A method for constructing a characteristic spectrum of a nasal congestion relieving rhinitis tablet preparation and a multi-index component detection method

Abstract

The application provides a characteristic spectrum construction method and multi-index component detection method of Tongqiao Biyan tablet preparation, and relates to the technical field of drug analysis structure.The standard characteristic spectrum construction method of Tongqiao Biyan tablet preparation provided by the application comprehensively characterizes the internal quality of Tongqiao Biyan tablet preparation, simultaneously determines the main index components (chlorogenic acid, verbascoside, imperatorin and isoimperatorin) related to drug efficacy in the standard characteristic spectrum, covers the components (xanthium, astragalus and radix angelicae alba) occupying the main efficacy position in the two paths of extraction and crushing, and perfects the excessively simple present execution quality standard of Tongqiao Biyan tablet preparation.The precision, stability and reproducibility of the characteristic spectrum method of Tongqiao Biyan tablet preparation are all good, the quality of Tongqiao Biyan tablet preparation can be more comprehensively controlled, so that the quality stability, consistency and controllability of the preparation can be better ensured.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of pharmaceutical analysis structure technology, and particularly relates to a method for constructing a characteristic chromatogram of a nasal congestion relieving tablet preparation and a method for detecting multiple index components. BACKGROUND

[0002] Chinese patent medicine is a kind of Chinese medicine preparation which uses Chinese medicine as medicinal materials and is based on the theory of traditional Chinese medicine. It is approved by relevant management departments for mass production and can be used for tonifying the body and preventing and treating diseases. However, the quality problem of Chinese patent medicine is an important reason for hindering its development. With the development of detection technology, the quality of Chinese patent medicine is evaluated and analyzed. In the process of comprehensively evaluating the quality and curative effect of Chinese medicine compound, the application of chromatographic characteristic chromatogram has attracted special attention. The source of characteristic chromatogram of Chinese medicine is developed from the concept of forensic identification. It is a characteristic chromatogram of multiple components in Chinese medicine expressed in the form of figures, tables and data by using modern analysis methods. It is a comprehensive and quantifiable identification mode with the characteristics of integrity and fuzziness. Its application control range is very wide, from the planting of Chinese medicinal materials, the processing of medicinal slices to the production process of Chinese patent medicine and the quality control of finished products. The widespread use of high performance liquid chromatography (HPLC) technology, the conventional quality control method of Chinese patent medicine usually controls a certain component or index component of a certain medicine (such as ministerial medicine and auxiliary medicine) in the prescription monarch medicine. However, this single index quantitative control cannot meet the characteristics of multiple components and multiple targets of Chinese patent medicine, and cannot fully reflect the quality status, so that the characteristic chromatogram has become the most commonly used chemical component information expression mode. The chromatographic characteristic chromatogram can reflect the changes of the chemical composition and quantity of Chinese medicine. The similarity evaluation of the characteristic chromatogram can accurately reflect the similarity of the contained components. The characteristic chromatogram technology of Chinese medicine can be used as a qualitative quality control method of Chinese patent medicine to characterize the overall quality of the sample.

[0003] The nasal congestion relieving tablet preparation has the effects of dispersing wind, consolidating the exterior, promoting the lung and unblocking the nasal passage. It is used for the treatment of nasal congestion which is sometimes severe and sometimes mild, nasal discharge of clear or turbid nasal discharge, and forehead headache caused by wind-heat accumulating in the lung and exterior deficiency. It is also used for the treatment of chronic rhinitis, allergic rhinitis and sinusitis with the above symptoms. The nasal congestion relieving tablet preparation has high curative effect and high market recognition. The nasal congestion relieving tablet preparation is composed of 7 kinds of Chinese medicines, including fried Xanthium sibiricum, prepared windproof plant, Astragalus membranaceus, radix angelicae dahuricae, lonicera, fried atractylodes and mint. Among them, Xanthium sibiricum is the monarch medicine which can disperse wind. Chlorogenic acid, as the main active substance of Xanthium sibiricum, has the effects of antibacterial, antiviral and anti-inflammatory. Astragalus membranaceus and radix angelicae dahuricae are the ministerial medicine. Astragalus membranaceus can tonify qi, raise yang, consolidate the exterior and stop sweating. Mucronulatol, as the main characteristic component of Astragalus membranaceus, has a hormone-like effect and plays a role in antibacterial and antiviral. Radix angelicae dahuricae can dispel wind and cold, unblock the nasal passage and relieve pain. The main components of radix angelicae dahuricae are imperatorin and isoimperatorin, which have the effects of antibacterial, antiasthmatic and anti-allergic. Prepared windproof plant and atractylodes are auxiliary medicine which can dispel wind, relieve the exterior, eliminate dampness and relieve pain. Atractylodes can tonify the spleen and kidney, consolidate the exterior and stop sweating. Mint can dispel wind and clear the head and eyes. The nasal congestion relieving tablet preparation.

[0004] The quality standard of Tongqiao Biyan tablet is Tongqiao Biyan tablet in the first part of Chinese Pharmacopoeia (2020 edition), which contains the characteristics, identification (fried atractylodes rhizome, white peony root control drug identification, astragaloside thin layer identification, xanthium sibiricum identification), imperatorin single component content determination, which cannot effectively control the internal quality of the product. In order to ensure the quality of the product, it is necessary to select reasonable and necessary control indicators and include them into the quality standard.

