Quality detection method of traditional Chinese medicine composition for acute and chronic pharyngitis

Abstract

The invention relates to a quality detection method of a traditional Chinese medicine composition for acute and chronic pharyngitis. The quality detection method comprises the steps of identifying components of the traditional Chinese medicine composition and determining the content of the traditional Chinese medicine composition, in the aspect of component identification, an ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry is adopted, and the method is high in resolution, high in quality precision and high in sensitivity, and can be used for rapidly and accurately identifying eight characteristic components from monarch drug scutellaria baicalensis, ministerial drugs honeysuckle and mint, adjuvant drug radix scrophulariae and conductant drug fructus chebulae immaturus at one time; the reliable technical support is provided for the definition of the material basis of the preparation; in the aspect of content determination, a high performance liquid chromatography method for simultaneously determining the components of baicalin, chlorogenic acid and gallic acid is established. Through verification, the method has the advantages of good precision, repeatability and stability, ideal sample recovery rate, simple and rapid operation and accurate and reliable result, is suitable for production process monitoring and finished product batch release inspection of the preparation, and can be used for quality control and technical reference of the traditional Chinese medicine granules.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical preparation analysis technology, and in particular to a method for quality testing of a traditional Chinese medicine composition used for acute and chronic pharyngitis. Background Technology

[0002] Traditional Chinese medicine (TCM) preparations, originating from a long history of clinical practice, play an irreplaceable role in disease prevention and treatment due to their unique advantages of holistic regulation and multi-target action. TCM granules, as a common dosage form, can address the challenges of oral liquids, such as poor stability, inconvenience in carrying, easy breakage, easy freezing, and poor taste, which hinder their widespread application in the military. Most TCM preparations are composed of multiple medicinal herbs, each of which is a complex chemical system containing numerous primary and secondary metabolites. When these herbs are decocted or extracted together, a series of physicochemical reactions, such as hydrolysis, polymerization, and oxidation, may occur between the components, resulting in a more complex chemical composition in the final preparation. This characteristic of "multi-component, synergistic effect," while potentially the basis for its efficacy, also makes it difficult to clearly define its chemical composition and identify the effective substance groups, posing significant challenges to subsequent quality control and evaluation.

[0003] Currently, the quality control of traditional Chinese medicine preparations, especially at the standard level, generally suffers from the problem of using only one indicative component and insufficient correlation with traditional Chinese medicine theory. Traditional quality control models often select only one or a few easily detectable chemical components as quality markers, which cannot comprehensively reflect the overall chemical information of compound preparations guided by the "principal, assistant, adjuvant, and guide" formulation theory.

[0004] Jin Sang Granules, based on traditional Chinese medicine theory, uses Scutellaria baicalensis and Isatis indigotica, bitter and cold in nature, to clear heat and detoxify the lungs, as the principal herbs; Lonicera japonica, Mentha haplocalyx, and Cicadae Periostracum, pungent and cool in nature, to disperse heat and relieve stagnation, assisting in clearing heat and detoxifying, as the assistant herbs; Scrophularia ningpoensis, salty, cold, and moistening, serves to clear heat and detoxify, and nourish yin and moisten the lungs, preventing excessive heat from damaging yin, as the adjuvant herbs; Terminalia chebula and Sterculia lychnophora clear the throat and benefit the lungs, promote lung function and open the voice, and together with the above herbs, can guide the medicinal properties directly to the affected area, thus serving as adjuvant herbs. Jin Sang Granules can be used for acute and chronic pharyngitis. However, current technology lacks a systematic quality control method corresponding to this complete formulation theory. How to establish a method that can simultaneously identify the characteristic components of each of the principal, assistant, adjuvant, and adjuvant herbs, and accurately quantify multiple key effective components, thereby scientifically and comprehensively evaluating the intrinsic quality of Jin Sang Granules, has become an urgent technical problem to be solved. Summary of the Invention

[0005] In view of the shortcomings of existing technologies, this invention proposes a quality testing method for traditional Chinese medicine granules used in acute and chronic pharyngitis.

