Method for determining oseltamivir isomer in oseltamivir phosphate product by using high performance liquid chromatography

Abstract

The invention discloses a method for determining isomers in an oseltamivir phosphate product by using high performance liquid chromatography. The chromatographic condition is that a Waters Symmetry C8 chromatographic column is used, a mobile phase is phosphate buffer solution-methanol-acetonitrile, the volume ratio is 620: 245: 135-700: 245: 135, isocratic elution is performed, the concentration of the phosphate buffer solution is 0.040 mol / L to 0.075 mol / L, the pH value is 6.25 to 6.40, the column temperature is 43 to 47 DEG C, the flow velocity is 1.1 to 1.3 ml / min, the detection wavelength is 207 nm, and the sample size is 60 microliters. According to the method, the isomer in the oseltamivir phosphate product can be rapidly and accurately detected, the content of the isomer can be calculated, the operation is simple, the reproducibility is good, the quality control of an oseltamivir phosphate preparation and an intermediate product thereof can be well realized, and a guarantee is provided for synthesis and preparation process optimization.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of pharmaceutical analysis and detection, and particularly relates to a method for determining oseltamivir isomers in oseltamivir phosphate products by high performance liquid chromatography. BACKGROUND

[0002] Oseltamivir phosphate, the chemical name of which is (3R, 4R, 5S)-4-acetylamino-5-amino-3-(1-ethylpropoxy)-1-cyclohexen-1-carboxylic acid ethyl ester phosphate, has a molecular formula of C 16 H 28 N2O4·H3PO4, the chemical structural formula of which is as follows:

[0003]

[0004] Oseltamivir phosphate can competitively bind to the active site of influenza virus neuraminidase, thereby reducing the spread of influenza A or B viruses by interfering with the release of viruses from infected host cells. Oseltamivir phosphate has the characteristics of specificity, high efficiency, safety, and small drug resistance, and is the most effective anti-viral influenza drug. It is commonly used in the treatment of influenza A and B in adults and children over 1 year old, and can also be used for prevention. Oseltamivir phosphate capsules are a type of preparation of such drugs.

[0005] Oseltamivir phosphate has multiple chiral centers in its structure, and oseltamivir phosphate has an RRS configuration. Its isomers are RRR-isomer, SSS-isomer, SRS-isomer, RSR-isomer, RSS-isomer, SRR-isomer, and SSR-isomer. In order to monitor the isomers in oseltamivir phosphate products, it is very important to establish an effective detection method for the quality control of the products. At present, the quality standards of oseltamivir phosphate, oseltamivir phosphate capsules and other dosage forms in various pharmacopoeias do not include the detection method of oseltamivir phosphate isomers. CN 109580850A discloses a high performance liquid chromatography method for separating and determining oseltamivir phosphate and specific impurities, but the above seven isomers are not simultaneously studied. At present, there is no literature report on the simultaneous study of the above isomers.

[0006] Therefore, the present application aims to provide an accurate and effective method for determining the isomers in oseltamivir phosphate products, so as to realize the quality control of oseltamivir phosphate raw materials and preparations. SUMMARY

[0007] The present application aims to provide an accurate and effective method for determining the isomers in oseltamivir phosphate products, so as to realize the quality control of oseltamivir phosphate raw materials and preparations.

[0008] The chemical structural formulas of oseltamivir and its seven isomers, and impurities II, III, IV and VII commonly found in oseltamivir products are shown in the following table:

[0009]

[0010]

[0011] The method of the present application comprises the following steps:

[0012] (1) preparing a system suitability solution;

[0013] Take the oseltamivir phosphate reference substance, SRR-isomer reference substance, RRR-isomer reference substance, SRS-isomer reference substance, and impurity IV reference substance in appropriate amounts, and prepare a solution containing 1 mg / ml of oseltamivir phosphate, 0.002 mg / ml of SRR-isomer, RRR-isomer, SRS-isomer, and impurity IV, using the solvent; wherein the preparation method of the solvent is: 0.003 mol / L aqueous phosphoric acid is mixed with methanol and acetonitrile to form a mixed solution with a volume ratio of 670:245:135;

[0014] (2) preparing a test sample solution;

[0015] Take the oseltamivir phosphate sample to be tested in appropriate amounts, and add the solvent to prepare a test sample solution containing 1 mg / ml of oseltamivir phosphate;

[0016] (3) detecting by high performance liquid chromatography

[0017] Inject the solvent, system suitability solution and test sample solution into the high performance liquid chromatograph in sequence, respectively, and record the chromatogram, and calculate the content of the isomers in the oseltamivir phosphate sample to be tested by the peak area normalization method; the calculation formula is:

[0018]

[0019] In the formula, A X is the peak area of the isomer peak in the test sample solution;

[0020] A 总 is the peak area of the main peak in the test sample solution.

