A method for establishing HPLC fingerprint spectrum of Lianhuaqingwen capsule based on chemical identification method

Abstract

The application discloses a method for establishing HPLC fingerprint spectrum of Lianhuaqingwen capsules based on chemical identification method, and is characterized in that the method adopts high performance liquid chromatography and specifically comprises the following steps: 1) preparation of reference solution: precisely take new chlorogenic acid, quercitrin, forsythia glycoside, amygdalin, emodin, chrysophanol, chlorogenic acid, forsythia glycoside A, rhein, glycyrrhizic acid reference, and add methanol to prepare a reference solution; 2) preparation of test sample solution: 3) chromatographic conditions: the mobile phase is 0.1% phosphoric acid aqueous solution (A)-acetonitrile (B), gradient elution; the detection wavelength is 207 nm, the column temperature is 30 DEG C, and the flow rate is 1.0 mL·min ‑1 ; 4) determination: precisely take the test sample solution and inject it into a high performance liquid chromatograph, and the HPLC fingerprint spectrum is obtained by HPLC determination. The HPLC fingerprint spectrum established by the application is helpful for further improving the quality evaluation system of Lianhuaqingwen capsules and can provide a reference for the quality control of Lianhuaqingwen capsules.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of traditional Chinese medicine quality control, and particularly relates to a method for establishing HPLC fingerprint of Lianhuaqingwen capsules based on chemical recognition. BACKGROUND

[0002] The prescription of Lianhuaqingwen capsules is composed of thirteen medicinal materials such as forsythia, honeysuckle, fried bitter apricot kernel, houttuynia cordata, rhubarb, licorice and patchouli, and has the effects of clearing away heat and toxic material, and relieving lung heat, and is clinically used for treating epidemic influenza with heat-toxin attacking lung, and symptoms such as fever, aversion to cold, etc.

[0003] The content determination of Lianhuaqingwen capsules only involves the monarch drug forsythia, and the content of forsythia in each capsule is not less than 0.17 mg calculated by forsythia glycoside (C 27 H 34 O 11 ) as the quality evaluation index. Since Lianhuaqingwen capsules contain multiple medicinal materials and have complex components, and forsythia glycoside mainly exists in the monarch drug forsythia, the overall quality characteristics may not be reflected by the single component control. In addition, in the existing literature reports on the quality evaluation of Lianhuaqingwen capsules, it has been confirmed by the application of network pharmacology and molecular docking technology that the components such as rhein, neochlorogenic acid, chlorogenic acid, amygdalin and glycyrrhizic acid contained in Lianhuaqingwen capsules all have pharmacological effects. At the same time, the chemical components mainly contained in the monarch drug forsythia, such as forsythia glycoside and forsythia ester glycoside A, have important pharmacological effects in the treatment of acute lung injury, etc. Therefore, it is necessary to further optimize the existing quality evaluation method of Lianhuaqingwen capsules by a multi-index quality evaluation mode, so as to comprehensively and integrally evaluate the quality of Lianhuaqingwen capsules.

[0004] The multi-index overall quality evaluation mode is consistent with the characteristics of traditional Chinese medicine in terms of multiple components, multiple targets and multiple pathways in playing curative effect, and therefore has become the development trend of the quality evaluation method of traditional Chinese medicine. Among them, the traditional Chinese medicine fingerprint can realize the further evaluation of the consistency and stability of product quality by providing the characteristic common peak spectrum with quality representation on the basis of sufficient cognition of the overall action of multiple components of traditional Chinese medicine. At the same time, with the help of chemical pattern recognition analysis method, the fingerprint can find the main markers causing the quality difference between different batches of products, and can provide valuable reference and guidance for the quality control of products. In the existing research reports on the fingerprint of Lianhuaqingwen capsules, most of the researches have not adopted the chemical pattern recognition method for further quality evaluation and comprehensive discriminant analysis of different batches of samples after the similarity evaluation of the fingerprint, and lack the analysis and tracing of the quality difference between different batches of samples, which may not provide more important reference and basis for the internal quality analysis and application of Lianhuaqingwen capsules. SUMMARY

