A method for improving the stability of determination results of tanshinone ⅡA content in Xinmuan tablets

Abstract

The application provides a preparation method of a sample solution for determining the content of tanshinone IIA in Xinyuan tablets, which comprises the following steps: 10 tablets of the product are taken, the film coating is removed, and the tablets are precisely weighed, finely ground, and taken as one tablet, which is precisely weighed, 30 ml of diethyl ether is added, and the mixture is shaken for 5 minutes, and then is placed in dark for 24 hours, filtered through nylon cloth, and the filter residue is washed with diethyl ether for 3 times, each time being 5-20 ml, and the mixture is shaken and filtered through the same nylon cloth, the diethyl ether solutions are combined, and are evaporated at room temperature until dry, the residue is dissolved in proper amount of methanol, and is quickly transferred into a 10 ml brown volumetric flask, which is shaken and filtered, and the sample solution is obtained. The application also provides a method for determining the content of tanshinone IIA in Xinyuan tablets based on the above preparation method.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of analytical chemistry and pharmaceutical quality control, and particularly relates to a method for improving the stability of determination results of tanshinone IIA in Xin'an tablets. BACKGROUND

[0002] Xin'an tablets are a new compound traditional Chinese medicine developed according to the prescription of Dr. Su Shurong, a famous traditional Chinese doctor in Shanghai. After years of application in Shanghai Huangpu District Central Hospital and other hospitals, it has been proved that Xin'an tablets have good effects on treating arrhythmia caused by coronary heart disease, such as ventricular premature beat, tachycardia, atrial fibrillation, etc. The prescription composition of Xin'an tablets comprises six traditional Chinese medicines, i.e. ginseng, astragalus, salvia miltiorrhiza, red peony root, ophiopogon and borneol. At present, the standard executed by the preparation is the national drug standard YBZ12852004-2009Z of the State Food and Drug Administration, which is collected in the 80th volume of "New Drug Registration Standard". The standard content includes thin-layer identification items of ginseng, astragalus, red peony root, borneol and salvia miltiorrhiza, and determination of contents of tanshinone IIA and salvianolic acid B. Under the item of determination of content of tanshinone IIA, the specific provisions are as follows:

[0003] "Chromatographic conditions and system suitability test: octadecylsilane-bonded silica gel as the filler; methanol-water (75:25) as the mobile phase; detection wavelength is 270 nm. The theoretical plate number calculated according to the tanshinone IIA peak should not be less than 2000.

[0004] Preparation of reference solution: precisely take 10 mg of tanshinone IIA reference substance, and place it in a 50 ml brown volumetric flask, add methanol to the scale, shake well; precisely take 5 ml, and place it in a 25 ml brown volumetric flask, add methanol to the scale, shake well, and obtain (40 μg of tanshinone IIA is contained in each 1 ml).

[0005] Preparation of test sample solution: take 10 tablets of the product, remove the film coating, precisely weigh, grind finely, take an amount equivalent to 1 tablet, precisely weigh, add 30 ml of diethyl ether, shake for 5 minutes, stand for 24 hours, filter, wash the filter residue with diethyl ether for 3 times, 5 ml each time, combine the diethyl ether liquid, evaporate to dryness, add an appropriate amount of methanol to dissolve and transfer to a 10 ml brown volumetric flask, shake well, filter, and obtain.

[0006] Determination method: precisely take 10 μl of the reference solution and the test sample solution respectively, inject into the liquid chromatograph, and determine, and obtain".

[0007] When preparing the sample solution according to the above statutory method, the filter paper commonly used in the field is used as the filter material. However, the inventors found in production practice that the content of tanshinone IIA in the sample to be tested is often unqualified in the laboratory test results, or because the content is less than the internal control standard, or the relative average deviation is too large (the relative average deviation is the ratio of the average deviation of the parallel test results to the average value, and the average deviation is the average value of the absolute value of the deviation of each parallel test result). According to the internal control standard, it belongs to one inspection unqualified. Therefore, repeated detection has to be carried out. However, it is puzzling that even after repeated measurement, both the content of tanshinone IIA and the relative average deviation of the content still cannot meet the internal control standard, and the test results of each time are unstable and have large dispersion. This not only prolongs the sample test cycle and causes the rise of enterprise cost; more seriously, because the test results are unstable and cannot reflect the true quality of the product, it may even mislead the actual production, and determine the qualified product as unqualified, and determine the unqualified product as qualified. This not only causes waste of resources, but also affects the safety of clinical patients' medication.