[0006] The technical solution of this invention is as follows: A method for quality testing of a traditional Chinese medicine composition used for acute and chronic pharyngitis includes the following steps: S1. Ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry was used to identify whether the traditional Chinese medicine granules contained eight components: gallic acid, neochlorogenic acid, chlorogenic acid, corilagin, verbascoside, rosmarinic acid, baicalin, and wogonin. S2. The content of at least one of baicalin, chlorogenic acid and gallic acid in the traditional Chinese medicine composition was determined by high performance liquid chromatography; The traditional Chinese medicine composition is a preparation made from Scutellaria baicalensis, Lonicera japonica, Isatis indigotica, Mentha haplocalyx, Scrophularia ningpoensis, Sterculia lychnophora, Terminalia chebula, and Cicadae periostracum as raw materials.

[0007] Further, in step S1, the identification includes the following steps: S11. Take the Chinese herbal medicine composition, extract it with methanol by ultrasonication, centrifuge it, and take the supernatant to prepare a test solution for testing; S12. Dissolve the standards of the eight components to be identified in methanol solution and mix to obtain a mixed reference solution for testing; S13. Inject the sample under the set liquid chromatography and mass spectrometry conditions, and use ultra-high performance liquid chromatography in tandem quadrupole time-of-flight mass spectrometry to detect the test solution and the reference solution respectively; S14. The components in the traditional Chinese medicine composition are qualitatively identified by comparing the mass-to-charge ratio and retention time of each component in the test solution and the reference solution.

[0008] Further, in step S13, the conditions for liquid chromatography include: using a Saturn C18-AQ liquid chromatography column with dimensions of 2.1 × 100 mm and a diameter of 1.8 μm; mobile phase A being 0.05-0.2% formic acid-water; mobile phase B being 0.05-0.2% formic acid-acetonitrile; gradient elution; flow rate of 0.2-0.5 mL / min; injection volume of 1-10 μL; and column temperature of 30-40℃. The gradient elution conditions are shown in the table below. .

[0009] Further, in step S13, the mass spectrometry conditions are as follows: scanning is performed using an electrospray ionization source and an information-dependent acquisition mode; the collision gas is nitrogen; the nebulizer gas pressure is 20-99 psi; the scanning mass range is m / z 50-1500; the capillary voltage is 3000-5500 V for positive ion mode and -4500-0 V for negative ion mode; the declustering voltage for positive ion mode is 10-150 V and the collision energy is 20-100 V; the declustering voltage for negative ion mode is -150-0 V and the collision energy is -100-0 V; and the ion source temperature is 400-600℃.

[0010] Further, in step S2, the content determination includes the following steps: S21. Take the Chinese herbal medicine composition, extract it with methanol by ultrasonication, centrifuge it, and take the supernatant to prepare a test solution; S22. Take the standard of the component to be tested, dissolve and dilute it with methanol to prepare a series of reference solutions of different concentrations; S23. High performance liquid chromatography was used to detect a series of concentrations of reference solutions and test solutions; S24. Plot a standard curve with the concentration of the reference solution as the x-axis and the peak area as the y-axis to obtain a regression equation. Substitute the peak area of ​​the analyte in the test solution into the regression equation to calculate its content.

[0011] Further, in step S23, the high-performance liquid chromatography (HPLC) conditions include: using a ShimNex CS C18 column (4.6 mm × 250 mm, 5 μm), with 0.05-0.2% formic acid aqueous solution as mobile phase A and acetonitrile as mobile phase B, performing gradient elution at a flow rate of 0.5-1.0 mL / min, an injection volume of 5-20 μL, a column temperature of 30-40℃, and detection using a photodiode array detector in the wavelength range of 190-800 nm; the gradient elution conditions are shown in the table below: .

[0012] Furthermore, the Chinese medicinal materials in the Chinese herbal composition are, by weight, 1-3 parts of Scutellaria baicalensis, 1-3 parts of Lonicera japonica, 1-3 parts of Isatis indigotica, 1-3 parts of Mentha haplocalyx, 1-3 parts of Scrophularia ningpoensis, 1-3 parts of Sterculia lychnophora, 1-3 parts of Terminalia chebula, and 1-3 parts of Cicadae periostracum.

[0013] Furthermore, the traditional Chinese medicine composition is in the form of granules.