[0005] In order to solve the above technical problems, the purpose of the present application is to provide a method for establishing the HPLC fingerprint of Lianhuaqingwen Capsules based on chemical recognition method, and the HPLC-DAD method is used to establish the fingerprint of Lianhuaqingwen Capsules, which provides a useful supplement for the optimization of quality evaluation of Lianhuaqingwen Capsules, and provides a basis for pharmaceutical manufacturers to better control the quality uniformity and stability of Lianhuaqingwen Capsules, so as to ensure the safety and effectiveness of patients.

[0006] The technical scheme of the present application is realized as follows: a method for establishing the HPLC fingerprint of Lianhuaqingwen Capsules based on chemical recognition method, characterized in that high performance liquid chromatography is used, and the method specifically comprises the following steps:

[0007] 1) Preparation of reference solution: accurately weigh the reference substances of neochlorogenic acid, quercitrin, forsythoside, amygdalin, emodin, chrysophanol, chlorogenic acid, forsythoside A, rhein, and glycyrrhizic acid, and add methanol to prepare a reference solution;

[0008] 2) Preparation of test sample solution: take a proper amount of the content of Lianhuaqingwen Capsules, grind it finely, accurately weigh it, place it in a conical flask, accurately add methanol, shake well, weigh, ultrasonically treat, cool to room temperature, make up the weight loss, shake well, stand, and pass through a 0.22 μm organic phase microporous filter membrane to obtain a test sample solution;

[0009] 3) Chromatographic conditions: use a Shimadzu LC-20AT type high performance liquid chromatograph, the chromatographic column is an Agilent Zorbax SB-C 18 (250 mm x 4.6 mm, 5 μm), the mobile phase is 0.1% phosphoric acid aqueous solution (A) - acetonitrile (B), the gradient elution is 0~5 min, 5~7% B; 5~10 min, 7~8% B; 10~11 min, 8~9% B; 11~14 min, 9~12% B; 14~20 min, 12~20% B; 20~40 min, 20~32% B; 40~45 min, 32~45% B; 45~55 min, 45~60% B; 55~56 min, 60~90% B; 56~64 min, 90~90% B; the detection wavelength is 207 nm, the column temperature is 30℃, and the flow rate is 1.0 mL·min -1 ;

[0010] 4) Determination: accurately take the test sample solution and inject it into the high performance liquid chromatograph, and determine the fingerprint by high performance liquid chromatography.

[0011] In the above scheme: the control solution contains 1.45 mg of forsythoside A, 0.14 mg of quercitrin, 0.13 mg of chlorogenic acid, 0.30 mg of forsythoside A, 0.19 mg of neochlorogenic acid, 0.11 mg of amygdalin, 0.02 mg of rhein, 0.03 mg of emodin, 0.02 mg of chrysophanol, and 0.19 mg of glycyrrhizic acid per 1 mL.

[0012] In the above scheme: the content of the test solution is 1 g / 10 ml of methanol.

[0013] In the above scheme: the injection amount of the test solution is 10 ul.

[0014] In the above scheme: the obtained fingerprint has 40 common peaks, wherein peak 12, 15, 16, 21, 26, 30, 35, 36, 38, and 39 are identified as neochlorogenic acid, amygdalin, chlorogenic acid, forsythoside A, quercitrin, forsythoside, glycyrrhizic acid, rhein, emodin, and chrysophanol, respectively, and the average retention time is 17.7, 23.2, 23.4, 29.8, 34.8, 40.7, 52.9, 56.6, 61.8, and 63.9 minutes, respectively (the relative retention time is 0.312, 0.410, 0.413, 0.527, 0.615, 0.720, 0.935, 1.000, 1.092, and 1.129, respectively, with rhein as the reference peak).

