CN114577941A

CN114577941A - Fingerprint detection method for cough-relieving and phlegm-reducing traditional Chinese medicine

Info

Publication number: CN114577941A
Application number: CN202210224429.3A
Authority: CN
Inventors: 刘峰; 贺敬霞; 许刚; 陈衍斌; 王晓梅; 张鑫; 王青; 彭修娟; 党艳妮; 张建
Original assignee: Shaanxi Buchang Pharmaceuticals Co ltd
Current assignee: Shaanxi Buchang Pharmaceuticals Co ltd
Priority date: 2022-03-07
Filing date: 2022-03-07
Publication date: 2022-06-03
Anticipated expiration: 2042-03-07
Also published as: CN114577941B

Abstract

The invention provides a fingerprint detection method of a cough-relieving and sputum-reducing traditional Chinese medicine, wherein an UPLC method adopted by the method has the advantages of short detection time, low solvent consumption, 19-25 chromatographic peaks marked under different detection wavelengths, and 6 effective components such as apioside isoliquiritin, baicalin, wogonoside, baicalein, ammonium glycyrrhizinate and the like can be identified.

Description

Fingerprint detection method of cough-relieving and phlegm-reducing traditional Chinese medicine

Technical Field

The invention relates to a fingerprint spectrum detection method of a cough-relieving and sputum-reducing traditional Chinese medicine, belonging to the field of Chinese patent medicine quality detection methods.

Background

The traditional Chinese medicine for relieving cough and reducing sputum is the Kelu oral liquid, which is a unique patent of Shanxi step-size Limited company and is a large variety of Chinese patent medicines for step-size pharmacy. The traditional Chinese medicine is prepared from 1999, 2004 and approved by the State drug administration with the standard number of Z20027542 and the national drug Standard number of WS-11419(ZD-1419-, Preparation process, dripping pill, soft capsule, etc. The existing standard executed by the quality standard of the Kelu oral liquid product at present is WS-11419(ZD-1419) -2002, the components identified by thin-layer identification in the detection standard comprise baicalin, codeine phosphate and morphine reference substances, and the detection method comprises that each 1ml of ephedra contains ephedrine hydrochloride (C10H15No & HCl) which is not less than 65 mu g. However, the existing product detection method only detects single index components, so that the control requirements of the internal quality of a plurality of effective components of the traditional Chinese medicine compound cannot be met, and the research of a multi-index detection quality control method is very important.

The basic research of the drug effect substances of the compound traditional Chinese medicine is a key hotspot technical field of the modernization research of the traditional Chinese medicine, and is also the basis for determining the safety, effectiveness and quality control of the traditional Chinese medicine. For the traditional Chinese medicine at present, the unclear mechanism of action of the medicine is a restriction factor for restricting the modernization of the traditional Chinese medicine due to the complex active substance components. In recent years, the traditional Chinese medicine fingerprint spectrum is used as a modern comprehensive analysis means, can be used for analyzing the pharmacodynamic components and non-pharmacodynamic components of active ingredients, and accords with the overall appearance and systemic characteristics of the traditional Chinese medicine. And through basic research on active compounds, the method establishes control which can objectively reflect material basis and clarify integral composition, is an effective analysis method and is widely applied to control of the quality standard of the traditional Chinese medicine. The cough and dew oral liquid has good effect in clinical practice application and wide market prospect, and the inherent quality of the cough and dew oral liquid cannot be comprehensively controlled only by a single component, so that a traditional Chinese medicine quality control mode combining multi-index component analysis and chromatographic fingerprint is established on the basis of the original single component, and the problem of methodology for comprehensively controlling the quality of the traditional Chinese medicine on the whole is solved. The invention adopts UPLC analysis method to establish the fingerprint of the traditional Chinese medicine preparation of the Kelu oral liquid and carry out more comprehensive quality control on the identification and content determination of the determined components.

