Method for detecting glucose adduct in oseltamivir phosphate dry suspension

Abstract

The invention belongs to the technical field of oseltamivir phosphate dry suspension detection methods, and particularly relates to a detection method for a glucose adduct in an oseltamivir phosphate dry suspension. The method for detecting the glucose adduct in the oseltamivir phosphate dry suspension comprises the following steps: preparing a system applicability solution, a test solution, a reference solution and a blank auxiliary solution, carrying out liquid chromatography detection on the solutions, recording a chromatogram, and obtaining the content of an impurity V and an impurity VI in the test solution according to a main component external standard method. According to the method for detecting the glucose adduct in the oseltamivir phosphate dry suspension, high performance liquid chromatography is adopted for detecting impurities V and VI, primary degradation products and secondary degradation products can be accurately and effectively detected and calculated, the content of the degradation products can be monitored in real time, meanwhile, an isocratic elution method is adopted, and the content of the glucose adduct in the oseltamivir phosphate dry suspension can be accurately and effectively detected. The load of a high performance liquid chromatograph is reduced, the instrument loss is reduced, and the inspection cost is saved.

Description

Technical Field

[0001] This invention belongs to the technical field of detection methods for oseltamivir phosphate dry suspension, specifically relating to a method for detecting glucose adducts in oseltamivir phosphate dry suspension. Background Technology

[0002] Oseltamivir phosphate, Chinese name: (3R,4R,5S)-4-acetamido-5-amino-3-(1-ethylpropoxy)-1-cyclohexene-1-carboxylic acid ethyl ester phosphate, molecular weight: 410.400, molecular formula: C 16 H 31 N₂O₈P is generally a white or yellowish-white powder, and its molecular structure is shown below: .

[0003] The main function of oseltamivir phosphate is to inhibit viral release and reduce viral transmission. It works by inhibiting neuraminidase, blocking the release of the influenza virus from cells and thus preventing it from causing disease. It has a good therapeutic effect on influenza A and B in adults and children over one year old; it can also be used for the prevention of influenza A and B in adults and adolescents over thirteen years old. Generally, oseltamivir phosphate is more effective when taken within 48 hours of the onset of symptoms in patients with influenza A or B. Under normal circumstances, the dosage of oseltamivir phosphate for adults and adolescents over thirteen years old is approximately 75 mg twice daily for five consecutive days, which can relieve or even cure influenza A or B. Common dosage forms of oseltamivir phosphate include dry suspension, granules, and capsules.

[0004] The formulation of oseltamivir phosphate dry suspension indicates that sorbitol is an excipient in the dry suspension formulation. Sorbitol contains a small amount of residual glucose. Therefore, in the formulation process of oseltamivir phosphate dry suspension, if temperature and humidity conditions are not controlled or avoided in time, oseltamivir phosphate will undergo a Maillard reaction with the residual glucose in the excipient, thereby forming a glucose adduct. This will ultimately affect the purity and quality of oseltamivir phosphate. Therefore, it is of great significance to achieve quantitative analysis and detection of glucose adducts in oseltamivir phosphate dry suspension.

[0005] In oseltamivir phosphate dry suspension formulations, the amino group at position 5 of the oseltamivir phosphate structure undergoes a dehydration reaction with a reducing sugar to generate oseltamivir phosphate glucose adduct impurity V (a primary degradation product). The molecular formula of impurity V is C. 22 H 38 N2O9, with a molecular weight of 474.55, has the following structural formula: .

[0006] The synthetic route for impurity V is shown below: .

[0007] Oseltamivir phosphate glucose adduct impurity V continues to dehydrate under specific temperature and humidity conditions, generating oseltamivir phosphate glucose adduct impurity VI (a secondary degradation product). The molecular formula of impurity VI is C. 22 H 32 N₂O₆, with a molecular weight of 420.50, has the following structural formula: .

