A method for determining fingerprints of blood-activating, blood-stasis-removing, qi-promoting and pain-relieving drug preparations

Abstract

The present invention relates to the technical field of traditional Chinese medicine quality detection methods, and specifically to a method for determining the fingerprint of a drug preparation for promoting blood circulation and removing blood stasis, promoting qi and relieving pain. The method for determining the fingerprint of the drug preparation comprises the following steps performed in sequence: extracting a test sample from a drug preparation; detecting the test sample using high performance liquid chromatography to obtain a fingerprint; comparing the fingerprint with a standard fingerprint, and then evaluating the quality of the drug preparation. The standard fingerprint includes characteristic peaks of the following components: sodium danshensu, protocatechuic aldehyde, 3'-hydroxypuerarin, puerarin, 3'-methoxypuerarin, puerarin-7-xyloside, soybean glycoside, and tannic acid B. The technical solution can solve the technical problems of the complicated operation and limited accuracy of the detection method of the prior art, and can reduce the requirements of the detection process on the equipment, reduce the detection cost, meet the conventional detection needs of pharmaceutical companies, and have a relatively ideal promotion and application value.

Description

Technical Field

[0001] The invention relates to the technical field of traditional Chinese medicine quality detection methods, and in particular to a method for determining a fingerprint spectrum of a blood-activating, blood-stasis-removing, qi-promoting and pain-relieving drug preparation. Background Art

[0002] The blood-activating, blood-stasis-removing, qi-promoting and analgesic drug preparation of this scheme is composed of raw materials of salvia miltiorrhiza, kudzu root, panax notoginseng, hawthorn and costus root. The preparation method is as follows: take panax notoginseng, costus root and part of hawthorn and grind them into fine powder, add 60% ethanol to soak the remaining hawthorn and kudzu root for 30 minutes, reflux extraction twice, combine the alcohol extract, recover the ethanol and set aside; salvia miltiorrhiza is decocted twice with water, the decoction is combined, filtered, the filtrate is combined with the above-mentioned reserve liquid, mixed, concentrated to an appropriate amount, and the above-mentioned fine powder is added to make granules, capsules, pills, tablets, etc. The preparation has the effects of promoting blood circulation, removing blood stasis, promoting qi and relieving pain. It is used for chest tightness, pain, dizziness, headache, neck pain and other symptoms caused by qi stagnation and blood stasis. It is mostly used for patients with arrhythmia (premature beats) caused by coronary heart disease, angina pectoris and heart disease with the above-mentioned symptoms. There are many kinds of characteristic components in its preparation. Which characteristic components are suitable for preparation quality monitoring and how to ensure that various characteristic components can be effectively detected during the detection process are the problems that need to be solved first to achieve preparation quality monitoring. In addition, the solubility properties, ultraviolet absorption wavelengths and separation characteristics of the indicative ingredients in the preparation are different, and it is difficult to find a stable and reliable method to identify the characteristic ingredients in the preparation, that is, it is difficult to evaluate the quality stability of the characteristic ingredients in the preparation.

[0003] In order to evaluate the quality of drugs more effectively and accurately, it is urgent to develop a new fingerprint determination method that can conveniently, quickly and accurately detect the content of various characteristic ingredients in the preparation. Summary of the invention

[0004] The present invention aims to provide a method for determining the fingerprint of a blood-activating, blood-stasis-removing, qi-promoting and analgesic drug preparation, so as to solve the technical problems of the prior art detection methods that are complex in operation and have limited accuracy.

[0005] In order to achieve the above object, the present invention adopts the following technical scheme:

[0006] A method for determining the fingerprint spectrum of a blood-activating, blood-stasis-removing, qi-promoting and pain-relieving drug preparation comprises the following steps performed in sequence:

[0007] S1: The test sample is extracted from the drug preparation;

[0008] S2: Detecting the test sample using high performance liquid chromatography to obtain a fingerprint;

[0009] S3: comparing the fingerprint with the standard fingerprint to evaluate the quality of the drug preparation;

[0010] The standard fingerprint spectrum includes the following characteristic peaks: characteristic chromatographic peak 1 representing sodium danshensu, characteristic chromatographic peak 2 representing protocatechuic aldehyde, characteristic chromatographic peak 3 representing 3'-hydroxypuerarin, characteristic chromatographic peak 4 representing puerarin, characteristic chromatographic peak 5 representing 3'-methoxypuerarin, characteristic chromatographic peak 6 representing puerarin-7-xyloside, characteristic chromatographic peak 7 representing daidzein, and characteristic chromatographic peak 8 representing salvianolic acid B.

[0011] The present invention also provides an application of a method for determining the fingerprint spectrum of a blood-activating, blood-stasis-removing, qi-promoting and pain-relieving drug preparation in the evaluation of drug characteristic components.

