High performance liquid chromatography method for detecting related substances in oseltamivir phosphate product

Abstract

The invention discloses a high performance liquid chromatography method for detecting related substances in an oseltamivir phosphate product, and the chromatographic conditions are as follows: an octyl silane bonded silica gel column is used as a chromatographic column, 0.05 mol / L monopotassium phosphate buffer solution-methanol-acetonitrile is used as a mobile phase, the volume ratio is 620: 245: 135-700: 245: 135, the pH value of the 0.05 mol / L monopotassium phosphate buffer solution is regulated to 5.5-5.9 by using a 1mol / L potassium hydroxide solution, and the column temperature is controlled to be 30-60 DEG C; the flow velocity is 1.1-1.3 ml / min, the column temperature is 48-52 DEG C, the detection wavelength is 207 nm, and the sample injection volume is 15-30 [mu] l. The chromatographic method provided by the invention can effectively separate and detect oseltamivir phosphate and related substances in an oseltamivir phosphate product, is not interfered by auxiliary material peaks, can fully meet the requirements of detection of related substances and determination of decomposed products, well controls specific impurities and non-specific impurities in a sample, and improves the detection accuracy of oseltamivir phosphate. Therefore, the product quality of the oseltamivir phosphate capsule is controlled, and a guarantee is provided for synthesis and preparation process optimization.

Description

Technical Field

[0001] This invention relates to a high-performance liquid chromatography method for the detection of related substances in oseltamivir phosphate products, belonging to the field of pharmaceutical analysis and detection. Background Technology

[0002] Oseltamivir phosphate is an antiviral drug primarily used to treat influenza A and B in children over 1 year of age and adults. Its molecular formula is C1. 16 H 28 N₂O₄·H₃PO₄ has the following chemical structural formula:

[0003]

[0004] Oseltamivir phosphate capsules are a type of formulation of this class of drugs. Currently, the related substances methods listed in pharmacopoeias of various countries suffer from poor resolution or column instability. This article, by preparing the test sample with different solvents, determined a solvent that could make the test sample solution relatively stable. By investigating different chromatographic columns, mobile phase ratios, and pH values, good separation of impurities was achieved, column wear was reduced, and the quality control of oseltamivir phosphate capsules was well ensured. Summary of the Invention

[0005] The purpose of this invention is to provide a high-performance liquid chromatography method for determining related substances in oseltamivir phosphate products, which can be used for the quality control of oseltamivir phosphate products, especially oseltamivir phosphate capsules.

[0006] The technical solution of this invention is as follows:

[0007] A high-performance liquid chromatography (HPLC) method for the simultaneous determination of impurities I, II, III, IV, VII, K, and L in oseltamivir phosphate products is characterized by the following steps:

[0008] (1) Preparation of system suitability solution and reference solution

[0009] The system suitability solution and the reference solution were subjected to a system suitability test under the following chromatographic conditions to evaluate the separation effect and sensitivity:

[0010] System suitability solution: Take appropriate amounts of impurity I, impurity II, impurity III, impurity IV, impurity VII, impurity K and impurity L reference standards, dissolve and dilute them with solvent to prepare impurity reference standard stock solutions containing 15 μg / ml each of impurity I, impurity II, impurity IV, impurity VII, impurity K and impurity L, and 38 μg / ml of impurity III, for later use; Separately, take about 50 mg of povidone K30, place it in a 100 ml volumetric flask, dissolve and dilute it with solvent to prepare a solution containing 0.5 mg / ml, centrifuge, and take the supernatant as the povidone K30 stock solution for later use; Accurately measure 1 ml each of the above impurity reference standard stock solution and povidone K30 stock solution, place them in the same 10 ml volumetric flask, accurately weigh about 10 mg of oseltamivir phosphate reference standard (equivalent to about 7.61 mg of oseltamivir), dissolve it in the above 10 ml volumetric flask, dilute to the mark with solvent, and shake well to obtain the final solution;

[0011] Reference solution: Accurately weigh oseltamivir phosphate reference standard, and quantitatively dilute it with solvent to prepare a solution containing 1 μg / ml of oseltamivir phosphate (equivalent to approximately 0.761 μg of oseltamivir);

[0012] Accurately measure 30 μl of the system suitability solution and the reference solution, inject them into the liquid chromatograph, record the chromatograms, and calibrate the detection to ensure that the resolution between excipients and impurities, between oseltamivir main peak and impurities, and between impurities in the system suitability solution chromatogram is greater than 1.5; and that the signal-to-noise ratio of the oseltamivir peak in the reference solution chromatogram is not less than 10.

