Method for constructing specific chromatogram of Kangfugu anti-inflammatory suppository

Abstract

The invention relates to the technical field of traditional Chinese medicine analysis, in particular to a construction method of a Kangfugu anti-inflammatory suppository characteristic spectrum, which comprises the following steps: 1, preparing a reference substance solution; 2, preparing a test solution; 3, determining conditions; and 4, importing the chromatogram into a traditional Chinese medicine fingerprint spectrum to generate a control characteristic spectrum. According to the characteristic method, 13 characteristic peaks are defined, the method is good in repeatability and high in precision, the chemical component characteristics of the Kangfugu anti-inflammatory suppository are fully and integrally displayed, and the medicine quality of the Kangfugu anti-inflammatory suppository is integrally controlled.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine analysis, and particularly to a method for constructing a characteristic chromatogram of Kangfu Xiaoyan Suppository. Background Art

[0002] Kangfu Xiaoyan Suppository is composed of 8 traditional Chinese medicines such as Sophora flavescens, Patrinia scabiosaefolia, Viola philippica, Taraxacum mongolicum, etc. Pharmacodynamic experiments show that Kangfu Xiaoyan Suppository has both the effects of widely and significantly clearing heat and detoxifying, killing insects and promoting diuresis, removing stasis and relieving pain, and also has the effects of anti-pathogens, increasing the phagocytic function of white blood cells and anti-inflammatory, and at the same time has the efficacy of repairing damaged tissues, and is mainly used for the treatment of gynecological diseases. Kangfu Xiaoyan Suppository has significant curative effects on acute and chronic pelvic inflammatory diseases, inflammatory masses, endometritis, adnexitis, urinary tract infections, hemorrhoids, prostatitis and other diseases. In the first part of the Chinese Pharmacopoeia 2020 edition, its quality standards only include the quality control of matrine, aloin and hyodeoxycholic acid, and do not mention the components of the remaining medicinal materials. There is no research on the characteristic chromatogram of Kangfu Xiaoyan Suppository in patents, and there is only research on the determination of matrine content in the literature, so the overall quality of Kangfu Xiaoyan Suppository cannot be controlled.

[0003] Kangfu Xiaoyan Suppository is composed of 8 traditional Chinese medicines, and each medicinal material contains a variety of active ingredients (such as alkaloids, glycosides, organic acids, etc.). These components have large polarity differences and high structural similarities, resulting in difficult chromatographic separation, easy peak overlap or co-elution problems, affecting the accuracy of qualitative and quantitative analysis. Moreover, the solubility differences of different components are large, and it is necessary to determine the optimal extraction solvent, extraction method and time through repeated experiments to ensure the extraction efficiency and reproducibility.

[0004] In view of this, the present invention is particularly proposed. Summary of the Invention

[0005] In order to make up for the deficiencies in the prior art, the present invention provides a method for constructing a characteristic chromatogram of Kangfu Xiaoyan Suppository.

[0006] The present invention provides a method for constructing a characteristic chromatogram of Kangfu Xiaoyan Suppository, comprising the following steps: (1) Preparation of the reference substance solution: Take an appropriate amount of the reference substance, weigh it accurately, and dissolve it in methanol to prepare a solution containing 65 μg per 1 ml, shake well, and use it as the reference substance solution; the reference substances are: monocaffeoyl tartaric acid, chlorogenic acid, aesculetin, caffeic acid, chicoric acid, aloin; (2) Preparation of the test solution: Weigh 1 g of Kangfu Xiaoyan Suppository precisely, add 40 ml of a methanol-water-formic acid composite solvent with a ratio of 70:30:0.1; sonicate at 50 °C for 30 minutes with a sonication power of 200 W; centrifuge at 8000 rpm for 10 minutes, and take the supernatant for standby; select an Oasis MCX mixed-mode solid-phase extraction column, and sequentially equilibrate with 5 ml of methanol and 5 ml of water containing 0.1% formic acid to activate the extraction column; pass the extract, i.e., the supernatant obtained after centrifugation, through the column at a flow rate of 1 ml / min, wash away the fat-soluble impurities with 5 ml of a methanol-water-formic acid solution with a ratio of 20:80:0.1, elute the acidic components with 5 ml of a methanol-water-formic acid solution with a ratio of 70:30:0.1, change to 5 ml of a methanol-water-ammonia solution with a ratio of 70:30:2 to elute the basic components, collect the eluates of the acidic and basic components, concentrate by nitrogen blowing to 2 ml, and filter through a 0.22 μm filter membrane to obtain the test solution; (3) Determination: Inject the reference solution and the test solution into a high-performance liquid chromatograph under the following chromatographic conditions, inject 10 μl each, and record the chromatogram; the chromatographic conditions are as follows: use octadecylsilane-bonded silica gel as the filler; use a 0.1% trifluoroacetic acid-80% acetonitrile aqueous solution as mobile phase A, and use tetrahydrofuran:methanol:water with a volume ratio of 10:38:45 as mobile phase B, and perform elution according to the specified gradient; the column temperature is 30 °C; the flow rate is 1 ml per minute; use an ultraviolet-visible (UV-Vis) detector, and the detection wavelength is 300 - 340 nm; The elution gradient conditions are as follows: 0 - 15 min, mobile phase A: mobile phase B, volume ratio changes from 5:95 to 10:90; 15 - 25 min, mobile phase A: mobile phase B, volume ratio changes from 10:90 to 14:86; 25 - 27 min, mobile phase A: mobile phase B, volume ratio changes from 14:86 to 17:83; 27 - 28 min, mobile phase A: mobile phase B, volume ratio changes from 17:83 to 22:78; 28 - 50 min, mobile phase A: mobile phase B, volume ratio changes from 22:78 to 23:77; 50 - 65 min, mobile phase A: mobile phase B, volume ratio changes from 23:77 to 55:45; 65 - 68 min, mobile phase A: mobile phase B, volume ratio changes from 55:45 to 5:95; 68 - 80 min, mobile phase A: mobile phase B, volume ratio is 5:95; (4) Generating a reference characteristic chromatogram: Using the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicines" formulated by the Pharmacopoeia Commission of the People's Republic of China, select the chromatographic peaks that exist in the chromatograms of different batches of Kangfu Xiaoyan Suppositories as common peaks, and use the average value calculation method to generate the reference characteristic chromatogram of Kangfu Xiaoyan Suppositories.