[0021] The conditions of high performance liquid chromatography are as follows:

[0022] Chromatographic column: using octylsilane bonded silica gel as the filler;

[0023] The mobile phase is phosphate buffer-methanol-acetonitrile with a volume ratio of 620:245:135 to 700:245:135.

[0024] The column temperature is 43-47℃.

[0025] The flow rate is 1.1-1.3 ml / min.

[0026] The injection volume is 60 μl.

[0027] The detection wavelength is 207 nm.

[0028] Preferably, the column temperature is 45℃ and the flow rate is 1.2 ml / min.

[0029] Preferably, the volume ratio of the above-mentioned mobile phase phosphate buffer-methanol-acetonitrile is 670:245:135, and the elution is isocratic.

[0030] Preferably, the above-mentioned phosphate is potassium dihydrogen phosphate with a concentration of 0.05 mol / L and a pH value of 6.3.

[0031] Preferably, the above-mentioned chromatographic column has a specification of 4.6 mm x 250 mm and a packing particle size of 5 μm.

[0032] Preferably, the above-mentioned chromatographic column is a Waters Symmetry C8 chromatographic column.

[0033] Preferably, the above-mentioned oseltamivir phosphate sample to be tested is any one selected from the group consisting of oseltamivir phosphate raw material, oseltamivir phosphate preparation intermediate product and oseltamivir phosphate preparation.

[0034] The present application determines the chromatographic conditions for effectively separating oseltamivir phosphate and its isomers by exploring chromatographic conditions, and verifies that the method of the present application has the advantages of good specificity, good resolution and good reproducibility, and has important significance for product quality control and stability sample detection.

[0035] The method of the present application is suitable for detecting oseltamivir phosphate raw material, oseltamivir phosphate preparation including oseltamivir phosphate preparation intermediate product and oseltamivir phosphate preparation finished product. The detection method of the present application is accurate, simple to operate and easy to obtain reagents, and can fully meet the separation requirements of oseltamivir phosphate and each isomer, better control the content of isomers in the sample to be tested, ensure product quality, and has strong practicality in actual quality control work. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1 Figure 1 is the superimposed chromatogram of Example 1 chromatographic condition comparison No. 1;

[0037] Figure 2 Figure 2 is the superimposed chromatogram of Example 1 chromatographic condition comparison No. 2;

[0038] Figure 3 Chromatogram of spiked sample solution at 40°C for column temperature selection in Example 1;

[0039] Figure 4 Chromatogram of spiked sample solution at 45°C for column temperature selection in Example 1;

[0040] Figure 5 Chromatogram of spiked sample solution at 45°C for column temperature selection in Example 1;

[0041] Figure 6 Chromatogram of RRR-isomer limit of quantitation solution at 15 μl for injection volume selection in Example 1;

[0042] Figure 7 Chromatogram of RRR-isomer limit of quantitation solution at 60 μl for injection volume selection in Example 1;

[0043] Figure 8 Chromatogram of system suitability solution at 620:245:135 for mobile phase ratio in Example 1;

[0044] Figure 9 Chromatogram of system suitability solution at 670:245:135 for mobile phase ratio in Example 1;

[0045] Figure 10 Chromatogram of blank solution in specificity test of Example 2;

[0046] Figure 11 Chromatogram of excipient control solution in specificity test of Example 2;

[0047] Figure 12 Chromatogram of system suitability solution in specificity test of Example 2;

[0048] Figure 13 Chromatogram of spiked sample solution in specificity test of Example 2. DETAILED DESCRIPTION

[0049] Various exemplary embodiments of the present application will now be described in detail, with reference to the drawings, which are provided as illustrative examples so as to enable those skilled in the art to practice the present application. However, these exemplary embodiments of the present application are not intended to limit the scope of the present application, and they are not intended to limit the scope of the claims herein.

