A method for establishing a high performance liquid chromatography characteristic spectrum of anti-thrombotic regeneration pills based on double-label linear method and a method for determining the content of its components

Abstract

The invention relates to a method for establishing a high-performance liquid phase characteristic spectrum of anti-thrombotic regeneration pills based on a double-label linear method and a method for determining the content of components, and belongs to the field of drug analysis. The method for establishing a high-performance liquid phase characteristic spectrum of anti-thrombotic regeneration pills based on a double-label linear method comprises the following steps: S1: preparing a reference solution; S2: preparing a test solution; S3: determining chromatographic conditions; S4: collecting chromatograms of the reference solution and multiple batches of test solution, analyzing the chromatograms of each test solution, calibrating 11 common peaks, and identifying characteristic peaks of three components; S5: using daidzein and physaccharin methyl ether as double-label reference substances, adopting a double-label linear correction method, and using two reference substances to effectively predict and locate other nine characteristic peaks, and having good performance in scientific accuracy and durability, and being relatively economical. The method is conducive to relatively comprehensive and effective control of the quality of the anti-thrombotic regeneration pills.

Description

Technical Field

[0001] The present invention relates to the field of drug analysis, and in particular to a method for establishing a high performance liquid phase characteristic spectrum of anti-thrombotic regeneration pills based on a double-label linear method and a method for determining the content of components. Background Art

[0002] Anti-thrombotic Rejuvenation Pills are composed of various ingredients such as red ginseng, astragalus, arisaema, pangolin (scalded), bezoar, borneol, leech (scalded), musk, salvia miltiorrhiza, Panax notoginseng, rhubarb, earthworm, storax, scorpion, kudzu root, dactyloides, angelica, Achyranthes bidentata, Polygonum multiflorum, black-necked snake, peach kernel, cinnabar, safflower, earthworm, gastrodia, asarum, clematis, cardamom, licorice, etc. It has the effects of promoting blood circulation and removing blood stasis, relaxing tendons and unblocking collaterals, extinguishing wind and calming spasms, and can be used to treat symptoms such as numbness of hands and feet, difficulty in walking, paralysis, crooked mouth and eyes, and slurred speech during the recovery period of stroke sequelae.

[0003] The chemical composition of anti-thrombotic regeneration pills is complex. At present, the quality standard of anti-thrombotic regeneration pills is mainly based on thin layer identification items. The characteristic spectrum inspection method and standard have not yet been established. It is impossible to comprehensively reflect the quality of anti-thrombotic regeneration pills and cannot guarantee comprehensive and accurate control of the quality of anti-thrombotic regeneration pills. Summary of the invention

[0004] In order to make up for the deficiencies of the prior art, the present invention provides a method for establishing an HPLC characteristic spectrum of anti-thrombotic regeneration pills based on a double-label linear method, which is conducive to more comprehensive and effective control of the quality of anti-thrombotic regeneration pills.

[0005] The technical solution adopted by the present invention to solve the above technical problems is:

[0006] A method for establishing a high-performance liquid phase characteristic spectrum of anti-thrombotic regeneration pills based on a double-label linear method comprises the following steps:

[0007] Preparation of S1 reference solution: accurately weigh appropriate amount of puerarin, daidzin and physcholin methyl ether reference substances, dissolve them in appropriate amount of 30% ethanol, 70% methanol and methanol respectively to prepare reference substance mother solution, then take 1ml of reference substance mother solution of puerarin, 0.5ml of daidzin and 3ml of physcholin methyl ether respectively in the same volumetric flask and add methanol to make up to volume to prepare mixed reference substance solution;

[0008] Preparation of S2 test solution: accurately weigh an appropriate amount of anti-thrombotic regeneration pill powder, dissolve it in methanol, weigh it, reflux extract, weigh it, make up the weight with methanol, shake it well, filter it, take the filtrate, pass it through a 0.22μm microporous filter membrane, and take the filtrate as the test solution;

[0009] Determination of S3 HPLC conditions: C18 chromatographic column, acetonitrile as mobile phase A, 0.05% phosphoric acid solution as mobile phase B, gradient elution conditions: 0-18min, 5%-12%A; 18-23min, 12% A; 23-24min, 12%-18%A; 24-39min, 18%-23%A; 39-43min, 23%-50%A; 43-75min, 50%-70%A; flow rate 0.2-0.32mL / min; column temperature 29-31℃; detection wavelength 286nm; injection volume 1μL;

[0010] S4 HPLC determination: according to the chromatographic conditions in step S3, chromatograms of the reference solution and multiple batches of test solution are collected, and the chromatograms are analyzed to confirm that there are 11 peaks in total, and the characteristic peaks of 3 components are identified, among which peak 1 is puerarin, peak 4 is daidzein, and peak 9 is physaccharin methyl ether;

[0011] S5 Determination of predicted retention time: Using multiple chromatographic columns of different brands and types, collect chromatograms of the mixed reference solution and the test solution according to the chromatographic conditions in step S3 to obtain standard retention times of 11 common peaks; using daidzein and physaccharin methyl ether as double-label reference substances, use double-label linear calibration method to obtain predicted retention times of the remaining peaks.

[0012] Furthermore, the preparation method of the test solution is specifically as follows: accurately weigh 0.3 g of anti-thrombotic regeneration pellets, add 25 mL of methanol, weigh the weight, reflux extraction for 1 h, weigh the weight, make up the weight with methanol, shake well, filter, take the filtrate, pass it through a 0.22 μm microporous filter membrane, and take the filtrate as the test solution.

