A method for establishing HPLC fingerprint of laryngopharyngeal clearing

Abstract

The present application provides a method for establishing a HPLC fingerprint of laryngopharyngeal clear, which has the characteristics of integrity, fuzziness, strong characteristic, etc., can comprehensively reflect the relationship between the chemical components of traditional Chinese medicine, and show the complete picture of the internal relationship of traditional Chinese medicine. It is a feasible mode for quality control of traditional Chinese medicine compound preparations. In addition, the HPLC fingerprint is used to control the quality of laryngopharyngeal clear. The advantages are that compared with the thin layer scanning method, the high performance liquid chromatograph has the advantages of high precision and accuracy, good stability, low cost, etc., and the method of the present invention is simple for laryngopharyngeal clear sample processing, and does not require complex operations such as hydrolysis and extraction like the current "Chinese Pharmacopoeia" laryngopharyngeal clear quality control items.

Description

Technical Field

[0001] The present application relates to the field of medical technology, and in particular to a method for establishing a HPLC fingerprint of laryngopharyngeal clear fluid. Background Art

[0002] Pharyngitis is divided into acute pharyngitis and chronic pharyngitis. Acute pharyngitis is an acute inflammation of the pharyngeal mucosa and submucosal tissues. Pharyngeal lymphoid tissues are often involved. The inflammation may be localized in the early stage, and may often involve the entire pharyngeal cavity as the disease progresses. It is more common in autumn and winter and the turn of winter and spring. The main manifestations are dryness, burning, pain in the pharynx, obvious pain when swallowing, congestion and swelling of the pharynx, etc. Chronic pharyngitis can be divided into chronic simple pharyngitis, chronic hypertrophic pharyngitis and atrophic pharyngitis. Among them, chronic simple pharyngitis is more common. The lesions are mainly in the mucosal layer, manifested as chronic congestion of the pharyngeal mucosa, hyperplasia of the mucosal and submucosal connective tissue, hypertrophy of the mucous glands, hypersecretion, and increased mucus secretion. Patients often cough up sticky phlegm in the pharynx, or feel a foreign body sensation in the pharynx, which cannot be coughed up or swallowed. The course of the disease is long, prone to relapse, and the symptoms are stubborn.

[0003] The current quality standard of Houyanqing oral liquid in the "Chinese Pharmacopoeia" requires that the sample be hydrolyzed and extracted with petroleum ether, and then separated by thin layer chromatography using thin layer chromatography scanning to determine the content of oleanolic acid in the hydrolyzate. However, the component obtained by hydrolysis is the aglycone of multiple saponins, not a single component, so it is impossible to fully control the quality of the preparation. In addition, the quantitative experiment using thin layer chromatography scanning is not good in terms of accuracy, stability or precision. The use of thin layer chromatography scanning for quantitative research is in a state of being gradually cancelled. In addition, the current method of determining the content of oleanolic acid by hydrolysis is aglycone of multiple saponins, not a single active indicator component of Houyanqing, so it is impossible to accurately control the quality of Houyanqing. Summary of the invention

[0004] The present application aims to solve the problem that the existing method determines the content of oleanolic acid in the hydrolyzate by thin layer chromatography scanning, but the component obtained by hydrolysis is the aglycone of multiple saponins, rather than a single component, so it is impossible to comprehensively control the quality of the preparation. Therefore, the quality control standard has the problem that the accuracy, stability and precision are not ideal. The present application provides a method for quality control of laryngopharyngeal clear with simple processing, high precision and accuracy, good stability and low cost, and a method for establishing HPLC fingerprint of laryngopharyngeal clear that provides a reference method for different batches of laryngopharyngeal clear in terms of quality control and overall evaluation.

