A method for constructing a characteristic spectrum of Kangfu Xiaoyan Suppository

Abstract

The present invention relates to the field of traditional Chinese medicine analysis technology, and more particularly to a method for constructing a characteristic spectrum for Kangfu Xiaoyan Suppositories, comprising the following steps: 1. preparing a reference solution; 2. preparing a test solution; 3. determining assay conditions; and 4. importing the chromatogram into a traditional Chinese medicine fingerprint to generate a control characteristic spectrum. This characteristic method identifies 13 characteristic peaks, demonstrates high repeatability and precision, and comprehensively and comprehensively displays the chemical composition characteristics of Kangfu Xiaoyan Suppositories, enabling comprehensive control of their quality.

Description

Technical Field

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

[0002] Kangfu Anti-inflammatory Suppositories are composed of 8 traditional Chinese medicines, including Sophora flavescens, Herba Patriniae, Viola yedoensis, and Taraxacum mongolicum. Pharmacodynamic tests have shown that Kangfu Anti-inflammatory Suppositories have both extensive and obvious effects of clearing away heat and detoxifying, killing insects and removing dampness, removing blood stasis and relieving pain, as well as anti-pathogens, increasing the phagocytic function of leukocytes and anti-inflammatory effects. At the same time, they also have the effect of repairing damaged tissues and are mainly used for the treatment of gynecological diseases. Kangfu Anti-inflammatory Suppositories have significant therapeutic effects on acute and chronic pelvic inflammatory disease, inflammatory masses, metritis, adnexitis, urinary tract infections, hemorrhoids, prostatitis and other diseases. The quality standards in the 2020 edition of the "Chinese Pharmacopoeia" only include quality control of matrine, aloin and hyodeoxycholic acid, and the ingredients of other medicinal materials are not mentioned. There is no research on the characteristic spectrum of Kangfu Anti-inflammatory Suppositories in the patent, and the literature only has research on the determination of matrine content, and the overall quality of Kangfu Anti-inflammatory Suppositories cannot be controlled.

[0003] Kangfu Xiaoyan Suppositories are composed of eight traditional Chinese medicines, each containing a variety of active ingredients (such as alkaloids, glycosides, and organic acids). These ingredients exhibit significant polarity differences and structural similarity, making chromatographic separation difficult and prone to peak overlap and coelution, impacting the accuracy of qualitative and quantitative analysis. Furthermore, the solubility of different ingredients varies significantly, requiring repeated experimentation to determine the optimal extraction solvent, method, and time to ensure extraction efficiency and reproducibility.

[0004] In view of this, the present invention is 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 map of Kangfu Xiaoyan Suppository.

[0006] The present invention provides a method for constructing a characteristic spectrum of Kangfu Xiaoyan Suppository, comprising the following steps:

[0007] (1) Preparation of reference solution: Take an appropriate amount of reference substance, weigh accurately, add methanol to make a solution containing 65 μg per 1 ml, shake well, and use it as the reference solution; the reference substances are: monocaffeoyltartaric acid, chlorogenic acid, esculetin, caffeic acid, chicoric acid, and aloin;

[0008] (2) Preparation of test solution: Take 1g of Kangfu Xiaoyan Suppository, weigh accurately, add 40ml of methanol-water-formic acid complex solvent with a ratio of 70:30:0.1; ultrasonicate at 50℃ for 30 minutes, ultrasonic power is 200W; centrifuge at 8000rpm for 10 minutes, and take the supernatant for later use; select Oasis An MCX mixed-mode solid-phase extraction column was balanced with 5 ml of methanol and 5 ml of water containing 0.1% formic acid in sequence to activate the extraction column; the extract, i.e., the supernatant obtained after centrifugation, was passed through the column at a flow rate of 1 ml / min, and fat-soluble impurities were washed away with 5 ml of methanol-water-formic acid in a ratio of 20:80:0.1, and acidic components were eluted with 5 ml of methanol-water-formic acid in a ratio of 70:30:0.1. The alkaline components were eluted with 5 ml of methanol-water-ammonia in a ratio of 70:30:2. The acidic and alkaline eluates were collected, concentrated to 2 ml with nitrogen blowdown, and filtered with a 0.22 μm filter membrane to obtain the test solution;