[0013] Furthermore, appropriate amounts of puerarin, daidzein, and physcion methyl ether reference substances were accurately weighed and dissolved in appropriate amounts of 30% ethanol, 70% methanol, and methanol, respectively, to prepare solutions with concentrations of 691.45, 484.62, and 77.22 μg•mL -1 The mother solution of the reference substance; take 1 ml of puerarin, 0.5 ml of daidzein and 3 ml of physaccharin methyl ether in the same 5 ml volumetric flask, add methanol to make up to 5 ml, and make the concentrations of 138.29, 48.46 and 46.33 μg•mL -1 of mixed reference solution.

[0014] Furthermore, in step S5, the specific method of using the double-label linear correction method to obtain the predicted retention time is: using the actual retention time of the chromatographic peaks of the double-label reference substances daidzein and physaccharin methyl ether as the ordinate and the standard retention time as the abscissa to obtain a linear fitting equation; substituting the standard retention time of the remaining 9 characteristic peaks into the equation to obtain the corresponding predicted retention time; wherein the standard retention time is the average retention time of the test sample collected on multiple chromatographic columns.

[0015] Furthermore, in step S5, the standard retention times of the 11 peaks are 12.167 min, 13.907 min, 15.395 min, 16.256 min, 22.324 min, 31.898 min, 33.792 min, 50.789 min, 53.465 min, 55.449 min, and 56.499 min, respectively.

[0016] Furthermore, in step S3, the flow rate is selected to be 0.3 mL / min; and the column temperature is selected to be 30°C.

[0017] The present application also provides a method for determining the content of ingredients in anti-thrombotic regeneration pills, comprising the following steps: performing liquid chromatography determination according to steps S1-S3 of the established method to obtain a chromatogram, and calculating the content of each ingredient, puerarin, daidzein, and physcion by peak area based on the external standard method.

[0018] The quality control method for Anti-thrombotic Rejuvenation Pills is as follows: prepare a double-label mixed reference solution of daidzein and physaccharin methyl ether according to the method of step S1, prepare a test solution according to the method of step S2, collect the chromatograms of the two according to the chromatographic conditions of step S3, and locate the chromatographic peak of each component to be tested by determining the predicted retention time, that is, observe whether each component has a peak at the predicted retention time.

[0019] The specific preparation method of the double-label mixed reference solution is as follows: take 0.5 ml of daidzein reference stock solution and 3 ml of physaccharin methyl ether reference stock solution in the same 5 ml volumetric flask, add methanol to make the volume to 5 ml, and make the concentrations of 48.46 and 46.33 μg•mL -1 of mixed reference solution.

[0020] The present invention adopts the above structure, and has the advantages of:

[0021] 1. The present invention discloses a method for establishing a high performance liquid chromatography characteristic spectrum of anti-thrombotic regeneration pills based on a double-label linear method. Soybean glucoside and physaccharin methyl ether are used as double-label reference substances. A double-label linear correction method is used to effectively predict and locate the chromatographic peaks of the other nine components using two reference substances. The method is based on multi-point linear regression correction. The method is significantly superior to the relative retention time method currently commonly used for chromatographic peak qualitative analysis in terms of the applicability of the chromatographic column and the accuracy of the prediction. The method greatly eliminates the problems of the relative retention time method in terms of the accuracy of the characteristic peak qualitative analysis and the applicability of the chromatographic column being insufficient. The method has good scientific accuracy and durability, is relatively economical, improves the simplicity of the experiment and reduces the cost. The method provides a reference and basis for the quality control of anti-thrombotic regeneration pills.

[0022] 2. This method has the advantages of simplicity, stability, high precision and good reproducibility.

[0023] 3. The characteristic spectrum obtained by the method established in this application has 11 effectively separated characteristic peaks. Therefore, the obtained characteristic spectrum can more effectively characterize the quality of the anti-thrombotic regeneration pills, thereby facilitating a more comprehensive monitoring of the quality of the drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The high performance liquid phase characteristic spectrum of the anti-thrombotic regeneration pill established by the present invention;

[0025] Figure 2 It is the characteristic spectrum of the sample solution and mixed reference substance of Anti-thrombotic Regeneration Pill;

[0026] Figure 3 This is the characteristic spectrum of the anti-thrombotic regeneration pill sample solution at a wavelength of 286nm;

[0027] Figure 4 It is the characteristic spectrum of the sample solution of anti-thrombotic regeneration pill at a wavelength of 200nm;

[0028] Figure 5 It is the characteristic spectrum of the anti-thrombotic regeneration pill sample solution at a wavelength of 240nm;

[0029] Figure 6 It is the characteristic spectrum of the sample solution of anti-thrombotic regeneration pill at a wavelength of 300nm;

[0030] Figure 7 The characteristic spectra of three batches of anti-thrombotic regeneration pill sample solutions;

[0031] Figure 8 The linear relationship of retention time on 15 different chromatographic columns. DETAILED DESCRIPTION

[0032] In order to clearly illustrate the technical features of the present solution, the present invention is described in detail below through specific implementation methods and in conjunction with the accompanying drawings. In the following description, many specific details are described to facilitate a full understanding of the present application. However, the present application can also be implemented in other ways different from those described herein. Therefore, the protection scope of the present application is not limited by the specific embodiments disclosed below.

