A method for constructing an HPLC characteristic chromatogram of a traditional Chinese medicine for nourishing liver and kidney

Abstract

The application provides a construction method of HPLC characteristic spectrum of a nourishing liver and kidney traditional Chinese medicine. The application has carried out systematic experimental research on the construction method of the HPLC characteristic spectrum of Gujiyangyin tablets, and the established method has good reproducibility and feasibility through the methodological verification of precision, stability, repeatability and the like. The method can simultaneously monitor multiple components and effectively control product quality. The application overcomes the deficiency of lacking quality detection methods in the present executed standard, and the one-sidedness of the detection method index in the prior art, which is strong in pertinence and small in coverage, the established characteristic spectrum can comprehensively reflect the complex chemical components and relative proportion of Gujiyangyin tablets, and is a quality control mode conforming to the characteristics of traditional Chinese medicine compound, which can effectively represent the drug quality.

Description

Technical Field

[0001] This invention belongs to the field of drug detection technology, specifically relating to a method for constructing HPLC characteristic chromatograms of a traditional Chinese medicine for nourishing the liver and kidneys. Background Technology

[0002] The different sources and dispersed production areas of Chinese medicinal herbs directly affect the quality of processed Chinese medicinal materials, leading to differences in the quality and clinical efficacy of Chinese patent medicines from different manufacturers and batches. Therefore, it is necessary to adopt effective and reasonable methods and technologies to control the quality of Chinese medicinal herbs and ensure product quality.

[0003] Guijia Yangyin Tablets are a renowned traditional Chinese medicine, composed of twenty-six ingredients including tortoise shell, raspberry, turtle shell, and salt-processed plantain seed. It nourishes yin and softens hardened masses, tonifies the liver and kidneys, and is used for arteriosclerosis, yin deficiency causing lower back pain, hypochondriac pain, dizziness, tinnitus, five-center heat (palms, soles, and chest), and coronary heart disease. The current standard is included in the Ministry of Health's Drug Standards for Traditional Chinese Medicine Compound Preparations, Volume VIII, standard number: WS3-B-1552-93. The quality standard includes items on appearance, microscopic identification, and routine tablet inspections. Due to the numerous ingredients and complex components in traditional Chinese medicine compound preparations, which are difficult to effectively detect due to mutual interference, existing quality inspection items cannot comprehensively monitor product quality. Establishing a characteristic spectrum of traditional Chinese medicine preparations will more comprehensively reflect the types and quantities of its chemical components, thus providing an overall description and evaluation of drug quality. The current quality standard only includes appearance, microscopic identification, and routine tablet inspections, resulting in a low level of quality control and failing to comprehensively and effectively control product quality. Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide a method for constructing HPLC characteristic chromatograms of traditional Chinese medicine for nourishing the liver and kidneys. The characteristic chromatograms established by the present invention can comprehensively reflect the complex chemical components and their relative proportions of Guijia Yangyin tablets. It is a quality control mode that conforms to the characteristics of traditional Chinese medicine compound prescriptions and can effectively characterize the quality of the medicine.

[0005] This invention provides a method for constructing HPLC characteristic chromatograms of traditional Chinese medicine for nourishing the liver and kidneys, comprising the following steps:

[0006] A) Dissolve the tortoise shell yin-nourishing tablets in a solvent to obtain the test solution;

[0007] B) Dissolve the reference substance in a solvent to obtain a reference substance solution, wherein the reference substance is selected from paeonol;

[0008] C) The test solution and the reference solution were determined by high performance liquid chromatography to obtain the HPLC characteristic chromatogram of Guijia Yangyin tablets and the chromatogram of the reference solution;

[0009] The chromatographic conditions for the high performance liquid chromatography method are as follows: the chromatographic column is a C18 column; mobile phase A is acetonitrile, mobile phase B is 0.1% glacial acetic acid aqueous solution, and gradient elution is used.

[0010] D) The components of the HPLC characteristic chromatogram of Guijia Yangyin tablets were qualitatively determined based on the chromatogram of the reference material.