[0013] Preferably, the solvent is a 0.003 mol / L phosphoric acid aqueous solution-methanol-acetonitrile with a volume ratio of 670:245:135.

[0014] (2) Preparation of test solution

[0015] Take an appropriate amount of oseltamivir phosphate sample, accurately weigh it, add an appropriate amount of solvent to dissolve it, and prepare a test solution containing 1 mg / ml of oseltamivir phosphate.

[0016] (3) Chromatographic conditions

[0017] The chromatographic column was an octylsilane-bonded silica column, with a mobile phase of 0.05 mol / L potassium dihydrogen phosphate buffer-methanol-acetonitrile at a volume ratio of 620:245:135 to 700:245:135. The pH of the 0.05 mol / L potassium dihydrogen phosphate buffer was adjusted to 5.5 to 5.9 with 1 mol / L potassium hydroxide solution. The flow rate was 1.1 to 1.3 ml / min, the column temperature was 48 to 52 °C, the detection wavelength was 207 nm, and the injection volume was 15 to 30 μl.

[0018] The chromatographic column has a size of 4.6 mm × 250 mm and a packing particle size of 5 μm.

[0019] Preferably, the chromatographic column is a Waters Xbridge C8 column.

[0020] Preferably, the mobile phase is 0.05 mol / L potassium dihydrogen phosphate buffer-methanol-acetonitrile, with a volume ratio of 670:245:135.

[0021] Preferably, the pH of the above potassium dihydrogen phosphate buffer solution is adjusted to 5.7 using a 1 mol / L potassium hydroxide solution.

[0022] Preferably, the injection volume is 30 μl.

[0023] Preferably, the sample to be tested is the contents of oseltamivir phosphate capsules.

[0024] (4) Determine the content of related substances in the sample by high performance liquid chromatography.

[0025] Inject the reference solution into the liquid chromatograph calibrated in step (1) above, adjust the detection sensitivity, then inject the test solution into the liquid chromatograph, perform high performance liquid chromatography determination under the chromatographic conditions described in step (3), and record the chromatogram.

[0026] (5) Calculate the content of each impurity in the sample to be tested.

[0027] Calculated by peak area using the principal component external standard method.

[0028] A qualified oseltamivir phosphate product shall contain no more than 0.2% of the labeled amount of oseltamivir for impurity I, no more than 0.2% for impurity II (with a correction factor of 0.4 relative to oseltamivir) for impurity II, no more than 0.5% for impurity III, no more than 0.2% for any other individual impurity (including impurities IV and VII), no more than 0.2% for any other individual impurity, no more than 0.5% for the total amount of other impurities, and no more than 1.5% for the total amount of impurities.

[0029] This invention utilizes high-performance liquid chromatography (HPLC) to simultaneously determine impurities I, II, III, IV, VII, K, and L in oseltamivir phosphate capsules. Through exploration of chromatographic conditions, the optimal conditions for effectively separating oseltamivir phosphate from its impurities were determined. This method achieves the simultaneous determination of the contents of impurities I, II, III, IV, VII, K, and L in oseltamivir phosphate, exhibiting good specificity, excellent resolution, and high sensitivity. It is of significant importance for product quality control and stability sample detection.

[0030] In this invention, "related substances" refers to impurities (including intermediates, byproducts, and other organic impurities) that may be generated or degraded during drug preparation and storage. Specifically, the related substances in the oseltamivir phosphate capsules of this invention include: impurity I, impurity II, impurity III, impurity IV, impurity VII, impurity K, and impurity L.