[0007] Preferably, the chromatographic column described in step (3) is: Alltima-C18 (4.6x250mm, 5um).

[0008] Preferably, the reference characteristic chromatogram generated in step (4) includes 13 common chromatographic peaks, specifically: peak 3 corresponding to monocaffeoyl tartaric acid, peak 4 corresponding to chlorogenic acid, peak 6 corresponding to aesculin, peak 7 corresponding to caffeic acid, peak 9 corresponding to chicoric acid, and peak 12 corresponding to aloin.

[0009] Preferably, with the chicoric acid chromatographic peak as the reference peak, the relative retention times of the 13 chromatographic peaks are: peak 1 is 0.19, peak 2 is 0.40, peak 3 is 0.50, peak 4 is 0.60, peak 5 is 0.63, peak 6 is 0.65, peak 7 is 0.69, peak 8 is 0.97, peak 10 is 1.11, peak 11 is 1.15, peak 12 is 1.22, and peak 13 is 1.49.

[0010] Advantages of the present invention: (1) The present invention has established an HPLC characteristic chromatogram for Kangfu Xiaoyan Suppositories, identified 13 characteristic peaks, and identified 6 components, which more fully and comprehensively demonstrates the chemical composition characteristics of Kangfu Xiaoyan Suppositories; (2) The sample pretreatment of the present invention is simple and rapid. The extraction method of the sample is investigated, and the types and proportions of the mobile phase are investigated. Within the investigated range, 13 characteristic peaks are presented in this chromatographic method, and the relative retention times of each peak vary little; (3) The present invention has carried out methodological verification on the constructed characteristic chromatogram method of Kangfu Xiaoyan Suppositories, including specificity, precision, repeatability, and stability experiments. In the results of each experiment, the RSD values of the relative retention times of each peak are all ≤2.0%, and the RSD values of the relative peak areas are all ≤10.0%. It shows that this characteristic chromatogram method is good, and this method can reflect the major chemical components in Kangfu Xiaoyan Suppositories. It can more efficiently and quickly control the overall quality of Kangfu Xiaoyan Suppositories. Description of the Drawings

[0011] The present invention will be further described below with reference to the accompanying drawings.

[0012] Figure 1 Chromatogram of wavelength investigation; wherein, S1: 300nm; S2: 320nm; S3: 340nm; Figure 2Specific HPLC chromatogram; among them, S1: negative; S2: caffeoyl tartaric acid control; S3: aesculetin control; S4: caffeic acid control; S5: chlorogenic acid control; S6: chicoric acid control; S7: aloin control; S8: test sample: 20221119; Figure 3 Precision HPLC chromatogram; among them, S1 - S6: precision 1 - precision 6; Figure 4 Repeatability HPLC chromatogram; among them, S1 - S6: repeatability 1 - repeatability 6; Figure 5 Stability HPLC chromatogram; among them, S1: 0h; S2: 2h; S3: 4h; S4: 8h; S5: 12h; S6: 24h; Figure 6 HPLC chromatograms of eight batches of test samples of Kangfu Xiaoyan Suppository; among them, S1: 20221119; S2: 20230209; S3: 20230311; S4: 20230415; S5: 20230608; S6: 20230704; S7: 20230817; S8: 20231102; Figure 7 Characteristic chromatogram of Kangfu Xiaoyan Suppository control. Detailed implementation mode