[0012] The principle and beneficial effects of this scheme are:

[0013] In the fingerprint analysis of Chinese patent medicine based on liquid chromatography, due to the complex composition of the medicine, it is usually necessary to separate multiple characteristic components in the same analysis cycle to characterize the content of the effective components of the Chinese patent medicine. In liquid chromatography, the gradient elution method is used for samples with complex components. Gradient elution is also called gradient elution or program elution. In the same analysis cycle, the concentration ratio of the mobile phase is continuously changed to a certain extent, so that the components with large differences in properties in a complex sample can achieve good separation purposes according to their respective characteristics. For the blood circulation and blood stasis, qi circulation and analgesic drug preparations of this scheme, its effective components are relatively complex, and there are many characteristic components used to characterize it, including: sodium danshensu, protocatechuic aldehyde, 3'-hydroxypuerarin, puerarin, 3'-methoxypuerarin, puerarin-7-xyloside, soybean glycoside, tannic acid B, etc. The inventor's previous research shows that the joint identification of the above-mentioned several characteristic components can ensure a comprehensive and effective drug quality evaluation. Since the composition of this preparation is complex and there are many characteristic components that need to be separated, the existing technology generally uses gradient elution to construct the fingerprint of the blood-activating, blood-stasis-removing, qi-promoting and analgesic drug preparation of this scheme. However, the use of gradient elution has the following disadvantages: (1) Gradient elution has high requirements for instrument configuration, usually requiring a quaternary pump or a binary pump; (2) Between each sample run, a certain equilibrium time needs to be set to allow the ratio of the mobile phase in the system to reach the initial mobile phase ratio; (3) Due to the complex method settings, such as the initial mobile phase ratio, the gradient change of the mobile phase, the end mobile phase ratio, the column oven temperature, etc. that affect the separation, the development and optimization of the method will be more complicated.

[0014] In order to improve the analysis efficiency and reduce the running cost, the inventor tried to change the gradient elution method to isocratic elution. When isocratic elution is used for chromatographic separation, the composition of the mobile phase composed of different solvents, such as the polarity, ionic strength, pH value, etc. of the mobile phase, remains unchanged during the whole separation process. Since the ratio of the mobile phase does not change when the isocratic elution method is running, it does not require high requirements for the instrument and only requires one unit pump to run. And since the ratio of the mobile phase is the same during each operation of the isocratic elution, when multiple samples are run by the same method, there is no need to set the equilibrium time between the samples to allow the system to return to the required mobile phase ratio. Although isocratic elution has the above-mentioned advantages, since there are too many characteristic components used to prepare the fingerprint spectrum of this preparation, if isocratic elution is used, the following adverse consequences will be brought about: the analysis time is long, the peak shape of some characteristic components is poor (not sharp enough), and it is difficult to fully analyze all 8 characteristic components (the separation between characteristic peaks is poor). In order to explore how to apply isocratic elution to the establishment of the preparation fingerprint of this scheme, the inventors conducted a large number of attempts and found that the mobile phase composition and preparation extraction method in the preparation process of the preparation fingerprint have a very critical influence on obtaining the ideal fingerprint.

[0015] Based on the above research, this technical solution has established a method for determining the fingerprint of blood-activating, blood-stasis-removing, qi-promoting and analgesic drug preparations. It can quickly and accurately obtain fingerprints for product quality identification and conduct quantitative or qualitative analysis of the characteristic components of the preparations while simplifying the operating procedures and reducing equipment costs.

[0016] The inventors conducted fingerprint tracing analysis on the preparations of historical production batches and found that the eight characteristic components, namely, danshensu sodium, protocatechuic aldehyde, 3'-hydroxypuerarin, puerarin, 3'-methoxypuerarin, puerarin-7-xyloside, daidzin, and salvianolic acid B, have certain stability in each batch and can be used as indicators for evaluating the quality of the preparations. The standard fingerprint of this solution includes the characteristic peaks of the above eight characteristic components. When evaluating the quality of the batch to be tested, it is only necessary to compare the fingerprint of the batch to be tested with the standard fingerprint.

[0017] Further, in S1, the test sample is prepared by the following method: using N,N-dimethylformamide as an extraction solvent, performing ultrasound-assisted extraction on the drug preparation to obtain the test sample.

[0018] The preferred technical parameters include: ultrasonic power of 250W, frequency of 40kHz, time of 30 minutes; and dosage ratio of the drug preparation to N,N-dimethylformamide of 0.5g:50mL.