[0005] A method for establishing a HPLC fingerprint of laryngopharyngeal clear liquid comprises the following steps:

[0006] S1. Preparation of test solution: accurately weigh laryngeal and pharyngeal clear fluid, add methanol solution for ultrasonic extraction, dilute to volume, mix thoroughly, and filter using a 0.45 μm microporous filter membrane to obtain the test solution;

[0007] S2. Preparation of reference solution: accurately weigh reference substances chlorogenic acid, ferulic acid, psyllium glycoside, β-ecdysterone, R-hyssostadone, S-hyssostadone, isochlorogenic acid A, and isochlorogenic acid C, and use methanol as solvent to prepare a mixed reference solution with a chlorogenic acid concentration of 0.11 mg / mL, a ferulic acid concentration of 0.30 mg / mL, a psyllium glycoside concentration of 0.27 mg / mL, a β-ecdysterone concentration of 0.08 mg / mL, a R-hyssostadone concentration of 0.05 mg / mL, a S-hyssostadone concentration of 0.06 mg / mL, a isochlorogenic acid A concentration of 0.35 mg / mL, and a isochlorogenic acid C concentration of 0.31 mg / mL;

[0008] S3. Chromatographic conditions: The chromatographic column is a C18 reverse phase column; the column temperature is 20-40°C, the detection wavelength is 250nm, the mobile phase A is acetonitrile, the mobile phase B is a 0.1% volume concentration of phosphoric acid aqueous solution, the total flow rate is 0.8-1mL / min; gradient elution is performed;

[0009] S4, determination: aspirate 20 μL of the test solution and the reference solution, inject them into the high performance liquid chromatograph, use the high performance liquid chromatography detection of step S3, match 24 common peaks, determine 8 characteristic peaks, and establish the laryngeal and pharyngeal clear fingerprint.

[0010] Preferably, in the method for preparing the test solution in step S1, the ratio of the test laryngeal clear solution to methanol is 1:10.

[0011] Preferably, in the method for preparing the test solution in step S1, the power of ultrasonic extraction is 250 W, the frequency is 40 kHz, and the extraction time is 1 hour.

[0012] Preferably, the preparation method of the reference substance solution in step S2 specifically includes: accurately weighing the reference substances chlorogenic acid, ferulic acid, plantain glycosides, β-ecdysterone, R-cyathosterone, S-cyathosterone, isochlorogenic acid A, and isochlorogenic acid C, using methanol as solvent, ultrasonically treating at a frequency of 40 kHz for 3 minutes, mixing and filtering with a 0.45 μm microporous filter membrane to obtain a mixed reference substance solution.

[0013] Preferably, the gradient elution described in the chromatographic conditions of step S3 is performed with the following volume concentration configuration:

[0014] From 0 to 8 min, mobile phase A was 10% acetonitrile solution;

[0015] At 8-25 min, mobile phase A was 10-13% acetonitrile solution;

[0016] At 25-35 min, mobile phase A was 13-15% acetonitrile solution;

[0017] At 35-50 min, mobile phase A was 15-20% acetonitrile solution;

[0018] At 50-55 min, mobile phase A was 20% acetonitrile solution;

[0019] At 55-75 min, mobile phase A was 20-25% acetonitrile solution;

[0020] At 75-85 min, mobile phase A was 25% acetonitrile solution;

[0021] At 90 min, mobile phase A was 10% acetonitrile solution.

[0022] Preferably, the chromatographic column described in the chromatographic conditions of step S3 has a column length of 150 mm, an inner diameter of 4.6 mm, and a particle size of 5 μm; the chromatographic column has a column length of 100 mm, an inner diameter of 2.1 mm, and a particle size of 1.7 μm.

[0023] Preferably, the eight characteristic peaks in step S4 are: peak 6 chlorogenic acid, peak 13 ferulic acid, peak 14 plantagoside, peak 15 β-ecdysterone, peak 17 R-cyperasterone, peak 18 S-cyperasterone, peak 20 isochlorogenic acid A and peak 22 isochlorogenic acid C; wherein peak 15 is a reference peak.

[0024] Preferably, the relative retention time tR of the No. 6 peak chlorogenic acid is 0.382;

[0025] The relative retention time tR of the peak 13 ferulic acid is 0.915;

[0026] The relative retention time tR of the No. 14 peak psyllium is 1.000;

[0027] The relative retention time tR of the peak No. 15 β-ecdysterone is 1.017;

[0028] The relative retention time tR of the peak No. 17, R-hydnosterone, is 1.063;

[0029] The relative retention time tR of the peak No. 18 S-hyacinthosterone is 1.092;

[0030] The relative retention time tR of the peak No. 20 isochlorogenic acid A is 1.260;

[0031] The relative retention time tR of the peak No. 22, isochlorogenic acid C, is 1.393.

