Method for establishing fingerprint of Kemin capsule

Abstract

The present invention relates to a method for establishing a fingerprint spectrum of a Kemin capsule. By examining different sample solution preparations and chromatographic conditions, the present invention determines the sample solution preparation method and detection conditions for the product's fingerprint spectrum, identifies four characteristic peaks, namely, liquiritin, benzoylmonaconitine, ammonium glycyrrhizate, and ephedra hydrochloride, and effectively reflects the material basis of the Kemin capsule. This method can serve as a basis for quality control of the Kemin capsule and is used to evaluate the authenticity, excellence, and stability of the preparation.

Description

Technical Field

[0001] The present invention relates to a fingerprint spectrum detection method for traditional Chinese medicine preparations, in particular to a method for establishing a fingerprint spectrum of a cough-mining capsule. Background Art

[0002] A traditional Chinese medicine fingerprint is a chromatogram or spectrum obtained through appropriate processing and analytical methods, which can identify the chemical characteristics of a traditional Chinese medicine. Traditional Chinese medicine fingerprints are primarily used to evaluate the authenticity, quality, and stability of Chinese medicinal materials and semi-finished Chinese medicine preparations. They offer comprehensive and quantifiable results.

[0003] Kemin capsules contain aconite root, honeyed ephedra, and roasted liquorice root, and have antitussive properties. Currently, there is no method to comprehensively characterize the chemical characteristics of Kemin capsules. Summary of the Invention

[0004] In order to solve the technical problems existing in the prior art, the present invention provides a method for establishing a fingerprint spectrum of cough-mining capsules.

[0005] The technical solution adopted by the present invention to solve the above technical problems is:

[0006] The method for establishing the fingerprint of Kemin capsule comprises the following steps:

[0007] (1) Prepare the test solution: Take an appropriate amount of the sample content, grind it into powder, take 0.5 g of the fine powder, add 50 ml of methanol, sonicate, cool to room temperature, filter, and take the filtrate;

[0008] (2) Preparation of reference solution: Take an appropriate amount of reference substance and dissolve it in methanol to make a solution containing 0.1 mg per 1 ml, which serves as the reference solution;

[0009] (3) Chromatographic conditions and system suitability test: Octadecyl bonded silica gel was used as the filler; acetonitrile was used as mobile phase A, 0.1% phosphoric acid was used as mobile phase B, the flow rate was 1.0 ml per minute; the column temperature was 30°C; the resolution of liquiritin and adjacent chromatographic peaks should be greater than 1.5;

[0010] (4) Take 10 μL of the reference solution and the test solution respectively, inject them into the liquid chromatograph, measure, and record the chromatogram.

[0011] Furthermore, the cough-inducing capsule is composed of the following ingredients: aconite root (Heishunpian), honeyed ephedra, roasted licorice, and starch.

[0012] Furthermore, a gradient elution method was used, and the elution time and the ratio of the mobile phase were: 0 min, mobile phase A 10%, mobile phase B 90%; 60 min, mobile phase A 90%, mobile phase B 10%.

[0013] Furthermore, the chromatographic column is a Welch Xtimate@C18 or equivalent chromatographic column with a column length of 2.5 cm, an inner diameter of 4.6 mm, and a particle size of 5 μm.

[0014] Furthermore, the detection wavelength is 235 nm.

[0015] Furthermore, the ultrasonic treatment conditions were 250 W, 40 KHz, and 30 minutes.

[0016] Furthermore, the reference substances are ephedrine hydrochloride, pseudoephedrine hydrochloride, benzoylmesaconitine, benzoylaconitine, benzoylhypaconitine, liquiritin, and ammonium glycyrrhizate.

[0017] Furthermore, 12 characteristic peaks should be present in the fingerprint of the test solution, among which the retention times of peaks 3, 6, 8 and 12 are basically consistent with those of the reference substances ephedrine hydrochloride, glycyrrhizin, benzoylmesaconitine and ammonium glycyrrhizate; the peak corresponding to the retention time of the reference substance glycyrrhizin peak is designated as S peak, and the relative retention time of each characteristic peak and the S peak is calculated, which should be within ±10.0% of the specified value.