[0019] By adopting the above technical scheme, N,N-dimethylformamide is used as a solvent to extract the index components in the samples of blood-activating and stasis-removing, qi-promoting and analgesic drug preparations, and the index components can be fully extracted and used in the subsequent liquid chromatography detection process. There are many index components in the blood-activating and stasis-removing, qi-promoting and analgesic drug preparations (tanshinone sodium, protocatechuic aldehyde, 3'-hydroxypuerarin, puerarin, 3'-methoxypuerarin, puerarin-7-xyloside, soybean glycoside, and salvianolic acid B), and the solubility properties of each substance are different. It is difficult to find a solvent that can dissolve and extract all the index substances. The inventor tried a variety of solvents and finally found that only N,N-dimethylformamide can fully extract the 8 index components in the drug preparation, and can obtain a fingerprint spectrum with clear characteristic peak separation and meeting the requirements in the subsequent liquid chromatography detection. N,N-dimethylformamide has good adaptability to the fingerprint spectrum identification of this scheme. If it is replaced with other solvents, various index components cannot be extracted simultaneously and fully.

[0020] Further, in S2, the mobile phase of the high performance liquid chromatography consists of acetonitrile, tetrahydrofuran and acetic acid solution.

[0021] Furthermore, in S2, the volume ratio of acetonitrile, tetrahydrofuran and acetic acid solution is 40:40:20. Preferably, the mass percentage of the solute in the acetic acid solution is 0.4%.

[0022] Further, in S2, the filler of the HPLC column is octadecylsilane bonded silica gel, and the filler particle size is 3.5-5.0 μm. The preferred specifications of the HPLC column are: diameter 4.6 mm, length 7.5-25 cm, preferably 7.5 cm; the filler particle size is preferably 3.5 μm.

[0023] By adopting the above detection method, a standard fingerprint spectrum, a fingerprint spectrum of the sample to be tested and a fingerprint spectrum of the reference substance with sharp characteristic peaks, high theoretical plate numbers and good separation degree can be obtained.

[0024] Further, in S2, the detection wavelength of the HPLC detection is 287 nm, the column temperature is 40°C, the flow rate is 1 mL / min, and the injection volume is 10 to 20 μL.

[0025] Further, in S3, the retention time of characteristic chromatographic peak 1 is 5.5-5.8 min, the retention time of characteristic chromatographic peak 2 is 11.4-11.7 min, the retention time of characteristic chromatographic peak 3 is 17.1-17.4 min, the retention time of characteristic chromatographic peak 4 is 24.4-24.7 min, the retention time of characteristic chromatographic peak 5 is 27.7-27.9 min, the retention time of characteristic chromatographic peak 6 is 28.5-29.0 min, the retention time of characteristic chromatographic peak 7 is 29.1-29.5 min, and the retention time of characteristic chromatographic peak 8 is 41.5-41.9 min.

[0026] More specifically, in the present technical scheme, the total length of the standard fingerprint is 50 min; the average retention time of the characteristic chromatographic peak of peak 1 is 5.7 min, and the average peak area is 50360; the average retention time of the characteristic chromatographic peak of peak 2 is 11.6 min, and the average peak area is 83769; the average retention time of the characteristic chromatographic peak of peak 3 is 17.1 min, and the average peak area is 550224; the average retention time of the characteristic chromatographic peak of peak 4 is 24.5 min, and the average peak area is 1350121; the average retention time of the characteristic chromatographic peak of peak 5 is 27.7 min, and the average peak area is 371365; the average retention time of the characteristic chromatographic peak of peak 6 is 28.6 min, and the average peak area is 219346; the average retention time of the characteristic chromatographic peak of peak 7 is 29.3 min, and the average peak area is 364074; the average retention time of the characteristic chromatographic peak of peak 8 is 41.6 min, and the average peak area is 367234.

[0027] Furthermore, in parts by weight, the raw materials of the pharmaceutical preparation include: 10-20 parts of salvia miltiorrhiza, 10-20 parts of kudzu root, 7-12 parts of panax notoginseng, 10-20 parts of hawthorn, and 7-12 parts of costusroot.

[0028] Furthermore, the dosage form of the pharmaceutical preparation is any one of capsules, tablets, granules, oral liquid and pills.

[0029] The pharmaceutical preparations of this scheme include various dosage forms, such as capsules, tablets, granules, pills and oral liquids, etc. The fingerprint spectrum and identification method established in this scheme can be applied to the identification of the above-mentioned capsules, tablets, granules, pills and oral liquids.

[0030] In summary, the beneficial effects of this technical solution are summarized as follows:

[0031] (1) The liquid chromatography fingerprint identification pattern determination method provided by the present invention can obtain an index component identification fingerprint of a blood-activating, blood-stasis-removing, qi-promoting and analgesic drug preparation composition, which can reflect the index components in the drug. These components are respectively from danshensu sodium, protocatechuic aldehyde, 3'-hydroxypuerarin, puerarin, 3'-methoxypuerarin, puerarin-7-xyloside, soybean glycoside, and salvianolic acid B.

[0032] (2) The liquid chromatography fingerprint identification pattern determination method provided by the present invention has good specificity, precision, repeatability, solution stability and durability.