[0050] In the following detailed description of the application, the instruments, reagents, samples and controls used are as follows:

[0051] Instrument: Agilent 1260 series high performance liquid chromatograph;

[0052] Reagents: Potassium dihydrogen phosphate (chromatographically pure), methanol (chromatographically pure), acetonitrile (chromatographically pure), phosphoric acid (chromatographically pure), potassium hydroxide (analytically pure);

[0053] Column: Waters Symmetry C8, 4.6 mm x 250 mm, 5 μm;

[0054] Table 1 Samples

[0055]

[0056] Composition of Oseltamivir Phosphate Capsules and its production process:

[0057] I. Content: Oseltamivir Phosphate, pre-gelatinized starch, povidone K30, croscarmellose sodium, sodium stearyl fumarate, talc; Capsule shell: gelatin, yellow iron oxide, red iron oxide, black iron oxide, titanium dioxide; Reference to the Russian drug control authority - Tammifu - public information;

[0058] II. Production process: wet granulation, drying, granulation, total mixing and capsule filling, refer to "European drug control authority - Tammifu - 30 mg - scientific discussion" for specific operation.

[0059] The source, content and batch number of the standard are as follows:

[0060] Oseltamivir Phosphate:

[0061] Source: EDQM, content: 99.7%, batch number: 2.0.

[0062] Oseltamivir RRR-isomer reference substance:

[0063] Source: OST, content: 91.46%, batch number: U4O-1507003-06.

[0064] Oseltamivir SSS-isomer reference substance:

[0065] Source: OST, content: 96.09%, batch number: U4O-1507080-02.

[0066] Oseltamivir RSR-isomer reference substance:

[0067] Source: OST, content: 85.61%, batch number: U4O-1507004-06.

[0068] Oseltamivir SRS-isomer reference substance:

[0069] Source: OST, content: 93.43%, batch number: U4O-1507301-02.

[0070] Oseltamivir RSS-isomer reference standard:

[0071] Source: OST, content: 90.10%, 83.6%, batch number: U40-1507001-05, U40-1507001-06.

[0072] Oseltamivir SRR-isomer reference standard:

[0073] Source: OST, content: 96.7%, 95.9%, batch number: U40-1507012-02, U40-1507012-03.

[0074] Oseltamivir SSR-isomer reference standard:

[0075] Source: OST, content: 88.62%, batch number: U40-1507300-02.

[0076] Oseltamivir impurity II reference standard:

[0077] Chemical name: 3-hydroxy-4-acetylamino benzoic acid ethyl ester

[0078] Source: National Institutes for Food and Drug Control, content: 100%, batch number: 101383-202002.

[0079] Oseltamivir impurity III reference standard:

[0080] Chemical name: (3R, 4R, 5S)-4-acetylamino-5-amino-3-(1-ethylpropoxy)-1-cyclohexen-1-carboxylic acid Source: National Institutes for Food and Drug Control, content: 62.7%, batch number: 101384-202002.

[0081] Oseltamivir impurity IV reference standard:

[0082] Chemical name: (3R, 4R, 5S)-5-acetylamino-4-amino-3-(1-ethylpropoxy)-1-cyclohexen-1-carboxylic acid ethyl ester

[0083] Source: Haiwang Research Institute, content: 98.3%, batch number: 20210513.

[0084] Oseltamivir impurity VII reference standard:

[0085] Chemical name: (3R, 4R, 5S)-5-acetylamino-4-amino-3-(1-ethylpropoxy)-1-cyclohexen-1-carboxylic acid Source: EDQM, content: 100%, batch number: 3.0.

[0086] [Example 1] Determination of optimal separation and detection conditions of high performance liquid chromatography

[0087] I. Comparison of chromatographic conditions

[0088] 1.1.1 Preparation of the solution to be tested

[0089] RRR-isomer reference stock solution: about 4 mg of RRR-isomer reference was accurately weighed into a 20-ml volumetric flask, dissolved and diluted to the mark with solvent, and mixed to obtain the stock solution.

[0090] RRR-isomer reference solution: 1 ml of the RRR-isomer reference stock solution was accurately measured into a 100-ml volumetric flask, diluted to the mark with solvent, and mixed to obtain the solution.

[0091] Raw material test solution: about 50 mg of oseltamivir phosphate raw material was accurately weighed into a 50-ml volumetric flask, dissolved and diluted to the mark with solvent, and mixed to obtain the solution.

[0092] 1.1.2 Preparation of the mobile phase

[0093] The mobile phase was prepared according to Table 2.