[0005] The existing technology (Zhang Qian, Wang Shixiang, Chen Yongyong, et al. Simultaneous determination of five components in Tongqiao Biyan tablet by RP-HPLC [J]. Journal of Northwest University (Natural Science Edition), 2015, 45(002): 253-256.) discloses that five components, including zanthoxylin, hydrated oxypeucedanin, white angelica root, imperatorin and isoeucommarin, are determined at the same time. Zanthoxylin is the component of the prepared wind-preventing medicinal material, and the other four components are the components of white peony root medicinal material. However, the above-mentioned existing technology only tests the prepared wind-preventing medicinal material and the subordinate white peony root, and the detection results are not comprehensive, which cannot effectively control the internal quality of Tongqiao Biyan tablet preparation. SUMMARY

[0006] Therefore, the purpose of the present application is to provide a characteristic spectrum construction method and a multi-index component detection method of Tongqiao Biyan tablet preparation. The method provided by the present application overcomes the problems that the existing quality standard of Tongqiao Biyan tablet preparation is single, there is no qualitative and quantitative detection item, and the quality of the product cannot be accurately reflected. The method can comprehensively and comprehensively reflect the internal quality of Tongqiao Biyan tablet preparation, and ensure the uniformity, stability, safety and effectiveness of the product quality.

[0007] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical scheme:

[0008] The present application provides a standard characteristic spectrum construction method of Tongqiao Biyan tablet preparation, comprising the following steps:

[0009] Mix the Tongqiao Biyan tablet preparation with a solvent to obtain a test solution; the solvent comprises methanol and / or water;

[0010] The test solution and the control solution are respectively detected by high performance liquid chromatography to obtain the test liquid chromatogram and the control liquid chromatogram, respectively;

[0011] The control solution comprises chlorogenic acid control solution, calycosin-7-glucoside control solution, isoeucommarin control solution and imperatorin control solution;

[0012] The conditions of the high performance liquid chromatography detection include: the column packing includes octadecylsilane bonded silica gel; the mobile phase system includes mobile phase A and mobile phase B, the mobile phase A is acetonitrile, and the mobile phase B is 0.05-0.3v / v% formic acid aqueous solution; the elution mode is gradient elution, and the gradient elution program includes: 0-18min, the volume fraction of the mobile phase A is increased from 5% to 14%; 18-33min, the volume fraction of the mobile phase A is increased from 14% to 28%; 33-50min, the volume fraction of the mobile phase A is increased from 28% to 57%; 50-70min, the volume fraction of the mobile phase A is 57%;

[0013] The sample liquid chromatogram is introduced into a traditional Chinese medicine chromatographic characteristic atlas similarity evaluation system to generate a characteristic atlas common mode, and a standard characteristic atlas containing 18 characteristic peaks is obtained; by comparing the retention time of the standard characteristic atlas with that of the control sample liquid chromatogram, the standard characteristic atlas includes the characteristic peaks of chlorogenic acid, verbascoside, isoimperatorin and imperatorin.

[0014] Preferably, the solid-liquid ratio of the Tongqiao Biyan tablet preparation and the solvent is 1g:2-100mL.

[0015] Preferably, the mixing includes ultrasonic mixing, the ultrasonic mixing has a power of 300-6000W, a frequency of 30-50kHz, and a time of 10-60min.

[0016] Preferably, after the mixing, the obtained ultrasonic system is further filtered.

[0017] Preferably, the concentrations of the chlorogenic acid control sample solution, the verbascoside control sample solution and the isoimperatorin control sample solution are independently 30-50μg / mL, and the concentration of the imperatorin control sample solution is 10-30μg / mL.

[0018] Preferably, the mobile phase flow rate of the high performance liquid chromatography detection is 0.5-1.5mL / min, the detection wavelength is 220-350nm, the column temperature is 20-40℃, the theoretical plate number calculated according to imperatorin is ≥3000, and the injection amount is 5-20μL.

[0019] Preferably, the sample liquid chromatogram and the control sample liquid chromatogram are calculated by a traditional Chinese medicine chromatographic characteristic atlas similarity evaluation system software, and the similarity is all >0.9.

[0020] The application also provides a multi-index component detection method of the Tongqiao Biyan tablet preparation, which includes the following steps:

[0021] The Tongqiao Biyan tablet preparation to be detected is mixed with a solvent to obtain a sample liquid to be detected; the solvent includes methanol and / or water;

[0022] The sample liquid is detected by high performance liquid chromatography to obtain the detection results of multiple index components of the Tongqiao Biyan tablet preparation; the multiple index components include chlorogenic acid, verbascoside, imperatorin and isoimperatorin;

[0023] The conditions of the high performance liquid chromatography detection include: the chromatographic column filler includes octadecylsilane bonded silica gel; the mobile phase system includes mobile phase A and mobile phase B, the mobile phase A is acetonitrile, and the mobile phase B is 0.05-0.3v / v% formic acid aqueous solution; the elution mode is gradient elution, and the program of the gradient elution includes: 0-18min, the volume fraction of the mobile phase A is increased from 5% to 14%; 18-33min, the volume fraction of the mobile phase A is increased from 14% to 28%; 33-50min, the volume fraction of the mobile phase A is increased from 28% to 57%; and 50-70min, the volume fraction of the mobile phase A is 57%.

[0024] Preferably, the solid-liquid ratio of the Tongqiao Biyan tablet preparation and the solvent is 1g:2-100mL;

[0025] The mixing includes ultrasonic mixing, the ultrasonic mixing has a power of 300-6000W, a frequency of 30-50kHz, and a time of 10-60min;

[0026] After the mixing, the obtained ultrasonic system is further filtered.