[0008] CN117214369A discloses a liquid chromatography method for detecting related substances in oseltamivir phosphate dry suspension. The method involves first preparing a system suitability solution, a test solution, and a blank excipient solution, using a phosphoric acid solution-methanol-acetonitrile solvent. Chromatographic experiments are then performed using an octylsilane-bonded silica column. Mobile phase A is 0.05 mol / L potassium dihydrogen phosphate solution, and mobile phase B is methanol-acetonitrile. A specific gradient elution program is employed, coupled with a UV detector, at a flow rate of 1.2 mL / min, a column temperature of 45℃, and an injection volume of 75 μL. This method can simultaneously separate multiple impurities, including impurities I-V and VII, achieving effective separation of sodium benzoate from impurity III and oseltamivir phosphate from impurity V. It exhibits high sensitivity, good accuracy, excellent repeatability and robustness, and is easy to operate, enabling precise control of product quality. However, it requires a two-phase gradient elution method, with phase A being a pure salt phase, which is highly prone to equipment blockage, resulting in a high failure rate and significant damage to the chromatographic column, leading to high maintenance costs.

[0009] CN119959403A discloses a method for detecting impurities in oseltamivir phosphate dry suspension, employing high-performance liquid chromatography (HPLC) with a Waters Symmetry C8 column. The mobile phase is a phosphate buffer-ethanol-dimethyl sulfoxide (DMSO) solution with a volume ratio of 619-631:245:124-146, a pH of 6.0-6.2, and a concentration of 6.5-7.0 g / L. This method offers high specificity, good precision, linearity, accuracy, limit of quantitation, limit of detection, and robustness, enabling the simultaneous detection of multiple impurities in oseltamivir phosphate dry suspension, which is beneficial for improving drug safety. However, the use of the Waters Symmetry C8 column, a typical octylsilane-bonded silica gel column, can lead to column packing collapse due to the mobile phase ratio, resulting in a short column lifespan, disordered instrument peaks, high wear and tear, and increased testing costs.

[0010] In summary, existing methods for detecting impurities in oseltamivir phosphate dry suspensions, while designed to detect oseltamivir phosphate and related impurities, utilize chromatographic columns with octylsilane-bonded silica gel as the stationary phase (commonly referred to as C8 columns). However, these methods fail to consider the lifespan of such columns and do not systematically examine the selection of the mobile phase. These shortcomings result in a short column lifespan, increasing the frequency of deviations during testing and raising detection costs. Summary of the Invention

[0011] To overcome the problems existing in the prior art, this invention provides a method for detecting glucose adducts in oseltamivir phosphate dry suspension. High performance liquid chromatography (HPLC) is used to detect impurities V and VI, which can accurately and effectively detect and calculate primary and secondary degradation products. The content of degradation products can be monitored in real time. At the same time, the isocratic elution method is used to reduce the load on the HPLC instrument, reduce instrument wear and tear, and save on testing costs.

[0012] The method for detecting glucose adducts in oseltamivir phosphate dry suspension according to the present invention includes the following steps: (1) System suitability solution: Take oseltamivir phosphate, impurity V, and impurity VI reference standards, dilute to the mark with solvent to obtain system suitability solution; (2) Test solution: Take oseltamivir phosphate dry suspension and place it in a volumetric flask. Add solvent and dissolve by sonication. Dilute to the mark and filter to obtain the test solution. (3) Reference solution: Take oseltamivir phosphate reference standard, dilute to the mark with solvent, shake well to obtain reference solution; (4) Blank excipient solution: Take blank excipient, dilute it to the mark with solvent, shake well to obtain blank excipient solution; (5) Perform liquid chromatography on the above solution, record the chromatogram, and obtain the content of impurity V and impurity VI in the test solution according to the external standard method of principal component.

[0013] The solvent is a mixed solution composed of phosphoric acid aqueous solution, methanol and acetonitrile, with a volume ratio of (610-630):(240-250):(130-140), wherein the concentration of the phosphoric acid aqueous solution is 0.002-0.004 mol / L, preferably 0.003 mol / L.

[0014] In step (1), the concentrations of oseltamivir phosphate, impurity V, and impurity VI in the system suitability solution are 0.73-0.77 mg / mL, 3.8-4.2 μg / mL, and 1.3-1.7 μg / mL, respectively.

[0015] In step (2), the concentration of oseltamivir phosphate in the test solution is 73-77 mg / mL.

[0016] In step (3), the concentration of oseltamivir phosphate in the reference solution is 3.6-4.0 μg / mL.

