CN116148407A - Method for Establishing Fingerprint of Kemin Capsules - Google Patents

Abstract

The invention relates to a method for establishing fingerprints of Kemin capsules. The present invention determines the preparation method and detection conditions of the test solution for the fingerprint of the product through different test solution preparations and chromatographic conditions, and identifies liquiritin, benzoyl neoaconitine, ammonium glycyrrhizinate, and ephedra hydrochloride. The four characteristic peaks, such as Jian Jian, better reflect 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 quality of the preparation.

Description

Method for Establishing Fingerprint of Kemin Capsules

technical field

The invention relates to a method for detecting fingerprints of traditional Chinese medicine preparations, in particular to a method for establishing the fingerprints of Kemin capsules.

Background technique

Fingerprint of traditional Chinese medicine refers to the chromatogram or spectrum that can mark its chemical characteristics after proper treatment of traditional Chinese medicine and certain analytical methods. Traditional Chinese medicine fingerprints are mainly used to evaluate the authenticity, goodness and stability of the quality of Chinese medicinal materials and semi-finished products of traditional Chinese medicine preparations. It has comprehensive and quantifiable benefits.

Kemin Capsules include Fu Pian, Honey Ephedra, and Zhi Licorice, which have the effect of relieving cough. Currently there is no method to fully characterize the chemical characteristics of Kemin Capsules.

Contents of the invention

In order to solve the technical problems existing in the prior art, the present invention provides a method for establishing the fingerprint of Kemin capsule.

The technical solution adopted by the present invention to solve the problems of the technologies described above is:

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

(1) Preparation of the test solution: take an appropriate amount of the sample content, grind it finely, take 0.5 g of fine powder, add 50 ml of methanol, ultrasonically treat it, cool to room temperature, filter, and get the filtrate to obtain final product;

(2) Preparation of reference substance solution: take an appropriate amount of reference substance, add methanol to dissolve to make a solution containing 0.1mg per 1ml, and use it as the reference substance solution;

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

(4) Take 10 μL each of the reference substance solution and the test solution, inject it into a liquid chromatograph, measure it, record the chromatogram, and obtain it.

Furthermore, the Kemin Capsules are composed of the following components: Fu Pian (Heishun Pian), honey ephedra, roasted licorice, and starch.

Further, a gradient elution method was adopted, and the elution time and the mobile phase ratio were as follows: 0 min, mobile phase A 10%, mobile phase B 90%; 60 min, mobile phase A 90%, mobile phase B 10%.

Further, 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.

Further, the detection wavelength is 235nm.

Further, the ultrasonic treatment condition is 250W, 40KHz, 30 minutes.

Further, the reference substance is ephedrine hydrochloride, pseudoephedrine hydrochloride, benzoyl neoaconitine, benzoyl aconitine, benzoyl hypoaconitine, liquiritin, and ammonium glycyrrhizinate.

Further, there should be 12 characteristic peaks in the fingerprint of the test solution, among which peak 3, peak 6, peak 8, peak 12 and the reference substance ephedrine hydrochloride, liquiritin, benzoyl neoaconitine, glycyrrhizic acid The retention time of ammonium is basically the same; the peak corresponding to the peak retention time of the reference substance liquiritin is defined as the 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.

Further, the prescribed 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).

Further, for MARK peak matching, according to the similarity evaluation system of traditional Chinese medicine chromatographic fingerprints, the fingerprints of the test solution and the fingerprints of the reference substance are calculated by similarity, and the similarity is not less than 0.90.

Compared with the prior art, the beneficial effects of the present invention are:

Through the preparation of different test sample solutions and chromatographic conditions, the test solution preparation method and detection conditions for the fingerprint of this product were determined, and the identification of liquiritin, benzoyl neoaconitine, ammonium glycyrrhizinate, and ephedrine hydrochloride Four characteristic peaks, such as Kemin capsules, better reflect 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 quality of the preparation.

Description of drawings

The accompanying drawings constituting a part of the present application are used to provide further understanding of the present application, and the schematic embodiments and descriptions of the present application are used to explain the present application, and do not constitute an improper limitation of the present application.

Figure 1 is the chromatogram of Elution Condition 1.

Figure 2 is the chromatogram of Elution Condition 2.

Figure 3 is a chromatogram of Elution Condition 3.

