Construction method and application of characteristic chromatogram of Xuefuzhuyu tablets

Abstract

The invention relates to the technical field of traditional Chinese medicine analysis, provides a construction method and application of a Xuefuzhuyu tablet characteristic spectrum, and is characterized in that detection of a characteristic peak is realized through extraction, preparation and purification of an extract and specific gradient selection under a specific mobile phase condition. According to the method, seven chemical components in the Xuefuzhuyu tablet can be analyzed, five characteristic peaks including amygdalin, hydroxysafflor yellow A, paeoniflorin, naringin and ammonium glycyrrhizinate are identified, verification research on specificity, precision, repeatability and stability is carried out on the method, and it is proved that the method is accurate and reliable. The technical means is used as a method for overall evaluation of the Xuefuzhuyu tablets, and a quality control system of the Xuefuzhuyu tablets can be comprehensively improved.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine analysis, and particularly relates to a method for constructing a characteristic fingerprint of Xuefu Zhuyu tablets and its application. Background Art

[0002] Xuefu Zhuyu tablets are made from various medicinal herbs such as Bupleuri Radix, Angelicae Sinensis Radix, Rehmanniae Radix, Paeoniae Rubra Radix, Carthami Flos, Persicae Semen, Aurantii Fructus Immaturus Praeparatus, Glycyrrhizae Radix, Chuanxiong Rhizoma, Achyranthis Bidentatae Radix, Platycodonis Radix, etc. through processing. It can promote blood circulation to remove blood stasis and regulate qi to relieve pain, and is used for chest pain, headache of long duration, pain like pricking needles with a fixed location, internal heat and restlessness, palpitations and insomnia, irritability and anger caused by qi stagnation and blood stasis.

[0003] The 2020 edition of the Chinese Pharmacopoeia stipulates that the quality control standards for Xuefu Zhuyu tablets include: microscopic identification of Bupleuri Radix, Chuanxiong Rhizoma, Aurantii Fructus Immaturus, Carthami Flos; thin-layer identification of control medicinal materials of Aurantii Fructus Immaturus, Achyranthis Bidentatae Radix, Glycyrrhizae Radix, Platycodonis Radix and control substances of ferulic acid, naringin, β-ecdysterone, paeoniflorin; content determination stipulates that each pill of Xuefu Zhuyu tablets contains not less than 4.0 mg of Paeoniae Rubra Radix calculated as paeoniflorin (C 23 H 28 O 11 ), and not less than 8.7 mg of Aurantii Fructus Immaturus Praeparatus calculated as naringin (C 27 H 32 O 14 ). Since Xuefu Zhuyu tablets contain many medicinal herbs and have complex chemical components, it is difficult for a single component to fully reflect the chemical component characteristics of the preparation. Therefore, applying high-performance liquid chromatography to establish the characteristic fingerprint of Xuefu Zhuyu tablets can effectively control its quality and achieve an overall grasp of the drug components.

[0004] Currently, the quality standards of this preparation have been included in the Chinese Pharmacopoeia, and the quality standards are relatively complete. However, there is no item of characteristic fingerprint in the quality standards of Xuefu Zhuyu tablets in the Chinese Pharmacopoeia, and no other characteristic fingerprint detection methods for Xuefu Zhuyu tablets have been found in the existing technology. Therefore, the present invention has established a reliable and effective detection method for the characteristic fingerprint of Xuefu Zhuyu tablets, enabling better quality control on the basis of the original pharmacopoeia standards. Summary of the Invention

[0005] Aiming at the deficiencies in the research on the characteristic fingerprint of Xuefu Zhuyu tablets in the existing quality standards, the purpose of the present invention is to provide a method for constructing a characteristic fingerprint of Xuefu Zhuyu tablets and its application. This method can analyze 7 chemical components in Xuefu Zhuyu tablets and identify 5 characteristic peaks, aiming to solve the problem of the lack of an overall evaluation method for Xuefu Zhuyu tablets in the existing technology and comprehensively improve the quality control system of Xuefu Zhuyu tablets.