[0011] Preferably, in steps A) and B), the solvent is selected from methanol or an aqueous methanol solution.

[0012] Preferably, the test solution is prepared according to the following method:

[0013] After accurately weighing the tortoise shell yin-nourishing tablets, they were precisely added to the solvent, ultrasonically treated, cooled, and then the solvent was added to make up the volume. After mixing evenly, the mixture was filtered, and the filtrate was collected to obtain the test solution.

[0014] Preferably, the reference solution is prepared according to the following method:

[0015] Take an appropriate amount of paeonol reference standard, accurately weigh it, and add solvent to prepare a solution containing 0.1-0.3 mg per ml, thus obtaining the reference solution.

[0016] Preferably, the gradient elution specifically comprises:

[0017] 0–50 min, mobile phase A 5%–40%, mobile phase B 95%–60%;

[0018] 50–75 min, mobile phase A 40%–90%, mobile phase B 60%–10%;

[0019] 75–80 min, mobile phase A 90%–100%, mobile phase B 10%–0%.

[0020] Preferably, the chromatographic column is selected from Agilent ZORBAX SB-C18, with a column length of 250 mm, an inner diameter of 4.6 mm, and a particle size of 5 μm; the column temperature is 30–40 °C.

[0021] Preferably, the mobile phase flow rate is 0.9–1.1 ml / min, the detection wavelength is 250–270 nm, the injection volume is 5–15 μL, and the theoretical plate number calculated based on the paeonol peak is not less than 10,000.

[0022] Preferably, the HPLC characteristic chromatogram of Guijia Yangyin tablets is compared with that of traditional Chinese medicine chromatographic fingerprint chromatogram similarity evaluation software to determine the common characteristic peaks and generate a reference characteristic chromatogram consisting of 14 characteristic peaks, wherein peak 1 corresponds to protocatechuic acid, peak 3 corresponds to ellagic acid, peak 4 corresponds to salvianolic acid B, peak 6 corresponds to ferulic acid, peak 7 corresponds to paeonol, and peak 8 corresponds to schisandrol A.

[0023] Preferably, in the reference characteristic spectrum, the paeonol peak is used as the reference peak S peak, and the relative retention time of each characteristic peak and the S peak is calculated. The relative retention time should be within ±5% of a specified value, which is: 0.11-peak 1, 0.12-peak 2, 0.56-peak 3, 0.72-peak 4, 0.74-peak 5, 0.76-peak 6, 1.00-peak S, 1.22-peak 8, 1.28-peak 9, 1.31-peak 10, 1.39-peak 11, 1.49-peak 12, 1.51-peak 13, 1.55-peak 14.

[0024] Compared with the prior art, the present invention provides a method for constructing the HPLC characteristic chromatogram of a traditional Chinese medicine for nourishing the liver and kidneys, comprising the following steps: A) dissolving Guijia Yangyin tablets in a solvent to obtain a test solution; B) dissolving a reference substance in a solvent to obtain a reference solution, wherein the reference substance is selected from paeonol; C) determining the test solution and the reference solution by high performance liquid chromatography (HPLC) to obtain the HPLC characteristic chromatogram of Guijia Yangyin tablets and the chromatogram of the reference substance; D) qualitatively determining the components of the HPLC characteristic chromatogram of Guijia Yangyin tablets based on the chromatogram of the reference substance; the HPLC chromatographic conditions are as follows: the chromatographic column is a C18 column; mobile phase A is acetonitrile, mobile phase B is 0.1% glacial acetic acid aqueous solution, and gradient elution is used.