[0031] This invention provides a method applicable to the detection of oseltamivir phosphate products, particularly finished oseltamivir phosphate capsules. The method is accurate, simple to operate, and rapid, fully meeting the requirements for related substance testing and decomposition product determination. It effectively controls specific and non-specific impurities in the sample, ensuring product quality and demonstrating strong practicality in actual quality control work. Attached Figure Description

[0032] Figure 1 This is the chromatogram of the spiked test solution on the Waters Symmetry C8 column in Example 2.

[0033] Figure 2 This is the chromatogram of the spiked test solution on the Agilent ZORBAX Eclipse Plus C8 column in Example 2.

[0034] Figure 3 The image shows the chromatogram of the spiked test solution on the Agilent ZORBAX Eclipse XDB C8 column used in Example 2.

[0035] Figure 4 The chromatograms are of the spiked test solution from the Waters XBridge C8 column in Example 2 and the spiked test solution from the mobile phase ratio of 620:245:135 described in Example 3.

[0036] Figure 5 The chromatogram is of the spiked test solution with a mobile phase ratio of 650:245:135 in Example 3.

[0037] Figure 6 The chromatogram is of the spiked test solution with a mobile phase ratio of 670:245:135 in Example 3.

[0038] Figure 7 The chromatogram is of the spiked test solution with a mobile phase ratio of 700:245:135 in Example 3.

[0039] Figure 8 This is the chromatogram of the specific test solution with mobile phase pH 6.0 in Example 5.

[0040] Figure 9 This is the chromatogram of the specific test solution with mobile phase pH 5.7 in Example 5.

[0041] Figure 10 This is the chromatogram of the specific test solution with mobile phase pH 5.9 in Example 5.

[0042] Figure 11 This is the chromatogram of the specific test solution with a mobile phase pH of 5.5 in Example 5. Detailed Implementation

[0043] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0044] The reference standards, samples, reagents, and instruments used in the following specific implementation methods are as follows:

[0045] Instrument: Agilent 1260 series high performance liquid chromatograph;

[0046] Reagents: Potassium dihydrogen phosphate (chromatographic grade, Aladdin), potassium hydroxide (analytical grade, Aladdin), methanol, acetonitrile (chromatographic grade, CINC), phosphoric acid (chromatographic grade, Aladdin);

[0047] Chromatographic column: Waters Xbridge C8 column, 4.6 mm × 250 mm, 5 μm;

[0048] Sample: Oseltamivir phosphate capsules, self-developed formulation, all raw materials are commercially available: Contents: Oseltamivir phosphate, pregelatinized starch, povidone K30, croscarmellose sodium, stearyl fumarate, talc; Capsule shell: Gelatin, yellow iron oxide, red iron oxide, black iron oxide, titanium dioxide; Raw materials referenced from Russian Drugs and Medicines Agency - tammifu - public information; Production process: wet granulation, drying, sizing, total mixing and capsule filling, referencing "European Medicines Agency - tamiflu - 30mg - scientific discussion".

[0049] Excipient: Povidone K30 (Source: BASF New Materials Co., Ltd.) Table 1 Impurity Information

[0050]

[0051] [Example 1] Solvent Screening

[0052] 1.1 Preparation of test solution

[0053] Accurately weigh approximately 58 mg of the contents of oseltamivir phosphate capsules and place them in different 20 ml volumetric flasks. Add an appropriate amount of the following solvent to each flask and sonicate to dissolve. Dilute to the mark with the solvent and shake well to obtain the final product.

[0054] Solvent (1): Water-methanol-acetonitrile, volume ratio 620:245:135

[0055] Solvent (2): 0.003 mol / L phosphoric acid aqueous solution - methanol - acetonitrile, volume ratio 620:245:135

[0056] Solvent (3): Mobile phase, namely 0.05 mol / L potassium dihydrogen phosphate buffer - methanol - acetonitrile, volume ratio 620:245:135, the pH of 0.05 mol / L potassium dihydrogen phosphate buffer was adjusted to 6.0 with 1 mol / L potassium hydroxide solution.

[0057] 1.2 Measurement

[0058] Take 15 μl of each of the above three solvents to prepare the test solution, and collect and measure the samples at room temperature at 0 h and 22 h according to the following chromatographic conditions. The results are shown in Table 2.