Disclosure of Invention

According to the problems, the invention provides a fingerprint detection method of a cough-relieving and sputum-reducing traditional Chinese medicine, wherein the UPLC method adopted by the method has the advantages of short detection time, low solvent consumption, 19-25 chromatographic peaks marked under different detection wavelengths, and identification of 6 effective components such as apioside isoliquiritin, baicalin, wogonoside, baicalein, ammonium glycyrrhizinate, and the like.

The technical scheme provided by the invention is as follows:

a fingerprint detection method of a cough-relieving and sputum-reducing traditional Chinese medicine comprises the following steps:

preparing a test article solution: putting the traditional Chinese medicine into a measuring flask, adding water for dilution, fixing the volume to a scale mark, shaking up, and filtering for later use;

preparing a control solution: weighing apiose isoliquiritin, baicalin, wogonoside, baicalein and ammonium glycyrrhizinate as reference substances, diluting with methanol, fixing volume, placing scale mark, and performing ultrasonic dissolution to obtain reference substance solution;

the chromatographic conditions are as follows: a chromatographic column: octadecylsilane chemically bonded silica is used as a filler, and the mobile phase is acetonitrile (A) -0.1% phosphoric acid aqueous solution (B); gradient elution conditions are 0-5 min and 2-5% (A); 5-10 min, 5% -10% (A); 10-15 min, 10% -13% (A); 15-20 min, 13% -20% (A); 20-35 min, 20% -30% (A); 35-45 min, 30-40% (A); 45-50 min, 40% -20% (A); 50-55 min, 20% -2% (A), flow rate: 0.1-1 ml/min, detection column temperature: and (2) at 20-30 ℃, the detection wavelength is as follows: 200-400 nm;

fourthly, establishing a fingerprint map: the method comprises the steps of respectively absorbing a test solution and a reference solution, measuring by adopting a chromatographic condition method, preparing multiple batches of test solutions by adopting the step (1), respectively measuring according to the chromatographic conditions in the step (3), obtaining multiple batches of fingerprint spectrums, introducing traditional Chinese medicine fingerprint similarity evaluation software, and carrying out similarity evaluation.

Preferably, the number of times of dilution with water in the preparation of the test article solution is 5 to 50.

Preferably, the detection method comprises the following steps of preparing a reference substance solution, wherein the concentration of the apiose isoliquiritin is as follows: 200-400 mu g/mL, 300-400 mu g/mL of isoliquiritin, 70-150/mL of baicalin, 400-600 mu g/mL of wogonoside, 60-120 mu g/mL of baicalein and 120-180 mu g/mL of ammonium glycyrrhizinate.

Preferably, in the preparation of the control solution, the concentration of the apiose isoliquiritigenin is 311 mug/mL, the concentration of the isoliquiritigenin is 369.433 mug/mL, the concentration of the baicalin is 111.6 mug/mL, the concentration of the wogonin is 541.858 mug/mL, the concentration of the baicalein is 80.120 mug/mL, and the concentration of the ammonium glycyrrhizinate is 161.2 mug/mL.

Preferably, the detection method comprises the steps of: 0.1-0.4 ml/min.

Preferably, the detection method comprises the steps of: 0.2 ml/min.

Preferably, the chromatographic conditions of the step three of the detection method are as follows: the detection column temperature: the detection wavelength is 250nm and 354nm at 25 ℃.

Preferably, the chromatographic conditions of the step three of the detection method are as follows: the types of the chromatographic column are as follows: waters BEH C18.

Preferably, the detection method is characterized in that the fingerprint similarity of the multiple batches of sample solutions in the detection method step four is not less than 0.85.

The traditional Chinese medicine for relieving cough and reducing sputum can be a traditional Chinese medicine compound preparation with similar functional indications, such as fritillaria, loquat leaves, aster, ephedra, scutellaria and the like. The specific products corresponding to the traditional Chinese medicine of the invention are as follows: kelu oral liquid.