[0008] Because of the particularity of drugs, production and detection must be strictly carried out according to the existing standard. Therefore, even if a method with better stability has appeared in the prior art, such as using methanol solution as the extraction solvent to prepare the sample solution of the Danshen-containing traditional Chinese medicine (see the invention patent application with the publication number CN113671099A and the invention name "a detection method of Ziyedan capsule"), from the legal and compliant point of view of drug inspection, the drug inspection standard of the enterprise must carry out the content determination of tanshinone IIA of Xinmian tablet according to the existing drug standard, and cannot be arbitrarily changed. SUMMARY

[0009] In order to overcome the shortcomings of the prior art, the inventors have researched the preparation of the sample solution of tanshinone IIA content determination of Xinmian tablet under the drug standard YBZ12852004-2009Z without changing the existing statutory standard, in order to find the factors affecting the extraction transfer rate of tanshinone IIA and improve it. It is found that the extraction temperature of diethyl ether and the light avoidance time during extraction will affect the test results. However, more unexpectedly, the filter material of diethyl ether extract is also an important influencing factor.

[0010] Based on the above research and discovery, the present application provides a method for improving the stability of the determination results of the content of tanshinone IIA in Xinmian tablet.

[0011] Therefore, the present application adopts the following technical solution:

[0012] A preparation method of a sample solution for the determination of the content of tanshinone IIA in Xinmian tablet, comprising the following operations:

[0013] Take 10 tablets of the product, remove the film coating, finely weigh, take the equivalent of 1 tablet, precisely weigh, add 30 ml of diethyl ether, shake for 5 minutes, avoid light for 24 hours, filter with nylon cloth, filter the residue with diethyl ether 3 times, each time 5-20 ml, shake, filter on the same piece of nylon cloth, combine the diethyl ether liquid, evaporate at room temperature, add methanol to the residue to dissolve, quickly transfer to a 10 ml brown volumetric flask, shake well, filter, and obtain.

[0014] Preferably, when preparing the test solution, add 30 ml of diethyl ether, shake for 5 minutes, avoid light for 24 hours at 2-12°C.

[0015] More preferably, when preparing the test solution, add 30 ml of diethyl ether, shake for 5 minutes, avoid light for 24 hours at 2-8°C.

[0016] Preferably, after 24 hours of standing, the diethyl ether liquid is filtered with 100-300 mesh nylon cloth.

[0017] More preferably, after 24 hours of standing, the diethyl ether liquid is filtered with 100 mesh nylon cloth.

[0018] Preferably, the residue is washed with diethyl ether 3 times, each time 10-20 ml.

[0019] More preferably, the residue is washed with diethyl ether 3 times, each time 10-15 ml.

[0020] Most preferably, the residue is washed with diethyl ether 3 times, each time 10 ml.

[0021] The present application also aims to provide a method for determining the content of tanshinone IIA in Xinmian tablets, which is based on high performance liquid chromatography and comprises:

[0022] I. Chromatographic conditions and system suitability test

[0023] Octadecylsilane-bonded silica gel is used as the filler; methanol-water (75:25) is used as the mobile phase; and the detection wavelength is 270 nm. The theoretical plate number calculated according to the tanshinone IIA peak should not be less than 2000;

[0024] II. Preparation of the reference solution

[0025] Precisely weigh 10 mg of tanshinone IIA reference substance, place it in a 50 ml brown volumetric flask, add methanol to the mark, shake well; precisely take 5 ml, place it in a 25 ml brown volumetric flask, add methanol to the mark, shake well, and obtain 40 μg of tanshinone IIA per 1 ml;

[0026] III. Preparation of the test solution

[0027] Take 10 tablets of the product, remove the film coating, finely weigh, take the equivalent of 1 tablet, precisely weigh, add 30 ml of diethyl ether, shake for 5 minutes, avoid light for 24 hours, filter with nylon cloth, wash the filter residue with diethyl ether 3 times, each time 5-20 ml, shake, filter on the same piece of nylon cloth, combine the diethyl ether solution, evaporate at room temperature, add methanol to the residue to dissolve, quickly transfer to a 10 ml brown volumetric flask, shake well, filter, and you get;

[0028] IV. Assay

[0029] Precisely take 10 μl of the control solution and the test solution respectively, inject into the liquid chromatograph, and determine, and you get.

[0030] Preferably, in III, add 30 ml of diethyl ether, shake for 5 minutes, avoid light for 24 hours at 2-12°C.

[0031] More preferably, in III, add 30 ml of diethyl ether, shake for 5 minutes, avoid light for 24 hours at 2-8°C.

[0032] Preferably, in III, filter the diethyl ether solution with 100-300 mesh nylon cloth.

[0033] More preferably, in III, filter the diethyl ether solution with 100 mesh nylon cloth.

[0034] Preferably, in III, wash the filter residue with diethyl ether 3 times, each time 10-20 ml.

[0035] More preferably, in III, wash the filter residue with diethyl ether 3 times, each time 10-15 ml.

[0036] Most preferably, in III, wash the filter residue with diethyl ether 3 times, each time 10 ml.

