Quality control method for glycyrrhizin related substances in compound glycyrrhizin tablet

Abstract

The invention discloses a quality control method of glycyrrhizin related substances in a compound glycyrrhizin tablet. The related substances comprise a known impurity A, namely 24-hydroxyglycyrrhizic acid, and an impurity B, namely 18 alpha-glycyrrhizic acid. High performance liquid chromatography is adopted, octadecylsilane chemically bonded silica serves as filler of a chromatographic column, a mobile phase A is a mixed solution prepared from a monopotassium phosphate aqueous solution and methanol, the pH value of the mixed solution is adjusted to 2.0-3.0 through phosphoric acid, and a mobile phase B is acetonitrile. The method provided by the invention can effectively detect the known impurity A (24-hydroxyglycyrrhizic acid), the impurity B (18 alpha-glycyrrhizic acid) and other non-specific impurities generated by glycyrrhizin in the compound glycyrrhizin tablet, and peaks of other raw and auxiliary materials have no interference on detection of each impurity. The analysis method is good in specificity, high in separation degree and accurate in result, and the quality of glycyrrhizin in the compound glycyrrhizin tablet can be effectively controlled by adopting the method disclosed by the invention.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical analysis, and particularly relates to a quality control method for related substances of glycyrrhizin in compound glycyrrhizin tablets. Background Art

[0002] The original compound glycyrrhizin tablets (Compound Glycyrrhizin Tablets, trade name: Minone) is a compound preparation developed and produced by Minophagen Pharmaceutical Co., Ltd. in Japan. It was launched in Japan in 1948 and imported into China in 2000. The compound glycyrrhizin tablets contain components of glycyrrhizin, glycine, and methionine, and is a drug with multiple indications. It is suitable for the treatment of chronic liver diseases to improve liver function abnormalities, and can also be used for skin diseases such as eczema, dermatitis, and alopecia areata. During the development and research process of compound glycyrrhizin tablets, related substances are closely related to the quality, safety, and efficacy of drugs. The presence of related substances may reduce the efficacy of drugs and even cause toxic and side effects. Therefore, it is necessary to control the types and contents of related substances in drugs through appropriate detection and analysis methods to ensure the quality of drugs.

[0003] At present, the specific component contents in compound glycyrrhizin tablets may vary due to different production batches or manufacturers, but generally each tablet contains about 25 mg of glycyrrhizin. In the methods for detecting related substances of glycyrrhizin in compound glycyrrhizin tablets: In the existing literature (Tian Li. Research and Development of Compound Glycyrrhizin Tablets and Their in Vitro Metabolism [D]. Xinjiang Medical University, 2008.), an isocratic elution method using 0.025 mol / L sodium acetate solution (pH 3.5)-acetonitrile (59:41) as the mobile phase and a detection wavelength of 251 nm is disclosed. However, this method can only roughly calculate the maximum single impurity, the second largest single impurity, and the total impurity, and cannot accurately determine impurities at lower levels, and cannot truly reflect the quality of compound glycyrrhizin tablets, posing a risk to the evaluation of drug quality; In the European Pharmacopoeia EP11.5 or the British Pharmacopoeia BP2024 (referred to as Method 1) and the United States Pharmacopoeia USP-NF2024 (referred to as Method 2), the detection methods for related substances of the glycyrrhizin derivative ammonium glycyrrhizinate are both included, and isocratic elution is carried out under the condition of using a mixed solution of acetonitrile-acetic acid-water containing 38% acetonitrile as the mobile phase. However, the separation between the main peak and the adjacent impurity peaks is poor, and the types of impurities detected are few; The detection method for related substances of compound glycyrrhizin tablets included in the imported drug registration standard JX20150409 method (referred to as Method 3) uses a mixed solution of acetonitrile-acetic acid-water containing 34.4% acetonitrile as the mobile phase for isocratic elution. Although the separation between the main peak and the adjacent impurity peaks and between impurity peaks has improved, and the types of eluted impurities have also increased, the impurity peaks are still not completely separated, and the main peak has a poor peak shape, showing tailing and forking phenomena, which interfere with the research and detection of related substances.

