A method for establishing the fingerprint of chathai granules by high performance liquid chromatography and application thereof

Abstract

The application discloses a method for establishing a fingerprint of Chuantai granules by using high performance liquid chromatography and application thereof, and belongs to the technical field of compound traditional Chinese medicine preparation quality control management. The method comprises the following steps: preparing a test sample solution and a control sample solution, performing chromatographic analysis by using a high performance liquid chromatograph, recording a chromatogram, and performing similarity analysis and high resolution mass spectrometry analysis, so as to finally obtain the fingerprint of the Chuantai granules. The fingerprint comprises 10 common peaks, and the attribution analysis is performed to determine the sources of the common peaks. The method is simple, easy to operate, accurate, reliable, capable of confirming multiple components at one time, and capable of improving detection efficiency. The obtained fingerprint of the Chuantai granules can be used for simultaneously determining 10 components in the Chuantai granules, and provides a fast and efficient technical method for quality control of the Chuantai granules. The method can be used for accurately, clearly and objectively evaluating the quality of the Chuantai granules.

Description

Technical Field

[0001] This invention belongs to the field of quality control management technology for compound traditional Chinese medicine preparations, specifically relating to a method and application of establishing a fingerprint spectrum of Chuantai granules using high performance liquid chromatography. Background Technology

[0002] Chuantai Granules (National Drug Approval Number: Z20020081) is a traditional Chinese medicine compound preparation that integrates the essence of various Chinese medicinal herbs, demonstrating significant efficacy in the treatment of respiratory diseases. Chuantai Granules consist of eight Chinese medicinal herbs: Ephedra, Scutellaria baicalensis, Bitter Almond, Coltsfoot Flower, Prepared Pinellia ternata, Lycium barbarum, Cinnamon, and Licorice. Each herb plays an indispensable role, collectively forming the unique pharmacodynamic system of Chuantai Granules. Ephedra acts as the principal herb, dispersing external cold and relieving asthma; Scutellaria baicalensis, Bitter Almond, and Prepared Pinellia ternata act as assistant herbs, clearing internal heat and dispersing external cold, while simultaneously regulating spleen and stomach function and enhancing the body's disease resistance; while Coltsfoot Flower, Lycium barbarum, Cinnamon, and Licorice act as adjuvant herbs, tonifying the kidneys, nourishing qi, moistening the lungs, and resolving phlegm, further enhancing the therapeutic effect of Chuantai Granules. For complex diseases such as asthma, chronic bronchitis, and allergic cough, whose pathological mechanisms involve multiple physiological processes, including abnormal immune system responses, respiratory tract inflammation, and airway hyperresponsiveness, Chuantai granules can exert a synergistic therapeutic effect on these pathological mechanisms through multi-target regulation, thereby achieving a comprehensive therapeutic effect.

[0003] However, the efficacy of Chuantai granules does not stem from a single ingredient, but rather from the interaction and synergistic effect of multiple pharmacodynamic components. This complexity makes traditional quality control methods, such as single-component testing, insufficient to comprehensively and accurately reflect its overall quality and efficacy. This is because many minor components in Chuantai granules, although perhaps less abundant than the main component, may play a crucial role in the treatment process. These minor components may synergistically enhance the efficacy of the main component, or provide favorable conditions for the main component to exert its effects by regulating the body's physiological environment. Therefore, neglecting these minor components could lead to biased assessments of the overall efficacy of Chuantai granules.

[0004] With the widespread application of high-performance liquid chromatography (HPLC), new ideas and methods have been provided for the quality control of traditional Chinese medicine compound preparations. HPLC technology has advantages such as high separation efficiency, fast analysis speed, and high sensitivity, enabling the separation and detection of multiple components in complex systems. Therefore, establishing a fingerprint spectrum of Chuantai granules using HPLC technology has become an accurate, clear, and objective method for evaluating the quality of Chuantai granules. A fingerprint spectrum is a chromatogram or spectrum that can indicate the chemical characteristics of a substance, possessing high individual specificity and environmental stability. By comparing the fingerprint spectra of Chuantai granules from different batches or sources, the content changes of each component in Chuantai granules, the interaction relationships between 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, currently, no literature or patent reports have been found regarding the construction of an HPLC fingerprint spectrum for Chuantai granules. Summary of the Invention

[0005] To address the problem that traditional quality control methods are insufficient to comprehensively evaluate the overall quality and efficacy of the complex multi-component system of Chuantai granules, and given the lack of reports in existing patents and literature regarding the use of HPLC to construct fingerprint profiles for Chuantai granules, this invention aims to provide a method and application for establishing fingerprint profiles of Chuantai granules using high-performance liquid chromatography. Through the efficient separation and detection of HPLC, a fingerprint profile of Chuantai granules with individual specificity and stability can be established, intuitively reflecting the content and interaction relationships of each component in Chuantai granules. This provides an accurate, clear, and objective evaluation method for controlling its quality and ensuring clinical efficacy.