[0013] The present invention will be further described below in conjunction with the embodiments and the accompanying drawings. The instruments and equipment used in the embodiments are as follows: I. Instruments and test drugs 1.1 Instrument and equipment 。

[0014] 1.2 Test materials 。

[0015] 1.3 Control information 。

[0016] 1.4 Sample information 。

[0017] Investigation on the construction method of the characteristic chromatogram of Kangfu Xiaoyan Suppository in Example 1 1.1 Investigation on the preparation method of the test sample 1.1.1 Investigation on the extraction solvent of the test sample Chromatographic conditions Chromatographic column: Alltima-C18 (4.6x250mm, 5um); mobile phase A is 0.1% trifluoroacetic acid - 80% acetonitrile aqueous solution, mobile phase B is tetrahydrofuran: methanol: water (10:38:45), and gradient elution is carried out according to the regulations in the following table; column temperature is 30°C; detection wavelength is 320nm. The theoretical plate number calculated by the chicoric acid peak should be not less than 5000.

[0018] 。

[0019] Preparation of reference substance solution Weigh appropriate amounts of reference substances of monocaffeoyl tartaric acid, chlorogenic acid, aesculin, caffeic acid, chicoric acid, and aloin accurately, and dissolve them in methanol respectively to make solutions containing 65μg per 1ml. Shake well to obtain the reference substance solution.

[0020] Preparation of test solution Take 1g of Kangfu Xiaoyan Suppository (batch number: 20221119), weigh accurately, and add 40ml of methanol-water-formic acid solution with a ratio of 70:30:0.1, methanol-water-formic acid solution with a ratio of 50:50:0.1, and methanol-water solution with a ratio of 70:30 respectively; ultrasonicate at 50°C for 30 minutes with an ultrasonic power of 200W; centrifuge at 8000rpm for 10 minutes, and take the supernatant for standby; select Oasis MCX mixed-mode solid-phase extraction column, and sequentially equilibrate with 5ml of methanol and 5ml of water containing 0.1% formic acid to activate the extraction column; pass the extract through the column at a flow rate of 1ml / min, and wash off the fat-soluble impurities with 5ml of methanol-water-formic acid solution with a ratio of 20:80:0.1; elute the acidic components with 5ml of methanol-water-formic acid solution with a ratio of 70:30:0.1, and change to elute the basic components with 5ml of methanol-water-ammonia solution with a ratio of 70:30:2. Collect the eluents of acidic and basic components, concentrate them to 2ml by nitrogen blowing, and filter through a 0.22μm filter membrane to obtain the test solution; Determination method Precisely pipette 10μl of the reference substance solution and the test solution respectively, inject them into the liquid chromatograph, and record the chromatogram.

[0021] The results of solvent investigation show that when the extraction solvent contains 0.1% formic acid and the volume ratio of the extraction solvent is 70:30:0.1, the peak separation degree is better and the peak shape is better; when there is no formic acid in the extraction solvent, there are situations of poor peak shape and peak missing, and the corresponding effects cannot be achieved. Therefore, the methanol-water-formic acid solution with a volume ratio of 70:30:0.1 is selected as the extraction solvent.

[0022] Table 1 Results of relative retention time of solvent investigation 。

[0023] 1.1.2 Investigation of extraction time of test sample Preparation of Test Solution Take 1 g of Kangfu Xiaoyan Suppository (batch number: 20221119), accurately weigh it, add 40 ml of methanol-water-formic acid solution with a ratio of 70:30:0.1; ultrasonicate at 50 °C and 200 W and heat under reflux at 85 °C for 30 minutes respectively; centrifuge at 8000 rpm for 10 minutes, take the supernatant for standby; select Oasis MCX mixed-mode solid-phase extraction column, and sequentially equilibrate with 5 ml of methanol and 5 ml of water containing 0.1% formic acid to activate the extraction column; pass the extract through the column at a flow rate of 1 ml / min, and wash off the fat-soluble impurities with 5 ml of methanol-water-formic acid solution with a ratio of 20:80:0.1; elute the acidic components with 5 ml of methanol-water-formic acid solution with a ratio of 70:30:0.1, and change to elute the basic components with 5 ml of methanol-water-ammonia solution with a ratio of 70:30:2. Collect the eluates of acidic and basic components, concentrate to 2 ml by nitrogen blowing, and filter through a 0.22 μm filter membrane to obtain the test solution; The chromatographic conditions are the same as those in item "1.1.1"; the reference solution used is the reference solution in item "1.1.1".

[0024] Assay Method Precisely pipette 10 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and record the chromatogram.

[0025] The results of the extraction method investigation show that the peak resolution obtained by ultrasonic extraction is higher than that by heating under reflux extraction, and the peak shape is better. Therefore, ultrasonic extraction method is selected.