[0059] Chromatographic conditions: The column was an octylsilane-bonded silica column, and the mobile phase was 0.05 mol / L potassium dihydrogen phosphate buffer-methanol-acetonitrile at a volume ratio of 620:245:135. The pH of the 0.05 mol / L potassium dihydrogen phosphate buffer was adjusted to 6.0 with 1 mol / L potassium hydroxide solution. The flow rate was 1.2 ml / min, the column temperature was 50 ℃, and the detection wavelength was 207 nm.

[0060] Table 2 Solvent Screening

[0061]

[0062] Experimental results show that when the solvent is water-methanol-acetonitrile (volume ratio 620:245:135) and the mobile phase, the percentage of the main peak area of ​​oseltamivir decreases and the content of impurity I increases with the increase of standing time, indicating that oseltamivir is unstable and degrades under these two solvent conditions. When the solvent is 0.003 mol / L phosphoric acid aqueous solution-methanol-acetonitrile (volume ratio 620:245:135), impurity I remains basically unchanged within 22 hours, and oseltamivir is relatively stable. Therefore, 0.003 mol / L phosphoric acid aqueous solution-methanol-acetonitrile with a volume ratio of 620:245:135 is temporarily selected as the solvent.

[0063] [Example 2] Chromatographic column screening

[0064] 2.1 Preparation of spiked test solution

[0065] Take appropriate amounts of reference standards for impurities I, II, III, IV, and VII, dissolve and dilute them in a solvent (0.003 mol / L phosphoric acid aqueous solution-methanol-acetonitrile, volume ratio 620:245:135) to prepare a stock solution of impurity reference standards containing 13.2 μg / ml of impurity I, 3.7 μg / ml of impurity II, 7.2 μg / ml of impurity III, and 10 μg / ml each of impurities IV and VII; accurately weigh approximately 58 mg of oseltamivir phosphate capsule powder, place it in a 20 ml volumetric flask, add 2 ml of the above-mentioned stock solution of each impurity reference standard, sonicate to dissolve, dilute to the mark with the solvent, and shake well to obtain the final product.

[0066] 2.2 Measurement

[0067] Take 15 μl of the spiked test solution and collect and determine the sample under the following chromatographic conditions. The results are shown in Table 3.

[0068] Chromatographic conditions: The column was an octylsilane-bonded silica column; the mobile phase was 0.05 mol / L potassium dihydrogen phosphate buffer-methanol-acetonitrile at a volume ratio of 620:245:135; the pH of the 0.05 mol / L potassium dihydrogen phosphate buffer was adjusted to 6.0 with 1 mol / L potassium hydroxide solution; the flow rate was 1.2 ml / min; the column temperature was 50 ℃; and the detection wavelength was 207 nm.

[0069] Chromatographic column (1) Waters Symmetry C8, 4.6 mm × 250 mm, 5 μm;

[0070] Chromatographic column (2) Agilent ZORBAX Eclipse Plus C8, 4.6 mm × 250 mm, 5 μm;

[0071] Chromatographic column (3) Agilent ZORBAX Eclipse XDB C8, 4.6 mm × 250 mm, 5 μm;

[0072] Chromatographic column (4) Waters XBridge C8, 4.6 mm × 250 mm, 5 μm.

[0073] Table 3 Column Screening

[0074] serial number Column name Minimum resolution Number of impurities detected (1) Waters Symmetry C8 2.22 6 (2) Agilent ZORBAX Eclipse Plus C8 0.88 8 (3) Agilent ZORBAX Eclipse XDB C8 1.68 7 (4) Waters XBridge C8 1.56 8

[0075] Experimental results show that, using column (1), the resolution between each impurity and between the impurity and the main peak is greater than 2.0. However, the upper limit of the temperature of column (1) is 45℃, and the column temperature for the related substances of this product is 50℃. Therefore, the column packing is prone to collapse and damage during the use of the column. Using column (2), the resolution between impurity IV and the adjacent impurity peak is only 0.88, which is not completely separated. Both column (3) and column (4) can meet the detection requirements. Since the standard used in the pharmacopoeia is column (1), and the packing of column (4) has similar properties, column (4) is used to determine the related substances of this product. However, since the resolution between impurity VII and impurity III is 1.56, which basically meets the resolution requirements, the resolution between impurity VII and impurity III can be increased by adjusting the mobile phase ratio without affecting the applicability of other systems.