[0006] This invention is achieved through the following technical solution: This invention discloses a method for establishing a fingerprint spectrum of Chuantai granules using high-performance liquid chromatography, comprising the following steps: S1, Prepare a test solution of Chuantai granules using different batches of Chuantai granules as test samples; S2, using uridine, gallic acid, chlorogenic acid, vitexin, glycyrrhizin, hyperoside, baicalin, sennain A, cinnamic acid and glycyrrhizic acid as reference standards, prepare single reference standard solutions; S3, inject the test solution of Chuantai granules and the single reference solution into the high performance liquid chromatograph, perform chromatographic analysis under the same conditions, and record the corresponding chromatograms; In S3, the high-performance liquid chromatography (HPLC) conditions are as follows: Shim-pack VP-ODS column; mobile phase is acetonitrile-0.1% formic acid aqueous solution; gradient elution program is as follows: ; S4. The chromatograms of the test solution of Chuantai granules and the single reference solution obtained in S3 were imported into the Chinese medicine chromatographic fingerprint similarity evaluation system and similarity analysis was performed to confirm the reliability of the results. S5. Based on the chromatogram of the Chuantai granules test sample solution obtained in S3, high-resolution mass spectrometry analysis was performed to obtain the total ion chromatogram; based on the peak elution of the total ion chromatogram and the test sample chromatogram obtained in S3, data analysis was performed to obtain the mass spectrometry results of each chemical component. S6. The chromatograms of the test solution and the single reference solution of the Chuantai granules obtained in S3, the total ion chromatogram obtained in S5, and the mass spectra of each chemical component are compared to obtain the fingerprint spectrum of Chuantai granules composed of common characteristic peaks.

[0007] In S1, the contents of different batches of Chuantai granules were taken, methanol solution was added, ultrasonic extraction was performed, and the mixture was filtered through a microporous membrane to obtain the Chuantai granule test solution.

[0008] Furthermore, each 20 mL methanol solution contains 2.0 g of the contents of the acetaminophen granules. The mixture is extracted by ultrasonication, filtered, and filtered through a microporous membrane to obtain the acetaminophen granule test solution.

[0009] Furthermore, the volume concentration of the methanol solution is 80%.

[0010] Furthermore, ultrasonic extraction was performed for 30 minutes.

[0011] Furthermore, it is filtered twice using a 0.45 µm microporous membrane.

[0012] In S2, a single reference solution was prepared by dissolving uridine, gallic acid, chlorogenic acid, vitexin, glycyrrhizin, hyperoside, baicalin, sennain A, cinnamic acid, and glycyrrhizic acid reference standards in methanol.

[0013] Further, in S2, the single reference solution for uridine contained 0.69 mg of uridine per 1 mL of methanol; the single reference solution for gallic acid contained 0.62 mg of gallic acid per 1 mL of methanol; the single reference solution for chlorogenic acid contained 0.63 mg of chlorogenic acid per 1 mL of methanol; the single reference solution for vitexin contained 0.56 mg of vitexin per 1 mL of methanol; the single reference solution for glycyrrhizin contained 0.64 mg of glycyrrhizin per 1 mL of methanol; the single reference solution for hyperoside contained 0.67 mg of hyperoside per 1 mL of methanol; the single reference solution for baicalin contained 0.89 mg of baicalin per 1 mL of methanol; the single reference solution for sensolin A contained 0.56 mg of sensolin A per 1 mL of methanol; and the single reference solution for cinnamic acid contained 0.76 mg of cinnamic acid per 1 mL of methanol. mg; in the single reference solution of glycyrrhizic acid, each 1 mL of methanol solution contains 0.75 mg of glycyrrhizic acid.

[0014] The high-performance liquid chromatography (HPLC) has a detection wavelength of 210~290nm, a column temperature of 25~35℃, and a flow rate of 0.6~1.0mL / min.

[0015] Furthermore, the high-performance liquid chromatography (HPLC) uses a detection wavelength of 275 nm, a column temperature of 30 °C, a flow rate of 0.6 mL / min, and an injection volume of 20 μL.

[0016] S4 specifically involves: importing the chromatograms of the Chuantai granules test solution and reference solution obtained in S3 into the Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System; selecting chromatographic peaks present in the chromatograms of different batches of Chuantai granules test solutions as common peaks, generating a reference chromatogram of Chuantai granules using 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 a similarity result table between the chromatograms of different batches of Chuantai granules test solutions and the common peak patterns; and confirming the reliability of the results based on the similarity result table and the chromatograms of the Chuantai granules test solutions.

[0017] 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℃, auxiliary gas temperature 300℃, scanning mode is full scan mode, and mass-to-charge ratio scanning range m / z is 100-1500.

[0018] S6 specifically involves comparing the chromatograms of the Chuantai granules test solution and the single reference solution obtained in S3 with the total ion chromatogram and the mass spectrometry results of the chemical components obtained by high-resolution mass spectrometry in S5. Using baicalin at peak 27 as the reference peak, peak 2 is identified as uridine, peak 5 as gallic acid, peak 13 as chlorogenic acid, peak 18 as vitexin, peak 19 as glycyrrhizin, peak 20 as hyperoside, peak 31 as phloroglucinol A, peak 33 as cinnamic acid, and peak 35 as glycyrrhizic acid, thus obtaining the fingerprint spectrum of Chuantai granules.