[0037] As a preferred embodiment, the present application provides a method for determining the content of tanshinone IIA in Xinya'an tablets, which is based on high performance liquid chromatography and comprises:

[0038] I. Chromatographic conditions and system suitability test

[0039] Use octadecylsilane-bonded silica gel as the filler; use methanol-water (75:25) as the mobile phase; and the detection wavelength is 270 nm. The theoretical plate number should not be less than 2000 according to the tanshinone IIA peak;

[0040] II. Preparation of the control solution

[0041] Accurately weigh 10 mg of tanshinone IIA reference substance into a 50 ml brown volumetric flask, add methanol to the calibration mark, shake well; accurately pipette 5 ml into a 25 ml brown volumetric flask, add methanol to the calibration mark, shake well, and obtain (40 μg of tanshinone IIA is contained in every 1 ml).

[0042] III. Preparation of test solution

[0043] Take 10 tablets of the product, remove the film coating, accurately weigh, finely grind, take one tablet, accurately weigh, add 30 ml of diethyl ether, shake for 5 minutes, stand at 2-12 ℃ for 24 hours in the dark, filter with a 100 mesh nylon cloth, wash the filter residue with diethyl ether for 3 times, 10 ml each time, shake, filter on the same nylon cloth, combine the diethyl ether solution, evaporate at room temperature, add an appropriate amount of methanol to dissolve, quickly transfer to a 10 ml brown volumetric flask, shake well, filter, and obtain.

[0044] IV. Determination method

[0045] Accurately pipette 10 μl of the reference solution and the test solution respectively, inject into the liquid chromatograph, and determine, and obtain.

[0046] The current standard for Xinyuanan tablets is the national drug standard YBZ12852004-2009Z of the State Food and Drug Administration, which is recorded in the 80th volume of New Drug Registration Standards. Under the premise of strictly complying with the legal standards, the inventors have carried out research on various factors that may affect the test results in the content detection process. On the basis of a large number of experiments, the key influencing factors are found, and the preparation process of the test solution is optimized, thereby improving the stability and one-time inspection pass rate of the determination of tanshinone IIA content in Xinyuanan tablets at one time, not only reducing the related cost of enterprises, but also ensuring the safety of clinical medication. BRIEF DESCRIPTION OF DRAWINGS

[0047] The application will be further described below in combination with the drawings.

[0048] Figure 1 Shown are the HPLC chromatograms of the reference substance and the test substance obtained in Example 1. In the figure, A is the HPLC chromatogram of the test substance, and B is the HPLC chromatogram of the reference substance. DETAILED DESCRIPTION

[0049] The determination of tanshinone IIA in Xinmian tablet in the medicine standard YBZ12852004-2009Z is carried out by high performance liquid chromatography. The chromatographic conditions and the preparation of test solution are specified in the standard. In the chromatographic conditions, only the filler (stationary phase) of the chromatographic column is specified, and the specific model of the chromatographic column is not specified. The performance of the chromatographic column produced by different manufacturers is different even if the filler is the same. In the preparation of test solution, the operations such as ether extraction, filtration of ether extract, washing of the residue after filtration and the like are involved. Generally, the temperature of solvent extraction has an influence on the extraction of target components, and the higher the extraction temperature, the more favorable for the extraction of components. Therefore, the inventors have studied the influence of ether extraction temperature on the extraction of tanshinone IIA. It is unexpectedly found that low temperature is favorable for the extraction of tanshinone IIA. The inventors have also found that light is an important factor affecting the extraction and transfer of tanshinone IIA during the ether extraction process, and the chromatographic column has no significant influence on the determination of tanshinone IIA.

[0050] The filter material commonly used in the art is filter paper, and the adsorption rate of the filter material to target components is rarely studied. Bai Yuguo et al. have reported that the adsorption rates of different filter materials to tanshinone IIA sodium sulfonate injection are different. Among them, the adsorption rate of mixed cellulose ester membrane is the lowest, only 0.97%, and the adsorption rate of nylon cloth is as high as 15.10% (Bai Yuguo, et al. Adsorption of tanshinone IIA sodium sulfonate and breviscapine on disposable filter membrane [J]. Medical Journal of China, May 2011, 30 (5): 663-665). The inventors have studied the influence of different filter materials on the determination of tanshinone IIA in ether extract of Xinmian tablet. Unexpectedly, nylon cloth has the least adsorption to tanshinone IIA in ether extract of Xinmian tablet.

[0051] Based on the above research, the present application provides a method for improving the determination accuracy of tanshinone IIA in Xinmian tablet, which comprises preparing a test solution by the following method:

[0052] Take 10 tablets of the product, remove the film coating, accurately weigh, finely grind, take an equivalent of 1 tablet, accurately weigh, add 30 ml of ether, shake for 5 minutes, avoid light, stand for 24 hours, filter with nylon cloth, wash the residue with ether for 3 times, shake, filter on the same piece of nylon cloth, combine the ether solution, evaporate at room temperature, add an appropriate amount of methanol to dissolve, quickly transfer to a 10 ml brown volumetric flask, shake well, filter, and obtain the test solution.