Figure 4 is a chromatogram of Elution Condition 4.

Figure 5 is a chromatogram of Elution Condition 5.

Figure 6 is a chromatogram of Elution Condition 6.

Figure 7 is a comparative chromatogram of different solvent extractions (from bottom to top: S1 is purified water; S2 is 70% methanol; S3 is 90% methanol; S4 is 100% methanol).

Fig. 8 is a comparative chromatogram taken at different ultrasonic times (30 min for S1, 45 min for S2, and 60 min for S3).

Figure 9 is a chromatogram of a reference substance (wavelength: 235).

Figure 10 is the chromatogram of the reference substance (wavelength: 237).

Figure 11 is the chromatogram of the reference substance (wavelength: 210).

Figure 12 is the UV absorption distribution of the reference substance and the test solution at 235nm.

Figure 13 is the UV absorption distribution of the reference substance and the test solution at 237nm.

Fig. 14 is the ultraviolet absorption distribution of reference substance and need testing solution at 210nm.

Figure 15 is a comparative illustration of characteristic peaks attributable to chromatographic peaks (from bottom to top: need testing solution, ephedrine hydrochloride, pseudoephedrine hydrochloride, liquiritin, benzoyl neoaconitine, benzoyl hypoaconitine , benzoyl aconitine, ammonium glycyrrhizinate).

Fig. 16 is the comparative figure of 6 parts of test solution fingerprints.

Figure 17 is a liquid chromatogram (235nm) of Kemin Capsules.

Figure 18 is a characteristic diagram of Kemin capsule.

Figure 19 is the fingerprints of Kemin Capsules test product and control.

Detailed ways

In order to enable those skilled in the art to understand the technical solution of the present disclosure more clearly, the technical solution of the present patent will be described in detail below in conjunction with specific embodiments. It should be pointed out that the following detailed description is exemplary and intended to provide further explanation to the present application.

The present invention refers to "Chinese Pharmacopoeia" 2020 Edition General Rule 9101 "Guiding Principles for Analytical Method Verification" and "Technical Requirements for Fingerprint Research of Traditional Chinese Medicine Injections", and mainly uses Kemin Capsule Zhongfu Tablet (Heishun Tablet), Honey Ephedra, and Zhigancao. Composition was used as the index for investigation. The fingerprints of Kemin Capsules were established by HPLC detection, and the fingerprints were evaluated with the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" V2.0 formulated by the National Pharmacopoeia Commission.

Prescription composition: Fu Pian (Heishun Pian), honey ephedra, roasted licorice, excipients: starch.

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

The experimental equipment is as follows:

Table 1 Experimental Instruments

Comprehensively analyze the chemical components of the content determination of the prescription composition medicinal materials, combined with the material basis of the actual preparation medicinal materials of this product and the measurement conditions of the above table statistics, tentatively determine the following fingerprint HPLC conditions for the screening of test sample processing (refer to "Chinese Pharmacopoeia" Glycyrrhizae content assay method), specific conditions are as follows:

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

Mobile phase: use 0.1% phosphoric acid water as mobile phase B and acetonitrile as mobile phase A, and wash with gradient as shown in the table below.

Table 2 Elution program

Flow rate: 1.0ml/min;

Column temperature: 30°C;

Detection wavelength: 235nm, 237nm, 210nm.

The optimization of embodiment 1 fingerprint chromatogram liquid phase elution condition

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

peaks or common peaks, it is necessary to optimize the elution mode of the monitored chromatographic conditions in order to separate or wash the components to the greatest extent,

The specific test plan is designed as follows:

Preparation of the test sample solution: Tentative: Take about 0.5 g of the fine powder of the sample content (batch number: 20190701), accurately weigh it, and prepare 3 parts of the Erlenmeyer flask with stoppers, and accurately add 70% methanol, 100% methanol, 50ml of pure water, sonicated (250W, 40KHz) for 30 minutes, cooled to room temperature, replenished with the corresponding solvent, filtered, and the filtrate was obtained.