[0027] Preferably, the flow rate of the mobile phase of the high performance liquid chromatography detection is 0.5-1.5mL / min, the detection wavelength is 220-350nm, the column temperature is 20-40℃, the theoretical plate number calculated according to imperatorin is ≥3000, and the injection amount is 5-20μL.

[0028] The test solution of the Tongqiao Biyan tablet preparation is detected by high performance liquid chromatography, and an octadecylsilane bonded silica gel column is used as the filler of the chromatographic column, the mobile phase A is acetonitrile, the mobile phase B is 0.05%-0.3% formic acid aqueous solution, a gradient elution mode is used and the gradient elution procedure is controlled, and 18 characteristic peaks in the characteristic chromatogram of the Tongqiao Biyan tablet preparation are obtained, wherein the 18 characteristic peaks include the characteristic peaks of four substances of chlorogenic acid, verbascoside, imperatorin and isoimperatorin. The standard characteristic chromatogram construction method of the Tongqiao Biyan tablet preparation provided by the application comprehensively characterizes the internal quality of the Tongqiao Biyan tablet preparation, simultaneously determines the main index components (chlorogenic acid, verbascoside, imperatorin and isoimperatorin) related to the drug efficacy in the standard characteristic chromatogram, covers the components of Xanthium sibiricum, Astragalus and Angelica dahurica etc. which occupy the main efficacy position in the two paths of extraction and crushing, and perfects the too simple existing quality standard of the Tongqiao Biyan tablet preparation. The precision, stability and reproducibility of the Tongqiao Biyan tablet preparation characteristic chromatogram method are good, the quality of the Tongqiao Biyan tablet preparation can be more comprehensively controlled, so that the quality stability, consistency and controllability of the preparation can be better ensured. The method provided by the application overcomes the problems that the existing quality standard of the Tongqiao Biyan tablet preparation is single, there is no qualitative and quantitative detection item, and the quality of the product cannot be accurately reflected.

[0029] The application establishes a synchronous detection method of multiple index components of the Tongqiao Biyan tablet preparation, the method has the advantages of strong specificity and simple operation, and has good stability, precision, reproducibility and sample recovery rate, and the methodological investigation test result is good, the contents of four main effective components (chlorogenic acid, verbascoside, imperatorin and isoimperatorin) in the Tongqiao Biyan tablet preparation can be comprehensively detected, the method can be used as the quality control method of the Tongqiao Biyan tablet preparation, and the effectiveness, safety and stability of the Tongqiao Biyan tablet preparation can be evaluated completely and accurately, the quality of the product can be controlled more accurately and comprehensively, and the quality and curative effect of the product can be ensured. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 The standard characteristic chromatogram of the Tongqiao Biyan tablet preparation has 18 common peaks, the peak No. 15 is the characteristic peak of imperatorin (S), the peak No. 5 is chlorogenic acid, the peak No. 7 is verbascoside, and the peak No. 17 is isoimperatorin;

[0031] Figure 2 It is the superimposed chromatogram of 10 batches of Tongqiao Biyan tablet preparations, wherein R is the control characteristic chromatogram;

[0032] Figure 3 It is Figure 2 the control characteristic chromatogram of R;

[0033] Figure 4 It is the chromatogram of the specificity investigation result, sucrose (negative). DETAILED DESCRIPTION

[0034] The application provides a standard characteristic spectrum construction method of Tongqiao Biyan tablet preparation, and comprises the following steps:

[0035] The Tongqiao Biyan tablet preparation is mixed with a solvent to obtain a test sample solution; the solvent comprises methanol and / or water.

[0036] The test sample solution and the control sample solution are respectively subjected to high performance liquid chromatography detection to respectively obtain a test sample liquid chromatogram and a control sample liquid chromatogram.

[0037] The control sample solution comprises chlorogenic acid control sample solution, verbascoside control sample solution, isoimperatorin control sample solution and imperatorin control sample solution.

[0038] The high performance liquid chromatography detection conditions comprise that the chromatographic column filler comprises octadecylsilane bonded silica gel; a mobile phase system comprises mobile phase A and mobile phase B, the mobile phase A is acetonitrile, and the mobile phase B is 0.05-0.3v / v% formic acid aqueous solution; an elution mode is gradient elution, and the gradient elution program comprises the following steps: 0-18min, the volume fraction of the mobile phase A is increased from 5% to 14%; 18-33min, the volume fraction of the mobile phase A is increased from 14% to 28%; 33-50min, the volume fraction of the mobile phase A is increased from 28% to 57%; and 50-70min, the volume fraction of the mobile phase A is 57%.

[0039] The test sample liquid chromatogram is introduced into a traditional Chinese medicine chromatographic characteristic spectrum similarity evaluation system to generate a characteristic spectrum common mode, and a standard characteristic spectrum containing 18 characteristic peaks is obtained; the standard characteristic spectrum is compared with the retention time of the control sample liquid chromatogram, and the standard characteristic spectrum comprises the characteristic peaks of chlorogenic acid, verbascoside, isoimperatorin and imperatorin.

[0040] Unless otherwise specified, the materials and equipment used in the application are commercially available in the art.

[0041] The Tongqiao Biyan tablet preparation is mixed with a solvent to obtain a test sample solution.