[0019] The above method for establishing the fingerprint spectrum of Chuantai granules using high performance liquid chromatography (HPLC) is used to obtain the fingerprint spectrum of Chuantai granules, and then the fingerprint spectrum results of Chuantai granules are subjected to attribute analysis.

[0020] Furthermore, the attribution analysis of the 10 common peaks in the fingerprint spectrum of Chuantai granules revealed that peak 2, uridine, originated from Pinellia ternata; peak 5, gallic acid, and peak 20, hyperoside, originated from Tussilago farfara; peak 13, chlorogenic acid, originated from Tussilago farfara and Lycium barbarum; peak 18, vitexin, originated from Ephedra sinica; peak 19, glycyrrhizin, and peak 35, glycyrrhizic acid, originated from Glycyrrhiza uralensis; peak 27, baicalin, and peak 31, mesona chinensis A, originated from Scutellaria baicalensis; and peak 33, cinnamic acid, originated from cinnamon.

[0021] The present invention provides a fingerprint spectrum of acetylcholine particles obtained by the above method.

[0022] This invention provides a method for detecting the quality of cyclophosphamide granules, which utilizes the aforementioned cyclophosphamide granule fingerprint spectrum to detect the quality of cyclophosphamide granule samples.

[0023] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a method for establishing a fingerprint spectrum of Chuantai granules using high-performance liquid chromatography (HPLC). This method is simple, easy to implement, highly reproducible, accurate, and reliable. Under the same chromatographic conditions, at least 10 components can be identified simultaneously, saving time, reducing testing costs, and improving detection efficiency. HPLC enables the separation and detection of multiple components in Chuantai granules. Both major and minor components can be represented in the fingerprint spectrum, ensuring the comprehensiveness and accuracy of the evaluation. By comparing chromatograms and mass spectra, the chemical components corresponding to each peak in the chromatogram are identified, constructing a fingerprint spectrum of Chuantai granules with clear chemical component information. This fingerprint spectrum exhibits high individual specificity and environmental stability, and can be used not only for the quality control of Chuantai granules but also to provide strong support for its efficacy research and clinical application.

[0024] Furthermore, attribution analysis can further confirm the source 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.

[0025] The fingerprint spectrum of Chuantai granules obtained by this invention exhibits high individual specificity and stability, providing an accurate, clear, and objective evaluation method for the quality control of Chuantai granules. Fingerprint spectra of Chuantai granules from different batches or sources can be compared through similarity analysis, thereby promptly identifying quality differences and ensuring product stability and consistency. The fingerprint spectrum visually reflects the content and proportion of each component in Chuantai granules, enabling timely detection and correction of deviations in the production process. This helps ensure the clinical efficacy and medication safety of Chuantai granules, improving patient satisfaction and trust.

[0026] The method for detecting the quality of Chuantai granules provided by this invention differs from existing standards, which only measure a single component or herb in the drug. This invention utilizes the fingerprint spectrum of Chuantai granules for detection, successfully characterizing 35 common peaks and accurately identifying 10 of them. This enables comprehensive detection of multiple herbs and active ingredients in Chuantai granules simultaneously, avoiding the errors and limitations of traditional methods and thus improving the accuracy and reliability of quality testing. By comparing the sample fingerprint spectrum with the standard fingerprint spectrum, the quality of the sample can be directly judged to ensure compliance with requirements, achieving standardization and normalization of quality control. This helps reduce the impact of human factors on quality control and improves the stability and controllability of the production process. It accurately assesses the quality of Chuantai granules, ensuring that the content and proportion of its active ingredients meet standards, thus helping to guarantee the clinical efficacy and medication safety of Chuantai granules and reducing the occurrence of poor efficacy or adverse reactions due to quality problems. Attached Figure Description