[0009] (3) Determination: The reference solution and the test solution were injected into a high performance liquid chromatograph under the following chromatographic conditions, with 10 μl of each sample injected, and the chromatogram was recorded; the chromatographic conditions were: octadecylsilane bonded silica gel as the filler; 0.1% trifluoroacetic acid-80% acetonitrile aqueous solution as the mobile phase A, tetrahydrofuran:methanol:water with a volume ratio of 10:38:45 as the mobile phase B, and elution was performed according to the specified gradient; the column temperature was 30°C; the flow rate was 1 ml per minute; the UV-Vis detector had a detection wavelength of 300-340 nm;

[0010] The elution gradient conditions were:

[0011] From 0 to 15 min, the volume ratio of mobile phase A:phase B changed from 5:95 to 10:90;

[0012] From 15 to 25 min, the volume ratio of mobile phase A:phase B changed from 10:90 to 14:86;

[0013] From 25 to 27 min, the volume ratio of mobile phase A:phase B changed from 14:86 to 17:83;

[0014] From 27 to 28 minutes, the volume ratio of mobile phase A:phase B changed from 17:83 to 22:78;

[0015] From 28 to 50 min, the volume ratio of mobile phase A:phase B changed from 22:78 to 23:77;

[0016] From 50 to 65 min, the volume ratio of mobile phase A:phase B changed from 23:77 to 55:45;

[0017] From 65 to 68 min, the volume ratio of mobile phase A:phase B was changed from 55:45 to 5:95;

[0018] 68-80 min, mobile phase A:B, volume ratio 5:95;

[0019] (4) Generate a control characteristic spectrum: Use the “Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System” developed by the Chinese Pharmacopoeia Committee to 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 control characteristic spectrum of Kangfu Xiaoyan Suppositories.

[0020] Preferably, the chromatographic column in step (3) is: Alltima-C18 (4.6x250mm, 5um).

[0021] Preferably, the control characteristic spectrum generated in step (4) includes 13 common chromatographic peaks, specifically: peak 3 corresponding to monocaffeoyltartaric acid, peak 4 corresponding to chlorogenic acid, peak 6 corresponding to esculentin, peak 7 corresponding to caffeic acid, peak 9 corresponding to chicoric acid, and peak 12 corresponding to aloin.

[0022] Preferably, the characteristic peak is based on the chicoric acid chromatographic peak as the reference peak, and 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.

[0023] Beneficial effects of the present invention:

[0024] (1) The present invention established an HPLC characteristic spectrum for Kangfu Xiaoyan Suppositories, identified 13 characteristic peaks, and identified 6 components, which fully and comprehensively demonstrated the chemical composition characteristics of Kangfu Xiaoyan Suppositories;

[0025] (2) The sample pretreatment of the present invention is simple and rapid. The sample extraction method was investigated, and the type and ratio of the mobile phase were investigated. Within the investigated range, 13 characteristic peaks were present in the chromatographic method, and the relative retention time of each peak had a small change;

[0026] (3) The method for characterizing Kangfu Xiaoyan Suppositories was validated by methodology, including specificity, precision, repeatability, and stability tests. The RSD values ​​of the relative retention times of the peaks in each experimental result were all ≤2.0%, and the RSD values ​​of the relative peak areas were all ≤10.0%. This indicates that the characterizing method is good and can reflect the major chemical components in Kangfu Xiaoyan Suppositories. This method can more efficiently and quickly control the overall quality of Kangfu Xiaoyan Suppositories. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The present invention will be further described below with reference to the accompanying drawings.

[0028] Figure 1 Wavelength inspection chromatogram; among them, S1: 300nm; S2: 320nm; S3: 340nm;

[0029] Figure 2 Specific HPLC chromatogram; S1: negative; S2: monocaffeoyltartaric acid control; S3: aesculetin control; S4: caffeic acid control; S5: chlorogenic acid control; S6: chicoric acid control; S7: aloin control; S8: test sample: 20221119;

[0030] Figure 3 Precision HPLC chromatogram; where S1-S6: precision 1-precision 6;

[0031] Figure 4 Repeatability HPLC chromatogram; where S1-S6: repeatability 1-repeatability 6;

[0032] Figure 5 Stability HPLC chromatogram; wherein, S1: 0h; S2: 2h; S3: 4h; S4: 8h; S5: 12h; S6: 24h;

[0033] Figure 6 HPLC chromatograms of eight batches of Kangfu Xiaoyan Suppositories test samples; among them, S1: 20221119; S2: 20230209; S3: 20230311; S4: 20230415; S5: 20230608; S6: 20230704; S7: 20230817; S8: 20231102;

[0034] Figure 7 Comparative characteristic spectrum of Kangfu Anti-inflammatory Suppository. DETAILED DESCRIPTION

[0035] The present invention will be further described below with reference to the following examples and accompanying drawings. The instruments and equipment used in the examples are as follows:

[0036] 1. Instruments and reagents

[0037] 1.1 Instruments and Equipment

[0038] .