[0017] In step (4), the concentration of blank excipient in the blank excipient solution is 23-27 μg / mL. The blank excipient is other substances contained in the dry suspension besides the active ingredient oseltamivir phosphate. The formulation and proportion of other substances remain unchanged.

[0018] The detection conditions for the liquid chromatography were as follows: a Luna C8 column (4.6 mm × 250 mm, 5 μm) was used; the mobile phase was 0.05 mol / L potassium dihydrogen phosphate buffer-methanol-acetonitrile; the injection volume was 8-12 μL, the flow rate was 1.0-1.4 mL / min, the detection wavelength was 205-209 nm, the column temperature was 48-52℃, and isocratic elution was used.

[0019] In the mobile phase, the volume ratio of 0.05 mol / L potassium dihydrogen phosphate buffer, methanol, and acetonitrile is (610-630):(345-355):(25-35). The pH of the 0.05 mol / L potassium dihydrogen phosphate buffer needs to be adjusted to 5.5-6.5 with 1 mol / L potassium hydroxide solution before use.

[0020] Preferably, the method for detecting glucose adducts in the oseltamivir phosphate dry suspension includes the following steps: (1) System suitability solution: Take oseltamivir phosphate, impurity V and impurity VI reference standards and dilute to the mark with 0.003 mol / L phosphoric acid aqueous solution-methanol-acetonitrile solution (620:245:135). In the system suitability solution, the concentrations of oseltamivir phosphate, impurity V and impurity VI reference standards are 0.75 mg / mL, 4 μg / mL and 1.5 μg / mL, respectively. (2) Test solution: Take oseltamivir phosphate dry suspension and place it in a volumetric flask. Add 0.003 mol / L phosphoric acid aqueous solution-methanol-acetonitrile solution (620:245:135), sonicate to dissolve, and dilute to the mark. After filtration, the test solution is obtained. The concentration of oseltamivir phosphate, the active component in the oseltamivir phosphate dry suspension, is 75 mg / mL. (3) Reference solution: Take oseltamivir phosphate reference standard, dilute to the mark with 0.003 mol / L phosphoric acid aqueous solution-methanol-acetonitrile solution (620:245:135), and shake well. The concentration of oseltamivir phosphate reference standard is 3.8 μg / mL. (4) Blank excipient solution: Other substances in the dry suspension except for the active ingredient oseltamivir phosphate are used as blank excipients. They are diluted to the mark with 0.003 mol / L phosphoric acid aqueous solution-methanol-acetonitrile solution (620:245:135), shaken well, and the concentration of the blank excipient is 25 μg / mL. (5) Perform liquid chromatography on the above solution, record the chromatogram, and obtain the content of impurity V and impurity VI in the test solution according to the external standard method of principal component; wherein, the detection conditions of liquid chromatography are as follows: Luna C8 column (4.6mm×250mm, 5μm), mobile phase: 0.05mol / L potassium dihydrogen phosphate buffer-methanol-acetonitrile (620:350:30); injection volume 10μl, flow rate 1.2mL / min, detection wavelength 207nm; column temperature 50℃, isocratic elution method; the pH value of 0.05mol / L potassium dihydrogen phosphate buffer is adjusted to 6.0 with 1mol / L potassium hydroxide solution before use.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The method for detecting glucose adducts in oseltamivir phosphate dry suspension of the present invention uses high performance liquid chromatography to confirm the presence of primary degradation products and secondary degradation products in oseltamivir phosphate dry suspension, making the analysis results of oseltamivir phosphate dry suspension more accurate and effective.

[0022] (2) The method for detecting glucose adducts in oseltamivir phosphate dry suspension of the present invention can accurately and effectively detect and calculate primary and secondary degradation products, and can monitor the content of degradation products in real time. At the same time, the isocratic elution method reduces the load on the high performance liquid chromatograph, reduces instrument wear and tear, and saves testing costs. Attached Figure Description

[0023] Figure 1 The HPLC chromatogram of the forced degradation experiment obtained by the method provided in this invention; Figure 2 The HPLC chromatogram of the specific system suitability solution obtained in Example 1; Figure 3 The HPLC chromatogram obtained in Example 2 is used to verify the accuracy of the oseltamivir phosphate dry suspension glucose adduct. Figure 4 The HPLC chromatogram of oseltamivir phosphate reference solution was obtained in Example 2; Figure 5 This is a superimposed HPLC chromatogram of the test solution obtained in Example 3; Figure 6 The HPLC chromatogram of the self-made formulation (batch number: 2307006) obtained in Example 4 is shown below. Figure 7 This is the HPLC chromatogram of the reference preparation (batch number: B8086B01) obtained in Example 4. Detailed Implementation