[0042] In the application, the solvent comprises methanol and / or water, preferably methanol-water, preferably 30-70% of methanol in the methanol-water, more preferably 40-60%, and further preferably 50%.

[0043] In the application, the solid-liquid ratio of the Tongqiao Biyan tablet preparation and the solvent is preferably 1g:2-100mL, more preferably 1g:10-50mL, and further preferably 1g:25mL.

[0044] In the present application, the mixing preferably comprises ultrasonic mixing, the power of which is preferably 300-6000 W, more preferably 400-3000 W, and further preferably 500-1000 W; the frequency of which is preferably 30-50 kHz, more preferably 35-45 kHz, and further preferably 40 kHz; and the time of which is preferably 10-60 min, more preferably 20-50 min, and further preferably 30-40 min.

[0045] After the mixing, the present application preferably further comprises passing the obtained ultrasonic system through a 0.45 μm filter membrane to obtain a test sample solution.

[0046] After obtaining the test sample solution, the present application separately detects the test sample solution and a control sample solution by high performance liquid chromatography to obtain a test sample liquid chromatogram and a control sample liquid chromatogram, respectively; the control sample solution comprises a chlorogenic acid control sample solution, a calycosin-7-glucoside control sample solution, a homoeriodictyol control sample solution, and a imperatorin control sample solution.

[0047] In the present application, the concentration of the chlorogenic acid control sample solution, the calycosin-7-glucoside control sample solution, and the homoeriodictyol control sample solution is independently preferably 30-50 μg / mL, more preferably 40-50 μg / mL, and further preferably 45-50 μg / mL; and the concentration of the imperatorin control sample solution is preferably 10-30 μg / mL, more preferably 15-25 μg / mL, and further preferably 15-20 μg / mL. In the present application, the solvent in the control sample solution preferably comprises methanol.

[0048] In the present application, the conditions of the high performance liquid chromatography detection comprise: the chromatographic column filler comprises octadecylsilane bonded silica gel; the mobile phase system comprises mobile phase A and mobile phase B, the mobile phase A is acetonitrile, and the mobile phase B is 0.05-0.3 v / v% formic acid aqueous solution, preferably 0.1-0.2 v / v% formic acid aqueous solution; the flow rate of the mobile phase is preferably 0.5-1.5 mL / min, more preferably 1-1.2 mL / min; the detection wavelength is preferably 220-350 nm, more preferably 280-300 nm; the column temperature is preferably 20-40℃, more preferably 30-35℃; the theoretical plate number calculated according to imperatorin is preferably ≥3000; the injection volume is preferably 5-20 μL, more preferably 10-15 μL; and the elution mode is gradient elution, the program of which comprises: 0-18 min, the volume fraction of the mobile phase A is increased from 5% to 14%; 18-33 min, the volume fraction of the mobile phase A is increased from 14% to 28%; 33-50 min, the volume fraction of the mobile phase A is increased from 28% to 57%; and 50-70 min, the volume fraction of the mobile phase A is 57%.

[0049] After obtaining the test sample liquid chromatogram, the test sample liquid chromatogram is introduced into the traditional Chinese medicine chromatographic characteristic spectrum similarity evaluation system to generate a characteristic spectrum common mode, and a standard characteristic spectrum containing 18 characteristic peaks is obtained; by comparing the retention time of the standard characteristic spectrum with that of the control sample liquid chromatogram, the standard characteristic spectrum includes the characteristic peaks of chlorogenic acid, calycosin-7-glucoside, isoimperatorin and imperatorin. In the present application, the test sample liquid chromatogram is preferably calculated by the traditional Chinese medicine chromatographic characteristic spectrum similarity evaluation system software, and the common peaks with a similarity of greater than 0.9 are the standard characteristic spectrum of the Tongqiao Biyan tablet preparation. In the present application, the traditional Chinese medicine chromatographic characteristic spectrum similarity evaluation system software is preferably the traditional Chinese medicine chromatographic characteristic spectrum similarity evaluation system software (2012 edition) of the State Pharmacopoeia Commission.

[0050] In the present application, by comparing and analyzing the standard characteristic spectrum with the control sample retention time of the control sample liquid chromatogram, it is found that the peak No. 15 in the standard characteristic spectrum is the characteristic peak of imperatorin (S), the peak No. 5 is chlorogenic acid, the peak No. 7 is calycosin-7-glucoside, and the peak No. 17 is isoimperatorin.

[0051] The present application also provides a multi-index component detection method for the Tongqiao Biyan tablet preparation, comprising the following steps:

[0052] The Tongqiao Biyan tablet preparation to be tested is mixed with a solvent to obtain a sample liquid to be tested; the solvent comprises methanol and / or water;

[0053] The sample liquid to be tested is subjected to high performance liquid chromatography detection to obtain the multi-index component detection result of the Tongqiao Biyan tablet preparation; the multi-index components include chlorogenic acid, calycosin-7-glucoside, imperatorin and isoimperatorin; the conditions of the high performance liquid chromatography detection include that the column filler comprises octadecylsilane bonded silica gel; the mobile phase system comprises mobile phase A and mobile phase B, the mobile phase A is acetonitrile, and the mobile phase B is 0.05-0.3 v / v % methanol aqueous solution; the elution mode is gradient elution, and the program of the gradient elution comprises: 0-18 min, the volume fraction of the mobile phase A is increased from 5 % to 14 %; 18-33 min, the volume fraction of the mobile phase A is increased from 14 % to 28 %; 33-50 min, the volume fraction of the mobile phase A is increased from 28 % to 57 %; and 50-70 min, the volume fraction of the mobile phase A is 57 %.