[0033] (3) The liquid chromatography fingerprint identification pattern determination method provided by the present invention can control the intrinsic quality of the product, improve the quality control level, and better ensure the quality of the product. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is the reference substance spectrum of Example 1 of the present invention.

[0035] Figure 2 This is the spectrum of the Pueraria lobata reference medicinal material of Example 1 of the present invention.

[0036] Figure 3 This is the standard fingerprint established in Example 1 of the present invention.

[0037] Figure 4 This is the fingerprint of the test sample obtained using methanol as the extraction solvent in Experimental Example 1 of the present invention.

[0038] Figure 5 This is the fingerprint of the test sample obtained using ethyl acetate as the extraction solvent in Experimental Example 1 of the present invention.

[0039] Figure 6 This is the fingerprint of the test sample obtained using toluene as the extraction solvent in Experimental Example 1 of the present invention.

[0040] Figure 7 This is the fingerprint of the test sample obtained by testing using a chromatographic column (4.6 mm×25 cm, 3.5 μm) in Experimental Example 2 of the present invention.

[0041] Figure 8 This is the fingerprint of the test sample obtained by testing using a chromatographic column (4.6 mm×25 cm, 5.0 μm) in Experimental Example 2 of the present invention.

[0042] Fig. 9 This is the fingerprint of the test sample obtained by testing using a chromatographic column (4.6 mm×15 cm, 3.5 μm) in Experimental Example 2 of the present invention.

[0043] Fig.10This is the fingerprint of the test sample obtained by testing using a chromatographic column (4.6 mm×15 cm, 5.0 μm) in Experimental Example 2 of the present invention.

[0044] Fig.11 This is the fingerprint of the test sample obtained by testing using a chromatographic column (4.6 mm×7.5 cm, 3.5 μm) in Experimental Example 2 of the present invention.

[0045] Fig.12 This is the fingerprint of the test sample obtained by testing using a chromatographic column (4.6 mm×7.5 cm, 5.0 μm) in Experimental Example 2 of the present invention.

[0046] Fig.13 This is the fingerprint of the negative sample of Salvia miltiorrhiza in Experimental Example 3 of the present invention.

[0047] Fig.14 This is the fingerprint of the negative test sample of Pueraria lobata in Experimental Example 3 of the present invention. DETAILED DESCRIPTION

[0048] The present invention is further described in detail below in conjunction with the examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following examples and experimental examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used can be obtained from commercial channels. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art.

[0049] Embodiment 1:

[0050] Taking a blood-activating, blood-stasis-removing, qi-promoting and pain-relieving drug preparation (capsule, specification: 0.31g) composition as an example, the fingerprint establishment method and standard fingerprint are described. The prescription of the preparation is: 294g of salvia miltiorrhiza, 294g of kudzu root, 19.6g of notoginseng, 294g of hawthorn, and 19.6g of costus root.

[0051] 1. Instruments and test drugs

[0052] Shimadzu liquid chromatograph (LC-2030); Shimadzu electronic balance (ATY224); test preparation (self-produced, batch number 210301); acetonitrile and tetrahydrofuran (chromatographic grade, Chengdu Kelon Reagent Factory); acetic acid and N,N-dimethylformamide (AR Chengdu Kelon Reagent Factory); sodium danshensu reference substance, protocatechuic aldehyde reference substance, salvianolic acid B reference substance, puerarin reference substance and pueraria reference medicinal material (China Food and Biological Products Control Institute).

[0053] 2. Methods and Results

[0054] (1) Chromatographic conditions: Shimadzu liquid chromatograph (LC-2030);

[0055] Chromatographic column: octadecylsilane bonded silica gel as filler (Shimadzu, 4.6 mm x 7.5 cm, 3.5 μm);

[0056] Mobile phase: acetonitrile:tetrahydrofuran:0.4% acetic acid solution (40:40:20);

[0057] Detection wavelength: 287nm;

[0058] Column temperature: 40°C;

[0059] Flow rate: 1 mL / min;

[0060] Injection volume: 10~20μL.

[0061] (2) Preparation method of control reference solution: Take 0.5 g of Pueraria root control medicinal material, place it in a stoppered conical flask, add 50 ml of N,N-dimethylformamide, ultrasonically treat for 30 minutes, shake well, filter, and take the filtrate as the control medicinal material reference solution.

[0062] (3) Preparation method of reference substance: Take appropriate amount of sodium danshensu reference substance, protocatechuic aldehyde reference substance, salvianolic acid B reference substance and puerarin reference substance, weigh accurately, add N,N-dimethylformamide to prepare a mixed solution containing 40-60 μg sodium danshensu, 15-25 μg protocatechuic aldehyde, 80-120 μg salvianolic acid B and 140-160 μg puerarin per 1 ml.