[0032] Preferably, in the method for preparing the test sample in step S1, the Houyanqing is any one of Houyanqing granules, Houyanqing granules (without sucrose) or Houyanqing oral liquid.

[0033] Preferably, the preparation method of the test sample in step S1 specifically comprises the following steps: taking the laryngopharyngeal clearing granules or laryngopharyngeal clearing granules (without sucrose) under the item of filling amount difference, mixing, grinding, accurately weighing according to the proportion, placing in a stoppered conical flask, accurately adding methanol, sealing, weighing, ultrasonically treating for 1 hour, cooling, weighing again, supplementing the lost weight with methanol, shaking, filtering, and taking the filtrate to obtain.

[0034] Accurately measure the throat and pharynx clearing oral liquid in proportion, put it in a container, add methanol, shake well, centrifuge, take the supernatant, filter, and take the filtrate to obtain the product.

[0035] Compared with the prior art, the beneficial effects of this application are as follows:

[0036] The present application provides a method for establishing a HPLC fingerprint of laryngopharyngeal clear, which has the characteristics of integrity, fuzziness, strong characteristic, etc., can comprehensively reflect the relationship between the chemical components of traditional Chinese medicine, and show the complete picture of the internal relationship of traditional Chinese medicine. It is a feasible mode for quality control of traditional Chinese medicine compound preparations. In addition, the HPLC fingerprint is used to control the quality of laryngopharyngeal clear. The advantages are that compared with the thin layer scanning method, the high performance liquid chromatograph has the advantages of high precision and accuracy, good stability, low cost, etc., and the method of the present invention is simple for laryngopharyngeal clear sample processing, and does not require complex operations such as hydrolysis and extraction like the current "Chinese Pharmacopoeia" laryngopharyngeal clear quality control items. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following briefly introduces the drawings required for describing the embodiments.

[0038] Figure 1 is the blank solvent chromatogram;

[0039] Figure 2-A It is the superimposed fingerprint of 16 batches of laryngopharyngeal clear;

[0040] Figure 2-B It is a comparison chart of 16 batches of laryngopharyngeal clear;

[0041] Figure 3 is the mixed control standard chromatogram;

[0042] Figure 4 It is the total ion current chromatogram (TIC) of UPLC-Q-TOF-MS in the laryngeal and pharyngeal clear negative ion mode;

[0043] Figure 5is the total ion current chromatogram (TIC) of UPLC-Q-TOF-MS in negative ion mode of mixed reference standard;

[0044] Figure 6 It is the instrument precision chromatogram;

[0045] Figure 7 is the sample stability chromatogram;

[0046] Figure 8 is the sample repeatability chromatogram;

[0047] Fig. 9 It is the chromatogram of mixed reference solution;

[0048] Fig.10 This is the HPLC standard fingerprint of the throat and throat clearing oral solution of this application;

[0049] Fig.11 It is the common peak retention time and peak area of ​​16 batches of throat and pharynx clear fingerprints;

[0050] Fig.12 This is the similarity evaluation result of 16 batches of laryngopharyngeal clear fingerprints. DETAILED DESCRIPTION

[0051] The present invention will be further described below in conjunction with specific embodiments and accompanying drawings:

[0052] 1. Chromatographic conditions

[0053] Shimadzu HPLC instrument (LC-20A binary high pressure gradient instrument, SPD-20A UV detector), chromatographic column is Agilent-C18 column (250mm×4.6mm, 5μm); flow rate is 1.0mL / min; detection wavelength is 250nm; column temperature is 30℃; injection volume is 20μL; mobile phase A is 0.1% phosphoric acid water, mobile phase B is acetonitrile, and the gradient elution program of laryngeal and pharyngeal clear fluid is shown in Table 1.