[0033] 1. Instruments and reagents

[0034] Agilent UHPLC-1290 ultra-high performance liquid chromatograph, Agilent; DRS Origin software (China Food and Drug Inspection Institute); XS105DU 1 / 100,000 electronic analytical balance, METTLER TOLEDO; BSA224S-CW 1 / 10,000 electronic analytical balance, Sartorius Scientific Instruments (Beijing) Co., Ltd.; KQ-800VSM digital ultrasonic cleaner, Kunshan Ultrasonic Instrument Co., Ltd.

[0035] Puerarin reference substance (batch number 110752-202217, content 96.8%), soybean glycoside reference substance (batch number 111738-202305, content 96.3%), and physcion reference substance (batch number 110758-202218, content 98.9%) were all purchased from China Food and Drug Inspection Institute; purified water, Hangzhou Wahaha Group Co., Ltd.; phosphoric acid, HPLC grade, batch number 20210710, Tianjin Komiou Chemical Reagent Co., Ltd.; acetonitrile, HPLC grade, batch number AHI010101205, Yuexu Technology (Shanghai) Co., Ltd.; methanol, AR grade, batch number: 20230516, Tianjin Fuyu Fine Chemical Co., Ltd.

[0036] 2. Method for establishing the HPLC characteristic spectrum of anti-thrombotic regeneration pills and method for determining the content of components

[0037] 2.1 Establishment Method

[0038] 2.1.1 Preparation of reference solution: Accurately weigh appropriate amount of puerarin, daidzein, and physcion methyl ether reference substances, and dissolve them in appropriate amount of 30% ethanol, 70% methanol, and methanol, respectively, to prepare solutions with concentrations of 691.45, 484.62, and 77.22 μg•mL -1 The mother solution of the reference substance; take 1 ml of puerarin, 0.5 ml of daidzein and 3 ml of physaccharin methyl ether in the same 5 ml volumetric flask, add methanol to make up to 5 ml, and make the concentrations of 138.29, 48.46 and 46.33 μg•mL -1 of mixed reference solution.

[0039] 2.1.2 Preparation of test solution: Accurately weigh 0.3 g of anti-thrombotic regeneration pellets, add 25 mL of methanol, weigh the weight, reflux and extract for at least 1 hour, weigh the weight, make up the weight with methanol, shake well, filter, take the filtrate, pass it through a 0.22 μm microporous filter membrane, and the filtrate is the test solution.

[0040] 2.1.3 The chromatographic conditions for HPLC determination of the test solution and the mixed reference solution are as follows: C18 chromatographic column, 15 chromatographic columns are numbered Col.1~Col.15, and the specific specifications, brands and models are shown in Table 1; acetonitrile is used as mobile phase A, 0.05% phosphoric acid solution is used as mobile phase B, and the gradient elution conditions are: 0-18min, 5%-12%A; 18-23min, 12%A; 23-24min, 12%-18%A; 24-39min, 18%-23%A; 39-43min, 23%-50%A; 43-75min, 50%-70%A; the flow rate is 0.2-0.32mL / min; the column temperature is 29-31℃; the detection wavelength is 286nm; the injection volume is 1μL.

[0041]

[0042] 2.1.4 HPLC determination: Collect the chromatograms of the test solution and the mixed reference solution, and import the chromatograms of multiple batches of test solution into the Chinese medicine chromatographic fingerprint similarity evaluation system for analysis (see Figure 7 ), the characteristic spectrum is obtained by the average value algorithm. Figure 1 The characteristic peaks of puerarin (peak 1), peak 2, peak 3, daidzein (peak 4), peak 5, peak 6, peak 7, peak 8, physcion (peak 9), peak 10, and peak 11 were calibrated by spectral analysis.

[0043] 2.1.5 Determination of predicted retention time: Using 15 chromatographic columns of different brands and types, the standard retention times of puerarin (peak 1), peak 2, peak 3, daidzein (peak 4), peak 5, peak 6, peak 7, peak 8, physaccharin methyl ether (peak 9), peak 10, and peak 11 were obtained as 12.167 min, 13.907 min, 15.395 min, 16.256 min, 22.324 min, 31.898 min, 33.792 min, 50.789 min, 53.465 min, 55.449 min, and 56.499 min, respectively. Among them, the standard retention time is the average retention time of the test sample collected on multiple chromatographic columns;

[0044] The actual retention time of the double-labeled reference substance was collected on another chromatographic column, and the double-label linear correction method was adopted, with the actual retention time of the chromatographic peaks of the double-labeled reference substances daidzein and physcion as the ordinate and the standard retention time as the abscissa to obtain the linear fitting equation; the standard retention times of puerarin (peak 1), peak 2, peak 3, peak 5, peak 6, peak 7, peak 8, peak 10, and peak 11 were substituted into the equation to obtain the corresponding predicted retention times.

[0045] 2.2 Quality Control Methods of Antithrombotic Regeneration Pills

[0046] When performing quality control on the anti-thrombotic regeneration pills, the chromatograms of the double-labeled mixed reference solution of daidzein and physcion methyl ether and the test solution were collected, and the chromatographic peaks of each component to be tested were located by determining the predicted retention time, that is, observing whether each component peaked at the predicted retention time. The specific preparation method of the double-labeled mixed reference solution is as follows: 0.5 ml of daidzein reference solution mother solution and 3 ml of physcion methyl ether reference solution mother solution were taken in the same 5 ml volumetric flask, and methanol was added to make the volume to 5 ml, and the concentrations were 48.46 and 46.33 μg•mL -1 The preparation method of the test solution is as follows: 2.2.