[0063] (4) The test sample preparation method is as follows: take the contents of the capsule of the preparation to be tested, take out about 0.5 g and accurately weigh it, place it in a stoppered conical flask, accurately add 50 mL of N,N-dimethylformamide, seal it, weigh it, and treat it with ultrasound (power 250 W, frequency 40 kHz) for 30 minutes. Take it out, let it cool, weigh it again, make up the lost weight with N,N-dimethylformamide, shake it well, filter it, and take the filtrate to obtain it.

[0064] (5) Determination: Take 10 to 20 μL (10 μL in this protocol) of each of the reference solution, control reference solution and test solution and inject them into a liquid chromatograph for determination.

[0065] (6) Results: The fingerprints of 15 batches of a blood-activating, blood-stasis-removing, qi-promoting and analgesic drug preparation were obtained, as well as the fingerprints of the reference solution and the reference product (typical fingerprints are shown in Figure 1 , Figure 2 , Figure 3 ). The 15 batches of fingerprints were compared with the reference solution and the reference product, and the common characteristic peaks were obtained as the standard fingerprint. The characteristic peaks were sodium danshensu, protocatechuic aldehyde, 3'-hydroxypuerarin, puerarin, 3'-methoxypuerarin, puerarin-7-xyloside, soybean glycoside, and salvianolic acid B. The standard fingerprint is as follows Figure 3As shown, the total length of its fingerprint is about 50 minutes; the retention time of the characteristic chromatographic peak of sodium danshensu (marked as chromatographic peak 1) is 5.5-5.8 minutes; the retention time of the characteristic chromatographic peak of protocatechuic aldehyde (marked as chromatographic peak 2) is 11.4-11.7 minutes; the average retention time of the characteristic chromatographic peak of 3'-hydroxypuerarin (marked as chromatographic peak 3) is 17.1-17.4 minutes; the retention time of the characteristic chromatographic peak of puerarin (marked as chromatographic peak 4) is 24.4-2 4.7min; the retention time of the characteristic chromatographic peak of 3'-methoxypuerarin (marked as chromatographic peak 5) is 27.7-27.9min; the retention time of the characteristic chromatographic peak of puerarin-7-xyloside (marked as chromatographic peak 6) is 28.5-29.0min; the retention time of the characteristic chromatographic peak of daidzein (marked as chromatographic peak 7) is 29.1-29.5min; the retention time of the characteristic chromatographic peak of salvianolic acid B (marked as chromatographic peak 8) is 41.5-41.9min.

[0066] In the quality review analysis, the liquid chromatography identification fingerprints of the index components of the production batches over the years were summarized, and all showed chromatographic peaks consistent with the standard fingerprint, indicating that the determination method of the liquid chromatography identification fingerprint of the index components of this product is durable. Danshensu sodium, protocatechuic aldehyde, 3'-hydroxypuerarin, puerarin, 3'-methoxypuerarin, puerarin-7-xyloside, soybean glycoside, and salvianolic acid B are stable in the preparation and can be used to characterize the quality of the preparation.

[0067] Embodiment 2:

[0068] The test sample of this embodiment is a blood-activating, blood-stasis-removing, qi-promoting and pain-relieving drug preparation (tablets, specification 0.31g), the prescription is the same as that of Example 1, and the rest of the detection methods are the same as those of Example 1. The preparation method of the test sample is: grind the substance to be tested, take about 0.5g and accurately weigh it, put it in a stoppered conical flask, accurately add 50ml of N,N-dimethylformamide, seal it, weigh the weight, ultrasonically treat (power 250W, frequency 40kHz) for 30 minutes, take it out, cool it, weigh it again, make up the lost weight with N,N-dimethylformamide, shake it well, filter it, and take the filtrate to obtain it. After testing, the fingerprint spectrum of the test sample is consistent with that of Example 1. Figure 3 The characteristic peaks of the standard fingerprint are consistent.

[0069] Embodiment 3:

[0070] The test sample of the embodiment is a drug preparation for promoting blood circulation, removing blood stasis, promoting qi and relieving pain (pills, specification 0.31g). The prescription is the same as that of Example 1, and the rest of the detection methods are the same as those of Example 1. The preparation method of the test sample is the same as that of Example 2. After detection, the fingerprint of the test sample is consistent with that of Example 1. Figure 3 The characteristic peaks of the standard fingerprint are consistent.