[0039] 1.2 Test materials

[0040] .

[0041] 1.3 Reference Material Information

[0042] .

[0043] 1.4 Sample Information

[0044] .

[0045] Example 1 Investigation of the method for constructing the characteristic spectrum of Kangfu Xiaoyan Suppository

[0046] 1.1 Investigation of test sample preparation methods

[0047] 1.1.1 Investigation of the extraction solvent of the test sample

[0048] Chromatographic conditions

[0049] Chromatographic column: Alltima-C18 (4.6 x 250 mm, 5 μm); mobile phase A: 0.1% trifluoroacetic acid-80% acetonitrile in water; mobile phase B: tetrahydrofuran: methanol: water (10:38:45); gradient elution as specified in the table below; column temperature: 30°C; detection wavelength: 320 nm. The number of theoretical plates calculated based on the chicoric acid peak should be no less than 5000.

[0050] .

[0051] Preparation of reference solution

[0052] Take appropriate amount of monocaffeoyltartaric acid, chlorogenic acid, esculetin, caffeic acid, chicoric acid, and aloin reference substances, accurately weigh them, add methanol to make solutions containing 65ug per 1ml, shake well, and use them as reference solutions.

[0053] Preparation of test solution

[0054] Take 1g of Kangfu Xiaoyan Suppository (batch number: 20221119), accurately weigh it, 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℃ for 30 minutes, ultrasonic power of 200W; centrifuge at 8000rpm for 10 minutes, and take the supernatant for use; select Oasis An MCX mixed-mode solid-phase extraction column was equilibrated with 5 ml of methanol and then 5 ml of water containing 0.1% formic acid to activate the extraction column. The extract was passed through the column at a flow rate of 1 ml / min, and fat-soluble impurities were washed away with 5 ml of methanol-water-formic acid in a ratio of 20:80:0.1. The acidic components were eluted with 5 ml of methanol-water-formic acid in a ratio of 70:30:0.1, and the alkaline components were eluted with 5 ml of methanol-water-ammonia in a ratio of 70:30:2. The acidic and alkaline eluates were collected, concentrated to 2 ml with nitrogen blowdown, and filtered with a 0.22 μm filter membrane to obtain the test solution.

[0055] Assay

[0056] Accurately pipette 10 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and record the chromatogram.

[0057] Solvent investigations revealed that peak separation and peak shape were better when the extraction solvent contained 0.1% formic acid and the volume ratio was 70:30:0.1. However, when the extraction solvent lacked formic acid, peak shape was poor and peaks were missing, failing to achieve the desired effect. Therefore, a methanol-water-formic acid solution with a volume ratio of 70:30:0.1 was selected as the extraction solvent.

[0058] Table 1 Results of relative retention time of solvent investigation

[0059] .

[0060] 1.1.2 Investigation of extraction time of test samples

[0061] Preparation of test solution

[0062] Take 1g of Kangfu Xiaoyan Suppository (batch number: 20221119), weigh accurately, add 40ml of methanol-water-formic acid solution with a ratio of 70:30:0.1; heat and reflux at 50℃, 200W ultrasound and 85℃ for 30 minutes; centrifuge at 8000rpm for 10 minutes, and take the supernatant for later use; select Oasis An MCX mixed-mode solid-phase extraction column was equilibrated with 5 ml of methanol and then 5 ml of water containing 0.1% formic acid to activate the extraction column. The extract was passed through the column at a flow rate of 1 ml / min, and fat-soluble impurities were washed away with 5 ml of methanol-water-formic acid in a ratio of 20:80:0.1. The acidic components were eluted with 5 ml of methanol-water-formic acid in a ratio of 70:30:0.1, and the alkaline components were eluted with 5 ml of methanol-water-ammonia in a ratio of 70:30:2. The acidic and alkaline eluates were collected, concentrated to 2 ml with nitrogen blowdown, and filtered with a 0.22 μm filter membrane to obtain the test solution.

