Establishment of HPLC Fingerprint of Chuanbei Zhisou Mixture

Abstract

The application belongs to the technical field of pharmaceutical analysis, and particularly relates to a method for establishing a high performance liquid chromatography (HPLC) fingerprint of Chuanbei Zhisou mixture. The method comprises the following steps: (1) preparing a test sample solution; (2) preparing a control sample solution; (3) preparing a control medicinal material solution; and (4) HPLC determination. The test sample solution, the control sample solution and the control medicinal material solution are injected into a HPLC instrument under optimized chromatographic conditions, and chromatograms are recorded. The HPLC chromatograms of multiple batches of test sample solutions are analyzed to obtain a control fingerprint of the Chuanbei Zhisou mixture, and the relative retention times of all common peaks are calculated. The method has the advantages of high compound separation degree and good repeatability. The fingerprint provided by the method can be used to determine characteristic compounds in the Chuanbei Zhisou mixture, and can effectively characterize and comprehensively monitor the quality of the Chuanbei Zhisou mixture.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of pharmaceutical analysis, and particularly relates to a method for establishing a high performance liquid chromatography (HPLC) fingerprint of Chuanbei Zhisou mixture. BACKGROUND

[0002] Chuanbei Zhisou mixture is recorded in the Third Edition of Traditional Chinese Medicine Prescription of Ministry of Public Health Pharmaceutical Standards: WS3-B-0489-91, which is composed of 8 kinds of traditional Chinese medicinal materials, i.e., 150 g of radix asteris, 150 g of perilla leaf, 10 g of fritillaria cirrhosa, 150 g of platycodon grandiflorum, 20 g of rhizoma pinelliae praeparatum, 50 g of glycyrrhiza, 40 ml of armeniacae amarae semen, and 2 ml of menthae hylata, and has the effects of relieving the exterior and expelling the lung, relieving cough and reducing sputum, and is often used for treating symptoms of external cough.

[0003] Chuanbei Zhisou mixture has many medicinal ingredients and complex chemical components, and the quality control research is less. The quality standard of Chuanbei Zhisou mixture recorded in the standard only controls the quality by physico-chemical identification of alkaloids and saponins, pH value, relative density and mixture, and the quality standard is low, which cannot comprehensively reflect the internal quality of Chuanbei Zhisou mixture and cannot guarantee the comprehensive control of the quality of Chuanbei Zhisou mixture. SUMMARY

[0004] In order to solve the above problems, the method for establishing the HPLC fingerprint of Chuanbei Zhisou mixture is provided, which has the advantages of high compound separation degree and good repeatability. The fingerprint provided by the method can be used to determine the characteristic compounds in Chuanbei Zhisou mixture, which can effectively characterize and comprehensively monitor the quality of Chuanbei Zhisou mixture.

[0005] The application provides a method for establishing the HPLC fingerprint of Chuanbei Zhisou mixture, which comprises the following steps:

[0006] (1) Preparation of test solution:

[0007] A certain volume of Chuanbei Zhisou mixture is taken in a container, an appropriate amount of methanol is added for dilution, shaking, filtration, and the filtrate is taken as the test solution;

[0008] (2) Preparation of control solution:

[0009] A certain weight of rosmarinic acid, ammonium glycyrrhizinate, p-hydroxybenzoic acid and caffeic acid is taken in a volumetric flask, an appropriate amount of methanol is added and ultrasonic treatment is performed, cooling, shaking, and filtering to obtain the control solution; wherein rosmarinic acid is used as the reference substance;

[0010] (3) Preparation of control medicinal material solution:

[0011] Take a certain weight of Ziyuan, Qingsuxie, Jiegeng, Gancao, Qingshanxia and Chuanbeimu respectively, and prepare single medicinal material solution according to the process procedure. Take a certain volume of single medicinal material solution and place it in a container, add an appropriate amount of methanol, shake well, filter, and take the filtrate as the control medicinal material solution;

[0012] (4) High performance liquid chromatography determination:

[0013] Take an appropriate amount of test solution, control solution and control medicinal material solution respectively, inject into the high performance liquid chromatograph, and record the chromatogram. Analyze the high performance liquid chromatograms of multiple batches of test solution to obtain the control fingerprint of Chuanbeizicuo mixture, and calculate the relative retention time of each common peak.