[0004] The structural formulas of the main components and impurities in compound glycyrrhizin tablets are as follows:

[0005]

[0006] Therefore, to ensure the safety and effectiveness of compound glycyrrhizin tablets, it is necessary to develop a detection method for related substances of glycyrrhizin with good specificity, high sensitivity, accuracy and precision, so as to realize the quality control of related substances. Summary of the Invention

[0007] In order to overcome the deficiencies in the above technologies, the purpose of the present invention is to provide a method for detecting related substances of glycyrrhizin in compound glycyrrhizin tablets. The method provided by the present invention can ensure the effectiveness and safety of compound glycyrrhizin tablets, and enable the quality of compound glycyrrhizin tablets to be effectively controlled during the production process. The system suitability of this method is stable, the blank solvent does not interfere with the detection of each impurity and the main peak. The reference solution is injected continuously for 5 times, and the relative standard deviation (RSD) of the main peak area is 0.18% (<2.0%), and the separation degree between the main peak and known impurities and adjacent peaks is good.

[0008] In order to achieve the above purpose, the technical scheme adopted by the present invention is as follows:

[0009] The present invention discloses a method for detecting related substances of glycyrrhizin in compound glycyrrhizin tablets, wherein the related substances include known impurity A (24-hydroxyglycyrrhizic acid) and impurity B (18α-glycyrrhizic acid).

[0010] The method for detecting related substances of glycyrrhizin in the compound glycyrrhizin tablets of the present invention adopts high performance liquid chromatography, and the chromatographic conditions are as follows: the chromatographic column is packed with octadecylsilane-bonded silica gel, mobile phase A is a mixed solution made of potassium dihydrogen phosphate aqueous solution adjusted to pH 2.0 - 3.0 with phosphoric acid and methanol, mobile phase B is acetonitrile. Set the detection wavelength, column temperature, flow rate, injection volume and running time, and perform gradient elution. The program of the gradient elution includes:

[0011] Time (min) Mobile phase A (%) Mobile phase B (%) 0 60 40 20 70 30 60 50 50 65 70 30 75 60 40

[0012] In some embodiments, due to the acidic mobile phase, acid-resistant chromatographic columns of different brands are screened. When the chromatographic column is Ultimate LP-C18 (4.6 mm × 250 mm, 5 μm) column, the detection of each substance peak is not interfered.

[0013] In some embodiments, the concentration of potassium dihydrogen phosphate aqueous solution in mobile phase A is 0.08 mol / L, and the volume ratio with methanol is 60:10.

[0014] In some embodiments, the flow rate is 1.0 ml / min, the injection volume is 40 μl, and the running time is 75 min.

[0015] In some embodiments, when the detection wavelength is selected from 251 nm - 257 nm and the column temperature is selected from 28 - 32 °C, by using the detection method of the present invention, the related substances of glycyrrhizin can be effectively separated, thus ensuring the quality control of compound glycyrrhizin tablets.

[0016] Preferably, the detection wavelength is selected from 254 nm and the column temperature is selected from 30 °C.

[0017] The present invention further discloses a specific method for detecting the related substances of glycyrrhizin in compound glycyrrhizin tablets, which includes the following steps:

[0018] (1) Preparation of solutions

[0019] Preparation of blank solvent: The blank solvent is the diluent, which is a mixed solution of ethanol - ultrapure water, where the volume ratio of ethanol to ultrapure water is 529:471. Measure the corresponding volumes of absolute ethanol and ultrapure water and mix them to obtain the diluent.

[0020] Preparation of test solution: Weigh precisely the powdered compound glycyrrhizin tablets that have been ground fine, add the blank solvent to make a solution containing about 0.25 mg / ml of glycyrrhizin, centrifuge, take the supernatant and filter it through a 0.45 μm organic filter head, and take the subsequent filtrate as the test solution.