[0014] The application of the quality testing method in the quality control of traditional Chinese medicine compositions for treating acute and chronic pharyngitis.

[0015] Compared with the prior art, the present invention has at least the following advantages: This invention relates to a quality testing method for a traditional Chinese medicine composition used for acute and chronic pharyngitis. The method includes steps for identifying the components of the traditional Chinese medicine composition and determining its content. For component identification, ultra-high performance liquid chromatography (UHPLC) tandem quadrupole time-of-flight mass spectrometry (QTF-MS) is employed. This method features high resolution, high quality precision, and high sensitivity, enabling rapid and accurate simultaneous identification of eight characteristic components from the principal herb Scutellaria baicalensis, the assistant herb Lonicera japonica, Mentha haplocalyx, the adjuvant herb Scrophularia ningpoensis, and the guiding herb Terminalia chebula, providing reliable technical support for clarifying the material basis of the preparation. For content determination, a high-performance liquid chromatography method for simultaneously determining three key indicator components—baicalin, chlorogenic acid, and gallic acid—has been established. This method has been validated, demonstrating good precision, repeatability, and stability, ideal recovery rate, simple and rapid operation, and accurate and reliable results. It is suitable for monitoring the production process and releasing finished batches of this preparation, and can be used for quality control and technical reference of the traditional Chinese medicine granules. Attached Figure Description

[0016] To more clearly illustrate the specific embodiments of the present invention, the accompanying drawings used in the description of the specific embodiments or the prior art will be briefly introduced below.

[0017] Figure 1 The total ion chromatogram of the test solution in the information-dependent acquisition (IDA) mode in Embodiment 1 of the present invention is shown. Figure 2 The ion chromatogram of the reference solution in the first embodiment of the present invention is extracted in information-dependent acquisition (IDA) mode; Figure 3 This is a comparison diagram of the secondary ion characteristics of baicalin in the test solution and the reference solution in Example 1 of the present invention; Figure 4 This is a comparison diagram of the secondary ion characteristics of baicalin in the test solution and the reference solution in Example 1 of the present invention; Figure 5 This is a comparison diagram of the secondary ion characteristics of Coraligines in the test solution and the reference solution in Example 1 of the present invention; Figure 6 This is a comparison diagram of the secondary ion characteristics of gallic acid in the test solution and the reference solution in Example 1 of the present invention; Figure 7 This is a comparison diagram of the secondary ion characteristics of chlorogenic acid in the test solution and the reference solution in Example 1 of the present invention; Figure 8 This is a comparison diagram of the secondary ion characteristics of neochlorogenic acid in the test solution and the reference solution in Example 1 of the present invention; Figure 9 This is a comparison diagram of the secondary ion characteristics of verbascoside in the test solution and the reference solution in Example 1 of the present invention; Figure 10 This is a comparison diagram of the secondary ion characteristics of rosmarinic acid in the test solution and the reference solution in Example 1 of the present invention; Figure 11 The image shows the separation chromatograms of baicalin, chlorogenic acid, and gallic acid in Example 2 of this invention. Figure 12 This is a standard curve diagram of baicalin, chlorogenic acid, and gallic acid in Example 3 of the present invention.

[0018] The accompanying drawings are for illustrative purposes only and should not be construed as limiting the scope of this patent. To better illustrate this embodiment, some components in the drawings may be omitted, enlarged, or reduced, and do not represent the actual dimensions of the product. It is understandable to those skilled in the art that some well-known structures and their descriptions may be omitted in the drawings. The same or similar reference numerals correspond to the same or similar components. The terms describing positional relationships in the drawings are for illustrative purposes only and should not be construed as limiting the scope of this patent. Detailed Implementation

[0019] The present invention will now be described in further detail. It should be noted that the following specific embodiments are only used to further illustrate the present invention and should not be construed as limiting the scope of protection of the present invention. Those skilled in the art can make some non-essential improvements and adjustments to the present invention based on the above application content.

[0020] This invention provides a general and / or specific description of the materials and experimental methods used in the experiments. Unless otherwise specified, all experimental or testing methods are conventional methods; all reagents or instruments used, unless otherwise specified, are commercially available conventional products prepared or used using conventional methods.