[0014] The chromatographic conditions of the high performance liquid chromatograph are as follows:

[0015] The mobile phase includes mobile phase A and mobile phase B, mobile phase A is acetonitrile, and mobile phase B is phosphoric acid solution with a volume concentration of 0.1%; the gradient elution program is as follows:

[0016] 0-10min, the volume concentration of mobile phase A increases from 5% to 12%, and the volume concentration of mobile phase B decreases from 95% to 88%;

[0017] 10-20min, the volume concentration of mobile phase A increases from 12% to 20%, and the volume concentration of mobile phase B decreases from 88% to 80%;

[0018] 20-30min, the volume concentration of mobile phase A increases from 20% to 22%, and the volume concentration of mobile phase B decreases from 80% to 78%;

[0019] 30-35min, the volume concentration of mobile phase A increases from 22% to 25%, and the volume concentration of mobile phase B decreases from 78% to 75%;

[0020] 35-60min, the volume concentration of mobile phase A increases from 25% to 55%, and the volume concentration of mobile phase B decreases from 75% to 45%;

[0021] 60-65min, the volume concentration of mobile phase A decreases from 55% to 5%, and the volume concentration of mobile phase B increases from 45% to 95%.

[0022] Preferably, in the chromatographic conditions, the column specifications are Agilent Eclipse Plus C18, 250mm x 4.6mm, 5μm; the flow rate is 1mL / min; the column temperature is 25℃; the detection wavelength is 240nm; and the injection volume is 5μL.

[0023] Preferably, in the chromatographic conditions, the theoretical plate number calculated according to rosmarinic acid peak is not less than 20000.

[0024] Preferably, the preparation of the test solution is as follows: 10ml of Chuanbei Zhicuo mixture is accurately pipetted into a 20ml volumetric flask, methanol is added to the calibration line, shaken well, filtered, and the filtrate is taken, thereby obtaining the mixture.

[0025] Preferably, the control fingerprint of Chuanbei Zhicuo mixture comprises 16 common peaks, peaks 7, 8, 14, 15 and 16 are licorice characteristic peaks, and peak 10 is a characteristic peak of radix asteris.

[0026] Preferably, peak 13 is rosmarinic acid, peak 2 is p-hydroxybenzoic acid, peak 3 is caffeic acid, and peak 16 is ammonium glycyrrhizinate.

[0027] Preferably, the relative standard deviations (RSD) of the relative retention times of the 16 common peaks are all less than 3%.

[0028] The similarity of the Chuanbei Zhicuo mixture fingerprint is calculated with reference to the control fingerprint of Chuanbei Zhicuo mixture, and the similarity of the Chuanbei Zhicuo mixture fingerprint is all above 0.9.

[0029] The beneficial effects of the present application include but are not limited to:

[0030] 1. The method for establishing the high performance liquid chromatography (HPLC) fingerprint of Chuanbei Zhicuo mixture according to the present application is based on the drug effects of the components contained in Chuanbei Zhicuo mixture, and the main components of perilla leaf, radix asteris and licorice, such as rosmarinic acid, ammonium glycyrrhizinate, p-hydroxybenzoic acid and caffeic acid, are used as research indicators to study the fingerprint of Chuanbei Zhicuo mixture, and a method for establishing and detecting the fingerprint of Chuanbei Zhicuo mixture is provided, which provides a reference and basis for the quality control of Chuanbei Zhicuo mixture; the method is simple, stable, highly precise and reproducible.

[0031] 2. The fingerprint of Chuanbei Zhicuo mixture established according to the method for establishing the HPLC fingerprint of Chuanbei Zhicuo mixture has 16 characteristic peaks that are effectively separated, and therefore the obtained fingerprint can effectively represent the quality of Chuanbei Zhicuo mixture and is conducive to the comprehensive monitoring of the quality of the drug.

