Method for detecting oseltamivir phosphate impurities

Abstract

The invention discloses a detection method of oseltamivir phosphate impurities. The detection method comprises the following steps: (1) preparing a test solution; (2) preparing a system applicable solution; (3) preparing a blank auxiliary material solution; (4) preparing a contrast test solution; and (5) testing method. The method can separate and detect various impurities in an oseltamivir phosphate sample, can effectively separate povidone K30 in oseltamivir phosphate capsule auxiliary materials, improves the separation degree of oseltamivir phosphate and the impurities, has high sensitivity, and can better control the product quality of oseltamivir phosphate capsules.

Description

Technical Field

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

[0002] Impurities are closely related to the safety of clinical use of drugs. If the impurities in drugs are not detected and controlled by effective methods, they will have a potential impact on clinical safety. Therefore, it is very important to develop a reasonable and effective drug impurity detection method to control impurities in drugs. Affected by influenza A and B infectious diseases, the quality of its effective therapeutic drug oseltamivir phosphate is related to the safety of people's lives, and it is urgent to accurately determine the relevant substances in its drugs. Some impurities may be generated during the production process of oseltamivir phosphate, which may affect the efficacy and safety of the drug. In the process of preparing oseltamivir phosphate into dry suspensions, capsules and other dosage forms, the oseltamivir phosphate capsule preparation contains the following excipients: povidone K30, pregelatinized starch, sodium stearyl fumarate and other components. The added excipients and the degradation products of the excipients will have certain interference with the main drug. The active impurities in the excipients will cause the degradation of the drug product even at a trace level, thereby affecting the stability and quality of the drug. However, the existing technology usually does not test or control certain impurities in the excipients.

[0003] At present, the detection of oseltamivir phosphate impurities is basically detected by high performance liquid chromatography. When using high performance liquid chromatography for detection, it is rare to separate and detect auxiliary materials, and the resolution of the liquid chromatograph is affected by the selection of separation columns, the setting of separation conditions and column packing, etc. In some cases, the effect of the separation column is very poor, and the resolution will also be greatly reduced; if the separation conditions are improper, such as too fast flow rate, unstable temperature, unbalanced elution conditions, etc., it will also cause the resolution to decline and the separation effect to be not obvious. At the same time, due to the different effects of column packing, the same substance may have certain differences in the test results on different days, thereby causing the quality repeatability to decrease. Therefore, it is very critical to select suitable separation columns, detection conditions, and elution conditions for the result of chromatographic analysis. The present invention uses reasonable optimization measures to improve the effect of liquid chromatography detection, so that it can not only detect the content of impurities in the production process of oseltamivir capsules and can also detect the content of reference auxiliary material preparations, and improve the reliability of the analysis results. Summary of the invention

[0004] In view of this, the present invention provides a method for detecting impurities of oseltamivir phosphate. By optimizing liquid chromatography conditions, the detection method has stability and excellent separation, and can accurately identify and quantify relevant impurities in the production of oseltamivir phosphate capsules, thereby achieving accurate control of product quality.

[0005] The technical means adopted by the present invention are as follows: a method for detecting impurities of oseltamivir phosphate, comprising the following steps:

[0006] (1) Preparation of test solution: Accurately weigh an appropriate amount of oseltamivir phosphate, dissolve it in solvent, and shake to volume;

[0007] (2) Preparation of system suitability solution: Accurately weigh appropriate amounts of impurity I, impurity II, impurity III, impurity A, impurity G, povidone K30 and oseltamivir phosphate reference substance, dissolve them in solvent, and shake to volume;

[0008] (3) Preparation of blank excipient solution: Weigh an appropriate amount of blank excipient of oseltamivir phosphate capsules, dissolve and dilute with solvent;

[0009] (4) Preparation of control test solution: Accurately weigh an appropriate amount of test solution, dissolve it in solvent and shake to volume;

[0010] (5) Test method: Take equal amounts of the test solution, system suitability solution, and control test solution and inject them into the liquid chromatograph, and record the chromatogram;

[0011] Wherein, gradient elution is used for elution;

[0012] In the gradient elution, the mobile phase includes mobile phase A and mobile phase B. Mobile phase A: phosphate buffer solution, which is an aqueous solution of 15-16.5 g / L potassium dihydrogen phosphate and 10-12.5 g / L anhydrous disodium hydrogen phosphate. Mobile phase B: methanol-acetonitrile. The gradient elution procedure is as follows:

[0013] From 0 to 10 minutes, the volume fraction of mobile phase A is maintained at 90%, and the volume fraction of mobile phase B is maintained at 10%; from 10 to 25 minutes, the volume fraction of mobile phase A gradually changes to 70%, and the volume fraction of mobile phase B gradually changes to 30%; from 25 to 40 minutes, the volume fraction of mobile phase A gradually changes to 40%, and the volume fraction of mobile phase B gradually changes to 60%; from 40 to 55 minutes, the volume fraction of mobile phase A gradually changes to 90%, and the volume fraction of mobile phase B gradually changes to 10%; from 55 to 60 minutes, the volume fraction of mobile phase A is maintained at 90%, and the volume fraction of mobile phase B is maintained at 10%.