[0094] Table 2 Comparison of chromatographic conditions

[0095]

[0096] 1.1.3 Determination by high-performance liquid chromatography

[0097] The above solution was collected and determined according to the mobile phase, column, and the following chromatographic conditions in Table 2.

[0098] The flow rate was 1.2 ml / min, the column temperature was 50°C, the detection wavelength was 207 nm, and the injection volume was 15 μl.

[0099] 1.1.4 Experimental results

[0100] The results are shown in Table 2 and Figures 1-2 .

[0101] The results show that, using chromatographic condition No. 1, the retention time of the RRR-isomer peak is basically the same as that of oseltamivir, and they cannot be completely separated. As can be seen from the superimposed chromatogram of the RRR-isomer reference solution and the raw material test solution (attached Figure 1 ), the RRR-isomer is wrapped into the oseltamivir chromatographic peak; using chromatographic condition No. 2, the RRR-isomer peak appears at 22.705 min, and as can be seen from the superimposed chromatogram of the RRR-isomer reference solution and the raw material test solution (attached Figure 2 ), the oseltamivir peak does not interfere with the determination of the RRR-isomer, and therefore chromatographic condition No. 2 is temporarily selected as the isomer detection condition for the product.

[0102] II. Selection of column temperature

[0103] This example intends to screen the column temperature, and the column temperature of 40℃ and 45℃ are selected for investigation.

[0104] 1.2.1 Preparation of the solution to be tested

[0105] System suitability solution stock solution: Take 5 mg of oseltamivir phosphate reference substance, 5 mg of oseltamivir phosphate impurity III reference substance, 4 mg of impurity VII reference substance, 4 mg of impurity IV reference substance, and 4 mg of oseltamivir phosphate impurity II reference substance into the same 100 ml volumetric flask, add an appropriate amount of solvent to dissolve and dilute to the mark, and shake well to obtain the solution.

[0106] Raw material spiked test sample solution: Take about 100 mg of oseltamivir phosphate raw material, accurately weigh, and place it in a 100 ml volumetric flask. Add 6.8 mg of povidone K30, 1 ml of system suitability stock solution, and 1 ml of RRR-isomer stock solution. Add an appropriate amount of solvent to dissolve and dilute to the mark, and shake well to obtain the solution.

[0107] Spiked test sample solution: Take appropriate amounts of oseltamivir phosphate capsule contents, RSS-isomer reference substance, SRR-isomer reference substance, SSR-isomer reference substance, RRR-isomer reference substance, SSS-isomer reference substance, SRS-isomer reference substance, and RSR-isomer reference substance, accurately weigh, and add solvent to dissolve and dilute quantitatively to prepare a solution containing about 1 mg of oseltamivir phosphate per 1 ml, about 0.01 mg of SSR-isomer, RRR-isomer, SSS-isomer, SRS-isomer, and RSR-isomer each, about 0.02 mg of RSS-isomer, and about 0.0005 mg of SRR-isomer.

[0108] 1.2.2 Determination by high performance liquid chromatography

[0109] Take the above solution and adjust the column temperature according to the following chromatographic conditions for collection and determination.

[0110] Mobile phase: 0.05 mol / L potassium dihydrogen phosphate buffer (pH value adjusted to 6.3 with 1 mol / L potassium hydroxide solution) - methanol - acetonitrile (620:245:135)

[0111] Chromatographic column: Waters Symmetry C8 (4.6 mm x 250 mm, 5 μm)

[0112] Flow rate: 1.2 ml / min, detection wavelength: 207 nm, injection volume: 15 μl

[0113] Table 3 Column Temperature Comparison Results

[0114]

[0115]

[0116] 1.2.3 Experimental Results

[0117] The test results are shown in Table 3 and Figures 3-5 As shown.

[0118] The results showed that, under column temperatures of 40℃ and 45℃, the resolution between known impurities and between the main peak and adjacent impurities in the chromatograms of the spiked sample solution was greater than 1.5. The tailing factor of the main peak was not different. Under the condition of 45℃, the theoretical plate number and the resolution between the RRR-isomer peak and the main peak were better than those under the condition of 40℃. Therefore, 45℃ was selected as the column temperature for chromatography.

[0119] Since the Waters Symmetry C8 (4.6 mm × 250 mm, 5 μm) column is a non-chiral column, the RSS-isomer peak and SRR-isomer peak, the oseltamivir peak and SSR-isomer peak, the RRR-isomer peak and SSS-isomer peak, and the RSR-isomer peak and SRS-isomer peak eluted at the same position. Among them, the SSR-isomer peak with the same retention time as the oseltamivir peak was detected by other methods, and the other isomers were controlled by this method by combined detection. Under the condition of 45 °C, the resolution between the main component and each isomer in the spiked test solution, as well as between each isomer, was greater than 1.5.