[0018] Furthermore, the specified values ​​are: 0.33 (peak 1), 0.40 (peak 2), 0.51 (peak 3), 0.94 (peak 4), 0.97 (peak 5), 1.0 (peak 6, S), 1.28 (peak 7), 1.31 (peak 8), 1.35 (peak 9), 1.38 (peak 10), 1.53 (peak 11), and 2.04 (peak 12).

[0019] Furthermore, MARK peak matching was performed and according to the similarity evaluation system of traditional Chinese medicine chromatographic fingerprint, the fingerprint of the test solution and the fingerprint of the reference solution were calculated with a similarity of not less than 0.90.

[0020] Compared with the existing technology, the beneficial effects of the present invention are:

[0021] The experiment determined the test solution preparation method and detection conditions of the product's fingerprint through different test solution preparation and chromatographic conditions, and identified four characteristic peaks including glycyrrhizin, benzoylmonaconitine, ammonium glycyrrhizate, and ephedra hydrochloride, which better reflected the material basis of Kemin Capsules and can be used as the basis for quality control of Kemin Capsules to evaluate the authenticity, excellence and stability of the preparation quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The drawings in the specification, which constitute a part of this application, are used to provide further understanding of this application. The illustrative embodiments of this application and their descriptions are used to explain this application and do not constitute improper limitations on this application.

[0023] Figure 1 This is the chromatogram of elution condition 1.

[0024] Figure 2 This is the chromatogram of elution condition 2.

[0025] Figure 3 This is the chromatogram of elution condition 3.

[0026] Figure 4 This is the chromatogram of elution condition 4.

[0027] Figure 5 This is the chromatogram of elution condition 5.

[0028] Figure 6 This is the chromatogram of elution condition 6.

[0029] Figure 7 Comparative chromatograms of extractions with different solvents (from bottom to top: S1 is purified water; S2 is 70% methanol; S3 is 90% methanol; S4 is 100% methanol).

[0030] Figure 8 Comparative chromatograms were taken for different ultrasonic times (S1 for 30 min, S2 for 45 min, and S3 for 60 min).

[0031] Figure 9 This is the chromatogram of the reference substance (wavelength: 235).

[0032] Figure 10 This is the chromatogram of the reference substance (wavelength: 237).

[0033] Figure 11 This is the chromatogram of the reference substance (wavelength: 210).

[0034] Figure 12 is the UV absorption distribution of the reference and test solutions at 235 nm.

[0035] Figure 13 is the UV absorption distribution of the reference and test solutions at 237 nm.

[0036] Figure 14 is the UV absorption distribution of the reference and test solutions at 210 nm.

[0037] Figure 15 This is a diagram showing the comparison of chromatographic peaks attributing characteristic peaks (from bottom to top: test solution, ephedrine hydrochloride, pseudoephedrine hydrochloride, liquiritin, benzoylmeaconitine, benzoylhypaconitine, benzoylaconitine, and ammonium glycyrrhizate).

[0038] Figure 16 The figure shows the fingerprint comparison of 6 test solution.

[0039] Figure 17This is the liquid chromatogram of Kemin Capsules (235nm).

[0040] Figure 18 This is the characteristic diagram of Kemin Capsule.

[0041] Figure 19 These are the fingerprints of the test and control samples of Kemin Capsules. DETAILED DESCRIPTION

[0042] In order to enable those skilled in the art to more clearly understand the technical solution of the present disclosure, the technical solution of this patent will be described in detail below in conjunction with specific embodiments. It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of this application.

[0043] This invention refers to the General Chapter 9101 "Guidelines for Validation of Analytical Methods" and "Technical Requirements for Research on Fingerprints of Traditional Chinese Medicine Injections" of the 2020 edition of the "Chinese Pharmacopoeia", and takes the main ingredients of Kemin Capsules such as Aconite Pian (Heishunpian), Honey Ephedra, and Roasted Licorice as investigation indicators. High-performance liquid chromatography is used for detection to establish the fingerprint of Kemin Capsules, and the fingerprint evaluation is performed using the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" V2.0 version formulated by the National Pharmacopoeia Committee.

[0044] Prescription composition: Aconite root (Heishunpian), honeyed ephedra, roasted licorice, auxiliary material: starch.

[0045] Sample information: Kemin Capsules, production date: 20190701; production batch number: 20190701; production unit: Shandong Mingren Freda Pharmaceutical Co., Ltd.