Monitor chromatographic conditions:

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

Mobile Phase A: Acetonitrile

Mobile phase B: 0.1% phosphoric acid solution

Flow rate: 1.0ml/min

Column temperature: 30°C

The following gradient elution conditions were designed and tested:

Table 3 Elution condition 1

Table 4 Elution condition 2

Table 5 Elution condition 3

Table 6 Elution Condition 4

Table 7 Elution Condition 5

Table 8 Elution condition 6

Determination method: Precisely measure 10 μl of the above test solution, inject them into the liquid chromatograph respectively according to the above elution conditions ①～③, and record the typical chromatograms. The results are shown in Figure 1-6:

Results: From the above experiments, it can be seen that compared with the above elution conditions, when elution is carried out according to the predetermined elution condition 1 of licorice, no obvious characteristic peaks appear, and when elution is carried out according to the elution condition 2 isocratic, good separation is not achieved. Effect, when eluted according to elution condition 4, the resolution of several characteristic peaks at 40-45min is poor, and the response value is low; when eluted according to elution condition 5, the resolution of characteristic peaks at 18-20min is poor, according to gradient 6 When the elution time is removed, it is basically impossible to achieve good separation. When the elution condition 3 is followed, the fingerprint of the test solution of the test product shows that the resolution of each peak is better, and the response of the peak is better than other elution conditions. The analysis time is moderate, so temporarily In this study, the mobile phase system elution condition 3 was used as the determination condition of the capsule fingerprint.

Embodiment 2 test sample processing and solution preparation investigation

Kemin Capsule is a capsule preparation. The choice of solvent 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 content is as follows;

Table 9: Sample solution preparation scheme for experiments

Prepare the test solution separately.

Determination method: respectively accurately measure each 10 μ l of the test solution, inject liquid chromatograph respectively, measure according to the law according to the fingerprint monitoring condition optimized in Example 1, and record the chromatogram. On the Chinese medicine similarity evaluation software, 70% The chromatograms of methanol, 100% methanol, purified water, and 90% methanol dissolved samples were imported respectively, and the similarity was evaluated, and the degree of separation and the response value of characteristic peaks were observed.

The comparison map is shown in Figure 7.

Result: As can be seen from the above comparison spectrum information, when the solvent is purified water (S1 in the figure), the response values of features 7 and 9 are too low, so this solvent is not selected; when the solvent is 70% methanol (S2 in the figure) , the peak of the third characteristic peak (ephedrine hydrochloride) cannot appear, so this solvent is not selected for use; The number of chromatographic peaks and the separation between each peak are good, and the response value of each peak is higher than that of the other two solvents, so the two solvents (90% and 100% methanol) can be used as the extraction solvent of this capsule. 100% methanol is used as the extraction solvent for the contents of the capsule.

Ultrasound time selection

According to the preparation scheme of the sample solution used in the experiment in Table 9, the time selection of the extraction ultrasound was investigated, and the test solution was prepared respectively. On the Chinese medicine similarity evaluation software, the chromatograms of 100% methanol dissolved samples were respectively imported to evaluate their similarity, and the resolution and characteristic peak response value were observed.

The comparison map is shown in Figure 8.

Results: From the above comparison spectrum information, it can be seen that when the ultrasonic time is selected as 30, 45, and 60 methanol (S1, S2, S3 in the figure), there is little difference between the response value and the resolution of the characteristic peaks, so the ultrasonic time of 30 minutes is selected as Extraction conditions for the contents of this capsule.

In summary, the sample pretreatment method is as follows: take 0.5g of the fine powder of the sample content (batch number: 20190701), accurately weigh it, put it in a stoppered Erlenmeyer flask, add 50ml of 100% methanol precisely, and ultrasonicate (250W, 40KHz) for 30 After 10 minutes, cool to room temperature, add 100% methanol to make up the weight, filter, and take the subsequent filtrate to obtain the final product.

Embodiment 3 The positioning of the main material basis of Kemin capsule

Experimental plan: take an appropriate amount of the reference substance in the table below, and prepare the concentration of the positioning solution as shown in the table below

Table 10 Research reference substance solution information table

Determination method: Take 10 μl of the above-mentioned reference substance solution, inject them into the liquid chromatograph respectively, measure according to the optimized conditions, record the chromatogram, and the chromatogram is shown in Figure 9-11:

Experimental result: from the result of above-mentioned each reference substance solution location, it can be known that ephedrine hydrochloride and pseudoephedrine hydrochloride completely overlap in its peak time of this detection condition, so ephedrine hydrochloride or pseudoephedrine hydrochloride can be positioned when the position is determined (this research is all defined as ephedrine hydrochloride); the response of benzoylaconitine and benzoylaconitine is relatively low, which is not conducive to the identification of the characteristic peaks of the particle fingerprint of this product; in order to more effectively reflect the material characteristics of this product, this study mainly investigates Substances that respond better at specific wavelengths are used as characteristic peaks of fingerprints. Therefore, the selection of a specific wavelength must be further optimized.