[0076] [Example 3] Adjustment of mobile phase ratio

[0077] Take 15 μl of the spiked test solution described in "Example 2 Column Screening" and collect and measure it according to the following chromatographic conditions. The results are shown in Table 4.

[0078] Chromatographic conditions: The column used was a Waters XBridge C8 column. The mobile phase was 0.05 mol / L potassium dihydrogen phosphate buffer-methanol-acetonitrile, with the volume ratio adjusted as shown in Table 4. The pH of the 0.05 mol / L potassium dihydrogen phosphate buffer was adjusted to 6.0 with 1 mol / L potassium hydroxide solution. The flow rate was 1.2 ml / min, the column temperature was 50 ℃, and the detection wavelength was 207 nm.

[0079] Table 4 Adjustment of Mobile Phase Ratio

[0080] Mobile phase ratio Main peak retention time (min) Main peak tailing factor Minimum resolution of chromatographic peaks 620∶245∶135 11.199 1.75 1.56 650∶245∶135 13.860 1.75 1.99 670∶245∶135 14.598 1.79 2.09 700∶245∶135 14.993 2.16 2.24

[0081] Experimental results show that when the mobile phase ratio is adjusted to 670:245:135, the tailing factor of the main peak is 1.79, and the resolution between impurity VII and impurity III is 2.09. Compared with the ratios of 650:245:135 and 620:245:135, the resolution between the impurities is higher, and there is no significant difference in the tailing factor of the main peak. When the mobile phase ratio is 700:245:135, the tailing factor of the main peak is larger. Therefore, considering all factors, the provisional ratio of 670:245:135 is adopted for the determination of related substances of this product, and the solvent volume ratio is also adjusted accordingly to 670:245:135.

[0082] [Example 4] Injection Volume Adjustment

[0083] 4.1 Preparation of reference solution

[0084] Accurately weigh 5.0 mg of oseltamivir phosphate reference standard and place it in a 100 ml volumetric flask. Add solvent (0.003 mol / L phosphoric acid aqueous solution-methanol-acetonitrile, volume ratio 670:245:135), sonicate to dissolve and dilute to the mark, and shake well. Accurately measure 1 ml and place it in a 100 ml volumetric flask, dilute to the mark with the solvent, and shake well. Then accurately measure 5 ml and place it in a 10 ml volumetric flask, dilute to the mark with the solvent, and shake well to obtain the final product.

[0085] 4.2 Measurement

[0086] Take 15 μl or 30 μl of the reference solution and collect and determine the sample according to the following chromatographic conditions. The results are shown in Table 5.

[0087] Chromatographic conditions: The column used was a Waters XBridge C8 column, with a mobile phase of 0.05 mol / L potassium dihydrogen phosphate buffer-methanol-acetonitrile at a volume ratio of 670:245:135. The pH of the 0.05 mol / L potassium dihydrogen phosphate buffer was adjusted to 6.0 with 1 mol / L potassium hydroxide solution. The flow rate was 1.2 ml / min, the column temperature was 50 ℃, and the detection wavelength was 207 nm.

[0088] Table 5 Injection Volume Adjustment

[0089] Injection volume Limit of Quantification Concentration Equivalent to the concentration of the test sample solution 15μl 0.5μg / ml 0.05％ 30μl 0.25μg / ml 0.025％

[0090] Experimental results show that when the injection volume is adjusted from 15 μl to 30 μl, the limit of quantitation (LOQ) of oseltamivir phosphate is 0.25 μg / ml, which is equivalent to 0.025% of the concentration of the test solution. This is not higher than the reporting limit (0.05%) specified in the standard of this product and can be accurately quantified. Therefore, it can be used as the LOQ concentration.