[0071] Experimental Example 1: Selection of Extraction Solvent for Test Samples

[0072] The inventors have conducted a large number of tests on the extraction solvents of the test samples and found that only by using N,N-dimethylformamide as the extraction reagent of the test sample can it be guaranteed that the 8 characteristic components in the fingerprint spectrum all have characteristic peaks with ideal peak shapes. Taking methanol, ethyl acetate, toluene and DMF as extraction reagents as examples, the influence of different extraction reagents on the quality of the fingerprint spectrum is demonstrated. Take a blood-activating, blood-stasis-removing, qi-promoting and analgesic drug preparation (capsule) in Example 1 of the same batch, respectively select methanol, ethyl acetate, toluene and DMF (N,N-dimethylformamide) as the dissolving and extracting solvent, ultrasonically apply for 30 minutes under ice bath conditions, let it stand, take the supernatant and filter, take the filtrate as the test sample solution (4 kinds), and perform liquid chromatography detection according to the method in Example 1 to obtain the fingerprint spectrum. The peak of sodium danshensu in the liquid chromatography fingerprint spectrum of methanol extract is not obvious ( Figure 4 ); The peak of protocatechuic aldehyde in the liquid chromatography fingerprint of the ethyl acetate extract was not obvious ( Figure 5 ); The peak of salvianolic acid B in the liquid chromatography fingerprint of toluene extract is not obvious ( Figure 6 ); DMF extraction effect is very good, each component can be effectively extracted ( Figure 3 ), indicating that using DMF as the extraction solvent can ensure that each characteristic peak is obvious and easy to identify.

[0073] Experimental Example 2: Selection of Chromatographic Column

[0074] A blood-activating, blood-stasis-removing, qi-promoting and pain-relieving drug preparation (capsule) from the same batch as in Example 1 was used for a chromatographic column selection experiment. The experimental process is shown in Example 1.

[0075] 1. Selection of chromatographic column specifications

[0076] (1) A chromatographic column with octylsilane bonded silica as filler was selected. Shimadzu chromatographic columns with column lengths of 7.5 cm, 15 cm and 25 cm, inner diameters of 4.6 mm, and stationary phase particle sizes of 3.5 μm and 5 μm were selected for testing. The results showed that the columns with stationary phase particle sizes of 3.5 and 5 μm could not completely separate the target peaks, especially the separation effect between 3'-methoxypuerarin, puerarin-7-xyloside, and daidzein was poor, indicating that the chromatographic column with octylsilane bonded silica as filler had a poor separation effect on the target peaks of the preparation.

[0077] (2) Select octadecylsilane bonded silica gel as the filler of the chromatographic column, and select Shimadzu chromatographic columns with column lengths of 7.5 cm, 15 cm and 25 cm, inner diameters of 4.6 mm, and stationary phase particle sizes of 3.5 μm and 5 μm for the test (see Figure 7 to Figure 12), the separation effect of the column with a stationary phase particle size of 5μm is not as good as that of the column with a stationary phase particle size of 3.5μm. For example, when using a column with a stationary phase particle size of 5μm, the separation effect between 3'-methoxypuerarin, puerarin-7-xyloside, daidzein and nearby impurities is not good, or the peaks of some characteristic components are not obvious, or the peaks of some characteristic components are slightly less sharp. The target peaks of the 3.5μm column can achieve baseline separation, and the theoretical plate number can reach more than 10,000, but the longer the column, the longer the running time, which is not conducive to rapid detection; the test results show that the column with a length of 7.5cm, an inner diameter of 4.6mm, and a stationary phase particle size of 3.5μm can meet the measurement requirements, save detection time, and improve efficiency.

[0078] 2. Chromatographic column brand selection

[0079] Currently, the chromatographic columns on the market are mainly from brands such as Shimadzu, Waters, Agilent, Thermo Fisher, Daicel, Yuexu, YMC, and Shiseido. Therefore, octadecylsilane bonded silica gel from various brands was selected as the filler (chromatographic column specifications: 4.6 mm × 7.5 cm, stationary phase particle size 3.5 μm) to examine the durability of the method.

[0080] Table 1: Column brand selection results

[0081]

[0082] Our company has carried out corresponding chromatographic column brand screening tests. The chromatographic columns selected include Shimadzu, Waters, Agilent, Thermo Fisher, Daicel, Yuexu, YMC, Shiseido, etc. The brand specifications are all 4.6mm×7.5cm, 3.5μm. The screening results confirm that these conventional chromatographic columns can separate the index components of the product, with high separation degree and theoretical plate number, and can meet the identification requirements of one test and multiple evaluations. The chromatographic columns have good durability.

[0083] Experimental Example 3: Specificity Test

[0084] 1. Solution preparation

[0085] (1) Preparation of reference solution: Take 0.5 g of Pueraria root reference drug, place it in a stoppered conical flask, add 50 ml of N,N-dimethylformamide, ultrasonically treat for 30 minutes, shake well, filter, and take the filtrate as the reference drug solution (see typical test results). Figure 2 ).

[0086] (2) Preparation of reference substances: Take appropriate amounts of sodium danshensu reference substance, protocatechuic aldehyde reference substance, salvianolic acid B reference substance, and puerarin reference substance, weigh them accurately, and add N,N-dimethylformamide to prepare a mixed solution containing 40-60 μg sodium danshensu, 15-25 μg protocatechuic aldehyde, 80-120 μg salvianolic acid B, and 140-160 μg puerarin per 1 ml. (Typical test results are shown in Figure 1 ).