[0053] Preferably, the test solution is prepared by the following method:

[0054] Take 10 tablets of the product, remove the film coating, accurately weigh, finely grind, take the equivalent of 1 tablet, accurately weigh, add 30 ml of ethyl ether, shake for 5 minutes, stand in the dark at 2-12°C for 24 hours, filter with a 100-300 mesh nylon cloth, wash the residue with ethyl ether 3 times, each time 5-20 ml, shake, filter on the same piece of nylon cloth, combine the ethyl ether solution, evaporate at room temperature, add methanol to the residue to dissolve, quickly transfer to a 10 ml brown volumetric flask, shake well, filter, and you get it.

[0055] In addition, the application also provides a method for determining the content of tanshinone IIA in Xinmian tablet, which is based on high performance liquid chromatography and comprises the following steps:

[0056] I. Chromatographic conditions and system suitability test

[0057] Take octadecylsilane-bonded silica gel as the filler, take methanol-water (75:25) as the mobile phase, and take 270 nm as the detection wavelength. The theoretical plate number should not be less than 2000 according to the tanshinone IIA peak;

[0058] II. Preparation of the control solution

[0059] Accurately weigh 10 mg of tanshinone IIA control, put it into a 50 ml brown volumetric flask, add methanol to the mark, shake well, accurately take 5 ml, put it into a 25 ml brown volumetric flask, add methanol to the mark, shake well, and you get it, 40 μg of tanshinone IIA per 1 ml;

[0060] III. Preparation of the test solution

[0061] Take 10 tablets of the product, remove the film coating, accurately weigh, finely grind, take the equivalent of 1 tablet, accurately weigh, add 30 ml of ethyl ether, shake for 5 minutes, stand in the dark for 24 hours, filter with a nylon cloth, wash the residue with ethyl ether 3 times, filter on the same piece of nylon cloth, combine the ethyl ether solution, evaporate at room temperature, add methanol to the residue to dissolve, quickly transfer to a 10 ml brown volumetric flask, shake well, filter, and you get it;

[0062] IV. Determination method

[0063] Accurately take 10 μl of the control solution and the test solution respectively, inject them into the liquid chromatograph, and determine, and you get it.

[0064] The application will be described below with reference to specific examples. Those skilled in the art can understand that these examples are only used to illustrate the application, and do not limit the scope of the application in any way.

[0065] The experimental methods in the following examples are all conventional methods unless otherwise specified. The raw materials, reagents and materials used in the following examples are all commercially available products unless otherwise specified. Among them, part of the instruments and materials are purchased as follows.

[0066] Waters H-Class high performance liquid chromatograph (equipped with automatic sampler, PDA detector, Empore 3.0 chromatographic workstation) of Waters Corporation of USA; chromatographic column: Waters ACQUITY BEH C18 (100 mm x 2.1 mm, 1.7 μm), ACQUITY BEH Shield RP18 (100 mm x 2.1 mm, 1.7 μm); BSA224S type one-in-ten-thousand balance (Sartorius Corporation of Germany); MSA6.6S-0CE-DM type one-in-one-hundred-thousand balance (Sartorius Corporation of Germany).

[0067] The samples of Xinmaian tablets were all produced by Guangzhou Baiyun Mountain and Hutchison Whampoa Pharmaceutical Co., Ltd.; tanshinone IIA (batch number: 110766-202022) reference substance was purchased from China Institute for Drug Control. Acetonitrile was chromatographically pure (Merck Corporation of Germany); formic acid was chromatographically pure (Kemiyou Corporation); methanol was analytically pure (Guangzhou Chemical Reagent Factory).

[0068] Study Example 1 Investigation of Factors Influencing Determination of Content of Tanshinone IIA in Xinmaian

[0069] In this study example, the drug standard YBZ12852001-2009Z was used as the basis to study various possible factors influencing determination of content of tanshinone IIA in Xinmaian tablets. When studying each influencing factor, the method for determination of content of tanshinone IIA was as follows:

[0070] I. Chromatographic conditions

[0071] Liquid chromatograph: Waters H-Class high performance liquid chromatograph (equipped with automatic sampler, PDA detector, Empore 3.0 chromatographic workstation) of Waters Corporation of USA;

[0072] Chromatographic column: BEH C18 (100 mm x 2.1 mm, 1.7 μm);

[0073] Mobile phase: methanol-water (75:25);

[0074] Detection wavelength was 270 nm.

[0075] II. Preparation of reference substance solution

[0076] Precisely weigh 10 mg of tanshinone IIA reference substance, place in a 50 ml brown volumetric flask, add methanol to the calibration mark, shake well; precisely take 5 ml, place in a 25 ml brown volumetric flask, add methanol to the calibration mark, shake well, to obtain a reference substance solution containing 40 μg of tanshinone IIA per 1 ml;

[0077] III. Preparation of Test Solution

[0078] The specific studies are described below.