[0027] Figure 1 This is the HPLC fingerprint of the Chuantai granules of the present invention; Figure 2 This is a chromatogram obtained by optimizing the extraction method during the preparation of the test sample solution according to the present invention; Figure 3 This is a chromatogram obtained by optimizing the extraction solvent during the preparation of the test sample solution according to the present invention; Figure 4 The full-wavelength 3D scan image (190nm~800nm) of the DAD sample of this invention; Figure 5 This is a chromatogram obtained by optimizing the detection wavelength under chromatographic conditions according to the present invention; Figure 6 This is a chromatogram obtained by optimizing column temperature under chromatographic conditions according to the present invention; Figure 7 This is a chromatogram obtained by optimizing the flow rate under chromatographic conditions according to the present invention; Figure 8This is a chromatogram obtained by optimizing the mobile phase composition under chromatographic conditions according to the present invention; Figure 9 This is a chromatogram obtained by optimizing the injection volume under chromatographic conditions according to the present invention; Figure 10 This is a chromatogram obtained by optimizing the elution procedure under chromatographic conditions according to the present invention; Figure 11 This is the chromatogram of uridine in this invention; Figure 12 This is the chromatogram of gallic acid in this invention; Figure 13 This is the chromatogram of chlorogenic acid in this invention; Figure 14 This is the chromatogram of vitexin of the present invention; Figure 15 This is the chromatogram of glycyrrhizin in this invention; Figure 16 This is the chromatogram of hyperoside in this invention; Figure 17 This is the chromatogram of baicalin of the present invention; Figure 18 This is the chromatogram of the thousand-layer paper glycoside A of the present invention; Figure 19 This is the chromatogram of cinnamic acid in this invention; Figure 20 This is the glycyrrhizic acid chromatogram of the present invention; Figure 21 This is the total ion flow chromatogram of the Chuantai particles of the present invention in positive ion mode; Figure 22 This is the total ion flow chromatogram of the Chuantai granules of the present invention in negative ion mode; Figure 23 This is the mass spectrum of protocatechuic acid in this invention; Figure 24 This is a mass spectrum of the oak bark from the present invention; Figure 25 This is the mass spectrum of glycyrrhizin / isoglycyrrhizin of the present invention; Figure 26 This is the mass spectrum of hyperoside / isoquercetin of the present invention; Figure 27 This is the mass spectrum of baicalin of the present invention; Figure 28 This is a spectral image of licorice root from the present invention; Figure 29 This is the mass spectrum of baicalin / phloem A glycoside of the present invention; Figure 30 This is the mass spectrum of Scutellaria baicalensis in this invention; Figure 31 This is the glycyrrhizic acid mass spectrum of the present invention; Figure 32 This is the C-mass spectrum of licorice flavonoids in this invention; Figure 33 This is the mass spectrum of glycyrrhizin ketone in this invention; Figure 34 This is the mass spectrum of hemi-glycyrrhizin isoflavone B of the present invention; Figure 35 This is the glycyrrhizin mass spectrum of the present invention; Figure 36 This is the mass spectrum of apigenin / apigenin isoglycyrrhizin of the present invention; Figure 37 This is the mass spectrum of lycine from the present invention; Figure 38 This is the citric acid mass spectrum of the present invention; Figure 39 This is the chlorogenic acid mass spectrum of the present invention; Figure 40 This is the mass spectrum of amygdalin from the present invention; Figure 41 This is the mass spectrum of Scutellaria baicalensis in this invention; Figure 42 This is a mass spectrum of Vitex negundo of the present invention; Figure 43 This is the mass spectrum of baicalin / norbaicalin of the present invention; Figure 44 This is the mass spectrum of rutin in this invention; Figure 45 The fingerprint spectrum of 15 batches of Chuantai granules tested according to this invention. Detailed Implementation

[0028] To further understand the present invention, the present invention will be described below with reference to embodiments. These descriptions are only for further explaining the features and advantages of the present invention and are not intended to limit the claims of the present invention.

[0029] It should be noted that the terms "first," "second," etc., in the specification, claims, and accompanying drawings of this invention are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that such data can be interchanged where appropriate so that the embodiments of the invention described herein can be implemented in orders other than those illustrated or described herein. Furthermore, the terms "comprising" and "having," and any variations thereof, are intended to cover a non-exclusive inclusion; for example, a process, method, system, product, or apparatus that comprises a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to such processes, methods, products, or apparatus.

[0030] This invention provides a method for establishing a fingerprint spectrum of Chuantai granules using high performance liquid chromatography, comprising the following steps: S1, weigh different batches of Chuantai granules, add 80% methanol solution, and extract by ultrasonication to obtain Chuantai granule test solution; S2, take uridine, gallic acid, chlorogenic acid, vitexin, glycyrrhizin, hyperoside, baicalin, sensodeoxylin A, cinnamic acid, and glycyrrhizic acid, dissolve them in methanol to obtain a single reference solution; S3, inject the test solution of Chuantai granules and the single reference solution into the high performance liquid chromatograph for chromatographic analysis, perform the detection under the same conditions, and record the corresponding chromatograms; S4. The test solution was subjected to high-resolution mass spectrometry analysis to obtain a total ion chromatogram. The total ion chromatogram was imported into Xcalibur software, and data analysis was performed based on the peak elution of the chromatogram of the test solution to obtain the mass spectrometry results of each chemical component. Through high-resolution mass spectrometry analysis of the material basis of Chuantai granules, a total of 23 chemical components were resolved. Combined with the HPLC-MS component analysis results of the extracts of eight medicinal materials such as ephedra, scutellaria, and bitter almond in the formulation of the present invention in the previous stage, the 23 chemical components were assigned to specific components. The results showed that protocatechuic acid and vitexin were derived from ephedra; baicalin, baicalin-containing baicalin, baicalin A, baicalin, baicalin, baicalin, and no-baicalin baicalin were derived from scutellaria; quercetin, amygdalin, and rutin were derived from bitter almond; quercetin, chlorogenic acid, and rutin were derived from coltsfoot flower; citric acid and chlorogenic acid were derived from pinellia tuber; quercetin, citric acid, and rutin were derived from wolfberry; and glycyrrhizin, isoglycyrrhizin, glycyrrhizic acid, glycyrrhizin C, glycyrrhizin glabridin, hesoglycyrrhizin B, glycyrrhizin, apigenin, and apigenin isoglycyrrhizin were derived from licorice. S5. Import the chromatograms of different batches of Chuantai granules test solution and reference solution into the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Version A); select the chromatographic peaks present in the chromatograms of different batches of Chuantai granules test solution as common peaks, generate the reference chromatogram of Chuantai granules using 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 different batches of Chuantai granules test solution and the common peak pattern; confirm the reliability of the results based on the similarity result table and the chromatograms of Chuantai granules test solution.

