A method for detecting impurities in compound bergenin tablets

Abstract

A method for detecting impurities in compound bergenin tablets comprises the following steps: preparing a test solution: weighing a predetermined amount of compound bergenin powder to be tested, placing it in a volumetric flask, adding a solvent, and completely dissolving the compound bergenin powder to be tested to obtain a test solution; preparing a reference solution: taking predetermined amounts of impurities A, B, C, D, E, and bergenin, dissolving and diluting them with a solvent to form a reference solution containing 10 μg of bergenin and 5 μg of each impurity per milliliter; preparing a system suitability solution: taking predetermined amounts of bergenin, chlorpheniramine maleate, impurities A, B, C, D, and E, dissolving and diluting them with a solvent to form a mixed solution containing 1 mg of bergenin, 16 μg of chlorpheniramine maleate, and 5 μg of each impurity per milliliter; and gradient elution. The present invention detects the impurity components and contents of compound bergenin tablets by establishing a high-performance liquid chromatography and a principal component self-reference method with a correction factor.

Description

Technical Field

[0001] The present invention relates to the technical field of drug detection, and in particular to a method for detecting impurities in compound bergenin tablets. Background Art

[0002] Compound Bergenin Tablets have antitussive and expectorant properties and are used for chronic bronchitis. This compound preparation contains 125mg of bergenin and 2mg of chlorpheniramine maleate. Bergenin, a dihydroisocoumarin compound, has anti-inflammatory, analgesic, and antitussive and antiasthmatic properties. It is commonly used as an active ingredient in antitussive and anti-inflammatory drugs for the treatment of coughs, acute and chronic tracheitis, and duodenal ulcers. Chlorpheniramine maleate has a strong histamine H1 receptor blocking effect, which can alleviate respiratory symptoms caused by allergies.

[0003] Because the impurities in Compound Bergenin Tablets have similar structures, impurity peaks easily overlap when tested using HPLC, making it impossible to accurately detect the specific components and amounts of the impurities. Only the total impurity content or the bergenin content in Compound Bergenin Tablets can be detected. For example, the invention patent application number 01410813386.8, entitled "Method for Testing the Quality of Compound Bergenin Tablets," also uses HPLC analysis, but can only detect the bergenin content, not the individual impurity components and amounts. Summary of the Invention

[0004] In view of this, it is necessary to provide a method for detecting impurities in compound bergenin tablets using high performance liquid chromatography analysis, which can quickly detect and accurately measure the impurity components and contents of compound bergenin tablets.

[0005] A method for detecting impurities in compound bergenin tablets comprises the following steps:

[0006] Step S1: preparing a test solution: weighing a predetermined amount of the compound bergenin powder to be tested, placing it in a volumetric flask, adding a solvent, and completely dissolving the compound bergenin powder to be tested to obtain a test solution;

[0007] Step S2: preparing a reference solution: taking predetermined amounts of impurity A, impurity B, impurity C, impurity D, impurity E, and bergenin, dissolving them in a solvent, and diluting them into a reference solution containing 10 μg of bergenin and 5 μg of each impurity per ml;

[0008] Impurity A: 3,5-dihydroxy-4-methoxy-2-C-β-D-glucoside;

[0009] Impurity B: norbergenin;

[0010] Impurity C: 4-O-galloylbergenin;

[0011] Impurity D: 11-O-galloylbergenin;

[0012] Impurity E: 11-O-4-hydroxybenzoylbergenin;

[0013] Step S3: Preparing a system suitability solution: taking predetermined amounts of bergenin, chlorpheniramine maleate, impurity A, impurity B, impurity C, impurity D, and impurity E, dissolving and diluting with a solvent to prepare a mixed solution containing 1 mg of bergenin, 16 μg of chlorpheniramine maleate, and 5 μg of each impurity per milliliter;

[0014] Step S4: Gradient elution:

[0015] Octadecylsilane bonded silica gel is used as filler;

[0016] Methanol was used as mobile phase A;

[0017] Phosphate buffer was used as mobile phase B.