[0046] The experimental instruments are as follows:

[0047] Table 1 Experimental instruments

[0048]

[0049]

[0050] After comprehensive analysis of the chemical components of the medicinal materials used in the prescription, combined with the material basis of the medicinal materials used in the actual preparation of this product and the determination conditions listed in the above table, the following fingerprint HPLC conditions are tentatively set for screening the test sample processing (referring to the determination method of licorice content in the "Chinese Pharmacopoeia"). The specific conditions are as follows:

[0051] Chromatographic column: Welch Xtimate@C18 (4.6 mm × 250 mm, 5 μm);

[0052] Mobile phase: Use 0.1% phosphoric acid water as mobile phase B and acetonitrile as mobile phase A, and wash according to the gradient shown in the table below.

[0053] Table 2 Elution program

[0054]

[0055] Flow rate: 1.0 ml / min;

[0056] Column temperature: 30°C;

[0057] Detection wavelengths: 235nm, 237nm, 210nm.

[0058] Example 1 Optimization of liquid phase elution conditions for fingerprint

[0059] Considering that the prescription of this product has many medicinal flavors and complex ingredients, in order to identify the characteristic fingerprint of the material basis of this product as much as possible and effectively

[0060] If there are peaks or common peaks, it is necessary to optimize the elution mode of the monitored chromatographic conditions in order to separate or elute the components to the greatest extent.

[0061] The specific experimental design is as follows:

[0062] Preparation of test sample solution: tentative: take about 0.5 g of the fine powder of the sample content (batch number: 20190701), accurately weigh, prepare 3 portions in total, place in a stoppered conical flask, accurately add 70% methanol, 100% methanol, and 50 ml of pure water, ultrasonically treat (250W, 40KHz) for 30 minutes, cool to room temperature, make up with the corresponding solvent, filter, and take the filtrate to obtain.

[0063] Monitor chromatographic conditions:

[0064] Chromatographic column: octadecyl bonded silica gel as filler (Welch Xtimate@C18; 4.6 mm × 250 mm, 5 μm);

[0065] Mobile phase A: acetonitrile

[0066] Mobile phase B: 0.1% phosphoric acid solution

[0067] Flow rate: 1.0ml / min

[0068] Column temperature: 30°C

[0069] Design the following gradient elution conditions and conduct the experiment:

[0070] Table 3 Elution Conditions 1

[0071]

[0072] Table 4 Elution Conditions 2

[0073]

[0074] Table 5 Elution Conditions 3

[0075]

[0076] Table 6 Elution Conditions 4

[0077]

[0078] Table 7 Elution Conditions 5

[0079]

[0080] Table 8 Elution Conditions 6

[0081]

[0082] Determination method: Accurately measure 10 μl of the above test solution and inject it into the liquid chromatograph according to the above elution conditions ① to ③ respectively, and record the typical chromatogram. The results are as follows Figure 1-6 :

[0083] Results: From the above experiments, it can be seen that when the elution conditions are compared, when elution is carried out according to the predetermined elution condition 1 of licorice, no obvious characteristic peaks appear. When elution is carried out according to the isocratic elution condition 2, a good separation effect is not achieved. When elution is carried out according to the elution condition 4, the separation of several characteristic peaks at 40-45min is poor, and the response value is low. When elution is carried out according to the elution condition 5, the separation of characteristic peaks at 18-20min is poor. When elution is carried out according to the gradient 6, good separation is basically impossible to achieve. When elution is carried out according to the elution condition 3, the fingerprint of the test solution of the test sample shows that the separation of each peak is good, and the response of the peak is better than that of other elution conditions. The analysis time is moderate. Therefore, this study tentatively uses the mobile phase system elution condition 3 as the fingerprint determination condition of this capsule.

[0084] Example 2 Experimental sample processing and solution preparation

[0085] Kemin Capsules are capsule preparations. The selection of solvents mainly considers the consistency with the solvent used in the process preparation and the extraction method. The test sample processing method and solution preparation plan are designed. The specific contents are as follows;

[0086] Table 9: Sample solution preparation scheme for the experiment

[0087]

[0088] Prepare the test sample solutions separately.

[0089] Determination method: Accurately measure 10 μl of each test solution and inject it into the liquid chromatograph respectively. According to the fingerprint spectrum monitoring conditions optimized in Example 1, measure according to the law and record the chromatogram. In the traditional Chinese medicine similarity evaluation software, import the chromatograms of samples dissolved in 70% methanol, 100% methanol, purified water, and 90% methanol respectively, evaluate their similarity, and observe the separation and the size of the characteristic peak response value.

