as cinnamic acid, and peak 35 as glycyrrhizic acid, and the fingerprint of Chuantai granules was obtained ( Fig.45 ).

[0064] At the same time, the present invention uses the automatically generated reference spectrum R to generate a common chromatographic peak pattern, and the analysis and calculation show that the common chromatographic peaks of 15 batches of Chuan Tai Granules test samples have relatively good similarity, indicating that the fingerprint spectrum of Chuan Tai Granules established by this method can well detect the quality of Chuan Tai Granules and 15 batches of Chuan Tai Granules. The results are shown in Table 10.

[0065] Table 10: Similarity between each batch of samples and the common chromatographic peak pattern

[0066] Example 2 Methodological study of feature profile detection methods.

[0067] (1) Precision study The test solution prepared by the method of Example 1 was taken and analyzed according to the detection method of Example 1. The sample was injected 6 times in parallel with an injection volume of 10 μL. Hyperoside was used as the reference peak. The peak area and relative retention time were analyzed and the RSD value was calculated. The results showed that the RSD of the relative retention time was less than 0.122%, and the RSD of the relative peak area was less than 0.890%, indicating that the parallel injection precision of the equipment was good.

[0068] Table 10: Peak areas and retention times for precision studies

[0069] (2) Stability study The test solution prepared by the method of Example 1 was analyzed according to the detection method of Example 1, and the sample was injected and analyzed at different times of 0, 2, 6, 12, 18, and 24 h, with an injection volume of 10 μL. Hyperoside was used as the reference peak, and the peak area and retention time of the common peak of 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 0.668%, and the RSD of the relative peak area was less than 0.803%, indicating that the chromatographic peak of the test solution of Chuantai Granules within 24 h had almost no change, and the stability was good.

[0070] Table 11: Stability study peak areas and retention times

[0071] (3) Reproducibility study Six batches of sample solutions were prepared according to the test solution method in Example 1. Referring to the chromatographic conditions of Example 1, the injection volume was 10 μL, and hyperoside was used as the reference peak. The peak area and retention time of the common peaks of the sample HPLC fingerprint were analyzed and the RSD values ​​were calculated. The results showed that the RSD of the relative retention time was less than 0.534%, and the RSD of the relative peak area was less than 0.496%, indicating that the sample chromatographic peak reproducibility was good and the repeatability of the method was good.

[0072] Table 12: Peak areas and retention times for repeatability studies

[0073] The above experimental results show that the method and application of establishing the fingerprint of Chuantai granules using high performance liquid chromatography technology provided by the present invention have the characteristics of good stability, high precision and good repeatability, can comprehensively and objectively evaluate the quality of Chuantai granules, and provide quality assurance for clinical efficacy.

[0074] The above embodiments are only exemplary embodiments of the present invention and are not intended to limit the present invention. The protection scope of the present invention is defined by the claims. The above contents are only used as examples and explanations of the concept of the present invention. Various modifications or supplements made by technicians in the technical field to the described specific embodiments or replacements in similar ways shall fall within the protection scope of the present invention as long as they do not deviate from the concept of the invention or exceed the scope defined by the claims.

Claims

1. A method for establishing a fingerprint of Chuantai granules using high performance liquid chromatography, characterized in that: The following steps are involved: S1, using different batches of Chuantai granules as test samples to prepare Chuantai granules test sample solutions; S2, prepare a single reference solution with uridine, gallic acid, chlorogenic acid, vitexin, liquiritin, hyperoside, baicalin, melaleuca A glycoside, cinnamic acid and glycyrrhizic acid reference substances; S3, injecting the Chuantai granules test solution and the single reference solution into a high performance liquid chromatograph, respectively, performing chromatographic analysis under the same conditions, and recording the corresponding chromatograms; The HPLC determination conditions are as follows: Shim-pack VP-ODS column; the mobile phase is acetonitrile-0.1% formic acid aqueous solution, and the gradient elution procedure is as follows: S4, importing the chromatogram of the test solution of Chuantai Granules and the chromatogram of the single reference solution obtained in S3 into the Chinese medicine chromatographic fingerprint similarity evaluation system and performing similarity analysis to confirm the reliability of the results; S5, performing high-resolution mass spectrometry analysis on the chromatogram of the Chuantai granules test sample solution obtained in S3 to obtain a total ion flow diagram; performing data analysis on the total ion flow diagram and the peak conditions of the test sample chromatogram to obtain a mass spectrometry result diagram of each chemical component; S6, comparing the chromatograms of the test sample and the reference sample obtained in S3, the total ion flow diagram obtained in S5, and the mass spectra of each chemical component, to obtain a fingerprint of the Chuantai granules consisting of common characteristic peaks.