[0047] 2.3 Content determination

[0048] Perform liquid chromatography determination according to 2.1.1-2.1.3 to obtain a chromatogram, and calculate the content of puerarin, daidzein, and physcion by peak area based on the external standard method.

[0049] 3. Selection of detection wavelength

[0050] Take 0.3g of anti-thrombotic regeneration pill powder, prepare the test solution according to the preparation method of the test solution, accurately weigh 1μL of each of the above 4 test solutions, inject into ultra-high performance liquid chromatography for detection, the detection wavelengths are 286nm, 200nm, 240nm and 300nm respectively, and the test results are shown in Figure 3-6 Based on the number of chromatographic peaks, chromatographic peak response values, and whether the baseline is stable, 286 nm was finally selected as the detection wavelength for the characteristic spectrum of Anti-thrombotic Regeneration Pills.

[0051] 4. Optimization experiment of test solution preparation conditions

[0052] 4.1 Selection of extraction method

[0053] Take an appropriate amount of anti-thrombotic regeneration pill powder, accurately weigh 2 portions of 0.3010g and 0.3014g respectively, then place them in stoppered conical bottles, accurately add 25mL of methanol, observe reflux extraction for 1h and ultrasonic extraction (300W, 40KHz) for 1h, cool, add weight, shake well, filter, and take the filtrate as the test solution. Accurately weigh 1μL of each of the above two test solutions, inject into the ultra-high performance liquid chromatograph, record the chromatogram for 75min, and use the number of chromatographic peaks and the main peak area as evaluation indicators. The results are shown in Table 2.

[0054]

[0055] From the results in Table 2, it can be seen that the main peak chromatographic peak area of ​​the reflux sample is basically larger than that of the ultrasonic sample, so the reflux extraction method is selected.

[0056] 4.2 Investigation of extraction solvent

[0057] Take an appropriate amount of anti-thrombotic regeneration pill powder, accurately weigh 4 portions, 0.3010g, 0.3016g, 0.3010g and 0.3009g respectively, and place them in stoppered conical bottles, and accurately add 25% methanol, 50% methanol, 75% methanol and 25mL of methanol, reflux extraction for 1h, investigate different solvents, cool, add weight, shake well, filter, and take the filtrate as the test solution. Accurately weigh 1μL of each of the above 4 test solutions, inject into the ultra-high performance liquid chromatograph, record the chromatogram for 75min, and analyze with the number of chromatographic peaks and the main peak area as evaluation indicators. The results are shown in Table 3.

[0058]

[0059] From the results in Table 3, it can be seen that the number of chromatographic peaks when 25% methanol and 50% methanol are used as extraction solvents is small, and the number of chromatographic peaks and the main peak area when methanol is used as the extraction solvent are greater than those of 75% methanol, so methanol is selected as the extraction solvent.

[0060] 4.3 Selection of extraction time

[0061] Take an appropriate amount of anti-thrombotic regeneration pill powder, accurately weigh 3 portions, 0.3010g, 0.3010g and 0.3016g respectively, and place them in stoppered conical bottles, accurately add 25mL of methanol, observe extraction for 0.5h, reflux extraction for 1h, and reflux extraction for 1.5h, cool, add weight, shake well, filter, and take the filtrate as the test solution. Accurately weigh 1μL of each of the above 3 test solutions, inject into the ultra-high performance liquid chromatograph, record the chromatogram for 75min, and analyze with the number of chromatographic peaks and the peak area of ​​the main peak as evaluation indicators. The results are shown in Table 4.

[0062]

[0063] From the results in Table 4, it can be seen that there is no significant difference in the number of chromatographic peaks and the main peak area when refluxing for 0.5 h, 1 h and 1.5 h. Taking into account that refluxing for 1 h is shorter and the extraction is more complete, the extraction time is selected as 1 h.

[0064] 4.4 Selection of sample weight

[0065] Take an appropriate amount of anti-thrombotic regeneration pill powder, accurately weigh 3 portions, 0.2021g, 0.3010g and 0.4007g respectively, and place them in stoppered conical bottles, accurately add 25mL of methanol, reflux extraction for 1h, investigate different sample amounts, cool, add weight, shake well, filter, and take the filtrate as the test solution. Accurately weigh 1μL of each of the above 3 test solutions, inject into the ultra-high performance liquid chromatograph, record the chromatogram for 75min, and analyze with the number of chromatographic peaks and the main peak area divided by the sampling amount as evaluation indicators. The results are shown in Table 5.

[0066]

[0067] From the results in Table 5, it can be seen that the extraction effects of sample weights of 0.2g, 0.3g and 0.4g are equivalent. Considering that the response of 0.3g sample weight is higher, 0.3g sample weight is selected.

[0068] Based on the above test results, the specific preparation method of the test solution of the characteristic spectrum of the anti-thrombotic regeneration pills was determined as follows: accurately weigh 0.3 g of the anti-thrombotic regeneration pills, add 25 mL of methanol, weigh the weight, reflux extraction for 1 h, weigh the weight, make up the weight with methanol, shake well, filter, take the filtrate, pass it through a 0.22 μm microporous filter membrane, and the filtrate is the test solution.