[0024] The technical solution of the present invention will be further described below with reference to the embodiments. The equipment used in the preparation method of the present invention can all be equipment known in the art. Unless otherwise specified, the raw and auxiliary materials used in the present invention are all commercially available. The self-made preparation used is produced by Shandong Xinhua Pharmaceutical Co., Ltd.; the reference preparation is purchased from Roche Pharmaceuticals (Switzerland) Ltd.

[0025] A confirmation experiment was conducted using a forced degradation method to verify the reaction between oseltamivir phosphate and glucose (i.e., residual glucose in the excipient sorbitol), thereby obtaining impurities V and VI. The specific steps included: (1) Take 0.2g of oseltamivir phosphate as the blank group, and take another 0.2g of oseltamivir phosphate and 0.2g of glucose (simulating the residual glucose in the excipient sorbitol) as the experimental group; (2) Dissolve the experimental group and blank group in step (1) respectively in a solvent (0.003mol / L phosphoric acid aqueous solution-methanol-acetonitrile solution (620:245:135)) to prepare test solution, perform liquid chromatography analysis, and record the content of substances; (3) The experimental group and the blank group were placed at 50℃ and 90%RH for 30 days, and the test solution was prepared again. The solution was then analyzed by liquid chromatography. The chromatograms obtained are shown in the figure below. Figure 1 As shown, the content of substances was recorded. The summary table of the content of substances obtained in the above process is shown in Table 1. The relative retention time is the ratio of the elution time of the corresponding impurity peak to the elution time of the main peak oseltamivir phosphate.

[0026] Table 1. Degradation scheme test results

[0027] According to Table 1, the results of the 30-day test of raw materials and glucose in the experimental group (high temperature 50℃, high humidity 90%RH) show that oseltamivir phosphate can react with glucose after long-term storage, thereby generating impurities. The relative retention time is 2.39 min. Impurity VI (secondary degradation product of glucose adduct) was detected, with a detection amount of 0.17%; impurity V (primary degradation product of glucose adduct) was detected at 7.21%.