[0006] Characteristic Fingerprint Detection: A method for constructing a characteristic fingerprint of Xuefu Zhuyu tablets, comprising the following steps: The Xuefu Zhuyu tablets sample was added to a solvent, then refluxed for extraction, cooled, shaken well, filtered, evaporated to dryness, dissolved, passed through a polyamide column for elution, the eluate was evaporated to dryness and dissolved to obtain the test solution. The test solution was detected by high performance liquid chromatography (HPLC), and the index components in the characteristic chromatogram of the test solution were assigned and located according to the retention time or relative retention time, so as to obtain the characteristic chromatogram of the pharmaceutical preparation. The specific steps are as follows: (1) Preparation of the reference solution: Weigh the reference substances of paeoniflorin, naringin, ammonium glycyrrhizinate, hydroxysafflor yellow A, and amygdalin, dissolve them in a solvent and make up the volume to obtain the reference solution. (2) Preparation of the test solution: Take 1 g of this product, pulverize it, add 2 - 5 g of column chromatography silica gel, grind evenly, add 40 - 70 ml of a methanol - acetone mixed solution with a weight ratio of 3:7, then heat the mixed solution under reflux for 45 minutes. Let the product after heating under reflux cool down, shake well, filter through a 0.45 μm nylon membrane, evaporate the methanol - acetone mixed filtrate to dryness. Then dissolve the dried precipitate in hot water, and pass the dissolved solution through a 5 g polyamide column. First, elute with 30 - 50 ml of water, then elute with 30 - 50 ml of 70% methanol with the pH adjusted to 6 - 6.5 with hydrochloric acid. Then collect the 70% methanol eluate, evaporate it to dryness, dissolve the residue in 5 ml of a solution of N,N - dimethylformamide:methanol:0.04 mol / L citric acid solution with a volume ratio of 0.1:0.9:9 to obtain the test solution. (3) Determination: The test solution and the reference solution in step (1) were respectively determined by HPLC to obtain the characteristic chromatogram of the test solution and the characteristic chromatogram of the reference solution. The characteristic chromatogram of the test solution was compared with the characteristic chromatogram of the reference solution in terms of retention time for qualitative analysis, and the index components in the characteristic chromatogram of the test solution were assigned and located, so as to obtain the characteristic chromatogram of the pharmaceutical preparation. The conditions for HPLC are as follows: The chromatographic column is packed with octadecylsilane chemically bonded silica gel (column length is 250 mm, inner diameter is 4.6 mm, particle size is 5 μm); The mobile phase A is N,N - dimethylformamide - methanol (1:9), and the mobile phase B is 0.04 mol / L citric acid solution; The column temperature is 35 °C; The detection wavelength of the ultraviolet - visible detector is 237 nm. The theoretical plate number calculated based on the naringin peak should be not less than 5000. The gradient elution is as follows: 0 - 5 min, the volume ratio of mobile phase A to B is 10:90; 5 - 19 min, the volume ratio of mobile phase A to B changes from 10:90 to 19:81; 19 - 28 min, the volume ratio of mobile phase A to B changes from 19:81 to 26:74; 28 - 39 min, the volume ratio of mobile phase A to B changes from 26:74 to 32:68; From 39 to 55 min, mobile phase A: B, volume ratio changes from 32:68 to 55:45; From 55 to 70 min, mobile phase A: B, volume ratio changes from 55:45 to 30:70; From 70 to 75 min, mobile phase A: B, volume ratio changes from 30:70 to 10:90; From 75 to 80 min, mobile phase A: B, volume ratio is 10:90.

