Impurity detection method of oseltamivir phosphate dry suspension

Abstract

The invention provides an impurity detection method of an oseltamivir phosphate dry suspension. According to the impurity detection method of the oseltamivir phosphate dry suspension, high performance liquid chromatography is adopted for detection, and a chromatographic column is Waters Symmetry C8; the mobile phase is phosphate buffer solution-ethanol-dimethyl sulfoxide with the volume ratio of (619-631): (245): (124-146), the pH value of the phosphate buffer solution is 6.0-6.2, and the concentration of the phosphate buffer solution is 6.5-7.0 g / L. The impurity detection method developed for the oseltamivir phosphate dry suspension has the advantages of high specificity, high precision, high linearity, high accuracy, high quantitation limit, high detection limit and high durability, various impurities of the oseltamivir phosphate dry suspension can be well detected at the same time, and the medicine safety can be improved.

Description

Technical Field

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

[0002] Oseltamivir phosphate is an antiviral drug widely used for influenza virus infection, especially in the prevention and treatment of influenza A and B viruses. Oseltamivir phosphate dry suspension, indications include the treatment of influenza A and B in adults and children 2 weeks and older (oseltamivir phosphate can effectively treat influenza A and B, but there is not much clinical application data for influenza B). Patients should use it within 48 hours of the first onset of symptoms. It is used for the prevention of influenza A and B in patients aged 1 year and above.

[0003] Oseltamivir phosphate dry suspension is gradually and widely used in the treatment of influenza virus infection, and the quality and safety of the drug are extremely important. Impurities in oseltamivir phosphate dry suspension not only affect the efficacy of the drug, but may also cause adverse reactions and endanger the health of patients. Therefore, further development and optimization of impurity detection methods for oseltamivir phosphate dry suspension is crucial to ensure product quality and patient safety. Summary of the invention

[0004] In view of this, the present invention provides a method for detecting impurities in an oseltamivir phosphate dry suspension.

[0005] The technical scheme of the present invention is achieved as follows: a method for detecting impurities of an oseltamivir phosphate dry suspension, using high performance liquid chromatography for detection, a chromatographic column: Waters Symmetry C8; a mobile phase: a phosphate buffer-ethanol-dimethyl sulfoxide (DMSO) with a volume ratio of 619-631:245:124-146, a pH value of the phosphate buffer of 6.0-6.2, and a concentration of the phosphate buffer of 6.5-7.0 g / L.

[0006] Furthermore, the preparation method of the phosphate buffer is as follows: potassium dihydrogen phosphate is weighed, dissolved and diluted with water, the pH value is adjusted to 6.0-6.2 with 0.8-1.2 mol / L potassium hydroxide solution, and filtered to obtain a phosphate buffer with a concentration of 6.5-7.0 g / L.

[0007] Furthermore, the injection volume is 14-16 μl.

[0008] Furthermore, the chromatographic column has a specification of 4.6 mm×250 mm, 5 μm or a chromatographic column with equivalent performance.

[0009] Furthermore, detector: ultraviolet detector, detection wavelength: 205-209 nm.

[0010] Furthermore, flow rate: 1.1-1.3 ml / min; column temperature: 48-52°C.

[0011] Further, the system suitability solution was prepared by weighing impurity A reference substance, impurity C reference substance, impurity D reference substance, impurity E reference substance, impurity G reference substance, impurity Z reference substance, oseltamivir sugar adduct 1 reference substance and oseltamivir phosphate reference substance, placing them in a volumetric flask, adding diluent, sonicating to dissolve, and diluting with diluent, the concentration of each reference substance was 1-3 μg / ml.

[0012] Furthermore, the reference solution is prepared by taking the oseltamivir phosphate reference substance, accurately weighing it, placing it in a volumetric flask, adding a diluent, sonicating it to dissolve it, and diluting it with the diluent to prepare an oseltamivir phosphate concentration of 4 to 6 μg / ml.

[0013] Further, the test solution is prepared: take the product, pour out the contents, mix evenly, take the powder, accurately weigh, put it in a volumetric bottle, add diluent, sonicate, shake while sonicating, cool, dilute to the scale with diluent, shake well, take the solution and centrifuge it at 9000-11000rpm for 8-12min, take the supernatant and filter it, and prepare oseltamivir with a concentration of 0.7-0.8mg / ml.