[0021] Preparation of self - reference solution: Precisely transfer the test solution and dilute it 20 times with the diluent to prepare a test solution of 0.0125 mg / ml.

[0022] (2) Chromatographic conditions

[0023] Chromatographic column: Ultimate LP - C18 4.6 mm × 250 mm, 5 μm,

[0024] Mobile phase A: A mixed solution of 0.08 mol / L potassium dihydrogen phosphate aqueous solution (pH 2.5) - methanol, where the volume ratio of potassium dihydrogen phosphate aqueous solution to methanol is 60:10,

[0025] Mobile phase B: Acetonitrile,

[0026] Flow rate: 1.0 ml / min,

[0027] Column temperature: 30 °C,

[0028] Detection wavelength: 254 nm,

[0029] Injection volume: 40 μl,

[0030] Running time: 75 min,

[0031] Gradient elution program:

[0032]

[0033]

[0034] (3) Sample injection and determination

[0035] Inject 40 μl of blank solvent, self-control solution, and test solution into the liquid chromatograph in sequence, record the chromatogram. Calculate the contents of relevant substances in the test sample according to the self-control method based on the chromatographic peak areas of each relevant substance obtained. The calculation formula is:

[0036]

[0037] ; The relevant substances include: impurity A: 24-hydroxyglycyrrhizic acid, impurity B: 18α-glycyrrhizic acid.

[0038] Furthermore, in some embodiments, the correction factor is 1 and the dilution factor of the control solution is 20.

[0039] Compared with the prior art, the beneficial effects achieved by the present invention are as follows:

[0040] The method described in the present invention can effectively detect the known impurities A (24-hydroxyglycyrrhizic acid), impurity B (18α-glycyrrhizic acid) produced by glycyrrhizin in compound glycyrrhizin tablets and other non-specific impurities, and the peaks of other excipients do not interfere with the detection of each impurity.

[0041] This analytical method has good specificity, high resolution, and accurate results. Using the method disclosed in the present invention can effectively control the quality of glycyrrhizin in compound glycyrrhizin tablets. Brief description of the drawings

[0042] The specification drawings forming a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation to the present invention.

[0043] Figure 1 It is the liquid chromatogram of method 1 in Example 1;

[0044] Figure 2 It is the liquid chromatogram of method 2 in Example 1;

[0045] Figure 3 It is the liquid chromatogram of method 3 in Example 1;

[0046] Figure 4 It is the liquid chromatogram of the system suitability in Example 3;

[0047] Figure 5 It is the liquid chromatography analysis chart for the specificity of Example 4, where (a) is the blank solvent, (b) is the negative control solution, (c) is the localization solution of impurity A, (d) is the localization solution of impurity B, and (e) is the test solution.

[0048] Figure 6 It is the result of the comparison between the chromatographic columns of Welch Materials and Shiseido in Comparative Example 1.

[0049] Reference numerals in the drawings: 1: The peak of the main component glycyrrhizin; 2: The peak of the related substance impurity A; 3: The peak of the related substance impurity B. Detailed implementation manners

[0050] It should be noted that the following detailed description is exemplary and is intended to provide further illustration of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0051] Reproduction of the method in the prior art in Example 1

[0052] The methods 1-3 in the prior art shown in Table 1 were respectively reproduced. Since the column temperature was not specified in each method, the column temperature was temporarily set at 30 °C, and the running time of Method 2 was tentatively set to be 3 to 4.5 times the retention time of glycyrrhizin, and the related substances of glycyrrhizin in compound glycyrrhizin tablets were detected. The obtained chromatograms are as Figures 1 - 3 shown. The impurity peaks were not completely separated, and the methods in the prior art could not accurately and effectively monitor the contents of each impurity, indicating that there is room for improvement.