[0015] Beneficial effects: the HPLC fingerprint of the Lianhuaqingwen capsule sample is established by combining the chemical pattern recognition method, 40 common peaks are determined, 10 of which are identified, and the similarity of the sample is greater than 0.947. Compared with the existing Lianhuaqingwen capsule fingerprint research, the number of common peaks determined by the method of the present application is more, and the identified common peaks all have important pharmacological activities. The HPLC fingerprint established by the present application is helpful to further improve the quality evaluation system of Lianhuaqingwen capsule, and can provide reference for the quality control of Lianhuaqingwen capsule. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 is the HPLC fingerprint and control fingerprint (R) of 13 batches of Lianhuaqingwen capsules.

[0017] Figure 2 It is the chromatogram of the sample.

[0018] Figure 3 It is the chromatogram of the mixed control.

[0019] Figure 4 It is the cluster analysis tree diagram of 13 batches of Lianhuaqingwen capsules.

[0020] Figure 5Score plot of 40 common peaks in 13 batches of Lianhuaqingwen capsules.

[0021] Figure 6 Score plot of main components in 13 batches of Lianhuaqingwen capsules.

[0022] Figure 7 Score scatter plot of OPLS-DA.

[0023] Figure 8 Score scatter plot of OPLS-DA.

[0024] Figure 9 Score scatter plot of OPLS-DA. DETAILED DESCRIPTION

[0025] The application will be further described below in conjunction with the accompanying drawings and examples.

[0026] Example 1

[0027] 1. Experimental instruments

[0028] Shimadzu LC-20AT type high performance liquid chromatograph (Japan Shimadzu Corporation); SQP type 100,000th electronic analytical balance (Sartorius Scientific Instruments Co., Ltd.); LS220A type 10,000th electronic analytical balance (Plysis Co., Ltd.); KQ-2200B type ultrasonic cleaner (Jiangsu Kunshan Ultrasonic Instrument Co., Ltd.); Milli-Q type ultrapure water instrument (Millipore Corporation, USA).

[0029] 2. Materials and reagents

[0030] The control substance neochlorogenic acid (batch number DSTDX001503, content 98.0 %) was purchased from Chengdu Desit Technology Co., Ltd.; the control substance quercitrin (batch number MUST-21111917, content 99.2 %) was purchased from Chengdu Manxite Technology Co., Ltd.; the control substance forsythrin (batch number 21041221, content 98.3 %) and amygdalin (batch number 21110935, content 98.6 %) were purchased from Shanghai Tongtian Biotechnology Co., Ltd.; the control substance emodin (batch number 19080203, content 99.3 %) and chrysophanol (batch number 481-74-3, content 98.2 %) were purchased from Chengdu Pu Feide Biotechnology Co., Ltd.; the control substance chlorogenic acid (batch number 110325, content 98.3 %) was purchased from Sichuan Weikeqi Biotechnology Co., Ltd.; the control substance forsythrin A (batch number 10059, content 97.8 %) was purchased from Shanghai Shidand Standard Technology Service Co., Ltd.; the control substance rhein (batch number SR8100, content 98.2 %) was purchased from Beijing Solaybao Technology Co., Ltd.; and the control substance glycyrrhizic acid (batch number AMU488, content 98.5 %) was purchased from Shanghai Bide Pharmaceutical Technology Co., Ltd. Methanol and acetonitrile were chromatographic grade, and phosphoric acid was analytical grade. Thirteen batches of Lianhua Qingwen capsules (from a domestic manufacturer, batch numbers: B2102150, B2102154, B2102009, B2102186, B2102166, B2102148, B2102078, B2101161, B2102162, B2101315, B2102175, B2101162, B2101153, which are denoted as S1-S13 in turn).

[0031] 3. Method

[0032] 3.1 Preparation of the mixed control substance solution A proper amount of each control substance was accurately weighed, and methanol was added to prepare a mixed solution containing 1.45 mg of forsythin A, 0.14 mg of quercitrin, 0.13 mg of chlorogenic acid, 0.30 mg of forsythrin, 0.19 mg of neochlorogenic acid, 0.11 mg of amygdalin, 0.02 mg of rhein, 0.03 mg of emodin, 0.02 mg of chrysophanol and 0.19 mg of glycyrrhizic acid per 1 mL, thereby obtaining the mixed control substance solution.