[0031] S6. Based on the chromatograms of the test solution and the single reference solution of Chuantai granules, and comparing them with the total ion chromatogram obtained by high-resolution mass spectrometry and the mass spectrometry results of the chemical components, using baicalin (peak 27) as the reference peak, peak 2 was identified as uridine, peak 5 as gallic acid, peak 13 as chlorogenic acid, peak 18 as vitexin, peak 19 as glycyrrhizin, peak 20 as hyperoside, peak 31 as sensodeoxylin A, peak 33 as cinnamic acid, and peak 35 as glycyrrhizic acid. The fingerprint chromatogram of Chuantai granules was thus obtained. (See Appendix) Figure 1 .

[0032] The preferred method for preparing the test solution of Chuantai granules in S1 is as follows: weigh 2.0 g of Chuantai granules from 15 batches, place them in a stoppered conical flask, add 20 mL of 80% methanol solution, extract by ultrasonication for 30 min, filter, evaporate to dryness, add 5 mL of water to redissolve, and filter the solution twice through a 0.45 µm microporous membrane to obtain the test solution of Chuantai granules.

[0033] The preferred method for preparing the standard solution in S2 is as follows: accurately weigh uridine, gallic acid, chlorogenic acid, vitexin, glycyrrhizin, hyperoside, baicalin, sennain A, cinnamic acid, and glycyrrhizic acid separately and dissolve them in pure methanol to prepare a single reference solution containing 0.69 mg of uridine, 0.62 mg of gallic acid, 0.63 mg of chlorogenic acid, 0.56 mg of vitexin, 0.64 mg of glycyrrhizin, 0.67 mg of hyperoside, 0.89 mg of baicalin, 0.56 mg of sennain A, 0.76 mg of cinnamic acid, and 0.75 mg of glycyrrhizic acid per mL.

[0034] The liquid chromatography conditions in S3 were as follows: Column: Shim-pack VP-ODS (250 mm × 4.6 mm, 5 μm) column; Detector: Diode array detector, detection wavelength: 275 nm; 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: Liquid Chromatography Elution Procedure

[0035] This invention provides an optimized process for establishing a fingerprint spectrum of Chuantai granules using high-performance liquid chromatography: (1) Optimization of test solution preparation Experiments were conducted to investigate different extraction methods (ultrasound, reflux, maceration, and ultrasound followed by filtration, with the filtrate evaporated and then reconstituted with 5 mL of water). The results are shown in the appendix. Figure 2 As shown, after analysis, the final extraction method was determined to be ultrasonic followed by filtration. The chromatogram obtained by reconstituted the filtrate with 5 mL of water after rotary evaporation showed relatively comprehensive composition and good separation. Therefore, ultrasonic followed by filtration and reconstitution with 5 mL of water was chosen as the method for further extraction. This method efficiently releases the target components through ultrasonic extraction, removes impurities through filtration, and further purifies by rotary evaporation, concentration, and reconstitution. The final chromatogram obtained shows comprehensive composition and excellent separation, indicating high extraction efficiency, low impurity interference, simple operation, and controllable solvent usage. This invention compared the extraction effects of different extraction solvents (50% methanol solution, 80% methanol solution, 90% methanol solution, pure methanol solution, water, ethanol, and acetonitrile). The results are shown in the appendix. Figure 3 As shown, the results revealed that when 80% methanol solution was used as the extraction solvent, the extract had the most chromatographic information and the highest component content, indicating that it was significantly superior to other solvents in terms of extraction efficiency and component coverage. Compared with other solvents, 80% methanol solution has both polar and non-polar properties, which can more comprehensively dissolve and extract diverse target substances while reducing impurity interference, and has high efficiency, universality and practicality.

[0036] This invention compared the extraction effects of different extraction times (30 min, 45 min, 1 h), and the results are shown in the appendix. Figure 4 As shown, the results showed that the chromatogram obtained by ultrasonic extraction for 30 min had the highest component content, the best peak shape, and high separation. Therefore, 30 min was selected as the ultrasonic extraction time.