[0028] Example 1: Specificity Experiment The method for detecting glucose adducts in oseltamivir phosphate dry suspension includes the following steps: (1) Blank excipient solution: Take 2.5g of blank excipient, place it in a 100ml volumetric flask, add 0.003mol / L phosphoric acid aqueous solution-methanol-acetonitrile solution (620:245:135) and mix. After shaking and sonicating, dilute to the mark, shake well and filter to obtain the solution. Impurity I stock solution: Weigh 2.3 mg of impurity I reference standard accurately, place it in a 50 ml volumetric flask, add 0.003 mol / L phosphoric acid aqueous solution-methanol-acetonitrile solution (620:245:135) to dissolve and dilute to the mark, shake well, and use as impurity I stock solution; Impurity II stock solution: Weigh 3 mg of impurity II reference standard accurately, place it in a 100 ml volumetric flask, add 0.003 mol / L phosphoric acid aqueous solution-methanol-acetonitrile solution (620:245:135) to dissolve and dilute to the mark, shake well, and use as impurity II stock solution; Impurity III stock solution: Weigh 6 mg of impurity III reference standard accurately, place it in a 50 ml volumetric flask, add 0.003 mol / L phosphoric acid aqueous solution-methanol-acetonitrile solution (620:245:135) to dissolve and dilute to the mark, shake well, and use as impurity III stock solution; Impurity IV stock solution: Weigh 3 mg of impurity IV reference standard accurately, place it in a 100 ml volumetric flask, add 0.003 mol / L phosphoric acid aqueous solution-methanol-acetonitrile solution (620:245:135) to dissolve and dilute to the mark, shake well, and use as impurity IV stock solution; Impurity V stock solution: Weigh 2 mg of impurity V reference standard accurately, place it in a 25 ml volumetric flask, add 0.003 mol / L phosphoric acid aqueous solution-methanol-acetonitrile solution (620:245:135) to dissolve and dilute to the mark, shake well, and use as impurity V stock solution; Impurity VI stock solution: Weigh 3 mg of impurity VI reference standard accurately, place it in a 100 ml volumetric flask, add 0.003 mol / L phosphoric acid aqueous solution-methanol-acetonitrile solution (620:245:135) to dissolve and dilute to the mark, shake well, and use as impurity VI stock solution; (2) Preparation of specific system suitability solution: Weigh 7.5 mg of oseltamivir phosphate reference standard accurately, place it in a 10 ml volumetric flask, add 1 ml of impurity I stock solution, 1.3 ml of impurity II stock solution, 0.5 ml of impurity III stock solution, 1.3 ml of impurity IV stock solution, 0.5 ml of impurity V stock solution, and 0.5 ml of impurity VI stock solution, dissolve and dilute to the mark with 0.003 mol / L phosphoric acid aqueous solution-methanol-acetonitrile solution (620:245:135), shake well, and the specific system suitability solution is obtained; (3) Perform liquid chromatography analysis on the blank excipient solution from step (1) and the solution obtained from step (2). The liquid chromatography conditions are as follows: Luna C8 column, 4.6 mm × 250 mm, 5 μm; mobile phase: 0.05 mol / L potassium dihydrogen phosphate buffer (adjusted to pH 6.0 with 1 mol / L potassium hydroxide solution) - methanol - acetonitrile (volume ratio 620:350:30); injection volume 10 μL, flow rate 1.2 mL / min, detection wavelength 207 nm; column temperature 50 ℃, isocratic elution. The obtained liquid chromatogram is as follows Figure 2 As shown in Table 2, the results of the specificity test of oseltamivir phosphate and its various impurities are as follows. The resolution between the impurity peaks in the blank excipient and the test sample and the main component oseltamivir phosphate peak is not less than 1.5, and the resolution between oseltamivir phosphate and each impurity is greater than 1.2. The purity of each component peak meets the requirements, that is, the purity of each component is less than its corresponding threshold.

[0029] Table 2 Results of Specificity Test

[0030] Example 2 Accuracy Experiment The method for detecting glucose adducts in oseltamivir phosphate dry suspension includes the following steps: (1) Reference solution: Weigh 3.8 mg of oseltamivir phosphate reference standard accurately, place it in a 100 ml volumetric flask, dissolve and dilute to the mark with 0.003 mol / L phosphoric acid aqueous solution-methanol-acetonitrile solution (620:245:135), shake well, then accurately measure 1 ml, place it in a 10 ml volumetric flask, dissolve and dilute to the mark with solvent, shake well, and the solution is ready; Impurity V stock solution: Weigh 2 mg of impurity V reference standard accurately, place it in a 25 ml volumetric flask, add 0.003 mol / L phosphoric acid aqueous solution-methanol-acetonitrile solution (620:245:135) to dissolve and dilute to the mark, shake well, and use as impurity V stock solution; Impurity VI stock solution: Weigh 3 mg of impurity VI reference standard accurately, place it in a 100 ml volumetric flask, dissolve and dilute to the mark with 0.003 mol / L phosphoric acid aqueous solution-methanol-acetonitrile solution (620:245:135), shake well, and the solution is ready. (2) Test solution: Preparation of suspension: Take 3 bottles of this product (0.36g of active ingredient oseltamivir phosphate / bottle, the weight of 3 bottles is equivalent to 1.08g of active ingredient oseltamivir phosphate), add 55ml of water to each, slowly invert, and shake slowly to the left and right; take 13.5g of suspension (equivalent to 75mg of oseltamivir phosphate), accurately weigh it, put it in a 100ml volumetric flask, measure 5ml of impurity V stock solution and impurity VI stock solution (equivalent to 100% limit), add 0.003mol / L phosphoric acid aqueous solution-methanol-acetonitrile solution, shake and sonicate, and dilute to the mark, shake well, filter, and take the filtrate to obtain the test solution; (3) Perform liquid chromatography analysis on the reference solution obtained in step (1) and the test solution obtained in step (2). The liquid chromatography conditions are as follows: Luna C8 column, 4.6 mm × 250 mm, 5 μm; mobile phase: 0.05 mol / L potassium dihydrogen phosphate buffer (adjusted to pH 6.0 with 1 mol / L potassium hydroxide solution) - methanol - acetonitrile (volume ratio 620:350:30); injection volume 10 μL, flow rate 1.2 mL / min, detection wavelength 207 nm; column temperature 50 ℃, isocratic elution. The obtained liquid chromatogram is as follows Figures 3-4 As shown, a linear experiment was conducted based on the above experiment. With a suspension volume of 13.5 g (background amount), the addition amounts of impurity V and impurity VI stock solutions were adjusted to achieve limits of 20%, 100%, and 150%, respectively. The average recovery rates of primary degradation products (impurity V) and secondary degradation products (impurity VI) in the test solution were found to be in the range of 90%-110%, and the RSD of the recovery rate at this concentration was <10.0%, indicating good accuracy. The results are shown in Tables 3 and 4 below.