[0014] Furthermore, the diluent is a phosphoric acid solution-ethanol-dimethyl sulfoxide in a volume ratio of 619-631:245:124-146, and the concentration of the phosphoric acid solution is 0.35-0.4 g / L.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] (1) The impurity detection method developed for oseltamivir phosphate dry suspension of the present invention has strong specificity, good precision, linearity, accuracy, quantitative limit and detection limit, and durability, and can better detect multiple impurities of oseltamivir phosphate dry suspension at the same time, which is beneficial to improving drug safety.

[0017] (2) The detection method of the present invention requires a relatively small amount of impurity reference substance, which is beneficial for reducing the cost of drug detection while ensuring the accuracy of detection. DETAILED DESCRIPTION

[0018] In order to better understand the technical content of the present invention, specific embodiments are provided below to further illustrate the present invention.

[0019] Unless otherwise specified, the experimental methods used in the embodiments of the present invention are all conventional methods.

[0020] Unless otherwise specified, the materials, reagents, etc. used in the embodiments of the present invention can be obtained from commercial sources.

[0021] Impurity information

[0022]

[0023] Example 1 Related Substance Analysis Method 1 Solution Preparation

[0024] 1 mol / L potassium hydroxide solution: Weigh about 5.6 g of potassium hydroxide, dissolve it in water and dilute it to 100 ml.

[0025] pH 6.1 phosphate buffer: weigh about 6.8 g of potassium dihydrogen phosphate, dissolve it in water and dilute it to 1 L, adjust the pH value to 6.1±0.1 with 1 mol / L potassium hydroxide solution, and filter it to obtain.

[0026] Mobile phase: pH 6.1 phosphate buffer-ethanol-dimethyl sulfoxide (620:245:135, v / v / v), mixed evenly according to the proportion, and degassed by ultrasonication.

[0027] 0.38g / L phosphoric acid solution: weigh 0.45g of 85%wt phosphoric acid, dilute to 1L with water, and mix well.

[0028] Diluent: 0.38 g / L phosphoric acid solution-ethanol-dimethyl sulfoxide (620:245:135, v / v / v), mix evenly according to the proportion, and degas by ultrasonication.

[0029] Preparation of system suitability solution (SST): Weigh about 2 mg of impurities A, C, D, E, G, Z, oseltamivir sugar adduct 1 and oseltamivir phosphate reference substance, place in a 100 ml volumetric flask, add diluent, dissolve by ultrasound, and dilute to scale with diluent. Accurately measure 2 ml, place in a 20 ml volumetric flask, and dilute to scale with diluent.

[0030] Preparation of reference solution: Take about 20 mg of oseltamivir phosphate reference substance, accurately weigh it, place it in a 200 ml volumetric flask, add an appropriate amount of diluent, dissolve it by ultrasound, dilute it to the scale with diluent, and shake it well. Accurately measure 1 ml, place it in a 20 ml volumetric flask, dilute it to the scale with diluent, and shake it well. (The concentration of oseltamivir is about 3.8 μg / ml, and the conversion coefficient of oseltamivir phosphate to oseltamivir is 0.761).

[0031] Preparation of test solution: Take 2 bottles of this product, pour out the contents, mix evenly, take about 1270 mg of powder (equivalent to 38 mg of oseltamivir), accurately weigh, put into a 50 ml volumetric bottle, add appropriate amount of diluent, sonicate for about 5 minutes, shake while sonicating, cool, and dilute to the scale with diluent.

[0032] Shake well, take an appropriate amount of solution and centrifuge (10000rpm, 10min), take the supernatant and filter it with a 0.45μm PVDF filter membrane, discard 2ml of the initial filtrate, and take the subsequent filtrate as the test solution. (The test solution should be prepared within 1 hour after opening the bottle and mixing, and the concentration of oseltamivir is about 0.75mg / ml).

[0033] 2 Chromatographic system

[0034] Chromatographic column: Waters Symmetry C8 4.6mm×250mm, 5μm or chromatographic column with equivalent performance; detector: UV detector (wavelength: 207nm); column temperature: 50℃; flow rate: 1.2ml / min; injection volume: 15μl; running time: 45 minutes (25 minutes for reference solution).

[0035] 3 System suitability requirements:

[0036] In the system suitability solution chromatogram, the order of peaks is impurity A, impurity C, impurity D, impurity E, oseltamivir, oseltamivir sugar adduct 1, impurity G and impurity Z. The separation degree between impurity A and impurity C should be no less than 1.0.