[0037] (2) Optimization of chromatographic conditions This invention employs a diode array detector to investigate the detection wavelength, performing a full wavelength scan of the sample from 190 to 800 nm. (See appendix) Figure 5 As shown in the figure, the results show that the chromatogram contains the most comprehensive information and the baseline is stable when the detection wavelength is 275 nm. Therefore, this method is selected as the detection wavelength condition. This invention screens column temperatures (25℃, 30℃, 35℃), see appendix. Figure 6 As shown, the results showed that the peak elution was optimal and the separation of each component was better when the column temperature was maintained at 30℃. Therefore, the column temperature of 30℃ was finally selected.

[0038] This invention screens for flow rates (0.6 mL / min, 0.8 mL / min, 1.0 mL / min), see appendix. Figure 7 As shown, the results showed that the peak elution was optimal and the separation of each component was good when the flow rate was kept at 0.6 mL / min. Therefore, the flow rate of 0.6 mL / min was finally selected. This invention screens injection volumes (10 μL, 15 μL, 20 μL), see appendix. Figure 9 As shown in the results, the peak elution was best when the injection volume was 20 μL, and the separation of each component was better. Therefore, the final injection volume was 20 μL. This invention compares the elution effects of several different elution systems, including 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, and acetonitrile-0.1% phosphoric acid, under different elution gradients. See attached figure. Figure 8 As shown, the results showed that the separation effect of each component in the Chuantai granules was better when acetonitrile-0.1% formic acid was used as the mobile phase. Therefore, acetonitrile-0.1% formic acid was finally selected as the mobile phase.

[0039] After determining the optimal mobile phase composition, this invention screened the optimal gradient elution program through numerous experiments. Some of the elution programs are as follows: Table 2: Elution Procedure 1

[0040] Table 3: Washing Procedure 2

[0041] Table 4: Elution Procedure 3

[0042] Table 5: Washing Procedure 4

[0043] Table 6: Washing Procedure 5

[0044] Table 7: Elution Procedure 6

[0045] Test results as follows Figure 10 As shown. (Through) Figure 10 It can be seen that elution program 6 has good resolution, high peak height, stable baseline, and complete chromatographic information. Therefore, elution program 6 is selected as the optimal elution program.

[0046] The embodiments of the present invention will be described in detail below with reference to the examples. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0047] The instruments and reagents used in the examples are as follows: Experimental equipment: The instruments used in this invention are shown in Table 8.

[0048] Table 8: Instruments and reagents used in this invention

[0049] Drugs and reagents: The different batches of Chuantai granules used in this invention were all purchased from the market and were produced by Shaanxi Kanghui Pharmaceutical Co., Ltd. (batch numbers: 201201, 210903, 210904, 210907, 211212, 211213, 211215, 220102, 220103, 220104, 220107, 221203, 221205, 221207, 221209).

[0050] The reagents used in this invention are shown in Table 9.

[0051] Table 9: Reagents used in this invention

[0052] Reference standards: uridine reference standard (batch number: 100635-201902, purity: 99.9%), gallic acid reference standard (batch number: 110831-202407, purity: 90.8%), chlorogenic acid reference standard (batch number: 110753-202119, purity: 96.3%), vitexin reference standard (batch number: 111687-202306, purity: 99.5%), glycyrrhizin reference standard (batch number: 111610-202209, purity: 95.2%), hypericin. The reference standards for baicalin (batch number: 111521-202310: 94.7%), baicalin (batch number: 110715-202223: 97.2%), and cinnamic acid (batch number: 110786-202305, purity: 99.8%) were all purchased from the National Institutes for Food and Drug Control; the reference standards for sennain A (batch number: PS011092: 98%) and glycyrrhizic acid (batch number: 20070203, purity: 98.27%) were purchased from Chengdu Pufeide Biotechnology Co., Ltd.

[0053] Example 1 This embodiment provides a method for establishing a fingerprint spectrum of Chuantai granules using high performance liquid chromatography, including the following steps: S1, Prepare a test solution of Chuantai granules using different batches of Chuantai granules as test samples; Weigh 2.0 g of each of 15 batches of Chuantai granules, place them in a stoppered conical flask, add 20 mL of 80% methanol solution, extract by ultrasonication for 30 min, filter, evaporate to dryness, add 5 mL of water to reconstitute, and filter the solution twice through a 0.45 µm microporous membrane to obtain the Chuantai granules test solution.

[0054] S2, a single reference solution was prepared using uridine, gallic acid, chlorogenic acid, vitexin, glycyrrhizin, hyperoside, baicalin, sennain A, cinnamic acid and glycyrrhizic acid as reference standards.

[0055] Accurately weigh uridine, gallic acid, chlorogenic acid, vitexin, glycyrrhizin, hyperoside, baicalin, sennain A, cinnamic acid, and glycyrrhizic acid into methanol to prepare single reference solutions containing 0.69 mg uridine, 0.62 mg gallic acid, 0.63 mg chlorogenic acid, 0.56 mg vitexin, 0.64 mg glycyrrhizin, 0.67 mg hyperoside, 0.89 mg baicalin, 0.56 mg sennain A, 0.76 mg cinnamic acid, and 0.75 mg glycyrrhizic acid per mL.