[0031] Table 3. Experimental Results of Impurity V Accuracy

[0032] Table 4. Experimental Results of Impurity VI Accuracy

[0033] Example 3 According to the detection method for glucose adducts in oseltamivir phosphate dry suspension described in Examples 1-2, three batches of self-made formulations and one batch of reference formulations that had been stored for a long time (temperature: 25℃±2℃, humidity: 60%±5%RH) were periodically tested to evaluate the tolerability of the method.

[0034] Test solution: Take three batches of self-made formulation and one batch of reference formulation (the method is the same as the preparation method of suspension in Example 2, equivalent to 75 mg of oseltamivir phosphate), place them in a 100 mL volumetric flask, add solvent and sonicate to dissolve, take out, cool and dilute with solvent to the mark, shake well, filter with a 0.22 μm filter membrane, discard 2 mL, and take the filtrate. One injection of each of the above-mentioned test solutions was administered into the liquid chromatograph, and the chromatogram was recorded. The liquid chromatographic conditions were as follows: Luna C8 column, 4.6 mm × 250 mm, 5 μm; mobile phase: 0.05 mol / L potassium dihydrogen phosphate buffer (adjusted to pH 6.0 with 1 mol / L potassium hydroxide solution) - methanol - acetonitrile (volume ratio 620:350:30); injection volume 10 μL, flow rate 1.2 mL / min, detection wavelength 207 nm; column temperature 50 ℃, isocratic elution. Figure 5 The chromatograms of the self-made formulation and the reference formulation after 24 months of storage are shown in Table 5 below. The detection results of each sample from 0 to 24 months are shown in Table 5 below.

[0035] Table 5. Results of Impurity V and Impurity VI determination in Example 3.

[0036] Example 4 According to the detection method for glucose adducts in oseltamivir phosphate dry suspension described in Examples 1-2, three batches of self-made formulations and one batch of reference formulations that had been stored for a long time (temperature: 30℃±2℃, humidity: 65%±5%RH) were periodically tested to evaluate the tolerability of the method.

[0037] Test solution: Take three batches of self-made formulation and one batch of reference formulation (the method is the same as the preparation method of suspension in Example 2, equivalent to 75 mg of oseltamivir phosphate), place them in a 100 mL volumetric flask, add solvent and sonicate to dissolve, take out, cool and dilute with solvent to the mark, shake well, filter with a 0.22 μm filter membrane, discard 2 mL, and take the filtrate. One injection of each of the above-mentioned test solutions was administered into the liquid chromatograph, and the chromatogram was recorded. The liquid chromatographic conditions were as follows: Luna C8 column, 4.6 mm × 250 mm, 5 μm; mobile phase: 0.05 mol / L potassium dihydrogen phosphate buffer (adjusted to pH 6.0 with 1 mol / L potassium hydroxide solution) - methanol - acetonitrile (volume ratio 620:350:30); injection volume 10 μL, flow rate 1.2 mL / min, detection wavelength 207 nm; column temperature 50 ℃, isocratic elution. Figures 6-7 The chromatograms of the self-made formulation 2307006 and the reference formulation B8089B01 after 24 months of storage are shown in Table 6 below. The detection results of each sample from 0 to 24 months are shown in Table 6 below.