[0025] This invention presents a systematic experimental study on the construction of HPLC characteristic chromatograms for Guijia Yangyin tablets. The established method has been validated through methodological verification of precision, stability, and repeatability, demonstrating good reproducibility and feasibility. This method can simultaneously monitor multiple components, effectively controlling product quality. This invention overcomes the shortcomings of current standards lacking quality testing methods, and the limitations of existing technologies in terms of the specificity and coverage of testing indicators. The established characteristic chromatograms comprehensively reflect the complex chemical components and their relative proportions of Guijia Yangyin tablets, representing a quality control model that aligns with the characteristics of traditional Chinese medicine compound formulas and effectively characterizes drug quality. Attached Figure Description

[0026] Figure 1 The HPLC characteristic chromatogram of Tortoise Shell Yin-Nourishing Tablets in Example 1;

[0027] Figure 2 This is a common pattern diagram of the HPLC characteristic chromatograms of 10 batches of Tortoise Shell Yin-Nourishing Tablets in Example 5;

[0028] Figure 3 This is a comparative characteristic chromatogram of the tortoise shell yin-nourishing tablets in Example 5;

[0029] Figure 4 Chromatogram was selected for the mobile phase of Comparative Example 1, Tortoise Shell Yin-Nourishing Tablets;

[0030] Figure 5 Chromatograms were selected based on the mobile phase ratio for Comparative Example 2, Tortoise Shell Yin-Nourishing Tablets.

[0031] Figure 6 Chromatograms of different types of columns for Comparative Example 3, Tortoise Shell Nourishing Yin Tablets;

[0032] Figure 7 The chromatogram of the detection wavelength (240nm) for Comparative Example 5, Tortoise Shell Yin-Nourishing Tablets, was selected.

[0033] Figure 8 The chromatogram of the detection wavelength (280nm) for Comparative Example 6, Guijia Yangyin Tablets, was selected. Detailed Implementation

[0034] This invention provides a method for constructing HPLC characteristic chromatograms of traditional Chinese medicine for nourishing the liver and kidneys, comprising the following steps:

[0035] A) Dissolve the tortoise shell yin-nourishing tablets in a solvent to obtain the test solution;

[0036] B) Dissolve the reference substance in a solvent to obtain a reference substance solution, wherein the reference substance is selected from paeonol;

[0037] C) The test solution and the reference solution were determined by high performance liquid chromatography to obtain the HPLC characteristic chromatogram of Guijia Yangyin tablets and the chromatogram of the reference solution;

[0038] D) The components of the HPLC characteristic chromatogram of Guijia Yangyin tablets were qualitatively determined based on the chromatogram of the reference material;

[0039] The chromatographic conditions for the high performance liquid chromatography method are as follows: the chromatographic column is a C18 column; mobile phase A is acetonitrile, mobile phase B is 0.1% glacial acetic acid aqueous solution, and gradient elution is used.

[0040] The present invention first prepares the test solution and the reference solution, and there are no special restrictions on the order of preparation of the two.

[0041] The test solution is prepared according to the following method:

[0042] After accurately weighing the tortoise shell yin-nourishing tablets, they were precisely added to the solvent, ultrasonically treated, cooled, and then the solvent was added to make up the volume. After mixing evenly, the mixture was filtered, and the filtrate was collected to obtain the test solution.

[0043] The mass-to-volume ratio of the tortoise shell yin-nourishing tablets to the solvent is 1-3 g: 12.5-37.5 ml. The solvent is selected from methanol or an aqueous methanol solution, and the volume concentration of the aqueous methanol solution is ≥50%. This invention does not impose special limitations on the conditions for the ultrasonic treatment, but preferably includes: an ultrasonic power of 600 W, a frequency of 40 kHz, and an ultrasonic treatment time of 20-40 minutes.

[0044] The reference solution was prepared according to the following method:

[0045] Accurately weigh an appropriate amount of paeonol reference standard, add solvent to prepare a solution containing 0.1–0.3 mg per ml, thus obtaining the reference solution. The solvent is selected from methanol or an aqueous methanol solution, wherein the volume concentration of the aqueous methanol solution is ≥50%.

[0046] Then, the test solution and the reference solution were determined by high performance liquid chromatography, respectively, to obtain the HPLC characteristic chromatogram of Guijia Yangyin tablets and the chromatogram of the reference solution;

[0047] The chromatographic conditions for the high performance liquid chromatography method are as follows: the chromatographic column is a C18 column; mobile phase A is acetonitrile, mobile phase B is 0.1% glacial acetic acid aqueous solution, and gradient elution is used.