[0014] Furthermore, the pH value of mobile phase A was adjusted to 6-7 with aqueous ammonia.

[0015] Furthermore, the volume content of acetonitrile in mobile phase B is 65-75%.

[0016] Furthermore, the solvent is phosphate buffer solution-methanol-acetonitrile, and the volume ratio of the phosphate buffer solution to methanol-acetonitrile is 55:45 to 60:40.

[0017] Furthermore, the liquid chromatography conditions are set as follows:

[0018] Chromatographic column: octadecylsilane bonded silica gel as filler;

[0019] Detection wavelength: 207nm;

[0020] Flow rate: 0.5~1.0ml / min;

[0021] Column temperature: 40-45°C;

[0022] Injection volume: 20 μl.

[0023] Furthermore, the concentration of the test solution is 0.8-1.5 mg / ml.

[0024] Furthermore, the concentration of the control test solution is 8-15 μg / ml.

[0025] Furthermore, the system suitability solution is configured to contain 0.01 mg of impurity I, impurity II, impurity III, impurity A, impurity G, povidone K30 and oseltamivir phosphate reference solution per 1 mL.

[0026] The beneficial effects of the present invention are as follows: by optimizing the liquid chromatography conditions, the auxiliary material impurities can be effectively detected, and the method has the characteristics of good specificity, high sensitivity and high separation efficiency, and is suitable for the detection of related substances in the production of oseltamivir phosphate capsules, and can detect the auxiliary material povidone in the capsule preparation, and analyze and control the quality of the drug; the detection method of the present invention has good stability and good separation degree, and can accurately qualitatively and quantitatively detect the related substances in oseltamivir phosphate, so as to objectively, accurately and comprehensively evaluate its quality, and provide a basis for formulating the quality standard of oseltamivir phosphate. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The figure is an impurity detection chromatogram of a method for detecting impurities in oseltamivir phosphate. DETAILED DESCRIPTION

[0028] The principles and features of the present invention are described below. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0029] Example 1

[0030] 1. Instruments and reagents:

[0031] The chromatograph was 1260 liquid chromatograph, from Nanjing Kejie Instrument Co., Ltd.

[0032] Oseltamivir phosphate capsules: specifications 75mg, Tamiflu.

[0033] Oseltamivir phosphate reference substance: mass fraction is 99%, Hunan Eurasia Pharmaceutical Co., Ltd.;

[0034] The structural formulas of impurity I, impurity II, impurity III, EP impurity A, impurity G, and povidone K30 are as follows:

[0035]

[0036]

[0037] 2. The liquid chromatography conditions are set as follows:

[0038] Chromatographic column: octadecylsilane bonded silica gel as filler;

[0039] Detection wavelength: 207nm;

[0040] Flow rate: 1.0ml / min;

[0041] Column temperature: 45°C;

[0042] Injection volume: 20 μl;

[0043] The elution was performed by gradient elution; mobile phase A: phosphate buffer solution, potassium dihydrogen phosphate 16g / L and aqueous solution containing anhydrous disodium hydrogen phosphate 12g / L, pH value adjusted to 7, mobile phase B: methanol-acetonitrile (30:70). The elution method is shown in Table 1 below:

[0044] Table 1

[0045] Time / min Mobile phase A / % Mobile phase B / % 0～10 90 10 10～25 90→70 10→30 25～40 70→40 30→60 40～55 40→90 60→10 55～60 90 10

[0046] 3. Configuration of related solutions

[0047] Test solution: 50 mg of oseltamivir phosphate test sample was measured and accurately weighed. It was placed in a 50 ml volumetric flask. 25 ml of solvent was added for ultrasonic dissolution and dilution. The volume was adjusted to the mark with solvent and shaken well. The ultrasonic conditions were 150 W for 2 to 3 min. The solution concentration was 1 mg / ml.

[0048] System suitability solution: Measure impurity I, impurity II, impurity III, impurity A, impurity G, povidone K30 and oseltamivir phosphate reference substance, dissolve in solvent and shake well to prepare solutions containing 0.01 mg of impurity I, impurity II, impurity III, impurity A, impurity G, povidone K30 and oseltamivir phosphate reference substance per 1 mL of solution.

[0049] Preparation of blank excipient solution: weigh an appropriate amount of blank excipient of oseltamivir phosphate capsules, dissolve and dilute with solvent;

[0050] Control test solution: Measure 1 ml of the above test solution, place it in a 100 ml volumetric flask, add solvent to dilute to the scale, shake well, and obtain oseltamivir phosphate control test solution with a solution concentration of 10 μg / ml.