[0079] IV. Assay Method

[0080] Accurately pipette 1 μl of the control solution and the test solution into the liquid chromatograph (as an ultra-high performance liquid chromatograph is used, the sample size is reduced from 10 μl to 1 μl in a normal high performance liquid chromatograph), and determine, to obtain.

[0081] In the following studies, the sample of Xinman is not the same batch. Therefore, it is understood that although all are referred to as "Sample 1" or "Sample 2", the content of tanshinone IIA determined in each study is not consistent. What is significant for this study is the effect of different factors or factor levels on the determination of the content of the same sample in each study.

[0082] 2.1 Effect of Different Filter Materials

[0083] The current standard requires that the ether extract be filtered, but does not specify the filter material. Three common filter materials, filter paper, cotton, and nylon cloth, are used to prepare the test solution. The specific operation is as follows:

[0084] Take 10 tablets of Xinman, remove the film coating, accurately weigh, finely grind, and take the equivalent of 1 tablet, accurately weigh, add 30 ml of ether, shake for 5 minutes, and stand at room temperature for 24 hours. Filter through filter paper, cotton, and 100-mesh nylon cloth, wash the residue with ether 3 times, 5 ml each time, filter through the same filter material, combine the ether solution, and evaporate at room temperature. Add an appropriate amount of methanol to dissolve the residue, quickly transfer to a 10-ml brown volumetric flask, shake well, filter, and obtain.

[0085] The content determination results are shown in Table 1.

[0086] Table 1 Content Determination Results of Tanshinone IIA with Different Filter Materials

[0087]

[0088] The results in the above table suggest that the most commonly used filter paper has the most adsorption of tanshinone IIA, and the nylon cloth has the least adsorption. Therefore, the filter material is preferably nylon cloth.

[0089] 2.1 Effect of Ether Extraction Temperature

[0090] In general, the higher the temperature, the more beneficial it is for the dissolution of plant components in solvent extraction. The existing standard does not specify the temperature for ether extraction, which is usually carried out at room temperature. Therefore, ether extraction is carried out at 4°C, 8°C, 12°C, and room temperature, respectively. The specific operation is as follows:

[0091] Take 10 tablets of Xinyuan tablets, remove the film coating, accurately weigh, finely grind, and take an equivalent of 1 tablet, accurately weigh, add 30 ml of diethyl ether, shake for 5 minutes, and stand at 4°C, 8°C, 12°C, and room temperature (28°C) for 24 hours. Filter with 100 mesh nylon cloth, wash the residue with diethyl ether 3 times, each time with 5 ml, combine the diethyl ether liquid, and evaporate at room temperature. Add an appropriate amount of methanol to the residue to dissolve, quickly transfer to a 10 ml brown volumetric flask, shake well, and filter to obtain the product.

[0092] The test results are shown in Table 2.

[0093] Table 2 shows the content of tanshinone IIA in the test sample determined decreases with the increase of extraction temperature. It is indicated that diethyl ether is more beneficial to the dissolution of tanshinone IIA under low temperature conditions. It is preliminarily judged that the diethyl ether extraction temperature is lower than 12°C.

[0094]

[0095] Table 2 shows that as the extraction temperature increases, the content of tanshinone IIA in the test sample determined decreases. It is indicated that diethyl ether is more beneficial to the dissolution of tanshinone IIA under low temperature conditions. It is preliminarily judged that the diethyl ether extraction temperature is lower than 12°C.

[0096] 2.2 Influence of different light avoidance time:

[0097] The current standard requires diethyl ether extraction for more than 24 hours, but does not specify whether it needs to be light-protected. In a solvent environment, components may be sensitive to light. Therefore, the test sample solution was prepared by standing for 24 hours under light-protected conditions for 0h, 10h, and 24h, respectively. The specific operation is as follows:

[0098] Take 10 tablets of Xinyuan tablets, remove the film coating, accurately weigh, finely grind, and take an equivalent of 1 tablet, accurately weigh, add 30 ml of diethyl ether, shake for 5 minutes, and stand at 4°C, 8°C, 12°C, and room temperature (28°C) for 24 hours. Filter with 100 mesh nylon cloth, wash the residue with diethyl ether 3 times, each time with 5 ml, combine the diethyl ether liquid, and evaporate at room temperature. Add an appropriate amount of methanol to the residue to dissolve, quickly transfer to a 10 ml brown volumetric flask, shake well, and filter to obtain the product.

[0099] The test results are shown in Table 3.

[0100] Table 3 shows the content of tanshinone IIA in the test sample determined decreases with the increase of extraction temperature. It is indicated that diethyl ether is more beneficial to the dissolution of tanshinone IIA under low temperature conditions. It is preliminarily judged that the diethyl ether extraction temperature is lower than 12°C.

[0101]

[0102] The results show that different light avoidance times have a great influence on the content when preparing the test sample solution by diethyl ether extraction. It is preferred to avoid light during the standing extraction process.