[0091] [Example 5] Adjustment of mobile phase pH

[0092] 5.1 Preparation of specific test solution

[0093] Accurately weigh approximately 5 mg each of impurity I, impurity II, impurity III, impurity IV, impurity L, and impurity K reference standards, and place them in the same 50 ml volumetric flask. Add an appropriate amount of solvent (0.003 mol / L phosphoric acid aqueous solution-methanol-acetonitrile, volume ratio 670:245:135) and sonicate to dissolve and dilute to the mark. Shake well to obtain the impurity reference standard stock solution. Accurately weigh approximately 170 mg of the contents of oseltamivir phosphate capsules and place them in a 100 ml volumetric flask. Add an appropriate amount of solvent and sonicate to dissolve. Accurately add 2 ml of each impurity reference standard stock solution, dilute to the mark with solvent, and shake well to obtain the final product.

[0094] 5.2 Measurement

[0095] Take 30 μl of the specific test solution and collect and determine it according to the following chromatographic conditions.

[0096] Chromatographic conditions: The column used was a Waters XBridge C8 column, and the mobile phase was 0.05 mol / L potassium dihydrogen phosphate buffer-methanol-acetonitrile with a volume ratio of 670:245:135. The pH of the 0.05 mol / L potassium dihydrogen phosphate buffer was adjusted with 1 mol / L potassium hydroxide solution as shown in Table 6. The flow rate was 1.2 ml / min, the column temperature was 50 ℃, and the detection wavelength was 207 nm.

[0097] Table 6 Screening by mobile phase pH value

[0098] pH value Main peak retention time (min) Minimum resolution 6.0 14.293 0.81 5.9 12.974 2.32 5.7 12.245 2.63 5.5 12.171 1.69

[0099] The experimental results are shown in Table 6 and Figures 8-11 The results show that when the mobile phase pH is 5.7, the resolution between each chromatographic peak is not less than 2.0, which meets the resolution requirement. Furthermore, when the pH changes by ±0.2, the resolution between each peak is greater than 1.5, indicating good robustness. Therefore, the final mobile phase pH is determined to be 5.7.

[0100] [Example 6] Comparison of Impurity Detection Amounts

[0101] The self-developed oseltamivir phosphate capsules were tested for impurities using the relevant substance detection methods for oseltamivir phosphate capsules listed in the pharmacopoeias of various countries (including import registration standards). The results were compared with the self-developed method to study the impurity detection. The details are as follows:

[0102] (1) Test solution: Take the contents of this product, grind it finely, mix it evenly, take about 170 mg, accurately weigh it, put it in a 100 ml volumetric flask, add an appropriate amount of solvent (0.003 mol / L phosphoric acid aqueous solution-methanol-acetonitrile, volume ratio 670:245:135) to dissolve and dilute to the mark, shake well, filter, and take the filtrate to obtain the test solution;

[0103] (2) Reference solution: Weigh about 4 mg of oseltamivir phosphate reference standard, place it in a 20 ml volumetric flask, add an appropriate amount of the solvent to dissolve and dilute to the mark, shake well, then accurately pipette 1 ml into a 200 ml volumetric flask, add solvent to dilute to the mark, prepare a solution containing about 1 μg of oseltamivir phosphate per ml, shake well, and the solution is ready.

[0104] (3) Control solution: Accurately measure 2 ml of the test solution and place it in a 100 ml volumetric flask. Dilute it to the mark with the solvent and shake well. Then accurately measure 5 ml of the solution and place it in a 50 ml volumetric flask. Dilute it to the mark with the solvent and shake well.

[0105] (4) Impurity reference solution: Take appropriate amounts of impurity I, impurity II and impurity III reference standards, weigh them accurately, dissolve them in the solvent and dilute quantitatively to prepare a solution containing approximately 2.2 μg of impurity I, 1.5 μg of impurity II and 3.5 μg of impurity III per ml;

[0106] (5) The solutions (1) to (4) were tested under the chromatographic conditions determined in Example 5. The experimental results are shown in Table 7.

[0107] Table 7 Comparison of Impurity Detection Amounts

[0108]

[0109] Experimental results show that the detection limits of impurities using the self-developed related substances method are comparable to those detected under the capsule quality standards in various pharmacopoeias (including imported registration standards).