[0120] III. Selection of Injection Volume

[0121] 1.3.1 Determination by High Performance Liquid Chromatography

[0122] Based on the conditions determined in points one and two above, namely: mobile phase: 0.05 mol / L potassium dihydrogen phosphate buffer (pH adjusted to 6.3 with 1 mol / L potassium hydroxide solution) - methanol - acetonitrile (620:245:135), chromatographic column: Waters Symmetry C8 (4.6 mm × 250 mm, 5 μm), flow rate: 1.2 ml / min, detection wavelength: 207 nm, injection volume: 15 μl, the detection limit and quantitation limit of RRR-isomer were initially explored.

[0123] Accurately weigh approximately 4 mg of RRR-isomer reference standard and place it in a 20 ml volumetric flask. Add an appropriate amount of solvent to dissolve and dilute to the mark, shake well, and continuously dilute with solvent to prepare a 2 μg / ml solution, which is used as the limit of quantitation solution ①. Accurately measure 15 μl of the limit of quantitation solution ① and inject it into the liquid chromatograph. Record the chromatogram (see attached figure). Figure 6 At this point, the signal-to-noise ratio of the RRR-isomer peak is 15.4, and the solution concentration is equivalent to 0.2% of the test sample solution concentration (1 mg / ml). The maximum daily dose of this product is 150 mg. According to the ICH Q3B guideline, the reporting limit for related substances is 0.1%, which cannot meet the purpose of accurate quantification at the reporting limit concentration. Therefore, it is proposed to increase the injection volume to 60 μl.

[0124] Accurately weigh approximately 4 mg of RRR-isomer reference standard and place it in a 20 ml volumetric flask. Add an appropriate amount of solvent to dissolve and dilute to the mark, shake well, and continuously dilute with solvent to prepare a solution of approximately 0.5 μg / ml, which is used as the limit of quantitation solution ②. Accurately measure 60 μl of the limit of quantitation solution ② and inject it into the liquid chromatograph. Record the chromatogram (see attached figure). Figure 7 ).

[0125] 1.3.2 Experimental Results

[0126] The test results are shown in Table 4 and Figure 6 , Figure 7 As shown.

[0127] Table 4 Selection of Injection Volume

[0128] Solution name Injection volume Signal-to-noise ratio Absolute concentration (ng / ml) Equivalent to test sample concentration (%) Peak area Limit of quantitation solution 2 60 μl 13.8 449.41 0.04 37.81494

[0129] The experimental results show that when the injection volume is increased to 60 μl, the quantitation limit concentration solution is equivalent to 0.05% of the concentration of the 1 mg / ml test sample solution, which can meet the purpose of accurate quantification when reporting the limit concentration. Therefore, the injection volume is determined to be 60 μl.

[0130] IV. Selection of Mobile Phase Ratio

[0131] 1.4.1 Preparation of the test solution

[0132] System suitability solution ①: Take appropriate amounts of oseltamivir phosphate reference standard, RRR-isomer reference standard, SRS-isomer reference standard and impurity IV reference standard, accurately weigh them, add solvent to dissolve them and quantitatively dilute to prepare a solution containing approximately 1 mg of oseltamivir phosphate and 2 μg each of RRR-isomer, SRS-isomer and impurity IV per 1 ml.

[0133] System suitability solution ②: Take appropriate amounts of oseltamivir phosphate reference standard, SRR-isomer reference standard, RRR-isomer reference standard, SRS-isomer reference standard and impurity IV reference standard, accurately weigh them, add solvent to dissolve them and quantitatively dilute to prepare a solution containing approximately 1 mg of oseltamivir phosphate and 2 μg each of SRR-isomer, RRR-isomer, SRS-isomer and impurity IV per 1 ml.