[0032] 3. Ten different batches of Chuanbei Zhicuo mixture are detected by using the method for establishing the HPLC fingerprint of Chuanbei Zhicuo mixture, and the RSD of the relative retention times of the chromatographic peaks at the same position is all less than 3%, and therefore the fingerprint established by using the method has good reliability. BRIEF DESCRIPTION OF DRAWINGS

[0033] The drawings described herein are used to provide a further understanding of the present application, and form a part of the present application. The illustrative embodiments of the present application and their descriptions serve to explain the present application and do not constitute an improper limitation of the present application. In the drawings:

[0034] Figure 1Chromatographic peak of the test sample of Chuanbei Zhicou mixture under the chromatographic conditions of the method of the present application;

[0035] Figure 2 Chromatographic peak of the reference substance under the chromatographic conditions of the method of the present application;

[0036] Figure 3 Chromatographic peak of the reference medicinal material under the chromatographic conditions of the method of the present application;

[0037] Figure 4 Investigation of the chromatogram under the condition that the mobile phase of the present application is composed of methanol and water;

[0038] Figure 5 Investigation of the chromatogram under the condition that the mobile phase of the present application is composed of acetonitrile and water;

[0039] Figure 6 Investigation of the chromatogram under the condition that the mobile phase of the present application is composed of acetonitrile and 0.1% phosphoric acid;

[0040] Figure 7 Investigation of the chromatogram under the condition that the fingerprint chromatogram of the present application is compared under different wavelengths;

[0041] Figure 8 Investigation of the chromatogram 1# under the elution gradient of the present application;

[0042] Figure 9 Investigation of the chromatogram 2# under the elution gradient of the present application;

[0043] Figure 10 Investigation of the chromatogram 3# under the elution gradient of the present application;

[0044] Figure 11 Detection chromatogram of the solution fingerprint chromatogram of Chuanbei Zhicou mixture of 10 batches of test samples of the present application;

[0045] Figure 12 Reference fingerprint chromatogram of Chuanbei Zhicou mixture generated by the average value calculation method of the present application. DETAILED DESCRIPTION

[0046] The present application will be further described below in combination with specific examples, but the scope of protection of the present application is not limited to these examples.

[0047] I. Instruments and reagents:

[0048] 1.1. Instruments

[0049] Agilent 1260 high performance liquid chromatograph; DAD detector; BSA224S-CW electronic analytical balance; HEAL FORCE NW (30K) VF water purifier;

[0050] 1.2. Reagents

[0051] Reference substance: rosmarinic acid, ammonium glycyrrhizinate, p-hydroxybenzoic acid, caffeic acid;

[0052] Reference medicinal materials: Radix Asteris, Perilla frutescens, Fritillaria cirrhosa, Platycodon grandiflorum, Glycyrrhiza uralensis, Rhizoma Pinelliae, all from Shandong Hongjitang Pharmaceutical Group Co., Ltd., and all meet the standards of Chinese Pharmacopoeia.

[0053] Test sample: The present application uses the self-made pilot sample of Chuanbei Zhisou Mixture, batch numbers: 240601, 240602, 240603, 240604, 240605, 240606, 240607, 240608, 240609, 240610.

[0054] Methanol and acetonitrile are chromatographically pure, purchased from Shanghai Yuhua Technology Co., Ltd., and water is ultrapure water, and the rest of the reagents are analytically pure.

[0055] II. Method for establishing high performance liquid chromatography fingerprint of Chuanbei Zhisou Mixture

[0056] 1) Preparation of reference substance solution: rosmarinic acid, ammonium glycyrrhizinate, p-hydroxybenzoic acid, and caffeic acid were precisely weighed and placed in 10 mL volumetric flasks, methanol was added for ultrasonic dissolution, cooled, constant volume, shaken, filtered, and the filtrate was taken as the reference substance solution.

[0057] Among the above-mentioned five compounds, the content of rosmarinic acid in Chuanbei Zhisou Mixture is relatively stable, and the retention time in the chromatogram is moderate and the separation degree is good, therefore, rosmarinic acid is selected as the reference substance, and the corresponding rosmarinic acid chromatographic peak in the subsequent is selected as the reference peak.

[0058] 2) Preparation of reference medicinal material solution: 15 g of Radix Asteris, 15 g of Perilla frutescens, 15 g of Platycodon grandiflorum, 50 g of Glycyrrhiza uralensis, 20 g of Rhizoma Pinelliae, and 10 g of Fritillaria cirrhosa were taken and prepared into single medicinal material solutions according to the process regulations, 10 mL of the single medicinal material solution was taken and placed in a container, an appropriate amount of methanol was added, shaken, filtered, and the filtrate was taken as the reference medicinal material solution.