[0007] Furthermore, in the present invention, (1) A total of 7 characteristic peaks are presented in the characteristic chromatogram of Xuefu Zhuyu tablets. Taking the 5th peak as the S peak, calculate the relative retention times of peak 1, peak 2, peak 3, peak 4, peak 6, and peak 7 with respect to the S peak, which should be within the range of ±10% of the specified values. The specified values are: 0.47 (peak 1), 0.53 (peak 2), 0.62 (peak 3), 0.69 (peak 4), 1.07 (peak 6), 1.76 (peak 7); (2) Compare the standard characteristic chromatogram of the Xuefu Zhuyu tablets with the characteristic chromatogram of the reference substance solution, and locate and determine that peak 1 is amygdalin, peak 2 is hydroxysafflor yellow A, peak 4 is paeoniflorin, peak 5 is naringin, and peak 7 is ammonium glycyrrhizinate; (3) The preparation of the test sample is as follows: Take 1 g of this product, pulverize it, add 2 g of column chromatography silica gel, grind evenly, add 40 ml of a methanol-acetone mixed solution with a weight ratio of 3:7, heat under reflux for 45 minutes, cool, shake well, filter through a 0.45 μm nylon filter membrane, evaporate the filtrate to dryness, dissolve it in hot water, pass through a 5 g polyamide column, first elute with 30 ml of water, then elute with 30 ml of 70% methanol adjusted to pH 6 - 6.5 with hydrochloric acid, collect the 70% methanol eluate, evaporate to dryness, and dissolve the residue in 5 ml of a solution of N,N-dimethylformamide: methanol: 0.04 mol / L citric acid solution with a volume ratio of 0.1:0.9:9 to obtain the solution.

[0008] In the second aspect, the present invention provides the application of the above method in the quality rating of Xuefu Zhuyu tablets.

[0009] Compared with the prior art, the advantages of the present invention are: (1) A detection method for the characteristic chromatogram of Xuefu Zhuyu tablets provided by the present invention. This method can separate and detect the characteristic components of Xuefu Zhuyu tablets, more comprehensively reflect the component information of Xuefu Zhuyu tablets, and realize applications such as quality control of the characteristic chromatogram of Xuefu Zhuyu tablets, monitoring the production of pharmaceutical preparations, and identifying the authenticity of pharmaceutical preparations; (2)The detection method of the characteristic chromatogram of Xuefu Zhuyu tablets provided by the present invention conducts specificity, precision, stability, and reproducibility test studies using relative retention time as an index during the process of establishing the determination method of the characteristic chromatogram of this pharmaceutical preparation. A total of 7 common characteristic peaks are confirmed, ensuring the stability and adaptability of the method. Description of the Drawings

[0010] Figure 1 It is a specificity chromatogram; among them, S1: negative control chromatogram; S2: test sample chromatogram; Figure 2 It is a reference substance chromatogram; among them, S1: test sample chromatogram; S2 - S6 are amygdalin, hydroxysafflor yellow A, paeoniflorin, naringin, and ammonium glycyrrhizinate in sequence; Figure 3 It is the control characteristic chromatogram of Xuefu Zhuyu tablets. Specific Embodiments

[0011] The present invention will be further elaborated below in combination with specific embodiments. These embodiments are only used to illustrate the present invention and not to limit the protection scope of the present invention.

[0012] Experimental Example 1: Characteristic Chromatogram 1. Instruments, reagents, and test samples Instruments: High-performance liquid chromatograph (Thermo U3000); electronic balance (Sartorius Scientific Instruments (Beijing) Co., Ltd., BSA224S-CW (one ten-thousandth)); chromatographic column (Agilent TC C18, 250mm * 4.6mm 5μm); digital display constant temperature water bath (DRHH-S6, Shanghai Chengjie).

[0013] Reagents: Methanol (Merck, I1228907236); acetonitrile (Merck, JB095730); N, N-dimethylformamide (Macklin, C17477721); citric acid (Yantai Shuangshuang Chemical Industry Co., Ltd., 20210901); phosphoric acid (Macklin, C12485051); water (self-made, purified water); hydrochloric acid (Yantai Shuangshuang Chemical Industry Co., Ltd., 20221101).