[0103] 2.4 Influence of different diethyl ether elution volumes:

[0104] The current standard stipulates that the filter residue of diethyl ether extraction solution needs to be washed with diethyl ether after filtration. Although the amount of diethyl ether used for washing is stipulated as 5 ml / time, there may be incomplete washing. Therefore, the test sample solution was prepared by washing with 5, 10, 15 and 20 ml of diethyl ether each time, and the specific operation was as follows:

[0105] Take 10 Xinyuan tablets, remove the film coating, accurately weigh, finely grind, and take the equivalent of 1 tablet, accurately weigh, add 30 ml of diethyl ether, shake for 5 minutes, and place in the refrigerator (2-8°C) for 24 hours in the dark. Filter through a 100-mesh nylon cloth, and wash the filter residue with diethyl ether for 3 times, each time using 5 ml, 10 ml, 15 ml and 20 ml of diethyl ether respectively, shake, and filter on the same nylon cloth. Combine the diethyl ether solution, and evaporate at room temperature. Add an appropriate amount of methanol to dissolve the residue, quickly transfer to a 10-ml brown volumetric flask, shake well, and filter to obtain the test sample solution.

[0106] The determination results are shown in Table 4.

[0107] Table 4 Determination results of tanshinone IIA content under different diethyl ether washing volume conditions

[0108]

[0109] In the current standard, 5 ml of diethyl ether is used for washing each time, and the washing is not sufficient. When the amount of diethyl ether used for washing each time exceeds 10 ml, the content does not increase substantially. Therefore, 10 ml of diethyl ether is preferably used for washing each time.

[0110] Comprehensive investigation of test sample solution preparation process conditions

[0111] Based on the above research example 1, in order to further clarify the comprehensive influence of diethyl ether immersion temperature, diethyl ether immersion light avoidance time and nylon cloth mesh size on the extraction and transfer of tanshinone IIA in Xinyuan, L9(34) orthogonal experiment was carried out, and the content of tanshinone IIA in Xinyuan tablets obtained under the same chromatographic conditions in research example 1 was determined and analyzed. The experimental arrangement is shown in Table 5, and the results are shown in Table 6. 4 ) orthogonal experiment, and the content of tanshinone IIA in Xinyuan tablets obtained under the same chromatographic conditions in research example 1 was determined and analyzed. The experimental arrangement is shown in Table 5, and the results are shown in Table 6.

[0112] From the range analysis of Table 6, R 避光时长 > R 浸渍温度 > R 尼龙布目数 , the three influencing factors in order of significance are: light avoidance time > immersion temperature > nylon cloth mesh size, that is, the light avoidance time has the greatest influence on the content of tanshinone IIA, the immersion temperature is second, and the nylon cloth mesh size has the least influence; and R 空 > R 尼龙布目数 , indicating that the nylon cloth mesh size has a smaller influence on the test determination results than the experimental error. In summary, the optimal scheme for preparing the test sample solution is C3B2A1, that is, the diethyl ether immersion light avoidance time is 24 h, the immersion temperature is 4°C, and the diethyl ether immersion liquid is filtered through a 100-mesh nylon cloth.

[0113] Table 5 Orthogonal test factor level table

[0114]

[0115] Table 6 Orthogonal test results and analysis

[0116]

[0117] According to the orthogonal experiment results, take the same batch of Xinmaian tablets, according to the determined optimal test solution preparation method, repeat the preparation of test solution 6 times, determine the content of tanshinone IIA, and the average value is taken. The above verification experiment results are shown in Table 7.

[0118] Table 7 Content of tanshinone IIA in Xinmaian tablets under the optimal preparation process conditions (mg / g)

[0119]

[0120] The RSD of 6 determination results is less than 3%, which meets the internal control standard.

[0121] Example 1 Determination of tanshinone IIA content in Xinmaian tablets

[0122] 1. Determination method of tanshinone IIA content in Xinmaian tablets

[0123] Chromatographic conditions: octadecylsilane-bonded silica gel as filler; methanol-water (75:25) as mobile phase; detection wavelength is 270 nm. The theoretical plate number should not be less than 2000, calculated by tanshinone II A peak.

[0124] The liquid chromatograph and chromatographic column used are the same as in Research Example 1.

[0125] Preparation of reference solution: accurately weigh 10 mg of tanshinone II A reference substance into a 50 ml brown volumetric flask, add methanol to the mark, shake well; accurately take 5 ml into a 25 ml brown volumetric flask, add methanol to the mark, shake well, and you get it (every 1 ml contains 40 μg of tanshinone II A).

[0126] Preparation of test solution: take 10 tablets of the product, remove the film coating, accurately weigh, grind finely, take equivalent to 1 tablet, accurately weigh, add 30 ml of diethyl ether, shake for 5 minutes, stand in the refrigerator for 24 hours, filter with 100 mesh nylon cloth, wash the filter residue with diethyl ether 3 times, each time 10 ml, shake, filter on the same piece of nylon cloth, combine the diethyl ether liquid, evaporate, add an appropriate amount of methanol to dissolve, quickly transfer to a 10 ml brown volumetric flask, shake well, filter, and you get it.