[0059] 3) Preparation of test sample solution:

[0060] 10 ml of Chuanbei Zhisou Mixture was precisely transferred into a 20 ml volumetric flask, methanol was added to the calibration line, shaken, filtered, and the filtrate was taken as the test sample solution.

[0061] 4) Detection: 5 μL of the reference substance solution, the reference medicinal material solution, and the test sample solution were injected into the high performance liquid chromatograph, and the chromatographic conditions were as follows:

[0062] Column specification: C18 chromatographic column, 250 mm x 4.6 mm, 5 μm;

[0063] Mobile phase: the mobile phase comprises mobile phase A and mobile phase B; wherein A is acetonitrile, and the mobile phase B is a phosphoric acid solution with a volume concentration of 0.1%;

[0064] Flow rate: 1 mL / min;

[0065] Column temperature: 25℃;

[0066] Detection wavelength: 240 nm;

[0067] Injection volume: 5 μL;

[0068] Gradient elution procedure:

[0069] 0-10 min, the volume concentration of mobile phase A is increased from 5% to 12%, and the volume concentration of mobile phase B is decreased from 95% to 88%;

[0070] 10-20 min, the volume concentration of mobile phase A is increased from 12% to 20%, and the volume concentration of mobile phase B is decreased from 88% to 80%;

[0071] 20-30 min, the volume concentration of mobile phase A is increased from 20% to 22%, and the volume concentration of mobile phase B is decreased from 80% to 78%;

[0072] 30-35 min, the volume concentration of mobile phase A is increased from 22% to 25%, and the volume concentration of mobile phase B is decreased from 78% to 75%;

[0073] 35-60 min, the volume concentration of mobile phase A is increased from 25% to 55%, and the volume concentration of mobile phase B is decreased from 75% to 45%;

[0074] 60-65 min, the volume concentration of mobile phase A is decreased from 55% to 5%, and the volume concentration of mobile phase B is increased from 45% to 95%.

[0075] The high performance liquid chromatography determination results are as follows Figure 1 、 Figure 2 and Figure 3 .

[0076] III. Chromatogram establishment

[0077] 3.1 Selection of chromatographic conditions

[0078] 3.1.1 Investigation of mobile phase composition

[0079] The detection wavelength is the same as that in the preparation method of the test sample in “II”, and the fingerprint spectrum under different mobile phase compositions is investigated. The mobile phase compositions are methanol-water, acetonitrile-water and acetonitrile-0.1% phosphoric acid, respectively. The chromatogram of 120 min is recorded, and the results are shown in Figure 4 、 Figure 5 、 Figure 6 .

[0080] By Figures 4-6 It can be seen that when the mobile phase composition is methanol and water, the fingerprint baseline is not flat, when the mobile phase composition is acetonitrile and water, the fingerprint baseline is smoother than that of methanol, but the peak separation is not good, when the mobile phase composition is acetonitrile and 0.1% phosphoric acid, the fingerprint baseline is smooth and the peak separation is good, so the mobile phase composition is acetonitrile-0.1% phosphoric acid.

[0081] 3.1.2 Selection of detection wavelength

[0082] Except for the detection wavelength condition, the basic steps of chromatographic detection of the test sample are the same. The wavelength range of the diode array detector is 190-400 nm. By comparing the chromatograms of the sample at different wavelengths, it can be seen that when the wavelength is greater than 300 nm or less than 210 nm, the response value of the glycyrrhizic acid chromatographic peak is small, and when the wavelength is 260 nm, the response value of the chromatographic peak of the auxiliary sorbic acid potassium is too high. Finally, according to the principles of the number of chromatographic peaks, the response value of the chromatographic peak, whether the baseline is stable, etc., 240 nm is selected as the detection wavelength of the fingerprint of Chuanbei Zhicuo mixture. The results are shown in Figure 7 .

[0083] 3.1.3 Elution gradient investigation

[0084] Except for the elution gradient condition, the rest of the chromatographic conditions and the preparation of the test sample solution are the same as in "two". The gradient elution conditions are carried out according to Table 1, Table 2 and Table 3 respectively.

