Method for detecting related substances in emtricitabine-propofol-tenofovir disoproxil fumarate tablets

Abstract

The invention relates to the technical field of drug analysis and detection, and particularly discloses a method for detecting related substances in emtricitabine, propiophenol and tenofovir disoproxil fumarate tablets. According to the method, an octadecyl silane bonded silica gel chromatographic column is adopted, a mixed solution of a phosphate buffer solution and a tetrahydrofuran-acetonitrile solution in different volume ratios is taken as a mobile phase A and a mobile phase B respectively, and a gradient elution mode is adopted by a dual-wavelength high-performance liquid chromatography; the method can realize effective separation of a plurality of impurities in the emtricitabine, propofol and tenofovir disoproxil fumarate tablet, and accurately detect the condition of the impurities in the emtricitabine, propofol and tenofovir disoproxil fumarate tablet. The methodology research and verification of specificity, sensitivity and the like of the method provided by the invention find that the method provided by the invention is sensitive, accurate and relatively good in reproducibility, and accurate and quantitative detection of the related substances in the emtricitabine, propofol and tenofovir disoproxil tablet can be realized by using a simple, convenient and rapid method; and a reliable guarantee is provided for improving and better controlling the quality of the emtricitabine, propofol and tenofovir disoproxil fumarate tablet product.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical analysis and detection technology, and in particular to a method for detecting related substances in emtricitabine acetonide and tenofovir disoproxil fumarate tablets. Background Technology

[0002] Emtricitabine-tenofovir disoproxil fumarate tablets are a combination nucleoside reverse transcriptase inhibitor composed of emtricitabine (FTC) and tenofovir alafenamide (TAF). Clinically, it is primarily used for the treatment of human immunodeficiency virus (HIV-1) infection and antiviral therapy for chronic hepatitis B (CHB). This formulation, through the synergistic effect of its two active ingredients, can effectively inhibit viral nucleic acid synthesis and possesses advantages such as strong antiviral activity, a high resistance barrier, and low nephrotoxicity and bone toxicity. It has become a first-line drug for the treatment of HIV infection and CHB.

[0003] During the production, storage, and transportation of pharmaceutical preparations, the generation and control of related substances directly affect the safety and efficacy of medication. Related substances in emtricitabine and tenofovir disoproxil fumarate tablets mainly originate from three pathways: First, residual intermediates and byproducts from the synthesis of the active pharmaceutical ingredient (API), such as 5-fluorocytosine derivatives in emtricitabine synthesis and phosphoramide ester impurities in tenofovir disoproxil fumarate preparation; second, degradation products generated during the formulation manufacturing process due to the degradation of the main component caused by conditions such as temperature, humidity, and light, such as cytosine analogs that may be generated from emtricitabine under acidic conditions, and tenofovir (TFV) and its derivatives generated from the hydrolysis of ester bonds in tenofovir disoproxil fumarate; third, interaction products between excipients and the main component, such as antioxidants and fillers in excipients that may chemically react with emtricitabine or tenofovir disoproxil fumarate to generate new impurities.

[0004] Currently, there is no systematic detection scheme for multiple known potential impurities in emtricitabine-tenofovir disoproxil fumarate tablets. Most methods only detect impurities of a single main component, failing to comprehensively assess the overall quality risk of the formulation. Therefore, developing a detection method that can simultaneously separate and accurately quantify emtricitabine, tenofovir disoproxil fumarate, and multiple potential related substances, with higher specificity, sensitivity, and analytical efficiency, has become an urgent need to ensure the safety of emtricitabine-tenofovir disoproxil fumarate tablets and promote the improvement of formulation quality. This method is of great significance for the production quality control and clinical application of this drug. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a method for detecting related substances in emtricitabine acetonide and tenofovir disoproxil fumarate tablets.

[0006] To solve the above-mentioned technical problems, the technical solution provided by the present invention is as follows:

[0007] A method for detecting related substances in emtricitabine acetonide and tenofovir disoproxil fumarate tablets, employing dual-wavelength high-performance liquid chromatography (HPLC), with the following chromatographic conditions:

[0008] Column: Octadecylsilane-bonded silica gel column;

[0009] DAD detector, with detection wavelengths of 258nm~262nm and 238nm~242nm;

[0010] Mobile phase A: a mixed solution of phosphate buffer and tetrahydrofuran-acetonitrile solution with a volume ratio of 98-99:2-1;

[0011] Mobile phase B: a mixture of phosphate buffer and tetrahydrofuran-acetonitrile solution in a volume ratio of 47.5–52.5:52.5–47.5;

[0012] The elution method is gradient elution, and the elution procedure for gradient elution is as follows:

[0013] 0 min, 99% mobile phase A, 1% mobile phase B;

[0014] 5 min, 97% mobile phase A, 3% mobile phase B;

[0015] 8 min, 74% mobile phase A, 26% mobile phase B;

[0016] 19 min, 70% mobile phase A, 30% mobile phase B;

[0017] 26 min, 0% mobile phase A, 100% mobile phase B;

[0018] 28 min, 0% mobile phase A, 100% mobile phase B;

[0019] 30 min, 99% mobile phase A, 1% mobile phase B;

[0020] 35 min, 99% mobile phase A, 1% mobile phase B.

[0021] Compared to existing technologies, the method for detecting related substances in emtricitabine acetonitrile tablets provided by this invention employs an octadecylsilane-bonded silica gel column, using mixed solutions of phosphate buffer and tetrahydrofuran-acetonitrile solution at different volume ratios as mobile phase A and mobile phase B, respectively. Through gradient elution using dual-wavelength high-performance liquid chromatography, it can effectively separate multiple impurities in emtricitabine acetonitrile tablets and accurately detect impurities. Furthermore, methodological studies and validations of specificity and sensitivity have shown that the method provided by this invention is sensitive, accurate, and reproducible, enabling accurate quantitative detection of related substances in emtricitabine acetonitrile tablets using a simple and rapid method. This provides a reliable guarantee for improving and better controlling the quality of emtricitabine acetonitrile tablets and is of great significance for improving medication safety.

[0022] It should be noted that the related substances in emtricitabine and tenofovir disoproxil fumarate tablets include impurities found in both emtricitabine and tenofovir disoproxil fumarate. Specifically, impurities in emtricitabine include 5-FC, FTU, cyclic FTU1, cyclic FTU2, FTC-carboxylic acid, FTC-II, FTC-IV, lamivudine, FTC-thioacetate, and FTC-disulfide; the structures of each impurity are shown in Table 1. Impurities in tenofovir disoproxil fumarate include fumaric acid, PMPA, monophenyl PMPA, phenol, PMPA anhydride, PMPA monoamide, TAF diastereomer 1, GS-7339, PMPA diamide, and TAF diastereomer 2; the structures of each impurity are shown in Table 2.

[0023] Table 1. Structural formulas of impurities related to emtricitabine (FTC)

[0024]

[0025]

[0026] Table 2. Structural formulas of impurities related to tenofovir disoproxil fumarate (TAF).