[0053] Table 1 Methods 1-3 in the prior art

[0054]

[0055]

[0056] Development process of the related substance detection method in Example 2

[0057] Selection of the elution mode: Based on the fact that the isocratic elution mode adopted in the prior art cannot balance the peak shape and resolution, gradient elution was adopted to improve the separation ability in the case of multi-component samples or impurity interference.

[0058] Selection of the mobile phase: Referring to Method 2, the mobile phase A was an aqueous solution of glacial acetic acid (where the dosage ratio of glacial acetic acid to water was 1:61), and the mobile phase B was acetonitrile for gradient elution. By continuously adjusting the elution gradient of the mobile phase, good separation of the main peak and the known impurities from the adjacent peaks could still not be achieved.

[0059] The mobile phase A was changed to a mixed solution of potassium dihydrogen phosphate aqueous solution - methanol, and the mobile phase B remained acetonitrile. When the concentration of the potassium dihydrogen phosphate aqueous solution was 0.08 mol / L, by adjusting the pH value of the potassium dihydrogen phosphate aqueous solution, the retention behavior of the sample components on the chromatographic column was changed. It was found that adjusting the pH value of the potassium dihydrogen phosphate aqueous solution to 2.0 - 3.0 with phosphoric acid could significantly improve the separation effect (Table 2). According to the separation degree results of sample detection, the final gradient elution method was confirmed as follows:

[0060] Time (min) Mobile phase A (%) Mobile phase B (%) 0 60 40 20 70 30 60 50 50 65 70 30 75 60 40

[0061] In addition, the volume ratio of the potassium dihydrogen phosphate aqueous solution to methanol in the mobile phase A was further adjusted.

[0062] Table 2 Separation conditions of different mobile phases

[0063]

[0064]

[0065] Selection of detection wavelength: By comparing the ultraviolet absorption curves of glycyrrhizin and related substances, it was known that the maximum absorption wavelengths of most related substances were closer to 254 nm, and the unknown impurities in the test solution at 251 - 257 nm could be effectively detected.

[0066] Selection of column temperature: The inventors investigated the separation effects of glycyrrhizin and its impurities at different temperatures (20 °C, 30 °C, and 35 °C) under the same gradient. The results showed that the separation effect of each compound was the best at 30 °C, and it met the requirements at 28 - 32 °C.

[0067] The related substance detection method described in the present invention was verified as follows:

[0068] Example 3 System suitability test

[0069] (1) Preparation of solutions

[0070] Blank solvent: Ethanol - ultrapure water (529:471). Measure 529 ml of absolute ethanol and 471 ml of water and mix them to obtain.

[0071] Test solution: Take 20 tablets of compound glycyrrhizin, weigh accurately, grind them finely, weigh accurately about 0.146 g (equivalent to about 25 mg of glycyrrhizin), place it in a 100 ml volumetric flask, add an appropriate amount of blank solvent, shake well, sonicate for 40 min to completely dissolve the main component, cool to room temperature, dilute to the mark with blank solvent, shake well, filter through a 0.45 μm organic filter membrane, discard 2 ml of the initial filtrate, and take the subsequent filtrate as the test solution.

[0072] Self-control solution: Accurately measure 5 ml of the test solution, transfer it to a 100-ml volumetric flask, dilute it to the mark with the blank solvent, and shake well to obtain the solution.

[0073] (2) Chromatographic conditions

[0074] The adopted chromatographic conditions are shown in Table 3.

[0075] Table 3 Chromatographic conditions

[0076]

[0077] (3) Injection and determination

[0078] After the liquid chromatography system is stable, inject the blank solvent (≥1 injection), the self-control solution (5 injections), and the test solution respectively, and record the chromatogram.

[0079] Table 4 System suitability results

[0080]

[0081] The results show that as Figure 4 , the separation between the impurity and the main peak is good. And as shown in Table 4, when the control solution is continuously injected 5 times, the RSD of the main peak area is 0.18% (<2.0%), meeting the system suitability requirements.