[0063] The chromatographic conditions are the same as those in item “1.1.1”; the reference solution is the reference solution in item “1.1.1”.

[0064] Assay

[0065] Accurately pipette 10 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and record the chromatogram.

[0066] The results of the extraction method investigation showed that ultrasonic extraction had higher peak separation and better peak shape than heating reflux extraction, so ultrasonic extraction was selected.

[0067] Table 2 Results of relative retention time of extraction method

[0068] .

[0069] 1.2 Investigation of chromatographic conditions

[0070] 1.2.1 Wavelength Investigation

[0071] Chromatographic conditions

[0072] Chromatographic column: Alltima-C18 (4.6 x 250 mm, 5 μm); mobile phase A: 0.1% trifluoroacetic acid-80% acetonitrile in water, mobile phase B: tetrahydrofuran: methanol: water (10:38:45), gradient elution as specified in the table below; column temperature: 30°C; detection wavelengths: 300 nm, 320 nm, and 340 nm. The number of theoretical plates calculated based on the chicoric acid peak should be no less than 5000.

[0073] .

[0074] Preparation of reference solution

[0075] Take appropriate amount of monocaffeoyltartaric acid, chlorogenic acid, esculetin, caffeic acid, chicoric acid, and aloin reference substances, accurately weigh them, add methanol to make solutions containing 65ug per 1ml, shake well, and use them as reference solutions.

[0076] Preparation of test solution

[0077] Take 1g of Kangfu Xiaoyan Suppository, weigh it accurately, add 40ml of methanol-water-formic acid composite solvent with a ratio of 70:30:0.1; ultrasonicate at 50℃ for 30 minutes, ultrasonic power is 200W; centrifuge at 8000rpm for 10 minutes, and take the supernatant for use; select OasisMCX mixed mode solid phase extraction column, and balance with 5ml of methanol and 5ml of water containing 0.1% formic acid in turn to activate the extraction column; pass the extract through the column at a flow rate of 1ml / min, and wash away fat-soluble impurities with 5ml of methanol-water-formic acid with a ratio of 20:80:0.1; elute the acidic component with 5ml of methanol-water-formic acid with a ratio of 70:30:0.1, and use 5ml of methanol-water-ammonia water with a ratio of 70:30:2 to elute the alkaline component, collect the acidic and alkaline component eluates, concentrate to 2ml with nitrogen blow, and filter with 0.22μm filter membrane to obtain the test solution.

[0078] Assay

[0079] Accurately pipette 10 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and record the chromatogram.

[0080] The results show that 13 characteristic peaks can be detected at wavelengths between 300nm and 340nm, and the RSD of the relative retention times is less than 1.0%. Therefore, the wavelength between 300nm and 340nm can meet the characteristic spectrum detection requirements. Figure 1 .

[0081] Table 3 Results of relative retention time of wavelength investigation

[0082] .

[0083] 1.2.2 Investigation of mobile phase types

[0084] Chromatographic Conditions 1: Column: Alltima-C18 (4.6 x 250 mm, 5 μm); mobile phase A: 0.1% trifluoroacetic acid-80% acetonitrile in water; mobile phase B: tetrahydrofuran: methanol: water (10:38:45); gradient elution as specified in the table below; column temperature: 30°C; detection wavelength: 320 nm. The number of theoretical plates calculated based on the chicoric acid peak should be no less than 5000.

[0085] .

[0086] Chromatographic Conditions 2: Column: Alltima-C18 (4.6 x 250 mm, 5 μm); mobile phase A: 80% acetonitrile in water, mobile phase B: tetrahydrofuran: methanol: water (10:38:45), gradient elution as specified in the table below; column temperature: 30°C; detection wavelength: 320 nm. The theoretical plate number calculated based on the chicoric acid peak should be no less than 5000.

[0087] .

[0088] Chromatographic Conditions 3: Column: Alltima-C18 (4.6 x 250 mm, 5 μm); mobile phase A: 0.1% trifluoroacetic acid-80% acetonitrile in water, mobile phase B: 0.2% phosphoric acid in water, gradient elution as specified in the table below; column temperature: 30°C; detection wavelength: 320 nm. The theoretical plate number calculated based on the chicoric acid peak should be no less than 5000.

[0089] .

[0090] Preparation of reference solution

[0091] Take appropriate amount of monocaffeoyltartaric acid, chlorogenic acid, esculetin, caffeic acid, chicoric acid, and aloin reference substances, accurately weigh them, add methanol to make solutions containing 65ug per 1ml, shake well, and use them as reference solutions.