[0027]

[0028]

[0029]

[0030] It should be noted that the phosphate buffer and tetrahydrofuran-acetonitrile solution in mobile phase A and mobile phase B are the same, except that the volume ratio of phosphate buffer and tetrahydrofuran-acetonitrile solution in mobile phase A and mobile phase B is different.

[0031] Preferably, in both mobile phase A and mobile phase B, the phosphate buffer solution is a potassium dihydrogen phosphate-dipotassium hydrogen phosphate solution with a concentration of 0.018 mol / L to 0.022 mol / L and a pH of 6.7 to 7.1; the volume ratio of tetrahydrofuran to acetonitrile is 68 to 72:32 to 28.

[0032] More preferably, in both mobile phase A and mobile phase B, the concentration of the phosphate buffer is 0.02 mol / L, the pH is 6.9, and the volume ratio of tetrahydrofuran to acetonitrile is 70:30.

[0033] Preferably, in mobile phase A, the volume ratio of phosphate buffer to tetrahydrofuran-acetonitrile solution is 99:1.

[0034] Preferably, in mobile phase B, the volume ratio of phosphate buffer and tetrahydrofuran-acetonitrile solution is 50:50.

[0035] Preferably, the chromatographic column is a Waters BEH-C18 XP, 150mm*3.0mm, 2.5μm.

[0036] The optimal column specifications and mobile phase can ensure excellent peak shape, resolution, and detection sensitivity for each component, with minimal baseline interference. This facilitates the effective separation of various impurities in emtricitabine acetonide tenofovir disoproxil fumarate tablets, resulting in accurate, reliable, and reproducible results.

[0037] Preferably, the flow rate is 0.5 mL / min to 0.7 mL / min, and the column temperature is 20℃ to 30℃.

[0038] More preferably, the flow rate is 0.6 mL / min and the column temperature is 25 °C.

[0039] Preferably, the detection wavelengths are 260nm and 240nm.

[0040] Preferably, the injection volume is 10 μL.

[0041] Optimized detection conditions enable higher separation between various impurities in emtricitabine acetonide tenofovir disoproxil fumarate tablets, ensuring effective detection of impurities and thus achieving effective and accurate control of the content of related substances in emtricitabine acetonide tenofovir disoproxil fumarate tablets.

[0042] It should be noted that the detection method provided by this invention is applicable to the detection of related substances in commercially available emtricitabine acetonide and tenofovir disoproxil fumarate tablets (I) and (II). Emtricitabine acetonide and tenofovir disoproxil fumarate tablets (I) contain 200 mg of emtricitabine and 10 mg of tenofovir disoproxil fumarate per tablet. Emtricitabine acetonide and tenofovir disoproxil fumarate tablets (II) contain 200 mg of emtricitabine and 25 mg of tenofovir disoproxil fumarate per tablet.

[0043] The detection method provided by this invention can simultaneously detect multiple impurities in emtricitabine and tenofovir disoproxil fumarate, accurately detect the impurities in emtricitabine and tenofovir disoproxil fumarate tablets, provide a reliable method for quality control of emtricitabine and tenofovir disoproxil fumarate tablets, and help improve patient medication safety, thus having high practical value. Detailed Implementation

[0044] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0045] Example 1

[0046] 1.1 Materials and Methods:

[0047] Instruments: High performance liquid chromatograph, ultraviolet detector, volumetric flask, electronic balance.

[0048] Reagents: potassium dihydrogen phosphate, phosphate buffer (pH 6.9), tetrahydrofuran, acetonitrile, 5-FC, FTU, emtricitabine, PMPA, PMPA anhydride, monophenyl PMPA, PMPA monoamide, phenol, GS-7339, tenofovir alafenamide fumarate reference standard.

[0049] 1.2 Solution Preparation

[0050] 0.02 mol / L phosphate buffer (pH 6.0): Dissolve 10.88 g of potassium dihydrogen phosphate in 1000 mL of 0.02 mol / L potassium dihydrogen phosphate solution (pH 6.9), add water to bring the volume to 5000 mL, mix well, and the pH value should be 6.0 ± 0.1.

[0051] Solvent A: 0.02 mol / L phosphate buffer (pH 6.0) - 70% tetrahydrofuran in acetonitrile solution at a volume ratio of 99:1.

[0052] Reference solution: Weigh appropriate amounts of emtricitabine and tenofovir alafenamide fumarate reference standards accurately, dissolve them in solvent A and dilute quantitatively to prepare a solution containing 20 μg of emtricitabine and 2.5 μg of tenofovir alafenamide per 1 mL.

[0053] System suitability solution: Accurately weigh 5-FC, FTU, emtricitabine, PMPA, PMPA anhydride, monophenyl PMPA, PMPA monoamide, phenol, GS-7339 and tenofovir alafenamide fumarate reference standards, dissolve in solvent A and quantitatively dilute to prepare a solution containing approximately 5 μg of each in 1 mL.

[0054] Sensitivity solution: Accurately measure 2 mL of the reference solution and place it in a 10 mL volumetric flask. Dilute to the mark with solvent A and shake well.

[0055] Test solution: Take 10 tablets of emtricitabine acetonide tenofovir disoproxil fumarate (II), place them in a 500 mL volumetric flask, add an appropriate amount of solvent A, shake for 60 min to dissolve them, dilute to the mark with solvent A, shake well, filter, and take the filtrate to obtain the test solution.

[0056] 1.3 Conditions for high performance liquid chromatography:

[0057] Column: Waters BEH-C18 XP (150mm*3.0mm, 2.5μm);

[0058] Mobile phase A: 0.02 mol / L phosphate buffer (pH 6.9) with a volume ratio of 99:1 (take 6.00 g of potassium dihydrogen phosphate and 8.20 g of dipotassium hydrogen phosphate, add 4000 mL of water to dissolve and mix well. The pH value should be 6.9 ± 0.1 to obtain) - 70% tetrahydrofuran acetonitrile solution;

[0059] Mobile phase B: 0.02 mol / L phosphate buffer (pH 6.9) - 70% tetrahydrofuran in acetonitrile solution (v / v, 50:50);

[0060] Flow rate: 0.6 mL / min;

[0061] Detection wavelengths: 260nm, 240nm;

[0062] Column temperature, 25℃;

[0063] Injection volume: 10 μL.

[0064] Elution was performed according to the following gradient procedure:

[0065]

[0066]

[0067] System suitability requirements: In the system suitability solution chromatograms, the resolution between the PMPA anhydride and emtricitabine peaks, and between the GS-7339 and tenofovir alafenamide peaks, should meet the requirements; the resolution between the emtricitabine and FTU peaks should be no less than 2.0. In the sensitivity solution chromatograms, the signal-to-noise ratio of the emtricitabine and tenofovir alafenamide peaks should not be less than 10.