[0037] The reference solution was injected 5 times continuously and the chromatogram was recorded. The RSD of the oseltamivir peak area was ≤5.0%.

[0038] After the system suitability is passed, each test solution is sampled once, and the control solution (reference solution) is sampled once every 12 hours and at the end of the sequence, and the chromatogram is recorded. The peak area of ​​the control solution is compared with the average peak area of ​​oseltamivir in the reference solution, and the recovery rate should be between 90.0% and 110.0%.

[0039] Calculation formula:

[0040] Impurity content (%) = (impurity peak area × reference sample weight × reference content × test sample dilution factor × 0.761 × F) ÷ (reference peak area × test sample weight × reference sample dilution factor × 3%)

[0041] Where:

[0042] 3%: Oseltamivir in prescriptions

[0043] 0.761: Conversion coefficient of oseltamivir phosphate to oseltamivir;

[0044] F: impurity correction factor, including impurity C: 0.74; impurity D: 0.37; impurity E: 1.1; impurity G: 1.0; impurity Z: 1.1.

[0045] Result determination: In the chromatogram of the test solution [minus the excipient peak before the retention time of about 0.16 relative to the main peak], if there is an impurity peak, calculate it with oseltamivir reference substance according to the external standard method, and the impurity limit is as follows:

[0046] name limit Impurity C 0.5％ Impurity D 0.2％ Impurity E 0.2％ Impurity G 0.5％ Impurity Z 0.6％ Other single impurities 0.2％ Total amount of other impurities 0.5％ Total impurities (except oseltamivir sugar adduct 1) 2.0％ Reporting Limits 0.05％

[0047] Note: Impurity A is controlled as an unknown impurity and is only used for system suitability tests; Oseltamivir sugar adduct 1 is not controlled under the related substances item, only integrated and located, and the impurity content is not calculated.

[0048] The above analysis method was verified and the results are as follows:

[0049] 1. Exclusivity

[0050] There is no interference between the peak positions of oseltamivir and impurities C, D, E, and G in the diluent and blank excipient spectra; in the selective solution, the separation between the known impurity peaks is as follows: the separation between impurity C and impurity D is 27.76, the separation between impurity D and impurity E is 2.93, the separation between impurity E and oseltamivir is 16.56, the separation between oseltamivir and GA-1 is 3.20, and the separation between GA-1 and impurity G is 9.45. Therefore, the known impurity peaks and the known impurity peaks and oseltamivir peaks can be clearly separated (R>1.5), with good specificity.

[0051] 2. Precision (repeatability)

[0052] The average contents of impurity C, impurity D and impurity G in the six replicate solutions prepared in parallel were 0.47%, 0.22% and 0.47% respectively, and within the range of 0.2%≤x<0.5%, the RSDs were 0.87%, 2.55% and 1.35% respectively, all less than 10.0%; the average content of impurity E was 0.17%, and within the range of 0.1%≤X<0.2%, the RSD was 4.39%<20.0%, indicating good repeatability.

[0053] 3. Precision (intermediate precision)

[0054] The average contents of impurity C, impurity D and impurity G in the 6 parallel prepared intermediate precision solutions were 0.49%, 0.21% and 0.46%, respectively. In the range of 0.2%≤x<0.5%, the RSDs were 0.84%, 0.00% and 1.13%, respectively, all <15.0%. The average content of impurity E was 0.17%. In the range of 0.1%≤x<0.2%, the RSD was 2.43%<30.0%, all meeting the validation acceptance criteria. The RSDs of impurity C, D and G in the 12 solutions for repeatability and intermediate precision were 1.99%, 3.55% and 1.92%, respectively, all <15.0%. The ERSD of impurity was 3.55%<30.0%, all meeting the validation acceptance criteria, and the intermediate precision was good.