[0090] Comparison charts such as Figure 7 .

[0091] Results: From the above comparative chromatographic information, it can be seen that when purified water is selected as the solvent (S1 in the figure), the response values ​​of features 7 and 9 are too low, so this solvent is not selected; when 70% methanol is selected as the solvent (S2 in the figure), the third characteristic peak (ephedrine hydrochloride) cannot be seen, so this solvent is not selected; when 90% methanol (S3 in the figure) or 100% methanol (S4 in the figure) is selected as the solvent, the number of chromatographic peaks in the test sample and the separation between peaks are good, and the response values ​​of each peak are higher than the other two solvents. Therefore, both solvents (90% and 100% methanol) can be used as the extraction solvent for this capsule. This experimental study uses 100% methanol as the extraction solvent for the contents of this capsule.

[0092] Ultrasound timing

[0093] The extraction ultrasonic time selection was investigated according to the experimental sample solution preparation scheme in Table 9, and the test sample solutions were prepared. The chromatograms of the 100% methanol-dissolved samples were imported into the Chinese medicine similarity evaluation software to evaluate their similarity and observe the resolution and characteristic peak response values.

[0094] Comparison charts such as Figure 8 .

[0095] Results: From the above comparative chromatographic information, it can be seen that when the ultrasonic time is selected as 30, 45, and 60 methanol (S1, S2, and S3 in the figure), the response value and separation degree of the characteristic peak are not much different, so the ultrasonic time of 30 min is selected as the extraction condition for the contents of this capsule.

[0096] In summary, the sample pretreatment method is as follows: take 0.5 g of the fine powder of the sample content (batch number: 20190701), accurately weigh it, place it in a stoppered conical flask, accurately add 50 ml of 100% methanol, ultrasonically treat (250 W, 40 kHz) for 30 minutes, cool to room temperature, add 100% methanol to make up the weight, filter, and take the filtrate.

[0097] Example 3 Positioning of the main material basis of Kemin Capsules

[0098] Experimental plan: Take an appropriate amount of the reference substance in the following table and prepare the positioning solution concentration as shown in the following table

[0099] Table 10 Information of reference solution for research

[0100]

[0101]

[0102] Determination method: Take 10 μl of the above reference solution and inject it into the liquid chromatograph respectively. Determine according to the optimized conditions and record the chromatogram. Figure 9-11 :

[0103] Experimental Results: The results of the positioning of the reference solutions above show that the peak times of ephedrine hydrochloride and pseudoephedrine hydrochloride completely overlap under these detection conditions. Therefore, when determining the position, either ephedrine hydrochloride or pseudoephedrine hydrochloride (both identified as ephedrine hydrochloride in this study) can be located. Benzoylaconitine and benzoylaconitine exhibit relatively low responses, making them unsuitable for identifying the characteristic peaks of the fingerprint of this product's particles. To more effectively characterize the material properties of this product, this study primarily examined substances with good responses at specific wavelengths as characteristic peaks of the fingerprint. Therefore, further optimization of the specific wavelength is necessary.

[0104] Example 4 Optimization of the UV Absorption Wavelength of the Fingerprint

[0105] In order to ensure the effective traceability and monitoring of the particle material basis of this product, the optimal characteristic ultraviolet absorption wavelength of the material basis of Kemin Capsules was explored; this study combined the detection conditions under the corresponding medicinal materials in the first volume of the 2020 edition of the "Chinese Pharmacopoeia". The ultraviolet absorption of the drug basis of this product is mainly concentrated near 235nm, 210nm, and 237nm wavelengths; to ensure the comprehensiveness of the material basis detection, feature positioning was used to examine the characteristics of the chromatograms at different wavelengths to confirm the required wavelengths for fingerprint spectrum monitoring in this study.

[0106] The test solution and reference solution prepared after optimization were injected into the liquid chromatograph respectively, and the detection wavelengths were mainly recorded at 235nm, 210nm, and 237nm. The experimental results are as follows: Figure 12-14 :

[0107] Conclusion: From the above experiments, it can be seen that when the fingerprint is detected at wavelengths of 210nm and 237nmm, the characteristic indicators of the drugs in the prescription of this product, such as ephedrine hydrochloride, pseudoephedrine hydrochloride, benzoylaconitine, liquiritin, ammonium glycyrrhizate, etc., all respond; but under the wavelength of 235nm, the ultraviolet characteristics of the basic substances in the sample respond better and the separation is higher, so it is confirmed that the ultraviolet monitoring wavelength of this study is 235nm.