[0068] Limits: If the chromatogram of the test solution recorded at a wavelength of 240 nm shows a peak with the same relative retention time as emtricitabine ring FTU1 and ring FTU2, the content shall be calculated by external standard method using emtricitabine as a reference standard and the corrected peak area (multiplied by the correction factor). Ring FTU1 shall not exceed 0.50% of the labeled amount of emtricitabine, and ring FTU2 shall not exceed 0.50% of the labeled amount of emtricitabine. If the chromatogram of the test solution recorded at a wavelength of 260 nm shows a peak with a relative retention time consistent with other known degradation products of emtricitabine (excluding process impurities), the content should be calculated using the external standard method with emtricitabine as a reference and the corrected peak area (multiplied by a correction factor). FTU should not exceed 0.50% of the labeled amount of emtricitabine, and 5-FC should not exceed 0.20% of the labeled amount of emtricitabine. If the chromatogram shows a peak with a relative retention time consistent with known degradation products of tenofovir alafenamide (excluding process impurities), the content should be calculated using the external standard method with tenofovir alafenamide fumarate as a reference and the corrected peak area (multiplied by a correction factor). PMPA should not exceed 3.00% of the labeled amount of tenofovir alafenamide, and PMPA anhydride should not exceed 2% of the labeled amount of tenofovir alafenamide. The content of PMPA monophenyl compounds shall not exceed 0.75% of the labeled amount of tenofovir alafenamide; the content of PMPA monoamide compounds shall not exceed 1.00% of the labeled amount of tenofovir alafenamide; and the content of phenol shall not exceed 2.00% of the labeled amount of tenofovir alafenamide. If other non-specific degradation product peaks are observed, and their UV absorption spectra are consistent with those of emtricitabine or tenofovir alafenamide reference standards, their content shall be calculated using the external standard method with the corresponding reference standard, and shall not exceed 0.20% of the labeled amount of emtricitabine or tenofovir alafenamide. If their UV absorption spectra are inconsistent with those of the two reference standards mentioned above, their content shall be calculated using the peak area method with the external standard method with tenofovir alafenamide fumarate as the reference standard, and shall not exceed 0.20% of the labeled amount of tenofovir alafenamide. The total amount of emtricitabine degradation products shall not exceed 1.2% of the labeled amount of emtricitabine; the total amount of tenofovir alafenamide degradation products shall not exceed 6.0% of the labeled amount of tenofovir alafenamide (impurity peaks with peak areas smaller than the corresponding main peak areas in the sensitivity test solution shall be ignored).

[0069] The relative retention times and correction factors of each impurity in emtricitabine and tenofovir disoproxil fumarate are shown in Tables 3 and 4.

[0070] Table 3. Relative retention times and correction factors for emtricitabine (FTC) related impurities.

[0071]

[0072]

[0073] Table 4. Relative retention times and correction factors for impurities related to tenofovir alafenamide (TAF)

[0074]

[0075]

[0076] Example 2

[0077] Methodological validation:

[0078] 2.1 Specificity

[0079] Solvent A: 0.02 mol / L phosphate buffer (pH 6.0) - 70% tetrahydrofuran in acetonitrile solution at a volume ratio of 99:1.

[0080] Reference solution: Weigh appropriate amounts of emtricitabine and tenofovir alafenamide fumarate reference standards, dissolve them in solvent and dilute quantitatively to prepare a solution containing approximately 0.4 mg of emtricitabine and 50 μg of tenofovir alafenamide per 1 mL. Accurately measure 1 mL of the solution and place it in a 20 mL volumetric flask. Dilute to the mark with solvent A and shake well to obtain the reference solution.

[0081] Sensitivity solution: Accurately measure 2 mL of the above reference solution and place it in a 10 mL volumetric flask. Dilute to the mark with solvent A and shake well to obtain the sensitivity solution.

[0082] 5-FC reference solution: Accurately weigh 5 mg of 5-FC reference standard, place it in a 20 mL volumetric flask, dissolve and dilute to the mark with solvent A, and shake well to obtain the 5-FC reference standard stock solution; then accurately measure 0.2 mL of the 5-FC reference standard stock solution, place it in a 5 mL volumetric flask, dilute to the mark with solvent A, and shake well to obtain the solution.

[0083] Tenofovir alafenamide fumarate (TAF) reference standard positioning solution: Take an appropriate amount of tenofovir alafenamide fumarate reference standard, accurately weigh it, dissolve it in solvent A and quantitatively dilute it to prepare a solution containing about 0.1 mg per 1 mL, shake well, and the solution is ready.

[0084] Emtricitabine (FTC) reference standard positioning solution: Accurately weigh 5 mg of emtricitabine reference standard, place it in a 10 mL volumetric flask, add solvent A to dissolve and dilute to the mark, shake well, and the solution is ready.

[0085] Lamivudine reference solution: Accurately weigh 6 mg of lamivudine reference standard, place it in a 20 mL volumetric flask, add solvent A to dissolve and dilute to the mark, shake well, and the solution is ready.

[0086] FTU reference solution: Accurately weigh 10 mg of FTU reference standard, place it in a 50 mL volumetric flask, add solvent A to dissolve and dilute to the mark, shake well, and the solution is ready.

[0087] Cyclic FTU1 reference solution: Accurately weigh 5 mg of cyclic FTU1 reference standard, place it in a 10 mL volumetric flask, add solvent A to dissolve and dilute to the mark, and use it as the stock solution of cyclic FTU1 impurity reference standard. Then accurately measure 0.2 mL, place it in a 5 mL volumetric flask, dilute to the mark with solvent A, and shake well to obtain the solution.

[0088] Cyclic FTU2 reference solution: Accurately weigh 5 mg of cyclic FTU2 reference standard, place it in a 10 mL volumetric flask, add solvent A to dissolve and dilute to the mark, and use it as the stock solution of cyclic FTU2 impurity reference standard. Then accurately measure 0.2 mL, place it in a 5 mL volumetric flask, dilute to the mark with solvent A, and shake well to obtain the solution.

[0089] PMPA reference solution: Accurately weigh 5 mg of PMPA reference standard, place it in a 10 mL volumetric flask, dissolve and dilute to the mark with solvent A, shake well, and use it as a PMPA impurity reference standard stock solution. Then accurately take 0.2 mL of the solution, place it in a 5 mL volumetric flask, dilute to the mark with solvent A, shake well, and the solution is ready.

[0090] PMPA anhydride reference solution: Accurately weigh 5 mg of PMPA reference standard, place it in a 10 mL volumetric flask, dissolve and dilute to the mark with solvent A, shake well, and use it as a PMPA anhydride impurity reference standard stock solution. Then accurately measure 0.2 mL of the solution, place it in a 5 mL volumetric flask, dilute to the mark with solvent A, shake well, and the solution is ready.

[0091] Monophenyl PMPA reference solution: Accurately weigh 5 mg of monophenyl PMPA reference standard, place it in a 10 mL volumetric flask, dissolve and dilute to the mark with solvent A, shake well, and use it as a stock solution of monophenyl PMPA impurity reference standard. Then accurately measure 0.2 mL of the solution, place it in a 5 mL volumetric flask, dilute to the mark with solvent A, shake well, and the solution is ready.