[0048] Preferably, the chromatographic column is selected from Agilent ZORBAX SB-C18, with a column length of 250 mm, an inner diameter of 4.6 mm, and a particle size of 5 μm; the column temperature is 30 to 40 °C, preferably 30, 35, or 40 °C, or any value between 30 and 40 °C.

[0049] The flow rate of the mobile phase is 0.9 to 1.1 ml / min, preferably 0.9, 1.0, 1.1, or any value between 0.9 and 1.1 ml / min.

[0050] The gradient elution specifically refers to:

[0051] 0–50 min, mobile phase A 5%–40%, mobile phase B 95%–60%;

[0052] 50–75 min, mobile phase A 40%–90%, mobile phase B 60%–10%;

[0053] 75–80 min, mobile phase A 90%–100%, mobile phase B 10%–0%.

[0054] The injection volume for detection is 5–15 μL, preferably 5, 10, 15, or 5–15 μL;

[0055] The detection wavelength is 250–270 nm, preferably 250, 260, or 270 nm, or any value between 250 and 270 nm. The optimal ultraviolet absorption wavelength of this invention is 260 nm. Using the method of this invention, the Guijia Yangyin tablets were measured with a DAD detector. Based on the detection wavelength at which all chromatographic peaks showed maximum absorption in the three-dimensional scan, chromatograms were retrieved at each wavelength. When the detection wavelength was 260 nm, the number of characteristic peaks was relatively large, the area of ​​each peak was high, and the separation effect between the characteristic peaks was also the best.

[0056] The theoretical plate number, calculated based on the paeonol peak, should be no less than 10,000.

[0057] Several batches of Guijia Yangyin tablets were analyzed by HPLC to obtain HPLC characteristic chromatograms. The HPLC characteristic chromatograms of Guijia Yangyin tablets were compared using traditional Chinese medicine chromatographic fingerprint similarity evaluation software to identify common characteristic peaks. A control characteristic chromatogram consisting of 14 characteristic peaks was generated, where peak 1 corresponds to protocatechuic acid, peak 3 to ellagic acid, peak 4 to salvianolic acid B, peak 6 to ferulic acid, peak 7 to paeonol, and peak 8 to schisandrol A.

[0058] In the comparative characteristic spectrum, using the paeonol peak as the reference peak S peak, the relative retention time of each characteristic peak and the S peak is calculated. The relative retention time should be within ±5% of the specified value, which is as follows: 0.11-peak 1, 0.12-peak 2, 0.56-peak 3, 0.72-peak 4, 0.74-peak 5, 0.76-peak 6, 1.00-peak S, 1.22-peak 8, 1.28-peak 9, 1.31-peak 10, 1.39-peak 11, 1.49-peak 12, 1.51-peak 13, 1.55-peak 14.

[0059] This invention presents a systematic experimental study on the construction of HPLC characteristic chromatograms for Guijia Yangyin tablets. The established method has been validated through methodological verification of precision, stability, and repeatability, demonstrating good reproducibility and feasibility. This method can simultaneously monitor multiple components, effectively controlling product quality. This invention overcomes the shortcomings of current standards lacking quality testing methods, and the limitations of existing technologies in terms of the specificity and coverage of testing indicators. The established characteristic chromatograms comprehensively reflect the complex chemical components and their relative proportions of Guijia Yangyin tablets, representing a quality control model that aligns with the characteristics of traditional Chinese medicine compound formulas and effectively characterizes drug quality.

[0060] To further understand the present invention, the method for constructing the HPLC characteristic chromatogram of the liver and kidney tonifying traditional Chinese medicine provided by the present invention will be described below with reference to the embodiments. The scope of protection of the present invention is not limited by the following embodiments.

[0061] Example 1: A method for constructing the HPLC characteristic chromatogram of a traditional Chinese medicine for nourishing the liver and kidneys.