[0087] (3) The test sample preparation method is as follows: take about 0.5 g of the capsule content, accurately weigh it, place it in a stoppered conical flask, accurately add 50 ml of N,N-dimethylformamide, seal it, weigh it, ultrasonically treat it (power 250 W, frequency 40 kHz) for 30 minutes, take it out, cool it, weigh it again, make up the lost weight with N,N-dimethylformamide, shake it well, filter it, and take the filtrate to obtain (see typical test results). Figure 3 ).

[0088] (4) Preparation of negative test solution without Danshen: Take about 0.5 g of the content of the capsule without Danshen, weigh it accurately, put it in a stoppered conical flask, accurately add 50 ml of N,N-dimethylformamide, stopper it tightly, weigh it, and treat it with ultrasound (power 250 W, frequency 40 kHz) for 30 minutes. Take it out, let it cool, weigh it again, make up the lost weight with N,N-dimethylformamide, shake it well, filter it, and take the filtrate to obtain (see typical test results). Fig.13 ).

[0089] (5) Preparation of negative test solution without Pueraria root: Take about 0.5 g of the contents of the capsule without Pueraria root, weigh accurately, place in a stoppered conical flask, accurately add 50 ml of N,N-dimethylformamide, stopper, weigh, ultrasonically treat (power 250 W, frequency 40 kHz) for 30 minutes, take out, cool, weigh again, make up the lost weight with N,N-dimethylformamide, shake well, filter, and take the filtrate to obtain (see typical test results). Fig.14 ).

[0090] 2. Detection: refer to the chromatographic conditions of Example 1 for sample injection detection

[0091] 3. Research results: The negative solution does not produce a peak at the characteristic peak, which means that the negative solution does not interfere with the peak of the indicator component and has good specificity.

[0092] Experimental Example 4: Precision Test

[0093] The same batch of blood-activating, blood-stasis-removing, qi-promoting and pain-relieving drug preparations (capsules) were used to prepare the test solution according to the preparation method of the test solution, and the sample was injected 6 times for determination (refer to Example 1). The determination results are shown in Table 2-3. The results show that the chromatographic peaks corresponding to the standard reference fingerprint spectrum can appear in the test sample, and the RSD of the retention time and peak area of ​​each main peak is less than 3.0%, indicating that the precision of the determination is good.

[0094] Table 2: Statistical results of retention time in precision test

[0095]

[0096] Table 3: Statistical results of main peak area in precision test

[0097]

[0098] Experimental Example 5: Repeatability Test

[0099] The test solution was prepared according to the preparation method of the test solution. 6 minutes of the test solution was prepared in the same way, and the results were calculated according to the method (refer to Example 1). The relative deviation (RSD) of the peak area corresponding to the standard control fingerprint was calculated. The results are shown in Table 4. The research results show that the main peak of each identification substance can correspond to the standard fingerprint, and the relative deviation of the area is less than 3.0%, indicating that the method has good repeatability.

[0100] Table 4: Results of the repeatability test study

[0101]

[0102]

[0103] Experimental Example 6: Stability Test

[0104] Take a blood-activating, blood-stasis-removing, qi-promoting and analgesic drug preparation (capsule), prepare a test solution according to the preparation method of the test solution, measure the fingerprint spectrum once at 0, 2.5, 5, 10, 17.5, and 25 hours according to the law (refer to Example 1), a total of 6 measurements, calculate the relative deviation (RSD) of the corresponding peak area of ​​the fingerprint spectrum, and the results are shown in Table 5. The research results show that the main peak of each identified substance can correspond to the standard fingerprint spectrum, and the relative deviation of the main peak area of ​​the corresponding substance in the fingerprint spectrum at each time point is less than 3.0%, indicating that the test solution is stable within 25 hours.

[0105] Table 5: Solution stability study results

[0106]

[0107] Experimental Example 7: Durability Test

[0108] This experimental example tests the mobile phase used in high performance liquid chromatography detection. The experimental conditions refer to Example 1, and the mobile phase used is referred to Table 6. The calculation method of the separation between adjacent characteristic peaks is: separation (R) = 2[t(R2)-t(R1)] / (W1+W2); where t(R2) is the retention time of the latter of the two adjacent peaks; t(R1) is the retention time of the former of the two adjacent peaks; W1 and W2 are the peak base widths of the two adjacent peaks.

