A characteristic chromatogram construction method and a component content determination method of Jingzhuqingjie granules

Abstract

The application belongs to the technical field of pharmaceutical analysis, and particularly relates to a characteristic spectrum construction method and a component content determination method of Jingzhu Qingjie granules. The high performance liquid chromatography method is used for determination, and the conditions of the high performance liquid chromatography method include that octadecylsilane bonded silica gel is used as a filler of a chromatographic column; acetonitrile is used as a mobile phase A, and a 0.05-0.2% phosphoric acid aqueous solution is used as a mobile phase B, and gradient elution is performed. The method has strong specificity, good repeatability and durability, high stability and high accuracy; the constructed characteristic spectrum and the detection method can quickly and comprehensively realize the purpose of combined control of multiple components in Jingzhu Qingjie granules.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical analysis technology, and in particular relates to a method for constructing characteristic spectra of Jingzhu Qingjie granules and a method for determining the content of its components. Background Technology

[0002] Currently, there is a lack of quality testing methods for Jingzhu Qingjie Granules. Therefore, it is necessary to establish an HPLC method for the determination of the content and characteristic chromatograms of Jingzhu Qingjie Granules, so as to provide a basis for effectively controlling and comprehensively evaluating the quality of Jingzhu Qingjie Granules. Summary of the Invention

[0003] This invention aims to solve at least one of the technical problems existing in the prior art. To this end, this invention proposes a method for constructing characteristic spectra and a method for determining the component content of Jingzhu Qingjie granules, providing assurance for the quality control of Jingzhu Qingjie granules.

[0004] In one aspect, the present invention provides a method for constructing a characteristic spectrum of Jingzhu Qingjie granules, comprising: determining the common peaks by high performance liquid chromatography of a reference solution and a test solution, respectively; The conditions for the high-performance liquid chromatography method include: The chromatographic column was packed with octadecylsilane-bonded silica gel. Acetonitrile was used as mobile phase A, and a 0.05-0.2% (v / v) aqueous solution of phosphoric acid was used as mobile phase B for gradient elution. The conditions for gradient elution are as follows:

[0005] The percentage sign in the table means volume percentage.

[0006] According to some embodiments of the present invention, the mobile phase B is a phosphoric acid aqueous solution with a volume percentage of 0.1%.

[0007] According to some embodiments of the present invention, the chromatographic column is a ZORBAX Eclipse XDB-C18.

[0008] According to some embodiments of the present invention, the chromatographic column has dimensions of 4.6 mm × 250 mm and a diameter of 5 μm.

[0009] According to some embodiments of the present invention, the conditions of the high performance liquid chromatography method further include: the flow rate of the mobile phase is 0.5~1.0 mL / min; the column temperature of the chromatographic column is 30~35℃, preferably 30℃; the injection volume is 5~20 μL; and the detection wavelength is 280 nm.

[0010] According to some embodiments of the present invention, the detector for the high-performance liquid chromatography is an ultraviolet detector.

[0011] According to some embodiments of the present invention, the theoretical plate number of the high performance liquid chromatography, calculated based on the forsythoside A peak, is not less than 2000.

[0012] According to some embodiments of the present invention, the construction method further includes the preparation of a test solution; the preparation step includes: mixing the test sample with a first solvent, and ultrasonically extracting to obtain the test solution.

[0013] According to some embodiments of the present invention, the mass-to-volume ratio of the test sample to the first solvent is 0.5-50 mg:1 mL. Preferably, the first solvent is selected from alcohol solvents, water, or an alcohol-water mixture. More preferably, the alcohol solvent is selected from methanol or ethanol, and the alcohol-water mixture is an aqueous methanol solution with a volume percentage of 30-70% (e.g., 50%) or an aqueous ethanol solution with a volume percentage of 30-70% (e.g., 50%).

[0014] According to some embodiments of the present invention, the mass concentration of the test solution is 0.5~50 mg / mL.

[0015] According to some embodiments of the present invention, the ultrasound conditions include at least one of the following i-iii: i. Power: 400~600W; ii. Frequency: 30~50kHz; iii. Time: 20~40min.

[0016] According to some embodiments of the present invention, the construction method further includes the preparation of a reference solution, the preparation step comprising: mixing the reference with a second solvent to obtain the reference solution.

[0017] According to some embodiments of the present invention, the reference standard includes forsythoside A, chicoric acid, 5-hydroxymethylfurfural, forsythoside I, arctiin, fraxin, and caffeic acid.

[0018] According to some embodiments of the present invention, the mass concentration of the reference solution is 5~300 µg / mL.

[0019] According to some embodiments of the present invention, the second solvent is selected from alcohol solvents, water, or a mixture of alcohol and water. More preferably, the alcohol solvent is selected from methanol or ethanol, and the mixture of alcohol and water is an aqueous solution of methanol or an aqueous solution of 30-70% (e.g., 50%) by volume.

[0020] According to some embodiments of the present invention, the characteristic spectrum includes eight characteristic peaks, wherein the peak corresponding to the forsythoside A peak is designated as peak 5, and peak 5 is used as a reference peak. The relative retention times of the other characteristic peaks and the reference peak are calculated, and the relative retention times should be within ±10% of a specified value. Peaks 1, 2, 3, 4, 6, and 7 correspond to the 5-hydroxymethylfurfural peak, fraxin B peak, caffeic acid peak, forsythoside I peak, chicoric acid peak, and arctiin peak, respectively. The specified values ​​are: peak 1: 0.18, peak 2: 0.42, peak 3: 0.45, peak 4: 0.79, peak 6: 1.10, peak 7: 1.41, and peak 8: 1.74.

[0021] Another aspect of the present invention provides a method for determining the content of Jingzhu Qingjie granules, which is determined by high performance liquid chromatography (HPLC), wherein the conditions of the HPLC method include: The chromatographic column was packed with octadecylsilane-bonded silica gel. Acetonitrile was used as mobile phase A, and a 0.05-0.2% (v / v) aqueous solution of phosphoric acid was used as mobile phase B for gradient elution. The conditions for gradient elution are as follows:

[0022] The percentage sign in the table means volume percentage.

[0023] According to some embodiments of the present invention, the external standard method is used for determination.

[0024] According to some embodiments of the present invention, the mobile phase B is a phosphoric acid aqueous solution with a volume percentage of 0.1%.

[0025] According to some embodiments of the present invention, the chromatographic column is a ZORBAX Eclipse XDB-C18.