[0018] The solution to be detected is subjected to gradient elution, wherein:

[0019] 0-17 minutes, mobile phase A is 10%-60%, mobile phase B is 90%-40%;

[0020] 17-22 minutes, mobile phase A: 60%, mobile phase B: 40%;

[0021] 22-23 minutes, mobile phase A is 60%-10%, mobile phase B is 40%-90%;

[0022] 23-30 minutes, mobile phase A is 10%, mobile phase B is 90%.

[0023] In the above-mentioned method for detecting impurities in compound bergenin tablets, preferably, the solvent includes methanol and phosphate buffer, and the volume ratio of methanol to phosphate buffer is 1:9; wherein the phosphate buffer is prepared by the following method: take 11.5g of ammonium dihydrogen phosphate, dissolve it in water, add 1ml of phosphoric acid, and dilute it to 1000ml with water.

[0024] The above-mentioned method for detecting impurities in compound bergenin tablets preferably further comprises step S5, obtaining linear equations of each component, wherein:

[0025] The linear equation of bergenin is: A=16988C+1142.2, R 2 =0.9999;

[0026] The linear equation of impurity A is: A = 1929.3C-262.18, R 2 =1;

[0027] The linear equation of impurity B is: A = 13382C + 1224.5, R 2 =0.9994;

[0028] The linear equation of impurity C is: A = 22074C + 1623.5, R 2 =0.9996;

[0029] The linear equation of impurity D is: A = 22238C + 1963.5, R 2 =0.9997;

[0030] The linear equation of impurity E is: A = 21013C + 13.746, R 2 =1.

[0031] The correction factor can be obtained through the linear equation of each component, thereby calculating the content of each component.

[0032] Beneficial Effects: This method utilizes a high-performance liquid chromatography (HPLC) and principal component self-reference method with a correction factor to detect the impurities and their contents in compound bergenin tablets, enabling qualitative and quantitative analysis. This eliminates the need for impurity reference materials, addressing the difficulty of obtaining impurity reference materials, while also reducing the high cost of purchasing reference materials and shortening testing time. Results demonstrate that the established method is rapid and accurate, providing a basis for quality control of compound bergenin tablets. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is the system suitability chromatogram. Among them, 1. Maleic acid, 2. Impurity A, 3. Impurity B, 4. Bergenin, 5. Impurity C, 6. Impurity D, 7. Chlorpheniramine, 8. Impurity E.

[0034] Figure 2 The chromatogram of the reference substance is shown in Figure 1. Impurity A, 2. Impurity B, 3. Bergenin, 4. Impurity C, 5. Impurity D, and 6. Impurity E.

[0035] Figure 3 The chromatogram of the test sample is shown below: 1. Maleic acid, 2. Impurity A, 3. Impurity B, 4. Bergenin, 5. Impurity C, 6. Impurity D, 7. Chlorpheniramine, and 8. Impurity E.

[0036] Figure 4 The chromatogram of the control solution. DETAILED DESCRIPTION

[0037] To more clearly illustrate the technical solutions of the embodiments of the present invention, the solutions of the present invention will be described in detail below with reference to the accompanying drawings in the embodiments. Obviously, the following description includes some embodiments of the present invention, and it is obvious that those skilled in the art can derive other implementation methods based on these embodiments without inventive effort.

[0038] A method for detecting impurities in compound bergenin tablets comprises the following steps:

[0039] Step S1: preparing a test solution: weighing a predetermined amount of the compound bergenin powder to be tested, placing it in a volumetric flask, adding a solvent, and completely dissolving the compound bergenin powder to be tested to obtain a test solution;

[0040] Step S2: preparing a reference solution: taking predetermined amounts of impurity A, impurity B, impurity C, impurity D, impurity E, and bergenin, dissolving them in a solvent, and diluting them into a reference solution containing 10 μg of bergenin and 5 μg of each impurity per ml;

[0041] 5 μg of each impurity means 5 μg of each of impurity A, impurity B, impurity C, impurity D, and impurity E.