[0038] Table 6. Results of Impurity V and Impurity VI determination in Sample Placement, Example 4

[0039] In summary, the method for detecting glucose adduct impurities in oseltamivir phosphate dry suspension described in this invention can effectively detect oseltamivir phosphate and glucose adduct impurities. It is simple, effective, and the components do not interfere with each other, exhibiting good specificity. Secondly, the isocratic elution method in the chromatographic conditions reduces the load on the chromatographic instrument. The selected mobile phase is suitable for the chromatographic column (Luna C8) with octylsilane-bonded silica gel as the stationary phase, reducing the wear and tear on the instrument and column consumables, and saving testing costs. Finally, the accuracy of impurity V and impurity VI obtained under this detection method is good, making the detection results of oseltamivir glucose adduct impurities more accurate and reliable. It can effectively detect and quantify primary degradation impurity V and secondary degradation impurity VI of oseltamivir glucose adduct, and can monitor the content of oseltamivir phosphate glucose adduct degradation impurities in real time, ensuring the clinical efficacy of oseltamivir phosphate dry suspension.

Claims

1. A method for detecting glucose adducts in oseltamivir phosphate dry suspension, characterized in that, Includes the following steps: (1) System suitability solution: Take oseltamivir phosphate, impurity V, and impurity VI reference standards, dilute to the mark with solvent to obtain system suitability solution; (2) Test solution: Take oseltamivir phosphate dry suspension and place it in a volumetric flask. Add solvent and dissolve by sonication. Dilute to the mark and filter to obtain the test solution. (3) Reference solution: Take oseltamivir phosphate reference standard, dilute to the mark with solvent, shake well to obtain reference solution; (4) Blank excipient solution: Take blank excipient, dilute it to the mark with solvent, shake well to obtain blank excipient solution; (5) Perform liquid chromatography on the above solution, record the chromatogram, and obtain the content of impurity V and impurity VI in the test solution according to the external standard method of principal component.

2. The method for detecting glucose adducts in oseltamivir phosphate dry suspension according to claim 1, characterized in that, The solvent is a mixed solution consisting of an aqueous solution of phosphoric acid, methanol, and acetonitrile, with a volume ratio of (610-630):(240-250):(130-140).

3. The method for detecting glucose adducts in oseltamivir phosphate dry suspension according to claim 2, characterized in that, The concentration of the phosphoric acid aqueous solution is 0.002-0.004 mol / L.

4. The method for detecting glucose adducts in oseltamivir phosphate dry suspension according to claim 1, characterized in that, In step (1), the concentrations of oseltamivir phosphate, impurity V, and impurity VI in the system suitability solution are 0.73-0.77 mg / mL, 3.8-4.2 μg / mL, and 1.3-1.7 μg / mL, respectively.

5. The method for detecting glucose adducts in oseltamivir phosphate dry suspension according to claim 1, characterized in that, In step (2), the concentration of oseltamivir phosphate in the test solution is 73-77 mg / mL.

6. The method for detecting glucose adducts in oseltamivir phosphate dry suspension according to claim 1, characterized in that, In step (3), the concentration of oseltamivir phosphate in the reference solution is 3.6-4.0 μg / mL.

7. The method for detecting glucose adducts in oseltamivir phosphate dry suspension according to claim 1, characterized in that, In step (4), the concentration of blank excipient in the blank excipient solution is 23-27 μg / mL.

8. The method for detecting glucose adducts in oseltamivir phosphate dry suspension according to claim 1, characterized in that, The detection conditions for liquid chromatography were as follows: a Luna C8 column was used, and the mobile phase was: 0.05 mol / L potassium dihydrogen phosphate buffer-methanol-acetonitrile; injection volume 8-12 μl, flow rate 1.0-1.4 mL / min, detection wavelength 205-209 nm; column temperature 48-52℃, isocratic elution method.

9. The method for detecting glucose adducts in oseltamivir phosphate dry suspension according to claim 8, characterized in that, In the mobile phase, the volume ratio of 0.05 mol / L potassium dihydrogen phosphate buffer, methanol, and acetonitrile is (610-630):(345-355):(25-35).

10. The method for detecting glucose adducts in oseltamivir phosphate dry suspension according to claim 9, characterized in that, Before use, the pH of 0.05 mol / L potassium dihydrogen phosphate buffer solution needs to be adjusted to 5.5-6.5 with 1 mol / L potassium hydroxide solution.

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