[0021] The experimental equipment involved in this invention is as follows.

[0022] Experimental apparatus: ExionLC ultra-high performance liquid chromatograph: Spectroscopy Labs, Inc., USA; LX-40D XR high performance liquid chromatograph: Shimadzu, Japan; AB X500B high resolution mass spectrometer: AB SCIEX, Inc., USA; SPD-M40 photodiode array detector: Shimadzu, Japan; BT25S 0.0001 g electronic balance: Sartorius Group, Germany; Saturn C18-AQ liquid chromatography column: 2.1 × 100 mm, 1.8 μm, Aichiming Technology Co., Ltd.; ShimNex CS C18 liquid chromatography column: 4.6 mm × 250 mm, 5 μm, Shimadzu, Japan; Mingche-D24UV ultrapure water system: Merck Millipore Group, France; Allegra X-30R high-speed refrigerated centrifuge: Beckman Coulter, Inc., USA.

[0023] Experimental reagents: Acetonitrile: chromatographic grade, Honeywell Trading (Shanghai) Co., Ltd.; Methanol: chromatographic grade, Honeywell Trading (Shanghai) Co., Ltd.; All other reagents were of analytical grade.

[0024] Experimental subjects: The traditional Chinese medicine granules described in this patent are produced by the Army Special Medical Center of the Chinese People's Liberation Army. Specific preparation method: Take 1-3 parts of Scutellaria baicalensis, 1-3 parts of Lonicera japonica, 1-3 parts of Isatis indigotica, 1-3 parts of Scrophularia ningpoensis, 1-3 parts of Sterculia lychnophora, 1-3 parts of Terminalia chebula, and 1-3 parts of Cicadae periostracum. Add 8-12 times the volume of water and soak for 1-3 hours. Decoct, adding 1-3 parts of peppermint slices 10 minutes before the end of decoction. After decoction, filter while hot using gauze and collect the filtrate. Decoct a total of 3 times, 1-3 hours each time. Combine the filtrate and the seawater extract of Sterculia lychnophora to obtain an aqueous extract of the traditional Chinese medicine granules. Concentrate the aqueous extract under reduced pressure to obtain a clear extract with a relative density of 1.05-1.30 (45-70℃, measured hot). Dissolve steviol glycosides in the clear extract, ensuring complete dissolution; the mass fraction of steviol glycosides is 2-5%. Determine the mass ratio of the solid content of the extract (1-5 parts) to the excipients (1-5 parts). When preparing granules, first dry mix the excipients pregelatinized starch (1-5 parts) and dextrin (1-5 parts) for 10-30 min, then add micronized silica gel (0.5-3%) to the mixed excipients and continue mixing for 5-20 min. Perform one-step granulation in a multifunctional fluidized bed, spraying the extract into the fluidized excipients in the form of atomization. The granulation conditions are: air inlet temperature 50-75℃, material temperature 35-60℃, fan frequency 15-45 HZ, and pump speed 3-10 rpm. After granulation, remove the granules, sieve and granulate to obtain the Chinese medicine granules.

[0025] Example 1: Identification Method of Effective Components in Traditional Chinese Medicine Granules The identification method includes the following steps: S1. Sample pretreatment: S11. Preparation of the test solution: Accurately weigh approximately 1 g of Jin Sang granules (a traditional Chinese medicine), place it in a 50 mL volumetric flask, add an appropriate amount of 50% methanol, sonicate for 30 min, allow to cool to room temperature, add 50% methanol to the mark, shake well, filter, and collect the filtrate. Transfer the filtrate to a centrifuge tube, centrifuge at 14000 rpm for 10 min, and collect the supernatant to obtain the test solution. Take 100 μL of the test solution, dilute it 10 times with 50% methanol solution, and filter through a 0.22 μm organic filter membrane to obtain the test solution, ready for analysis. S12. Preparation of reference solutions: Gallic acid, neochlorogenic acid, chlorogenic acid, corilagin, verbascoside, rosmarinic acid, baicalin, and wogonin reference standards were dissolved in methanol by sonication to prepare solutions with a concentration of 1 mg / mL, thus obtaining eight reference stock solutions with a concentration of 1 mg / mL. Equal volumes of the eight reference stock solutions were mixed and then diluted with methanol to obtain a mixed reference solution with a concentration of 1 μg / mL. The solution was filtered through a 0.22 μm organic filter membrane to obtain the reference solution for testing. S2. Detection was performed using ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry: S21. Condition settings: A Saturn C18-AQ liquid chromatography column with specifications of 2.1 × 100 mm and 1.8 μm was used. The mobile phase A was 0.1% formic acid-water and the mobile phase B was 0.1% formic acid-acetonitrile. Gradient elution was used with a flow rate of 0.3 mL / min, an injection volume of 5 μL, and a column temperature of 40℃. The gradient elution conditions are shown in Table 1. Table 1. HPLC gradient elution procedure for identifying eight active ingredients in Jin Sang granules. .