[0054] In the present application, the preparation method of the sample liquid to be tested is the same as that of the test sample solution; the conditions of the high performance liquid chromatography detection are the same as those in the process of the standard characteristic spectrum construction method, which will not be described here.

[0055] In the present application, the multi-index component detection result comprises qualitative detection result and quantitative detection result. In the present application, the qualitative detection result is preferably obtained by using fingerprint method.

[0056] In the present application, the quantitative detection result is preferably obtained by using standard curve method, specifically, the chromatographic peak area of the multi-index component of the sample liquid to be detected is brought into the standard regression equation to obtain the quantitative detection result of the multi-index component.

[0057] In the present application, the method for obtaining the standard curve regression equation preferably comprises the following steps: detecting the linear mixed control solution according to the aforementioned conditions of ultra-high performance liquid chromatography detection to obtain the chromatographic peak area of the multi-index component, taking the concentration of the standard working solution as the independent variable and the chromatographic peak area of the standard curve solution as the dependent variable to draw a standard curve and obtain a standard curve regression equation. In the present application, the control in the linear mixed control solution preferably comprises chlorogenic acid control, verbascoside control, imperatorin control and isoimperatorin control; the concentration of the linear mixed control solution is preferably 1.5-105 μg / mL, specifically preferably 1.5-2.5 μg / mL, 4.5-5.5 μg / mL, 9.5-10.5 μg / mL, 49-53 μg / mL and 98-105 μg / mL; the solvent in the linear mixed control solution preferably comprises methanol.

[0058] In the present application, when the content of chlorogenic acid (C 16 H 18 O9) in the Tongqiao Biyan tablet preparation (the specification is 0.3 g / tablet) is ≥0.24 mg / tablet, the content of verbascoside (C 22 H 22 O 10 ) is ≥0.06 mg / tablet, the content of imperatorin (C 16 H 14 O4) is ≥0.08 mg / tablet and the content of isoimperatorin (C 16 H 14 O4) is ≥0.075 mg / tablet, the quality of the Tongqiao Biyan tablet preparation meets the requirements.

[0059] In order to further illustrate the present application, the method for constructing the characteristic spectrum of the Tongqiao Biyan tablet preparation and the method for detecting the multi-index component are described in detail below in combination with examples, but they should not be understood as limiting the protection scope of the present application.

[0060] In the following examples, the conditions for high performance liquid chromatography detection: the chromatographic column is octadecylsilane bonded silica gel as the filler of the chromatographic column, with acetonitrile as the mobile phase A, with 0.2v / v% formic acid-water as the mobile phase B, the flow rate of the mobile phase is 1mL / min, the detection wavelength is 300nm, the column temperature is 30℃, the injection volume is 10μL, the gradient elution is used, and the best elution gradient is determined according to the chromatographic peak performance, separation effect and retention time of each component, as shown in Table 1.

[0061] Table 1 mobile phase elution gradient

[0062] Time (min) Mobile phase A volume fraction / % Mobile phase B volume fraction / % 0 5 95 18 14 86 33 28 72 50 57 43 70 57 43

[0063] Example 1

[0064] Establishment of preparation characteristic spectrum of Tongqiao Biyan tablet

[0065] Preparation of reference solution: accurately weigh chlorogenic acid reference substance, dissolve in methanol, and dilute to obtain 50μg / mL chlorogenic acid reference solution. According to the preparation method of chlorogenic acid reference solution, 50μg / mL verbascoside reference solution, 50μg / mL imperatorin reference solution and 15μg / mL isoimperatorin reference solution are prepared respectively.

[0066] Preparation of test solution: accurately weigh 2g of Tongqiao Biyan tablet preparation, place it in a conical flask with a plug, accurately add 50mL of 50v / v% methanol-water, tightly plug, weigh, ultrasonically treat for 30min, take out, cool to room temperature, re-weigh, make up the weight loss with 50v / v% methanol-water, shake well, pass through a 0.45μm filter membrane, and the filtrate is the test solution.

[0067] Inject the reference solution and the test solution into the liquid chromatograph for detection, respectively, to obtain the reference spectrum and the test spectrum.

[0068] According to the traditional Chinese medicine chromatographic characteristic spectrum similarity evaluation system, the similarity of the test characteristic spectrum and the control characteristic spectrum is calculated, and the similarity should not be less than 0.9.

[0069] 1. Methodology investigation

[0070] 1.1 Precision

[0071] Take the same test solution, inject it 6 times in succession, and determine it by high performance liquid chromatography, calculate the relative retention time and relative peak area RSD value of each common peak in the chromatogram, and the results are shown in Table 2.