[0092] PMPA monoamide reference solution: Accurately weigh 5 mg of PMPA monoamide reference standard, place it in a 10 mL volumetric flask, dissolve and dilute to the mark with solvent A, shake well, and use it as the PMPA monoamide impurity reference standard stock solution. Then accurately measure 0.2 mL, place it in a 5 mL volumetric flask, dilute to the mark with solvent A, shake well, and the solution is ready.

[0093] Phenol reference solution: Accurately weigh 5 mg of phenol reference standard, place it in a 10 mL volumetric flask, dissolve and dilute to the mark with solvent A, shake well, and use it as the phenol impurity reference standard stock solution. Then accurately measure 0.2 mL of the stock solution, place it in a 5 mL volumetric flask, dilute to the mark with solvent A, shake well, and the solution is ready.

[0094] PMPA diamide reference solution: Accurately weigh 5 mg of PMPA diamide reference standard, place it in a 10 mL volumetric flask, add solvent A to dissolve and dilute to the mark, shake well, and use it as the mother solution of PMPA diamide impurity reference standard. Then accurately measure 0.2 mL, place it in a 5 mL volumetric flask, dilute to the mark with solvent A, shake well, and the solution is obtained.

[0095] GS-7339 reference solution: Accurately weigh 5 mg of GS-7339 reference standard, place it in a 10 mL volumetric flask, add solvent A to dissolve and dilute to the mark, shake well, and use it as the GS-7339 reference standard stock solution. Then accurately measure 0.2 mL of the stock solution, place it in a 5 mL volumetric flask, dilute to the mark with solvent A, shake well, and the solution is ready.

[0096] TAF diastereomer 1 reference solution: Accurately weigh 5 mg of diastereomer 1 reference standard, place it in a 10 mL volumetric flask, dissolve and dilute to the mark with solvent A, shake well, and use it as the stock solution of diastereomer 1 reference standard. Then accurately measure 0.2 mL of the stock solution, place it in a 5 mL volumetric flask, dilute to the mark with solvent A, shake well, and the solution is obtained.

[0097] TAF diastereomer 2 reference solution: Accurately weigh 5 mg of diastereomer 2 reference standard, place it in a 10 mL volumetric flask, add solvent A to dissolve and dilute to the mark, shake well, and use it as the stock solution of diastereomer 2 reference standard. Then accurately measure 0.2 mL, place it in a 5 mL volumetric flask, dilute to the mark with solvent A, shake well, and the solution is obtained.

[0098] Fumaric acid reference solution: Accurately weigh 5 mg of fumaric acid reference standard, place it in a 10 mL volumetric flask, dissolve and dilute to the mark with solvent A, shake well, and use it as the fumaric acid reference standard stock solution. Then accurately measure 0.2 mL of the stock solution, place it in a 5 mL volumetric flask, dilute to the mark with solvent A, shake well, and the solution is ready.

[0099] FTC-Disulfide Reference Solution: Accurately weigh 5 mg of disulfide reference standard, place it in a 10 mL volumetric flask, dissolve and dilute to the mark with solvent A, shake well, and use it as the stock solution of disulfide reference standard. Then accurately measure 0.2 mL, place it in a 5 mL volumetric flask, dilute to the mark with solvent A, shake well, and the solution is obtained.

[0100] FTC-carboxylic acid reference solution: Accurately weigh 5 mg of FTC-carboxylic acid reference standard, place it in a 10 mL volumetric flask, dissolve and dilute to the mark with solvent A, shake well, and use it as the FTC-carboxylic acid reference standard stock solution. Then accurately measure 0.2 mL of the stock solution, place it in a 5 mL volumetric flask, dilute to the mark with solvent A, shake well, and the solution is ready.

[0101] Emtricitabine Impurity II (FTC-II) Reference Solution: Accurately weigh 5 mg of emtricitabine impurity II reference standard, place it in a 10 mL volumetric flask, dissolve and dilute to the mark with solvent A, and shake well to obtain the emtricitabine impurity II reference standard stock solution. Then accurately measure 0.2 mL of the stock solution, place it in a 5 mL volumetric flask, dilute to the mark with solvent A, and shake well to obtain the final solution.

[0102] Emtricitabine Impurity IV (FTC-IV) Reference Solution: Accurately weigh 5 mg of emtricitabine impurity IV reference standard, place it in a 10 mL volumetric flask, dissolve and dilute to the mark with solvent A, and shake well to obtain the emtricitabine impurity IV reference standard stock solution. Then accurately measure 0.2 mL of the stock solution, place it in a 5 mL volumetric flask, dilute to the mark with solvent A, and shake well to obtain the final solution.

[0103] FTC-thioacetate reference solution: Accurately weigh 5 mg of emtricitabine thioacetate reference standard, place it in a 10 mL volumetric flask, dissolve and dilute to the mark with solvent A, shake well, and use it as the emtricitabine thioacetate reference standard stock solution. Then accurately measure 0.2 mL of the stock solution, place it in a 5 mL volumetric flask, dilute to the mark with solvent A, shake well, and the solution is obtained.

[0104] Mixed impurity reference solution: Accurately measure 0.1 mL each of the TAF reference standard stock solution, TAF impurity stock solutions, FTC reference standard stock solution, and FTC impurity stock solutions, and 1 mL of FTU reference stock solution, place them in the same 10 mL volumetric flask, dissolve and dilute to the mark with solvent A, and shake well to obtain the solution.

[0105] Raw materials + solutions of various impurities: Accurately weigh 80 mg of FTC raw material and 11.2 mg of TAF raw material, measure 0.2 mL each of the TAF impurity mother liquor and the FTC impurity mother liquor and 2 mL of the FTU control mother liquor, put them in the same 20 mL volumetric flask, dissolve and dilute to the mark with solvent A, shake well, and the solution is ready.

[0106] TAF raw material solution: Accurately weigh 11.2 mg of TAF raw material, place it in a 20 mL volumetric flask, add solvent A to dissolve and dilute to the mark, shake well, and the solution is ready.

[0107] FTC raw material solution: Accurately weigh 40mg of FTC raw material, place it in a 10mL volumetric flask, add solvent A to dissolve and dilute to the mark, shake well, and the solution is ready.

[0108] Test solution: Take 10 tablets of emtricitabine acetaminophen tenofovir tablets (II), place them in a 500mL volumetric flask, add an appropriate amount of solvent A, shake for 60min, cool, dilute to the mark with solvent, shake well, filter, and continue to collect the filtrate to obtain the test solution.

[0109] Blank excipient solution: Accurately weigh 132 mg of blank excipient, place it in a 50 mL volumetric flask, add an appropriate amount of solvent A, shake for 60 min, cool, dilute to the mark with solvent, shake well, filter, and continue the filtrate.