[0051] Preparation of solvent: phosphate buffer solution-methanol-acetonitrile, the volume ratio of phosphate buffer solution to methanol-acetonitrile is 60:40.

[0052] 4. Specificity test

[0053] Take the test solution, system suitability solution and blank excipient solution and inject them into the liquid chromatograph for gradient elution. Record the chromatogram. The results are shown in Table 2. The chromatogram is as follows: Figure 1 shown.

[0054] Table 2 Separation test results

[0055]

[0056]

[0057] The results showed that, according to the chromatogram analysis, there was no chromatographic peak in the test solution and the blank excipient solution that interfered with the peaks of the main component and impurities I, II, III, A, G, povidone K30 and other substances, and the specificity was good. The minimum separation between impurities and between impurities and the main component was 1.84, and the minimum theoretical plate number of each chromatographic peak was 6913, proving that the chromatographic conditions can effectively separate oseltamivir phosphate from other impurities.

[0058] Example 2 Forced degradation of main components

[0059] According to the destructive test methods in the literature, light, high temperature, acid, alkali, oxidation and high humidity conditions are used to destroy the test solution. The specific steps are:

[0060] (1) The contents of oseltamivir phosphate are placed at 65°C for 30 days, and then the test solution is prepared to obtain a high temperature destruction sample.

[0061] (2) The contents of oseltamivir phosphate were irradiated under strong light of 5000 lx for 30 days, and then the test solution was prepared to obtain a light-damaged sample.

[0062] (3) Add 1 mL of 1 mol / L hydrochloric acid solution to the sample solution and shake well. Place the solution at 65°C for 5 h, then add an alkali solution to neutralize the solution. Then, add a solvent to make up the volume and shake well to obtain a strong acid destruction sample.

[0063] (4) Add 1 mL of 1 mol / L sodium hydroxide solution to the sample solution and shake well. Place the solution at 65°C for 5 h, then add acid solution to neutralize it. Then, add solvent to make up to volume and shake well to obtain a strong base destruction sample.

[0064] (5) Add 1 mL of 30% hydrogen peroxide solution to the sample solution and shake well. Leave the solution at room temperature for 5 h, then add solvent to make up the volume and shake well to obtain an oxidatively damaged sample.

[0065] (6) The contents of oseltamivir phosphate were placed in an environment of 26°C and 85% humidity for 30 days, and then the test solution was prepared and the sample was destroyed by high humidity.

[0066] The above-mentioned damaged samples were tested by chromatograph injection, and the test results were recorded in Table 3 below.

[0067]

[0068]

[0069] In general, the number of impurity peaks in samples damaged by light, high humidity, high temperature, strong acid, strong alkali and oxidation increased less, indicating that oseltamivir phosphate was relatively stable under the above-mentioned damage conditions, and the chromatographic peaks of the degradation impurities could be effectively separated from the main component, indicating that the method had a strong ability to detect the degradation impurities of the main component and had good specificity.

[0070] Example 3 Detection Limit and Quantification Limit

[0071] Accurately measure an appropriate amount of system suitability solution to prepare 1.0 μg / mL, gradually dilute with solvent, and inject for detection. 3 times S / N=3 and 10 times S / N=10 of the baseline noise are used as the detection limit and quantification limit of the corresponding target compound, respectively. The test results are shown in Table 4 below.

[0072]

[0073]

[0074] It can be seen from the above table that the mass concentration of the detection limit of the relevant impurities is lower than the mass concentration of the quantification limit, and the set mass concentration of the quantification limit is not higher than the control limit of 10μg / mL of the impurities in the system suitability solution, thereby meeting the quantitative analysis requirements of oseltamivir phosphate related impurities.

[0075] Example 4 Precision Test

[0076] The above-prepared oseltamivir phosphate test sample was measured and measured 6 times in parallel. The results are shown in Table 5 below.

[0077]

[0078] The sample was injected six times in parallel, and the RSD of the total amount of impurities was 2.7%, indicating that the method had good precision and met the analytical requirements of different experiments.

[0079] Example 5 Accuracy test:

[0080] 10 mg of the contents of oseltamivir phosphate capsules were weighed, dissolved in a solvent, and impurity reference solutions of low, medium and high mass concentrations were added, and the solution was made up to volume with the solvent and shaken well. Another oseltamivir phosphate solution without impurities was prepared, and the sample was injected according to the chromatographic conditions. The mass fraction of impurities in each solution was determined, and the recovery rate was calculated. The results are shown in Table 6 below.

[0081]

[0082]

[0083] It can be seen from the above table that the spiked recoveries of the above 6 related substances are 97.2% to 99.6%, indicating that the detection method has good accuracy in the detection results of the 6 related substances in oseltamivir phosphate.