[0072] Table 2 precision test relative retention time results

[0073] Peak number 1 2 3 4 5 6 Mean value RSD value / % 1 0.125 0.125 0.125 0.125 0.125 0.125 0.125 0.06 2 0.159 0.159 0.159 0.159 0.159 0.159 0.159 0.06 3 0.214 0.214 0.214 0.214 0.214 0.214 0.214 0.06 4 0.264 0.264 0.264 0.264 0.263 0.263 0.264 0.03 5 0.322 0.322 0.322 0.322 0.321 0.321 0.322 0.05 6 0.470 0.470 0.470 0.470 0.470 0.470 0.470 0.03 7 0.522 0.522 0.522 0.522 0.522 0.522 0.522 0.03 8 0.554 0.554 0.554 0.554 0.554 0.554 0.554 0.02 9 0.590 0.590 0.589 0.589 0.589 0.589 0.589 0.01 10 0.726 0.726 0.726 0.726 0.726 0.726 0.726 0.01 11 0.739 0.739 0.739 0.739 0.739 0.739 0.739 0.01 12 0.813 0.813 0.813 0.813 0.813 0.813 0.813 0.01 13 0.862 0.862 0.862 0.862 0.862 0.862 0.862 0.01 14 0.917 0.917 0.917 0.917 0.917 0.917 0.917 0.00 15 1.000 1.000 1.000 1.000 1.000 1.000 1.000 0.00 16 1.023 1.023 1.023 1.023 1.022 1.022 1.023 0.01 17 1.052 1.052 1.051 1.051 1.051 1.051 1.051 0.01 18 1.063 1.063 1.063 1.063 1.063 1.063 1.063 0.01

[0074] Conclusion: The relative retention time RSD of each common peak is less than 3%, indicating good precision.

[0075] 1.2 Stability

[0076] Take the same test solution, respectively, at 0h, 4h, 8h, 12h, 16h, 20h and 24h, sample, high performance liquid chromatography determination, calculate the relative retention time and relative peak area RSD value of each common peak in the chromatogram, the results are shown in Table 3.

[0077] Table 3 Stability test relative retention time results

[0078]

[0079] Conclusion: The relative retention time and relative peak area RSD of each common peak are less than 3%, and the test solution is stable within 24h.

[0080] 1.3 Reproducibility

[0081] Take the same batch of test samples, prepare 6 test sample solutions according to the above method, and perform high performance liquid chromatography determination, calculate the relative retention time and relative peak area RSD value of each common peak in the chromatogram, the results are shown in Table 4.

[0082] Table 4 Reproducibility test relative retention time results

[0083]

[0084]

[0085] Conclusion: The relative retention time and relative peak area RSD of each common peak are less than 3%, indicating that the detection method provided by the present application has good reproducibility.

[0086] 2, Establishment of characteristic chromatogram common mode and identification of part of common peaks

[0087] According to the preparation method of the test solution of the above test sample, prepare 10 batches of test sample solutions of Tongqiao Biyan tablets, and sample according to the above chromatographic conditions, and perform high performance liquid chromatography determination. The chromatogram of 10 batches of samples is introduced into the "Chinese medicine chromatographic characteristic chromatogram similarity evaluation system software (2012 edition)" of the State Pharmacopoeia Commission, and the characteristic chromatogram common mode is generated, and 18 characteristic peaks are calibrated, as shown in Figure 1 The results show that, by comparing and analyzing the retention time of the control, peak No. 15 is the isoimperatorin characteristic peak (S), peak No. 5 is chlorogenic acid, peak No. 7 is calycosin glucoside, and peak No. 17 is isoeucommunarin.

[0088] 3, Comparison of characteristic chromatogram similarity of 10 batches of Tongqiao Biyan tablet preparations

[0089] The median method was used to establish the common mode of the 10 batches of Tongqiao Biyan tablet preparation spectrum. Each characteristic spectrum was compared with the control characteristic spectrum to obtain the similarity of each spectrum with the control characteristic spectrum. The analysis results are shown in Table 5 and Figures 2-3 , Figure 2 The control characteristic spectrum of R is shown in Figure 2. Figure 3 The control characteristic spectrum of R is shown in Figure 2. Figure 2 The control characteristic spectrum of R is shown in Figure 2.

[0090] Table 5 Similarity results of characteristic spectra of 10 batches of Tongqiao Biyan tablet preparation and control

[0091]

[0092] Conclusion: The similarity of the characteristic spectra of the 10 batches of Tongqiao Biyan tablet preparation is good, all above 0.90, meeting the requirements.

[0093] Example 2

[0094] Preparation of mixed control solution: The chlorogenic acid control, the calycosin-7-glucoside control, the imperatorin control, and the isoimperatorin control were precisely weighed, dissolved and constant volume with methanol to obtain a mixed control solution with a concentration of 100 μg / mL.

[0095] Preparation of linear mixed control solution: The mixed control solution was stepwise diluted with methanol as diluent to obtain linear mixed control solutions 1-5. The concentrations of each control are shown in Table 6:

[0096] Table 6 Concentrations of linear mixed control solutions (μg / mL)

[0097] Linear mixture control solution Chlorogenic acid Calycosin-7-glucoside Imperatorin Isoimperatorin 1 2.0051 2.0931 1.9728 2.0865 2 5.01274 5.2327 4.9321 5.21625 3 10.02548 10.46542 9.8642 10.4325 4 50.1274 52.3271 49.3209 52.1625 5 100.2548 104.6542 98.6418 104.3250

[0098] The test sample solution was prepared according to Example 1.

[0099] The control sample solution and the test sample solution were precisely pipetted and injected into the high performance liquid chromatograph for determination to obtain the contents of chlorogenic acid (C 16 H 18 O9) ≥ 0.24 mg, calycosin-7-glucoside (C 22 H 22 O 10 ) ≥ 0.06 mg, imperatorin (C 16 H 14 O4) ≥ 0.08 mg, and isoimperatorin (C 16 H 14 O4) ≥ 0.075 mg in each tablet of Tongqiao Biyan tablet preparation.