[0042] Impurity A: 3,5-dihydroxy-4-methoxy-2-C-β-D-glucoside;

[0043] Impurity B: norbergenin;

[0044] Impurity C: 4-O-galloylbergenin;

[0045] Impurity D: 11-O-galloylbergenin;

[0046] Impurity E: 11-O-4-hydroxybenzoylbergenin;

[0047] Step S3: Preparing a system suitability solution: taking predetermined amounts of bergenin, chlorpheniramine maleate, impurity A, impurity B, impurity C, impurity D, and impurity E, dissolving and diluting with a solvent to prepare a mixed solution containing 1 mg of bergenin, 16 μg of chlorpheniramine maleate, and 5 μg of each impurity per milliliter;

[0048] Step S4: Gradient elution:

[0049] Octadecylsilane bonded silica gel is used as filler;

[0050] Methanol was used as mobile phase A;

[0051] Phosphate buffer was used as mobile phase B.

[0052] The solution to be detected is subjected to gradient elution, wherein:

[0053] 0-17 minutes, mobile phase A is 10%-60%, mobile phase B is 90%-40%;

[0054] 17-22 minutes, mobile phase A: 60%, mobile phase B: 40%;

[0055] 22-23 minutes, mobile phase A is 60%-10%, mobile phase B is 40%-90%;

[0056] 23-30 minutes, mobile phase A is 10%, mobile phase B is 90%;

[0057] The above percentages are by volume. The sum of the volume ratios of mobile phase A and mobile phase B during the elution process is 1. During the elution process, from 0 to 17 minutes, mobile phase A was uniformly changed from 10% at 0 minute to 60% at 17 minutes, and mobile phase B was uniformly changed from 90% at 0 minute to 40% at 17 minutes. Similarly, from 22 to 23 minutes, mobile phase A was uniformly changed from 60% at 22 minutes to 10% at 23 minutes, and mobile phase B was uniformly changed from 40% at 22 minutes to 90% at 23 minutes.

[0058] In a preferred embodiment, the solvent includes methanol and phosphate buffer, and the volume ratio of methanol to phosphate buffer is 1:9; wherein the phosphate buffer is prepared by the following method: take 11.5g of ammonium dihydrogen phosphate, add an appropriate amount of water to dissolve it, add 1ml of phosphoric acid, and dilute it to 1000ml with water.

[0059] The solutions of the present invention will be described below with reference to specific embodiments.

[0060] The instruments used in the present invention include Shimadzu LC-40D high performance liquid chromatograph and Sartorius MCA66S-3CCN-D one millionth electronic balance.

[0061] In the reference substance, impurity A comes from Desit Biotechnology, batch number: DST241018-381, content: 98.25%;

[0062] Impurity B comes from Xili Biotechnology, batch number: BBP60551, content: 98%;

[0063] Impurity C comes from Xili Bio, batch number: BBP03294, content: 96%,

[0064] Impurity D was obtained from Sichuan Weikeqi Biotechnology Co., Ltd., batch number: WP24070311, content: 99.03%;

[0065] Impurity E comes from Desit Bio, batch number: DST240926-537, content: 96.07%.

[0066] The chromatographic column used in the present invention is Phenylephant Gemini C 18 (5μm, 4.6×250mm).

[0067] The methanol, phosphoric acid and ammonium dihydrogen phosphate in the solvent were all of HPLC grade, and the water was purified water.

[0068] Example 1

[0069] Prepare phosphate buffer: Dissolve 11.5 g of ammonium dihydrogen phosphate in water, add 1 ml of phosphoric acid, and dilute to 1000 ml with water.

[0070] Prepare the test solution: weigh 0.1 g of compound bergenin tablets using an electronic balance, pour into a 100 ml volumetric flask, add 10 ml of methanol, shake to dissolve the compound bergenin tablets, dilute to 100 ml with phosphate buffer, shake well and filter, and the filtrate is the test solution.

[0071] Prepare the control solution: Measure 1 ml of the test solution, place it in a 100 ml volumetric flask, dilute to 100 ml with solvent, and shake well.

[0072] Preparation of reference solution: Take appropriate amount of impurity A, impurity B, impurity C, impurity D, impurity E and bergenin reference substance, dissolve them with solvent and dilute them into a mixed reference solution containing 10 μg of bergenin and 5 μg of each impurity per ml.