[0085] Table 1 Solvent elution program 1

[0086]

[0087] Table 2 Solvent elution program 2

[0088]

[0089] Table 3 Solvent elution program 3

[0090]

[0091]

[0092] Precisely take 5 μL of the test sample solution, inject it into the high performance liquid chromatograph, and record the chromatogram. The chromatogram is shown in Figures 8-10 According to the test results, it can be seen that when the elution program is 2#, the adjacent peaks of rosmarinic acid are not separated. When the elution program is 3#, the fingerprint baseline is not flat and most of the peaks are not separated, so the elution program 1# is selected as the elution condition of the fingerprint.

[0093] Four, optimization of test sample solution preparation conditions

[0094] 4.1 Selection of extraction solvent

[0095] 5ml, 10ml, 15ml of Chuanbei Zhicou Mixture were respectively taken out accurately and placed in 20ml volumetric flasks, methanol was added to the calibration line, shaken, filtered, and the filtrate was taken as the test sample solution. 5μL of each of the above three test sample solutions was respectively taken out accurately and injected into the high performance liquid chromatograph, and the chromatogram was recorded for 65min. The number of chromatographic peaks and peak area were used as evaluation indexes. The test results showed that the number of chromatographic peaks of each extraction solvent had no obvious difference. When 50% methanol was used for extraction, the peak area was moderate, and 50% methanol was finally determined as the extraction solvent.

[0096] 4.2 Selection of extraction time

[0097] 10ml of Chuanbei Zhicou Mixture was taken out accurately and placed in a 20ml volumetric flask, and methanol was added. The ultrasonic extraction (300W, 40KHz) for 0min, ultrasonic extraction (300W, 40KHz) for 15min and ultrasonic extraction (300W, 40KHz) for 30min were investigated, and the filtrate was taken as the test sample solution.

[0098] 5μL of each of the above three ultrasonic extraction test sample solutions was respectively taken out accurately and injected into the high performance liquid chromatograph, and the chromatogram was recorded for 65min. The number of chromatographic peaks and peak area were used as evaluation indexes for analysis. The test results showed that different ultrasonic times had no significant influence on the number of peaks and peak area, and the extraction time was finally determined to be 0min.

[0099] Based on the above test results, the preparation method of the Chuanbei Zhicou Mixture fingerprint test sample solution was determined as follows: 10ml of Chuanbei Zhicou Mixture was taken out accurately and placed in a 20ml volumetric flask, methanol was added to the calibration line, shaken, filtered, and the filtrate was taken as the test sample solution.

[0100] Five, methodological verification of the fingerprint establishment method

[0101] 5.1 Precision experiment

[0102] The precision experiment of the Chuanbei Zhicou Mixture fingerprint establishment method provided by the application was carried out, and the process was as follows:

[0103] Preparation of test sample solution: 10ml of Chuanbei Zhicou Mixture was taken out accurately and placed in a 20ml volumetric flask, methanol was added to the calibration line, shaken, filtered, and the filtrate was taken as the test sample solution. The test sample solution was injected into the instrument for 6 times in parallel, and the relative retention time and relative peak area of the main chromatographic peaks (accounting for more than 1% of the total peak area) were calculated, and the results were shown in Tables 4 and 5.

[0104] Table 4 Precision test results (relative retention time)

[0105]

[0106] Table 5 Precision Investigation Results (Relative Peak Area)

[0107]

[0108] 5.2 Solution Stability Test

[0109] The solution stability test was performed on the method for establishing the fingerprint spectrum of the Chuanbei Zhisou mixture provided by the present application, and the process was as follows:

[0110] Preparation of the test sample solution: 10 ml of the Chuanbei Zhisou mixture was accurately transferred into a 20 ml volumetric flask, methanol was added to the calibration line, shaken well, filtered, and the filtrate was taken, to obtain the test sample solution.

[0111] The test sample solution was sampled at 0 h, 2 h, 4 h, 8 h, 12 h, and 24 h, respectively, and the fingerprint chromatogram was recorded. A total of 6 time points were determined, and the relative retention time and relative peak area of the main chromatographic peaks (more than 1% of the total peak area) were calculated. The results are shown in Tables 6 and 7, and the results show that the test sample solution is stable under room temperature conditions within 24 hours.