The batch of traditional Chinese medicine fingerprint chromatogram determination is usually not less than 8 batches, and the determination batch is generally 10-30 batches.

In the technical fingerprint spectrum research scheme, the inventor of the application carries out corresponding optimization on the preparation step (1) of the test solution, the detection method step (3) and the fingerprint spectrum establishment step (4) through a large amount of test and exploration, and obtains the quality detection method which is reliable in stability and simple and convenient to operate. In order to highlight the innovation of the technical scheme of the invention, the preferable partial screening scheme in the research technology of the project is provided as follows.

1. Preparation of test solution

In the experiment, 20%, 40%, 60% and 80% alcohol precipitation solutions are adopted for investigation in 24h, 48h and 72h of the traditional Chinese medicine in the step (1), and the sample solution is diluted and is extracted by adopting ultrasonic for 10min, 30min and 60min for investigation.

The results show that: the alcohol precipitation concentration and the alcohol precipitation time have no great influence on the number and content of chromatographic peaks, the column effect of a chromatographic column and the like, and meanwhile, different ultrasonic times have no obvious change on index components, and the optimized preparation method of the test sample is direct dilution sample injection analysis.

And further performing dilution investigation on the samples, respectively performing stock solution dilution 2 times, 5 times, 10 times and 50 times screening, and adopting the stock solution of the test sample diluted 2 times and 10 times, wherein partial index peak responses are too high or lower, and finally, 5 times and 50 times are preferably selected as the preparation of the test sample solution.

2. Screening of chromatographic conditions in the fingerprint of the invention

2.1 Mobile phase chromatography Condition optimization

In the experiment of the invention, acetonitrile-0.1% formic acid water, acetonitrile-0.1% phosphoric acid water and acetonitrile-dipotassium hydrogen phosphate water with different PH values are preferred for the mobile phase in the chromatographic condition in the step (3). The separation degree of chromatographic peaks is better along with the increase of the ph value by adopting the dipotassium phosphate aqueous solution, however, although the ph is in the acid-base range of the C18 chromatographic column, the column efficiency of the liquid phase chromatographic column is greatly changed, the service life of the chromatographic column is greatly reduced, and under the same conditions, the types, the separation degrees and the numbers of chromatographic peaks of the dipotassium phosphate aqueous solution are better than those of formic acid, and the final preference is that: 0.1% phosphoric acid solution was used for the isolation study of the oral liquid of Kelu.

2.2 selection of detection wavelength

Further, the detection wavelength in the step (3) of the invention is preferably selected, the sensitivity and absorption of partial peaks of chromatographic peaks under the wavelengths of 230nm, 250nm, 280nm, 354nm and the like are better through UPLC full-wavelength scanning, the partial chromatographic peak response value under the single wavelength of 250nm or 354nm is low, and the wavelength detection of 250nm and 354nm is preferably selected in comprehensive consideration in order to more comprehensively evaluate the quality of the cough and dew oral liquid by the chromatographic method.

2.3 determination of index Components in Kelu according to the present invention

In the process of establishing the fingerprint spectrum in the step (4), effective components contained in the traditional Chinese medicine are measured and analyzed by looking up relevant data, sample introduction is carried out in the step (3), and 5 chemical index components are identified according to retention time and an ultraviolet spectrogram of a test solution and a reference solution, wherein the chemical index components are apioside isoliquiritin, baicalin, wogonoside, baicalein and ammonium glycyrrhizinate respectively. The effective components are as follows: the baicalin, wogonoside and baicalein are related to the heat clearing effect of the traditional Chinese medicine, and the effective components of apiose isoliquiritin, isoliquiritin and licoflavone in ammonium glycyrrhizinate are also related to the phlegm eliminating and cough relieving effect of the traditional Chinese medicine.