[0026] The mass spectrometry conditions were as follows: an electrospray ionization source and information-dependent acquisition mode were used for scanning; nitrogen was used as the collision gas; the nebulizer gas pressure was 50 psi; the scanning mass range was m / z 100-1500; the capillary voltage was 5500 V for positive ion mode and -4500 V for negative ion mode; the declustering voltage for positive ion mode was 80 V; the collision energy was 35±15 V; the declustering voltage for negative ion mode was -80 V; the collision energy was -35±15 V; and the ion source temperature was 550℃. S22. Sample loading and detection: The test solution and the reference solution are injected under the set liquid chromatography and mass spectrometry conditions, and the ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry is used for detection; S3. Perform qualitative analysis on the test solution based on the mass-to-charge ratio and retention time obtained in step S2 to identify the effective components of the traditional Chinese medicine granules.

[0027] The results of the identification are as follows Figure 1 and Figure 2 As shown, Figure 1 and Figure 2 In the figure, A represents negative ion peaks and B represents positive ion peaks. Based on the characteristics of the compounds such as peak time, isotope abundance ratio, first-order mass deviation, and second-order ion fragments, the effective components of some of the medicinal materials in the Chinese medicine granules were identified. Among them, some of the medicinal materials included the principal drug Scutellaria baicalensis, the assistant drugs Lonicera japonica and Mentha haplocalyx, the adjuvant drug Scrophularia ningpoensis, and the guiding drug Terminalia chebula. The results are shown in Table 2. according to Figure 1 , Figure 2 As shown in Table 2, the effective components in this traditional Chinese medicine granule include gallic acid, neochlorogenic acid, chlorogenic acid, corilagin, verbascoside, rosmarinic acid, baicalin, and wogonin, thus confirming that the medicinal materials in the formula of this traditional Chinese medicine granule include Scutellaria baicalensis as the principal ingredient, Lonicera japonica and Mentha as the assistant ingredients, Scrophularia ningpoensis as the adjuvant ingredient, and Terminalia chebula as the guiding ingredient. In this embodiment, the secondary ion characteristic diagrams are all based on the negative ion mode.

[0028] Table 2. Mass spectrometric characteristics of eight active ingredients in Jin Sang granules Among them, baicalin and wogonin are used for the identification of the principal ingredient, Scutellaria baicalensis, such as... Figure 3 and Figure 4 As shown (A represents the reference solution, B represents the test solution); gallic acid and corilagin are mainly used for the identification of the drug *Terminalia chebula*, such as... Figure 5 and Figure 6 As shown ( Figure 5 and Figure 6 (In the diagram, A represents the reference solution and B represents the test solution). Chlorogenic acid and neochlorogenic acid are mainly used for the identification of the medicinal herb honeysuckle, such as... Figure 7 and Figure 8 As shown (A represents the reference solution, B represents the test solution); verbascoside is used for the identification of the adjuvant herb Scrophularia ningpoensis, such as... Figure 9 As shown (A represents the reference solution, B represents the test solution); rosmarinic acid is mainly used for the identification of the herb peppermint, such as... Figure 10 As shown (A represents the reference solution, B represents the test solution); In summary, this embodiment establishes a method for identifying the components of this traditional Chinese medicine, identifies eight characteristic components in the granules derived from the "principal, assistant, adjuvant, and guide" medicinal flavors, and clarifies the material basis of the preparation.