[0110] System suitability solution: Accurately measure 0.5 mL of FTU reference stock solution, 0.2 mL each of FTC reference stock solution, 5-FC, PMPA, PMPA anhydride, monophenyl PMPA, PMPA monoamide, phenol, GS-7339 and TAF reference stock solution, place them in the same 20 mL volumetric flask, dilute to the mark with solvent A, and shake well to obtain the solution.

[0111] Accurately measure 10 μl of each of the above solutions and inject them into a liquid chromatograph equipped with a DAD detector, and record the chromatograms. The experimental results are shown in Tables 5 to 7.

[0112] Table 5 Results of Specificity Examination

[0113]

[0114]

[0115] Table 6 Results of Mixed Impurity Investigation (Principal Components at Limit Concentrations)

[0116]

[0117]

[0118] Table 7 Results of Mixed Impurity Investigation (Principal Components at Limit Concentrations)

[0119]

[0120]

[0121] Conclusion: The main component and all impurities showed good absorption at 260 nm wavelength. The solvent and blank excipients did not interfere with the determination. The separation degree between the main component and each impurity was greater than 1.5.

[0122] 2.2 Linear Range

[0123] Five concentration points were selected relatively uniformly within the impurity quantitation limit range to 200% of the limit concentration. A linear regression was performed using the least squares method with peak area as a function of concentration, and the correlation coefficient r was calculated. The results are shown in Tables 8 to 20.

[0124] Table 8 Results of the investigation of 5-FC linear relationship

[0125]

[0126] Conclusion: 5-FC showed a significant linear relationship with its peak response value in the range of 16.300 μg / mL to 0.064 μg / mL. The linear equation was: A = 34192C - 265.07, the correlation coefficient r = 1.0000, and the intercept of the linear regression line was equivalent to 0.10% of the limit concentration response value.

[0127] Table 9 Results of the investigation into the linear relationship of PMPA

[0128]

[0129] Conclusion: PMPA showed a significant linear relationship with its peak response value in the range of 30.017 μg / mL to 0.117 μg / mL. The linear equation was: A = 50510C + 2736, the correlation coefficient was r = 1.0000, and the intercept of the linear regression line was equivalent to 0.36% of the limit concentration response value.

[0130] Table 10. Examination of Lamiff's Fixed Linearity Relationship

[0131]

[0132] Conclusion: Lamivudine reference solution showed a significant linear relationship with its peak response value in the concentration range of 15.940 μg / mL to 0.031 μg / mL. The linear equation was: A = 33802C - 355, the correlation coefficient r = 1.0000, and the intercept of the linear regression line was equivalent to 0.13% of the limit concentration response value.

[0133] Table 11 Results of the linear relationship investigation of PMPA anhydride

[0134]

[0135] Conclusion: PMPA anhydride showed a significant linear relationship with its peak response value in the range of 23.853 μg / mL to 0.093 μg / mL. The linear equation was: A = 33276C + 688, the correlation coefficient r = 1.0000, and the intercept of the linear regression line was equivalent to 0.17% of the limit concentration response value.

[0136] Table 12 Results of the FTC linear relationship examination

[0137]

[0138] Conclusion: FTC showed a significant linear relationship with its peak response value in the range of 39.952 μg / mL to 0.078 μg / mL. The linear equation was: A = 23435C + 2693, with a correlation coefficient r = 1.0000. The intercept of the linear regression line was equivalent to 0.57% of the limit concentration response value.

[0139] Table 13 Results of the FTU Linearity Examination

[0140]

[0141] Conclusion: FTU showed a significant linear relationship with its peak response value in the range of 39.702 μg / mL to 0.155 μg / mL. The linear equation was: A = 29817C + 732, the correlation coefficient was r = 1.0000, and the intercept of the linear regression line was equivalent to 0.12% of the limit concentration response value.

[0142] Table 14 Results of the investigation into the linear relationship of the FTU2 ring

[0143]

[0144] Conclusion: Cyclic FTU2 showed a significant linear relationship with its peak response value in the range of 39.706 μg / mL to 0.620 μg / mL. The linear equation was: A = 4751C + 2141, the correlation coefficient r = 1.0000, and the intercept of the linear regression line was equivalent to 2.34% of the limit concentration response value.

[0145] Table 15 Results of the investigation into the linear relationship of the FTU1 ring.

[0146]

[0147] Conclusion: Cyclic FTU1 showed a significant linear relationship with its peak response value in the range of 40.396 μg / mL to 0.631 μg / mL. The linear equation was: A = 3635C + 363, the correlation coefficient was r = 1.0000, and the intercept of the linear regression line was equivalent to 0.49% of the limit concentration response value.

[0148] Table 16 Results of linearity investigation of monophenyl PMPA

[0149]

[0150] Conclusion: Monobenzene PMPA showed a significant linear relationship with its peak response value in the range of 7.498 μg / mL to 0.029 μg / mL. The linear equation was: A = 35228C + 520, with a correlation coefficient r = 1.0000. The intercept of the linear regression line was equivalent to 0.39% of the limit concentration response value.

[0151] Table 17 Results of linearity analysis of PMPA monoamides

[0152]

[0153] Conclusion: PMPA monoamide showed a significant linear relationship with its peak response value in the range of 9.757 μg / mL to 0.152 μg / mL. The linear equation was: A = 20010C + 912, the correlation coefficient was r = 0.9999, and the intercept of the linear regression line was equivalent to 0.92% of the limit concentration response value.

[0154] Table 18 Results of linearity investigation of phenol

[0155]

[0156] Conclusion: Phenol showed a significant linear relationship with its peak response value in the range of 21.093 μg / mL to 0.659 μg / mL. The linear equation was: A = 8290.7C + 273.1, with a correlation coefficient r = 0.9999. The intercept of the linear regression line was equivalent to 0.31% of the limit concentration response value.

[0157] Table 19 Results of Linearity Examination for GS-7339

[0158]

[0159] Conclusion: The isomer showed a significant linear relationship with its peak response value in the range of 2.006 μg / mL to 0.063 μg / mL. The linear equation was: A = 19219C - 69.875, the correlation coefficient r = 0.9998, and the intercept of the linear regression line was equivalent to 0.37% of the limit concentration response value.

[0160] Table 20 Results of TAF Linear Relationship Examination

[0161]

[0162] Conclusion: TAF showed a significant linear relationship with its peak response value in the range of 4.883 μg / mL to 0.076 μg / mL. The linear equation was: A = 26229C + 6880.6, the correlation coefficient r = 0.9997, and the intercept of the linear regression line was equivalent to 9.94% of the limit concentration response value.

[0163] 2.3 Sensitivity

[0164] The linear solution was serially diluted with solvent. The lowest detection limit was defined as the concentration where the peak height was more than 3 times the baseline noise, and the quantitation limit was defined as the concentration where the peak height was more than 10 times the baseline noise. The quantitation limit solution was injected six times consecutively, and the RSD of the retention time was calculated. The results are shown in the table below.

[0165] The linear solution was serially diluted with solvent. The lowest detection limit was defined as the concentration where the peak height was more than three times the baseline noise, and the quantitation limit was defined as the concentration where the peak height was more than ten times the baseline noise. The quantitation limit solution was injected six times consecutively, and the RSD of the retention time was calculated. The results are shown in Tables 21 and 22.