[0054] Table 1 Laryngopharyngeal clear gradient elution procedure

[0055]

[0056] 2. Sample preparation

[0057] 1) Preparation of mixed reference substances

[0058] Accurately weigh 1.1 mg of chlorogenic acid reference substance, 3.0 mg of ferulic acid reference substance, 2.7 mg of plantagoside reference substance, 0.80 mg of β-ecdysterone reference substance, 0.50 mg of R-cyathosterone reference substance, 0.60 mg of S-cyathosterone reference substance, 3.5 mg of isochlorogenic acid A, and 3.1 mg of isochlorogenic acid C, place in a 10 mL volumetric flask, add methanol to make up to volume, mix by ultrasonication for 3 min, and pass through a 0.45 μm microporous filter membrane to obtain a mixed reference solution.

[0059] 2) Preparation of test solution

[0060] Take an appropriate amount of laryngopharyngeal clearing granules, grind them into powder with a mortar, accurately weigh 1.00 g and place it in a stoppered conical flask, add 10 mL of methanol, cover the flask and seal it with a sealing film, weigh the mass, extract it with ultrasound at 40 kHz for 1 h, weigh the mass, make up the lost mass with methanol, shake well and filter it through a 0.45 μm microporous filter membrane to obtain the product.

[0061] Example 1

[0062] 1. Blank control experiment

[0063] Take the methanol solution, pass it through a 0.45μm microporous filter membrane, and determine the chromatogram according to the chromatographic conditions in step 1). The test results are as follows: Figure 1 shown.

[0064] 2. Establishment of the common pattern of laryngopharyngeal clear fingerprint

[0065] Take 16 batches of Houyanqing granules, prepare the test solution according to the preparation method in step 2), and measure according to the chromatographic conditions in step 1). Use the Chinese medicine chromatographic fingerprint similarity evaluation system software (2012 edition), take the sample spectrum of batch number S9 (20220205) as the reference spectrum, use the average method, and the time window width is 0.5 to obtain the superimposed fingerprints of 16 batches of Houyanqing. Figure 2-A , and its reference spectrum is shown in 2-B; through software analysis, chromatographic peaks with good separation, stable retention time and peak area were selected, and common peaks were determined by comparing the retention time of each peak between different batches. HPLC chromatograms of 16 batches of laryngeal pharyngeal clear finally determined 24 common peaks, such as Figure 2-B The retention time and peak area of ​​the common peaks of different batches of samples are shown in Fig.11 The retention time of each common peak is stable and has fingerprint characteristics, which can be preliminarily proposed as the index component group of laryngopharyngeal clear. Fig.11 The results show that the RSD values ​​of the common peak retention time of the chromatograms of the throat and throat clear samples from different batches are all less than 3%, which meets the technical requirements of the fingerprint spectrum. The above 24 common peaks can be identified as stable characteristic peaks of the throat and throat clear fingerprint spectrum. However, the peak areas of the chromatographic peaks between different batches have large differences, and the RSD values ​​of the common peak areas are between 28% and 69%.

[0066] Characteristic peak identification

[0067] Using the mixed control chromatogram ( Figure 3 ) and laryngeal pharyngeal clear fingerprint chromatogram ( Figure 2-B ) were compared, and the target peak was verified by combining ultra high performance liquid chromatography-mass spectrometry (UPLC-Q-TOF-MS) technology.

[0068] UPLC-Q-TOF-MS chromatographic conditions: Waters UHPLC BEHC18 (100

[0069] mm×2.1mm, 1.7µm); the mobile phase was 0.1% formic acid water (A) and acetonitrile (B), gradient elution, elution program: 0~2.5min, 10%B; 2.5~8.5min, 10%~13%B; 8.5~12min, 13%~15%B; 12~17min, 15%~20%B; 17~18.5min, 20%B; 18.5~25min, 20%~25%B; 25~28min, 25%B; 28~29min, 25%~90%B; 29~30min, 90%~10%B. Column temperature 35℃, flow rate 0.3mL / min, injection volume 2μL.

[0070] The UPLC-Q-TOF-MS mass spectrometry conditions are as follows: XevoG2-SQTOFESI ion source, ionization mode is negative ion mode (ESI-), negative ion capillary voltage is 3.0 kV, sample cone voltage is 40 V, ion source temperature is 120 ° C, desolvation temperature is 400 ° C, nebulizer gas temperature is 400 ° C, cone gas volume flow rate is 50 L / h, solvent volatilization volume flow rate is 800 L / h, and mass scanning range is m / z50-1200. In negative ion mode, the low collision energy is 20 eV and the high collision energy is 50 eV. The liquid-mass spectrometry system is controlled by Masslynx4.1 software, and data acquisition and spectral processing are performed. The UPLC-Q-TOF-MS total ion current chromatograms (TIC) of laryngeal clear and mixed control standards in negative ion mode are shown in the figure below. Figure 4 , Figure 5 .