[0092] Preparation of test solution

[0093] Take 1g of Kangfu Xiaoyan Suppository, weigh it accurately, add 40ml of methanol-water-formic acid composite solvent with a ratio of 70:30:0.1; ultrasonicate at 50℃ for 30 minutes, ultrasonic power is 200W; centrifuge at 8000rpm for 10 minutes, and take the supernatant for use; select OasisMCX mixed mode solid phase extraction column, and balance with 5ml of methanol and 5ml of water containing 0.1% formic acid in turn to activate the extraction column; pass the extract through the column at a flow rate of 1ml / min, and wash away fat-soluble impurities with 5ml of methanol-water-formic acid with a ratio of 20:80:0.1; elute the acidic component with 5ml of methanol-water-formic acid with a ratio of 70:30:0.1, and use 5ml of methanol-water-ammonia water with a ratio of 70:30:2 to elute the alkaline component, collect the acidic and alkaline component eluates, concentrate to 2ml with nitrogen blow, and filter with 0.22μm filter membrane to obtain the test solution.

[0094] Assay

[0095] Accurately pipette 10 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and record the chromatogram.

[0096] The results showed that when elution was performed with 0.1% trifluoroacetic acid-80% acetonitrile aqueous solution as mobile phase A and tetrahydrofuran:methanol:water (10:38:45) as mobile phase B, the test sample chromatogram presented a total of 13 characteristic peaks, with good peak separation and excellent peak shape; when elution was performed with 80% acetonitrile aqueous solution as mobile phase A and tetrahydrofuran:methanol:water (10:38:45) as mobile phase B or with 0.1% trifluoroacetic acid-80% acetonitrile aqueous solution as mobile phase A and 0.2% phosphoric acid solution as mobile phase B, all peaks were missing and the corresponding effect could not be achieved.

[0097] Table 4 Relative retention time results of mobile phase type investigation

[0098] .

[0099] 1.2.3 Investigation of mobile phase ratio

[0100] Chromatographic conditions

[0101] Chromatographic column: Alltima-C18 (4.6 x 250 mm, 5 μm); mobile phase A: 0.1% trifluoroacetic acid-80% acetonitrile in water; mobile phase B: tetrahydrofuran: methanol: water (10:38:45); gradient elution as specified in the table below; column temperature: 30°C; detection wavelength: 320 nm. The number of theoretical plates calculated based on the chicoric acid peak should be no less than 5000.

[0102] Mobile phase ratio 1:

[0103] .

[0104] Mobile phase ratio 2:

[0105] .

[0106] Mobile phase ratio 3:

[0107] .

[0108] Preparation of reference solution

[0109] Take appropriate amount of monocaffeoyltartaric acid, chlorogenic acid, esculetin, caffeic acid, chicoric acid, and aloin reference substances, accurately weigh them, add methanol to make solutions containing 65ug per 1ml, shake well, and use them as reference solutions.

[0110] Preparation of test solution

[0111] Take 1g of Kangfu Xiaoyan Suppository, weigh it accurately, add 40ml of methanol-water-formic acid composite solvent with a ratio of 70:30:0.1; ultrasonicate at 50℃ for 30 minutes, ultrasonic power is 200W; centrifuge at 8000rpm for 10 minutes, and take the supernatant for use; select OasisMCX mixed mode solid phase extraction column, and balance with 5ml of methanol and 5ml of water containing 0.1% formic acid in turn to activate the extraction column; pass the extract through the column at a flow rate of 1ml / min, and wash away fat-soluble impurities with 5ml of methanol-water-formic acid with a ratio of 20:80:0.1; elute the acidic component with 5ml of methanol-water-formic acid with a ratio of 70:30:0.1, and use 5ml of methanol-water-ammonia water with a ratio of 70:30:2 to elute the alkaline component, collect the acidic and alkaline component eluates, concentrate to 2ml with nitrogen blown, and filter with 0.22μm filter membrane to obtain the test solution.

[0112] Assay

[0113] Accurately pipette 10 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and record the chromatogram.

[0114] The results showed that when eluted with a mobile phase ratio of 1, the test sample chromatogram showed a total of 13 characteristic peaks, with good peak separation and excellent peak shape; when eluted with a mobile phase ratio of 2 or a mobile phase ratio of 3, all peaks were missing and the corresponding effect was not achieved.