[0166] Table 21 Results of the Limit of Quantitation Study

[0167]

[0168]

[0169] Table 22 Results of the Detection Limit Study

[0170] Concentration (ng / mL) Signal-to-noise ratio PMPA 29.3 5.78、8.25 PMPA anhydride 23.3 4.28、6.21 Monophenyl PMPA 7.3 8.83、14.20 PMPA monoamide 38.1 3.28、2.92 phenol 329.6 6.04、7.85 5-FC 15.9 3.13、5.32 FTU 38.8 4.23、6.79 FTC-carboxylic acid 15.5 6.02、6.06 Lamivudine 15.6 7.73、7.68 FTC-II 31.9 7.22、6.89 GS-7339 63.7 7.43、6.88 FTU2 Ring 310.2 5.30、5.41 Ring FTU1 315.6 4.24、4.32

[0171] Conclusions: The limit of quantification (LOQ) for PMPA was 58.63 ng / mL, with an RSD of less than 2% for retention time across six determinations; the limit of detection (LOD) was 29.3 ng / mL. The LQ for PMPA anhydride was 46.59 ng / mL, with an RSD of less than 2% for retention time across six determinations; the LOD was 23.3 ng / mL. The LQ for monophenyl PMPA was 14.64 ng / mL, with an RSD of less than 2% for retention time across six determinations; the LOD was 7.3 ng / mL. The LQ for PMPA monoamide was... The limit of quantitation (LOQ) for phenol was 152.4 ng / mL, with an RSD of less than 2% for retention time across 6 assays; the limit of detection (LOD) was 38.1 ng / mL. The limit of quantitation (LOQ) for phenol was 659.1 ng / mL, with an RSD of less than 2% for retention time across 6 assays; the LOD was 329.6 ng / mL. The limit of quantitation (LOQ) for 5-FC was 31.84 ng / mL, with an RSD of less than 2% for retention time across 6 assays; the LOD was 15.9 ng / mL. The limit of quantitation (LOQ) for FTU was 77.54 ng / mL, with a retention time of less than 2% across 6 assays. The RSD of the four assays was less than 2%; the limit of detection was 38.8 ng / mL. The limit of quantitation for FTC-carboxylic acid was 30.98 ng / mL, and the RSD of the retention time over six assays was less than 2%; the limit of detection was 15.5 ng / mL. The limit of quantitation for lamivudine was 31.13 ng / mL, and the RSD of the retention time over six assays was less than 2%; the limit of detection was 15.6 ng / mL. The limit of quantitation for FTC-II was 63.87 ng / mL, and the RSD of the retention time over six assays was less than 2%; the limit of detection was 3... The limit of quantitation (LOQ) for GS-7339 was 127.35 ng / mL, with an RSD of less than 2% for retention time across 6 assays; the limit of detection (LOD) was 63.7 ng / mL. The LOQ for cyclic FTU2 was 620.41 ng / mL, with an RSD of less than 2% for retention time across 6 assays; the LOD was 310.2 ng / mL. The LOQ for cyclic FTU1 was 631.19 ng / mL, with an RSD of less than 2% for retention time across 6 assays; the LOD was 315.6 ng / mL.

[0172] 2.4 Precision

[0173] Repeated examination

[0174] Solvent A: 0.02 mol / L phosphate buffer (pH 6.0) - 70% tetrahydrofuran in acetonitrile solution at a volume ratio of 99:1.

[0175] Reference solution: Weigh appropriate amounts of emtricitabine and tenofovir alafenamide fumarate reference standards, dissolve them in solvent A and dilute quantitatively to prepare a solution containing approximately 0.4 mg of emtricitabine and 50 μg of tenofovir alafenamide per 1 mL. Accurately measure 1 mL of the solution and place it in a 20 mL volumetric flask. Dilute to the mark with solvent and shake well.

[0176] Test solution: Take emtricitabine acetonide tenofovir tablets (II) powder, and prepare 6 parallel recovery solutions with 100% impurity limit concentration according to the accuracy section. Take the filtrate.

[0177] Accurately measure 10 μL each of the reference solution and the test solution, and inject them sequentially into a liquid chromatograph equipped with a DAD detector. Record the chromatograms, and calculate the recovery rate and RSD of each impurity. The results are shown in Table 23.

[0178] Table 23 Repeatability Test Results - Emtricitabine & Tenofovir Tablets (II)

[0179] impurities Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 mean RSD% 5-FC 97.70 98.49 97.70 98.11 97.95 98.26 98.04 0.32 PMPA 99.17 99.14 98.96 99.23 98.51 99.81 99.14 0.42 PMPA anhydride 101.5 101.5 101.2 101.4 100.6 102.1 101.4 0.49 FTU 99.73 97.94 99.29 98.21 99.04 98.77 98.83 0.68 Monobenzene PMPA 100.8 99.39 99.89 99.72 98.96 99.74 99.74 0.60 PMPA monoamide 91.27 90.81 89.94 91.21 90.60 91.29 99.85 0.58 phenol 103.8 103.9 102.4 102.8 101.7 105.3 103.3 1.24 GS-7339 96.94 96.95 95.90 99.35 97.88 97.61 97.43 1.19

[0180] Conclusion: The RSD of the results of the six test solutions were all less than the limit (10%), indicating good repeatability.

[0181] Intermediate precision test

[0182] Six test samples were tested by different analysts in the same laboratory at different times, following the method described in the repeatability section above. The recoveries and RSDs of each impurity were calculated. The results are shown in Table 24.

[0183] Table 24 Intermediate Precision Study Results - Emtricitabine & Propofol Tenofovir Tablets (II)

[0184]

[0185]

[0186] Conclusion: In the intermediate precision test, the RSDs of all 6 test solutions were less than the limit (10%), indicating good repeatability. Combined with the repeatability data from the 6 sets, the RSDs of all 12 test solutions were less than the limit (20%), indicating that the intermediate precision test met the requirements.

[0187] 2.5 Accuracy

[0188] Solvent: 0.02 mol / L phosphate buffer (pH 6.0) - 70% tetrahydrofuran in acetonitrile solution (99:1).

[0189] Mother liquor for impurity recovery rate: Accurately weigh 4 mg of impurity 5-FC, place it in a 50 mL volumetric flask, dissolve and dilute to the mark with solvent, shake well, and use it as mother liquor for recovery rate 1; accurately weigh 10 mg of impurity FTU, place it in a 50 mL volumetric flask, dissolve and dilute to the mark with solvent, shake well, and use it as mother liquor for recovery rate 2; accurately weigh 7.5 mg of impurity PMPA, 6.25 mg of PMPA anhydride, and 5 mg of phenol, place them in the same 50 mL volumetric flask, and then accurately transfer 3.75 mL of monobenzene PMPA mother liquor, 5 mL of PMPA monoamide mother liquor, and 1 mL of GS-7339 mother liquor under the specificity investigation item (1), dissolve and dilute to the mark with solvent, shake well, and use it as mother liquor for recovery rate 3.