[0014] Reference substances: Ferulic acid reference substance (National Institutes for Food and Drug Control), batch number: 110773 - 201915; Naringin reference substance (National Institutes for Food and Drug Control), batch number: 110722 - 202116; Paeoniflorin reference substance (National Institutes for Food and Drug Control), batch number: 110736 - 202145; Amygdalin reference substance (National Institutes for Food and Drug Control), batch number: 110820 - 202109; Reference substance of hydroxysafflor yellow A (National Institutes for Food and Drug Control), batch number: 111637-202111; Sample: Xuefu Zhuyu tablets (self-made), batch number: 20220810.

[0015] 2. Preparation of reference solution Weigh appropriate amounts of paeoniflorin reference substance, naringin reference substance, ammonium glycyrrhizinate reference substance, hydroxysafflor yellow A reference substance, and amygdalin reference substance accurately, dissolve them in methanol to prepare a mixed solution containing 30 μg of paeoniflorin, 30 μg of naringin, 30 μg of ammonium glycyrrhizinate, 30 μg of hydroxysafflor yellow A, and 1 mg of amygdalin per 1 ml. Shake well to obtain the reference substance reference solution.

[0016] 3. Preparation of test solution Take 1 g of this product, pulverize it, add 2 g of column chromatography silica gel, grind evenly, add 40 ml of methanol-acetone mixed solution with a weight ratio of 3:7, heat under reflux for 45 minutes, cool, shake well, filter through a 0.45 μm nylon filter membrane, evaporate the filtrate to dryness, dissolve it in hot water, pass through a 5 g polyamide column, first elute with 30 ml of water, then elute with 30 ml of 70% methanol adjusted to pH 6-6.5 with hydrochloric acid, collect the 70% methanol eluate, evaporate it to dryness, dissolve the residue in 5 ml of N,N-dimethylformamide:methanol:0.04 mol / L citric acid solution with a volume ratio of 0.1:0.9:9 to obtain the solution.

[0017] 4. Chromatographic conditions and system suitability test Use octadecylsilane chemically bonded silica gel as the filler (column length is 250 mm, inner diameter is 4.6 mm, particle size is 5 μm); use N,N-dimethylformamide-methanol (1:9) as mobile phase A and 0.04 mol / L citric acid solution as mobile phase B, and perform gradient elution according to the regulations in the following table; the column temperature is 35 °C; the detection wavelength is 237 nm. The number of theoretical plates calculated based on the naringin peak should not be less than 5000.

[0018] 。

[0019] 5. Specificity Take an appropriate amount of this product, and prepare the test solution and the negative control solution without medicinal flavor according to the proposed method for preparing the test solution. Pipette 10 μL of each of the above two solutions and the reference solution, and inject them into the liquid chromatograph. The test results show that the negative has no interference, see attachment Figure 1 、 Figure 2 。

[0020] 6. Precision Take the test sample, weigh 1 g, prepare 1 portion of the test sample solution. Under the above chromatographic conditions, inject samples continuously for 6 times, record the chromatogram at 237 nm, determine the relative retention time of each chromatographic peak, and calculate the relative standard deviation. The RSD values of the relative retention time of each chromatographic peak are all less than 2.0%, and the RSD values of the relative peak area are all less than 2.0%, indicating good repeatability. The results are shown in the following table.

[0021] Table 1 Results of relative retention time test 。

[0022] Table 2 Results of relative peak area investigation 。

[0023] 7. Repeatability Take the test sample, weigh approximately 1 g, prepare 6 portions of the test sample solution. Under the above chromatographic conditions, inject samples for 6 times, record the chromatogram at 237 nm, determine the relative retention time of each chromatographic peak, and calculate the relative standard deviation. The RSD values of the relative retention time of each chromatographic peak are all less than 2.0%, and the RSD values of the relative peak area are all less than 2.0%, indicating good repeatability. The results are shown in the following table.

[0024] Table 3 Results of relative retention time test 。

[0025] Table 4 Results of relative peak area investigation 。

[0026] 8. Stability Take the test sample, weigh approximately 1 g, prepare the test sample solution. Under the above chromatographic conditions, inject samples at 0, 2, 4, 8, 12, and 24 h respectively, record the chromatogram at 237 nm, determine the relative retention time of each chromatographic peak, and calculate the relative standard deviation. The RSD values of the relative retention time of each chromatographic peak are all less than 2.0%, and the RSD values of the relative peak area are all less than 2.0%, indicating that the test sample solution is stable within 24 h. The results are shown in the following table.