[0026] Table 2 Results of Relative Retention Time of Extraction Method Investigation 。

[0027] 1.2 Investigation of Chromatographic Conditions 1.2.1 Wavelength Investigation Chromatographic Conditions Chromatographic column: Alltima-C18 (4.6x250 mm, 5 μm); use 0.1% trifluoroacetic acid-80% acetonitrile aqueous solution as mobile phase A, and use tetrahydrofuran:methanol:water (10:38:45) as mobile phase B, and perform gradient elution according to the regulations in the following table; the column temperature is 30 °C; the detection wavelengths are 300 nm, 320 nm, and 340 nm. The number of theoretical plates calculated based on the chicoric acid peak should not be less than 5000.

[0028] 。

[0029] Preparation of Reference Solution Weigh appropriate amounts of single caffeoyl tartaric acid, chlorogenic acid, aesculin, caffeic acid, chicoric acid, and aloin reference substances accurately. Dissolve them separately in methanol to prepare solutions containing 65 μg per 1 ml. Shake well to obtain the reference substance solutions.

[0030] Preparation of the test solution Take 1 g of Kangfu Xiaoyan Suppository, weigh accurately, add 40 ml of a methanol-water-formic acid composite solvent with a ratio of 70:30:0.1; sonicate at 50 °C for 30 minutes with a sonication power of 200 W; centrifuge at 8000 rpm for 10 minutes, and take the supernatant for standby; select an Oasis MCX mixed-mode solid-phase extraction column, and sequentially wash and activate the extraction column with 5 ml of methanol and 5 ml of water containing 0.1% formic acid; pass the extract through the column at a flow rate of 1 ml / min, and wash away the fat-soluble impurities with 5 ml of a methanol-water-formic acid solution with a ratio of 20:80:0.1; elute the acidic components with 5 ml of a methanol-water-formic acid solution with a ratio of 70:30:0.1, and change to 5 ml of a methanol-water-ammonia solution with a ratio of 70:30:2 to elute the basic components. Collect the eluates of the acidic and basic components, concentrate them to 2 ml by nitrogen blowing, and filter through a 0.22 μm filter membrane to obtain the test solution.

[0031] Determination method Precisely pipette 10 μl each of the reference substance solution and the test solution, inject them into the liquid chromatograph, and record the chromatogram.

[0032] The results show that 13 characteristic peaks can be detected at wavelengths ranging from 300 nm to 340 nm, and the RSD of the relative retention time is less than 1.0%. Therefore, wavelengths ranging from 300 nm to 340 nm can meet the requirements for detecting the characteristic chromatogram. The results are shown in the appendix Figure 1 。

[0033] Table 3 Results of relative retention time for wavelength investigation 。

[0034] 1.2.2 Investigation of mobile phase types Chromatographic condition 1: Chromatographic column: Alltima-C18 (4.6 x 250 mm, 5 μm); use 0.1% trifluoroacetic acid - 80% acetonitrile aqueous solution as mobile phase A, and use tetrahydrofuran:methanol:water (10:38:45) as mobile phase B, and perform gradient elution according to the regulations in the following table; column temperature is 30 °C; detection wavelength is 320 nm. The number of theoretical plates calculated based on the chicoric acid peak should be not less than 5000.

[0035] 。

[0036] Chromatographic conditions 2: Column: Alltima-C18 (4.6x250mm, 5um); mobile phase A is 80% acetonitrile aqueous solution, mobile phase B is tetrahydrofuran: methanol: water (10:38:45), gradient elution is carried out according to the regulations in the following table; column temperature is 30°C; detection wavelength is 320nm. The theoretical plate number calculated by the chicoric acid peak should be not less than 5000.

[0037] 。

[0038] Chromatographic conditions 3: Column: Alltima-C18 (4.6x250mm, 5um); mobile phase A is 0.1% trifluoroacetic acid - 80% acetonitrile aqueous solution, mobile phase B is 0.2% phosphoric acid solution, gradient elution is carried out according to the regulations in the following table; column temperature is 30°C; detection wavelength is 320nm. The theoretical plate number calculated by the chicoric acid peak should be not less than 5000.

[0039] 。

[0040] Preparation of reference substance solution Weigh appropriate amounts of reference substances of monocaffeoyl tartaric acid, chlorogenic acid, aesculin, caffeic acid, chicoric acid, and aloin, accurately weigh them, and dissolve them in methanol respectively to make solutions containing 65μg per 1ml, shake well, and use them as reference substance solutions.