[0100] 1. Methodology investigation

[0101] 1.1 Specificity Investigation

[0102] The interference of the adjuvant (sucrose) on the target peak was investigated to evaluate the strength of the specificity of the target component, and the results are shown in Table 1. Figure 4 Results: The adjuvant had no interference on the index peak, indicating that the specificity of the detection method provided by the application was good.

[0103] 1.2 Linear relationship investigation

[0104] 10 μL of each concentration of the mixed control solution was precisely injected into the high performance liquid chromatograph for detection, the chromatogram was recorded, the peak area was taken as the vertical coordinate (Y), and the control product concentration was taken as the horizontal coordinate (X), a standard curve was drawn, the linear regression equation and the correlation coefficient r value were calculated, and the results are shown in Table 7.

[0105] Table 7 Linear results

[0106]

[0107]

[0108] The results show that the linear relationship of chlorogenic acid, calycosin-7-glucoside, imperatorin, and isoimperatorin is good within the above concentration range.

[0109] 1.3 Precision

[0110] The same test sample solution was continuously injected for 6 times, and high performance liquid chromatography detection was performed, and the results are shown in Table 8.

[0111] Table 8 Precision test results

[0112]

[0113] Results: The content RSD of chlorogenic acid, calycosin-7-glucoside, imperatorin, and isoimperatorin is less than 3%, indicating good precision.

[0114] 1.4 Stability

[0115] The same test sample solution was detected at 0 h, 2 h, 4 h, 8 h, 12 h, 18 h, and 24 h, respectively, and the content RSD value of each component was calculated, and the results are shown in Table 9.

[0116] Table 9 Stability test results

[0117]

[0118] Results: The stability of the four components in the test sample solution is good within 24 h.

[0119] 1.5 Reproducibility

[0120] Take the same batch of test samples, prepare 6 test sample solutions, and perform high performance liquid chromatography detection to determine the RSD values of the contents of each component. The results are shown in Table 10.

[0121] Table 10 Repetitive test results

[0122]

[0123] The content RSD is less than 2%, indicating that the method has good repeatability.

[0124] 1.6 Recovery rate

[0125] Precisely weigh about 1.0 g of the known content of chlorogenic acid, calycosin-7-glucoside, imperatorin, and isoimperatorin in the Touqiangbiyan tablet preparation, a total of 6, and place it in a measuring flask. Precisely add 1 mL of the control sample solution containing chlorogenic acid (concentration 1.3025 mg / mL), calycosin-7-glucoside (concentration 0.3045 mg / mL), imperatorin (concentration 0.4316 mg / mL), and isoimperatorin (concentration 0.3642 mg / mL), a total of 6, and dilute to the calibration mark with 50 v / v% methanol-water, tightly cap, weigh, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 min, remove, cool to room temperature, re-weigh, make up the weight loss with 50 v / v% methanol-water, shake well, filter, and the filtrate is the spiked test sample solution. Perform high performance liquid chromatography detection to calculate the spiked recovery rate of the four indicators, and the results are shown in Tables 11-14.

[0126] Table 11 Recovery rate test results of chlorogenic acid

[0127]

[0128] Table 12 Recovery rate test results of calycosin-7-glucoside

[0129]

[0130] Table 13 Recovery rate test results of imperatorin

[0131]

[0132] Table 14 Recovery rate test results of isoimperatorin

[0133]

[0134] The average spiked recovery rate of the four indicator components is 99.24%, 97.77%, 100.36%, and 94.43%, respectively, and the RSD values are all less than 3%, indicating that the detection method provided by the application has high accuracy.

[0135] 1.7 Detection limit and quantification limit

[0136] The control solution and the blank solvent were injected into the liquid chromatograph respectively, the noise peak height of the standard solution and the absorption peak height of the blank solution were recorded, and the signal peak height ratio of the standard and the blank solvent with the same retention time was calculated. The 3:1 fold signal-to-noise ratio was the detection limit, and the 10:1 fold signal-to-noise ratio was the quantitative limit. The results are shown in Table 15.

[0137] Table 15 Detection limit and quantitative limit

[0138] Item Chlorogenic acid Calycosin-7-glucoside Imperatorin Isoimperatorin Limit of detection (pg / mL) 0.02 0.07 0.03 0.09 Limit of quantification (pg / mL) 0.07 0.22 0.08 0.28

[0139] Example 3

[0140] Sample determination results

[0141] The Tongqiao Biyan tablets of each batch were taken, and the sample solution was prepared according to the method of Example 1, and the high performance liquid chromatography detection was carried out to determine the contents of chlorogenic acid, calycosin-7-glucoside, imperatorin and isoimperatorin. The results are shown in Table 16.

[0142] Table 16 Content determination results of different batches of Tongqiao Biyan tablet preparations

[0143]

[0144] In the multi-index component detection and determination method provided by the present application, the limit of each index is set to 80% of the average value of the content of each index as the minimum limit. The results are converted into tablets, and the content of chlorogenic acid is 0.24 mg / tablet, the content of calycosin-7-glucoside is 0.06 / tablet, the content of imperatorin is 0.08 mg / tablet, and the content of isoimperatorin is 0.075 mg / tablet. These are the set limit values of each index. In the future, more batches will be determined to improve the limit values.

[0145] Although the above examples have made a detailed description of the present application, it is only a part of the embodiments of the present application, not all the embodiments, and other embodiments can be obtained without creative labor according to the embodiments of the present application, which all belong to the protection scope of the present application.

