A method for establishing gas phase chromatogram of anti-thrombus reconstituted pill and a method for determining effective component content

Abstract

The application relates to a method for establishing a gas phase characteristic map of an anti-thrombus reconstituted pill and a method for determining the content of effective components, the method for establishing the gas phase characteristic map of the anti-thrombus reconstituted pill comprises the following steps: S1, preparation of a reference solution; S2, preparation of a test solution; S3, gas phase determination, chromatographic conditions: an HP-5 chromatographic column is adopted, nitrogen is used as carrier gas, the flow rate is 1.0 mL.min ‑1 , the constant flow rate, the split ratio is 10:1; the injection amount is 1 muL, the injection port temperature is 200 DEG C; the gradient temperature rising condition: the initial temperature is 100 DEG C and maintained for 10 min; then the temperature is raised to 170 DEG C at a speed of 10 DEG C.min ‑1 , and maintained for 10 min; then the temperature is raised to 250 DEG C at a speed of 3 DEG C.min ‑1 , and maintained for 10 min; the detector temperature is 270 DEG C, the air flow rate is 300 mL.min ‑1 , the hydrogen flow rate is 30 mL.min ‑1 , and the tail blow flow rate is 15 mL.min ‑1 ; S4, chromatograms of the reference solution and multiple batches of test solutions are collected, chromatograms of the multiple batches of test solutions are analyzed, 10 common peaks are determined, and characteristic peaks of 5 components are identified. The method is beneficial to comprehensively and effectively controlling the quality of the anti-thrombus reconstituted pill.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical testing, and in particular to a method for establishing a gas phase characteristic spectrum of an antithrombotic reconstructive pill and a method for determining the content of its active ingredients. Background Technology

[0002] Antithrombotic Regeneration Pills are composed of various ingredients including red ginseng, astragalus, arisaema cum bile, pangolin scales (processed), bezoar, borneol, leech (processed), musk, salvia miltiorrhiza, notoginseng, rhubarb, earthworm, styrax, scorpion, kudzu root, pangolin scales, angelica, achyranthes, polygonum multiflorum, zaocys dhumnades, peach kernel, cinnabar, safflower, ground beetle, gastrodia elata, asarum, clematis chinensis, cardamom, and licorice. They have the effects of promoting blood circulation and removing blood stasis, relaxing muscles and tendons, and calming wind and relieving spasms. They can be used to treat symptoms such as numbness in the hands and feet, difficulty walking, paralysis, facial paralysis, and slurred speech during the recovery period of stroke sequelae.

[0003] The chemical composition of antithrombotic reconstructive pills is complex. Currently, the quality standards for antithrombotic reconstructive pills mainly rely on thin-layer chromatography identification. There is no established method or standard for characteristic chromatographic examination, which cannot comprehensively reflect the quality of antithrombotic reconstructive pills and cannot guarantee comprehensive quality control. Summary of the Invention

[0004] To overcome the shortcomings of the prior art, this invention provides a method for establishing the gas phase characteristic spectrum of antithrombotic reconstructive pills and a method for determining the content of effective components, which is beneficial for more comprehensive and effective control of the quality of antithrombotic reconstructive pills.

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

[0006] A method for establishing the gas phase characteristic spectrum of antithrombotic reconstituted pellets includes the following steps:

[0007] Preparation of S1 reference solution: Accurately weigh appropriate amounts of isoborneol, borneol, benzyl cinnamate, 3-phenylpropanol cinnamate, and cinnamate reference standards, and dissolve them in an appropriate amount of anhydrous ethanol to prepare reference solutions of a certain concentration.

[0008] Preparation of S2 test solution: Accurately weigh an appropriate amount of antithrombotic reconstructive pill powder, dissolve it in methanol, weigh it, extract it by ultrasound, weigh it, add methanol to make up the weight, shake it well, filter it, take the filtrate, filter it through a 0.22μm microporous membrane, and take the filtrate to obtain the test solution.