The detection method has the beneficial effects that:

firstly, the UPLC is optimized, and the UPLC fingerprint of the traditional Chinese medicine preparation of the cough syrup oral liquid is established, and the fingerprint marks 25 common peaks under 250nm and identifies 4 chromatographic peaks. 19 common peaks are marked at 354nm, 5 chromatographic peaks are identified, and the separation degree of each characteristic chromatographic peak is good, so that the method can be used for comprehensive quality evaluation of the cough-relieving traditional Chinese medicine.

② the preferable UPLC chromatographic method can simultaneously measure 6 components under the same chromatographic condition, and passes the method verification and sample content measurement. The result of the precision test shows that the RSD value of the retention time of the chromatographic common peak of the test solution of the invention is 0.02 percent, and the RSD value of the relative peak area of the common peak is 0.67 to 1.35 percent, thus showing that the precision of the instrument is good. The stability test shows that the measured retention time RSD value of the common peak is 0.19-0.26, and the RSD value of the relative peak area of the common peak is 0.27-2.27%. The test solution is stable within 24 h. The repeatability test shows that the RSD value of the retention time of the common peak is 0.08-0.13%, and the RSD value of the relative peak area of the common peak is 1.07-2.20%. Indicating that the operating method has good repeatability. The detection result shows that the fingerprint detection method is stable and reliable, has good repeatability and stable index components. Can meet the requirements of fingerprint technology and preparation quality control, and provides reference for the quality control of the Kelu oral liquid.

And thirdly, according to the liquid phase detection results of 10 batches, the similarity between samples of each batch at 354nm is 0.991-1.000 by taking S1 as a reference chromatogram, and the similarities between the samples of each batch at 250nm and the reference chromatogram are 1.000, 0.999, 1.000, 0.999, 0.996, 1.000, 0.999 and 0.998 respectively, which all accord with the fingerprint quality control regulation. Therefore, the similarity of the fingerprint detection method measured by the detection method of the invention is more than 0.991.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.

FIG. 1-250nm 10 batches of fingerprint spectra;

FIG. 2-354nm 10 batches fingerprint pattern;

FIG. 3 is a liquid chromatogram of a test solution of a cough-relieving and sputum-reducing traditional Chinese medicine of the present invention, wherein the chromatogram peak is 1-apiose isoliquiritin, the chromatogram peak is 2-isoliquiritin, the chromatogram peak is 3-baicalin, the chromatogram peak is 4-wogonoside, the chromatogram peak is 5-baicalein, and the chromatogram peak is 6-ammonium glycyrrhizinate;

figure 4-control solution chromatogram.

Detailed Description

In order to more fully understand the practice of the present invention, an experimental example is set forth below, and the present invention is further illustrated by the following exemplary examples.

Example 1 fingerprint detection method of the invention

1.1 instruments and reagents

Waters H-class ultra high performance liquid chromatograph (Waters corporation, USA); KUDOU ultrasound instrument (shanghai kodao ultrasound instruments ltd); MS430TS electronic balance (mettler-tollido instruments shanghai limited); XPE205 electronic balance (mettler-tollidods instruments shanghai ltd); merck Milli-Q water purifier (Merck chemical technology Shanghai Co., Ltd.); methanol, acetonitrile [ GR selmer feishel technologies (china) ltd ]; methanol (chemical reagent of AR national chemical group limited), phosphoric acid (red rock reagent factory of river east, tianjin city, AR).

1.2 materials

Wogonoside (batch: MUST-20111601, purity not less than 99.06% Chengdu Manster Biotech Co., Ltd.); ammonium glycyrrhizinate (batch number: MUST-21061007, purity not less than 99.06% Chengdu Manster Biotech Co., Ltd.); isoliquiritigenin (batch number: MUST-21051501, purity not less than 99.31% Chengdu Manster Biotech Co., Ltd.); apigenin (batch: PS012516, purity more than or equal to 99.87% Chengdu Pusi Biotech limited); baicalin (batch No. 110715-201720, China food and drug testing institute with purity of more than or equal to 93.5%); baicalein (batch number: 1115-201720, purity more than or equal to 98.5 percent in China institute for food and drug testing); kelu oral liquid (Shanxi step size pharmaceutical Co., Ltd., batch number details in Table 1 below).