[0056] S3. Inject the Chuantai granules test solution and the single reference solution separately into the high-performance liquid chromatograph (HPLC) and perform chromatographic analysis under the same conditions, recording the corresponding chromatograms (see Appendix). Figure 11 ~Appendix Figure 20 ); The liquid chromatography conditions were as follows: column: Shim-pack VP-ODS (250 mm × 4.6 mm, 5 μm) column; detector: diode array detector, detection wavelength: 275 nm; 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.

[0057] S4. Import the chromatograms of different batches of test sample solutions and the chromatogram of a single reference solution obtained in S3 into the chromatographic fingerprint similarity evaluation system for traditional Chinese medicine and perform similarity analysis to confirm the reliability of the results.

[0058] The chromatograms of 15 batches of Chuantai granules test solutions and reference solutions were imported into the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (Version 2012 A). Chromatographic peaks present in all chromatograms of different batches of Chuantai granules test solutions were selected as common peaks. A reference chromatogram of Chuantai granules was generated using the average value method, and the relative retention time and relative peak area of ​​each common peak were calculated. After data import, multi-point correction, and data matching, similarity analysis was performed. A similarity result table between the chromatograms of different batches of Chuantai granules test solutions and the common peak patterns was obtained and exported. The reliability of the results was confirmed based on the similarity result table and the chromatograms of the Chuantai granules test solutions.

[0059] S5, based on the chromatograms of different batches of test sample solutions obtained in S3, high-resolution mass spectrometry analysis was performed to obtain the total ion chromatogram ( Figures 21-22 Based on the total ion chromatogram and the peak elution data of the test sample obtained in S3, mass spectrometry results of each chemical component were obtained. Figures 23-44 ).

[0060] The high-resolution mass spectrometry detection conditions were: electrospray ionization, spray voltage 3500 V, sheath gas flow rate 40 arb, auxiliary gas flow rate 10 arb, capillary temperature 300℃, auxiliary gas temperature 300℃, full scan mode, and mass-to-charge ratio scan range m / z of 100-1500.

[0061] The test solution was subjected to high-resolution mass spectrometry analysis to obtain a total ion chromatogram. The total ion chromatogram was then imported into Xcalibur software. Data analysis was performed based on the peak elution of the chromatogram of the test solution to obtain the mass spectrometry results of each chemical component. S6. The chromatograms of the test sample and the reference sample obtained in S3, the total ion chromatogram obtained in S4, and the mass spectra of each chemical component are compared to obtain the fingerprint spectrum of the Chuantai particles composed of common characteristic peaks.

[0062] Using baicalin (peak 27) as a reference peak, peak 2 was identified as uridine, peak 5 as gallic acid, peak 13 as chlorogenic acid, peak 18 as vitexin, peak 19 as glycyrrhizin, peak 20 as hyperoside, peak 31 as styracin A, peak 33 as cinnamic acid, and peak 35 as glycyrrhizic acid, thus obtaining the fingerprint spectrum of Chuantai granules. Figure 45 ).

[0063] Meanwhile, this invention uses an automatically generated reference chromatogram R to generate a common chromatographic peak mode. Analysis and calculation show that the common chromatographic peaks of 15 batches of Chuantai granules have relatively good similarity, indicating that the fingerprint chromatogram of Chuantai granules established by this method can effectively detect the quality of Chuantai granules and 15 batches of Chuantai granules. The results are shown in Table 10.

[0064] Table 10: Similarity between each batch of samples and the common chromatographic peak pattern

[0065] Example 2 Methodological research on feature map detection methods.

[0066] (1) Precision study The test solution prepared by the method in Example 1 was analyzed according to the detection method in Example 1. Six parallel injections were performed 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 device is good.

[0067] Table 10: Peak area and retention time in precision studies

[0068] (2) Stability study The test solution prepared by the method in Example 1 was analyzed according to the detection method in Example 1. Injections were performed at different times (0, 2, 6, 12, 18, and 24 hours) with an injection volume of 10 μL. Hyperoside was used as the reference peak. The peak area and retention time of the common peaks in the HPLC fingerprint of the sample were analyzed, and the RSD value was calculated. The results showed that the RSD of the relative retention time was less than 0.668%, and the RSD of the relative peak area was less than 0.803%, indicating that the chromatographic peaks of the Chuantai granules test solution showed almost no change within 24 hours, demonstrating good stability.

[0069] Table 11: Peak area and retention time in stability studies

[0070] (3) Reproducibility studies Six batches of sample solutions were prepared according to the test solution method in Example 1. Under the chromatographic conditions of Example 1, the injection volume was 10 μL. Hyperoside was used as the reference peak. The peak area and retention time of the common peaks in the HPLC fingerprint of the samples were analyzed and the RSD value was calculated. The results showed that the RSD of the relative retention time was less than 0.534% and the RSD of the relative peak area was less than 0.496%, indicating that the sample chromatographic peaks had good reproducibility and the method had good repeatability.