[0009] S3 was analyzed by gas chromatography under the following conditions: an HP-5 capillary column was used, nitrogen was used as the carrier gas, and the flow rate was 1.0 mL / min. -1 Constant flow rate, split ratio of 10:1; injection volume of 1 μL, injection port temperature of 200℃; gradient temperature conditions: initial temperature of 100℃, maintained for 10 min; then increased at 10℃·min. -1The temperature was increased to 170℃ at a rate of [temperature] and maintained for 10 min; then increased at a rate of 3℃·min. -1 The temperature was increased to 250℃ and maintained for 10 min; the detector temperature was 270℃ and the air flow rate was 300 mL / min. -1 Hydrogen flow rate 30 mL / min -1 The tail gas flow rate is 15 mL·min -1 ;

[0010] S3 collected chromatograms of the reference solution and multiple batches of test solutions, and imported the multiple batches of test solutions into the Chinese medicine chromatographic fingerprint similarity evaluation system for analysis. There were a total of 10 peaks, and the characteristic peaks of 5 components were identified. The characteristic chromatogram of the antithrombotic regeneration pill can be obtained by averaging algorithm.

[0011] Furthermore, in step S2, the specific preparation method of the test solution is as follows: accurately weigh 0.2g of antithrombotic reconstructive pill powder, add 5mL of methanol, weigh, extract ultrasonically for 1h, weigh, add methanol to make up the weight, shake well, filter, take the filtrate, and filter it through a 0.22μm microporous membrane to obtain the solution.

[0012] Furthermore, in step S1, the specific preparation method of the reference solution is as follows: accurately weigh appropriate amounts of isoborneol, borneol, benzyl cinnamate, 3-phenylpropyl cinnamate, and cinnamyl cinnamate reference standards, dissolve them separately in an appropriate amount of anhydrous ethanol, and prepare solutions with concentrations of 1.6340, 1.7609, 0.9857, 21.3836, and 0.9869 mg·mL. -1 The reference solution.

[0013] Furthermore, among the five characteristic peaks in the gas phase characteristic spectrum of the antithrombotic pill, peak 1 is isoborneol, peak 2 is borneol, peak 6 is benzyl cinnamate, peak 7 is 3-phenylpropanoic cinnamate, and peak 9 is cinnamate.

[0014] Furthermore, using peak 7 (3-phenylpropyl cinnamic acid) as the characteristic peak, the relative retention times of other peaks were: 0.180, 0.188, 0.503, 0.519, 0.724, 0.840, 1.009, 1.069, and 1.128, with relative deviations within ±5%.

[0015] Furthermore, the HP-5 capillary column has dimensions of 30m × 250μm and a diameter of 0.25μm.

[0016] This application also provides a method for determining the content of components in antithrombotic reconstructive pills. The method involves performing gas chromatography according to steps S1-S3 of the method to obtain a chromatogram, and calculating the content of the active ingredient to be measured based on the peak area using the external standard method.

[0017] The advantages of this invention are:

[0018] 1. The method for establishing the high-performance liquid chromatography characteristic spectrum of the antithrombotic reconstructive pill in this application, using isoborneol, borneol, benzyl cinnamate, 3-phenylpropionic acid cinnamate, and cinnamyl cinnamate as research indicators, conducted gas phase characteristic spectrum research, and provided the corresponding characteristic spectrum establishment method and effective component determination method, providing a reference and basis for the quality control of the antithrombotic reconstructive pill.

[0019] 2. This method has advantages such as simplicity, stability, high precision, and good reproducibility.

[0020] 3. The characteristic spectrum obtained by the method established in this application has 10 effectively separated characteristic peaks. Therefore, the obtained characteristic spectrum can effectively characterize the quality of the antithrombotic pill, which is conducive to the comprehensive monitoring of drug quality. Attached Figure Description

[0021] Figure 1 The gas phase characteristic spectrum of the antithrombotic reconstituted pellet established for this invention;

[0022] Figure 2 This is a specificity analysis diagram of the gas phase characteristic spectrum of antithrombotic reconstituted pellets;

[0023] Figure 3 These are the gas phase characteristic spectra of multiple batches of antithrombotic reconstructive pill sample solutions. Detailed Implementation

[0024] To clearly illustrate the technical features of this solution, the invention will be described in detail below through specific embodiments and in conjunction with the accompanying drawings. Many specific details are set forth in the following description to provide a thorough understanding of this application; however, this application may also be implemented in other ways different from those described herein. Therefore, the scope of protection of this application is not limited to the specific embodiments disclosed below.