Claims

1. A high-performance liquid chromatography method for detecting related substances in oseltamivir phosphate products, wherein the related substances are impurity I, impurity II, impurity III, impurity IV, impurity VII, impurity K, and impurity L, with the following structural formulas: characterized in that The detection method includes the following steps: (1) Preparation of system-adaptive solution Take appropriate amounts of reference standards for impurities I, II, III, IV, VII, K, and L, dissolve them in solvent, and quantitatively dilute them to prepare a stock solution of impurity reference standards containing 15 μg / ml each of impurities I, II, IV, VII, K, and L, and 38 μg / ml of impurity III, for later use. Separately, dissolve and dilute povidone K30 in the solvent to prepare a solution containing 0.5 mg / ml, centrifuge, and take the supernatant as a stock solution of povidone K30 for later use; Accurately measure 1 ml each of the impurity reference standard stock solution and the povidone K30 stock solution, place them in the same container, then accurately weigh about 10 mg of oseltamivir phosphate reference standard, add it to the container to dissolve, and dilute with the solvent to 10 ml. Shake well to obtain the system adaptability solution. (2) Preparation of oseltamivir phosphate reference solution Accurately weigh the oseltamivir phosphate reference standard, and then quantitatively dilute it with the solvent to prepare a solution containing 1 μg / ml of oseltamivir phosphate, thus obtaining the oseltamivir phosphate reference standard solution; (3) Preparation of test solution Accurately weigh the sample to be tested, and quantitatively dilute it with the solvent to prepare a solution containing 1 mg / ml of oseltamivir phosphate, thus obtaining the test solution; (4) Determine the content of related substances in the sample by high performance liquid chromatography. The oseltamivir phosphate reference solution was injected into the liquid chromatograph, and the detection sensitivity was adjusted. Then, the test solution was injected into the liquid chromatograph, and high-performance liquid chromatography (HPLC) was performed under the following chromatographic conditions. The chromatogram was recorded. The HPLC conditions included: Chromatographic column: Octylsilane-bonded silica gel liquid chromatography column, the column size is 4.6mm×250mm, and the packing particle size is 5μm; Mobile phase: 0.05 mol / L potassium dihydrogen phosphate buffer-methanol-acetonitrile, volume ratio 620:245:135 to 700:245:135, wherein the pH of the 0.05 mol / L potassium dihydrogen phosphate buffer is adjusted to 5.5 to 5.9 with 1 mol / L potassium hydroxide solution; Detection wavelength: 207nm; Flow rate: 1.1–1.3 ml / min; Column temperature: 48~52℃; Injection volume: 15–30 μl; (5) Calculate the content of each impurity in the sample to be tested. Calculated by peak area using the principal component external standard method.

2. The method of claim 1, wherein, The solvent is a mixture of 0.003 mol / L phosphoric acid aqueous solution, methanol, and acetonitrile to form a mixed solution with a volume ratio of 670:245:

135.

3. The method of claim 1, wherein, The chromatographic column used was a Waters XBridge C8.

4. The method of claim 1, wherein, In the mobile phase, the volume ratio of 0.05 mol / L potassium dihydrogen phosphate buffer-methanol-acetonitrile is 670:245:135, wherein the pH of the 0.05 mol / L potassium dihydrogen phosphate buffer is adjusted to 5.7 with 1 mol / L potassium hydroxide solution.

5. The method of claim 1, wherein, The injection volume is 30 μl.

6. The method of claim 1, wherein, The chromatographic column is Waters XBridge C8 chromatographic column, the volume ratio of the mobile phase 0.05 mol / L potassium dihydrogen phosphate buffer-methanol-acetonitrile is 670:245:135, the 0.05 mol / L potassium dihydrogen phosphate buffer is adjusted to pH 5.7 with 1 mol / L potassium hydroxide solution, the flow rate is 1.2 ml / min, the column temperature is 50 DEG C, and the injection volume is 30 μl.

7. The method of claim 1, wherein, The sample to be detected is the content of the oseltamivir phosphate capsule.

Images