The optimization of embodiment 4 fingerprint spectrum ultraviolet absorption wavelength

In order to ensure the effective traceability and monitoring of the substance basis of the granules of this product, and to explore the ultraviolet absorption wavelength of the best characteristic of the substance basis of Kemin Capsules; this research experiment combines the detection conditions under the corresponding medicinal materials in the first part of the "Chinese Pharmacopoeia" 2020 edition, The ultraviolet absorption of the drug base of this product is mainly concentrated around the wavelengths of 235nm, 210nm, and 237nm; in order to ensure the comprehensiveness of the material base detection, feature positioning is used to investigate the characteristics of the chromatograms at different wavelengths, and to confirm the required wavelengths for fingerprint monitoring in this study.

Take the test solution and the reference solution prepared after optimizing the conditions and inject them into the liquid chromatograph respectively. The main recorded detection wavelengths are 235nm, 210nm and 237nm. The experimental results are shown in Figure 12-14:

Conclusion: It can be known from the above experiments that when the fingerprints are detected under the conditions of 210nm and 237nmm, the characteristic indicators in the prescription components of this product, such as far ephedrine hydrochloride, pseudoephedrine hydrochloride, benzoyl aconitine, liquiritin, and glycyrrhizic acid Ammonium, etc. have a response; but under the condition of 235nm wavelength, the ultraviolet characteristic response of each substance in the sample is better, and the resolution is higher, so it is confirmed that the ultraviolet monitoring wavelength of this research condition is 235nm.

Embodiment 5 durability experiment

By fine-tuning the eluent flow rate (±0.1ml/min), column temperature (±5°C) and other conditions to evaluate the influence of the method on the measurement results, it is required that under the above-mentioned fine-tuning conditions, the theoretical plate number of S peak (liquiritin) Not less than 5000, compared with the original conditions, the relative retention time of the S peak must not exceed 10.0% of the retention time of the S peak under the original conditions (retention time: 13.684min) (retention time range: 12.316min～15.053min);

Need test solution: Take 0.5g of the sample content and grind it into fine powder, accurately weigh it, put it in a stoppered conical flask, add 50ml of 100% methanol accurately, ultrasonicate (250W, 40KHz) for 30 minutes, cool to room temperature, filter, Take the continued filtrate.

Blank solution: 100% methanol solution

The durability test fine-tuning test condition scheme is as follows:

Table 11 Durability fine-tuning condition scheme

Determination method: Precisely measure 10 μl each of the above-mentioned blank solution and the test solution, inject them into the liquid chromatograph respectively, and measure according to the law according to the durability fine-tuning test plan and the chromatographic conditions to be verified. The results are as follows:

Table 12 Durability test results

Results: According to the above test data, when fine-tuning the mobile phase flow rate in the range of 0.9ml/min～1.1ml/min, the number of theoretical plates of S peak (liquiritin) is 59049～71106, and the retention time is 13.016～14.479min; When the column temperature is in the range of 25℃～35℃, the number of theoretical plates 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 difference in the positioning of the characteristic peak Influence, the S peak (Liquiritin) theoretical plate number is all greater than 5000, and the S peak (Liquiritin) relative retention time is all within 10.0% of the S peak retention time under the original condition; Show that when fine-tuning the liquid chromatography condition parameters in the above range , the method has good durability.

According to the above research results, the conditions for the fingerprint determination of Kemin Capsules are summarized as follows:

Chromatographic column: use octadecyl bonded silica gel as filler (eg: Welch Xtimate@C18; 4.6mm×250mm, 5μm or equivalent column efficiency);

Mobile phase: use 0.1% phosphoric acid water as mobile phase B and acetonitrile as mobile phase A, and perform gradient elution in the following table.

Table 13 Gradient elution

Flow rate: 1.0ml/min

Column temperature: 30°C;

Injection volume: 10μl;

Detection wavelength: 235nm;

Need test solution: Take 0.5g of the sample content and grind it into fine powder, accurately weigh it, put it in a stoppered conical flask, add 50ml of 100% methanol accurately, ultrasonicate (250W, 40KHz) for 30 minutes, cool to room temperature, filter, Take the continued filtrate.