[0134] 1.4.2 Determination by High Performance Liquid Chromatography

[0135] Referring to the chromatographic conditions determined above, namely: mobile phase: 0.05 mol / L potassium dihydrogen phosphate buffer (pH adjusted to 6.3 with 1 mol / L potassium hydroxide solution) - methanol - acetonitrile (620:245:135), chromatographic column: Waters Symmetry C8 (4.6 mm × 250 mm, 5 μm), flow rate 1.2 ml / min, detection wavelength 207 nm, injection volume 60 μl, and accurately inject 60 μl of system suitability solution ① into the liquid chromatograph and record the chromatogram; referring to the chromatographic conditions determined above, the mobile phase ratio was adjusted to 0.05 mol / L potassium dihydrogen phosphate solution (pH adjusted to 6.3 with 1 mol / L potassium hydroxide solution) - methanol - acetonitrile (670:245:135), and 60 μl of system suitability solution ② was accurately injected into the liquid chromatograph and the chromatogram was recorded.

[0136] Table 5 Results of Mobile Phase Proportion Adjustment

[0137]

[0138]

[0139] 1.4.3 Experimental Results

[0140] The test results are shown in Table 5 and Figure 8 , Figure 9 As shown.

[0141] The results showed that when the ratio of the mobile phase 0.05 mol / L potassium dihydrogen phosphate solution (pH adjusted to 6.3 with 1 mol / L potassium hydroxide solution) - methanol - acetonitrile was 670:245:135, the separation between the SRS-isomer peak and the impurity IV peak was better. Therefore, the mobile phase ratio was adjusted to 670:245:135.

[0142]

Example 2

[0143] I. Exclusivity

[0144] 2.1.1 Preparation of the test solution

[0145] Blank solvent: 0.003 mol / L phosphoric acid solution - methanol - acetonitrile, volume ratio 670:245:135.

[0146] Excipient control solution: Weigh approximately 70 mg of blank excipient for oseltamivir phosphate capsules according to the prescription ratio, place it in a 100 ml volumetric flask, add an appropriate amount of solvent, sonicate to dissolve and dilute to the mark, shake well, filter, and collect the filtrate.

[0147] Stock solutions of impurity reference standards: Weigh approximately 4 mg each of SRR-isomer reference standard, RRR-isomer reference standard, SRS-isomer reference standard, impurity IV reference standard, impurity VII reference standard, oseltamivir phosphate impurity III reference standard, and oseltamivir phosphate impurity II reference standard. Place them in separate 20 ml volumetric flasks, add an appropriate amount of solvent, sonicate to dissolve and dilute to the mark, and shake well to obtain the corresponding impurity reference standard stock solutions.

[0148] Spiked test solution ①: Weigh approximately 170 mg of the contents of oseltamivir phosphate capsules accurately, place in a 100 ml volumetric flask, add an appropriate amount of solvent, and sonicate to dissolve. Then add 2.5 ml of impurity III reference stock solution and 1 ml of the other impurity reference stock solutions mentioned above, dilute to the mark with solvent, shake well, filter, and collect the filtrate to obtain the test solution.

[0149] System suitability solution: Weigh approximately 10 mg of oseltamivir phosphate reference standard accurately, place it in a 10 ml volumetric flask, add an appropriate amount of solvent, and sonicate to dissolve. Add 0.1 ml of stock solutions of SRR-isomer, RRR-isomer, SRS-isomer, and impurity IV reference standard, respectively. Dilute to the mark with solvent, shake well, filter, and collect the filtrate.

[0150] 2.1.2 Determination by High Performance Liquid Chromatography

[0151] Accurately measure 60 μl each of blank solvent, excipient control solution, system suitability solution and spiked test solution ①, inject them into the liquid chromatograph, and perform detection according to the chromatographic conditions summarized in Example 1, and record the chromatogram.

[0152] Table 6 Specificity Results

[0153]

[0154] 2.1.3 Experimental Results

[0155] The test results are shown in Table 6. Figures 10-13 As shown.

[0156] The results showed that the blank solvent and blank excipients exhibited no absorption at the SRR-isomer peak (approximately 18.2 min), oseltamivir peak (approximately 24.6 min), RRR-isomer peak (approximately 26.5 min), and SRS-isomer peak (approximately 35.6 min), indicating no interference with isomer determination. In the chromatogram of the spiked test solution ①, the minimum resolution between the main peak and adjacent impurity peaks was 2.02, and the resolution between the RRR-isomer peak and the SRS-isomer peak was 10.13, both greater than 1.5. The resolutions between other impurities were all greater than 1.0, indicating that known impurities did not interfere with isomer determination.