[0062] Instruments: UltiMate 3000 high performance liquid chromatograph, ultraviolet DAD detector; MS205DU analytical balance (Shanghai Mettler Toledo Instruments Co., Ltd.), etc.

[0063] Reagents: Methanol, glacial acetic acid, etc. are analytical grade, acetonitrile is chromatographic grade, and water is purified water.

[0064] Test reagents: protocatechuic acid, ellagic acid, salvianolic acid B, ferulic acid, paeonol, and schisandrol A (all purchased from the National Institutes for Food and Drug Control).

[0065] Methods and Results

[0066] (1) Preparation of test solution: Take an appropriate amount of tortoise shell nourishing yin tablets, grind them into a fine powder, take about 2g, weigh it accurately, add 25ml of methanol accurately, weigh it, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool it, make up the lost weight with methanol, shake it well, filter it, and take the filtrate to obtain the test solution.

[0067] (2) Preparation of reference solution: Take an appropriate amount of paeonol reference standard, weigh it accurately, and add methanol to prepare a solution containing 0.2 mg per ml.

[0068] (3) Determination: An Agilent ZORBAX SB-C18 column (250 mm column length, 4.6 mm inner diameter, 5 μm particle size) was used; the detection wavelength was 260 nm; the column temperature was 35 °C; and the flow rate was 1.0 ml / min. The theoretical plate number, calculated based on the paeonol peak, should not be less than 10,000. Acetonitrile was used as mobile phase A, and 0.1% glacial acetic acid aqueous solution was used as mobile phase B. The gradient elution time and mobile phase ratio are shown in Table 1.

[0069] Table 1. Characteristic spectrum of Guijia Yangyin tablets: mobile phase time and gradient

[0070] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～50 5→40 95→60 50～75 40→90 60→10 75～80 90→100 10→0

[0071] Accurately pipette 10 μL each of the reference solution and the test solution into the high-performance liquid chromatograph (HPLC), and determine the HPLC characteristic chromatogram of the tortoise shell yin-nourishing tablets according to the HPLC method. Figure 1 As shown.

[0072] Example 2: A method for constructing the HPLC characteristic chromatogram of a traditional Chinese medicine for nourishing the liver and kidneys.

[0073] Instruments: UltiMate 3000 high performance liquid chromatograph, ultraviolet DAD detector; MS205DU analytical balance (Shanghai Mettler Toledo Instruments Co., Ltd.), etc.

[0074] Reagents: Methanol, glacial acetic acid, etc. are analytical grade, acetonitrile is chromatographic grade, and water is purified water.

[0075] Test reagents: protocatechuic acid, ellagic acid, salvianolic acid B, ferulic acid, paeonol, and schisandrol A (all purchased from the National Institutes for Food and Drug Control).

[0076] Methods and Results

[0077] (1) Preparation of test solution: Take an appropriate amount of tortoise shell nourishing yin tablets, grind them into a fine powder, take about 1g, weigh it accurately, add 12.5ml of 50% methanol accurately, weigh it, sonicate (power 600W, frequency 40kHz) for 20 minutes, cool it, make up the lost weight with 50% methanol, shake it well, filter it, and take the filtrate to obtain the test solution.

[0078] (2) Preparation of reference solution: Take an appropriate amount of paeonol reference standard, weigh it accurately, and add methanol to prepare a solution containing 0.1 mg per ml.

[0079] (3) Determination: An Agilent ZORBAX SB-C18 column (250 mm column length, 4.6 mm inner diameter, 5 μm particle size) was used; the detection wavelength was 250 nm; the column temperature was 30 °C; and the flow rate was 0.9 ml / min. The theoretical plate number, calculated based on the paeonol peak, should not be less than 10,000. Acetonitrile was used as mobile phase A, and 0.1% glacial acetic acid aqueous solution was used as mobile phase B. The gradient elution time and mobile phase ratio were the same as in Table 1.

[0080] Accurately pipette 5 μL each of the reference solution and the test solution, inject them into the high-performance liquid chromatograph, and determine them by high-performance liquid chromatography to obtain the HPLC characteristic chromatogram of Guijia Yangyin tablets.