[0073] Prepare system suitability solution: Take appropriate amounts of bergenin, chlorpheniramine maleate, impurity A, impurity B, impurity C, impurity D, and impurity E reference substances, dissolve them in methanol, and dilute with solvent to prepare a mixed solution containing 1 mg of bergenin, 16 μg of chlorpheniramine maleate, and 5 μg of each impurity per ml.

[0074] The system suitability solution, reference solution, test solution and reference solution were injected into the liquid chromatograph respectively for gradient elution and determination. Among them, octadecylsilane bonded silica gel was used as the filler, the filler particle size was 5 μm, the inner diameter was 4.6 mm, and the length was 250 mm. Methanol was used as mobile phase A and phosphate buffer was used as mobile phase B. During the gradient elution process,

[0075] 0-17 minutes, mobile phase A is 10%-60%, mobile phase B is 90%-40%;

[0076] 17-22 minutes, mobile phase A: 60%, mobile phase B: 40%;

[0077] 22-23 minutes, mobile phase A is 60%-10%, mobile phase B is 40%-90%;

[0078] 23-30 minutes, mobile phase A is 10%, mobile phase B is 90%;

[0079] The measurement wavelength was 272 nm, the injection volume was 10 μl, the sample plate temperature was 4 degrees Celsius, and the chromatographic temperature was 30 degrees Celsius.

[0080] The chromatogram after determination is as follows Figures 1 to 4 .

[0081] Obtain correction factors and relative retention times;

[0082] Take bergenin and various impurity reference substances, dilute them with solvent to prepare a series of linear solutions, and perform determination. Draw a standard curve with concentration C (μg / mL) as the abscissa and peak area A as the ordinate to obtain a linear equation.

[0083] in,

[0084] The linear equation of bergenin is: A=16988C+1142.2, R 2 =0.9999;

[0085] The linear equation of impurity A is: A = 1929.3C-262.18, R 2 =1;

[0086] The linear equation of impurity B is: A = 13382C + 1224.5, R 2 =0.9994;

[0087] The linear equation of impurity C is: A = 22074C + 1623.5, R 2 =0.9996;

[0088] The linear equation of impurity D is: A = 22238C + 1963.5, R 2 =0.9997;

[0089] The linear equation of impurity E is: A = 21013C + 13.746, R 2 =1;

[0090] The slope of the linear equation between bergenin and the component to be tested is used as the correction factor (f), that is, f = k s / k i , k s is the slope of the linear equation for bergenin, k i The slope of the linear equation of the measured component. The correction factors of impurity A, impurity B, impurity C, impurity D, and impurity E are 8.8053, 1.2695, 0.7696, 0.7639, and 0.8085, respectively.

[0091] Similarly, the relative retention time of the component to be measured is R = R i / R s , where R iis the retention time of the component to be analyzed, R s is the retention time of bergenin, combined with Figure 2 The relative retention times of impurity A, impurity B, impurity C, impurity D, and impurity E are 0.5619, 0.7649, 1.2245, 1.4464, and 1.7397, respectively.

[0092] The content of each component to be measured (W 本法 ),Right now

[0093]

[0094] Among them, A i is the peak area of ​​the component to be measured, f is the correction factor, A 对照 is the peak area of ​​the control solution.

[0095] The content of each component to be tested (W 外标 ) were used to compare the results of the present invention with those of the external standard method, and the relative deviation SMD was calculated to determine the accuracy of the results of the present invention.

[0096] Right now

[0097]

[0098] The results are shown in Table 1.

[0099] Table 1: Results of determination of impurity content in samples (%)

[0100]

[0101] It can be seen from Table 1 that the SMDs are all less than 5.0%, indicating that there is no significant difference between the determination results of this method and the determination results of the external standard method, and the results are accurate and reliable.

[0102] The above disclosure is only a preferred embodiment of the present invention, and it is certainly not intended to limit the scope of the present invention. A person skilled in the art can understand that all or part of the processes of the above embodiment and equivalent changes made in accordance with the claims of the present invention are still within the scope of the invention.