[0027] Table 5 Results of relative retention time investigation 。

[0028] Table 6 Results of relative peak area investigation 。

[0029] 9. Determination of characteristic peaks Seven characteristic peaks should be presented in the chromatogram of the test sample. Among them, five peaks should correspond to the retention times of the corresponding reference substance peaks of the reference substances respectively. Taking the peak corresponding to the naringin reference substance peak as the S peak, calculate the relative retention times of peak 1, peak 2, peak 3, peak 4, peak 6, and peak 7 with respect to the S peak, which should be within the range of ±10% of the specified values. The specified values are: 0.47 (peak 1), 0.53 (peak 2), 0.62 (peak 3), 0.69 (peak 4), 1.07 (peak 6), 1.76 (peak 7). See the attached reference characteristic chromatogram Figure 3 。

[0030] Experimental Example 2: Screening of characteristic chromatogram conditions 1. Instruments, reagents and test samples Instruments: High performance liquid chromatograph (Thermo U3000); Electronic balance (Sartorius Scientific Instruments (Beijing) Co., Ltd., BSA224S-CW (one ten-thousandth)); Chromatographic column (Agilent TC C18, 250mm * 4.6mm 5μm); Digital display constant temperature water bath (DRHH-S6, Shanghai Chengjie).

[0031] Reagents: Methanol (Merck, I1228907236); Acetonitrile (Merck, JB095730); N, N-dimethylformamide (Maclean, C17477721); Citric acid (Yantai Shuangshuang Chemical Industry Co., Ltd., 20210901); Phosphoric acid (Maclean, C12485051); Water (self-made, purified water); Hydrochloric acid (Yantai Shuangshuang Chemical Industry Co., Ltd., 20221101).

[0032] Reference substances: Ferulic acid reference substance (National Institutes for Food and Drug Control), batch number: 110773-201915; Naringin reference substance (National Institutes for Food and Drug Control), batch number: 110722-202116; Paeoniflorin reference substance (National Institutes for Food and Drug Control), batch number: 110736-202145; Amygdalin reference substance (National Institutes for Food and Drug Control), batch number: 110820-202109; Hydroxysafflor yellow A reference substance (National Institutes for Food and Drug Control), batch number: 111637-202111; Sample: Xuefu Zhuyu tablets (self-made), batch number: 20220810.

[0033] 2. Preparation of reference substance solution Weigh appropriate amounts of paeoniflorin reference substance, naringin reference substance, ammonium glycyrrhizinate reference substance, hydroxysafflor yellow A reference substance, and amygdalin reference substance accurately. Dissolve them in methanol to prepare a mixed solution containing 30 μg of paeoniflorin, 30 μg of naringin, 30 μg of ammonium glycyrrhizinate, 30 μg of hydroxysafflor yellow A, and 1 mg of amygdalin per 1 ml. Shake well to obtain the reference substance solution.

[0034] 3. Preparation of the test solution Take 1 g of this product, pulverize it, add 2 g of column chromatography silica gel, grind evenly, add 40 ml of a methanol-acetone mixed solution with a weight ratio of 3:7, heat under reflux for 45 minutes, cool, shake well, filter through a 0.45 μm nylon filter membrane, evaporate the filtrate to dryness, dissolve it in hot water, pass it through a 5 g polyamide column, first elute with 30 ml of water, then elute with 30 ml of 70% methanol adjusted to pH 6 - 6.5 with hydrochloric acid, collect the 70% methanol eluate, evaporate it to dryness, dissolve the residue in 5 ml of a solution of N,N-dimethylformamide:methanol:0.04 mol / L citric acid solution with a volume ratio of 0.1:0.9:9 to obtain the solution.