[0071] Table 12: Peak area and retention time in repeatability studies

[0072] The above experimental results show that the method and application of establishing fingerprint chromatograms of Chuantai granules using high performance liquid chromatography provided by the present invention have the characteristics of good stability, high precision, and good repeatability. It can comprehensively and objectively evaluate the quality of Chuantai granules and provide quality assurance for clinical efficacy.

[0073] The above embodiments are merely exemplary models of the present invention and are not intended to limit the present invention. The scope of protection of the present invention is defined by the claims. The above content is only used as an example and illustration of the concept of the present invention. Various modifications or additions made by those skilled in the art to the described specific embodiments, or substitutions made in a similar manner, as long as they do not deviate from the inventive concept or exceed the scope defined by the claims, shall fall within the scope of protection of the present invention.

Claims

1. A method for establishing the fingerprint of Chuan Tai granules by high performance liquid chromatography, characterized in that, Includes the following steps: S1. Take the contents of different batches of Chuantai granules, add methanol solution, extract by ultrasonication, filter, filter through a microporous membrane to obtain Chuantai granule test solution. S2, using uridine, gallic acid, chlorogenic acid, vitexin, glycyrrhizin, hyperoside, baicalin, sennain A, cinnamic acid and glycyrrhizic acid as reference standards, prepare single reference standard solutions; S3, inject the test solution of Chuantai granules and the single reference solution into the high performance liquid chromatograph, perform chromatographic analysis under the same conditions, and record the corresponding chromatograms; The high-performance liquid chromatography (HPLC) conditions were as follows: Shim-pack VP-ODS column; mobile phase: acetonitrile-0.1% formic acid aqueous solution; gradient elution program as follows: ； S4. The chromatograms of the test solution of Chuantai granules and the single reference solution obtained in S3 were imported into the Chinese medicine chromatographic fingerprint similarity evaluation system and similarity analysis was performed to confirm the reliability of the results. S5. Based on the chromatogram of the Chuantai granules test solution obtained in S3, high-resolution mass spectrometry analysis was performed to obtain the total ion chromatogram; based on the peak elution of the total ion chromatogram and the test sample chromatogram, data analysis was performed to obtain the mass spectrometry results of each chemical component; S6. The chromatograms of the test sample and the reference sample obtained in S3, the total ion chromatogram obtained in S5, and the mass spectra of each chemical component are compared to obtain the fingerprint spectrum of the Chuantai particles composed of common characteristic peaks. 2.The method for establishing the fingerprint of Chuan Tai granules by high performance liquid chromatography according to claim 1, characterized in that, In S2, a single reference solution was prepared by dissolving uridine, gallic acid, chlorogenic acid, vitexin, glycyrrhizin, hyperoside, baicalin, sennain A, cinnamic acid, and glycyrrhizic acid reference standards in methanol. 3.The method for establishing the fingerprint of Chuan Tai granules by high performance liquid chromatography according to claim 1, characterized in that, The high-performance liquid chromatography (HPLC) detection wavelength is 210~290nm, the column temperature is 25~35℃, and the flow rate is 0.6~1.0 mL / min.

4. The method for establishing the fingerprint of Chuan Tai granules by high performance liquid chromatography according to claim 1, characterized in that, S4 specifically involves: importing the chromatograms of different batches of Chuantai granules test solutions and reference solutions obtained in S2 into the Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System; selecting chromatographic peaks present in all batches of Chuantai granules test solutions as common peaks, generating a reference chromatogram of Chuantai granules using 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 a similarity result table between the chromatograms of different batches of Chuantai granules test solutions and the common peak patterns; and confirming the reliability of the results based on the similarity result table and the chromatograms of Chuantai granules test solutions.

5. The method for establishing the fingerprint of Chuan Tai granules by high performance liquid chromatography 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℃, auxiliary gas temperature 300℃, scanning mode is full scan mode, and mass-to-charge ratio scanning range m / z is 100-1500.

6. The method for establishing the fingerprint of Chuan Tai granules by high performance liquid chromatography according to claim 1, characterized in that, S6 specifically involves comparing the chromatograms of the test sample and the reference sample obtained in S3 with the total ion chromatogram and the mass spectrometry results of the chemical components obtained by high-resolution mass spectrometry in S5. Using baicalin at peak 27 as the reference peak, peak 2 is identified as uridine, peak 5 as gallic acid, peak 13 as chlorogenic acid, peak 18 as vitexin, peak 19 as glycyrrhizin, peak 20 as hyperoside, peak 31 as phloroglucinol A, peak 33 as cinnamic acid, and peak 35 as glycyrrhizic acid, thus obtaining the fingerprint chromatogram of Chuantai granules.

7. The method for establishing the fingerprint of Chuan Tai granules by high performance liquid chromatography according to claim 1, characterized in that, After obtaining the fingerprint spectrum of Chuantai granules, an attribution analysis was performed on the fingerprint spectrum results of Chuantai granules.

8. A method for detecting the quality of Chuantai granules, characterized in that, The quality of a cyclophosphamide particle sample is determined by using the fingerprint spectrum of cyclophosphamide particles established by the method described in any one of claims 1 to 7.

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