[0069] 5 Methodological investigation of feature maps

[0070] 5.1 Repeatability test: Take an appropriate amount of anti-thrombotic regeneration pill powder, accurately weigh 0.3010g, 0.3002g, 0.3001g, 0.3005g, 0.3008g, 0.3002g, and place in a stoppered conical flask. Add 25mL of methanol to the conical flask, heat and reflux for 1h, cool, reweight, shake well, filter, and take the filtrate to obtain the test solution. Accurately aspirate the test solution and the reference peak solution (with peaks 4 and 9 as reference peaks), respectively, use Col.1 chromatographic column, inject into ultra-high performance liquid chromatography, perform gradient elution according to the chromatographic conditions provided, and record the chromatogram. Calculate the RSD of the retention time and peak area of ​​peaks 1 to 11. The results are shown in Tables 6 and 7.

[0071]

[0072]

[0073] It can be seen from Tables 6 and 7 that the RSD of the retention time is less than 2%, and the RSD of the peak area is less than 2%, indicating that the method has good repeatability.

[0074] 5.2 Solution stability test

[0075] Take an appropriate amount of anti-thrombotic regeneration pill powder, accurately weigh 0.3000g, put it in a stoppered conical flask, add 25mL of methanol to the conical flask, heat and reflux for 1h, cool, add weight, shake well, filter, and take the filtrate to obtain the test solution. The test solution was injected at 0h, 2h, 4h, 8h, 12h, and 24h, respectively, using Col.1 chromatographic column, recording the chromatogram, measuring 6 time points in total, calculating the retention time and peak area RSD of peak 1 to peak 11, and the results are shown in Table 8 and Table 9.

[0076]

[0077]

[0078] The results in Tables 8 and 9 show that the retention time RSD is less than 2% and the peak area RSD is less than 3%, indicating that the test solution has good stability within 24 hours at room temperature.

[0079] 5.3 Intermediate precision experiment

[0080] Take an appropriate amount of anti-thrombotic regeneration pill powder, accurately weigh 0.3005g, 0.3008g, 0.3007g, 0.3006g, 0.3009g, 0.3001g, put it in a stoppered conical bottle, add 25mL of methanol, heat and reflux for 1h, cool, add weight, shake well, filter, and take the filtrate to obtain the test solution. Using Col.1 chromatographic column, the test solution was detected by another ultra-high performance liquid chromatograph, and the retention time and peak area RSD of peaks 1 to 11 were calculated. The results are shown in Tables 10 and 11.

[0081]

[0082]

[0083] It can be seen from the table that the RSD of the main peak retention time is less than 2%, and the RSD of the peak area is less than 2%, indicating that the intermediate precision is good.

[0084] 5.4 Durability test at different flow rates

[0085] Take an appropriate amount of anti-thrombotic regeneration pill powder, accurately weigh 0.3000g, put it in a stoppered conical flask, add 25mL of methanol to the conical flask, heat and reflux for 1h, cool, add weight, shake well, filter, and take the filtrate to obtain the test solution. Accurately aspirate the test solution and the reference peak solution, respectively, use the Col.1 chromatographic column, inject into the ultra-high performance liquid chromatograph, the flow rate is 0.2 mL / min, 0.3 mL / min and 0.32 mL / min, respectively, and the rest of the conditions are gradient eluted according to the chromatographic conditions provided, and record the chromatogram. Calculate the RSD of the retention time and peak area of ​​peaks 1 to 11, and the results are shown in Tables 12 and 13.

[0086]

[0087]

[0088] It can be seen from Tables 12 and 13 that the RSD of the retention time of the chromatographic peaks at different flow rates is less than 1%, and the RSD of the peak area is less than 4%, indicating that the flow rate has little effect on the retention time and peak area of ​​the chromatographic peaks.

[0089] 5.5 Durability test at different column temperatures

[0090] Take an appropriate amount of anti-thrombotic regeneration pill powder, accurately weigh 0.3000g, put it in a stoppered conical flask, add 25mL of methanol to the conical flask, heat and reflux for 1h, cool, add weight, shake well, filter, and take the filtrate to obtain the test solution. Accurately aspirate the test solution and the reference peak solution respectively, use the Col.1 chromatographic column, inject into the ultra-high performance liquid chromatograph, the column temperature is 29℃, 30℃ and 31℃ respectively, and the other conditions are gradient eluted according to the chromatographic conditions provided, and record the chromatogram. Calculate the retention time and peak area of ​​peaks 1 to 11, and the results are shown in Tables 14 and 15.

[0091]

[0092]

[0093] It can be seen from Tables 14 and 15 that the RSD of the chromatographic peak retention time under different column temperature conditions is less than 1%, and the RSD value of the peak area is less than 2%, indicating that the column temperature has little effect on the retention time and peak area of ​​the chromatographic peak.

[0094] 6. Qualitative study of double-label linear method

[0095] 6.1 Calculation of standard retention time and suitability of chromatographic columns

[0096] Take the test solution and measure it with 15 different chromatographic columns in Table 1 to obtain the measured retention times of 11 components. Calculate the average value to obtain the standard retention time (SRT) as the abscissa, which are peak 1: 12.167 min, peak 2: 13.907 min, peak 3: 15.395 min, peak 4: 16.256 min, peak 5: 22.324 min, peak 6: 31.898 min, peak 7: 33.792 min, peak 8: 50.789 min, peak 9: 53.465 min, peak 10: 55.449 min, peak 11: 56.499 min, with the measured retention time (t R ) as the ordinate to obtain the regression fitting curve of the chromatographic column, see Table 16. The linear fitting degree of the chromatographic column of the 11 components in the sample is good, and the correlation coefficients are all greater than 0.999, indicating that the linear relationship of the retention time of the 11 components on the chromatographic column is good.