[0082] Specificity test of Example 4

[0083] (1) Solution preparation

[0084] Blank solvent: The same as in Example 3.

[0085] Negative control solution: Accurately weigh about 0.121 g of the negative control powder (a mixed powder of other components without ammonium glycyrrhizinate S component), transfer it to a 100-ml volumetric flask, add an appropriate amount of the blank solvent, shake well, ultrasonicate for 40 min to completely dissolve, cool to room temperature, dilute to the mark with the blank solvent, shake well, and filter to obtain the solution.

[0086] Impurity A stock solution: Accurately weigh an appropriate amount of Impurity A (24-hydroxyglycyrrhizic acid) reference substance, transfer it to a volumetric flask, dissolve and dilute to the mark with an appropriate amount of the blank solvent, and shake well to obtain the solution. (Containing about 25 μg / ml of Impurity A)

[0087] Impurity B stock solution: Accurately weigh an appropriate amount of Impurity B (18α-glycyrrhizic acid) reference substance, transfer it to a volumetric flask, dissolve and dilute to the mark with an appropriate amount of the blank solvent, and shake well to obtain the solution. (Containing about 25 μg / ml of Impurity B)

[0088] Test solution: The same as in Example 3.

[0089] (2) Chromatographic conditions

[0090] The chromatographic conditions adopted are the same as those in Table 3.

[0091] (3) Injection and determination

[0092] After the liquid chromatography system is stable, take the blank solvent (≥1 injection), negative control solution (1 injection), localization solution (each 1 injection), and test solution (1 injection) for injection respectively, and record the chromatogram.

[0093] The results show that, as Figure 5 shown, there is no interference from the blank solvent and the negative control solution at the peak positions of the main peak and its impurities. As shown in Table 5, the minimum resolution between the main peak and the known impurities in the test solution and the adjacent peaks is 3.33 > 1.5, and the purity angle of the main peak in the test solution is less than the purity threshold, and the peak purity of the main peak meets the requirements. This method has good specificity.

[0094] Table 5 Localization test of known impurities

[0095]

[0096] Example 5 Quantitative limit and detection limit test (1) Solution preparation Blank solvent: The same as in Example 3.

[0097] Stock solution of impurity A: The same as in Example 4. Stock solution of impurity B: The same as in Example 4.

[0098] Stock solution of the main component: Weigh an appropriate amount of ammonium glycyrrhizinate reference substance accurately, place it in a volumetric flask, dissolve it with the blank solvent and dilute it to the scale, shake well, and obtain it. (Containing about 25 μg / ml of glycyrrhizin).

[0099] Quantitative limit solution: Accurately measure 2 ml each of the stock solution of impurity A, the stock solution of impurity B, and the stock solution of the main component, place them in the same 100-ml volumetric flask, dilute it to the scale with the blank solvent, and shake well to obtain it. (Containing about 0.5 μg / ml of impurity A / impurity B / glycyrrhizin).

[0100] Detection limit solution: Accurately measure 3 ml of the quantitative limit solution and place it in a 10-ml volumetric flask, dilute it to the scale with the blank solvent, and shake well to obtain it.

[0101] (2) Chromatographic conditions

[0102] The chromatographic conditions adopted are the same as those in Table 2.

[0103] (3) Injection and determination

[0104] After the liquid chromatography system is stable, take the blank solvent (≥1 injection), quantitative limit (6 injections), and detection limit (1 injection) for injection respectively, and record the chromatogram.

[0105] Table 6 Results of quantitative limit and detection limit of the main component and each impurity

[0106]

[0107] The results are shown in Table 6. In the quantitation limit solution, the minimum intensity ratio S / N of the main component and each impurity peak to the noise peak signal is 12.5 (>10); in the detection limit solution, the minimum intensity ratio S / N of the main component and each impurity peak to the noise peak signal is 3.1 (>3); the maximum percentage of the quantitation limit equivalent to the main component concentration is 0.198% (<ignoring limit (0.2%)).