[0041] Preparation of test solution Take 1g of Kangfu Xiaoyan Suppository, accurately weigh it, add 40ml of methanol-water-formic acid composite solvent with a ratio of 70:30:0.1; ultrasonicate at 50°C for 30 minutes, with an ultrasonic power of 200W; centrifuge at 8000rpm for 10 minutes, take the supernatant for standby; select Oasis MCX mixed-mode solid-phase extraction column, and sequentially activate the extraction column with 5ml of methanol and 5ml of water containing 0.1% formic acid; pass the extract through the column at a flow rate of 1ml / min, and wash away the fat-soluble impurities with 5ml of methanol-water-formic acid with a ratio of 20:80:0.1; elute the acidic components with 5ml of methanol-water-formic acid with a ratio of 70:30:0.1, and change to elute the basic components with 5ml of methanol-water-ammonia with a ratio of 70:30:2, collect the acidic and basic component eluates, concentrate them by nitrogen blowing to 2ml, and filter through a 0.22μm filter membrane to obtain the test solution.

[0042] Determination method Precisely pipette 10μl of each of the reference substance solution and the test solution respectively, inject them into the liquid chromatograph, and record the chromatogram.

[0043] The results showed that when using 0.1% trifluoroacetic acid - 80% acetonitrile aqueous solution as mobile phase A and tetrahydrofuran:methanol:water (10:38:45) as mobile phase B for elution, there were 13 characteristic peaks in the chromatogram of the test sample, and the peak separation was good and the peak shape was excellent; when using 80% acetonitrile aqueous solution as mobile phase A and tetrahydrofuran:methanol:water (10:38:45) as mobile phase B or using 0.1% trifluoroacetic acid - 80% acetonitrile aqueous solution as mobile phase A and 0.2% phosphoric acid solution as mobile phase B for elution, each peak was missing and the corresponding effects could not be achieved.

[0044] Table 4 Results of relative retention time for investigation of mobile phase types 。

[0045] 1.2.3 Investigation of mobile phase ratio Chromatographic conditions Chromatographic column: Alltima - C18 (4.6x250mm, 5um); using 0.1% trifluoroacetic acid - 80% acetonitrile aqueous solution as mobile phase A and tetrahydrofuran:methanol:water (10:38:45) as mobile phase B, perform gradient elution according to the regulations in the following table; column temperature is 30°C; detection wavelength is 320nm. The number of theoretical plates calculated based on the chicoric acid peak should be not less than 5000.

[0046] Mobile phase ratio 1: 。

[0047] Mobile phase ratio 2: 。

[0048] Mobile phase ratio 3: 。

[0049] Preparation of reference substance solution Weigh appropriate amounts of reference substances of monocaffeoyl tartaric acid, chlorogenic acid, aesculin, caffeic acid, chicoric acid, and aloin accurately, and dissolve them in methanol respectively to prepare solutions containing 65μg per 1ml, shake well, and use them as reference substance solutions.

[0050] Preparation of test sample solution Take 1 g of Kangfu Xiaoyan Suppository, accurately weigh it, add 40 ml of a methanol-water-formic acid composite solvent with a ratio of 70:30:0.1; ultrasonicate at 50 °C for 30 minutes with an ultrasonic power of 200 W; centrifuge at 8000 rpm for 10 minutes, and take the supernatant for standby; select an Oasis MCX mixed-mode solid-phase extraction column, and sequentially equilibrate it with 5 ml of methanol and 5 ml of water containing 0.1% formic acid to activate the extraction column; pass the extract through the column at a flow rate of 1 ml / min, and wash away the fat-soluble impurities with 5 ml of methanol-water-formic acid with a ratio of 20:80:0.1; elute the acidic components with 5 ml of methanol-water-formic acid with a ratio of 70:30:0.1, and change to 5 ml of methanol-water-ammonia water with a ratio of 70:30:2 to elute the basic components. Collect the eluates of the acidic and basic components, concentrate them to 2 ml by nitrogen blowing, and filter through a 0.22 μm filter membrane to obtain the test solution.

[0051] Determination method Precisely pipette 10 μl each of the reference solution and the test solution, inject them into the liquid chromatograph, and record the chromatogram.

[0052] The results showed that when eluting with mobile phase ratio 1, a total of 13 characteristic peaks appeared in the chromatogram of the test sample, and the peak separation was good and the peak shape was excellent; when eluting with mobile phase ratio 2 or mobile phase ratio 3, each peak was missing and the corresponding effects could not be achieved.

[0053] Table 5 Results of relative retention time for investigation of mobile phase ratio .

[0054] Example 2 Methodology verification of the characteristic chromatogram of Kangfu Xiaoyan Suppository 2.1 Specificity Chromatographic conditions Chromatographic column: Alltima-C18 (4.6 x 250 mm, 5 μm); use 0.1% trifluoroacetic acid - 80% acetonitrile aqueous solution as mobile phase A, and use tetrahydrofuran:methanol:water (10:38:45) as mobile phase B, and perform gradient elution according to the regulations in the following table; column temperature is 30 °C; detection wavelength is 320 nm. The number of theoretical plates calculated based on the chicoric acid peak should be not less than 5000.