[0055] 4. Linear

[0056] In the concentration range of 0.39248μg / ml to 5.88715μg / ml, the concentration of oseltamivir showed a good linear relationship with the peak area, with a correlation coefficient of 0.9922>0.990, and the Y-axis intercept was equivalent to 7.29%<10% of the peak area of ​​0.5% concentration; in the concentration range of 0.38401μg / ml to 5.76009μg / ml, the concentration of impurity C showed a good linear relationship with the peak area, with a correlation coefficient of 1.0000>0.990, and the Y-axis intercept was equivalent to 0.24%<10% of the peak area of ​​the limit concentration; in the concentration range of 0.39310μg / ml to 2.35862μg / ml, the concentration of impurity D showed a good linear relationship with the peak area, with a correlation coefficient of 1.0000>0.990, and the Y-axis intercept was equivalent to 0.24%<10% of the peak area of ​​the limit concentration. The coefficient is 0.9995>0.990, and the Y-axis intercept is equivalent to 3.37%<25% of the peak area of ​​the limit concentration; the concentration of impurity E is in the range of 0.38399μg / ml~2.30391μg / ml, and the concentration and peak area show a good linear relationship, the correlation coefficient is 0.9997>0.990, and the Y-axis intercept is equivalent to 2.09%<25% of the peak area of ​​the limit concentration; the concentration of impurity G is in the range of 0.37819μg / ml~5.67289μg / ml, and the concentration and peak area show a good linear relationship, the correlation coefficient is 0.9996>0.990, and the Y-axis intercept is equivalent to 1.72%<10% of the peak area of ​​the limit concentration; all meet the verification acceptance criteria. The correction factors of impurities C, D, E, and G are 0.74, 0.37, 1.1, and 1.0, respectively. In summary, the test results show good linearity.

[0057] 5. Limit of quantification and detection limit

[0058] The linear stock solution was diluted step by step to make the S / N ratio between oseltamivir and each impurity peak ≈ 10, which was the quantification limit. The quantification limit solution was diluted to make the S / N ratio between oseltamivir and each impurity peak ≈ 3, which was the detection limit. The peak area RSD of the quantification limit solution was < 20%.

[0059] The quantitative limits of impurity C, impurity D, impurity E, impurity G and oseltamivir were 0.019 μg / ml, 0.075 μg / ml, 0.19 μg / ml, 0.38 μg / ml and 0.23 μg / ml, respectively;

[0060] The detection limit concentrations of impurity C, impurity D, impurity E, impurity G and oseltamivir were 0.0063 μg / ml, 0.025 μg / ml, 0.063 μg / ml, 0.13 μg / ml and 0.077 μg / ml, respectively.

[0061] In summary, the test results showed good limits of quantification and detection.

[0062] 6. Accuracy

[0063] R-1: The content of each known impurity is <0.2%, the recovery is in the range of 98.71% to 115.60%, and the RSD is in the range of 0.50% to 6.71%, all of which meet the validation acceptance criteria;

[0064] R-2: The contents of impurity C and impurity G were within the range of 0.5% ≤ X < 5%, the recovery rates were within the range of 99.07% to 101.42%, and the RSDs were 0.15% and 0.88%, respectively; the contents of impurity D and impurity E were within the range of 0.2% ≤ X < 0.5%, the recovery rates were within the range of 100.85% to 107.15%, and the RSDs were 0.76% and 0.97%, respectively, all meeting the validation acceptance criteria;

[0065] R-3: The contents of impurity C and impurity G are in the range of 0.5% ≤ x < 5%, the recoveries are in the range of 99.62% to 101.92%, and the RSDs are 0.10% and 1.00%, respectively; the contents of impurity D and impurity E are in the range of 0.2% ≤ x < 0.5%, the recoveries are in the range of 100.50% to 109.35%, and the RSDs are 1.46% and 0.37%, respectively, all meeting the validation acceptance criteria.

[0066] The above R-1, R-2 and R-3 are respectively low, medium and high concentrations.

[0067] In summary, the test results show good accuracy.

[0068] 7. Durability

[0069] When changing the flow rate (1.2±0.1ml / min), column temperature (50±2℃), pH value of buffer salt in mobile phase (pH6.1±0.1), organic phase ratio in mobile phase (dimethyl sulfoxide±11%) and using different chromatographic columns, the contents of impurities C, D and G in the test solution and the corresponding impurity contents under the target conditions were within the range of 0.2%≤x<0.5%, and the RSDs were all less than 15.0%, which met the validation acceptance criteria; the contents of impurity E in the test solution and the corresponding impurity contents under the target conditions were within the range of 0.1%≤x<0.2%, and the RSDs were all less than 30.0%, which met the validation acceptance criteria. This indicates that when the chromatographic conditions change within a certain range, there is no significant effect on the determination results of impurities C, D, E and G, indicating that the chromatographic conditions of this method have good durability.