Claims

1. A method for detecting impurities in compound bergenin tablets, characterized by: The following steps are involved: Step S1: preparing a test solution: weighing a predetermined amount of the compound bergenin powder to be tested, placing it in a volumetric flask, adding a solvent, and completely dissolving the compound bergenin powder to be tested to obtain a test solution; Step S2: preparing a reference solution: taking predetermined amounts of impurity A, impurity B, impurity C, impurity D, impurity E, and bergenin, dissolving them in a solvent, and diluting them into a reference solution containing 10 μg of bergenin and 5 μg of each impurity per ml; Impurity A: 3,5-dihydroxy-4-methoxy-2-C-β-D-glucoside; Impurity B: norbergenin; Impurity C: 4-O-galloylbergenin; Impurity D: 11-O-galloylbergenin; Impurity E: 11-O-4-hydroxybenzoylbergenin; Step S3: Preparing a system suitability solution: taking predetermined amounts of bergenin, chlorpheniramine maleate, impurity A, impurity B, impurity C, impurity D, and impurity E, dissolving and diluting with a solvent to prepare a mixed solution containing 1 mg of bergenin, 16 μg of chlorpheniramine maleate, and 5 μg of each impurity per milliliter; Step S4: gradient elution of the test solution: Octadecylsilane bonded silica gel is used as filler; Methanol was used as mobile phase A; Phosphate buffer was used as mobile phase B. Gradient elution was performed, wherein 0-17 minutes, mobile phase A is 10%-60%, mobile phase B is 90%-40%; 17-22 minutes, mobile phase A: 60%, mobile phase B: 40%; 22-23 minutes, mobile phase A is 60%-10%, mobile phase B is 40%-90%; 23-30 minutes, mobile phase A is 10%, mobile phase B is 90%.

2. The method for detecting impurities in compound bergenin tablets according to claim 1, wherein: The solvent includes methanol and phosphate buffer, and the volume ratio of methanol to phosphate buffer is 1:9; wherein the phosphate buffer is prepared by the following method: taking 11.5g of ammonium dihydrogen phosphate, dissolving it in water, adding 1ml of phosphoric acid, and diluting it to 1000ml with water.

3. The method for detecting impurities in compound bergenin tablets according to claim 1, characterized in that: The step S5 is also included to obtain the linear equation of each component. Specifically, Take bergenin and various impurity reference substances, dilute them with solvent to prepare a series of linear solutions, and perform the determination. Draw a standard curve with concentration C as the abscissa and peak area A as the ordinate to obtain a linear equation. The linear equation of bergenin is: A=16988C+1142.2, R 2 =0.9999; The linear equation of impurity A is: A = 1929.3C-262.18, R 2 =1; The linear equation of impurity B is: A = 13382C + 1224.5, R 2 =0.9994; The linear equation of impurity C is: A = 22074C + 1623.5, R 2 =0.9996; The linear equation of impurity D is: A = 22238C + 1963.5, R 2 =0.9997; The linear equation of impurity E is: A = 21013C + 13.746, R 2 =1.

4. The method for detecting impurities in compound bergenin tablets according to claim 3, wherein: The correction factors of impurity A, impurity B, impurity C, impurity D, and impurity E are 8.8053, 1.2695, 0.7696, 0.7639, and 0.8085, respectively.

5. The method for detecting impurities in compound bergenin tablets according to claim 3, wherein: The relative retention times of impurity A, impurity B, impurity C, impurity D and impurity E are 0.5619, 0.7649, 1.2245, 1.4464 and 1.7397, respectively.

6. The method for detecting impurities in compound bergenin tablets according to claim 3, wherein: The method further includes step S6, calculating the content of the component to be tested: Among them, A i is the peak area of ​​the component to be measured, f is the correction factor, A 对照 is the peak area of ​​the control solution.

7. The method for detecting impurities in compound bergenin tablets according to claim 1, wherein: In step S4, the filler particle size is 5 μm, the inner diameter is 4.6 mm, and the length is 250 mm.

Images