[0109] Table 6: Mobile phase selection results

[0110]

[0111] In the process of trying to establish a fingerprint spectrum detection method for the drug, the inventors first used conventional mobile phase conditions in the prior art for isocratic elution, such as acetonitrile + 0.05% phosphoric acid, methanol + 0.5% acetic acid solution, and acetonitrile aqueous solution (these mobile phase conditions are usually used for gradient elution in the prior art, with gradient mixing of organic phase and aqueous phase). It was found that isocratic elution under the above mobile phase conditions always has different degrees of poor peak shape and poor separation between target components. The inventors tested and studied a large number of candidate mobile phases and found that the mobile phase of acetonitrile: tetrahydrofuran: 0.4% acetic acid solution (40:40:20) can obtain the most ideal fingerprint spectrum effect, with sharp peak shape, ideal separation (the separation between adjacent characteristic peaks is much greater than 1.5), and ideal theoretical plate number. For details, see Figure 3 . Compared with the prior art, the mobile phase of the present technical solution is composed of tetrahydrofuran, acetonitrile and 0.4% acetic acid solution, among which tetrahydrofuran is introduced into the mobile phase for the first time by the inventor, and the unexpected technical effect is obtained. When tetrahydrofuran or pure acetonitrile is used alone, there will be a situation where sodium danshensu does not peak, and the separation between the target peaks is extremely poor; when acetic acid solution is used alone as the mobile phase, although sodium danshensu can peak, the peak shape is not good, and other characteristic components do not peak, so the formed HPLC spectrum cannot be used as a standard spectrum. However, after tetrahydrofuran, acetonitrile and acetic acid solution are used together as the mobile phase, not only each characteristic component peaks, but also the sodium danshensu peak shape is ideal, and the theoretical plate number of each characteristic peak can reach more than 10,000, which meets the requirements of fingerprint spectrum detection.

[0112] The above is only an embodiment of the present invention, and the common knowledge such as the known specific technical solutions and / or characteristics in the solution is not described in detail here. It should be pointed out that for those skilled in the art, without departing from the technical solution of the present invention, several modifications and improvements can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. A method for determining the fingerprint of a blood-activating, blood-stasis-removing, qi-promoting and analgesic drug preparation, characterized in that: The method includes the following steps in sequence: S1: extracting a test sample from a drug preparation; the test sample is prepared by the following method: using N,N-dimethylformamide as an extraction solvent, performing ultrasound-assisted extraction on the drug preparation to obtain the test sample; the raw materials of the drug preparation include: 10-20 parts of Salvia miltiorrhiza, 10-20 parts of Pueraria lobata, 7-12 parts of Panax notoginseng, 10-20 parts of Crataegus pinnatifida, and 7-12 parts of Aucklandia lappa; S2: Detecting the sample using high performance liquid chromatography to obtain a fingerprint spectrum; the mobile phase of the high performance liquid chromatography is composed of acetonitrile, tetrahydrofuran and acetic acid solution; the volume ratio of acetonitrile, tetrahydrofuran and acetic acid solution is 40:40:20; The filler of the HPLC column is octadecylsilane bonded silica gel, and the filler particle size is 3.5-5.0 μm; S3: comparing the fingerprint with the standard fingerprint to evaluate the quality of the drug preparation; The standard fingerprint spectrum includes the following characteristic peaks: characteristic chromatographic peak 1 representing sodium danshensu, characteristic chromatographic peak 2 representing protocatechuic aldehyde, characteristic chromatographic peak 3 representing 3'-hydroxypuerarin, characteristic chromatographic peak 4 representing puerarin, characteristic chromatographic peak 5 representing 3'-methoxypuerarin, characteristic chromatographic peak 6 representing puerarin-7-xyloside, characteristic chromatographic peak 7 representing daidzein, and characteristic chromatographic peak 8 representing salvianolic acid B.

2. The method for determining the fingerprint of a blood-activating, blood-stasis-removing, qi-promoting and analgesic drug preparation according to claim 1, characterized in that: In S2, the detection wavelength of HPLC detection was 287 nm, the column temperature was 40 °C, the flow rate was 1 mL / min, and the injection volume was 10-20 μL.

3. The method for determining the fingerprint of a blood-activating, blood-stasis-removing, qi-promoting and analgesic drug preparation according to claim 2, characterized in that: In S3, the retention time of characteristic chromatographic peak 1 is 5.5~5.8min, the retention time of characteristic chromatographic peak 2 is 11.4~11.7min, the retention time of characteristic chromatographic peak 3 is 17.1~17.4min, the retention time of characteristic chromatographic peak 4 is 24.4~24.7min, the retention time of characteristic chromatographic peak 5 is 27.7~27.9min, the retention time of characteristic chromatographic peak 6 is 28.5~29.0min, the retention time of characteristic chromatographic peak 7 is 29.1~29.5min, and the retention time of characteristic chromatographic peak 8 is 41.5~41.9min.

4. The method for determining the fingerprint of a blood-activating, blood-stasis-removing, qi-promoting and analgesic drug preparation according to claim 1, characterized in that: The dosage form of the pharmaceutical preparation is any one of capsules, tablets, granules, oral liquid and pills.

5. Application of the method for determining the fingerprint of a blood-activating, blood-stasis-removing, qi-promoting and analgesic drug preparation according to any one of claims 1 to 4 in the evaluation of drug characteristic components.

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