[0157] II. Limit of Detection and Limit of Quantification

[0158] Take appropriate amounts of oseltamivir phosphate, SRR-isomer reference standard, RRR-isomer reference standard and SRS-isomer reference standard respectively, prepare 0.2 mg / ml solutions with solvent, and then dilute them with solvent to prepare a series of solutions of different concentrations. Detect them according to the chromatographic conditions summarized in Example 1.

[0159] The detection limits were determined based on the corresponding concentrations at a signal-to-noise ratio of 3:1. The detection limit for oseltamivir phosphate was 0.2003 μg / ml, equivalent to 0.02% of the test solution concentration; the detection limit for SRR-isomer was 0.0979 μg / ml, equivalent to 0.01% of the test solution concentration; the detection limit for RRR-isomer was 0.0922 μg / ml, equivalent to 0.01% of the test solution concentration; and the detection limit for SRS-isomer was 0.0940 μg / ml, equivalent to 0.01% of the test solution concentration.

[0160] The limits of quantitation (LOQs) were determined at a signal-to-noise ratio (SNR) of 10:1. Five injections were performed, and the RSD values ​​were calculated. The LOQ concentrations for oseltamivir phosphate were 0.5007 μg / ml, equivalent to 0.05% of the test solution concentration; the LOQ concentrations for SRR-isomer were 0.4896 μg / ml, equivalent to 0.05% of the test solution concentration; the LOQ concentrations for RRR-isomer were 0.4611 μg / ml, equivalent to 0.05% of the test solution concentration; and the LOQ concentrations for SRS-isomer were 0.4701 μg / ml, equivalent to 0.05% of the test solution concentration. These values ​​are all lower than the reporting limit (0.1%), and the RSD values ​​for the peak areas were less than 10% for all five consecutive injections, meeting the acceptable LOQ criteria. The results are shown in Table 7.

[0161] Table 7 Results of Limit of Quantitation and Limit of Detection

[0162] III. Linearity and Range

[0163] Accurately measure 2.5 ml of the SRR-isomer, RRR-isomer, and SRS-isomer reference stock solutions under section 2.1.1 above, place them in a 50 ml volumetric flask, dilute to the mark with solvent, and shake well to obtain the linear stock solution of the isomers. Prepare 5 solutions of different concentrations with solvent. Accurately weigh an appropriate amount of oseltamivir phosphate reference standard, dilute with solvent to prepare a 1 mg / ml solution, and then dilute with solvent to prepare 5 solutions of different concentrations. Inject the above solutions separately and detect them according to the chromatographic conditions summarized in Example 1. Perform linear regression analysis with concentration as the abscissa and peak area as the ordinate to obtain the linear regression equation. The results are shown in Table 8.

[0164] Table 8 Results of Linearity and Range Measurements

[0165]

[0166]

[0167] The results showed that, under this method, oseltamivir phosphate, SRR-isomer, RRR-isomer, and SRS-isomer all exhibited good linearity within a certain concentration range.

[0168] IV. Precision

[0169] 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. Then add 1 ml each of the stock solutions of SRR-isomer, RRR-isomer, and SRS-isomer reference standards from section 2.1, dilute to the mark with solvent, and shake well. Prepare six parallel samples and inject them separately. Detect the isomers according to the chromatographic conditions summarized in Example 1. Calculate the isomer content in each sample. The experimental results are shown in Table 9.

[0170] Table 9 Precision Test Results

[0171]

[0172] The results showed that the RSD values ​​of the measured SRR-isomer, RRR-isomer, and SRS-isomer contents were all less than 10%, indicating that the method had good repeatability.

[0173] V. Durability Test

[0174] Robustness tests were conducted under varying column temperature (±2℃), flow rate (±0.1 ml / min), mobile phase buffer salt concentration (-20%–+50%), mobile phase pH (6.25–6.40), mobile phase buffer salt ratio (-5%–+3%), and different column serial numbers. A system suitability solution was prepared according to the method described in 2.1 above, and 60 μl was accurately measured and injected into the liquid chromatograph. The chromatographic conditions summarized in Example 1 were used for detection, and the results are shown in Table 10.

[0175] Table 10 Durability Test Results

[0176]

[0177] The results showed that when all parameters changed slightly, the separation between the oseltamivir main peak and the RRR-isomer was greater than 1.5, and the separation between the SRS-isomer and impurity IV was greater than 1.0, which met the system suitability requirements and indicated that the method had good robustness.