[0033] 3.2 Preparation of the test sample solution

[0034] After investigating the extraction solvent (100 % methanol, 70 % methanol, 50 % methanol, ethanol, ethyl acetate, etc.), the extraction method (heating reflux, ultrasonic), the extraction time (15, 30, 45, 60 min) and other factors, the method for preparing the sample was finally determined as follows: 1.0 g of Lianhua Qingwen capsule powder was ultrasonically extracted with 10 mL of methanol for 30 min, and all the test sample solutions were prepared under this condition to obtain the optimal extraction efficiency.

[0035] Take the contents of Jinhua Qingwen Capsules, grind finely, take about 1.0 g, accurately weigh, place in a conical flask, accurately add 10 mL of methanol, shake well, weigh, treat with ultrasound (frequency 40 Hz, power 100 W) for 30 min, cool to room temperature, make up the weight loss, shake well, stand, pass through a 0.22 μm organic phase microporous filter membrane to obtain the test sample solution.

[0036] 3.3 Chromatographic conditions

[0037] After investigating the effects of chromatographic column (Agilent Zorbax SB-C 18 , InertSustain C 18 , Hypersil BDS-C 18 ), mobile phase composition (methanol-water, acetonitrile-water, acetonitrile-0.1% phosphoric acid, acetonitrile-0.2% phosphoric acid), column temperature (29, 30, 31 ℃), flow rate (0.8, 1.0, 2.0 mL / min), detection wavelength (207, 225, 254, 270 nm) on the number of chromatographic peaks, peak shape and separation, it is confirmed that:

[0038] A Shimadzu LC-20AT type high performance liquid chromatograph was used, the chromatographic column was Agilent Zorbax SB-C 18 (250 mm x 4.6 mm, 5 μm), the mobile phase was 0.1% phosphoric acid aqueous solution (A)-acetonitrile (B), gradient elution was 0~5 min, 5~7% B; 5~10 min, 7~8% B; 10~11 min, 8~9% B; 11~14 min, 9~12% B; 14~20 min, 12~20% B; 20~40 min, 20~32% B; 40~45 min, 32~45% B; 45~55 min, 45~60% B; 55~56 min, 60~90% B; 56~64 min, 90~90% B. The detection wavelength was 207 nm, the column temperature was 30 ℃, the flow rate was 1.0 mL·min -1 , and the injection volume was 10 μL.

[0039] 3.4 Determination: accurately pipette the test sample solution into the high performance liquid chromatograph, determine by high performance liquid chromatography to obtain the fingerprint spectrum.

[0040] 4. Methodology investigation

[0041] 4.1 Precision test Take the same batch of capsule samples (S1), prepare the test solution, and continuously inject 6 times under the chromatographic conditions. Take rhein as the reference peak, record the relative retention time and relative peak area of each common chromatographic peak. The RSD of the relative retention time of each common peak is less than 1%, and the RSD of the relative peak area is less than 3%. Use the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)" software to analyze and process the chromatogram, and find that the similarity of the chromatograms obtained by continuous injection for 6 times is not less than 0.99 compared with the reference chromatogram, indicating that the instrument precision is good and meets the requirements of fingerprint determination.

[0042] 4.2 Reproducibility test

[0043] Take the same batch of capsule samples (S1), prepare 6 test solutions in parallel, and inject them under the chromatographic conditions. Record the area integral value of 40 common peaks, and calculate the RSD value, which is less than 4%, indicating that the method has good reproducibility.

[0044] 4.3 Stability requirements

[0045] Take the same batch of capsule samples (S1), and inject them under the chromatographic conditions at 0, 6, 12, 24, and 36 h after preparation. Record the area integral value of 40 common peaks, and the RSD value is less than 4%, indicating that the test solution is stable within 36 h.