2. The method for constructing the fingerprint of Chuantai granules according to claim 1 and its application in quality standards, characterized in that: In S1, the contents of different batches of Chuantai granules were taken, methanol solution was added, ultrasonic extraction was performed, filtration was performed, and microporous membrane filtration was performed to obtain Chuantai granule test solution.

3. The method and application of establishing a fingerprint spectrum of Chuantai granules using high performance liquid chromatography technology according to claim 1, characterized in that: In S2, uridine, gallic acid, chlorogenic acid, vitexin, liquiritin, hyperoside, baicalin, melaleuca alternifolia glycoside A, cinnamic acid and glycyrrhizic acid reference substances were dissolved in methanol solution to prepare a single reference substance solution.

4. The method for establishing a fingerprint of Chuantai granules using high performance liquid chromatography technology according to claim 1, characterized in that: The HPLC detection wavelength is 210-290 nm, the column temperature is 25-35° C., and the flow rate is 0.6-1.0 mL / min.

5. The method for establishing a fingerprint of Chuantai granules using high performance liquid chromatography technology according to claim 1, characterized in that: S4 specifically includes: importing the chromatograms of the test solutions and reference solutions of the different batches of Chuan Tai Granules obtained in S2 into the Chinese medicine chromatographic fingerprint similarity evaluation system; selecting the chromatographic peaks existing in the chromatograms of the test solutions of the different batches of Chuan Tai Granules as common peaks, generating the reference spectrum of the Chuan Tai Granules by the average value calculation method, and calculating the relative retention time and relative peak area of ​​each common peak; performing similarity analysis after data import, multi-point correction and data matching; obtaining and exporting the similarity result table between the chromatograms of the test solutions of the Chuan Tai Granules from different batches and the common peak pattern; confirming the reliability of the results based on the similarity result table and the chromatogram of the test solution of the Chuan Tai Granules.

6. The method for establishing a fingerprint of Chuantai granules using high performance liquid chromatography technology according to claim 1, characterized in that: In S5, the high-resolution mass spectrometry detection conditions are: electrospray ionization, spray voltage 3500 V, sheath gas flow rate 40 arb, auxiliary gas flow rate 10 arb, capillary temperature 300°C, auxiliary gas temperature 300°C, scanning mode is full scan mode, and mass-to-charge ratio scanning range m / z is 100-1500.

7. The method for establishing a fingerprint of Chuantai granules using high performance liquid chromatography technology according to claim 1, characterized in that: S6 is specifically as follows: according to the chromatograms of the test sample and the reference sample obtained in S3, combined with the total ion flow diagram obtained by high-resolution mass spectrometry in S5 and the mass spectrum result diagram of the chemical components, a comparison is performed, and peak 27 baicalin is used as the reference peak, and peak 2 in the chromatogram is identified as uridine, peak 5 as gallic acid, peak 13 as chlorogenic acid, peak 18 as vitexin, peak 19 as liquiritin, peak 20 as hyperoside, peak 31 as melaleuca alternifolia A glycoside, peak 33 as cinnamic acid, and peak 35 as glycyrrhizic acid, thereby obtaining the fingerprint of Chuantai Granules.

8. The method for establishing a fingerprint of Chuantai granules using high performance liquid chromatography according to claim 1, characterized in that: After obtaining the fingerprint of Chuantai Granules, the fingerprint results of Chuantai Granules were subjected to attribute analysis.

9. The fingerprint of Chuantai granules obtained by the method described in any one of claims 1 to 8.

10. A method for detecting the quality of Chuantai granules, characterized in that: The quality of the Chuan Tai Granule sample is detected using the Chuan Tai Granule fingerprint spectrum described in claim 9.

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