[0178] Summarize

[0179] In summary, the chromatographic conditions employed in this invention are as follows: a Waters Symmetry C8 column; a mobile phase of phosphate buffer-methanol-acetonitrile at a volume ratio of 620:245:135 to 700:245:135; isocratic elution; a phosphate buffer concentration of 0.040 mol / L to 0.075 mol / L; a pH of 6.25 to 6.40; a column temperature of 43 to 47 °C; a flow rate of 1.1 to 1.3 ml / min; a detection wavelength of 207 nm; and an injection volume of 60 μl. This method can rapidly and accurately separate and detect the six oseltamivir isomers in oseltamivir phosphate products and calculate the total content of these six isomers. The operation is simple. The optimal chromatographic conditions for separation were as follows: Waters Symmetry C8 column (4.6 mm × 250 mm, 5 μm), mobile phase of 0.05 mol / L potassium dihydrogen phosphate solution (pH adjusted to 6.3 with 1 mol / L potassium hydroxide solution)-methanol-acetonitrile, volume ratio 670:245:135, flow rate 1.2 ml / min, column temperature 45 ℃, detection wavelength 207 nm, and injection volume 60 μl.

Claims

1. A method for determining the content of oseltamivir isomers in oseltamivir phosphate products using high performance liquid chromatography, wherein the oseltamivir isomers are RSS-isomer, SRR-isomer, RRR-isomer, SSS-isomer, RSR-isomer, and SRS-isomer, and their structural formulas are shown below: Its features are, The method includes the following steps: (1) Prepare a system-suitable solution; Take appropriate amounts of oseltamivir phosphate reference standard, SRR-isomer reference standard, RRR-isomer reference standard, SRS-isomer reference standard, and impurity IV reference standard, and prepare a solution containing 1 mg / ml oseltamivir phosphate and 0.002 mg / ml each of SRR-isomer, RRR-isomer, SRS-isomer, and impurity IV reference standard using a solvent. This is the system suitability solution. The structural formula of impurity IV is shown in formula (I). The solvent is prepared by mixing 0.003 mol / L phosphoric acid aqueous solution with methanol and acetonitrile to form a mixed solution with a volume ratio of 670:245:

135. (2) Prepare the test solution; Take an appropriate amount of the oseltamivir phosphate sample to be tested, and add the solvent mentioned above to prepare a test solution containing oseltamivir phosphate at a concentration of 1 mg / ml. (3) Detection using high performance liquid chromatography The solvent, system suitability solution, and test solution were sequentially injected into the high-performance liquid chromatograph (HPLC), and the chromatograms were recorded. The content of the oseltamivir isomer in the oseltamivir phosphate test sample was calculated using the peak area normalization method. The HPLC conditions were as follows: Chromatographic column: octylsilane-bonded silica gel was used as the packing material, with dimensions of 4.6 mm × 250 mm and a particle size of 5 μm; Mobile phase: phosphate buffer-methanol-acetonitrile, volume ratio 620:245:135~700:245:135; column temperature: 43~47℃; Flow rate: 1.1–1.3 ml / min; Injection volume: 60 μl; Detection wavelength: 207nm.

2. The method according to claim 1, wherein the phosphate is potassium dihydrogen phosphate.

3. The method according to claim 2, wherein the concentration of the potassium dihydrogen phosphate buffer solution is 0.040 mol / L to 0.075 mol / L, and the pH value is 6.25 to 6.

40.

4. The method according to claim 1, wherein the mobile phase is a phosphate buffer-methanol-acetonitrile volume ratio of 670:245:

135.

5. The method according to claim 1, wherein the chromatographic column is a Waters Symmetry C8 column.

6. The method according to claim 1, wherein the chromatographic conditions are: Column temperature: 45℃; Flow rate: 1.2 ml / min; The phosphate buffer solution has a concentration of 0.05 mol / L and a pH value of 6.

3.

7. The method according to claim 1, wherein the chromatographic column is a Waters Symmetry C8 column, the phosphate buffer concentration is 0.05 mol / L and the pH value is 6.3; the mobile phase has a volume ratio of phosphate buffer-methanol-acetonitrile of 670:245:135 and a flow rate of 1.2 ml / min; and the column temperature is 45 °C.

8. The method according to claim 7, wherein the phosphate buffer is a 0.05 mol / L potassium dihydrogen phosphate solution with a pH of 6.

3.

9. The method according to any one of claims 1-8, characterized in that, The oseltamivir phosphate test sample is selected from any one or a combination of two or more of the following: oseltamivir phosphate raw material, oseltamivir phosphate preparation and its intermediate products.

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