[0026] According to some embodiments of the present invention, the chromatographic column has dimensions of 250 mm × 4.6 mm and 5 µm.

[0027] According to some embodiments of the present invention, the conditions of the high performance liquid chromatography method further include: the flow rate of the mobile phase is 0.5~1.0 mL / min; the column temperature is 30~35℃, preferably 30℃; the injection volume is 5~20 μL, preferably 10 μL; and the detection wavelength is 280 nm or 327 nm.

[0028] According to some embodiments of the present invention, the detector for the high-performance liquid chromatography is an ultraviolet detector.

[0029] According to some embodiments of the present invention, the theoretical plate number of the high performance liquid chromatography, calculated based on the forsythoside A peak, is not less than 2000.

[0030] According to some embodiments of the present invention, the content determination method further includes the preparation of a test solution; the preparation step includes: mixing the test sample with a first solvent, ultrasonically extracting, and obtaining the test solution.

[0031] According to some embodiments of the present invention, the mass-to-volume ratio of the test sample to the first solvent is 0.5-50 mg:1 mL. Preferably, the first solvent is selected from alcohol solvents, water, or an alcohol-water mixture. More preferably, the alcohol solvent is selected from methanol or ethanol, and the alcohol-water mixture is an aqueous methanol solution with a volume percentage of 30-70% (e.g., 50%) or an aqueous ethanol solution with a volume percentage of 30-70% (e.g., 50%).

[0032] According to some embodiments of the present invention, the mass concentration of the test solution is 0.5~50 mg / mL.

[0033] According to some embodiments of the present invention, the ultrasound conditions include at least one of the following i-iii: i. Power: 400~600W; ii. Frequency: 30~50kHz; iii. Time: 20~40min.

[0034] According to some embodiments of the present invention, the content determination method further includes the preparation of a reference solution, wherein the preparation step includes: mixing the reference standard with a second solvent to obtain the reference solution.

[0035] According to some embodiments of the present invention, the reference standard includes forsythoside A and arctiin.

[0036] According to some embodiments of the present invention, the mass concentration of the reference solution is 5~300 µg / mL.

[0037] According to some embodiments of the present invention, the second solvent is selected from alcohol solvents, water, or a mixture of alcohol and water. More preferably, the alcohol solvent is selected from methanol or ethanol, and the mixture of alcohol and water is an aqueous solution of methanol or an aqueous solution of 30-70% (e.g., 50%) by volume.

[0038] In another aspect, the present invention provides the application of the above-described content determination method or construction method in detecting the quality of Jingzhu Qingjie granules.

[0039] Beneficial effects: This invention provides a characteristic chromatogram and content determination method for Jingzhu Qingjie granules, identifying eight common peaks, including forsythoside A. The constructed HPLC characteristic chromatogram shows good separation of chromatographic peaks, rich characteristic chromatographic information, and good peak shape, enabling overall control of characteristic components in Jingzhu Qingjie granules and effectively ensuring the overall quality stability of Jingzhu Qingjie granules. Furthermore, this method is simple to operate and has advantages such as high precision, good stability, good repeatability, and high accuracy.

[0040] Other features and advantages of the invention will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the invention. Attached Figure Description

[0041] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which: Figure 1 This is a chromatogram showing the system suitability of Jingzhu Qingjie granules in Example 1; Figure 2 This is a chromatogram of the mobile phase used in Example 1. Figure 3 This is a chromatogram of the elution gradient investigated in Example 1; Figure 4 The characteristic spectrum of the blank solution in Example 1; Figure 5 These are the characteristic spectra of the reference solutions in Example 1; Figure 6 The characteristic chromatogram of the test solution of Jingzhu Qingjie Granules in Example 1; Figure 7 HPLC overlay chromatograms showing the peak assignments for each herb in Jingzhu Qingjie Granules; where JZKL: Jingzhu Qingjie Granules; JJ: Schizonepeta tenuifolia; FF: Saposhnikovia divaricata; SSG: Gypsum fibrosum; LQ: Forsythia suspensa; PGY: Taraxacum mongolicum; ZHDD: Viola yedoensis; DZY: Lophatherum gracile; LG: Phragmites communis; JG: Platycodon grandiflorus; CKXR: Roasted bitter almond; NBZ: Arctium lappa; JNJ: Chicken gizzard lining; JSZ: Roasted hawthorn; GC: Glycyrrhiza uralensis; Figure 8 Chromatogram of the retention time of the test sample of Jingzhu Qingjie Granules; Figure 9 This is a superimposed image of the characteristic spectra of three batches of Jingzhu Qingjie Granules. Among them, peak 1: 5-hydroxymethylfurfural; peak 2: fraxin; peak 3: caffeic acid; peak 4: forsythoside I; peak 5 (S): forsythoside A; peak 6: chicoric acid; peak 7: arctiin. Figure 10 The characteristic spectra of each reference solution in Comparative Example 1; Figure 11The characteristic chromatogram of the test solution of Jingzhu Qingjie Granules in Comparative Example 1 is shown below. Peak 1: 5-hydroxymethylfurfural; Peak 2: Fraxinus chinensis; Peak 3: Caffeic acid; Peak 4: Forsythoside I; Peak 5: Forsythoside A; Peak 6: Chicoric acid; Peak 7: Arctiin. Figure 12 The chromatogram is obtained by using the content determination method of Comparative Example 2. Detailed Implementation

[0042] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0043] Example 1: Establishment of a method for characterizing Jingzhu Qingjie granules 1. Instruments and reagents 1.1 Instruments Thermo Vanquish high-performance liquid chromatograph (Thermo Fisher Scientific, USA); Waters high-performance liquid chromatograph (Waters Corporation, USA); ME104E and MS1050U electronic balances (Mettler-Toledo Instruments (Shanghai) Co., Ltd.); KQ-5000DE CNC ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.); electric thermostatic water bath (Beijing Yongguangming Medical Instruments Co., Ltd.); ZORBAX Eclipse XDB-C18 (250mm × 4.6mm, 5µm) column.