[0071] By comparing the UPLC-Q-TOF-MS results of laryngopharyngeal serum with those of mixed control standards, as shown in Table 2, 8 characteristic peaks were finally identified, and the results are shown in Table 3.

[0072] Table 2 Identification of chemical components of laryngeal and pharyngeal clear solution in negative ion mode

[0073]

[0074] Table 3 Identification of characteristic peaks of laryngeal and pharyngeal clear

[0075]

[0076] Similarity evaluation

[0077] The fingerprints of 16 batches of Houyanqing were imported into the Chinese medicine chromatographic fingerprint similarity evaluation system software (2012 version) to calculate the similarity. The similarity evaluation results are shown in Fig.12 .

[0078] According to the similarity evaluation results of 16 batches of throat and pharynx clear fingerprints, it can be seen that compared with the control spectrum R, the similarity of each batch of throat and pharynx clear samples is above 0.89, and the highest similarity reaches 0.992. Among them, the number of samples with a similarity greater than 0.9 is 14, accounting for 87.5% of the total number. It can be preliminarily determined that the quality of each batch of samples is similar. The similarities of S11 and S16 samples are 0.893 and 0.896 respectively, and the similarity is close to 0.9. The reason for the reduction in similarity may be related to the batch differences caused by the origin, harvesting time, storage and transportation of each medicinal material in the throat and pharynx clear compound.

[0079] Precision test results and analysis

[0080] Take Houyanqing Granules (sample 9), prepare the sample according to the preparation method in step 2), and inject the sample 6 times continuously according to the "chromatographic conditions". The results show that the peak area RSD of the common peak is less than 3%, and the retention time RSD is less than 2%. The instrument precision experiment chromatogram is shown in Figure 6 , the results are shown in Table 4.

[0081] Table 4 Total peak retention time and peak area in laryngeal semen density experiment (n=6)

[0082]

[0083] The chromatograms of six consecutive injections were evaluated for similarity. The similarity evaluation results are shown in Table 5. The results show that the similarities are all greater than 0.99, indicating that the instrument has good precision.

[0084] Table 5 Similarity of laryngopharyngeal clearance density maps (n=6)

[0085]

[0086] Sample stability test results and analysis

[0087] Take Houyanqing Granules (Sample 9), prepare the same test solution (S9) according to the "Preparation of Test Solution", inject samples at 0, 2, 4, 8, 10, and 24 hours, and measure the samples according to the "Chromatographic Conditions". Calculate the RSD values ​​of the peak area and retention time of the common peak. The results show that the peak area RSD of the common peak is less than 3%, and the retention time RSD is less than 2%. The chromatogram of the sample stability experiment is shown in Figure 7, the results are shown in Table 6.

[0088] Table 6 Total peak retention time and peak area of ​​laryngopharyngeal clearance stability test (n=6)

[0089]

[0090] The obtained chromatogram was imported into similarity evaluation software for evaluation, and the results showed that the similarity was greater than 0.99. The results are shown in Table 7, indicating that the laryngopharyngeal clearing test solution had good stability when placed at room temperature for 24 hours.

[0091] Table 7 Similarity of laryngopharyngeal clearance stability profiles (n=6)

[0092]

[0093] Repeatability test results and analysis

[0094] Take the same batch of samples (sample 9), prepare 6 test solutions in parallel according to the "Preparation of test solution", inject and measure according to the "Chromatographic conditions", and calculate the peak area and retention time of the common peak. The results show that the peak area RSD of the common peak is less than 3%, and the retention time RSD is less than 3%. The chromatogram of the sample repeatability experiment is shown in Figure 8 , the results are shown in Table 8.

[0095] Table 8 Total peak retention time and peak area of ​​laryngopharyngeal clear repeatability experiment (n=6)

[0096]

[0097] The sample repeatability experimental chromatograms were imported into the similarity evaluation software for evaluation. The results showed that the similarity was greater than 0.99. The results are shown in Table 9, indicating that the method has good repeatability.