[0097]

[0098] 6.2 Selection of double-labeled compounds and method optimization

[0099] The selection of double-labeled compounds is crucial to the prediction accuracy. The principle of selecting double-labeled compounds is to try to select compounds that are at both ends of the retention time of the target compound and are cheap and easy to obtain. Combined with the optimization results of the double-labeled linear retention, the combination of daidzein and physophanol methyl ether is used as the double-labeled compound, and the column compliance rate reaches 100%. Taking the Col.1 column as an example, the standard retention time of the above two double-labeled compounds is used as the horizontal coordinate, and the measured retention time (daidzein: 16.445min, physophanol methyl ether: 53.571 min) is used as the vertical coordinate, that is, points (16.256, 16.445) and (53.465, 53.571). Connect the two points to determine a straight line: Y=0.9978X+0.2247. Substituting the SRT of the remaining 9 compounds into the linear equation, the absolute deviations of the predicted retention times of puerarin (peak 1), peak 2, peak 3, peak 5, peak 6, peak 7, peak 8, peak 10, and peak 11 were 0.316, 0.167, 0.066, 0.023, 0.039, 0.022, 0.100, 0.137, and 0.117 min, respectively. The absolute deviations were much less than 0.5 min, which met the qualitative requirements, indicating that the double-label selection on the Col. 1 column had a good effect in predicting retention time. Similarly, the retention time deviations of the 9 compounds on other chromatographic columns were calculated, and the results are shown in Table 17.

[0100]

[0101] 6.3 Comparison between the dual-label linear calibration method and the relative retention time method

[0102] The average values ​​of the relative retention times of peaks 1 (puerarin), 2, 3, 5 and 4, and 6, 7, 8, 10, 11 and 9 in the 15 chromatographic columns were calculated, and then the absolute deviation of the predicted relative retention time was calculated. As shown in Table 17, the absolute deviation of the predicted sample retention time by most double-label linear calibration methods was smaller than that by the relative retention time method, and the maximum absolute deviation was smaller, indicating that the double-label linear calibration method is more accurate in predicting the retention time of compounds and has stronger applicability.

[0103] 6.4 Qualitative verification of double-label linearity

[0104] After the measured retention times of all the components of three batches of Kangshen Zaizao Pills were determined on the chromatographic column Col.1 by the mixed reference substance external standard method, the double-standard linear correction method was used to predict the components in the samples and verify the applicability of the method. The chromatograms were imported into the DRS Origin software, the double-standard retention times were extracted, and the retention times of other target components were predicted using the software. The results showed that the predicted retention times were compared with the measured retention times, and both achieved relatively ideal prediction effects, △T R The average time was <0.5min, and the relative deviation was <0.6%. The method was feasible. The results are shown in Table 18.

[0105]

[0106]

[0107] 7. Methodological investigation of the determination of index component content

[0108] 7.1 Investigation of Linear Relationship

[0109] Using the stepwise dilution method, accurately measure appropriate amounts of puerarin, daidzein, and physcion methyl ether reference solution, place them in a 10 mL volumetric flask, add methanol to make up the volume, and dilute to obtain concentrations of 138.29, 48.46, and 46.33 μg mL, respectively. -1 Puerarin and daidzein were diluted 2, 4, 8, and 16 times, and physcion was diluted 2, 4, 8, 16, and 32 times, respectively, to obtain a mixed reference solution with a concentration gradient. 1 μL was accurately drawn from each solution and the Col.1 column was used for determination under the above chromatographic conditions. X , μg·mL -1 ) is the horizontal axis, and the peak area ( Y ) was used as the vertical coordinate, and the standard curve was drawn. The regression equation and linear range of each component were calculated. The results are shown in Table 19.

[0110]

[0111] It can be seen from the table that the three components of puerarin, daidzein and physcion methyl ether show a good linear relationship within their respective concentration ranges.

[0112] 7.2 Repeatability test

[0113] Take an appropriate amount of anti-thrombotic regeneration pill powder, accurately weigh 0.3010g, 0.3002g, 0.3001g, 0.3005g, 0.3008g, 0.3002g, put it in a stoppered conical flask, add 25mL of methanol to the conical flask, heat and reflux for 1h, cool, add weight, shake well, filter, and take the filtrate to obtain the test solution. Accurately aspirate the test solution, use Col.1 chromatographic column, inject it into the ultra-high performance liquid chromatograph, and perform gradient elution according to the chromatographic conditions provided. The results are detailed in Table 20.

[0114]

[0115] Through analysis and determination, the RSDs of puerarin, daidzein and physcion in 6 samples were 0.26%, 0.39% and 0.51%, respectively, indicating that the method has good repeatability.

[0116] 7.3 Stability test

[0117] Take an appropriate amount of anti-thrombotic regeneration pill powder, accurately weigh 0.3000g, put it in a stoppered conical flask, add 25mL of methanol to the conical flask, heat and reflux for 1h, cool, add weight, shake well, filter, and take the filtrate to obtain the test solution. The test solution was injected at 0h, 2h, 4h, 8h, 12h, and 24h, the peak area was determined, and the chromatogram was recorded. The results are detailed in Table 21.

[0118]

[0119] It can be seen from the table that the RSDs of puerarin, daidzein and physcion at different times are 0.64%, 2.02% and 3.00%, respectively, indicating that the test solution is stable within 24 hours.