[0108] Table 7 Results of the parallelism of the quantitation limits of the main component and each impurity

[0109]

[0110] The results are shown in Table 7. The maximum RSD of the peak areas for 6 consecutive portions is 1.51% (<10.0%), meeting the requirements.

[0111] Example 6 Linearity and Range Test

[0112] (1) Solution Preparation

[0113] Blank solvent: The same as in Example 3.

[0114] Linear stock solution: Weigh accurately appropriate amounts of the reference substance of impurity A, the reference substance of impurity B, and the reference substance of ammonium glycyrrhizinate, place them in the same volumetric flask, dissolve with the blank solvent and dilute to the mark, and shake well to obtain. (Containing about 75 μg / ml of impurity A and about 175 μg / ml of impurity B / glycyrrhizin)

[0115] Linear solution: Accurately measure appropriate amounts of the linear stock solution according to Table 8 and place them in the corresponding volumetric flasks, dilute to the mark with the blank solvent, and shake well to prepare linear solutions with corresponding concentrations.

[0116] Table 8 Linear Concentrations and Their Preparation Conditions

[0117]

[0118] (2) Chromatographic Conditions

[0119] The chromatographic conditions used are the same as those in Table 3.

[0120] (3) Injection and Determination

[0121] After the liquid chromatography system is stable, inject the blank solvent (≥1 injection) and the linear solutions at each concentration (1 injection at each concentration) respectively, and record the chromatograms. The linear results are shown in Figures 9 - 12.

[0122] Table 9 Linear Results of Impurity A

[0123]

[0124]

[0125] Table 10 Linear Results of Glycyrrhizin

[0126]

[0127] Table 11 Linear Results of Impurity B

[0128]

[0129] Table 12 Correction Factor

[0130]

[0131] Example 7 Accuracy Test (1) Solution Preparation Blank Solvent: The same as in Example 3.

[0132] Stock Solution of Impurity A: Weigh an appropriate amount of Impurity A reference substance precisely, place it in a volumetric flask, dissolve it with an appropriate amount of solvent and dilute to the mark, then shake well to obtain. (Containing about 25 μg / ml of Impurity A)

[0133] Stock Solution of Impurity B: Weigh an appropriate amount of Impurity B reference substance precisely, place it in a volumetric flask, dissolve it with dilute ethanol and dilute to the mark, then shake well to obtain. (Containing about 125 μg / ml of Impurity B)

[0134] Test Solution: The same as in Example 3.

[0135] Accuracy Solution: Take 20 tablets of Compound Glycyrrhizin, weigh them precisely, grind them finely, weigh precisely about 73 mg of the fine powder, place it in a 50 ml volumetric flask, add the blank solvent and sonicate for 40 min to dissolve, cool to room temperature, precisely measure 7.5 ml of the stock solution of Impurity A and 8.2 ml of the stock solution of Impurity B and place them in the above volumetric flask, dilute to the mark with the blank solvent, filter, and take the subsequent filtrate to obtain. Prepare 6 portions in parallel.

[0136] Control Solution: Precisely measure 5 ml each of the background test solution and the accuracy solution into different 100 ml volumetric flasks, dilute to the mark with dilute ethanol, and shake well to obtain.

[0137] (2) Chromatographic Conditions

[0138] The chromatographic conditions adopted are the same as those in Table 3.

[0139] (3) Injection and Determination

[0140] After the liquid chromatography system is stable, inject the blank solvent (1 injection), the background test solution (1 injection), the accuracy solution (1 injection each), and the control solution (1 injection each) respectively, and record the chromatogram. According to the chromatographic peak areas of the related substances obtained, calculate the contents of the related substances in the test sample by the self - reference method. The calculation formula is:

[0141]

[0142] Among them, the correction factor is 1, and the dilution multiple of the control solution is 20.