[0115] Table 5 Results of relative retention time of mobile phase ratio

[0116] .

[0117] Example 2: Methodological Verification of the Characteristic Spectrum of Kangfu Xiaoyan Suppositories

[0118] 2.1 Exclusivity

[0119] Chromatographic conditions

[0120] Chromatographic column: Alltima-C18 (4.6 x 250 mm, 5 μm); mobile phase A: 0.1% trifluoroacetic acid-80% acetonitrile in water; mobile phase B: tetrahydrofuran: methanol: water (10:38:45); gradient elution as specified in the table below; column temperature: 30°C; detection wavelength: 320 nm. The number of theoretical plates calculated based on the chicoric acid peak should be no less than 5000.

[0121] .

[0122] Preparation of reference solution

[0123] Take appropriate amount of monocaffeoyltartaric acid, chlorogenic acid, esculetin, caffeic acid, chicoric acid, and aloin reference substances, accurately weigh them, add methanol to make solutions containing 65ug per 1ml, shake well, and use them as reference solutions.

[0124] Preparation of test solution

[0125] Take 1g of Kangfu Xiaoyan Suppository, weigh it accurately, add 40ml of methanol-water-formic acid composite solvent with a ratio of 70:30:0.1; ultrasonicate at 50℃ for 30 minutes, ultrasonic power is 200W; centrifuge at 8000rpm for 10 minutes, and take the supernatant for use; select OasisMCX mixed mode solid phase extraction column, and balance with 5ml of methanol and 5ml of water containing 0.1% formic acid in turn to activate the extraction column; pass the extract through the column at a flow rate of 1ml / min, and wash away fat-soluble impurities with 5ml of methanol-water-formic acid with a ratio of 20:80:0.1; elute the acidic component with 5ml of methanol-water-formic acid with a ratio of 70:30:0.1, and use 5ml of methanol-water-ammonia water with a ratio of 70:30:2 to elute the alkaline component, collect the acidic and alkaline component eluates, concentrate to 2ml with nitrogen blown, and filter with 0.22μm filter membrane to obtain the test solution.

[0126] Preparation of negative solution

[0127] Take an appropriate amount of excipients, accurately weigh them, and add 40 ml of a methanol-water-formic acid complex 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 later use; use an Oasis MCX mixed-mode solid phase extraction column, and balance 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 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 switch to 5 ml of methanol-water-ammonia with a ratio of 70:30:2 to elute the alkaline components. Collect the acidic and alkaline eluates, concentrate to 2 ml with nitrogen blow-through, and filter with a 0.22 μm filter membrane to obtain the test solution.

[0128] Assay

[0129] Accurately pipette 10 μl of each of the negative solution, reference solution and test solution, inject them into the liquid chromatograph, and record the chromatogram.

[0130] The results showed that the negative solution had no interference and good specificity. Figure 2 .

[0131] 2.2 Precision

[0132] Take 1 g of Kangfu Xiaoyan Suppository (Batch No.: 20221119) and prepare the test solution according to the test solution preparation method under "2.1". Inject 6 times according to the chromatographic conditions under "2.1", record the chromatogram, measure the relative retention time and relative peak area of ​​each chromatographic peak, and calculate the RSD.

[0133] Table 6 Precision relative retention time results

[0134] .

[0135] Table 7 Precision relative peak area results

[0136] .

[0137] The results showed that the relative retention time RSD of each chromatographic peak was less than 1.0%, and the relative peak area RSD was less than 10.0%, indicating good precision. The chromatogram is attached. Figure 3 .

[0138] 2.3 Repeatability

[0139] Take 1 g of Kangfu Xiaoyan Suppository (batch number: 20221119), a total of 6 portions, and prepare the test solution according to the test solution preparation method under "2.1". Inject the sample according to the chromatographic conditions under "2.1", record the chromatogram, measure the relative retention time and relative peak area of ​​each chromatographic peak, and calculate the RSD.

[0140] Table 8 Repeatability relative retention time results

[0141] .

[0142] Table 9 Repeatability relative peak area results

[0143] .

[0144] The results showed that the relative retention time RSD of each chromatographic peak was less than 2.0%, and the relative peak area RSD was less than 10.0%, with good repeatability. The chromatogram is attached. Figure 4 .