[0190] 50% recovery solution: Accurately weigh 70 mg of [emtricitabine acetaminophen tenofovir tablets (II)] powder, place it in a 10 mL volumetric flask, then accurately add 0.5 mL of the impurity recovery mother liquor, add an appropriate amount of solvent, shake for 60 minutes, cool, dilute to the mark with solvent, shake well, filter, and collect the filtrate. Prepare 3 parallel solutions using the same method.

[0191] 100% recovery solution: Accurately weigh 70 mg of [emtricitabine acetaminophen tenofovir tablets (II)] powder, place it in a 10 mL volumetric flask, then accurately add 1 mL of the impurity recovery mother solution, add an appropriate amount of solvent, shake for 60 minutes, cool, dilute to the mark with solvent, shake well, filter, and collect the filtrate. Prepare 3 parallel solutions using the same method.

[0192] 150% recovery solution: Accurately weigh 70 mg of [emtricitabine acetaminophen tenofovir tablets (II)] powder, place it in a 10 mL volumetric flask, then accurately add 1.5 mL of the impurity recovery mother liquor, add an appropriate amount of solvent, shake for 60 minutes, cool, dilute to the mark with solvent, shake well, filter, and collect the filtrate. Prepare 3 parallel solutions using the same method.

[0193] Reference solution: Accurately measure 1 mL of the mother solution for each recovery rate, place it in a 10 mL volumetric flask, dilute to the mark with solvent, and shake well.

[0194] Accurately measure 10 μl each of the reference solution and the test solution, and inject them sequentially into the liquid chromatograph. Record the chromatograms, and calculate the recovery rate and RSD of each impurity. The results are shown in Tables 25 to 35.

[0195] Table 25 Results of 5-FC Accuracy Study

[0196]

[0197]

[0198] Table 26 Results of PMPA Accuracy Assessment

[0199]

[0200] Table 27 Results of the Accuracy Study of Lamivudine

[0201]

[0202] Table 28 Results of PMPA Anhydride Accuracy Test

[0203]

[0204] Table 29 FTU Accuracy Evaluation Results

[0205]

[0206] Table 30 Results of Accuracy Assessment of FTU2 Ring

[0207]

[0208] Table 31 Results of Accuracy Assessment of Ring FTU1

[0209]

[0210] Table 32 Results of the Accuracy Study of Monophenyl PMPA

[0211]

[0212] Table 33 Results of Accuracy Study of PMPA Monoamide Compounds

[0213]

[0214]

[0215] Table 34 Results of Phenol Accuracy Test

[0216]

[0217] Table 35 GS-7339 Accuracy Test Results

[0218]

[0219] Conclusion: The recovery rates of all impurities were between 80% and 120%, and the RSD of the nine recovery rate determination results were all less than 5%, indicating good method accuracy.

[0220] 2.6 Durability

[0221] Take emtricitabine acetonide tenofovir tablets (II) and prepare the test solution and reference solution according to the specificity section. Investigate the effects of minor variations in parameters such as flow rate, column temperature, mobile phase ratio, aqueous phase pH, detection wavelength and column on the determination results of related substances.

[0222] 1) Flow velocity investigation

[0223] Condition 1: 0.5 mL / min.

[0224] Condition 2: 0.7 mL / min.

[0225] Accurately measure 10 μL each of the reference solution, system suitability solution, and test solution, inject them into the liquid chromatograph, record the chromatograms, and examine the results as shown in Tables 36 and 37.

[0226] Table 36 Flow velocity test results - System suitability

[0227]

[0228] Table 37 Flow velocity test results - test samples

[0229]

[0230] Conclusion: Flow rate changes (±0.1 mL / min) had no significant impact on the determination of impurity content, and the method demonstrated good flow rate robustness.

[0231] 2) Column temperature investigation

[0232] Condition 1: 20℃.

[0233] Condition 2: 30℃.

[0234] Accurately measure 10 μl each of the reference solution, system suitability solution, and test solution, inject them into the liquid chromatograph, record the chromatograms, and examine the results as shown in Tables 38 and 39 below.

[0235] Table 38 Durability Column Temperature Test Results - System Suitability

[0236]

[0237] Table 39 Durability Test Results at Column Temperature - Test Samples

[0238]

[0239] Conclusion: The column temperature variation (±5℃) and impurity content both meet the requirements, indicating that the method has good column temperature durability.

[0240] 3) Investigation of the proportion of mobile phase B

[0241] Condition 1: Aqueous phase - organic phase (47.5:52.5).

[0242] Condition 2: Aqueous phase - organic phase (52.5:47.5).

[0243] Accurately measure 10 μl each of the reference solution, system suitability solution and test solution, inject them into the liquid chromatograph, record the chromatograms, and the results are shown in Tables 40 and 41.

[0244] Table 40 Results of the Durability Assessment of Mobile Phase B Proportion - System Suitability

[0245]

[0246] Table 41 Results of the Investigation on the Proportion of Mobile Phase B for Durability - Test Samples

[0247]

[0248] Conclusion: With slight variations (±2.5%) in the proportion of mobile phase B, the impurity content remained within the specified limits, demonstrating the good robustness of this method in terms of mobile phase proportions.

[0249] 4) Different detection wavelengths

[0250] Condition 1: 258nm.

[0251] Condition 2: 262nm.

[0252] Accurately measure 10 μl each of the reference solution, system suitability solution and test solution, inject them into the liquid chromatograph, record the chromatograms, and the results are shown in Tables 42 and 43.

[0253] Table 42 Durability Wavelength Test Results - System Suitability

[0254]

[0255] Table 43 Durability Wavelength Test Results - Test Samples

[0256]

[0257] Conclusion: Changes in detection wavelength (±2nm) have no significant impact on the determination of impurity content, and the method exhibits good wavelength robustness.

[0258] 5) pH value of aqueous phase

[0259] Condition 1: Aqueous phase pH 7.1.

[0260] Condition 2: Aqueous phase pH 6.7.

[0261] Accurately measure 10 μl each of the reference solution, system suitability solution, and test solution, inject them into the liquid chromatograph, record the chromatograms, and examine the results as shown in Tables 44 and 45.

[0262] Table 44 Results of Durability Testing in Aqueous Phase pH Values ​​- System Suitability

[0263]

[0264] Table 45 Results of Aqueous Phase pH Test on Durability of Test Samples

[0265]

[0266] Conclusion: Changes in the pH value of the aqueous phase (±0.2) had no significant impact on the determination of impurity content, and the method showed good robustness in terms of aqueous phase pH.

[0267] 2.7 Solution stability

[0268] Solvent A: 0.02 mol / L phosphate buffer (pH 6.0) - 70% tetrahydrofuran in acetonitrile solution (99:1).