[0025] 1. Instruments and reagents

[0026] 7890B Gas Chromatograph, Agilent Technologies; XS105DU 0.0001 ppm Electronic Analytical Balance, METTLERTOLEDO; BSA224S-CW 0.0001 ppm Electronic Analytical Balance, Sartorius Scientific Instruments (Beijing) Co., Ltd.; KQ-800VSM Digital Display Ultrasonic Cleaner, Kunshan Ultrasonic Instruments Co., Ltd.

[0027] Isoborneol reference substance (batch number CHB231212, content 95%, Chengdu Keluoma), borneol reference substance (batch number 110881-201709, content 99.6%, China Food and Drug Control Institute), benzyl cinnamate reference substance (batch number 180067-202302, content 98.08%, Shanghai Zerui-Hongyong), cinnamyl cinnamate-3-phenylpropyl ester reference substance (batch number CHB240125, content 98.0%, Chengdu Keluoma), cinnamyl cinnamate reference substance (batch number 13586, content 98.0%, Shanghai Zerui-Shidandeng); pure water, Hangzhou Wahaha Group Co., Ltd.; methanol, AR grade, batch number: 20230516, Tianjin Fuyu Fine Chemical Co., Ltd.; anhydrous ethanol, AR grade, batch number: 20230408, Tianjin Fuyu Fine Chemical Co., Ltd.

[0028] 2. Method for establishing gas phase characteristic spectrum of anti-thrombus reconstituted pills

[0029] Preparation of reference substance solution: accurately weigh appropriate amounts of isoborneol, borneol, benzyl cinnamate, cinnamyl cinnamate-3-phenylpropyl ester and cinnamyl cinnamate reference substances, respectively, dissolve in appropriate amounts of anhydrous ethanol to prepare reference substance solutions with concentrations of 1.6340, 1.7609, 0.9857, 21.3836 and 0.9869 mg·mL -1 .

[0030] Preparation of test substance solution: accurately weigh 0.2 g of anti-thrombus reconstituted pill powder, add 5 mL of methanol, weigh, ultrasonically extract for 1 h, weigh, supplement with methanol, shake well, filter, take the filtrate, pass through a 0.22 μm microporous filter membrane, and obtain.

[0031] The chromatographic conditions for gas chromatographic determination of the test substance solution and the reference substance solution are as follows: use an HP-5 (30 m x 250 μm, 0.25 μm) capillary column, use nitrogen as the carrier gas, the flow rate is 1.0 mL·min -1 , constant flow, the split ratio is 10:1; the injection volume is 1 μL, the injection port temperature is 200℃; the gradient temperature conditions are: the initial temperature is 100℃, maintain for 10 min; then increase the temperature to 170℃ at a rate of 10℃·min -1 , maintain for 10 min; then increase the temperature to 250℃ at a rate of 3℃·min -1 , maintain for 10 min; the detector temperature is 270℃, the air flow rate is 300 mL·min -1 , the hydrogen flow rate is 30 mL·min -1 , and the tail gas flow rate is 15 mL·min -1 .

[0032] From the gas chromatographic determination of a single reference substance solution (see Figure 2), of the five compounds, cinnamic acid-3-phenylpropyl ester in the content of anti-thrombotic pills is more stable, and the retention time in the atlas is moderate, and the separation degree is better, so the characteristic peak of cinnamic acid-3-phenylpropyl ester is selected as the reference peak. The mixed reference solution and the test solution are determined by gas chromatography, and it is confirmed that there are 10 common peaks, and the characteristic atlas of anti-thrombotic pills is obtained by average value algorithm, which is shown in Figure 1 . Five characteristic peaks are identified from Figure 1 , in which peak No. 1 is isoborneol, peak No. 2 is borneol, peak No. 6 is benzyl cinnamate, peak No. 7 is cinnamic acid-3-phenylpropyl ester, and peak No. 9 is cinnamyl cinnamate.

[0033] 3. Optimization experiment of test solution preparation conditions

[0034] 3.1 Selection of extraction method

[0035] Take anti-thrombotic pills powder, accurately weigh two portions of 0.2000g and 0.2011g, respectively, and place them in a conical flask with a stopper. Precisely add 5mL of methanol, investigate ultrasonic extraction for 15min and shaking extraction for 15min, cool, supplement weight, shake well, filter, take the filtrate as the test solution. Accurately weigh 1μL of each of the above two test solutions, inject into the gas chromatograph, record the chromatogram for 64min, and take the number of chromatographic peaks and the peak area of the main peak as the evaluation index. The results are shown in Table 1.