[0145] 2.4 Stability

[0146] Take 1 g of Kangfu Xiaoyan Suppository (batch number: 20221119) and prepare the test solution according to the test solution preparation method under "2.1". Inject the sample at 0h, 2h, 4h, 8h, 12h, and 24h according to the chromatographic conditions under "2.1". Record the chromatogram, determine the relative retention time and relative peak area of ​​each chromatographic peak, and calculate the RSD.

[0147] Table 10 Stability relative retention time results

[0148] .

[0149] Table 11 Stability relative peak area results

[0150] .

[0151] The results showed that the relative retention time RSD of each chromatographic peak was less than 1.0%, and the relative peak area RSD was less than 8.0%, indicating that the solution was stable. The chromatogram is attached. Figure 5 .

[0152] In summary, the characteristic spectrum has good specificity and no interference from negative samples, indicating that the characteristic detection method has strong specificity. In the precision test, the relative retention time RSDs were all less than 1.0%, and the relative peak area RSDs were all less than 10.0%, indicating good instrument precision. In the repeatability test, the relative retention time RSDs of each chromatographic peak were all less than 2.0%, and the relative peak area RSDs were all less than 10.0%, indicating good repeatability. In the stability test, the relative retention time RSDs of each chromatographic peak were all less than 1.0%, and the relative peak area RSDs were all less than 8.0%, indicating that the test solution was stable within 24 hours. This method has been well validated.

[0153] Example 3 Construction of the Control Characteristic Spectrum of Kangfu Xiaoyan Suppositories

[0154] Chromatographic conditions

[0155] Chromatographic column: Alltima-C18 (4.6 x 250 mm, 5 μm); mobile phase A: 0.1% trifluoroacetic acid-80% acetonitrile in water; mobile phase B: tetrahydrofuran: methanol: water (10:38:45); gradient elution as specified in the table below; column temperature: 30°C; detection wavelength: 320 nm. The number of theoretical plates calculated based on the chicoric acid peak should be no less than 5000.

[0156] .

[0157] Preparation of reference solution

[0158] Take appropriate amount of monocaffeoyltartaric acid, chlorogenic acid, esculetin, caffeic acid, chicoric acid, and aloin reference substances, accurately weigh them, add methanol to make solutions containing 65ug per 1ml, shake well, and use them as reference solutions.

[0159] Preparation of test solution

[0160] Take 1g of Kangfu Xiaoyan Suppository, weigh it accurately, add 40ml of methanol-water-formic acid composite solvent with a ratio of 70:30:0.1; ultrasonicate at 50℃ for 30 minutes, ultrasonic power is 200W; centrifuge at 8000rpm for 10 minutes, and take the supernatant for use; select OasisMCX mixed mode solid phase extraction column, and balance with 5ml of methanol and 5ml of water containing 0.1% formic acid in turn to activate the extraction column; pass the extract through the column at a flow rate of 1ml / min, and wash away fat-soluble impurities with 5ml of methanol-water-formic acid with a ratio of 20:80:0.1; elute the acidic component with 5ml of methanol-water-formic acid with a ratio of 70:30:0.1, and use 5ml of methanol-water-ammonia water with a ratio of 70:30:2 to elute the alkaline component, collect the acidic and alkaline component eluates, concentrate to 2ml with nitrogen blow, and filter with 0.22μm filter membrane to obtain the test solution.

[0161] Assay

[0162] Accurately pipette 10 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and record the chromatogram.

[0163] The chromatograms of the test samples of 8 batches of Kangfu Xiaoyan Suppositories were imported into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" and the chromatographic peaks present in the chromatograms of different batches of Kangfu Xiaoyan Suppositories were selected as common peaks; the average value calculation method was used to generate a reference characteristic spectrum, and the relative retention time and relative peak area of ​​each common peak were calculated. The results are shown in Tables 12 and 13, and Appendix Figures 6 and 7 .

[0164] Table 12 Relative retention time results of eight batches of Kangfu Xiaoyan Suppositories

[0165] .

[0166] Table 13 Relative peak area results of eight batches of Kangfu Xiaoyan Suppositories

[0167] .

[0168] The chromatograms of the eight batches of Kangfu Xiaoyan Suppositories samples all showed 13 common peaks. The RSDs of the relative retention times of the 13 common peaks were all less than 1.0%, and the RSDs of the relative peak areas were all less than 10.0%.