[0112] Table 6 Stability Investigation Results (Relative Retention Time)

[0113]

[0114] Table 7 Stability Investigation Results (Relative Peak Area)

[0115]

[0116]

[0117] 5.3 Reproducibility Test

[0118] The reproducibility test was performed on the method for establishing the fingerprint spectrum of the Chuanbei Zhisou mixture provided by the present application, and the process was as follows:

[0119] Preparation of the test sample solution: 10 ml of the Chuanbei Zhisou mixture was accurately transferred into a 20 ml volumetric flask, methanol was added to the calibration line, shaken well, filtered, and the filtrate was taken, to obtain the test sample solution.

[0120] The test sample solution was sampled at 0 h, 2 h, 4 h, 8 h, 12 h, and 24 h, respectively, and the fingerprint chromatogram was recorded. A total of 6 time points were determined, and the relative retention time and relative peak area of the main chromatographic peaks (more than 1% of the total peak area) were calculated. The results are shown in Tables 6 and 7, and the results show that the test sample solution is stable under room temperature conditions within 24 hours.

[0121] The test sample solution was sampled at 0 h, 2 h, 4 h, 8 h, 12 h, and 24 h, respectively, and the fingerprint chromatogram was recorded. A total of 6 time points were determined, and the relative retention time and relative peak area of the main chromatographic peaks (more than 1% of the total peak area) were calculated. The results are shown in Tables 6 and 7, and the results show that the test sample solution is stable under room temperature conditions within 24 hours.

[0122] Table 8 Reproducibility Investigation Results (Relative Retention Time)

[0123]

[0124] Table 9 Repetitive investigation results (relative peak area)

[0125]

[0126] From Table 8 and Table 9, it can be seen that the RSD of relative retention time and the RSD of relative peak area are both less than 3%, indicating that the method is good in repeatability.

[0127] Six, establishment of fingerprint

[0128] 6.1 Fingerprint detection of Chuanbei Zhisou mixture

[0129] Take Chuanbei Zhisou mixture with batch numbers 240601, 240602, 240603, 240604, 240605, 240606, 240607, 240608, 240609, and 240610.

[0130] Preparation of multiple batches of test product solution: precisely transfer 10 ml of Chuanbei Zhisou mixture of the above 10 batches into a 20 ml volumetric flask, add methanol to the calibration line, shake well, filter, and take the filtered solution as the test product solution; 240601, 240602, 240603, 240604, 240605, 240606, 240607, 240608, 240609, and 240610.

[0131] Precisely pipette each batch of test product solution into a high-performance liquid chromatograph, perform gradient elution according to the provided chromatographic conditions, and record the chromatogram; see Table 10 for the results of one batch of test product solution. Figure 1 Calculate the relative retention time of the main chromatographic peaks (more than 1% of the total peak area) (see Table 10).

[0132] Table 10 Fingerprint determination results of 10 batches of Chuanbei Zhisou mixture (relative retention time)

[0133]

[0134] 6.2 Determination of common peaks and obtaining of control fingerprint

[0135] Export the AIA format of the fingerprint of 10 batches of Chuanbei Zhisou mixture from the instrument and import it into the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System, see Appendix Figure 11 , select the chromatographic peaks present in the fingerprints of 10 batches of Chuanbei Zhisou mixture as common peaks. Use the average value calculation method to generate the control fingerprint of Chuanbei Zhisou mixture, see Appendix Figure 12 , calculate the similarity of each common peak, and the results are shown in Table 11.

[0136] Table 11 Similarity results of 10 batches of Chuanbei Zhisou mixture fingerprint

[0137]

[0138] The above test results show that, with the reference fingerprint as the reference to calculate the similarity, the similarity of the fingerprint of 10 batches of Chuanbei Zhisou mixture is above 0.99, and therefore the fingerprint of Chuanbei Zhisou mixture is temporarily determined as follows: the test product spectrum should be consistent with the reference fingerprint, and according to the similarity evaluation system of traditional Chinese medicine chromatographic fingerprint, the similarity of the test product fingerprint and the reference fingerprint should not be lower than 0.90 after similarity calculation.

[0139] The above only describes the embodiments of the present application, and the protection scope of the present application is not limited by these specific embodiments, but is determined by the claims of the present application. Various modifications and changes can be made to the present application by those skilled in the art. Any modification, equivalent replacement, improvement, etc. within the technical thought and principle of the present application should be included in the protection scope of the present application.