[0036] Table 1 Extraction method investigation results (peak area)

[0037]

[0038] From the results in Table 1, it can be seen that the peak area of the main peak of the ultrasonic sample is basically greater than that of the shaking sample, so the ultrasonic extraction method is selected.

[0039] 3.2 Investigation of extraction solvent

[0040] Take anti-thrombotic pills powder, accurately weigh four portions of 0.2005g, 0.2009g, 0.2002g, 0.2008g and 0.2004g, respectively, and place them in a conical flask with a stopper. Precisely add 5mL of ethyl acetate, diethyl ether, anhydrous ethanol, methanol and petroleum ether, respectively, ultrasonic extraction for 15min, investigate different solvents, cool, supplement weight, shake well, filter, take the filtrate as the test solution. Accurately weigh 1μL of each of the above five test solutions, inject into the gas chromatograph, record the chromatogram for 64min, and analyze with the number of chromatographic peaks and the peak area of the main peak as the evaluation index. The results are shown in Table 2.

[0041] Table 2 Extraction solvent investigation results (peak area)

[0042]

[0043] From the results of Table 2, it can be seen that the peak area of the main peak of methanol as the extraction solvent is greater than that of the other extraction solvents, and the response values of each peak are relatively high. Therefore, methanol is selected as the extraction solvent.

[0044] 3.3 Selection of extraction time

[0045] An appropriate amount of anti-thrombotic reconstituted pill powder was precisely weighed into four portions, 0.2000 g, 0.1999 g, 0.2004 g and 0.2003 g, respectively, and placed in a conical flask with a stopper. Methanol 5 mL was precisely added, and ultrasonic extraction was performed for 15 min, 30 min and 45 min, respectively. After cooling, the weight was supplemented, and the mixture was shaken and filtered. The filtrate was collected as the test sample solution. 1 μL of each of the above three test sample solutions was precisely weighed and injected into a gas chromatograph. The chromatogram was recorded for 64 min. The number of chromatographic peaks and the peak area of the main peak were used as evaluation indexes for analysis. The results are shown in Table 3.

[0046] Table 3 Results of extraction time investigation (peak area)

[0047]

[0048] From the results of Table 3, it can be seen that the response of ultrasonic extraction for 15 min is higher than that of 30 min and 45 min. Considering comprehensively, the extraction time of 15 min is selected.

[0049] 3.4 Selection of sample weight

[0050] An appropriate amount of anti-thrombotic reconstituted pill powder was precisely weighed into three portions, 0.1004 g, 0.2003 g and 0.3007 g, respectively, and placed in a conical flask with a stopper. Methanol 5 mL was precisely added, and ultrasonic extraction was performed for 15 min. Different sample weights were investigated. After cooling, the weight was supplemented, and the mixture was shaken and filtered. The filtrate was collected as the test sample solution. 1 μL of each of the above three test sample solutions was precisely weighed and injected into a gas chromatograph. The chromatogram was recorded for 64 min. The number of chromatographic peaks and the peak area of the main peak were used as evaluation indexes for analysis. The results are shown in Table 4.

[0051] Table 4 Results of sample weight investigation (peak area)

[0052]

[0053] From the results of Table 4, it can be seen that the response of 0.1 g sample weight is low and the error is large. The extraction effect of 0.2 g is better than that of 0.3 g, and the response of 0.2 g sample weight is higher than that of 0.1 g. Therefore, the sample weight of 0.2 g is selected.

[0054] Based on the above test results, the specific preparation method of the characteristic fingerprint of the test solution of the Anti-stasis Reconstructed Pill is as follows: 0.2 g of the Anti-stasis Reconstructed Pill powder is precisely weighed, 5 mL of methanol is added, the weight is recorded, ultrasonic extraction is performed for 1 h, the weight is recorded again, methanol is added to make up the weight, it is shaken uniformly, filtered, the filtrate is taken, and 0.22 μm microporous filter membrane is used for filtration, and the filtrate is obtained.