TABLE 1 different batches of Kelu oral liquid

1.3 preparation of test solutions

Placing 5mL of Kelu oral liquid in 25mL measuring flask and 50mL measuring flask respectively, adding water for dilution, diluting to constant volume to scale mark, shaking, and filtering with 0.2 μm filter head for use.

1.4 preparation of control solutions

Precisely weighing appropriate amount of apiose isoliquiritin, baicalin, wogonoside, baicalein, and ammonium glycyrrhizinate as reference substances, diluting with methanol, fixing volume, placing scale mark, and performing ultrasonic dissolution. The concentrations were 311. mu.g/mL, 369.433. mu.g/mL, 111.6. mu.g/mL, 541.858. mu.g/mL, 780.120. mu.g/mL and 161.2. mu.g/mL, respectively.

1.5 liquid chromatography conditions

The chromatographic column is Waters BEH C₁₈(2.1X 200mm, 1.7 μm); the mobile phase is acetonitrile (A) -0.1 percent phosphoric acid water solution (B); gradient elution conditions are 0-5 min and 2-5% (A); 5-10 min, 5% -10% (A); 10-15 min, 10% -13% (A); 15-20 min, 13% -20% (A); 20-35 min, 20% -30% (A); 35-45 min, 30-40% (A); 45-50 min, 40% -20% (A); 50-55 min, 20% -2% (A); column temperature: normal temperature; flow rate: 0.2 ml/min; the sample volume is 1 mu L; detection wavelength: 354nm and 250 nm.

2 methodological validation

2.1 precision

Taking batch 2(S2) Kelu oral liquid, preparing a test solution according to a method of '1.1', continuously injecting samples for 6 times under the '1.5 liquid phase chromatographic condition', taking a No. 13 peak (baicalin) as a reference, and measuring that the retention time RSD value of the common peak is 0.02%, and the RSD value of the relative peak area of the common peak is 0.67-1.35%. Indicating that the precision of the instrument is good.

2.2 stability

Taking batch 2(S2) Kelu oral liquid, preparing a test solution according to a method of '1.1', injecting samples under the chromatographic condition of '2' item for 0, 2, 4, 6, 8, 10, 12 and 24 hours, taking No. 13 peak (baicalin) as reference, and measuring that the retention time RSD value of the common peak is 0.19-0.26, and the RSD value of the relative peak area of the common peak is 0.27-2.27%. The test solution is stable within 24 h.

2.3 repeatability

Taking batch 2(S2) Kelu oral liquid, preparing 6 test sample solutions in parallel according to a method of '1.1', injecting samples under the chromatographic condition of '2', taking No. 13 peak (baicalin) as reference, and measuring that the retention time RSD value of the common peak is 0.08-0.13%, and the RSD value of the relative peak area of the common peak is 1.07-2.20%. Indicating that the operating method has good repeatability.

2.4 fingerprint establishment and chromatographic peak identification

According to the invention, 10 batches of sample diluted solutions were prepared according to the "1.3 method for preparing a test solution" and analyzed by sample injection under the "2" chromatographic conditions. And (3) introducing a 2012-version traditional Chinese medicine fingerprint similarity evaluation system, taking S1 as a reference map, generating a comparison fingerprint by adopting the average time window width of 0.1min, and performing automatic matching on chromatograms of different batches. The results are shown in detail in FIGS. 1 and 2. As can be seen from the attached figure 1 of the specification, 25 chromatographic peaks are marked at 250nm of high performance liquid chromatography. Through analysis and comparison, 4 chromatographic peaks can be identified, wherein the 13 th peak is baicalin, the 19 th peak is wogonoside, the 21 st peak is baicalein, and the 23 th peak is ammonium glycyrrhizinate. As can be seen from FIG. 2, 19 chromatographic peaks are marked at 354 nm. 5 chromatographic peaks are identified, wherein the chromatographic peak is No. 11 peak which is apiose isoliquiritin, No. 12 peak which is isoliquiritin, No. 13 peak which is baicalin, No. 17 peak which is wogonoside and No. 19 peak ammonium glycyrrhizinate.