[0120] 7.4 Intermediate precision experiment

[0121] Take an appropriate amount of anti-thrombotic regeneration pill powder, accurately weigh 0.3005g, 0.3008g, 0.3007g, 0.3006g, 0.3009g, and 0.3001g, place in a stoppered conical bottle, add 25mL of methanol, heat and reflux for 1h, cool, add weight, shake well, filter, and take the filtrate to obtain the test solution. The test solution is detected by another ultra-high performance liquid chromatograph. The relative retention time and relative peak area of ​​the main chromatographic peaks are shown in Table 22.

[0122]

[0123] As can be seen from the table, the RSDs of puerarin, daidzein and physaccharin methyl ether contents were 0.64%, 0.44% and 0.93%, respectively, indicating that the intermediate precision was good.

[0124] 7.5 Sample recovery test

[0125] Take 6 portions of anti-thrombotic regeneration pill powder with known content, each portion is about 0.15g, weigh accurately, put into a conical flask, add appropriate amount of puerarin, daidzein, and physaccharin methyl ether reference substances according to 50% of the content of each component, prepare the test solution according to the preparation method of the test sample, inject the sample, and determine according to the above-mentioned chromatographic conditions. Calculate the sample recovery rate and RSD. The results are shown in Table 23.

[0126]

[0127] It can be seen from the table that the average recoveries of puerarin, daidzein and physaccharin methyl ether were 94.93%, 101.48% and 91.38%, respectively, and the RSDs were 0.93%, 1.07% and 1.66%, respectively.

[0128] 7.6 Durability test

[0129] 7.6.1 Investigation of different flow rates

[0130] Take an appropriate amount of anti-thrombotic regeneration pill powder, accurately weigh 0.3002g, put it in a stoppered conical flask, add 25mL of methanol to the conical flask, heat and reflux for 1h, cool, add weight, shake well, filter, and take the filtrate to obtain the test solution. Accurately aspirate the test solution and the reference peak solution respectively, inject them into the ultra-high performance liquid chromatograph, and the flow rates are 0.2mL / min, 0.3 mL / min and 0.32 mL / min respectively. The rest of the conditions are gradient eluted according to the chromatographic conditions provided, and the chromatogram is recorded. Calculate the difference in sample content at different flow rates, and the results are shown in Table 24.

[0131]

[0132] It can be seen from the table that the RSDs of puerarin, daidzein and physaccharin methyl ether at different flow rates are 1.13%, 1.35% and 1.46%, respectively, indicating that the flow rate has little effect on the sample content.

[0133] 7.6.2 Investigation of different column temperatures

[0134] Take an appropriate amount of anti-thrombotic regeneration pill powder, accurately weigh 0.3002g, put it in a stoppered conical flask, add 25mL of methanol to the conical flask, heat and reflux for 1h, cool, add weight, shake well, filter, and take the filtrate to obtain the test solution. Accurately aspirate the test solution and the reference peak solution respectively, inject them into the ultra-high performance liquid chromatograph, the column temperature is 29 ℃, 30 ℃ and 31 ℃ respectively, and the other conditions are gradient eluted according to the chromatographic conditions provided, and record the chromatogram. Calculate the difference in sample content under different column temperatures, and the results are shown in Table 25.

[0135]

[0136] It can be seen from the table that the RSDs of puerarin, daidzein and physaccharin methyl ether at different column temperatures are 1.21%, 0.37% and 0.53%, respectively, indicating that the column temperature has little effect on the sample content.

[0137] 7.6.3 Investigation of different chromatographic columns

[0138] Take an appropriate amount of anti-thrombotic regeneration pill powder, accurately weigh 0.3000g, put it in a stoppered conical flask, add 25mL of methanol to the conical flask, heat and reflux for 1h, cool, add weight, shake well, filter, and take the filtrate to obtain the test solution. The test solution was accurately aspirated and injected into the ultra performance liquid chromatograph, and gradient elution was performed according to the above chromatographic conditions. The following chromatographic columns were selected for investigation: Waters ACQUITY UPLC BEH C18 (2.1×100mm, 1.7μm), Agilent Eclipse PlusC18 (2.1×100mm, 1.8μm), Waters ACQUITYUPLC CSH C18 (2.1×100mm, 1.6μm), WatersACQUITY UPLC BEH Shield RP18 C18 (2.1×100mm, 1.7μm). The above chromatographic columns are respectively referred to as column 1, column 2, column 3, and column 4. The results are shown in Table 26.

[0139]

[0140] It can be seen from the table that the RSDs of puerarin, daidzein and physcion under different chromatographic columns are 1.77%, 1.55% and 1.50%, respectively, indicating that the different chromatographic columns have little effect on the sample content.

[0141] 7.7 Content determination

[0142] Take 3 batches of anti-thrombotic regeneration pill powder, prepare double samples for each batch, accurately weigh 0.3005g, 0.3002g, 0.3003g, 0.3002g, 0.3004g, 0.3001g, put them in a stoppered conical flask, add 25mL of methanol to the conical flask, heat and reflux for 1h, cool, add weight, shake well, filter, and take the filtrate to obtain the test solution. Accurately pipette the test solution and inject it into the ultra-high performance liquid chromatograph, measure the peak area, and record the chromatogram. The results are detailed in Table 27.

[0143]

[0144] As shown in the table, the contents of puerarin in the three batches of Kangshuan Zaizao Pills were 2.977, 2.654, and 3.563 mg·g -1 The contents of daidzein and daidzein were 0.693, 0.735 and 0.633 mg·g, respectively. -1 The contents of physalisin methyl ether were 0.365, 0.405 and 0.338 mg·g -1 .