[0035] 4. Screening of chromatographic conditions (screening of mobile phase ratio and elution gradient) ① Use octadecylsilane chemically bonded silica gel as the filler (column length is 250 mm, inner diameter is 4.6 mm, particle size is 5 μm); use methanol as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, and perform gradient elution according to the regulations in the following table; column temperature is 35 °C; detection wavelength is 237 nm. The number of theoretical plates calculated based on the naringin peak should be not less than 5000.

[0036] 。

[0037] ② Use octadecylsilane chemically bonded silica gel as the filler (column length is 250 mm, inner diameter is 4.6 mm, particle size is 5 μm); use methanol as mobile phase A and 0.04 mol / L citric acid solution as mobile phase B, and perform gradient elution according to the regulations in the following table; column temperature is 35 °C; detection wavelength is 237 nm. The number of theoretical plates calculated based on the naringin peak should be not less than 5000.

[0038] 。

[0039] ③ Use octadecylsilane chemically bonded silica gel as the filler (column length is 250 mm, inner diameter is 4.6 mm, particle size is 5 μm); use N,N-dimethylformamide-methanol (1:8) as mobile phase A and 0.04 mol / L citric acid solution as mobile phase B, and perform gradient elution according to the regulations in the following table; column temperature is 35 °C; detection wavelength is 237 nm. The number of theoretical plates calculated based on the naringin peak should be not less than 5000.

[0040] 。

[0041] ④ Using octadecylsilane chemically bonded silica gel as the packing material (column length: 250 mm, inner diameter: 4.6 mm, particle size: 5 μm); using N,N-dimethylformamide-methanol (1:9) as mobile phase A and 0.04 mol / L citric acid solution as mobile phase B, perform gradient elution according to the regulations in the following table; column temperature is 35 °C; detection wavelength is 237 nm. The number of theoretical plates calculated based on the naringin peak should be not less than 5000.

[0042] 。

[0043] ⑤ Using octadecylsilane chemically bonded silica gel as the packing material (column length: 250 mm, inner diameter: 4.6 mm, particle size: 5 μm); using N,N-dimethylformamide-methanol (1:9) as mobile phase A and 0.04 mol / L citric acid solution as mobile phase B, perform gradient elution according to the regulations in the following table; column temperature is 35 °C; detection wavelength is 237 nm. The number of theoretical plates calculated based on the naringin peak should be not less than 5000.

[0044] 。

[0045] ⑥ Using octadecylsilane chemically bonded silica gel as the packing material (column length: 250 mm, inner diameter: 4.6 mm, particle size: 5 μm); using N,N-dimethylformamide-methanol (1:9) as mobile phase A and 0.04 mol / L citric acid solution as mobile phase B, perform gradient elution according to the regulations in the following table; column temperature is 35 °C; detection wavelength is 237 nm. The number of theoretical plates calculated based on the naringin peak should be not less than 5000.

[0046] 。

[0047] Summary: Through the screening of different mobile phases and elution gradients, more characteristic peaks with better resolution were obtained by using Method ④. The determined chromatographic conditions are as follows: Using octadecylsilane chemically bonded silica gel as the packing material (column length: 250 mm, inner diameter: 4.6 mm, particle size: 5 μm); using N,N-dimethylformamide-methanol (1:9) as mobile phase A and 0.04 mol / L citric acid solution as mobile phase B, perform gradient elution according to the regulations in the following table; column temperature is 35 °C; detection wavelength is 237 nm. The number of theoretical plates calculated based on the naringin peak should be not less than 5000.

[0048] 。

Claims

1. A method for constructing a characteristic fingerprint of Xuefu Zhuyu tablets, characterized in that: It includes the following steps: Add the Xuefu Zhuyu Tablet sample to a solvent, reflux extract, cool, shake well, filter, evaporate to dryness, dissolve, elute through a polyamide column, evaporate the eluate to dryness, and dissolve. The obtained test solution is detected by high performance liquid chromatography (HPLC). Then, based on the retention time or relative retention time, the index components in the characteristic chromatogram of the obtained test solution are assigned and located, so as to obtain the characteristic chromatogram of the pharmaceutical preparation.