[0108] Example 5 Durability Test

[0109] The degree of influence of the method on the determination results was evaluated by fine-tuning the eluent flow rate (±0.1 ml / min) and column temperature (±5°C). Under the above-mentioned fine-tuning conditions, the theoretical plate number of the S peak (liquiritin) was required to be no less than 5000, and the relative retention time of the S peak should not exceed 10.0% of the S peak retention time under the original conditions (retention time: 13.684 min) (retention time range: 12.316 min to 15.053 min).

[0110] Test solution: Grind 0.5 g of the sample content into fine powder, accurately weigh, and place in a stoppered conical flask. Accurately add 50 ml of 100% methanol, ultrasonically treat (250 W, 40 kHz) for 30 minutes, cool to room temperature, filter, and obtain the filtrate.

[0111] Blank solution: 100% methanol solution

[0112] The fine-tuning test conditions for the durability test are as follows:

[0113] Table 11 Durability fine-tuning condition scheme

[0114]

[0115] Determination method: Accurately measure 10 μl of the blank solution and the test solution, respectively, and inject them into the liquid chromatograph. According to the durability fine-tuning test plan and the chromatographic conditions to be verified, the determination is carried out in accordance with the law. The results are as follows:

[0116] Table 12 Durability test results

[0117]

[0118]

[0119] Results: From the above experimental data, it can be seen that when the mobile phase flow rate is fine-tuned in the range of 0.9ml / min~1.1ml / min, the theoretical plate number of S peak (liquiritin) is 59049~71106, and the retention time is 13.016~14.479min; when the column temperature is fine-tuned in the range of 25℃~35℃, the theoretical plate number of S peak (liquiritin) is 62949~67544, and the retention time is 13.275~14.310min; when the eluent flow rate and column temperature are fine-tuned, there is no significant effect on the positioning of the characteristic peaks, the theoretical plate number of S peak (liquiritin) is greater than 5000, and the relative retention time of S peak (liquiritin) is within 10.0% of the S peak retention time under the original conditions; this shows that when the liquid chromatography condition parameters are fine-tuned within the above range, the method has good durability.

[0120] According to the results of the above studies, the determination conditions of the fingerprint of Kemin Capsules are summarized as follows:

[0121] Chromatographic column: Use octadecyl bonded silica gel as filler (such as Welch Xtimate@C18; 4.6mm×250mm, 5μm or equivalent column efficiency);

[0122] Mobile phase: Use 0.1% phosphoric acid water as mobile phase B and acetonitrile as mobile phase A, and perform gradient elution according to the table below.

[0123] Table 13 Gradient elution

[0124]

[0125] Flow rate: 1.0ml / min

[0126] Column temperature: 30°C;

[0127] Injection volume: 10 μl;

[0128] Detection wavelength: 235nm;

[0129] Test solution: Grind 0.5 g of the sample content into fine powder, accurately weigh, and place in a stoppered conical flask. Accurately add 50 ml of 100% methanol, ultrasonically treat (250 W, 40 kHz) for 30 minutes, cool to room temperature, filter, and obtain the filtrate.

[0130] Determination method: Accurately measure 10μl of the test solution, inject it into the liquid chromatograph, determine it according to the method, and record the fingerprint chromatogram.

[0131] Example 6 Attribution of characteristic peaks

[0132] The known chemical components of Kemin Capsules mainly include ephedrine hydrochloride, benzoylaconitine, glycyrrhizin, ammonium glycyrrhizate, etc.

[0133] The test was conducted by positioning and identifying known components in the chromatogram of the test solution.

[0134] Positioning reference solution: Take appropriate amounts of ephedrine hydrochloride, benzoylaconitine, liquiritin, and ammonium glycyrrhizate reference substances, accurately weigh them, and dissolve them in methanol to make solutions containing approximately 0.1 mg of each substance per 1 ml, which serve as positioning reference solutions.