[0029] Example 2: Optimization of liquid phase separation conditions for the effective components of traditional Chinese medicine granules S1. Preparation of the test solution: Accurately weigh about 1 g of Jin Sang granules, place them in a 50 mL volumetric flask, add an appropriate amount of 50% methanol, sonicate for 30 min, let it cool to room temperature, add 50% methanol to the mark, shake well, filter, and take the filtrate; pipette the filtrate into a centrifuge tube, centrifuge at 14000 rpm for 10 min, and take the supernatant to obtain the test solution. Filter the solution through a 0.22 μm organic filter membrane to obtain the test solution, ready for testing. S2. Preparation of reference solutions: Baicalin, chlorogenic acid, and gallic acid reference standards were dissolved in methanol by sonication to prepare solutions with a concentration of 1 mg / mL, thus obtaining three reference stock solutions with a concentration of 1 mg / mL. Then, equal volumes of the reference stock solutions of the three components were taken and diluted with concentrated methanol to obtain mixed solutions containing baicalin, chlorogenic acid, and gallic acid, with final concentrations of 100 μg / mL, 80 μg / mL, 50 μg / mL, 20 μg / mL, 10 μg / mL, 8 μg / mL, and 5 μg / mL, respectively, for testing. S3. Chromatographic conditions: A ShimNex CS C18 liquid chromatography column with dimensions of 4.6 mm × 250 mm and a diameter of 5 μm was used. Mobile phase A was 0.1% formic acid-water, and mobile phase B was acetonitrile. Gradient elution was used at a flow rate of 1.0 mL / min, an injection volume of 10 μL, and a column temperature of 40℃. The gradient elution program for the mobile phase is shown in Table 3. Table 3. HPLC gradient elution procedure for determining the content of three indicative components in Jin Sang granules. S4. The photodiode array detector parameters are: full wavelength scanning is used, and the detection wavelength is 190-800nm, which is used to determine the optimal ultraviolet absorption wavelength of the compound; S5. Sample loading and detection: The test solution and reference solution are injected under the liquid chromatography and mass spectrometry conditions set above, and the Shimadzu photodiode array detector is used for detection and analysis.

[0030] The experimental results are shown in Table 4. The optimal ultraviolet absorption wavelengths of the three indicative components (baicalin, chlorogenic acid, and gallic acid) of the Chinese herbal granules within the linear relationship range were determined.

[0031] Table 4. Classification, medicinal material source, and optimal ultraviolet absorption wavelength of three indicative components in Jin Sang granules. Example 3: Establishment of Standard Curve and Linear Range The reference solution prepared in step S2 of Example 2 was taken and diluted sequentially with methanol to final concentrations of 100 μg / mL, 80 μg / mL, 50 μg / mL, 20 μg / mL, 10 μg / mL, 8 μg / mL and 5 μg / mL standard working solutions. Then, the samples were loaded onto a Shimadzu photodiode array detector for detection under the chromatographic conditions in steps S3 and S4 of Example 2.

[0032] Experimental results and analysis: such as Figure 11 and Figure 12As shown, rapid and well-separated chromatograms were obtained within 50 min. Standard curves for each active ingredient were plotted with the mass concentration of the standard working solution at each concentration as the x-axis and the peak area integral as the y-axis. Linear regression was performed to obtain the correlation coefficient and regression equation, as shown in Table 5. In this embodiment, standard curves for three indicator components were established, all of which showed excellent linearity (r > 0.999) in the concentration range of 5-100 μg / mL, verifying the quantitative capability of the content determination method and ensuring the accuracy of the calculation results.