Claims

1. A method for constructing a standard characteristic chromatogram of Tongqiao Biyan tablet formulation, comprising the following steps: mixing Tongqiao Biyan tablet formulation with solvent to obtain test sample solution; the solvent comprises methanol and water; the Tongqiao Biyan tablet formulation is composed of 7 traditional Chinese medicines, namely fried Xanthium sibiricum, Saposhnikovia divaricata, Astragalus membranaceus, Angelica dahurica, Lonicera japonica, fried Atractylodes lancea and Mentha haplocalyx; performing high performance liquid chromatography detection on the test sample solution and the control sample solution respectively to obtain test sample liquid chromatogram and control sample liquid chromatogram respectively; the control sample solution comprises chlorogenic acid control sample solution, genistin control sample solution, isoimperatorin control sample solution and imperatorin control sample solution; the conditions of high performance liquid chromatography detection comprise: the chromatographic column filler comprises octadecylsilane bonded silica gel; the mobile phase system comprises mobile phase A and mobile phase B, the mobile phase A is acetonitrile, and the mobile phase B is 0.05-0.3v / v% formic acid aqueous solution; the elution mode is gradient elution, and the program of gradient elution comprises: 0-18min, the volume fraction of the mobile phase A increases from 5% to 14%; 18-33min, the volume fraction of the mobile phase A increases from 14% to 28%; 33-50min, the volume fraction of the mobile phase A increases from 28% to 57%; 50-70min, the volume fraction of the mobile phase A is 57%; the detection wavelength is 280-300nm; introducing the test sample liquid chromatogram into traditional Chinese medicine chromatographic characteristic chromatogram similarity evaluation system to generate characteristic chromatogram common mode, and obtaining a standard characteristic chromatogram containing 18 characteristic peaks; by comparing the retention time of the standard characteristic chromatogram with that of the control sample liquid chromatogram, the standard characteristic chromatogram comprises the characteristic peaks of chlorogenic acid, genistin, isoimperatorin and imperatorin.

2. The standard feature map construction method of claim 1, wherein, The solid-liquid ratio of the Tongqiao Biyan tablet formulation and the solvent is 1g:2-100mL.

3. The standard feature map construction method according to claim 1 or 2, characterized in that, The mixing comprises ultrasonic mixing, and the ultrasonic mixing has a power of 300-6000W, a frequency of 30-50kHz and a time of 10-60min.

4. The standard feature map construction method of claim 3, wherein, After the mixing, the obtained ultrasonic system is further filtered.

5. The standard feature map construction method of claim 4, wherein, The concentration of the chlorogenic acid control sample solution, the genistin control sample solution and the isoimperatorin control sample solution is independently 30-50μg / mL, and the concentration of the imperatorin control sample solution is 10-30μg / mL. The solvent in the control sample solution comprises methanol.

6. The standard feature map construction method of claim 1, wherein, The flow rate of the mobile phase in the high performance liquid chromatography detection is 0.5-1.5mL / min, the column temperature is 20-40℃, the theoretical plate number calculated according to imperatorin is ≥3000, and the injection amount is 5-20μL.

7. The standard feature mapping method of claim 1, wherein, The test sample liquid chromatogram and the control sample liquid chromatogram are calculated by traditional Chinese medicine chromatographic characteristic chromatogram similarity evaluation system software, and the similarity is ≥0.

9.

8. A multi-index component detection method for a Tongqiao Rhinitis Tablet preparation, characterized in that, comprising the following steps: mixing the Tongqiao Biyan tablet formulation to be tested with solvent to obtain test sample liquid; the solvent comprises methanol and water; the Tongqiao Biyan tablet formulation is composed of 7 traditional Chinese medicines, namely fried Xanthium sibiricum, Saposhnikovia divaricata, Astragalus membranaceus, Angelica dahurica, Lonicera japonica, fried Atractylodes lancea and Mentha haplocalyx; The sample liquid is subjected to high performance liquid chromatography detection to obtain the detection results of multiple index components of the Tongqiao Biyan tablet preparation; the multiple index components include chlorogenic acid, verbascoside, imperatorin and isoimperatorin; The conditions of the high performance liquid chromatography detection include: the chromatographic column filler includes octadecylsilane bonded silica gel; the mobile phase system includes mobile phase A and mobile phase B, the mobile phase A is acetonitrile, and the mobile phase B is 0.05-0.3v / v% formic acid aqueous solution; the elution mode is gradient elution, and the program of the gradient elution includes: 0-18min, the volume fraction of the mobile phase A is increased from 5% to 14%; 18-33min, the volume fraction of the mobile phase A is increased from 14% to 28%; 33-50min, the volume fraction of the mobile phase A is increased from 28% to 57%; 50-70min, the volume fraction of the mobile phase A is 57%; and the detection wavelength is 280-300nm.

9. The multi-index composition detection method of claim 8, wherein, The solid-liquid ratio of the Tongqiao Biyan tablet preparation and the solvent is 1g:2-100mL; The mixing includes ultrasonic mixing, and the ultrasonic mixing is performed at a power of 300-6000W, a frequency of 30-50kHz and for 10-60min. After the mixing, the obtained ultrasonic system is further subjected to filtration.

10. The multi-index composition detection method of claim 8, wherein, The flow rate of the mobile phase of the high performance liquid chromatography detection is 0.5-1.5mL / min, the column temperature is 20-40℃, the theoretical plate number calculated according to imperatorin is ≥3000, and the injection amount is 5-20μL.

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