Determination method: Precisely measure 10 μl of the test solution, inject it into a liquid chromatograph, measure according to the law, and record the fingerprint chromatogram.

The assignment of embodiment 6 characteristic peak

The known chemical components in Kemin Capsules mainly include ephedrine hydrochloride, benzoylaconitine, liquiritin, ammonium glycyrrhizinate, etc.

In the test, the known components are identified in the chromatogram of the test solution.

Positioning reference substance solution: take appropriate amount of ephedrine hydrochloride, benzoylaconitine, liquiritin, and ammonium glycyrrhizinate reference substance respectively, weigh them accurately, add methanol to dissolve and make solutions containing about 0.1mg per 1ml respectively, As a positioning reference solution.

Determination method: Inject and measure according to the chromatographic conditions determined under the above item 5.0, compare with the peaks in the chromatogram of the test solution (batch number: 20190701), and carry out the attribution of the characteristic peaks. The results are shown in Figure 15:

Results: The identification of characteristic peaks in the chromatogram of the test solution indicated 7 known components. Among them, ephedrine hydrochloride, liquiritin, benzoyl neoaconitine, and ammonium glycyrrhizinate are more obvious and can be known as characteristic peaks; the rest of the substances are weakly detected and not regarded as characteristic peaks.

Embodiment 7 reference substance solution

The known components were detected in the test solution, with the peaks of "ephedrine hydrochloride, liquiritin, benzoyl neoaconitine, and ammonium glycyrrhizinate" relatively obvious, which were in the middle of the fingerprint feature spectrum, and liquiritin, benzoyl The peaks of neoaconitine and ammonium glycyrrhizinate can better reflect the separation effect of the spectrum, so they are used as references.

Preparation of reference solution: Take appropriate amount of reference substances of liquiritin, benzoyl neoaconitine, and ammonium glycyrrhizinate, weigh them accurately, add methanol to dissolve and make a solution containing about 0.1 mg per 1 ml, and use it as a reference solution.

Embodiment 8 fingerprint spectrum and technical parameter thereof

Sample fingerprinting

Take an appropriate amount of content in a batch of Kemin capsule samples for the test, prepare 6 parts of the test solution according to the preparation method of the test solution, and accurately measure 10 μl under the proposed chromatographic conditions, and inject them into the liquid chromatograph respectively Measure according to the law and record the chromatogram, the result is shown in Figure 16:

The above-mentioned fingerprint test data were imported into the similarity software of fingerprints of traditional Chinese medicine, and the peak matching was corrected by multi-point calibration of the similarity software of fingerprints of traditional Chinese medicines. Compared with the fingerprints of the 6 samples, the minimum similarity was 0.993, indicating that the repeatability of the fingerprints was good.

Calibration of common characteristic fingerprint peaks

Analyze and compare the chromatograms of 6 samples of the test solution from one batch of Kemin Capsules used in the test, and use the "Chinese Medicine Fingerprint Similarity Software" to calculate and determine their common peaks.

Results: More than 20 chromatographic peaks were separated in the chromatograms of 6 samples of Kemin Capsules used in the test, among which there were 12 characteristic peaks, and the area of common characteristic peaks accounted for more than 90% of the total peak area. As a reference peak, calculate the relative retention time, the calculation formula is as follows:

According to above-mentioned chromatogram peak table (Fig. 17), calculate relative retention time, demarcate characteristic peak, its result is as follows table:

Table 14 Chromatogram peak table

Based on the above experimental results, the characteristic spectrum of Kemin Capsules is determined: there should be 12 characteristic peaks in the characteristic spectrum of the test product, and the relative retention times between 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); peak 3: ephedrine hydrochloride, peak 6 (S): liquiritin, peak 8: benzoyl neoaconitine, peak 12: ammonium glycyrrhizinate.

Characteristic peak limit range:

As shown in Figure 18, there should be 12 characteristic peaks in the characteristic spectrum of the test product, wherein peak 3, peak 6, peak 8, peak 12 and the reference substance ephedrine hydrochloride, liquiritin, benzoyl neoaconitine, glycyrrhizic acid The retention times are basically the same; the peak corresponding to the peak retention time of the reference substance liquiritin is defined as the 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.