[0033] Table 5. Standard curves and linear ranges of three indicative components in Jin Sang granules. Example 4: Methodological investigation of the precision, repeatability, stability, and recovery rate of content determination. S1. Precision test: The reference solution prepared in step S2 of Example 2 was used as the sample. The sample was injected 6 times consecutively under the chromatographic conditions in steps S3 and S4 of Example 2, and the peak areas of the three components in the sample were measured. S2. Repeatability test: Six portions of Jin Sang granules were weighed in parallel and prepared in parallel using the preparation method in step S1 of Example 2 to obtain six test solutions; Six test solutions were taken and injected continuously under the chromatographic conditions in steps S3 and S4 of Example 2. The peak areas of the three components in the six test solutions were measured, and the RSD% values ​​of the mass fractions of gallic acid, baicalin, and wogonin were calculated. S3. Stability study: The test solution prepared in step S1 of Example 2 was placed at room temperature for 0h, 2h, 4h, 8h, 12h and 24h and injected into the sample under the chromatographic conditions in steps S3 and S4 of Example 2 respectively. The peak areas of the three components in the sample were measured at each time period. S4. Recovery rate test: Using the preparation method in step S1 of Example 2, accurately weigh about 0.5 g of Jin Sang granules, and add the corresponding amount of mixed reference solution according to 50% of gallic acid, baicalin and wogonin contained in the sample. The chromatographic conditions in steps S3 and S4 of Example 2 were used for sample loading and detection, and a standard curve was established using the method in Example 3 to analyze and obtain the concentration of each effective component in the test sample. The experimental results are shown in Table 6. In the precision test, the RSD of the three compounds obtained from the peak area was less than 1.34%, which proves that the method has good precision. In the repeatability test, the RSD of the three compounds obtained based on their mass fractions was less than 1.12%, proving that the method has good repeatability. In the stability study, the RSD of the three compounds obtained from the peak area was less than 1.39%, indicating that the six active ingredients have good stability within 24 hours. In the recovery study, the average recovery rates of the three compounds obtained from the mass fraction ranged from 100.21% to 101.49%, and the RSDs were all less than 0.75%, indicating high recovery rates.

[0034] Table 6. Precision, reproducibility, 24-hour stability, and recovery rate of three index components in Jin Sang granules. Example 5: A method for determining the content of active ingredients in traditional Chinese medicine granules. Experimental subjects: Three batches of Jin Sang Granules (produced by the Army Characteristic Medical Center of the Chinese People's Liberation Army) were randomly selected and labeled as A1, A2 and A3 respectively.

[0035] Experimental methods: S1. Preparation of test solutions: Accurately weigh approximately 1 g of Jin Sang granules A1, A2, and A3, place them in a 50 mL volumetric flask, add an appropriate amount of 50% methanol, sonicate for 30 min, allow to cool to room temperature, add 50% methanol to the mark, shake well, filter, and collect the filtrate. Transfer the filtrate to a centrifuge tube, centrifuge at 14000 rpm for 10 min, collect the supernatant to obtain the test solution stock solution, filter through a 0.22 μm organic filter membrane to obtain test solutions A1, A2, and A3, ready for testing. S2. Sample injection and analysis: Take the test solutions A1, A2, and A3, and then perform detection using the chromatographic conditions in steps S3 and S4 of Example 2; and calculate according to the regression equation in Example 3.

[0036] S3. Experimental Results: The contents of the three active ingredients in Jin Sang Granules A1, A2 and A3 are shown in Table 7. The average contents of baicalin, chlorogenic acid and gallic acid are 2.50 ± 0.17 mg / g, 0.74 ± 0.01 mg / g and 3.21 ± 0.06 mg / g, respectively.

[0037] Table 7. Results of content determination of three indicator components in three batches of Jin Sang granules This embodiment applies the established method to the testing of three batches of actual products. The results accurately show the content of the three indicator components in each batch, and the differences between batches are small, which fully demonstrates the application value and feasibility of the method in actual quality control.

[0038] In summary, the content detection method of the present invention has high sensitivity, good reproducibility, and accurate analysis. Furthermore, the pretreatment of the analyte is simple and the analysis is rapid, enabling rapid qualitative and quantitative quality control analysis of the traditional Chinese medicine granules described in the present invention.

[0039] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be covered within the scope of the claims and specification of the present invention.

Claims

1. A method for quality testing of a traditional Chinese medicine composition used for acute and chronic pharyngitis, characterized in that, Includes the following steps: S1. Ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry was used to identify whether the traditional Chinese medicine granules contained eight components: gallic acid, neochlorogenic acid, chlorogenic acid, corilagin, verbascoside, rosmarinic acid, baicalin, and wogonin. S2. The content of at least one of baicalin, chlorogenic acid and gallic acid in the traditional Chinese medicine composition was determined by high performance liquid chromatography; The traditional Chinese medicine composition is a preparation made from Scutellaria baicalensis, Lonicera japonica, Isatis indigotica, Mentha haplocalyx, Scrophularia ningpoensis, Sterculia lychnophora, Terminalia chebula, and Cicadae periostracum as raw materials.