[0127] Determination: accurately take 1 μl of the reference solution and the test solution respectively, inject into the liquid chromatograph, and determine, and you get it.

[0128] HPLC chromatograms of the reference and test samples are shown in Figure 1

[0129] 2. Methodological Investigation

[0130] 2.1 Investigation of Linearity: Reference solutions of different concentrations of tanshinone IIA were prepared, i.e. 0 mg / ml, 0.01074 mg / ml, 0.02149 mg / ml, 0.04298 mg / ml, 0.06447 mg / ml, 0.08596 mg / ml, 0.1074 mg / ml, 1 μL of each solution was injected into the liquid chromatograph for determination. The concentration of the reference was taken as the abscissa and the peak area as the ordinate to plot a curve, and the linear equation was:

[0131] Tanshinone IIA: Y = 1.89e+007X, r = 0.999897.

[0132] It was shown that tanshinone IIA had a good linear relationship in the range of 10-100 μg.

[0133] 2.2 Precision Test: Reference solution of tanshinone IIA at a concentration of 0.06447 mg / mL was taken and injected continuously for 6 times, the RSD of the average content of tanshinone IIA was 0.2% (n = 6). It was shown that the precision of the instrument was good.

[0134] 2.3 Stability Test: Sample 001 was taken and test sample solution was prepared according to the preparation method of test sample solution, and was injected at 0, 2, 4, 6, 8, 12, 24 and 48 hours, respectively, and was determined according to the above chromatographic conditions, the chromatographic peak of tanshinone IIA was integrated to obtain the peak area, and the results are shown in Table 8.

[0135] Table 8 Results of stability test

[0136]

[0137] The results of Table 8 show that the test sample solution is stable within 48 hours.

[0138] 2.4 Reproducibility Test: The same batch of sample 001 was taken and 6 test sample solutions were prepared, and were injected and analyzed according to the above chromatographic conditions, the average content of tanshinone IIA in the 6 test sample solutions was 2.19 mg / g, and the RSD was 1.8%, and the specific results are shown in Table 9.

[0139] Table 9 Results of reproducibility test

[0140]

[0141] The results of Table 9 show that the method has good reproducibility.

[0142] ​2.5 Spiking recovery test: 6 portions of Xinya An tablets (sample 001: tanshinone IIA content 2.19 mg / g) powder were weighed, 0.15 g per portion, accurately weighed, a certain amount of tanshinone IIA reference substance was accurately added, the test sample solution was prepared according to the above method, sample analysis was performed according to the above chromatographic conditions, the content of tanshinone IIA in each portion was determined, and the spiking recovery was calculated. The results are shown in Table 10.

[0143] Table 10 Spiking recovery test results

[0144]

[0145] The data in Table 10 show that the method has good accuracy.

[0146] 2.6 Effect of different chromatographic columns

[0147] Two kinds of chromatographic columns were used to determine (Waters ACQUITY BEH C18 and BEH Shield RP18), and the results are shown in Table 11.

[0148] Table 11 Determination results of tanshinone IIA content under different chromatographic columns (sample 001)

[0149]

[0150] The results show that the content of tanshinone IIA determined by different chromatographic columns for the same test sample solution has little difference, indicating that the method of the present application has good adaptability.

[0151] Comparative study of the method of the present application and the current standard method

[0152] To compare the difference between the method of the present application and the current standard in terms of accuracy and relative average deviation, the same batch of samples were selected, the content of tanshinone IIA was first determined by 6 experimenters according to the current drug standard, and then the determination was performed by these experimenters according to the method of the present application. Each sample was prepared by 2 parallel samples per person per time, and each parallel sample was injected twice; that is, each sample obtained 4 determination data results per time, the average value of the 4 data was taken as the content of tanshinone IIA, and the difference between the 4 data was expressed by the relative average deviation (the relative average deviation was the ratio of the average deviation to the average value, and the average deviation was the average value of the absolute values of each measurement deviation). The sample detection qualified standard: the content of tanshinone IIA is ≥1.35 mg / g, and the relative average deviation is ≤3.0%. The results are shown in Tables 12 and 13.

[0153] Table 12 Determination results of the current drug standard

[0154]

[0155] Table 12 shows that according to the current drug standard, one test is unqualified; even if repeated testing is carried out subsequently, the content limit and relative average deviation of tanshinone IIA each meet the requirements only once, and none of the two items reaches the enterprise internal control standard. In addition, the dispersion of the content determination results of tanshinone IIA is large.

[0156] Table 13 determination results of the content determination method of the present application

[0157]

[0158] The results of Table 13 show that after the optimization and improvement of the present application, one test is qualified, and the detection results are stable.