[0084] Example 6 Stability Test

[0085] The chromatograms of the system suitability solution, the test solution and the control test solution at 0, 8, 15, 18, 22, 30 and 50 h were compared to investigate the stability of the solutions. The results are shown in Table 7 below.

[0086]

[0087] It can be seen from the above table that within 50 hours, the difference of each impurity is about 0.1%, and the chromatographic peak area of ​​each impurity has no obvious change within 50 hours. At the same time, the test solution and the control test solution can be stored for a long time.

[0088] Through the above methodological validation, the validation results all meet the requirements of the Guiding Principles for Validation of Analytical Methods for Drug Quality Standards in the Appendix IV of the 2020 Edition of the Chinese Pharmacopoeia.

[0089] In summary, by optimizing the liquid chromatography conditions, the method has the characteristics of accuracy, high sensitivity and high separation efficiency, is suitable for the detection of related substances in the production of oseltamivir phosphate capsules, can detect the auxiliary material povidone in the capsule preparation, and analyze and control the quality of the drug; the detection method of the invention has good stability and good separation, and can accurately qualitatively and quantitatively detect the related substances in oseltamivir phosphate, thereby objectively, accurately and comprehensively evaluating its quality, and providing a basis for formulating the quality standard of oseltamivir phosphate.

[0090] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for detecting impurities in oseltamivir phosphate, characterized in that: The following steps are involved: (1) Preparation of test solution: Accurately weigh an appropriate amount of oseltamivir phosphate, dissolve it in solvent, and shake to volume; (2) Preparation of system suitability solution: Accurately weigh appropriate amounts of impurity I, impurity II, impurity III, impurity A, impurity G, povidone K30 and oseltamivir phosphate reference substance, dissolve them in solvent, and shake to volume; (3) Preparation of blank excipient solution: Weigh an appropriate amount of blank excipient of oseltamivir phosphate capsules, dissolve and dilute with solvent; (4) Preparation of control test solution: Accurately weigh an appropriate amount of test solution, dissolve it in solvent and shake to volume; (5) Test method: Take equal amounts of the test solution, system suitability solution, and control test solution and inject them into the liquid chromatograph, and record the chromatogram; Wherein, gradient elution is used for elution; In the gradient elution, the mobile phase includes mobile phase A and mobile phase B. Mobile phase A: phosphate buffer solution, which is an aqueous solution of 15-16.5 g / L potassium dihydrogen phosphate and 10-12.5 g / L anhydrous disodium hydrogen phosphate. Mobile phase B: methanol-acetonitrile. The gradient elution procedure is as follows: From 0 to 10 minutes, the volume fraction of mobile phase A is maintained at 90%, and the volume fraction of mobile phase B is maintained at 10%; from 10 to 25 minutes, the volume fraction of mobile phase A gradually changes to 70%, and the volume fraction of mobile phase B gradually changes to 30%; from 25 to 40 minutes, the volume fraction of mobile phase A gradually changes to 40%, and the volume fraction of mobile phase B gradually changes to 60%; from 40 to 55 minutes, the volume fraction of mobile phase A gradually changes to 90%, and the volume fraction of mobile phase B gradually changes to 10%; from 55 to 60 minutes, the volume fraction of mobile phase A is maintained at 90%, and the volume fraction of mobile phase B is maintained at 10%.

2. The method for detecting impurities of oseltamivir phosphate according to claim 1, characterized in that: The pH value of mobile phase A was adjusted to 6-7 with aqueous ammonia.

3. The method for detecting impurities of oseltamivir phosphate according to claim 1, characterized in that: The volume content of acetonitrile in mobile phase B is 65-75%.

4. The method for detecting impurities of oseltamivir phosphate according to claim 1, characterized in that: The solvent is phosphate buffer solution-methanol-acetonitrile, and the volume ratio of the phosphate buffer solution to methanol-acetonitrile is 55:45-60:

40.

5. The method for detecting impurities of oseltamivir phosphate according to claim 1, characterized in that: The HPLC conditions were set as follows: Chromatographic column: octadecylsilane bonded silica gel as filler; Detection wavelength: 207nm; Flow rate: 0.5~1.0ml / min; Column temperature: 40-45°C; Injection volume: 20μl.

6. The method for detecting impurities of oseltamivir phosphate according to claim 1, characterized in that: The concentration of the test solution is 0.8-1.5 mg / ml.

7. The method for detecting impurities of oseltamivir phosphate according to claim 1, characterized in that: The concentration of the control test solution was 8-15 μg / ml.

8. The method for detecting impurities of oseltamivir phosphate according to claim 1, characterized in that: The system suitability solution was prepared to contain 0.01 mg of impurity I, impurity II, impurity III, impurity A, impurity G, povidone K30 and oseltamivir phosphate reference solution per 1 mL.

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