[0044] 1.2 Drug Testing Methanol was analytical grade (Sinopharm Group Co., Ltd.); acetonitrile was chromatographic grade (Anhui Tiandi High Purity Solvent Co., Ltd.); phosphoric acid was chromatographic grade (Tianjin Kemeio Chemical Reagent Co., Ltd.); water was purified water (self-made); forsythoside A (batch number: 111810-202209, purity 96.4%, China National Institutes for Food and Drug Control); arctiin (batch number: 110819-202213, purity 99.0%, China National Institutes for Food and Drug Control); fraxetin (batch number: 110741-201708, purity 99.9%, China National Institutes for Food and Drug Control); caffeic acid (batch number: 110885-201703, purity 99.7%, China National Institutes for Food and Drug Control); chicoric acid (batch number: 111752-201703, purity 97.6%). China National Institutes for Food and Drug Control); 5-Hydroxymethylfurfural (batch number: 67-47-0, purity 89.2%, Shanghai Shidande Standard Technical Service Co., Ltd.); Forsythoside I (batch number: DSTDL011201, purity 99.05%, Chengdu Desite Biotechnology Co., Ltd.); Jingzhu Qingjie Granules are self-made by Hunan Chunguang Jiuhui Modern Chinese Medicine Co., Ltd., and are made from the following raw materials in weight percentage: Schizonepeta 3.95%, Saposhnikovia 3.95%, Bitter Almond 3.95%, Platycodon 3.95%, Chicken Gizzard Membrane 2.63%, Roasted Crataegus pinnatifida 6.58%, Forsythia 3.95%, Gypsum Fibrosum 26.31%, Arctium lappa 6.58%, Phragmites communis 13.16%, Lophatherum gracile 9.21%, Viola yedoensis 6.58%, Taraxacum mongolicum 6.58%, Stevia rebaudiana 1.31%, Glycyrrhiza uralensis 1.31%. Mix the Chinese medicinal materials evenly according to the above weight proportions, place them in a clay pot, add 150ml of water and soak for 30 minutes. First, bring to a boil over high heat, then simmer over low heat for 20 minutes. Filter out 50ml of the decoction. 70ml; add water to cover the raw materials, bring to a boil over high heat, then simmer over low heat for 15 minutes each time, filtering out 50ml of the liquid. 70ml; mix the two solutions and form into granules.

[0045] 2. Solution preparation Preparation of reference solutions: Accurately weigh appropriate amounts of forsythoside A, chicoric acid, 5-hydroxymethylfurfural, and forsythoside I reference standards, and add 50% methanol to prepare 30 µg / mL forsythoside A solution, 20 µg / mL chicoric acid solution, 20 µg / mL 5-hydroxymethylfurfural solution, and 20 µg / mL forsythoside I solution; accurately weigh appropriate amounts of arctiin, fraxetin, and caffeic acid reference standards, and add methanol to prepare 180 µg / mL arctiin solution, 10 µg / mL fraxetin solution, and 10 µg / mL caffeic acid solution.

[0046] Preparation of the test solution: Take the contents of the Jingzhu Qingjie granules under the content difference section, mix well, grind finely, take about 0.5g, weigh accurately, place in a stoppered conical flask, accurately add 25 mL of 50% methanol, weigh, extract by sonication (500 W, 40 kHz) for 30 minutes, cool, weigh again, make up the lost weight with 50% methanol, shake well, filter, and the test solution is obtained.

[0047] 3. Chromatographic conditions and system suitability test 3.1 Proposed chromatographic conditions The chromatographic column was a ZORBAX Eclipse XDB-C18 (4.6 mm × 250 mm, 5 μm), with acetonitrile as mobile phase A and 0.1% phosphoric acid aqueous solution as mobile phase B, using gradient elution as specified in the table below; the detection wavelength was 280 nm, the flow rate was 1.0 ml / min, the column temperature was 30 ℃, and the injection volume was 10 μL. The theoretical plate number, calculated based on the forsythoside A peak, should not be less than 2000.

[0048]

[0049] The percentage sign in the table means volume percentage.

[0050] 3.2 Determination Method Take 10 μL each of the reference solution and the test solution obtained under "2. Preparation of Solutions" and inject them into the high-performance liquid chromatograph. Perform characteristic chromatographic determination using the conditions under "3.1. Proposed Chromatographic Conditions". The system suitability meets the requirements, and the results are shown in Table 1 and... Figure 1 .

[0051] Table 1 System Suitability Test Results

[0052] 4. Investigation of chromatographic conditions 4.1 Investigation of the mobile phase Based on the established experimental conditions, with other conditions remaining unchanged, the changes in chromatographic peaks under three different mobile phases were investigated: A: acetonitrile-0.1% phosphoric acid; B: acetonitrile-water; C: acetonitrile-0.1% formic acid.

[0053] The results are as follows Figure 2 As shown, when acetonitrile-water is used as the mobile phase, peaks 2 and 3 have different shapes, and peak 6 is lost; when acetonitrile-formic acid is used as the mobile phase, peak 6 is lost; after comprehensive analysis, the mobile phase composition of the characteristic spectrum is determined to be acetonitrile (A) - 0.1% phosphoric acid (B).

[0054] 4.2 Examination of Gradients Based on the established experimental conditions, with other conditions remaining unchanged, the effects of different gradients on the chromatograms were investigated. The gradients are shown below: Gradient 1

[0055] Gradient 2

[0056] Gradient 3

[0057] The results are as follows Figure 3 As shown in Table 2, the chromatographic peak separation under gradients 1 and 2 is poor, and peak 6 of gradient 2 is lost. Taking all factors into consideration, gradient 3 is selected to construct the characteristic spectrum of Jingzhu Qingjie granules.

[0058] Table 2 Results of investigation at different gradients

[0059] 4.3 Examination of Extraction Methods Take two portions of Jingzhu Qingjie Granules, each about 0.5g, weigh them accurately, place them in a stoppered conical flask, add 25mL of 50% methanol accurately, weigh them, reflux one portion for 30 minutes, and sonicate the other portion (power 500W, frequency 40kHz) for 30 minutes, cool them, weigh them again, make up the weight loss with 50% methanol, shake well, filter, and you will get the product.

[0060] Characteristic chromatograms were determined using the conditions outlined in section 3.1, and appropriate extraction methods were selected based on the parameters of each characteristic peak (peak shape, separation degree, symmetry, total peak area, etc.). The results are shown in Table 3.

[0061] Table 3 Results of the investigation of different extraction methods

[0062] The results showed that there was no significant difference in the total peak area of ​​the chromatograms of the test solution extracted by reflux and ultrasonic extraction. Considering the convenience of the experiment, ultrasonic extraction was determined to be the extraction method for the test solution.