[0055] 4. Methodology investigation of the characteristic fingerprint establishment method

[0056] 4.1 Reproducibility test

[0057] The Anti-stasis Reconstructed Pill powder is taken in an appropriate amount, 0.2005 g, 0.2007 g, 0.2004 g, 0.2006 g, 0.2001 g and 0.2008 g are precisely weighed, and are placed in a conical flask with a stopper. Methanol is precisely added into the conical flask, 5 mL, ultrasonic treatment is performed for 15 min, cooling is performed, the weight is made up, shaking is uniformly performed, filtration is performed, the filtrate is taken, and 0.22 μm microporous filter membrane is used for filtration, and the filtrate is obtained. The test sample solution and the reference peak solution are precisely taken, are injected into a gas chromatograph, gradient elution is performed according to the provided chromatographic conditions, and a chromatogram is recorded. The relative retention time and the relative peak area of the main chromatographic peaks are calculated, and the results are shown in Tables 5 and 6.

[0058] Table 5 Reproducibility investigation results (relative retention time)

[0059]

[0060]

[0061] Table 6 Reproducibility investigation results (relative peak area)

[0062]

[0063] As can be seen from Tables 5 and 6, the RSD of the relative retention time is less than 2%, and the RSD of the relative peak area is less than 3%, which indicates that the method has good reproducibility.

[0064] 4.2 Solution stability test

[0065] The Anti-stasis Reconstructed Pill powder is taken in an appropriate amount, 0.2009 g is precisely weighed, and is placed in a conical flask with a stopper. Methanol is added into the conical flask, 5 mL, ultrasonic treatment is performed for 15 min, cooling is performed, the weight is made up, shaking is uniformly performed, filtration is performed, the filtrate is taken, and 0.22 μm microporous filter membrane is used for filtration, and the filtrate is obtained. The test sample solution is injected at 0 h, 2 h, 4 h, 8 h, 12 h, 16 h, 24 h and 30 h, a chromatogram is recorded, 8 time points are determined, the relative retention time and the relative peak area of the main chromatographic peaks are calculated, and the results are shown in Tables 7 and 8.

[0066] Table 7 Stability investigation results (relative retention time)

[0067]

[0068] Table 8 Stability Investigation Results (Relative Peak Area)

[0069]

[0070] The results of Tables 7 and 8 show that the RSD of relative retention time is less than 2%, and the RSD of relative peak area is less than 3%, indicating that the test solution is stable at room temperature for 30 hours.

[0071] 4.3 Intermediate Precision Experiment

[0072] Take an appropriate amount of anti-thrombotic reconstituted pill powder, accurately take 0.2005 g, 0.2008 g, 0.2007 g, 0.2006 g, 0.2009 g, and 0.2001 g, and place them in a conical flask with a stopper. Add 5 mL of methanol and ultrasonically treat for 15 min. Cool, supplement the weight, shake well, filter, and take the filtrate to obtain the test solution. The test solution is detected by another gas chromatograph (Agilent 7890B). The results of the relative retention time and the relative peak area of the main chromatographic peaks are shown in Tables 9 and 10.

[0073] Table 9 Precision Experiment Investigation Results (Relative Retention Time)

[0074]

[0075] Table 10 Precision Experiment Investigation Results (Relative Peak Area)

[0076]

[0077]

[0078] As shown in the tables, the RSD of the relative retention time of the main peak is less than 2%, and the RSD of the relative peak area is less than 3%, indicating that the intermediate precision is good.

[0079] 4.4 Different Chromatographic Columns

[0080] Take anti-thrombotic pill powder, accurately take 0.2006g, put into a conical flask with a stopper, add methanol 5mL, ultrasonic treatment for 15min, cool, supplement weight, shake evenly, filter, take the filtrate for test sample solution. Respectively, accurately take the test sample solution into the gas chromatograph, according to the above chromatographic conditions, gradient elution was carried out, and the following chromatographic column was used for investigation: HP-5(30x0.25mm, 0.25μm, SN:USN440242H), HP-5(30x0.25mm, 0.25μm, SN:USN465136J), HP-5MS(30x0.25mm, 0.25μm, SN:UST118632H), the above chromatographic column is abbreviated as chromatographic column 1, chromatographic column 2, chromatographic column 3. Calculate the relative retention time of the main chromatographic peak, and the results are shown in Table 11.

[0081] Table 11 Results of durability investigation of different chromatographic columns (relative retention time)

[0082]

[0083] As can be seen from the table, the retention time of chromatographic column 2 and chromatographic column 3 is within ±10% of the retention time of chromatographic column 1, indicating that the method has good reproducibility between different chromatographic columns.