[0081] Example 3: A method for constructing the HPLC characteristic chromatogram of a traditional Chinese medicine for nourishing the liver and kidneys.

[0082] Instruments: UltiMate 3000 high performance liquid chromatograph, ultraviolet DAD detector; MS205DU analytical balance (Shanghai Mettler Toledo Instruments Co., Ltd.), etc.

[0083] Reagents: Methanol, glacial acetic acid, etc. are analytical grade, acetonitrile is chromatographic grade, and water is purified water.

[0084] Test reagents: protocatechuic acid, ellagic acid, salvianolic acid B, ferulic acid, paeonol, and schisandrol A (all purchased from the National Institutes for Food and Drug Control).

[0085] Methods and Results

[0086] (1) Preparation of test solution: Take an appropriate amount of tortoise shell nourishing yin tablets, grind them into a fine powder, take about 3g, weigh accurately, add 37.5ml of 75% methanol accurately, weigh accurately, sonicate (power 600W, frequency 40kHz) for 40 minutes, cool, replenish the lost weight with 75% methanol, shake well, filter, and take the filtrate to obtain the test solution.

[0087] (2) Preparation of reference solution: Take an appropriate amount of paeonol reference standard, weigh it accurately, and add methanol to prepare a solution containing 0.3 mg per ml.

[0088] (3) Determination: An Agilent ZORBAX SB-C18 column (250 mm column length, 4.6 mm inner diameter, 5 μm particle size) was used; the detection wavelength was 270 nm; the column temperature was 40 °C; and the flow rate was 1.1 ml / min. The theoretical plate number, calculated based on the paeonol peak, should not be less than 10,000. Acetonitrile was used as mobile phase A, and 0.1% glacial acetic acid aqueous solution was used as mobile phase B. The gradient elution time and mobile phase ratio were the same as in Table 1.

[0089] Accurately pipette 15 μL each of the reference solution and the test solution, inject them into the high-performance liquid chromatograph, and determine them by high-performance liquid chromatography to obtain the HPLC characteristic chromatogram of Guijia Yangyin tablets.

[0090] Example 4: Methodological Investigation of Characteristic Spectrum Determination of Tortoise Shell Yin-Nourishing Tablets

[0091] Precision test: Tortoise shell yin-nourishing tablets were used to prepare a test solution according to the method described in Example 1. The solution was injected six times consecutively and tested according to the prescribed method. Results: The relative retention time (RSD) values ​​of all characteristic peaks in the six characteristic chromatograms were <2%, indicating good instrument precision.

[0092] Stability test: Tortoise shell yin-nourishing tablets were prepared into a test solution according to the method described in Example 1. The solution was tested at 0, 3, 6, 9, 12, and 24 hours. Results: The relative retention time (RSD) values ​​of all characteristic peaks in the characteristic chromatograms at each time point were <2%, indicating that the test solution was stable within 24 hours.

[0093] Repeatability test: Six test solutions were prepared from the same batch of tortoise shell yin-nourishing tablets according to the preparation method of the test solution in Example 1, and measured according to the method. Results: The relative retention time (RSD) values ​​of each characteristic peak in the six characteristic chromatograms were all <2%, indicating that the method has good repeatability.

[0094] The precision, stability, and repeatability of the methods in Examples 2 and 3 were examined, and the results were similar to those of the method in Example 1, showing good precision, stability, and repeatability.

[0095] Example 5: Constructing a comparative characteristic map of tortoise shell yin-nourishing tablets

[0096] Ten batches of Guijia Yangyin tablets were taken and analyzed according to the conditions of Example 2. The HPLC characteristic chromatograms of the ten batches of samples were obtained, such as... Figure 2 As shown. The similarity evaluation software for chromatographic fingerprints of traditional Chinese medicine was used to compare 10 batches of characteristic chromatograms, identify common characteristic peaks, and generate a control characteristic chromatogram consisting of 14 characteristic peaks, as shown. Figure 3 As shown in the figure. The reference peak is the paeonol peak (peak 7).