[0269] Reference solution: Weigh appropriate amounts of emtricitabine and tenofovir alafenamide fumarate reference standards, dissolve and quantitatively dilute with solvent A to prepare a solution containing approximately 0.4 mg emtricitabine and 50 μg tenofovir alafenamide per 1 mL. Accurately measure 1 mL, place it in a 20 mL volumetric flask, dilute to the mark with solvent A, and shake well.

[0270] Test solution: Take 10 tablets of emtricitabine propionol tenofovir tablets (II), place them in a 500mL volumetric flask, add an appropriate amount of solvent A, shake for 60min, cool, dilute to the mark with solvent A, shake well, filter, and take the filtrate.

[0271] After the test solution and reference solution were prepared, they were placed at 2-8℃. At 0h, 1h, 2h, 4h, 6h and 8h, 10μL of each solution was accurately measured and injected into the liquid chromatograph. The chromatograms were recorded, and the changes in the peak area of ​​the main component and each impurity over time were investigated. The results are shown in Tables 46 and 47.

[0272] Table 46 Results of Solution Stability Study - Peak Area of ​​Test Sample

[0273] # 0h 1h 2h 4h 6h 8h RSD% fumaric acid 463148 463151 464588 462554 462855 461319 0.23 5-FC 17248 17071 17353 17269 17328 17079 0.71 FTC Unknown Impurities 1 15888 15762 15764 15568 15718 15721 0.66 FTC Unknown Impurities 2 45428 46081 47220 47920 49222 50190 3.82 PMPA 39315 39207 39152 39139 39294 38955 0.33 FTC Unknown Impurities 3 N / A 7374 7373 6938 7254 7127 2.56 FTC Unknown Impurities 4 26058 26063 26542 26591 26688 26807 1.21 FTC 31107082 31601762 30535558 30549454 30407688 30791762 1.46 FTU 16261 16091 16530 16022 16193 16638 1.50 Monophenyl PMPA 5836 5959 6103 6171 6211 6373 3.11 FTC Unknown Impurities 5 22824 22744 22864 22679 22721 22594 0.43 FTC Unknown Impurities 6 89822 89038 89457 88789 89083 88770 0.46 TAF 14161755 14122863 14081413 14002513 14018149 13940634 0.59

[0274] Table 47 Results of Solution Stability Study - Peak Area of ​​Reference Standard

[0275] # 0h 1h 2h 4h 6h 8h RSD% FTC 463251 462558 461347 463143 462347 461991 0.16 TAF 65840 64108 61029 62448 64802 55621 5.93

[0276] Conclusion: During the investigation period, the RSD of the peak area of ​​the principal component and each impurity peak at each time point was less than 10%, and the solution was stable within 8 hours.

[0277] Comparative Example 1

[0278] Exclusivity Examination

[0279] By changing the elution procedure, we can examine the method specificity. The elution procedure is shown in the table below:

[0280]

[0281]

[0282] Other conditions were as described in Example 2.1, and the tests were conducted according to law. The results showed that the retention times of fumaric acid, 5-FC, FTC-carboxylic acid, FTC-II, PMPA, PMPA anhydride, FTC, FTC-IV, and FTU were significantly prolonged. Specifically, fumaric acid, 5-FC, FTC-carboxylic acid, FTC-II, PMPA, and PMPA anhydride in the mixed solution could not be effectively separated, and FTC and FTU could not be effectively separated. This demonstrates that changing the elution procedure of this invention will prevent the effective separation of related substances from emtricitabine acetaminophen tenofovir tablets (II).

[0283] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for detecting related substances in emtricitabine acetonide and tenofovir disoproxil fumarate tablets, characterized in that, The detection was performed using dual-wavelength high-performance liquid chromatography (HPLC), and the chromatographic conditions were as follows: Column: Octadecylsilane-bonded silica gel column; DAD detector, with detection wavelengths of 258nm~262nm and 238nm~242nm; Mobile phase A: a mixed solution of phosphate buffer and tetrahydrofuran-acetonitrile solution with a volume ratio of 98-99:2-1; Mobile phase B: a mixture of phosphate buffer and tetrahydrofuran-acetonitrile solution in a volume ratio of 47.5–52.5:52.5–47.5; The elution method is gradient elution, and the elution procedure for gradient elution is as follows: 0 min, 99% mobile phase A, 1% mobile phase B; 5 min, 97% mobile phase A, 3% mobile phase B; 8 min, 74% mobile phase A, 26% mobile phase B; 19 min, 70% mobile phase A, 30% mobile phase B; 26 min, 0% mobile phase A, 100% mobile phase B; 28 min, 0% mobile phase A, 100% mobile phase B; 30 min, 99% mobile phase A, 1% mobile phase B; 35 min, 99% mobile phase A, 1% mobile phase B.

2. The method for detecting related substances in emtricitabine acetonide and tenofovir disoproxil fumarate tablets as described in claim 1, characterized in that, In both mobile phase A and mobile phase B, the phosphate buffer solution is a potassium dihydrogen phosphate-dipotassium hydrogen phosphate solution with a concentration of 0.018 mol / L to 0.022 mol / L and a pH of 6.7 to 7.1; the volume ratio of tetrahydrofuran to acetonitrile is 68 to 72:32 to 28.

3. The method for detecting related substances in emtricitabine acetonide and tenofovir disoproxil fumarate tablets as described in claim 2, characterized in that, In both mobile phase A and mobile phase B, the concentration of the phosphate buffer is 0.02 mol / L, the pH is 6.9, and the volume ratio of tetrahydrofuran to acetonitrile is 70:

30.

4. The method for detecting related substances in emtricitabine acetonide and tenofovir disoproxil fumarate tablets as described in claim 1, characterized in that, In mobile phase A, the volume ratio of phosphate buffer to tetrahydrofuran-acetonitrile solution is 99:

1.

5. The method for detecting related substances in emtricitabine acetonide and tenofovir disoproxil fumarate tablets as described in claim 1, characterized in that, In mobile phase B, the volume ratio of phosphate buffer to tetrahydrofuran-acetonitrile solution is 50:

50.

6. The method for detecting related substances in emtricitabine acetonide and tenofovir disoproxil fumarate tablets as described in claim 1, characterized in that, The chromatographic column was a Waters BEH-C18 XP, 150mm*3.0mm, 2.5μm.

7. The method for detecting related substances in emtricitabine acetonide and tenofovir disoproxil fumarate tablets as described in claim 1, characterized in that, The flow rate was 0.5 mL / min to 0.7 mL / min, and the column temperature was 20℃ to 30℃.

8. The method for detecting related substances in emtricitabine acetonide and tenofovir disoproxil fumarate tablets as described in claim 7, characterized in that, The flow rate was 0.6 mL / min, and the column temperature was 25 °C.

9. The method for detecting related substances in emtricitabine acetonide and tenofovir disoproxil fumarate tablets as described in claim 1, characterized in that, The detection wavelengths are 260nm and 240nm.

10. The method for detecting related substances in emtricitabine acetonide and tenofovir disoproxil fumarate tablets as described in claim 1, characterized in that, The injection volume was 10 μL.

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