[0063] 4.4 Investigation of Extraction Solvents Take five portions of Jingzhu Qingjie Granules, each approximately 0.5g, weigh them precisely, place them in a stoppered conical flask, add 25mL of the corresponding solvent precisely, weigh them, extract by sonication for 30min, cool them, weigh them again, replenish the lost weight with the corresponding solvent, shake well, filter, and you will get the product.

[0064] Characteristic chromatograms were determined using the conditions outlined in section 3.1, and appropriate extraction solvents were selected based on the parameters of each characteristic peak (peak shape, separation degree, symmetry, total peak area, etc.). The results are shown in Table 4.

[0065] Table 4 Results of the investigation of different extraction solvents

[0066] The results showed that the extraction efficiency of different extraction solvents was basically the same. In order to ensure sufficient extraction and considering that organic solvents are easy to preserve and meet the requirements for content determination, the extraction solvent for preparing the test solution was determined to be 50% methanol.

[0067] 4.5 Examination of extraction time Take three portions of Jingzhu Qingjie Granules, each approximately 0.5g, weigh them precisely, place them in a stoppered conical flask, add 25mL of 50% methanol precisely, weigh them, and extract them by ultrasonication for 20, 30, and 40 minutes respectively. After cooling, weigh them again, replenish the lost weight with 50% methanol, shake well, filter, and you will get the product.

[0068] Characteristic chromatograms were determined using the conditions outlined in section 3.1, and the changes in chromatographic peaks (peak shape, separation efficiency, symmetry, total peak area, etc.) of the test solution prepared at different extraction times were compared to select an appropriate extraction time. The results are shown in Table 5.

[0069] Table 5 Results of the investigation at different extraction times

[0070] The results showed that there were no significant differences in the peak areas of each characteristic peak and the total peak area of ​​the chromatograms at different extraction times. This indicates that the peak areas of each characteristic peak in the test solution prepared at extraction times of 20-40 min were not different, and no other impurities appeared as the extraction time increased. Taking all factors into consideration, the extraction time was determined to be 30 min.

[0071] 4.6 Investigation into the amount of extraction solvent used Take three portions of Jingzhu Qingjie Granules, each approximately 0.5 g, and weigh them precisely. Place them in stoppered conical flasks and add 10 mL, 25 mL, and 50 mL of 50% methanol respectively. Weigh them and extract them by sonication for 30 minutes. After cooling, weigh them again and replenish the lost weight with 50% methanol. Shake well and filter to obtain the final product.

[0072] Characteristic chromatograms were determined using the conditions outlined in section 3.1, and the changes in chromatographic peaks (peak shape, separation efficiency, symmetry, total peak area, etc.) of the test solution prepared at different extraction times were compared to select an appropriate solvent volume. The results are shown in Table 6.

[0073] Table 6 Results of the investigation of different extraction solvent dosages

[0074] The results showed that, with a constant sampling volume, the total peak area of ​​the test sample solution decreased proportionally with increasing solvent volume, indicating that solvent volumes of 10 ml, 25 ml, and 50 ml had essentially the same extraction efficiency for the eight intended characteristic peaks in the test sample. Taking all factors into consideration, the extraction solvent volume for preparing the test sample solution was determined to be 25 ml.

[0075] 5. Identification of characteristic peaks Take 10 μL each of the reference solution and test solution obtained under "2. Preparation of Solutions" and 10 μL of blank solvent solution, respectively, and inject them into the liquid chromatograph. Perform characteristic chromatographic analysis under the conditions specified in "3.1. Proposed Chromatographic Conditions". Identify the characteristic peaks of Jingzhu Qingjie granules. The results are shown in [Figure 1]. Figures 4-6 .

[0076] Depend on Figure 4 It can be seen that in the negative control chromatogram, there are no chromatographic peaks at the retention time positions of the reference standard, indicating that the extraction solvent does not interfere with the determination of each characteristic peak, and the method is specific. Figure 5 The chromatograms of each reference solution are shown below. Figure 6 The chromatogram of the test solution of Jingzhu Qingjie granules showed 8 characteristic peaks. Figure 5 Each peak was identified using reference standards. Peak 1 corresponds to 5-hydroxymethylfurfural, peak 2 corresponds to fraxin, peak 3 corresponds to caffeic acid, peak 4 corresponds to forsythoside I, peak 5 corresponds to forsythoside A, peak 6 corresponds to chicoric acid, and peak 7 corresponds to arctiin.

[0077] Following the preparation method of Jingzhu Qingjie Granules, standard decoctions of each single herb in the prescription were prepared. The test solution was prepared according to the method described in section "2. Preparation of Solution". Characteristic chromatograms were determined using the conditions described in section "3.1. Proposed Chromatographic Conditions". The results were compared with those of the Jingzhu Qingjie Granules test sample, and the herb classification was performed. The results are shown in […]. Figure 7 See Table 7.

[0078] Table 7. Information on the Attribution of Characteristic Peaks and Herbal Flavors of Jingzhu Qingjie Granules

[0079] 6. Validation of the Feature Mapping Methodology 6.1 Double retention time Take the test solution prepared according to the method under "2 Preparation of Solution" and detect the characteristic spectrum of twice the retention time.

[0080] The results are as follows Figure 8 As shown, no other chromatographic peaks were observed in the test sample after 60 minutes, indicating that the method has good overall performance.

[0081] 6.2 Precision Test Take the same batch of Jingzhu Qingjie granules and prepare the test solution according to the method under "2. Preparation of Solution". Perform characteristic chromatographic determination under the conditions under "3.1. Proposed Chromatographic Conditions". Inject six times consecutively, record the chromatograms, and calculate the RSD of the relative retention time and relative peak area of ​​each characteristic peak. The results are shown in Tables 8 and 9.

[0082] Table 8. Results of Instrument Precision Test (Relative Retention Time)

[0083] Table 9. Results of Instrument Precision Test (Relative Peak Area)

[0084] The results show that the RSD of the relative retention time of the eight characteristic peaks ranges from 0.04% to 0.14%, and the RSD of the relative peak area ranges from 0.96% to 1.01%, all less than 3%, and the relative retention times all meet the requirements, indicating that the instrument precision of this method is good.

[0085] 6.3 Repeatability Test Take 6 portions of the same batch of Jingzhu granules and prepare test solutions according to the method under "2 Preparation of Solution". Use the conditions under "3.1 Proposed Chromatographic Conditions" to determine the characteristic chromatograms, record the chromatograms, and calculate the relative retention time and relative peak area RSD (%) of each characteristic peak. The experimental results are shown in Tables 10 and 11.