[0097] HPLC characteristic chromatograms of 10 batches of Guijia Yangyin tablets were established and analyzed using the 2004A version of the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine" published by the National Pharmacopoeia Commission. The resulting HPLC standard characteristic chromatogram of Guijia Yangyin tablets consisted of 14 characteristic peaks. Peak 1 was protocatechuic acid, peak 3 was ellagic acid, peak 4 was salvianolic acid B, peak 6 was ferulic acid, peak 7 (S) was paeonol, and peak 8 was schisandrol A.

[0098] Similarity analysis: The similarity between the characteristic chromatograms of 10 batches of test samples and the generated control characteristic chromatograms was calculated, and the results were all greater than 0.90. The similarity comparison results are shown in Table 2.

[0099] Table 2. Similarity evaluation results of HPLC characteristic chromatograms of Guijia Yangyin tablets

[0100] S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 R S1 1 0.985 0.96 0.979 0.988 0.987 0.992 0.993 0.982 0.969 0.984 S2 0.985 1 0.963 0.985 0.979 0.986 0.988 0.998 0.991 0.982 0.997 S3 0.96 0.963 1 0.979 0.988 0.955 0.968 0.998 0.963 0.982 0.959 S4 0.979 0.985 0.979 1 0.974 0.998 0.982 0.979 0.98 0.963 0.991 S5 0.988 0.979 0.988 0.974 1 0.957 0.982 0.977 0.984 0.991 0.975 S6 0.987 0.986 0.955 0.998 0.957 1 0.968 0.988 0.989 0.991 0.967 S7 0.992 0.988 0.968 0.982 0.982 0.968 1 0.958 0.963 0.977 0.981 S8 0.993 0.998 0.998 0.979 0.977 0.988 0.958 1 0.984 0.991 0.978 S9 0.982 0.991 0.963 0.98 0.984 0.989 0.963 0.984 1 0.995 0.969 S10 0.969 0.982 0.982 0.963 0.991 0.991 0.977 0.991 0.995 1 1 R 0.984 0.997 0.959 0.991 0.975 0.967 0.981 0.978 0.969 1 1

[0101] Comparative Example 1:

[0102] This comparative example is similar to Example 1, the only difference being that the mobile phase used is methanol-0.1% glacial acetic acid, and its characteristic chromatogram is shown below. Figure 4 Compared with the chromatogram obtained using acetonitrile-0.1% glacial acetic acid solution as the mobile phase, the chromatogram shows poor separation of the chromatographic peaks and unstable baseline.

[0103] Comparative Example 2:

[0104] This comparative example is similar to Example 1, except that the elution procedure is different. The elution procedure for this comparative example is as follows:

[0105] 0–35 min, Phase A: 10–40%, Phase B: 90–60%;

[0106] 35–60 min, Phase A: 40–90%, Phase B: 60–10%;

[0107] Feature maps are shown Figure 5 The results showed that the chromatographic peaks prior to 30 minutes in the chromatogram had poor separation and a small number of peaks.

[0108] Comparative Example 3:

[0109] This comparative example is similar to Example 1, the only difference being the manufacturer and model of the chromatographic column used. This comparative example uses a Kromasil 100-5-C18 column (250 mm length, 4.6 mm inner diameter, 5 μm particle size), and its characteristic chromatogram is shown below. Figure 6 .

[0110] The results showed that the separation of each chromatographic peak in the chromatogram of the test sample was poor, and the peak shape was also poor.

[0111] Comparative Example 4:

[0112] This comparative example is similar to Example 1, except that the solvent used to dissolve the sample is different; the solvent in this comparative example is ethanol.

[0113] The results showed that the number of chromatographic peaks in the test sample was relatively small.

[0114] Comparative Example 5:

[0115] This comparative example is similar to Example 1, the only difference being that the detection wavelength is 240nm, and its characteristic spectrum is shown below. Figure 7 Comparing this chromatogram with the chromatogram obtained using 260 nm as the detection wavelength, it is shown that the separation of the chromatographic peaks between 60 and 80 min in the chromatogram is poor.