Similarity evaluation

Import the fingerprint test data of one batch of Kemin Capsules (20190701) and the characteristic reference substance into the similarity software of traditional Chinese medicine fingerprints, and calculate the control fingerprints of the fingerprints of Kemin Capsules through the calculation of the similarity software of Chinese medicine fingerprints. The data is calculated based on the similarity of MARK peaks (characteristic peaks), and the results are shown in Figure 19 and the table below.

Table 15 Kemin Capsules and Feature Control Fingerprint Evaluation

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

Through the preparation of different test sample solutions and chromatographic conditions, the test solution preparation method and detection conditions for the fingerprint of this product were determined, and the identification of liquiritin, benzoyl neoaconitine, ammonium glycyrrhizinate, and ephedrine hydrochloride Four characteristic peaks, such as Kemin capsules, better reflect 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 quality of the preparation.

Finally, it should be noted that the above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, for those skilled in the art, it is still The technical solutions described in the foregoing embodiments may be modified, or part of them may be equivalently replaced. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention. Although the specific implementation of the present invention has been described above in conjunction with the accompanying drawings, it is not a limitation to the protection scope of the present invention. Those skilled in the art should understand that on the basis of the technical solution of the present invention, those skilled in the art do not need to pay creative work Various modifications or variations that can be made are still within the protection scope of the present invention.

Claims (10)

1. The method for establishing the fingerprint spectrum of the cough-sensitivity capsule is characterized by comprising the following steps of:

(1) Preparing a test solution: grinding the content of the sample into fine powder of 0.5g, adding 50ml of methanol, performing ultrasonic treatment, cooling to room temperature, filtering, and collecting the filtrate;

(2) Preparing a reference solution: taking a proper amount of reference substance, adding methanol to dissolve the reference substance to prepare a solution containing 0.1mg per 1ml, and taking the solution as a reference substance solution;

(3) Chromatographic conditions and system adaptation test: octadecyl bonded silica gel is used as a filler; acetonitrile is taken as a mobile phase A,0.1% phosphoric acid is taken as a mobile phase B, and the flow rate is 1.0ml per minute; column temperature is 30 ℃; the separation degree of the liquiritin from the adjacent chromatographic peak is more than 1.5;

(4) Respectively sucking 10 μl of reference solution and sample solution, injecting into liquid chromatograph, measuring, and recording chromatogram.

2. The method of claim 1, wherein the cough-sensitive capsule is composed of the following components: radix Aconiti lateralis Preparata, herba Ephedrae, radix Glycyrrhizae Preparata, and starch.

3. The method according to claim 1, wherein a gradient elution mode is adopted, and the elution time and the mobile phase ratio are as follows: 0min, mobile phase A10%, mobile phase B90%; 60min, mobile phase A90% and mobile phase B10%.

4. The method according to claim 1, wherein the chromatographic column is Welch Xtime@C18 or equivalent chromatographic column, the column length is 2.5cm, the inner diameter is 4.6mm, and the particle size is 5 μm.

5. The method of claim 1, wherein the detection wavelength is 235nm.

6. The method of claim 1, wherein the sonication conditions are 250w,40khz,30 minutes.

7. The method according to claim 1, wherein the reference substance is ephedrine hydrochloride, pseudoephedrine hydrochloride, benzoylmesaconine, glycyrrhizin, ammonium glycyrrhizate.

8. The method according to claim 1, wherein 12 characteristic peaks are present in the fingerprint of the sample solution, wherein peak 3, peak 6, peak 8, peak 12 are substantially consistent with retention times of ephedrine hydrochloride, glycyrrhizin, benzoylmesaconine, ammonium glycyrrhizate as reference substances; the peak corresponding to the retention time of the glycyrrhizin peak of the reference substance is defined as an S peak, and the relative retention time of each characteristic peak and the S peak is calculated and is within + -10.0% of the specified value.

9. The method according to claim 8, wherein the prescribed value is: 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).

10. The method according to claim 1, wherein the MARK peak is matched, and the similarity between the fingerprint of the sample solution and the fingerprint of the reference sample is calculated according to the similarity evaluation system of the chromatographic fingerprints of the traditional Chinese medicines, wherein the similarity is not lower than 0.90.

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