Claims

1. A method for establishing the high performance liquid chromatography fingerprint of Chuanbei Zhisou mixture, characterized in that, It comprises the following steps: (1) Preparation of test solution: Take a certain volume of Chuanbei Zhisou mixture into a container, add an appropriate amount of methanol for dilution, shake well, filter, and take the subsequent filtrate as the test solution; (2) Preparation of reference solution: Take a certain weight of rosmarinic acid, ammonium glycyrrhizinate, p-hydroxybenzoic acid and coffee acid, respectively, into a volumetric flask, add an appropriate amount of methanol and ultrasonic treatment, cool, shake well, filter, and then the reference solution is obtained; wherein, rosmarinic acid is used as the reference substance; (3) Preparation of control medicinal material solution: Take a certain weight of purple root, perilla leaf, platycodon, licorice, clear pinellia and fritillaria, respectively, and prepare single medicinal material solution according to the process procedure, take a certain volume of single medicinal material solution into a container, add an appropriate amount of methanol and shake well, filter, and take the subsequent filtrate as the control medicinal material solution; (4) High performance liquid chromatography determination: Take an appropriate amount of test solution, reference solution and control medicinal material solution into a high performance liquid chromatograph, and record the chromatogram; analyze the high performance liquid chromatograms of multiple batches of test solution to obtain the control fingerprint of Chuanbei Zhisou mixture, and calculate the relative retention time of each common peak; The chromatographic conditions of the high performance liquid chromatograph are as follows: The mobile phase comprises mobile phase A and mobile phase B, mobile phase A is acetonitrile, and mobile phase B is a 0.1% phosphoric acid solution by volume concentration; the gradient elution program is as follows: 0-10 min, the volume concentration of mobile phase A increases from 5% to 12%, and the volume concentration of mobile phase B decreases from 95% to 88%; 10-20 min, the volume concentration of mobile phase A increases from 12% to 20%, and the volume concentration of mobile phase B decreases from 88% to 80%; 20-30 min, the volume concentration of mobile phase A increases from 20% to 22%, and the volume concentration of mobile phase B decreases from 80% to 78%; 30-35 min, the volume concentration of mobile phase A increases from 22% to 25%, and the volume concentration of mobile phase B decreases from 78% to 75%; 35-60 min, the volume concentration of mobile phase A increases from 25% to 55%, and the volume concentration of mobile phase B decreases from 75% to 45%; 60-65 min, the volume concentration of mobile phase A decreases from 55% to 5%, and the volume concentration of mobile phase B increases from 45% to 95%; In the above chromatographic conditions, the column specifications are Agilent Eclipse Plus C18, 250mm×4.6mm, 5μm; the flow rate is 1mL / min; the column temperature is 25℃; the detection wavelength is 240nm; and the injection amount is 5μL.

2. The method for establishing the HPLC fingerprint of the Chuanbei Zhisu mixture according to claim 1, characterized in that, The preparation of the test solution is as follows: accurately transfer 10ml of Chuanbei Zhisou mixture into a 20ml volumetric flask, add methanol to the calibration line, shake well, filter, and take the subsequent filtrate.

3. The method for establishing the HPLC fingerprint of the Chuanbei Zhisu mixture according to claim 1, characterized in that, The control fingerprint of Chuanbei Zhisou mixture comprises 16 common peaks, peaks 7, 8, 14, 15 and 16 are the characteristic peaks of licorice, peak 10 is the characteristic peak of purple root.

4. The method for establishing the HPLC fingerprint of the Chuanbei Zhisu mixture according to claim 3, characterized in that, Peak 13 is rosmarinic acid, which is used as the reference peak, peak 2 is p-hydroxybenzoic acid, peak 3 is coffee acid, and peak 16 is ammonium glycyrrhizinate.

5. The method for establishing the HPLC fingerprint of the Chuanbei Zhisu mixture according to claim 3 or 4, characterized in that, The relative standard deviations RSD of the relative retention times of the 16 common peaks are all less than 3%. The similarity of the Fingerprint of Chuanbei Zicou Mixture was calculated by taking the Fingerprint of Chuanbei Zicou Mixture as the reference, and the similarity was above 0.9.

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