[0116] Comparative Example 6:

[0117] This comparative example is similar to Example 1, the only difference being that the detection wavelength is 280nm, and its characteristic spectrum is shown below. Figure 8 Compared to the chromatogram obtained using 260 nm as the detection wavelength, this chromatogram shows fewer chromatographic peaks.

[0118] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for constructing the HPLC characteristic chromatogram of a traditional Chinese medicine for nourishing the liver and kidneys, characterized in that, Includes the following steps: A) Dissolve the tortoise shell yin-nourishing tablets in a solvent to obtain the test solution; B) Dissolve the reference substance in a solvent to obtain a reference substance solution, wherein the reference substance is selected from paeonol; C) The test solution and the reference solution were determined by high performance liquid chromatography to obtain the HPLC characteristic chromatogram of Guijia Yangyin tablets and the chromatogram of the reference solution; The chromatographic conditions for the high performance liquid chromatography method are as follows: the chromatographic column is a C18 column; mobile phase A is acetonitrile, mobile phase B is 0.1% glacial acetic acid aqueous solution, and gradient elution is used. D) The components of the HPLC characteristic chromatogram of Guijia Yangyin tablets were qualitatively determined based on the chromatogram of the reference material; In steps A) and B), the solvent is selected from methanol or an aqueous methanol solution; In step C), the gradient elution specifically involves: 0~50 min, mobile phase A 5%~40%, mobile phase B 95%~60%; 50~75 min, mobile phase A 40%~90%, mobile phase B 60%~10%; 75~80 min, mobile phase A 90%~100%, mobile phase B 10%~0%; The chromatographic column was selected from Agilent ZORBAX SB-C18, with a column length of 250 mm, an inner diameter of 4.6 mm, and a particle size of 5 μm; The detection wavelength is 250~270nm; The HPLC characteristic chromatograms of Guijia Yangyin tablets were compared using the chromatographic fingerprint similarity evaluation software for traditional Chinese medicine. Common characteristic peaks were identified, and a control characteristic chromatogram consisting of 14 characteristic peaks was generated. Among them, peak 1 corresponds to protocatechuic acid, peak 3 corresponds to ellagic acid, peak 4 corresponds to salvianolic acid B, peak 6 corresponds to ferulic acid, peak 7 corresponds to paeonol, and peak 8 corresponds to schisandrol A.

2. The construction method according to claim 1, characterized in that, The test solution is prepared according to the following method: After accurately weighing the tortoise shell yin-nourishing tablets, add them precisely to the solvent, sonicate and cool, then add more solvent to make up the volume, mix evenly, filter, and take the filtrate to obtain the test solution.

3. The construction method according to claim 1, characterized in that, The reference solution was prepared according to the following method: Take an appropriate amount of paeonol reference standard, accurately weigh it, and add solvent to prepare a solution containing 0.1~0.3 mg per ml, thus obtaining the reference solution.

4. The construction method according to claim 1, characterized in that, The column temperature is 30~40℃.

5. The construction method according to claim 1, characterized in that, The flow rate of the mobile phase is 0.9~1.1 ml / min, and the injection volume is 5~15 μL; the theoretical plate number calculated based on the paeonol peak is not less than 10000.

6. The construction method according to claim 1, characterized in that, In the comparative characteristic spectrum, using the paeonol peak as the reference peak S peak, the relative retention time of each characteristic peak and the S peak is calculated. The relative retention time should be within ±5% of a specified value, where the specified value is: 0.11 - Peak 1, 0.12 - Peak 2, 0.56 - Peak 3, 0.72 - Peak 4, 0.74 - Peak 5, 0.76 - Peak 6, 1.00 - Peak 8, 1.22 - Peak 8, 1.28 - Peak 9, 1.31 - Peak 10, 1.39 - Peak 11, 1.49 - Peak 12, 1.51 - Peak 13, 1.55 - Peak 14.

Images