[0143] Table 13 Accuracy Results

[0144]

[0145]

[0146] The results in Table 13 show that the average recovery rate of impurity A is 89.3% (between 80.0% and 120.0%), and the RSD is 1.90% (<10.0%). The average recovery rate of impurity B is 92.4% (between 80.0% and 120.0%), and the RSD is 0.74% (<10.0%). All meet the requirements. The accuracy of this method is good.

[0147] Example 8 Durability Test

[0148] On the basis of Table 3, by slightly adjusting the chromatographic conditions, the effects of minor changes in the chromatographic conditions on the test results of known impurities, other individual impurities, and the total amount of impurities were investigated according to Table 14.

[0149] Precisely measure the solvent, test solution, and control solution, and inject them into the liquid chromatograph respectively, and record the chromatogram.

[0150] Solvent, test solution, and control solution: the same as in Example 3.

[0151] Table 14 Durability Test Conditions and Results

[0152]

[0153]

[0154] Note: For the spiked test solution, compared with the impurity content under the standard conditions for each changed condition,

[0155] When X < the ignore limit, the absolute value of the difference is not calculated;

[0156] When 0.20% ≤ X < 0.50%, the absolute value of the difference shall not exceed 0.10%;

[0157] When X ≥ 0.50%, the absolute value of the difference shall not exceed 0.20%;

[0158] X is the impurity content.

[0159] The verification results are shown in Table 14. When investigating different mobile phase pH values (pH 2.5 or 3.0) and different column temperatures (28°C, 32°C), under each condition, the absolute value of the difference between the known impurity content and the content under the standard conditions is less than ±20% of the known impurity limit concentration, the absolute value of the difference between the content of other individual impurities and the content under the standard conditions is less than ±20% of the limit concentration, and the absolute value of the difference between the total impurity amount and the standard conditions is less than ±20% of the limit concentration. The method has good durability when varying within the ranges of mobile phase pH (pH 2.5 - 3.0) and column temperature (28°C - 32°C).

[0160] Example 9

[0161] Adopt the chromatographic conditions provided in Example 3.

[0162] Test solution: Take 20 tablets of compound glycyrrhizin, weigh accurately, grind them finely, accurately weigh about 0.146 g (equivalent to about 25 mg of glycyrrhizin), place it in a 100 ml volumetric flask, add an appropriate amount of blank solvent, shake well, ultrasonicate for 40 min to completely dissolve the main component, cool to room temperature, dilute to the mark with blank solvent, shake well, filter through a 0.45 μm organic filter membrane, discard 2 ml of the initial filtrate, and take the subsequent filtrate as the test solution.

[0163] Self-control solution: Accurately measure 5 ml of the test solution, place it in a 100 ml volumetric flask, dilute to the mark with blank solvent, shake well, and you will get it.

[0164] The results of detecting multiple batches by the related substance detection method described in the present invention are as follows:

[0165] Table 9 Detection results of related substances in multiple batches of self-developed preparations

[0166]

[0167] Comparative Example 1, comparison of different brand chromatographic columns

[0168] Adopt the chromatographic conditions described in Example 3, and conduct experiments using two chromatographic columns respectively. The specifications of the chromatographic columns are as follows:

[0169] The specifications of the Shiseido chromatographic column are: CAPCELL PAK C18 (MGⅡ) 4.6 * 250 mm, 5 μm;

[0170] The specifications of the Welch chromatographic column are: Ultimate LP-C18 4.6 * 250 mm, 5 μm;

[0171] Take the test solution as the sample and inject samples respectively. The results are as Figure 6 shown.

[0172] Test solution: Take about 135 mg of ammonium glycyrrhizinate S (equivalent to 100 mg of glycyrrhizin), place it in a 100-ml volumetric flask, dissolve it with dilute ethanol and dilute to the mark, shake well to obtain the solution.

[0173] It can be seen that with the Shiseido chromatographic column, the unknown impurities with RRT 1.23 and RRT 1.24 could not be baseline separated. After replacing with the Welch chromatographic column, the peaks could be baseline separated. Therefore, the Welch chromatographic column was finally adopted in this method.