[0086] Table 10 Results of repeatability tests (relative retention time) (n=6)

[0087] Table 11 Results of repeatability tests (relative peak area) (n=6)

[0088] The results showed that the relative retention time (RSD) of the chromatographic peaks of the six test solutions ranged from 0.03% to 0.06%, and the relative peak area ranged from 1.29% to 1.57%, all less than 3%, and the relative retention times all met the requirements, indicating that the method has good repeatability.

[0089] 6.4 Stability Test The test solution was prepared according to the method described in section "2. Preparation of Solution". Characteristic chromatograms were determined using the conditions described in section "3.1. Proposed Chromatographic Conditions". Injections were performed at 0, 3, 6, 9, 12, and 24 hours. Chromatograms were recorded, and the relative retention time and relative peak area (RSD) of each characteristic peak were calculated to assess the stability of the test solution. The experimental results are shown in Tables 12 and 13.

[0090] Table 12 Results of stability test of the test sample solution (relative retention time)

[0091] Table 13 Results of the stability test of the test sample solution (relative peak area)

[0092] The results showed that within 24 hours, the relative retention time RSD of the eight characteristic peaks of the test solution ranged from 0.04% to 0.15%, and the relative peak area RSD ranged from 0.98% to 1.09%, which was less than 3%, indicating that the test solution was stable within 24 hours.

[0093] 6.5 Durability Take the same batch of *Jingzhu* granules and prepare the test solution according to the method under "2. Preparation of Solution". Use the conditions under "3.1. Proposed Chromatographic Conditions" to determine the characteristic chromatogram. Set the column temperature to 28℃, 30℃ and 32℃ for injection detection, record the chromatogram, calculate the relative retention time and relative peak area of ​​each characteristic peak, and investigate the effect of different column temperatures on the test results for the same mobile phase. The experimental results are shown in Tables 14 and 15.

[0094] Table 14 Results of tests at different column temperatures (relative retention time)

[0095] Table 15 Results of tests at different column temperatures (relative peak area)

[0096] The results showed that at a column temperature of (30±2)℃, the relative retention time RSD of the eight characteristic peaks ranged from 0.82% to 3.63%, which was less than 5%, and the relative peak area RSD ranged from 1.33% to 2.58%. However, no standard for the relative peak area was established. The column temperature robustness results met the methodological validation requirements.

[0097] Take the same batch of *Jingzhu* granules and prepare the test solution according to the method under "2. Preparation of Solution". Use the conditions under "3.1. Proposed Chromatographic Conditions" to determine the characteristic chromatogram. Set the column flow rate to 0.98 ml / min, 1.0 ml / min and 1.02 ml / min respectively for injection detection. Record the chromatograms and calculate the relative retention time and relative peak area RSD of each characteristic peak. Investigate the effect of different flow rates of the same mobile phase on the test results. The experimental results are shown in Tables 16 and 17.

[0098] Table 16 Results of tests at different flow rates (relative retention time)

[0099] Table 17 Results of tests at different flow rates (relative peak area)

[0100] The results showed that at a flow rate of (1.0±0.02) ml / min, the relative retention time RSD of the eight characteristic peaks ranged from 0.16% to 1.23%, which was less than 5%, and the relative peak area RSD ranged from 1.26% to 5.11%, which was quite different. However, no standard for relative peak area was established. The flow rate robustness results met the requirements for method validation.

[0101] 7. Sample Determination Three batches of Jingzhu Qingjie granules were taken, and test solutions were prepared according to the method described in section "2. Preparation of Solutions". The test solutions were accurately pipetted and subjected to characteristic chromatographic determination under the conditions described in section "3.1. Proposed Chromatographic Conditions". The HPLC characteristic chromatograms of the three batches of Jingzhu Qingjie granules were matched using the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine". Using peak 5 (forsythoside A) as the reference peak S, a control chromatogram was generated using the median method. A control characteristic chromatogram for Jingzhu Qingjie granules was established. The characteristic chromatograms of the three batches of granule samples and the control characteristic chromatogram are shown below. Figure 9 In addition, using the forsythoside A peak as the reference peak S, the relative retention times of peaks 1, 4, and 8 with peak S were calculated, and the results are shown in Table 18.

[0102] Table 18 Results of relative retention time determination of characteristic spectra of three batches of Jingzhu Qingjie granules

[0103] 8. Establishment of Feature Maps like Figure 1 As shown, the chromatogram of the test sample should show 8 characteristic peaks. Peaks 2, 3, 5, 6, and 7 should correspond to the retention times of the reference peaks of fraxin B, caffeic acid, forsythoside A, chicoric acid, and arctiin, respectively. The peak corresponding to the forsythoside A reference peak is the S peak. Calculate the relative retention times of each characteristic peak and the S peak. The relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.18 (peak 1), 0.42 (peak 2), 0.45 (peak 3), 0.79 (peak 4), 1.10 (peak 6), 1.41 (peak 7), and 1.74 (peak 8).

[0104] Comparative Example 1 Preparation of the test solution: Take an appropriate amount of the contents of Jingzhu Qingjie Granules, grind them into a fine powder, take about 1.0g, weigh it accurately, place it in a stoppered conical flask, add 25mL of 50% methanol accurately, weigh it, sonicate for 30min, cool it and make up the weight loss to obtain the test solution.

[0105] Preparation of reference solutions: Accurately weigh appropriate amounts of forsythoside A and chicoric acid reference standards, and add 50% methanol to prepare a solution containing 30 µg of forsythoside A and 20 µg of chicoric acid per ml. Accurately weigh appropriate amounts of arctiin, fraxetin, and caffeic acid reference standards, and add methanol to prepare a solution containing 180 µg of arctiin, 10 µg of fraxetin, and 10 µg of caffeic acid.

[0106] Chromatographic conditions and system suitability test: A Zhongpu Blue XR-C18 reversed-phase column (250 mm × 4.6 mm, 5 μm) was used. Acetonitrile (A) - 0.1% phosphoric acid aqueous solution (B) was used as the mobile phase, with gradient elution. The elution program was: 0–5 min, 7% A; 5–15 min, 7% → 20% A; 15–30 min, 20% → 30% A; 30–40 min, 30% → 40% A; 40–50 min, 40% A. Variable wavelength detection was used, with the wavelength switching program as follows: 0–11.5 min, 230 nm; 11.5–25 min, 330 nm; 25–33 min, 230 nm; 33–40 min, 280 nm; 40–50 min, 230 nm. The flow rate was 1.0 mL / min. -1 The column temperature was 35℃ and the injection volume was 10μL.