2.5 evaluation of similarity

From the experimental results in table 2, it is found that the similarity between the samples of 354nm batches is 0.991-1.000 using S1 as the reference chromatogram, and the similarity between the samples of 250nm batches and the reference chromatogram is 1.000, 0.999, 1.000, 0.999, 0.996, 1.000, 0.999, and 0.998, respectively, which all meet the fingerprint quality control regulations. The test result shows that the Kelu oral liquid produced by enterprises has small difference and stable quality, and the established UPLC fingerprint spectrum method is suitable for the quality evaluation and control of the Kelu oral liquid.

TABLE 2 similarity of samples from different batches

Example 2: the detection method of the invention measures the content

The preparation method of the sample solution and the reference solution is characterized in that the chromatographic conditions are as in chromatogram 1, the detection wavelengths in the chromatographic conditions are apioside isoliquiritin and isoliquiritin of 354nm, and baicalin, wogonoside, baicalein and ammonium glycyrrhizinate are 250 nm; the chromatograms are shown in FIG. 3 and FIG. 4 below.

1 methodological validation

1.1 Linear

Precisely absorbing a proper amount of each reference solution, placing the reference solutions in a measuring flask, adding methanol for dilution, fixing the volume to a scale mark, shaking up to obtain a series of linear reference solutions, injecting samples under the chromatographic condition of example 1, drawing a standard curve by taking a peak area Y as a vertical coordinate and a concentration X (mu g. mL < -1 >) as a horizontal coordinate, and calculating a regression equation, wherein the result is shown in Table 3.

Standard curve and linear range of 36 index components in table

1.2 precision

Precisely absorbing appropriate amount of each reference substance, mixing, continuously injecting sample for 6 times under the chromatographic condition of example 1, and calculating RSD values of 6 component peak areas of apiose isoliquiritin, baicalin, isoliquiritin, wogonoside, ammonium glycyrrhizinate and baicalein to be 0.90%, 0.73%, 1.25%, 0.67%, 1.35% and 1.10%, respectively. Indicating that the precision of the instrument is good.

1.3 stability

Taking the test solution and the reference solution of batch 2, respectively injecting samples for 0, 2, 4, 6, 8, 10, 12 and 24 hours under the chromatographic condition of example 1, measuring peak areas of the apiose isoliquiritin, the baicalin, the wogonoside, the baicalein and the ammonium glycyrrhizinate, calculating to obtain RSD values of the sample solution of 0.54%, 1.31%, 0.63%, 0.71%, 2.64% and 1.52%, and RSD values of the reference solution of 0.50%, 0.51%, 0.39%, 0.41%, 0.45% and 0.68%, which are all less than 3%, and indicating that the stability of the sample and the reference solution is better within 24 hours.

1.4 repeatability

Weighing the same batch of 2 Kelu oral liquid, preparing 6 test solution in parallel, measuring under the UPLC chromatographic condition, sequentially measuring 6 component peak areas, and obtaining RSD values of 2.64%, 2.71%, 2.33%, 2.56%, 2.40% and 2.29%, wherein the RSD values are respectively less than 3%, and the method has good repeatability.

1.5 sample recovery

6 parts of the same batch of Kelu oral liquid with known content and 2.5mL are taken and put into a 25mL measuring flask, corresponding reference substances are precisely added according to the level of 100 percent of the proportion of the components of the sample to be measured to prepare a sample solution, the sample adding recovery rate is calculated by measuring under the chromatographic condition of the example 1, the average sample adding recovery rate of each reference substance is measured to be 99.57 to 102.24 percent, and the RSD (n is 6) <3 percent, which shows that the method has good accuracy.