2. The method according to claim 1, wherein This method specifically includes the following steps: (1) Preparation of the reference solution: Dissolve the reference substances of paeoniflorin, naringin, ammonium glycyrrhizinate, hydroxysafflor yellow A, and amygdalin in a solvent and make up the volume to obtain a reference solution. (2) Preparation of the test solution: Take 1 g of this product, crush it, add 2 - 5 g of column chromatography silica gel, grind evenly, add 40 - 70 ml of a methanol - acetone mixed solution with a weight ratio of 3:7, then heat the mixed solution under reflux for 45 minutes. Let the product after heating under reflux cool down, shake well, filter through a 0.45 μm nylon filter membrane, evaporate the methanol - acetone mixed filtrate to dryness. Then dissolve the dried precipitate in hot water, and pass the dissolved solution through a 5 g polyamide column. First, elute with 30 - 50 ml of water, then elute with 30 - 50 ml of 70% methanol adjusted to pH 6 - 6.5 with hydrochloric acid. Then collect the 70% methanol eluate, evaporate it to dryness, and dissolve the residue in 5 ml of a solution of N,N - dimethylformamide:methanol:0.04 mol / L citric acid solution with a volume ratio of 0.1:0.9:9 to obtain the solution. (3) Determination: Use HPLC to separately determine the test solution and the reference solution in step (1) to obtain the characteristic chromatogram of the test solution and the characteristic chromatogram of the reference solution. Compare the retention time of the characteristic chromatogram of the test solution with that of the reference solution for qualitative analysis, and assign and locate the index components in the characteristic chromatogram of the test solution, so as to obtain the characteristic chromatogram of the pharmaceutical preparation. The conditions for HPLC are as follows: The chromatographic column is packed with octadecylsilane - bonded silica gel; N,N - dimethylformamide - methanol is used as mobile phase A, and 0.04 mol / L citric acid solution is used as mobile phase B; the column temperature is 35°C; the detection wavelength of the ultraviolet - visible detector is 237 nm, and the theoretical plate number calculated based on the naringin peak should be not less than 5000; the gradient elution program is as follows: 0 - 5 min, mobile phase A: mobile phase B, volume ratio is 10:90; 5 - 19 min, mobile phase A: mobile phase B, volume ratio changes from 10:90 to 19:81; 19 - 28 min, mobile phase A: mobile phase B, volume ratio changes from 19:81 to 26:74; 28 - 39 min, mobile phase A: mobile phase B, volume ratio changes from 26:74 to 32:68; 39 - 55 min, mobile phase A: mobile phase B, volume ratio changes from 32:68 to 55:45; 55 - 70 min, mobile phase A: mobile phase B, volume ratio changes from 55:45 to 30:70; 70 - 75 min, mobile phase A: mobile phase B, volume ratio changes from 30:70 to 10:90; 75 - 80 min, mobile phase A: mobile phase B, volume ratio is 10:90; There are 7 characteristic peaks in the characteristic chromatogram of the Xuefu Zhuyu tablets obtained in step (3). Taking peak 5 as the S peak, calculate the relative retention times of peaks 1, 2, 3, 4, 6, and 7 with respect to the S peak. The specified values are: peak 1 is 0.47, peak 2 is 0.53, peak 3 is 0.62, peak 4 is 0.69, peak 6 is 1.07, and peak 7 is 1.76; compare the standard characteristic chromatogram of the Xuefu Zhuyu tablets with the characteristic chromatogram of the reference substance solution, and locate and determine that peak 1 is amygdalin, peak 2 is hydroxysafflor yellow A, peak 4 is paeoniflorin, peak 5 is naringin, and peak 7 is ammonium glycyrrhizinate.

3. The method according to claim 1, wherein In the column of step (3), the column length is 250 mm, the inner diameter is 4.6 mm, and the particle size is 5 μm.

4. The method according to claim 1, characterized in that In the column of step (3), the volume ratio of N,N-dimethylformamide to methanol is 1:

9.

5. Application of the method according to any one of claims 1-4 in the quality rating of Xuefu Zhuyu tablets.

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