[0169] The chromatogram of the test sample showed 13 characteristic peaks, of which peaks 3, 4, 6, 7, 9, and 12 corresponded to the peaks of the monocaffeoyltartaric acid, chlorogenic acid, aesculetin, caffeic acid, chicoric acid, and aloin reference substances, respectively. The peak corresponding to the chicoric acid reference substance peak was the S peak. The relative retention times of each characteristic peak and the S peak were calculated, and their relative retention times should be within ±10% of the specified values. 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), and 1.49 (peak 13).

Claims

1. A method for constructing a characteristic spectrum of Kangfu Xiaoyan Suppository, characterized by: The following steps are involved: (1) Preparation of reference solution: Take the reference substance, weigh accurately, add methanol to make a solution containing 65 μg per 1 ml, shake well, and use it as the reference solution; the reference substances are: monocaffeoyltartaric acid, chlorogenic acid, esculetin, caffeic acid, chicoric acid, and aloin; (2) Preparation of the test solution: Take 1g of Kangfu Xiaoyan Suppository, weigh accurately, and add 40ml of methanol-water-formic acid composite solvent with a ratio of 70:30:0.1; centrifuge after ultrasonication, and take the supernatant for use; select a solid phase extraction column, and balance it with 5ml of methanol and 5ml of water containing 0.1% formic acid in turn to activate the extraction column; pass the extract, i.e. the supernatant after centrifugation, through the column at a flow rate of 1ml / min, and use 5ml of methanol-water-formic acid with a ratio of 20:80:0.1 to wash away fat-soluble impurities, use 5ml of methanol-water-formic acid with a ratio of 70:30:0.1 to elute the acidic components, and use 5ml of methanol-water-ammonia water with a ratio of 70:30:2 to elute the alkaline components, collect the acidic and alkaline eluates, concentrate to 2ml with nitrogen blowing, and filter with a 0.22μm filter membrane to obtain the test solution; (3) Determination: Inject the reference solution and the test solution into a high performance liquid chromatograph according to the following chromatographic conditions, inject 10 μl of each sample, and record the chromatogram; Chromatographic conditions were as follows: octadecylsilane bonded silica gel as the filler; 0.1% trifluoroacetic acid-80% acetonitrile aqueous solution as mobile phase A, and tetrahydrofuran:methanol:water (volume ratio:10:38:45) as mobile phase B, eluting according to the specified gradient; column temperature: 30°C; flow rate: 1 ml / min; UV-visible detector detection wavelength: 300-340 nm; The elution gradient conditions were: From 0 to 15 min, the volume ratio of mobile phase A:phase B changed from 5:95 to 10:90; From 15 to 25 min, the volume ratio of mobile phase A:phase B changed from 10:90 to 14:86; From 25 to 27 min, the volume ratio of mobile phase A:phase B changed from 14:86 to 17:83; From 27 to 28 minutes, the volume ratio of mobile phase A:phase B changed from 17:83 to 22:78; From 28 to 50 min, the volume ratio of mobile phase A:phase B changed from 22:78 to 23:77; From 50 to 65 min, the volume ratio of mobile phase A:phase B changed from 23:77 to 55:45; From 65 to 68 min, the volume ratio of mobile phase A:phase B was changed from 55:45 to 5:95; 68-80 min, mobile phase A:B, volume ratio 5:95; (4) Generate a control characteristic spectrum: 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 a control characteristic spectrum of Kangfu Xiaoyan Suppositories.

2. The method according to claim 1, characterized in that The ultrasonic conditions in step (2) are 50° C. for 30 minutes and an ultrasonic power of 200 W.

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

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

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

6. The method according to claim 1, characterized in that The control characteristic spectrum generated in step (4) includes 13 common chromatographic peaks, among which: peak 3 corresponds to monocaffeoyltartaric acid, peak 4 corresponds to chlorogenic acid, peak 6 corresponds to quercetin, peak 7 corresponds to caffeic acid, peak 9 corresponds to chicoric acid, and peak 12 corresponds to aloin.

7. The method according to claim 1, characterized in that The characteristic peaks were taken as reference peaks using the chicoric acid chromatographic peak. The relative retention times of the 13 chromatographic peaks were: peak 1 was 0.19, peak 2 was 0.40, peak 3 was 0.50, peak 4 was 0.60, peak 5 was 0.63, peak 6 was 0.65, peak 7 was 0.69, peak 8 was 0.97, peak 10 was 1.11, peak 11 was 1.15, peak 12 was 1.22, and peak 13 was 1.49.

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