2 determination of the content

10 batches of sample solutions are prepared according to the method of the embodiment 1 of the invention, the contents of 6 components in the Kelu oral liquid are determined under the same chromatographic conditions, and the determination results of the detected contents are shown in the following table 4.

TABLE 410 determination of the content of cough syrup oral liquid in batches (Unit:. mu.g/mL)

The test results in Table 4 show that the content ranges of the components of the 10 batches of Kelu oral liquid are 25.057-29.652 mu g/mL of apiose isoliquiritin, 26.404-32.072 mu g/mL of isoliquiritin, 824.457-1291.929 mu g/mL of baicalin, 319.541-557.251 mu g/mL of wogonin, 4.243-15.839 mu g/mL of baicalein and 250.566-479.037 mu g/mL of ammonium glycyrrhizinate, and the detection method limits the upper limit and the lower limit of the content limit of the effective components in the traditional Chinese medicine, and is favorable for controlling the internal quality of the traditional Chinese medicine.

Finally, it should be noted that: the present invention is not intended to be limited to the embodiments shown above, which are intended to be illustrative, instructive, and not restrictive. Those skilled in the art, having the benefit of this disclosure, will appreciate that various modifications, equivalents, and improvements may be made without departing from the spirit and scope of the invention.

Claims

1. A fingerprint detection method for a cough-relieving and sputum-reducing traditional Chinese medicine is characterized by comprising the following steps:

preparing a control solution: weighing apiose isoliquiritin, baicalin, wogonoside, baicalein, and ammonium glycyrrhizinate as reference substances, diluting with methanol, fixing volume, placing scale mark, and performing ultrasonic dissolution to obtain reference substance solution;

fourthly, establishing a fingerprint map: the test solution and the control solution are respectively absorbed in the steps of firstly, carrying out determination by adopting a chromatographic condition method in the steps of thirdly, preparing multiple batches of test solution in the step (1), respectively determining according to the chromatographic conditions in the step (3), obtaining multiple batches of fingerprints, importing traditional Chinese medicine fingerprint similarity evaluation software, and carrying out similarity evaluation.

2. The detection method according to claim 1, wherein the dilution with water in the preparation of the test solution is 5-50 times.

3. The detection method of claim 1, wherein the concentration of the apiosooisliquiritin in the preparation of the control solution is as follows: 200-400 mu g/mL, 300-400 mu g/mL of isoliquiritin, 70-150/mL of baicalin, 400-600 mu g/mL of wogonoside, 60-120 mu g/mL of baicalein and 120-180 mu g/mL of ammonium glycyrrhizinate.

4. The assay of claim 1, wherein the assay comprises the steps of preparing a control solution in which the concentration of apioside isoliquiritigenin is 311 μ g/mL, the concentration of isoliquiritigenin is 369.433 μ g/mL, the concentration of baicalin is 111.6 μ g/mL, the concentration of wogonin is 541.858 μ g mL, the concentration of baicalein is 80.120 μ g/mL, and the concentration of ammonium glycyrrhizinate is 161.2 μ g/mL.

5. The assay of claim 1, wherein the assay step is a chromatographic condition, wherein the flow rate: 0.1-0.4 ml/min.

6. The assay of claim 5, wherein the assay step is a chromatographic condition, wherein the flow rate: 0.2 ml/min.

7. The assay of claim 1, wherein the chromatographic conditions of assay step three are: the detection column temperature: the detection wavelength is 250nm and 354nm at 25 ℃.

8. The assay of claim 1, wherein the chromatographic conditions of assay step three are: the types of the chromatographic column are as follows: waters BEH C₁₈。

9. The detection method according to claim 1, wherein the fingerprint similarity of the multiple batches of sample solutions in the detection method step four is greater than or equal to 0.90.