[0046] 4.4 Establishment of liquid phase fingerprint

[0047] Take 13 batches of capsule samples, prepare the test solution according to the method in "2.1.2", and inject and determine it under the chromatographic conditions in "2.2". Record the chromatogram of each sample and import it into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)" software for processing. Take S1 (batch number B2102150) as the reference chromatogram, with a time window width of 0.1 min, and use the multi-point correction method to match the chromatogram. Generate the reference chromatogram (R) by the median method, and obtain the HPLC fingerprint of 13 batches of capsule samples, as shown in Figure 1 , and determine 40 common peaks.

[0048] Use Shimadzu LC-20AT high performance liquid chromatograph and DAD detector to analyze the mixed reference solution. According to the comparison of retention time and ultraviolet spectrum, 10 chromatographic peaks in 40 common peaks are identified, as shown in Figure 2 and 3peaks 12, 15, 16, 21, 26, 30, 35, 36, 38, 39 were identified as neochlorogenic acid, amygdalin, chlorogenic acid, forsythoside A, quercitrin, forsythoside, glycyrrhizic acid, rhein, emodin, chrysophanol, respectively, with retention times of 17.7, 23.2, 23.4, 29.8, 34.8, 40.7, 52.9, 56.6, 61.8, 63.9 min (with rhein as the reference peak, the relative retention times were 0.312, 0.410, 0.413, 0.527, 0.615, 0.720, 0.935, 1.000, 1.092, 1.129, respectively).

[0049] 4.5 Similarity evaluation of the fingerprint

[0050] The similarity of the fingerprint data of 13 batches of Lianhuaqingwen capsules was calculated by using the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)", and the results are shown in Table 1. The RSD of the retention time of each common peak was less than 1%, and the similarity between the 13 batches of Lianhuaqingwen capsules was 0.947-1, indicating that the quality similarity of different batches of Lianhuaqingwen capsules was high, and the chemical components contained were basically consistent, indicating that the established HPLC fingerprint method was suitable for the qualitative analysis of Lianhuaqingwen capsules.

[0051] Table 1 Similarity results of HPLC fingerprint of 13 batches of Lianhuaqingwen capsules

[0052]

[0053] 4.6 Chemical pattern recognition analysis

[0054] System clustering analysis (HCA) The peak area of 40 common peaks of 13 batches of Lianhuaqingwen capsule samples was used as the variable to establish the original data matrix, which was imported into SPSS 26.0 software, and the square Euclidean distance was used as the measure to evaluate the similarities and differences of Lianhuaqingwen capsules between different batches, and the results are shown in Figure 3 . The results showed that when the classification distance was 20, the 13 batches of Lianhuaqingwen capsule samples could be divided into 2 categories, S12 and S5 belonged to one category, and the remaining samples belonged to one category; when the classification distance was 15, all samples were divided into 3 categories, S12 was one category (G2), sample S5 was one category (G3), and the remaining samples were one category (G1), indicating that the differences in the peak area of common peaks between S12 and S5 samples and other samples were relatively large, while the differences in the quality of the remaining Lianhuaqingwen capsule samples were relatively small. The clustering results had certain correlation with the production date. Preliminary analysis showed that the partial quality differences caused by different production batches were possibly related to production process, transportation and storage of the drug, etc.

[0055] 4.7 Radar chart analysis Take the area size of 40 common peaks as index values, express them in the corresponding index axis of the radar chart in turn, and then connect the index value points on adjacent different axes with straight lines to form a radar chart with a certain shape, which is used to intuitively and visually reflect the quality differences and variation rules of different grouped samples in a two-dimensional plane.