[0084] 5. Methodology investigation of index component content determination

[0085] 5.1. Specificity investigation

[0086] According to the prescription ratio, except for borneol, the rest of the medicinal materials are prepared into borneol negative sample solution according to the pharmacopoeia method; according to the prescription ratio, except for styrax, the rest of the medicinal materials are prepared into styrax negative sample according to the pharmacopoeia method, and methanol, borneol negative sample, styrax negative sample and test sample solution are injected into the gas chromatograph, and the results are shown in Figure 2 .

[0087] As can be seen from the figure, the borneol negative sample has no isoborneol and borneol, and the styrax negative sample has no benzyl cinnamate, cinnamic acid-3-phenylpropyl ester and cinnamic acid cinnamate, and the method has good specificity.

[0088] 5.2. Investigation of linear relationship

[0089] Using stepwise dilution method, accurately take a certain amount of isoborneol, borneol, benzyl cinnamate, cinnamic acid-3-phenylpropyl ester and cinnamic acid cinnamate reference substance solution into 10mL volumetric flask, add anhydrous ethanol to constant volume, dilute to get the concentration of 490.2, 1760.93, 49.29, 534.59, 986.86μg·mL -1The control sample diluent was diluted 2, 4, 8, 16, and 32 times, respectively, to obtain 6 concentration gradient mixed control sample solutions, 1 μL of each was precisely pipetted, and the determination was performed according to the above chromatographic conditions. The standard curve was plotted with the control sample concentration (X, μg·mL -1 ) as the abscissa and the peak area (Y) as the ordinate. The regression equation and linear range of each component were calculated, and the results are shown in Table 12.

[0090] Table 12 Linear relationship investigation results

[0091]

[0092] As can be seen from the table, isoborneol, borneol, benzyl cinnamate, cinnamic acid-3-phenylpropyl ester, and cinnamic acid cinnamyl ester showed good linear relationships within their respective concentration ranges.

[0093] 5.3 Reproducibility test

[0094] 0.2005 g, 0.2007 g, 0.2004 g, 0.2006 g, 0.2001 g, and 0.2008 g of the anti-thrombus reconstituted pill powder were precisely weighed into 6 conical flasks with stoppers, 5 mL of methanol was added to each conical flask, and ultrasonic treatment was performed for 15 min. After cooling, the weight was supplemented, and the mixture was shaken and filtered. The filtrate was collected to obtain the test sample solution. The test sample solution was precisely pipetted into a gas chromatograph, gradient elution was performed according to the provided chromatographic conditions, and the results are shown in Table 13.

[0095] Table 13 Reproducibility test

[0096]

[0097]

[0098] Through analysis and determination, the RSDs of the contents of isoborneol, borneol, benzyl cinnamate, cinnamic acid-3-phenylpropyl ester, and cinnamic acid cinnamyl ester in the 6 samples were 1.89%, 1.96%, 1.07%, 0.35%, and 1.02%, respectively, indicating that the method had good reproducibility.

[0099] 5.4 Stability test

[0100] An appropriate amount of the anti-thrombus reconstituted pill powder was precisely weighed, 0.2009 g was precisely weighed into a conical flask with a stopper, 5 mL of methanol was added to the conical flask, ultrasonic treatment was performed for 15 min, the weight was supplemented after cooling, the mixture was shaken and filtered, and the filtrate was collected to obtain the test sample solution. The test sample solution was injected into the gas chromatograph at 0 h, 2 h, 4 h, 8 h, 12 h, 16 h, 24 h, and 30 h, the peak area was determined, and the chromatogram was recorded. The results are shown in Table 14.

[0101] Table 14 Stability test results

[0102]

[0103]

[0104] From the table, the RSD of isoborneol, borneol, benzyl cinnamate, cinnamic acid-3-phenylpropyl ester, cinnamyl cinnamate was 2.20%, 1.29%, 1.11%, 0.62%, 0.90% respectively, indicating that the test solution was stable within 30h.

[0105] 5.5 Intermediate precision experiment

[0106] Take anti-thrombotic reconstituted pill powder, accurately weigh 0.2005g, 0.2008g, 0.2007g, 0.2006g, 0.2009g, 0.2001g, and place them in a conical flask with a stopper. Add 5mL of methanol, ultrasonic treatment for 15min, cool, supplement weight, shake evenly, filter, and take the filtrate to obtain the test solution. The test solution was detected by another gas chromatograph (Agilent 7890B), and the content and RSD were calculated according to the above chromatographic conditions. See Table 15 for details.