[0055] .

[0056] Preparation of the reference solution Take appropriate amounts of reference substances of monocaffeoyl tartaric acid, chlorogenic acid, aesculin, caffeic acid, chicoric acid, and aloin, accurately weigh them, and separately dissolve them in methanol to prepare solutions containing 65 μg per 1 ml, shake well, and use them as the reference solution.

[0057] Preparation of the test solution Take 1 g of Kangfu Xiaoyan Suppository, accurately weigh it, add 40 ml of a methanol-water-formic acid composite solvent with a ratio of 70:30:0.1; ultrasonicate at 50 °C for 30 minutes with an ultrasonic power of 200 W; centrifuge at 8000 rpm for 10 minutes, and take the supernatant for standby; select an Oasis MCX mixed-mode solid-phase extraction column, and sequentially equilibrate with 5 ml of methanol and 5 ml of water containing 0.1% formic acid to activate the extraction column; pass the extract through the column at a flow rate of 1 ml / min, and wash away the fat-soluble impurities with 5 ml of a methanol-water-formic acid solution with a ratio of 20:80:0.1; elute the acidic components with 5 ml of a methanol-water-formic acid solution with a ratio of 70:30:0.1, and change to 5 ml of a methanol-water-ammonia solution with a ratio of 70:30:2 to elute the basic components. Collect the eluates of the acidic and basic components, concentrate them to 2 ml by nitrogen blowing, and filter through a 0.22 μm filter membrane to obtain the test solution.

[0058] Preparation of negative solution Take an appropriate amount of excipients, accurately weigh them, add 40 ml of a methanol-water-formic acid composite solvent with a ratio of 70:30:0.1; ultrasonicate at 50 °C for 30 minutes with an ultrasonic power of 200 W; centrifuge at 8000 rpm for 10 minutes, and take the supernatant for standby; select an Oasis MCX mixed-mode solid-phase extraction column, and sequentially equilibrate with 5 ml of methanol and 5 ml of water containing 0.1% formic acid to activate the extraction column; pass the extract through the column at a flow rate of 1 ml / min, and wash away the fat-soluble impurities with 5 ml of a methanol-water-formic acid solution with a ratio of 20:80:0.1; elute the acidic components with 5 ml of a methanol-water-formic acid solution with a ratio of 70:30:0.1, and change to 5 ml of a methanol-water-ammonia solution with a ratio of 70:30:2 to elute the basic components. Collect the eluates of the acidic and basic components, concentrate them to 2 ml by nitrogen blowing, and filter through a 0.22 μm filter membrane to obtain the test solution.

[0059] Determination method Precisely pipette 10 μl each of the negative solution, reference solution, and test solution, inject them into the liquid chromatograph, and record the chromatogram.

[0060] The results showed that the negative solution had no interference and the specificity was good. The results are shown in the appendix Figure 2 .

[0061] 2.2 Precision Take 1 g of Kangfu Xiaoyan Suppository (batch number: 20221119), prepare the test solution according to the preparation method of the test solution under item "2.1", inject 6 needles according to the chromatographic conditions under item "2.1", record the chromatogram, measure the relative retention time and relative peak area of each chromatographic peak, and calculate the RSD.

[0062] Table 6 Results of relative retention time for precision .

[0063] Table 7 Results of relative peak area for precision 。

[0064] The results showed that the RSD of the relative retention time of each chromatographic peak was less than 1.0%, and the RSD of the relative peak area was less than 10.0%, indicating good precision. The chromatogram is shown in the appendix Figure 3 。

[0065] 2.3 Repeatability Take 1 g of Kangfu Xiaoyan Suppository (batch number: 20221119), a total of 6 portions. Prepare the test solution according to the preparation method of the test solution under item "2.1", inject the sample according to the chromatographic conditions under item "2.1", record the chromatogram, determine the relative retention time and relative peak area of each chromatographic peak, and calculate the RSD

[0066] Table 8 Results of relative retention time for repeatability 。

[0067] Table 9 Results of relative peak area for repeatability 。

[0068] The results showed that the RSD of the relative retention time of each chromatographic peak was less than 2.0%, and the RSD of the relative peak area was less than 10.0%, indicating good repeatability. The chromatogram is shown in the appendix Figure 4 。

[0069] 2.4 Stability Take 1 g of Kangfu Xiaoyan Suppository (batch number: 20221119), prepare the test solution according to the preparation method of the test solution under item "2.1", and inject the sample according to the chromatographic conditions under item "2.1" at 0 h, 2 h, 4 h, 8 h, 12 h, and 24 h respectively. Record the chromatogram, determine the relative retention time and relative peak area of each chromatographic peak, and calculate the RSD