[24] The average peak areas of the 40 common peaks of the samples in G1, G2, G3 and the total batch were introduced into Origin 2018 software in turn, and radar chart analysis was performed, and the results are shown in Figure 5 . Figure 5 Figures A-D in the middle show the distribution of the average peak areas of the 40 common peaks in the total batch, G1, G2 and G3. By observing the shape of the radar chart, it is found that the four radar charts have certain similarity in the general outline, indicating that the quality of each group in G1-G3 is consistent with the overall quality. Then, B-D in Figure 5 are compared with A in Figure 5 , it is found that the shape of B in Figure 5 is most similar to that of A in 5, i.e., the common peak content level of the 11 batch samples in G1 is most similar to that of the total samples, and has good quality consistency; compared with A in Figure 5 , Figure 5 C-D in Figure 6 all show certain differences in shape, indicating that there may be certain differences between the 2 batch samples in G2 and G3 and the overall sample quality, which is basically consistent with the HCA classification result in “2.7.1”, verifying the accuracy and reliability of the HCA result. At the same time, E in

[0056] is obtained by superimposing the three radar charts of G1, G2 and G3, and the specific differences between the three radar charts of G1-G3 can be analyzed. It can be seen that the peak area of the 40 common peaks in G3 is larger than that in G1 and G2, indicating that the content of each component in sample S5 in G3 is higher than that in other samples; then by observing the two radar charts of G1 and G2, it is found that the content of components with peak numbers 7, 8, 11-13 and 17-21 gradually decreases, the content of components with peak numbers 15, 16 and 32 shows an upward trend, and the content of other components in G1 and G2 does not differ significantly, indicating that there is a slight difference in the content level of some components between the two groups. Figure 7The results showed that most of the sample points were within the 95% confidence interval, indicating that the 13 batches of Lianhuaqingwen capsules as a whole showed stable quality characteristics; the sum of the contribution rates of the first three principal components extracted was 83.58%, which could basically reflect the main characteristics of the peak areas of the 40 common peaks. At the same time, in the PCA score plot, adjacent sample points often showed high similarity in quality, so it can be seen that the quality of sample S5 is different from the other 12 batches of samples, which should be classified into a separate group, which is basically consistent with the HCA results; there is no obvious difference between the distribution of S12 sample points in G2 and the sample points in G1, which may be related to the different classification principles of PCA and HCA. Since S5 shows certain quality differences in both analysis methods, it is suggested that the manufacturer of this variety should pay attention to samples with large quality differences and trace the source of the quality differences in order to better ensure the uniformity of drug quality.

[0057] 4.8 Orthogonal partial least squares discriminant analysis (OPLS-DA) In order to better explain the contribution of the common peaks with larger difference between different batches of Lianhuaqingwen capsules, the peak areas of 40 common peaks of 13 batches of samples were introduced into SIMCA 13.0 software, and the data of 3 groups of samples were analyzed by OPLS-DA model which can enhance the separation between groups

[25] , respectively, the model evaluation score scatter plot, variable importance projection (Variable important in projection, VIP) value plot and loading scatter plot were obtained, see Figure 8 , most of the 13 batches of Lianhuaqingwen capsules fell within the 95% confidence interval, and were mainly divided into 3 groups, the OPLS-DA results were basically consistent with the PCA results; combined with Figure 9 , by taking VIP>1 and error bar range above X>0 as the screening criteria, 8 relatively large contribution chromatographic peaks were determined, in descending order of influence: peak 30 (forsythia glycoside), peak 21 (forystin A), peak 35 (glycyrrhizic acid), peak 24, peak 32, peak 5, peak 18, peak 40; from the loading scatter plot of ​ , it can be seen that the distances between the origins of these 8 chemical components are far apart, indicating that these components have a greater influence on the quality of different batches of Lianhuaqingwen capsules, and can be used as quality difference markers of Lianhuaqingwen capsules, at the same time, it shows that the efficacy of Lianhuaqingwen capsules is formed by the joint action of multiple active ingredients. Among the 8 components, the identified components such as forsythia glycoside and forystin A are mainly present in the monarch drug forsythia, which has good pharmacological activity [8-12] . It is recommended that the manufacturer of this variety should further pay attention to the above main markers in order to better ensure the uniformity of drug quality.