[0107] Table 15 Intermediate precision experiment content determination results

[0108]

[0109]

[0110] From the table, the RSD of isoborneol, borneol, benzyl cinnamate, cinnamic acid-3-phenylpropyl ester, cinnamyl cinnamate was 1.20%, 0.42%, 0.56%, 1.57%, 0.86%, indicating good intermediate precision.

[0111] 5.6 Sample recovery experiment

[0112] Take 6 portions of anti-thrombotic reconstituted pill powder with a known content of about 0.1g each, accurately weigh, and place them in a conical flask. Accurately add the appropriate amount of borneol, benzyl cinnamate, cinnamic acid-3-phenylpropyl ester, cinnamyl cinnamate reference substance according to 50% of the content of each component, and prepare the test solution according to the test preparation method. Inject into the gas chromatograph, and determine according to the above chromatographic conditions. Calculate the sample recovery rate and RSD, and the results are shown in Table 16.

[0113] Table 16 Sample recovery experiment

[0114]

[0115]

[0116] From the table, the average sample recovery rates of isoborneol, borneol, benzyl cinnamate, 3-phenylpropyl cinnamate, and cinnamyl cinnamate were 93.08%, 90.62%, 106.00%, 91.48%, and 101.14%, respectively, and the RSDs were 2.58%, 0.65%, 3.45%, 0.88%, and 1.23%, respectively, which met the requirements of the Pharmacopoeia.

[0117] 5.7 Different chromatographic column durability test

[0118] Take the anti-thrombotic reconstructed pill powder, precisely weigh 0.2006 g, and place it in a conical flask with a stopper. Add 5 mL of methanol to the conical flask, ultrasonically treat for 15 min, cool, supplement the weight, shake well, filter, and take the filtrate to obtain the test sample solution. Precisely inject the test sample solution into the gas chromatograph, perform gradient elution according to the above chromatographic conditions, and use the following chromatographic columns for investigation: HP-5 (30 x 0.25 mm, 0.25 μm, SN: USN440242H), HP-5 (30 x 0.25 mm, 0.25 μm, SN: USN465136J), and HP-5MS (30 x 0.25 mm, 0.25 μm, SN: UST118632H). The above chromatographic columns are referred to as chromatographic column 1, chromatographic column 2, and chromatographic column 3, respectively. Measure the peak area, record the chromatogram, and the results are shown in Table 17.

[0119] Table 17 Content determination under different chromatographic columns

[0120]

[0121]

[0122] From the table, the content differences of isoborneol, borneol, benzyl cinnamate, 3-phenylpropyl cinnamate, and cinnamyl cinnamate under different chromatographic columns had RSDs less than 2%, indicating that the method had good reproducibility between different chromatographic columns.

[0123] 6 Content determination of three batches of anti-thrombotic reconstructed pills

[0124] Take the powder of three batches of anti-thrombotic reconstructed pills, prepare double samples for each batch, precisely weigh 0.2003 g, 0.2003 g, 0.2004 g, 0.2005 g, 0.2004 g, and 0.2007 g, and place them in conical flasks with stoppers. Add 5 mL of methanol to the conical flasks, ultrasonically treat for 15 min, cool, supplement the weight, shake well, filter, and take the filtrate to obtain the test sample solution. Precisely inject the test sample solution into the gas chromatograph, measure the peak area, and record the chromatogram. The results are shown in Table 18.

[0125] Table 18 Content determination of three batches of anti-thrombotic reconstructed pills

[0126]

[0127]

[0128] From the table, the contents of isoborneol in the three batches of anti-thrombus reconstituted pills are 2.849, 2.999 and 2.498 mg·g -1 , the contents of borneol are 6.701, 6.526 and 6.141 mg·g -1 , the contents of benzyl cinnamate are 0.170, 0.201 and 0.219 mg·g -1 , the contents of cinnamic acid-3-phenylpropyl ester are 1.573, 1.292 and 1.402 mg·g -1 , the contents of cinnamic acid cinnamyl ester are 3.843, 3.823 and 4.033 mg·g -1 .