[0145] The above specific implementations cannot be used as limitations on the scope of protection of the present invention. For those skilled in the art, any replacement, improvement or change made to the implementations of the present invention falls within the scope of protection of the present invention. The parts not described in detail in the present invention are all known technologies to those skilled in the art.

Claims

1. A method for establishing a high-performance liquid phase characteristic spectrum of anti-thrombotic regeneration pills based on a double-label linear method, characterized in that: The steps include: Preparation of S1 reference solution: accurately weigh appropriate amount of puerarin, daidzin and physcholin methyl ether reference substances, dissolve them in appropriate amount of 30% ethanol, 70% methanol and methanol respectively to prepare reference substance mother solution, then take 1ml of reference substance mother solution of puerarin, 0.5ml of daidzin and 3ml of physcholin methyl ether respectively in the same volumetric flask and add methanol to make up to volume to prepare mixed reference substance solution; Preparation of S2 test solution: accurately weigh an appropriate amount of anti-thrombotic regeneration pill powder, dissolve it in methanol, weigh it, reflux extract, weigh it, make up the weight with methanol, shake it well, filter it, take the filtrate, pass it through a 0.22μm microporous filter membrane, and take the filtrate as the test solution; Determination of S3 HPLC conditions: C18 chromatographic column, acetonitrile as mobile phase A, 0.05% phosphoric acid solution as mobile phase B, gradient elution conditions: 0-18min, 5%-12%A; 18-23min, 12% A; 23-24min, 12%-18%A; 24-39min, 18%-23%A; 39-43min, 23%-50%A; 43-75min, 50%-70%A; flow rate 0.2-0.32mL / min; column temperature 29-31℃; detection wavelength 286nm; injection volume 1μL; S4 HPLC determination: according to the chromatographic conditions in step S3, the chromatograms of the mixed reference solution and multiple batches of test solution are collected, and the chromatograms are analyzed to confirm that there are 11 peaks in total, and the characteristic peaks of 3 components are identified, among which peak 1 is puerarin, peak 4 is daidzein, and peak 9 is physaccharin methyl ether; S5 Determination of predicted retention time: Using multiple chromatographic columns of different brands and types, collect chromatograms of the mixed reference solution and the test solution according to the chromatographic conditions in step S3 to obtain standard retention times of 11 common peaks; using daidzein and physaccharin methyl ether as double-label reference substances, use double-label linear calibration method to obtain predicted retention times of the remaining peaks.

2. The method for establishing the high performance liquid phase characteristic spectrum of anti-thrombotic regeneration pills based on the double-label linear method according to claim 1, characterized in that: The specific method for preparing the test solution is as follows: accurately weigh 0.3 g of anti-thrombotic regeneration pellets, add 25 mL of methanol, weigh the weight, reflux extraction for 1 hour, weigh the weight, make up the weight with methanol, shake well, filter, take the filtrate, pass it through a 0.22 μm microporous filter membrane, and the filtrate is the test solution.

3. The method for establishing the high performance liquid phase characteristic spectrum of anti-thrombotic regeneration pills based on the double-label linear method according to claim 1, characterized in that: Accurately weigh appropriate amounts of puerarin, daidzein, and physcion methyl ether reference substances, and dissolve them in appropriate amounts of 30% ethanol, 70% methanol, and methanol, respectively, to prepare solutions with concentrations of 691.45, 484.62, and 77.22 μg•mL -1 The mother solution of the reference substance; take 1 ml of puerarin, 0.5 ml of daidzein and 3 ml of physaccharin methyl ether in the same 5 ml volumetric flask, add methanol to make up to 5 ml, and make the concentrations of 138.29, 48.46 and 46.33 μg•mL -1 of mixed reference solution.

4. The method for establishing the high performance liquid phase characteristic spectrum of anti-thrombotic regeneration pills based on the double-label linear method according to claim 1, characterized in that: In step S5, the specific method of using the double-label linear correction method to obtain the predicted retention time is: using the actual retention time of the chromatographic peaks of the double-label reference substances daidzein and physcion as the ordinate and the standard retention time as the abscissa to obtain a linear fitting equation; substituting the standard retention times of the remaining 9 characteristic peaks into the equation to obtain the corresponding predicted retention time; wherein the standard retention time is the average retention time of the test sample collected on multiple chromatographic columns.

5. The method for establishing the high performance liquid phase characteristic spectrum of anti-thrombotic regeneration pills based on the double-label linear method according to claim 1, characterized in that: In step S5, the standard retention times of the 11 peaks are 12.167 min, 13.907 min, 15.395 min, 16.256 min, 22.324 min, 31.898 min, 33.792 min, 50.789 min, 53.465 min, 55.449 min, and 56.499 min, respectively.

6. The method for establishing the high performance liquid phase characteristic spectrum of anti-thrombotic regeneration pills based on the double-label linear method according to claim 1, characterized in that: In step S3, the flow rate is selected to be 0.3 mL / min and the column temperature is selected to be 30°C.

7. A method for determining the content of ingredients in anti-thrombotic regeneration pills, characterized in that: The method comprises the following steps: performing liquid chromatography determination according to steps S1-S3 of the method established in claims 1-6 to obtain a chromatogram, and calculating the contents of puerarin, daidzein and physcion by peak area based on an external standard method.

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