[0107] The characteristic peaks of Jingzhu Qingjie Granules were identified, and the results are shown in the figure. Figure 10 , Figure 11 See Table 19.

[0108] Table 19 Chromatographic detection results of Comparative Example 1

[0109] As shown in Table 19, the separation of peaks 3, 4, and 6 in the characteristic chromatograms determined by the chromatographic conditions provided in Comparative Example 1 is relatively poor. Furthermore, the detection method in Comparative Example 1 requires wavelength switching, indicating poor instrument applicability.

[0110] Example 2: Method for determining the content of Jingzhu Qingjie Granules Based on the method for constructing the characteristic spectrum of Jingzhu Qingjie granules in Example 1, Example 2 also conducted the following methodological investigation and established a method for determining the content of Jingzhu Qingjie granules, specifically including: 1. Instruments and reagents Same as "1 Instruments and reagents" under Example 1.

[0111] 2. Solution preparation Preparation of reference solutions: Take an appropriate amount of forsythoside A reference standard, accurately weigh it, and add 50% methanol to prepare a solution containing 30 µg per ml. Take an appropriate amount of arctiin reference standard, accurately weigh it, and add methanol to prepare a solution containing 180 µg per ml.

[0112] Preparation of the test solution: Take the contents of the Jingzhu Qingjie granules under the content difference section, mix well, grind finely, take about 0.5g, weigh accurately, place in a stoppered conical flask, accurately add 25 mL of 50% methanol, weigh, extract by sonication (500 W, 40 kHz) for 30 minutes, cool, weigh again, make up the lost weight with 50% methanol, shake well, filter, and the test solution is obtained.

[0113] 3. Chromatographic conditions and system suitability test 3.1 Proposed chromatographic conditions The chromatographic column was a ZORBAX Eclipse XDB-C18 (250 mm × 4.6 mm, 5 µm); acetonitrile was used as mobile phase A, and 0.1% phosphoric acid solution was used as mobile phase B, with gradient elution performed according to the following specifications: flow rate 1.0 ml / min; column temperature 30℃; detection wavelengths 327 nm (forsythoside A) and 280 nm (arbusculin); injection volume 10 μl. The theoretical plate number, calculated based on the forsythoside A peak, should be no less than 2000.

[0114]

[0115] 4. Investigation of chromatographic conditions 4.1 Investigation of extraction solvent Accurately weigh 0.5g of *Jingzhu* granules and place them in a stoppered conical flask. Divide the flask into three parallel portions and add 25ml each of 30% methanol, 50% methanol, 70% methanol, 50% ethanol, and water, respectively. Seal the flasks tightly, weigh them, and sonicate them (500W, 40kHz) for 30 minutes. After cooling, weigh them again to make up the lost weight. Shake well, filter, and collect the filtrate. Inject the sample according to the chromatographic conditions specified in section 3.1 and calculate the contents of forsythoside A and arctiin under different extraction solvent treatments.

[0116] The results showed that the contents of forsythoside A and arctiin were relatively similar under different extraction solvents. In order to save costs and ensure the stability of the test solution, 50% methanol was selected as the extraction solvent.

[0117] 4.2 Examination of Extraction Methods Accurately weigh 0.5g of Jingzhu Qingjie granules and place them in a stoppered conical flask. Accurately add 25ml of 50% methanol, seal tightly, and weigh. Perform two parallel extractions, using reflux and ultrasonic extraction methods respectively for 30min each. After cooling, weigh again to make up the lost weight, shake well, filter, and collect the filtrate. Inject and determine the contents of forsythoside A and arctiin under different extraction methods according to the chromatographic conditions in section 3.1.

[0118] The results showed that there was no significant difference in content between heating and reflux extraction and ultrasonic extraction. Considering the convenience of experimental operation, ultrasonic extraction was selected as the extraction method.

[0119] 4.3 Examination of extraction time Accurately weigh 0.5g of Jingzhu Qingjie granules and place them in a stoppered conical flask. Accurately add 25ml of 50% methanol, seal tightly, and weigh. Perform ultrasonic extraction on three parallel flasks for 20, 30, and 40 min respectively. After cooling, weigh again to make up the weight loss. Shake well, filter, and collect the filtrate. Inject and determine the contents of forsythoside A and arctiin under different extraction times according to the chromatographic conditions in section 3.1.

[0120] The results showed that the contents of forsythoside A and arctiin were not significantly different when extracted by ultrasound for 20, 30 and 40 min. The contents of both were slightly higher when the extraction time was 30 min. Therefore, the extraction time was determined to be 30 min.

[0121] 4.4 Investigation of Solvent Usage Accurately weigh 0.5g of Jingzhu Qingjie granules and place them in a stoppered conical flask. Divide the flask into three parallel portions and accurately add 10ml, 25ml, and 50ml of 50% methanol to each portion. Seal the flasks tightly and weigh them. Sonicate the flasks (500W, 40kHz) for 30 minutes, cool them, and weigh them again. Make up the lost weight with 50% methanol solution, shake well, filter, and collect the filtrate. Inject the flasks according to the chromatographic conditions specified in section 3.1 and calculate the content of forsythoside A and arctiin extracted with different solvent volumes.

[0122] The results showed that when the solvent volume reached 25 ml, forsythoside A and arctiin were basically completely extracted, so the solvent volume was determined to be 25 ml.

[0123] 5. Methodological Validation 5.1 Specificity Examination Take the test solution of Jingzhu Qingjie Granules, the reference solution of Forsythoside A, the reference solution of Arctiin, and the blank (50% methanol) solution, and determine them according to the chromatographic conditions under "3.1 Proposed Chromatographic Conditions".

[0124] The results showed that the chromatogram of the test sample exhibited a peak with the same retention time as the reference sample, and the blank showed no interference. The experiment demonstrates that this method has good specificity.