[0070] Table 10 Results of relative retention time for stability 。

[0071] Table 11 Results of relative peak area for stability 。

[0072] The results showed that the RSD of the relative retention time of each chromatographic peak was less than 1.0%, and the RSD of the relative peak area was less than 8.0%, indicating good stability of the solution. The chromatogram is shown in the appendix Figure 5 。

[0073] In summary, the specificity of this characteristic chromatogram is good, and there is no interference from negative samples, indicating that the characteristic detection method has strong specificity; in the precision test, the RSD of the relative retention time is less than 1.0%, and the RSD of the relative peak area is less than 10.0%, indicating good instrument precision; in the repeatability test, the RSD of the relative retention time of each chromatographic peak is less than 2.0%, and the RSD of the relative peak area is less than 10.0%, indicating good repeatability; in the stability test, the RSD of the relative retention time of each chromatographic peak is less than 1.0%, and the RSD of the relative peak area is less than 8.0%, so the test solution is stable within 24 hours. This method has been well verified by methodology.

[0074] Example 3 Construction of the Control Characteristic Chromatogram of Kangfu Xiaoyan Suppository Chromatographic Conditions Chromatographic column: Alltima-C18 (4.6x250mm, 5um); mobile phase A is 0.1% trifluoroacetic acid - 80% acetonitrile aqueous solution, and mobile phase B is tetrahydrofuran: methanol: water (10:38:45), and gradient elution is carried out according to the regulations in the following table; the column temperature is 30°C; the detection wavelength is 320nm. The theoretical plate number calculated based on the chicoric acid peak should not be less than 5000.

[0075] 。

[0076] Preparation of Reference Substance Solution Appropriately weigh a proper amount of reference substances of monocaffeoyl tartaric acid, chlorogenic acid, aesculin, caffeic acid, chicoric acid, and aloin, and accurately weigh them. Dissolve them in methanol respectively to make solutions containing 65μg per 1ml, shake well, and use them as reference substance solutions.

[0077] Preparation of Test Solution Take 1g of Kangfu Xiaoyan Suppository, accurately weigh it, add 40ml of a methanol - water - formic acid composite solvent with a ratio of 70:30:0.1; ultrasonicate at 50°C for 30 minutes, with an ultrasonic power of 200W; centrifuge at 8000rpm for 10 minutes, and take the supernatant for standby; select an Oasis MCX mixed-mode solid-phase extraction column, and sequentially use 5ml of methanol and 5ml of water containing 0.1% formic acid to balance and activate the extraction column; pass the extract through the column at a flow rate of 1ml / min, and wash off the fat-soluble impurities with 5ml of a methanol - water - formic acid solution with a ratio of 20:80:0.1; elute the acidic components with 5ml of a methanol - water - formic acid solution with a ratio of 70:30:0.1, and change to 5ml of a methanol - water - ammonia solution with a ratio of 70:30:2 to elute the basic components. Collect the eluates of the acidic and basic components, concentrate them to 2ml by nitrogen blowing, and filter through a 0.22μm filter membrane to obtain the test solution.

[0078] Determination Method Precisely pipette 10μl of each of the reference substance solution and the test solution, inject them into the liquid chromatograph, and record the chromatogram.

[0079] The chromatograms of 8 batches of Kangfu Xiaoyan Suppositories were imported into the "Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints". The chromatographic peaks that existed in the chromatograms of different batches of Kangfu Xiaoyan Suppositories were selected as common peaks; the reference characteristic fingerprint was generated by the average value calculation method, and the relative retention time and relative peak area of each common peak were calculated. The results are shown in Table 12 and Table 13, attached Figures 6 - 7 。

[0080] Table 12 Results of relative retention time of 8 batches of Kangfu Xiaoyan Suppository samples 。

[0081] Table 13 Results of relative peak area of 8 batches of Kangfu Xiaoyan Suppository samples 。

[0082] 13 common peaks were presented in the chromatograms of 8 batches of Kangfu Xiaoyan Suppository samples. The RSD of the relative retention time of the 13 common peaks was less than 1.0%, and the RSD of the relative peak area was less than 10.0%.

[0083] 13 characteristic peaks were presented in the test sample chromatogram. Among them, peak 3, peak 4, peak 6, peak 7, peak 9, and peak 12 corresponded to the reference substance peaks of monocaffeoyl tartaric acid, chlorogenic acid, aesculin, caffeic acid, chicoric acid, and aloin respectively; the peak corresponding to the reference substance peak of chicoric acid was the S peak. The relative retention time of each characteristic peak and the S peak was calculated, and its relative retention time should be within the range of ±10% of the specified value. The specified values are: 0.19 (peak 1), 0.40 (peak 2), 0.50 (peak 3), 0.60 (peak 4), 0.63 (peak 5), 0.65 (peak 6), 0.69 (peak 7), 0.97 (peak 8), 1.11 (peak 10), 1.15 (peak 11), 1.22 (peak 12), 1.49 (peak 13).