[0135] Determination method: Inject the sample and determine the peaks under the chromatographic conditions determined in 5.0 above. Compare the peaks with those in the chromatogram of the test solution (batch number: 20190701) to assign the characteristic peaks. The results are shown in Figure 15 :

[0136] Results: The characteristic peaks of the test solution chromatogram were identified and 7 known components were identified. Among them, ephedrine hydrochloride, liquiritin, benzoylmonaconitine, and ammonium glycyrrhizate were more obvious and could be used as characteristic peaks. The remaining substances were detected weakly and were not considered as characteristic peaks.

[0137] Example 7 Reference Solution

[0138] Among the known components detected in the test solution, the peaks of "ephedrine hydrochloride, glycyrrhizin, benzoylmesaconitine, and ammonium glycyrrhizate" are more obvious and are located in the middle position of the fingerprint characteristic spectrum. The peaks of glycyrrhizin, benzoylmesaconitine, and ammonium glycyrrhizate can better reflect the separation effect of the spectrum, so they are used as reference substances.

[0139] Preparation of reference solution: Take appropriate amount of glycyrrhizin, benzoylmonaconitine and ammonium glycyrrhizate reference substances, weigh accurately, and dissolve them in methanol to prepare a solution containing approximately 0.1 mg of each per 1 ml, which is used as the reference solution.

[0140] Example 8 Fingerprint and its technical parameters

[0141] Sample fingerprint mapping

[0142] Take an appropriate amount of the contents of one batch of Kemin capsule samples for testing, and prepare 6 test solutions according to the same method as the test solution. Under the proposed chromatographic conditions, accurately measure 10μl and inject them into the liquid chromatograph for determination according to the law, record the chromatogram, and the results are as follows. Figure 16 :

[0143] The above fingerprint test data were imported into the traditional Chinese medicine fingerprint similarity software. After multi-point correction peak matching of the traditional Chinese medicine fingerprint similarity software, the fingerprint spectra of 6 samples were compared, and the minimum similarity was 0.993, indicating that the fingerprint spectra had good repeatability.

[0144] Calibration of common characteristic fingerprint peaks

[0145] The chromatograms of 6 test sample solutions of one batch of experimental Kemin capsules were analyzed and compared, and the common peaks were determined by calculation using the "Chinese Medicine Fingerprint Similarity Software".

[0146] Results: The chromatograms of 6 test solutions of 1 batch of Kemin capsules were separated into more than 20 chromatographic peaks, of which there were 12 characteristic peaks in total. The area of ​​the characteristic peaks accounted for more than 90% of the total peak area. The relative retention time was calculated using liquiritin as the reference peak. The calculation formula is as follows:

[0147]

[0148] According to the above chromatogram peak table ( Figure 17 ), calculate the relative retention time, and calibrate the characteristic peaks. The results are shown in the following table:

[0149] Table 14 Chromatogram peak table

[0150]

[0151]

[0152] Based on the above experimental results, the characteristic spectrum of Kemin Capsules was determined: 12 characteristic peaks should be presented in the characteristic spectrum of the test sample, and the relative retention times of each characteristic peak and the liquiritin peak are as follows: 0.33 (peak 1), 0.40 (peak 2), 0.51 (peak 3), 0.94 (peak 4), 0.97 (peak 5), 1.0 (peak 6, S), 1.28 (peak 7), 1.31 (peak 8), 1.35 (peak 9), 1.38 (peak 10), 1.53 (peak 11), 2.04 (peak 12); among them, Peak 3: ephedrine hydrochloride, Peak 6 (S): liquiritin, Peak 8: benzoylmesaconitine, Peak 12: ammonium glycyrrhizate.

[0153] Characteristic peak limit range:

[0154] like Figure 18 , there should be 12 characteristic peaks in the characteristic spectrum of the test sample, among which the retention times of peaks 3, 6, 8 and 12 are basically consistent with those of the reference substances ephedrine hydrochloride, liquiritin, benzoylmesaconitine and glycyrrhizic acid; the peak corresponding to the retention time of the reference substance liquiritin peak is designated as S peak, and the relative retention time of each characteristic peak and S peak is calculated, which should be within ±10.0% of the specified value.

[0155] Similarity evaluation

[0156] The fingerprint test data of one batch of Kemin Capsules (20190701) and the characteristic reference substance were imported into the Chinese medicine fingerprint similarity software. The control fingerprint of the Kemin Capsules fingerprint was obtained by calculation using the Chinese medicine fingerprint similarity software. The fingerprint test data of each batch were calculated using the MARK peak (characteristic peak) similarity. The results are shown in the figure below. Figure 19 and the table below.