[0098] Table 9 Similarity of laryngopharyngeal clear repetitive patterns (n=6)

[0099]

[0100] Example 2

[0101] 1) Preparation of test solution: Take throat and pharynx clearing oral solution, shake well and filter through a 0.45 μm microporous filter membrane to obtain 10 batches of throat and pharynx clearing oral solution provided by Hunan Times Sunshine Pharmaceutical Co., Ltd. Acetonitrile was chromatographic grade, water was purified water, and the other reagents were of analytical grade.

[0102] Fingerprint establishment

[0103] The reference solution prepared in step 2 and the sample solution prepared in step 1) were respectively injected into the HPLC chromatography system, and the HPLC fingerprints of 10 batches of Houyanqing oral liquid were measured and analyzed and compared to obtain the HPLC standard fingerprint of Houyanqing oral liquid composed of their common characteristic peaks. Fig. 9 As shown in the figure, the HPLC standard fingerprint of Houyanqing oral liquid is as follows Fig.10 As shown, taking β-ecdysterone No. 15 as the reference peak, the relative retention times tR of the common peaks in the standard fingerprint were calculated to be 0.143, 0.204, 0.250, 0.307, 0.363, 0.382, 0.436, 0.454, 0.485, 0.506, 0.709, 0.732, 0.915, 1.000, 1.017, 1.036, 1.063, 1.092, 1.197, 1.260, 1.353, 1.393, 1.519, and 1.646, respectively.

[0104] Traditional Chinese medicine fingerprints have the characteristics of integrity, fuzziness, and strong characteristics. They can fully reflect the relationship between the chemical components of traditional Chinese medicines and show the complete picture of the internal relationship of traditional Chinese medicines. They are currently a feasible model for quality control of traditional Chinese medicine compound preparations. In addition, the HPLC fingerprint is used to control the quality of laryngopharyngeal clear. The advantages are that compared with the thin layer scanning method, the high performance liquid chromatography has the advantages of high precision and accuracy, good stability, low cost, etc., and the method of the present invention is simple to process laryngopharyngeal clear samples, and does not require complex operations such as hydrolysis and extraction as in the current "Chinese Pharmacopoeia" laryngopharyngeal clear quality control items.

[0105] This application is to establish the HPLC fingerprint of laryngeal pharyngeal clear fluid by reversed phase high performance liquid chromatography-ultraviolet detector. In theory, it can also be determined by ultra-high performance liquid chromatography-mass spectrometry (UPLC-Q-TOF-MS), reversed phase ultra-high performance liquid chromatography (UPLC) chromatography-ultraviolet detector, etc. In comparison, this design has the advantages and advantages of low instrument and equipment price, universal applicability (reverse phase high performance liquid chromatography-ultraviolet detector is the standard and conventional equipment of pharmaceutical companies), and low cost.

[0106] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for establishing a HPLC fingerprint of laryngopharyngeal clear, characterized in that: The following steps are involved: S1. Preparation of test solution: accurately weigh laryngeal and pharyngeal clear fluid, add methanol solution for ultrasonic extraction, dilute to volume, mix thoroughly, and filter using a 0.45 μm microporous filter membrane to obtain the test solution; S2. Preparation of reference solution: accurately weigh reference substances chlorogenic acid, ferulic acid, psyllium glycoside, β-ecdysterone, R-hyssostadone, S-hyssostadone, isochlorogenic acid A, and isochlorogenic acid C, and use methanol as solvent to prepare a mixed reference solution with a chlorogenic acid concentration of 0.11 mg / mL, a ferulic acid concentration of 0.30 mg / mL, a psyllium glycoside concentration of 0.27 mg / mL, a β-ecdysterone concentration of 0.08 mg / mL, a R-hyssostadone concentration of 0.05 mg / mL, a S-hyssostadone concentration of 0.06 mg / mL, a isochlorogenic acid A concentration of 0.35 mg / mL, and a isochlorogenic acid C concentration of 0.31 mg / mL; S3. Chromatographic conditions: The chromatographic column is a C18 reverse phase column; the column temperature is 20-40°C, the detection wavelength is 250nm, the mobile phase A is acetonitrile, the mobile phase B is a 0.1% volume concentration of phosphoric acid aqueous solution, the total flow rate is 0.8-1mL / min; gradient elution is performed; S4, determination: aspirate 20 μL of the test solution and the reference solution, inject them into the high performance liquid chromatograph, use the high performance liquid chromatography of step S3 to detect, match 24 common peaks, determine 8 characteristic peaks, and establish the throat and pharynx clear fingerprint spectrum; The gradient elution described in the chromatographic conditions of step S3 is performed with the following volume concentration configuration: 。 2. The method for establishing the HPLC fingerprint of laryngopharyngeal clearing according to claim 1, characterized in that: In the method for preparing the test solution in step S1, the ratio of the test laryngeal clear solution to methanol is 1:

10.

3. The method for establishing the HPLC fingerprint of laryngopharyngeal clearing according to claim 1, characterized in that: In the method for preparing the test solution in step S1, the power of ultrasonic extraction is 250 W, the frequency is 40 kHz, and the extraction time is 1 hour.

4. The method for establishing the HPLC fingerprint of laryngopharyngeal clearing according to claim 1, characterized in that: The preparation method of the reference substance solution in step S2 specifically includes: accurately weighing the reference substances chlorogenic acid, ferulic acid, plantain glycosides, β-ecdysterone, R-cyathosterone, S-cyathosterone, isochlorogenic acid A, and isochlorogenic acid C, using methanol as solvent, ultrasonicating at a frequency of 40 kHz for 3 minutes, mixing well, and filtering with a 0.45 μm microporous filter membrane to obtain a mixed reference substance solution.

5. The method for establishing the HPLC fingerprint of laryngopharyngeal clearing according to claim 1, characterized in that: The chromatographic column described in the chromatographic conditions of step S3 has a column length of 250 mm, an inner diameter of 4.6 mm, and a particle size of 5 μm.

6. The method for establishing the HPLC fingerprint of laryngopharyngeal clearing according to claim 1, characterized in that: The eight characteristic peaks in step S4 are: peak 6 chlorogenic acid, peak 13 ferulic acid, peak 14 plantagoside, peak 15 β-ecdysterone, peak 17 R-cyperasterone, peak 18 S-cyperasterone, peak 20 isochlorogenic acid A and peak 22 isochlorogenic acid C; wherein peak 15 is a reference peak.

7. The method for establishing the HPLC fingerprint of laryngopharyngeal clearing according to claim 6, characterized in that: The relative retention time tR of the chlorogenic acid of peak No. 6 is 0.382; The relative retention time tR of the peak 13 ferulic acid is 0.915; The relative retention time tR of the No. 14 peak psyllium is 1.000; The relative retention time tR of the peak No. 15 β-ecdysterone is 1.017; The relative retention time tR of the peak No. 17, R-hydnosterone, is 1.063; The relative retention time tR of the peak No. 18 S-hyacinthosterone is 1.092; The relative retention time tR of the peak No. 20 isochlorogenic acid A is 1.260; The relative retention time tR of the peak No. 22, isochlorogenic acid C, is 1.

393.

8. The method for establishing the HPLC fingerprint of laryngopharyngeal clearing according to claim 1, characterized in that: In the method for preparing the test sample in step S1, the throat clearing is any one of the throat clearing granules, sucrose-free throat clearing granules or throat clearing oral liquid.

9. The method for establishing the HPLC fingerprint of laryngopharyngeal clearing according to claim 8, characterized in that: The preparation method of the test sample in step S1 specifically comprises the following steps: taking the throat clearing granules or the throat clearing granules without sucrose under the item of filling amount difference, mixing, grinding, accurately weighing according to the proportion, placing in a stoppered conical bottle, accurately adding methanol, plugging, weighing, ultrasonic treatment for 1 hour, cooling, weighing again, supplementing the lost weight with methanol, shaking, filtering, and taking the filtrate to obtain; Or precisely measure the throat and pharynx clearing oral liquid in proportion, put it in a container, add methanol, shake well, centrifuge, take the supernatant, filter, and take the filtrate to obtain the product.

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