[0129] The above specific embodiments cannot be regarded as the limitation of the protection scope of the present application, and any alternative improvement or transformation made by the skilled in the art to the embodiments of the present application falls within the protection scope of the present application. The unexplained part of the present application is the known technology of the skilled in the art.

Claims

1. A method for establishing a gas phase pattern of an anti-thrombus reconstituted pill, characterized in that, It comprises the following steps: S1 Preparation of reference solution: accurately weigh a certain amount of isoborneol, borneol, benzyl cinnamate, 3-phenylpropyl cinnamate and cinnamyl cinnamate reference substances, respectively, add a certain amount of anhydrous ethanol to dissolve and prepare a certain concentration of reference solution; S2 Preparation of test solution: accurately weigh a certain amount of anti-stenosis reconstituted pill powder, dissolve in methanol, weigh, ultrasonic extraction, weigh, supplement with methanol, shake well, filter, take the filtrate, pass through a 0.22 μm microporous filter membrane, and take the filtrate to obtain the test solution; S3 was determined by gas chromatography, and the chromatographic conditions were as follows: an HP-5 capillary column was used, nitrogen was used as the carrier gas, the flow rate was 1.0 mL·min -1 , the constant flow rate, the split ratio was 10:1; the injection amount was 1 μL, the injection port temperature was 200℃; the gradient temperature rising condition was: the initial temperature was 100℃, maintained for 10 min; then increased to 170℃ at a rate of 10℃·min -1 , maintained for 10 min; then increased to 250℃ at a rate of 3℃·min -1 , maintained for 10 min; the detector temperature was 270℃, the air flow rate was 300 mL·min -1 , the hydrogen flow rate was 30 mL·min -1 , and the tail gas flow rate was 15 mL·min -1 ; S4 Collect the chromatograms of the reference solution and multiple batches of test solution, analyze the chromatograms of the multiple batches of test solution, determine 10 common peaks, and identify the characteristic peaks of 5 components.

2. The method for establishing the gas phase fingerprint spectrum of anti-thrombus remanufactured pills according to claim 1, characterized in that, In step S2, the specific preparation method of the test solution is as follows: accurately weigh 0.2 g of anti-stenosis reconstituted pill powder, add 5 mL of methanol, weigh, ultrasonic extraction for 1 h, weigh, supplement with methanol, shake well, filter, take the filtrate, pass through a 0.22 μm microporous filter membrane, and obtain the filtrate.

3. The method for establishing the gas phase fingerprint spectrum of anti-thrombus remanufactured pills according to claim 1, characterized in that, The specific preparation method of the control solution in step S1 is as follows: accurately weigh appropriate amount of isoborneol, borneol, benzyl cinnamate, 3-phenylpropyl cinnamate and cinnamyl cinnamate control samples, respectively, add appropriate amount of anhydrous ethanol to dissolve, and prepare control solutions with concentrations of 1.6340, 1.7609, 0.9857, 21.3836 and 0.9869 mg·mL -1 , respectively.

4. The method for establishing the gas phase fingerprint spectrum of anti-thrombus remanufactured pills according to claim 1, characterized in that, Among the 5 characteristic peaks of the gas chromatographic characteristic spectrum of anti-stenosis reconstituted pill, peak 1 is isoborneol, peak 2 is borneol, peak 6 is benzyl cinnamate, peak 7 is 3-phenylpropyl cinnamate, and peak 9 is cinnamyl cinnamate.

5. The method for establishing the gas phase fingerprint spectrum of anti-thrombus remanufactured pills according to claim 4, characterized in that, Taking peak 7, 3-phenylpropyl cinnamate, as the reference peak, the relative retention times of other peaks are: 0.180, 0.188, 0.503, 0.519, 0.724, 0.840, 1.009, 1.069, 1.128, and the relative deviation is within ±5%.

6. The method for establishing the gas phase fingerprint spectrum of anti-thrombus remanufactured pills according to claim 1, characterized in that, The specifications of the HP-5 capillary chromatographic column are 30 m x 250 μm, 0.25 μm.

7. A method for determining the content of effective components of an anti-thrombotic reconstituted pill, characterized by, According to steps S1-S3 of the method of claims 1-6, gas chromatography is determined to obtain a chromatogram, and the content of the measured effective component is calculated based on the external standard method through peak area.

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