Claims

1. A method for constructing a characteristic fingerprint of Kangfu Xiaoyan Suppository, characterized in that: It includes the following steps: (1) Preparation of the reference solution: Weigh the reference substances accurately, and dissolve them in methanol respectively to prepare solutions containing 65 μg per 1 ml, shake well, and use them as the reference solutions. The reference substances are: monocaffeoyl tartaric acid, chlorogenic acid, aesculin, caffeic acid, chicoric acid, and aloin; (2) Preparation of the test solution: Take 1 g of Kangfu Xiaoyan Suppository, weigh it accurately, add 40 ml of a methanol-water-formic acid composite solvent with a ratio of 70:30:0.1; after ultrasonic treatment and centrifugation, take the supernatant for standby; Select a solid-phase extraction column, and activate the extraction column by sequentially equilibrating with 5 ml of methanol and 5 ml of water containing 0.1% formic acid; Pass the extract, that is, the supernatant after centrifugation, through the column at a flow rate of 1 ml / min, wash away the fat-soluble impurities with 5 ml of a methanol-water-formic acid solution with a ratio of 20:80:0.1, elute the acidic components with 5 ml of a methanol-water-formic acid solution with a ratio of 70:30:0.1, and change to 5 ml of a methanol-water-ammonia water solution with a ratio of 70:30:2 to elute the basic components. Collect the eluates of the acidic and basic components, concentrate them to 2 ml by nitrogen blowing, and filter through a 0.22 μm filter membrane to obtain the test solution; (3) Determination: Inject the reference solution and the test solution into a high-performance liquid chromatograph under the following chromatographic conditions, inject 10 μl each, and record the chromatogram; The chromatographic conditions are: Using octadecylsilane-bonded silica gel as the filler; Using 0.1% trifluoroacetic acid - 80% acetonitrile aqueous solution as mobile phase A, and using tetrahydrofuran:methanol:water with a volume ratio of 10:38:45 as mobile phase B, and perform elution according to the specified gradient; The column temperature is 30°C; The flow rate is 1 ml per minute; The detection wavelength of the ultraviolet-visible detector is 300 - 340 nm; The elution gradient conditions are: 0 - 15 min, mobile phase A: mobile phase B, volume ratio changes from 5:95 to 10:90; 15 - 25 min, mobile phase A: mobile phase B, volume ratio changes from 10:90 to 14:86; 25 - 27 min, mobile phase A: mobile phase B, volume ratio changes from 14:86 to 17:83; 27 - 28 min, mobile phase A: mobile phase B, volume ratio changes from 17:83 to 22:78; 28 - 50 min, mobile phase A: mobile phase B, volume ratio changes from 22:78 to 23:77; 50 - 65 min, mobile phase A: mobile phase B, volume ratio changes from 23:77 to 55:45; 65 - 68 min, mobile phase A: mobile phase B, volume ratio changes from 55:45 to 5:95; 68 - 80 min, mobile phase A: mobile phase B, volume ratio is 5:95; (4) Generate the reference characteristic chromatogram: Select the chromatographic peaks that exist in the chromatograms of different batches of Kangfu Xiaoyan Suppository as the common peaks, and generate the reference characteristic chromatogram of Kangfu Xiaoyan Suppository by the average value calculation method.

2. The method according to claim 1, characterized in that, In step (2), the ultrasonic condition is ultrasonic treatment at 50°C for 30 minutes, and the ultrasonic power is 200 W.

3. The method according to claim 1, characterized in that, In step (2), the centrifugation condition is centrifugation at 8000 rpm for 10 minutes.

4. The method according to claim 1, characterized in that, In step (2), the solid-phase extraction column is Oasis MCX mixed-mode solid-phase extraction column.

5. The method according to claim 1, characterized in that In step (3), the chromatographic column is Alltima-C18 4.6x250mm, 5um.

6. The method according to claim 1, wherein The control characteristic chromatogram generated in step (4) includes 13 common chromatographic peaks, specifically: peak 3 corresponding to monocaffeoyl tartaric acid, peak 4 corresponding to chlorogenic acid, peak 6 corresponding to aesculin, peak 7 corresponding to caffeic acid, peak 9 corresponding to chicoric acid, and peak 12 corresponding to aloin.

7. The method according to claim 1, wherein Taking the chicoric acid chromatographic peak as the reference peak, the relative retention times of the 13 chromatographic peaks are as follows: peak 1 is 0.19, peak 2 is 0.40, peak 3 is 0.50, peak 4 is 0.60, peak 5 is 0.63, peak 6 is 0.65, peak 7 is 0.69, peak 8 is 0.97, peak 10 is 1.11, peak 11 is 1.15, peak 12 is 1.22, and peak 13 is 1.49.

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