[0125] 5.2 Examination of Linear Relationships Accurately weigh an appropriate amount of forsythoside A reference standard, add 50% methanol to prepare a solution containing 178.1472 μg / mL, and use this as the reference standard stock solution. Dilute this stock solution sequentially to prepare reference standard solutions with concentrations of 5.5671, 11.342, 22.2684, 44.5368, 89.0736, and 178.1472 µg / mL. Accurately pipette 10 μL of each of the above reference standard solutions and inject them sequentially into a high-performance liquid chromatograph (HPLC). Measure the peak area and perform a linear regression analysis on the peak area of ​​forsythoside A against the reference standard injection concentration (µg / mL). Plot a standard curve with reference standard concentration on the x-axis and peak area on the y-axis. The results are shown in Table 20. The regression equation is y = 17410.36264x + 8078.61717, R0. 2 =0.99997.

[0126] Table 20 Results of linear relationship investigation for the determination of forsythoside A content in Jingzhu Qingjie Granules

[0127] Accurately weigh an appropriate amount of arctiin reference standard and dissolve it in methanol to prepare a solution containing 413.6220 μg / ml. This solution is used as the reference standard stock solution and is subsequently diluted to prepare reference standard solutions with concentrations of 12.9257, 25.8514, 51.7028, 103.4055, 206.8110, and 413.6220 µg / ml. Accurately inject 10 μl of each of the above reference standard solutions into the high-performance liquid chromatograph (HPLC) and measure the peak area. Perform linear regression on the peak area of ​​arctiin with the reference standard injection concentration (µg / ml). Plot a standard curve with the reference standard concentration on the x-axis and the peak area on the y-axis. The results are shown in Table 21. The regression equation is y = 5497.32533x + 9346.21891, R² = 0.99995.

[0128] Table 21 Results of linear relationship investigation for the determination of arctiin content in Jingzhu Qingjie Granules

[0129] The results showed that, under the above chromatographic conditions, forsythoside A exhibited good linearity in the range of 5.5671–178.1472 μg / ml, and arctiin also showed good linearity in the range of 12.9257–413.6220 μg / ml.

[0130] 5.3 Repeatability Test Take 0.5g of the same batch of Jingzhu Qingjie granules and weigh 6 portions in total. Prepare 6 test solutions in parallel according to the method under "2 Preparation of Solution". Inject and determine the contents of forsythoside A and arctiin according to the conditions under "3.1 Proposed Chromatographic Conditions". The results are shown in Table 22.

[0131] Table 22 Repeatability test results of forsythoside A and arctiin in Jingzhu Qingjie Granules

[0132] The results showed that the average content of forsythoside A and the average content of arctiin in the Jingzhu granules were 1.3768 mg / g and 8.6096 mg / g respectively, with RSD less than 3%, indicating that the method had good repeatability.

[0133] 5.4 Precision Test Accurately pipette the same sample solution of Jingzhu Qingjie Granules and inject it 6 times according to the chromatographic conditions under “3.1 Proposed Chromatographic Conditions”. Measure the peak areas of forsythoside A and arctiin. The test results are shown in Table 23. The RSD of all samples is less than 2%, indicating that the instrument has good precision.

[0134] Table 23 Precision test results for the determination of forsythoside A and arctiin in Jingzhu Qingjie Granules

[0135] 5.5 Stability Test Accurately pipette the same sample solution of Jingzhu Qingjie Granules and inject it at 0, 3, 6, 9, 12 and 24 h according to the chromatographic conditions under “3.1 Proposed Chromatographic Conditions”. Measure the peak areas of forsythoside A and arctiin. The results are shown in Table 24. The RSD of all samples was less than 2%, indicating that the sample solution was stable within 24 h.

[0136] Table 24 Results of stability test for the content of forsythoside A and arctiin in Jingzhu granules

[0137] 5.6 Intermediate Precision Test Six test solutions were prepared in parallel by different personnel from the same batch of *Vitex negundo* granules according to the method described in section "2. Preparation of Solutions". These solutions were then injected into different high-performance liquid chromatographs (HPLC) under the chromatographic conditions described in section "3.1. Proposed Chromatographic Conditions" to determine the contents of forsythoside A and arctiin. The results are shown in Table 25. The results indicate that the average content of forsythoside A measured twice by different instruments was 1.3730 mg / g, and the average content of arctiin was 8.7297 mg / g, with RSDs less than 3%, indicating good intermediate precision of the method.

[0138] Table 25 Intermediate precision test results for the determination of forsythoside A and arctiin in Jingzhu Qingjie Granules

[0139] 5.7 Durability Test Take the same batch of *Jingzhu* granules and prepare the test solution according to the method under "2. Preparation of Solution". According to the chromatographic conditions under "3.1. Proposed Chromatographic Conditions", the flow rate was set to 0.98 ml / min, 1.0 ml / min and 1.02 ml / min respectively. The sample was injected for detection to investigate the effect of different flow rates of the same mobile phase on the test results. The results are shown in Table 26.

[0140] In addition, the same batch of Jingzhu Qingjie granules were used to prepare the test solution according to the method under "2 Preparation of Solution". The column temperature was set to 28℃, 30℃ and 32℃ respectively according to the chromatographic conditions under "3.1 Proposed Chromatographic Conditions". The sample was injected and detected to investigate the effect of different column temperatures on the test results. The results are shown in Table 26.

[0141] The results showed that when the flow rate varied within (1.0±0.02) ml / min, or when the column temperature varied within (30±2) ℃, the effect on the test results of the reference standard content in the sample was not significant, and the RSD was less than 5%, which met the requirements of method validation.

[0142] Table 26. Durability Study Results of Forsythoside A and Arctiin Content Determination in Jingzhu Qingjie Granules

[0143] 5.8 Recovery Test Three portions of the same batch of Jingzhu Qingjie granules (containing 1.3768 mg / g of forsythoside A and 8.6096 mg / g of arctiin) were accurately weighed, at amounts of 0.20, 0.25, and 0.30 g respectively. Forsythoside A and arctiin reference solutions were added in ratios of 0.8:1, 1:1, and 1.2:1, respectively, with three replicates for each portion. The test solution was prepared according to the method described in section "2. Preparation of Solution". The chromatographic conditions were determined according to section "3.1. Proposed Chromatographic Conditions", and the recovery rate was calculated. The results are shown in Table 27. The results show that the average recovery rate of forsythoside A was 98.36%, and the average recovery rate of arctiin was 101.23%, with RSDs less than 2%, indicating good recovery of the method.