[0157] Table 15 Kemin Capsule and characteristic control fingerprint evaluation

[0158]

[0159]

[0160] Results: The similarity between the fingerprint of Kemin Capsule and the MARK peak (characteristic peak) was not less than 0.90.

[0161] The experiment determined the test solution preparation method and detection conditions of the product's fingerprint through different test solution preparation and chromatographic conditions, and identified four characteristic peaks including glycyrrhizin, benzoylmonaconitine, ammonium glycyrrhizate, and ephedra hydrochloride, which better reflected the material basis of Kemin Capsules and can be used as the basis for quality control of Kemin Capsules to evaluate the authenticity, excellence and stability of the preparation quality.

[0162] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or make equivalent replacements for parts thereof. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention. Although the above describes the specific embodiments of the present invention in conjunction with the accompanying drawings, it is not intended to limit the protection scope of the present invention. Those skilled in the art should understand that, based on the technical solution of the present invention, various modifications or variations that can be made by those skilled in the art without creative work are still within the protection scope of the present invention.

Claims

1. A method for establishing a fingerprint of Kemin capsule, characterized in that: The steps include: (1) Prepare the test solution: Take an appropriate amount of the sample content, grind it into powder, take 0.5g of the fine powder, add 50ml of methanol, ultrasonically treat, cool to room temperature, filter, and take the filtrate; (2) Preparation of reference solution: Take an appropriate amount of reference substance and dissolve it in methanol to make a solution containing 0.1 mg per 1 ml, which serves as the reference solution; (3) Chromatographic conditions and system suitability test: octadecyl bonded silica gel was used as the filler; acetonitrile was used as the mobile phase A, 0.1% phosphoric acid was used as the mobile phase B, the flow rate was 1.0 ml per minute; the column temperature was 30°C; the resolution of glycyrrhizin and adjacent chromatographic peaks should be greater than 1.5; (4) Take 10µL of the reference solution and the test solution respectively, inject them into the liquid chromatograph, measure, and record the chromatogram; Gradient elution was used, and the elution time and mobile phase ratio were: 0 min, mobile phase A 10%, mobile phase B 90%; 60 min, mobile phase A 90%, mobile phase B 10%; The reference substances are ephedrine hydrochloride, pseudoephedrine hydrochloride, benzoylmeaconitine, benzoylaconitine, benzoylhypaconitine, liquiritin and ammonium glycyrrhizate.

2. The establishment method according to claim 1, characterized in that The cough-mining capsule is composed of the following components: aconite root, honeyed ephedra, roasted liquorice root and starch.

3. The establishment method according to claim 1, characterized in that The chromatographic column is Welch Xtimate@C18 with a column length of 2.5 cm, an inner diameter of 4.6 mm, and a particle size of 5 μm.

4. The establishment method according to claim 1, characterized in that The detection wavelength is 235 nm.

5. The establishment method according to claim 1, characterized in that: The ultrasonic treatment conditions were 250W, 40KHz, and 30min.

6. The establishment method according to claim 1, characterized in that: The fingerprint of the test solution should present 12 characteristic peaks, among which the retention times of peaks 3, 6, 8 and 12 are basically consistent with those of the reference substances ephedrine hydrochloride, liquiritin, benzoylmonoaconitine and ammonium glycyrrhizate; the peak corresponding to the retention time of the reference substance liquiritin peak is designated as S peak, and the relative retention time of each characteristic peak and S peak is calculated, which should be within ±10.0% of the specified value.

7. The establishment method according to claim 6, characterized in that: The specified values ​​are: 0.33-peak 1, 0.40-peak 2, 0.51-peak 3, 0.94-peak 4, 0.97-peak 5, 1.0-peak 6, S, 1.28-peak 7, 1.31-peak 8, 1.35-peak 9, 1.38-peak 10, 1.53-peak 11, 2.04-peak 12.

8. The establishment method according to claim 1, characterized in that: MARK peak matching, according to the Chinese medicine chromatographic fingerprint similarity evaluation system, the fingerprint of the test solution and the reference solution are calculated by similarity, and the similarity is not less than 0.90.

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