[0070] In summary, the impurity detection method developed for oseltamivir phosphate dry suspension of the present invention has strong specificity, good precision, linearity, accuracy, quantitative limit and detection limit and durability, and can better detect multiple impurities of oseltamivir phosphate dry suspension at the same time, which is beneficial to improving drug safety.

[0071] Comparative Example 1

[0072] The difference from Example 1 is that the amounts of ethanol and dimethyl sulfoxide in the mobile phase were adjusted. Specifically, the mobile phase was pH 6.1 phosphate buffer-ethanol-dimethyl sulfoxide (620:135:245, v / v / v). The results showed that the oseltamivir peak interfered with the adjacent impurity peak.

[0073] Comparative Example 2

[0074] The difference from Example 1 is that the pH value of the phosphate buffer in the mobile phase is adjusted. Specifically, the mobile phase is pH 7.0 phosphate buffer-ethanol-dimethyl sulfoxide (620:245:135, v / v / v). The results show that there is interference between adjacent impurity peaks.

[0075] Comparative Example 3

[0076] The difference from Example 1 is that dimethyl sulfoxide in the mobile phase is replaced by acetonitrile. The results show that the separation between the impurity peak and the main peak is significantly reduced, and overlapping peaks appear.

[0077] 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 principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for detecting impurities in oseltamivir phosphate dry suspension, characterized in that: High performance liquid chromatography was used for detection, with a chromatographic column of C8; the mobile phase was phosphate buffer-ethanol-dimethyl sulfoxide in a volume ratio of 619-631:245:124-146, the pH value of the phosphate buffer was 6.0-6.2, and the concentration of the phosphate buffer was 6.5-7.0 g / L.

2. The impurity detection method for an oseltamivir phosphate dry suspension according to claim 1, characterized in that: The preparation method of the phosphate buffer solution comprises the following steps: weighing potassium dihydrogen phosphate, adding water to dissolve and dilute, adjusting the pH value to 6.0-6.2 with 0.8-1.2 mol / L potassium hydroxide solution, filtering, and obtaining a phosphate buffer solution with a concentration of 6.5-7.0 g / L.

3. The impurity detection method for an oseltamivir phosphate dry suspension according to claim 1, characterized in that: Injection volume: 14-16 μl.

4. The impurity detection method for an oseltamivir phosphate dry suspension according to claim 1, characterized in that: The chromatographic column is Waters Symmetry C8; the chromatographic column specification is 4.6mm×250mm, 5μm or a chromatographic column with equivalent performance.

5. The impurity detection method for an oseltamivir phosphate dry suspension according to claim 1, characterized in that: Detector: UV detector, detection wavelength: 205~209nm.

6. The impurity detection method for an oseltamivir phosphate dry suspension according to claim 1, characterized in that: Flow rate: 1.1~1.3ml / min; column temperature: 48~52℃.

7. The impurity detection method for an oseltamivir phosphate dry suspension according to claim 1, characterized in that: Preparation of system suitability solution: Weigh impurity A reference substance, impurity C reference substance, impurity D reference substance, impurity E reference substance, impurity G reference substance, impurity Z reference substance, oseltamivir sugar adduct 1 reference substance and oseltamivir phosphate reference substance, place them in a volumetric flask, add diluent, sonicate to dissolve, and dilute with diluent. The concentration of each reference substance is 1-3 μg / ml.

8. The impurity detection method for an oseltamivir phosphate dry suspension according to claim 1, characterized in that: Preparation of reference solution: Take oseltamivir phosphate reference substance, accurately weigh it, place it in a volumetric flask, add diluent, dissolve it by ultrasonication, dilute it with diluent, and prepare oseltamivir phosphate with a concentration of 4-6μg / ml.

9. The impurity detection method for an oseltamivir phosphate dry suspension according to claim 1, characterized in that: Preparation of test solution: Take the product, pour out the contents, mix evenly, take the powder, accurately weigh, put it in a volumetric flask, add diluent, sonicate, shake while sonicating, cool, dilute to the scale with diluent, shake well, take the solution and centrifuge it at 9000-11000rpm for 8-12min, take the supernatant and filter it, and prepare oseltamivir with a concentration of 0.7-0.8mg / ml.

10. The impurity detection method of an oseltamivir phosphate dry suspension according to claims 7-9, characterized in that: The diluent is a phosphoric acid solution-ethanol-dimethyl sulfoxide in a volume ratio of 619-631:245:124-146, and the concentration of the phosphoric acid solution is 0.35-0.4 g / L.

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