[0144] Table 27 Results of the spiking recovery test for the content of forsythoside A and arctiin in Jingzhu Qingjie Granules

[0145] Comparative Example 2 Preparation of the test solution: Take an appropriate amount of sample, grind it into a fine powder, take about 0.8g of Jingzhu Qingjie granules, weigh it accurately, place it in an Erlenmeyer flask, add 50mL of 70% methanol aqueous solution accurately, weigh it, sonicate (power 300W, frequency 40kHz) for 30min, cool it, weigh it again, make up the amount lost with 70% methanol aqueous solution, shake it well, filter it through a 0.45μm microporous membrane, and take the filtrate to obtain the final product.

[0146] Preparation of the reference solution: Take an appropriate amount of forsythoside A reference standard, accurately weigh it, and add 50% methanol to prepare a solution containing 30 µg per ml. Take an appropriate amount of arctiin reference standard, accurately weigh it, and add methanol to prepare a solution containing 180 µg per ml.

[0147] Chromatographic conditions and system suitability test: Column: Agilent Zorbax SB-C18 (250 mm × 4.6 mm, 5 μm); Mobile phase: acetonitrile (A) - 0.1% phosphoric acid aqueous solution (B), gradient elution (0~20 min, 7% A → 14% A; 20~31 min, 14% A → 20% A; 31~40 min, 20% A → 30% A; 40~50 min, 30% A → 42% A; 50~60 min, 42% A → 60% A; 60~60.1 min, 60% A → 7% A; 60.1~65 min, 7% A); Flow rate: 1.0 mL·min⁻¹; Column temperature: 30℃; Detection wavelength: 278 nm (forsythoside A, 17~24 min), 326 nm (baicalin, 24~39 min), 278 nm (arbuscularin, 39~65 min); Injection volume: 10 μL.

[0148] The detection results of method 2 in Comparative Example 2 are as follows Figure 12 As shown in the chromatogram, the separation of forsythoside A from Jingzhu Qingjie Granules detected in the comparative document was poor. Furthermore, the detection method in the comparative document requires wavelength switching, a function not commonly found in liquid chromatography in some laboratories, resulting in poor instrument applicability.

[0149] The component contents measured by the detection methods of Example 2 and Comparative Example 2 are compared as shown in Table 28 below.

[0150] Table 28 Comparison of content from the two methods

[0151] According to the data in the table, the content values ​​determined by the chromatographic conditions and detection method provided in Comparative Example 2 are not significantly different from those determined in Example 2. However, the chromatograms show that the separation of forsythoside A from Jingzhu Qingjie Granules in Comparative Example 2 is poor. Furthermore, the detection method in Comparative Example 2 requires wavelength switching, a function not commonly found in liquid chromatography systems in some laboratories, resulting in poor instrument applicability.

[0152] The above description provides a detailed account of the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, unless otherwise specified, the embodiments of the present invention and the features thereof can be combined with each other.

Claims

1. A method for constructing a characteristic spectrum of Jingzhu Qingjie granules, characterized in that, The reference solution and the test solution were separately determined by high performance liquid chromatography, and the common peak was identified to obtain the solution. The conditions for the high-performance liquid chromatography method include: The chromatographic column was packed with octadecylsilane-bonded silica gel. Acetonitrile was used as mobile phase A, and a 0.05-0.2% (v / v) aqueous solution of phosphoric acid was used as mobile phase B for gradient elution. The conditions for gradient elution are as follows: From 0 to 15 min, the volume fraction of mobile phase A increased from 8% to 15%; from 15 to 32 min, the volume fraction of mobile phase A remained at 15%; from 32 to 40 min, the volume fraction of mobile phase A increased from 15% to 28%; from 40 to 47 min, the volume fraction of mobile phase A remained at 28%; and from 47 to 60 min, the volume fraction of mobile phase A increased from 28% to 80%.

2. The construction method according to claim 1, characterized in that, The test sample is mixed with a first solvent and extracted by ultrasonication to obtain the test sample solution; the mass concentration of the test sample solution is 0.5~50 mg / mL; and / or, The reference standard is mixed with a second solvent to obtain the reference standard solution; the mass concentration of the reference standard solution is 5~300µg / mL.

3. The construction method according to claim 2, characterized in that, The first solvent and the second solvent are each independently selected from alcohol solvents, water, or alcohol-water mixtures; the alcohol solvent is selected from methanol or ethanol, and the alcohol-water mixture is a methanol aqueous solution or an ethanol aqueous solution with a volume percentage of 30-70%.

4. The construction method according to claim 1, characterized in that, The conditions for the high-performance liquid chromatography method also include at least one of the following iv: i. Flow rate: 0.5~1.0 mL / min; ii. Column temperature: 25~35℃; iii. Injection volume: 5~20μL; iv. Detection wavelength: 280nm; v. The theoretical plate number, calculated based on the forsythoside A peak, shall not be less than 2000.

5. The construction method according to claim 1, characterized in that, The reference standards include forsythoside A, chicoric acid, 5-hydroxymethylfurfural, forsythoside I, arctiin, fraxin, and caffeic acid.

6. The construction method according to claim 1, characterized in that, The characteristic spectrum includes 8 characteristic peaks. Peak 5 is the peak corresponding to the forsythoside A peak. Peak 5 is used as a reference peak. The relative retention times of the other characteristic peaks and the reference peak are calculated. The relative retention times should be within ±10% of a specified value. The specified values ​​are: peak 1: 0.18, peak 2: 0.42, peak 3: 0.45, peak 4: 0.79, peak 6: 1.10, peak 7: 1.41, and peak 8: 1.

74.

7. A method for determining the content of Jingzhu Qingjie granules, characterized in that, The determination is performed by high-performance liquid chromatography, wherein the high-performance liquid chromatography includes the conditions described in claim 1.

8. The content determination method according to claim 7, characterized in that, The conditions for the high-performance liquid chromatography method also include at least one of the following iv: i. Flow rate: 0.5~1.0 mL / min; ii. Column temperature: 25~35℃; iii. Injection volume: 5~20μL; iv. Detection wavelength: 280 nm or 327 nm; v. The theoretical plate number, calculated based on the forsythoside A peak, shall not be less than 2000.

9. The content determination method according to claim 7, characterized in that, The reference standards used in the content determination method include forsythoside A and arctiin.

10. The application of the construction method as described in any one of claims 1 to 6 or the content determination method as described in any one of claims 7 to 9 in detecting the quality of Jingzhu Qingjie Granules.

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