Claims

1. A method for preparing a test solution for determining the content of tanshinone IIA in Xinyuan tablets, comprising the following operations: Take 10 tablets of the product, remove the film coating, accurately weigh, finely grind, take the equivalent of 1 tablet, accurately weigh, add 30 ml of diethyl ether, shake for 5 minutes, stand in the dark at 2-12°C for 24 hours, filter with 100-300 mesh nylon cloth, wash the residue with diethyl ether for 3 times, each time for 5-20 ml, shake, filter on the same piece of nylon cloth, combine the diethyl ether solution, evaporate at room temperature, add an appropriate amount of methanol to dissolve the residue, quickly transfer to a 10 ml brown volumetric flask, shake well, filter, and obtain.

2. The production method according to claim 1, characterized by, When preparing the test solution, add 30 ml of diethyl ether, shake for 5 minutes, stand in the dark at 2-8°C for 24 hours.

3. The production method according to claim 1 or 2, characterized by, After standing for 24 hours, filter the diethyl ether solution with 100 mesh nylon cloth.

4. The preparation method according to claim 1, characterized in that, Wash the residue with diethyl ether for 3 times, each time for 10-20 ml.

5. The production method according to claim 4, characterized by, Wash the residue with diethyl ether for 3 times, each time for 10-15 ml.

6. The production method according to claim 5, wherein Wash the residue with diethyl ether for 3 times, each time for 10 ml.

7. A method for determining the content of tanshinone IIA in Xinyuan tablets, the method is based on high performance liquid chromatography, comprising: I. Chromatographic conditions and system suitability test Use octadecylsilane-bonded silica gel as the filler; Use methanol-water 75:25 as the mobile phase; the detection wavelength is 270 nm; the theoretical plate number should not be less than 2000 calculated by tanshinone IIA peak; II. Preparation of reference solution Accurately weigh 10 mg of tanshinone IIA reference substance, place it in a 50 ml brown volumetric flask, add methanol to the mark, shake well; accurately take 5 ml, place it in a 25 ml brown volumetric flask, add methanol to the mark, shake well, and obtain, 1 ml contains 40 μg of tanshinone IIA; III. Preparation of test solution Take 10 tablets of the product, remove the film coating, accurately weigh, finely grind, take the equivalent of 1 tablet, accurately weigh, add 30 ml of diethyl ether, shake for 5 minutes, stand in the dark at 2-12°C for 24 hours, filter with 100-300 mesh nylon cloth, wash the residue with diethyl ether for 3 times, each time for 5-20 ml, shake, filter on the same piece of nylon cloth, combine the diethyl ether solution, evaporate at room temperature, add an appropriate amount of methanol to dissolve the residue, quickly transfer to a 10 ml brown volumetric flask, shake well, filter, and obtain; IV. Determination method Accurately take 10 μl of the reference solution and the test solution respectively, inject into the liquid chromatograph, and determine, and obtain.

8. The method of claim 7, wherein, In the III, add 30 ml of diethyl ether, shake for 5 minutes, stand in the dark at 2-8°C for 24 hours.

9. The method according to claim 7 or 8, characterized in that, In the III, filter the diethyl ether solution with 100 mesh nylon cloth.

10. The method of claim 7, wherein, In the III, wash the residue with diethyl ether for 3 times, each time for 10-20 ml.

11. The method of claim 10, wherein, In the III, wash the residue with diethyl ether for 3 times, each time for 10-15 ml.

12. A method for determining the content of tanshinone IIA in Xinyuan tablets, the method is based on high performance liquid chromatography, comprising: I. Chromatographic conditions and system suitability test Use octadecylsilane-bonded silica gel as the filler; Use methanol-water 75:25 as the mobile phase; the detection wavelength is 270 nm; the theoretical plate number should not be less than 2000 calculated by tanshinone IIA peak; II. Preparation of reference solution Accurately weigh 10 mg of tanshinone ⅡA reference substance into a 50-ml brown volumetric flask, add methanol to the calibration mark, shake well; accurately pipette 5 ml into a 25-ml brown volumetric flask, add methanol to the calibration mark, shake well, and thus a solution containing 40 μg of tanshinone ⅡA per 1 ml is obtained; III. Preparation of test solution Take 10 tablets of the product, remove the film coating, accurately weigh, finely grind, take the equivalent of 1 tablet, accurately weigh, add 30 ml of diethyl ether, shake for 5 minutes, stand at 2-12 ℃ for 24 hours, filter through a 100-mesh nylon cloth, wash the residue with diethyl ether for 3 times, 10 ml each time, shake, filter on the same nylon cloth, combine the diethyl ether solution, evaporate at room temperature, add an appropriate amount of methanol to dissolve the residue, quickly transfer into a 10-ml brown volumetric flask, shake well, filter, and thus the test solution is obtained; IV. Determination method Accurately pipette 10 μl of the reference solution and the test solution respectively into a liquid chromatograph, determine, and thus the determination is completed.

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