[0174] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for detecting glycyrrhizin-related substances in compound glycyrrhizin tablets, characterized in that: The related substances mentioned include known impurity A: 24-hydroxyglycyrrhizic acid, impurity B: 18α-glycyrrhizic acid; High performance liquid chromatography was used, wherein the chromatographic conditions were as follows: the chromatographic column was filled with octadecylsilane bonded silica gel, the mobile phase A was a mixed solution of potassium dihydrogen phosphate aqueous solution adjusted to pH 2.0-3.0 with phosphoric acid and methanol, and the mobile phase B was acetonitrile.

2. The method for detecting glycyrrhizin-related substances in compound glycyrrhizin tablets according to claim 1, characterized in that: The chromatographic column is Ultimate LP-C18, 4.6 mm×250 mm, 5 μm.

3. The method for detecting glycyrrhizin-related substances in compound glycyrrhizin tablets according to claim 1, characterized in that: The concentration of potassium dihydrogen phosphate aqueous solution in mobile phase A was 0.08 mol / L, and the volume ratio of potassium dihydrogen phosphate to methanol was 60:

10.

4. The method for detecting glycyrrhizin-related substances in compound glycyrrhizin tablets according to claim 1, characterized in that: The flow rate was 1.0 ml / min, the injection volume was 40 μl, and the running time was 75 min.

5. The method for detecting glycyrrhizin-related substances in compound glycyrrhizin tablets according to claim 1, characterized in that: The detection wavelength is selected from 251nm-257nm, and the column temperature is selected from 28-32°C.

6. The method for detecting glycyrrhizin-related substances in compound glycyrrhizin tablets according to claim 1, characterized in that: The detection wavelength is 254 nm and the column temperature is 30°C.

7. The method for detecting glycyrrhizin-related substances in compound glycyrrhizin tablets according to claim 1, characterized in that: Perform gradient elution. The procedure of gradient elution includes: 。 8. The method for detecting glycyrrhizin-related substances in compound glycyrrhizin tablets according to claim 1, characterized in that: The following steps are involved: (1) Solution preparation Preparation of blank solvent: Blank solvent is the diluent, which is an ethanol-ultrapure water mixed solution, wherein the volume ratio of ethanol to ultrapure water is 529:

471. The diluent is obtained by measuring the corresponding volumes of anhydrous ethanol and ultrapure water respectively; Preparation of test solution: accurately weigh the ground compound glycyrrhizin tablet powder, add blank solvent to make a solution containing 0.25±0.025mg / ml of glycyrrhizin, centrifuge, filter the supernatant with a 0.45μm organic filter, and take the filtrate as the test solution; Preparation of self-control solution: Accurately pipette the test solution and dilute it 20 times with diluent to prepare the self-control solution.

9. The method for detecting glycyrrhizin-related substances in compound glycyrrhizin tablets according to claim 1, characterized in that: The chromatographic conditions are: Chromatographic column: Ultimate LP-C18 4.6mm×250mm, 5μm, Mobile phase A: 0.08 mol / L potassium dihydrogen phosphate aqueous solution pH 2.5-methanol mixed solution, where the volume ratio of potassium dihydrogen phosphate aqueous solution to methanol is 60:10, Mobile phase B: acetonitrile, Flow rate: 1.0ml / min, Column temperature: 30°C, Detection wavelength: 254nm, Injection volume: 40 μl, Running time: 75 minutes, Gradient elution program: ； (3) Sampling measurement Inject 40 μl of blank solvent, self-control solution and test solution into the liquid chromatograph in sequence and record the chromatogram; calculate the content of each related substance in the sample according to the self-control method based on the chromatographic peak area of ​​each related substance obtained.

10. The method for detecting glycyrrhizin-related substances in compound glycyrrhizin tablets according to claim 9, characterized in that: The calculation formula is: The correction factor is 1, and the dilution factor of the control solution is 20.

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