2. The quality inspection method according to claim 1, characterized in that, In step S1, the identification includes the following steps: S11. Take the Chinese herbal medicine composition, extract it with methanol by ultrasonication, centrifuge it, and take the supernatant to prepare a test solution for testing; S12. Dissolve the standards of the eight components to be identified in methanol solution and mix to obtain a mixed reference solution for testing; S13. Inject the sample under the set liquid chromatography and mass spectrometry conditions, and use ultra-high performance liquid chromatography in tandem quadrupole time-of-flight mass spectrometry to detect the test solution and the reference solution respectively; S14. The components in the traditional Chinese medicine composition are qualitatively identified by comparing the mass-to-charge ratio and retention time of each component in the test solution and the reference solution.

3. The quality inspection method according to claim 2, characterized in that, In step S13, the liquid chromatography conditions include: using a Saturn C18-AQ liquid chromatography column with dimensions of 2.1 × 100 mm and a diameter of 1.8 μm; mobile phase A being 0.05-0.2% formic acid-water; mobile phase B being 0.05-0.2% formic acid-acetonitrile; gradient elution; flow rate of 0.2-0.5 mL / min; injection volume of 1-10 μL; and column temperature of 30-40℃. The gradient elution conditions are shown in the table below. 。 4. The quality inspection method according to claim 2, characterized in that, In step S13, the mass spectrometry conditions are as follows: an electrospray ionization source and an information-dependent acquisition mode are used for scanning; the collision gas is nitrogen; the nebulizer gas pressure is 20-99 psi; the scanning mass range is m / z 50-1500; the capillary voltage is 3000-5500 V for positive ion mode and -4500-0 V for negative ion mode; the declustering voltage for positive ion mode is 10-150 V and the collision energy is 20-100 V; the declustering voltage for negative ion mode is -150-0 V and the collision energy is -100-0 V; and the ion source temperature is 400-600℃.

5. The quality inspection method according to claim 1, characterized in that, In step S2, the content determination includes the following steps: S21. Take the Chinese herbal medicine composition, extract it with methanol by ultrasonication, centrifuge it, and take the supernatant to prepare a test solution; S22. Take the standard of the component to be tested, dissolve and dilute it with methanol to prepare a series of reference solutions of different concentrations; S23. High performance liquid chromatography was used to detect a series of concentrations of reference solutions and test solutions; S24. Plot a standard curve with the concentration of the reference solution as the x-axis and the peak area as the y-axis to obtain a regression equation. Substitute the peak area of ​​the analyte in the test solution into the regression equation to calculate its content.

6. The quality inspection method according to claim 5, characterized in that, In step S23, the high-performance liquid chromatography (HPLC) conditions include: using a ShimNex CS C18 column (4.6 mm × 250 mm, 5 μm), with 0.05-0.2% formic acid aqueous solution as mobile phase A and acetonitrile as mobile phase B, performing gradient elution at a flow rate of 0.5-1.0 mL / min, an injection volume of 5-20 μL, a column temperature of 30-40℃, and detection using a photodiode array detector in the wavelength range of 190-800 nm; the gradient elution conditions are shown in the table below. 。 7. The quality inspection method according to any one of claims 1-6, characterized in that, The Chinese medicinal materials in the composition are, by weight, 1-3 parts of Scutellaria baicalensis, 1-3 parts of Lonicera japonica, 1-3 parts of Isatis indigotica, 1-3 parts of Mentha haplocalyx, 1-3 parts of Scrophularia ningpoensis, 1-3 parts of Sterculia lychnophora, 1-3 parts of Terminalia chebula, and 1-3 parts of Cicadae periostracum.

8. The quality inspection method according to any one of claims 1-6, characterized in that, The traditional Chinese medicine composition is in granule form.

9. The application of the quality testing method according to any one of claims 1-8 in the quality control of traditional Chinese medicine compositions for treating acute and chronic pharyngitis.

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