[0058] The present application combines chemical pattern recognition method to establish the HPLC fingerprint of 13 batches of Lianhuaqingwen capsules samples, 40 common peaks are determined, 10 common peaks are identified, and the similarity of the samples is greater than 0.947. Compared with the existing Lianhuaqingwen capsule fingerprint research, the number of common peaks determined by the method established in the present application is more, and the identified common peaks all have important pharmacological activity, and combined with chemical pattern recognition analysis, 13 batches of Lianhuaqingwen capsule samples can be divided into 3 categories, and 8 main markers causing the quality difference between different batches of samples are found. The HPLC fingerprint established in the present application is helpful to further improve the quality evaluation system of Lianhuaqingwen capsules, and can provide reference for the quality control of Lianhuaqingwen capsules.

[0059] The present application is not limited to the above-mentioned embodiments, and those skilled in the art can understand that various changes, modifications, replacements and variations can be made to these embodiments without departing from the principles and purposes of the present application, and the scope of the present application is defined by the claims and their equivalents.

Claims

1. A method for establishing HPLC fingerprint of Lianhuaqingwen capsule based on chemical recognition method, characterized in that, The high performance liquid chromatography comprises the following steps: 1) Preparation of the reference solution: precisely take the reference substances of neochlorogenic acid, quercitrin, forsythoside, amygdalin, emodin, chrysophanol, chlorogenic acid, forsythoside A, rhein, glycyrrhizic acid, and add methanol to prepare the reference solution; 2) Preparation of the sample solution: take the content of Lianhuaqingwen capsules, grind them, precisely weigh, put them in a conical flask, precisely add methanol, shake, weigh, cool to room temperature, make up the weight, shake, stand, pass through a 0.22 μm organic phase microporous filter membrane to obtain the sample solution; 3) Chromatographic conditions: Shimadzu LC-20AT type high performance liquid chromatograph, chromatographic column Agilent Zorbax SB-C 18 , specification 250 mm x 4.6 mm, 5 μm, mobile phase A 0.1% phosphoric acid aqueous solution, mobile phase B acetonitrile, gradient elution 0~5 min, 5~7% B; 5~10 min, 7~8% B; 10~11 min, 8~9% B; 11~14 min, 9~12% B; 14~20 min, 12~20% B; 20~40 min, 20~32% B; 40~45 min, 32~45% B; 45~55 min, 45~60% B; 55~56 min, 60~90% B; 56~64 min, 90~90% B; detection wavelength 207 nm, column temperature 30℃, flow rate 1.0 mL·min -1 ; 4) Determination: precisely take the sample solution and inject it into the high performance liquid chromatograph, determine according to the high performance liquid chromatography method, and obtain the fingerprint spectrum.

2. The method for establishing the HPLC fingerprint of Lianhuaqingwen Capsules based on chemical recognition method according to claim 1, characterized in that: The reference solution contains 1.45 mg of forsythoside A, 0.14 mg of quercitrin, 0.13 mg of chlorogenic acid, 0.30 mg of forsythoside A, 0.19 mg of neochlorogenic acid, 0.11 mg of amygdalin, 0.02 mg of rhein, 0.03 mg of emodin, 0.02 mg of chrysophanol, and 0.19 mg of glycyrrhizic acid per 1 mL.

3. The method for establishing the HPLC fingerprint spectrum of Lianhuaqingwen Capsules based on chemical identification method according to claim 2, characterized in that: The content of the sample solution is 1 g / 10 ml of methanol.

4. The method for establishing the HPLC fingerprint spectrum of Lianhuaqingwen Capsules based on chemical identification method according to claim 3, characterized in that: The injection amount of the sample solution is 10 ul.

5. The method for establishing the HPLC fingerprint spectrum of Lianhuaqingwen Capsules based on chemical identification method according to claim 4, characterized in that: The obtained fingerprint spectrum has 40 common peaks, wherein peak 12, 15, 16, 21, 26, 30, 35, 36, 38, and 39 are identified as neochlorogenic acid, amygdalin, chlorogenic acid, forsythoside A, quercitrin, forsythoside, glycyrrhizic acid, rhein, emodin, and chrysophanol, and the average retention times are 17.7, 23.2, 23.4, 29.8, 34.8, 40.7, 52.9, 56.6, 61.8, and 63.9 minutes, respectively.

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