Detection method of tenofovir alafenamide isomers in fumarate

Abstract

The present invention relates to the technical field of drug analysis and detection, and specifically to a method for detecting isomers of tenofovir alafenamide fumarate. The method for detecting isomers of tenofovir alafenamide fumarate comprises: chromatographic column: chiral chromatographic column; mobile phase: n-hexane, anhydrous ethanol, methanol, diethylamine; detection wavelength: 260nm; column temperature: 30°C; flow rate: 1.0ml / min; solution preparation; high performance liquid chromatography detection; the chiral chromatographic column is a normal phase chromatographic column with a derivative of amylose as the stationary phase; the volume ratio of n-hexane, anhydrous ethanol, and methanol of the mobile phase is 65:25:10, and the volume of diethylamine is 0.1% of the total volume of the first three. The present invention provides a method for detecting isomers of tenofovir alafenamide fumarate, which effectively separates isomer impurities, has high sensitivity and good specificity, accurately evaluates the isomer content of raw materials, and is of great significance to the safety of clinical medication.

Description

Technical Field

[0001] The present invention relates to the technical field of drug analysis and detection, and in particular to a method for detecting tenofovir alafenamide fumarate isomers. Background Art

[0002] Isomers refer to compounds with the same chemical formula but different structures. Although the chemical composition of isomers is the same, the atoms are arranged in different ways in space, which makes their physical, chemical properties or toxicity very different. In the process of drug development, all chiral compounds need to be studied for their isomeric impurities. Since isomers only differ in spatial structure and have basically no difference in polarity, it is difficult to meet the separation requirements on conventional reversed-phase high-performance liquid chromatography (RP-HPLC) and common chromatographic columns. Generally, the retention time on RP-HPLC overlaps or the separation is less than 1.5. It is common to use normal phase chromatography and chiral chromatographic columns to study them so that their separation is greater than 1.5, which meets the system suitability requirements.

[0003] When the common normal phase chromatography method is used to detect isomers, the mobile phase is mostly organic solvents such as n-hexane, anhydrous ethanol, and isopropanol, which are used to separate one or two isomers. Tenofovir alafenamide fumarate (TAF) contains three chiral centers in its structure, and there will be configuration flipping during the synthesis process. Theoretically, there are 8 isomers (including the main component). According to the research on tenofovir alafenamide fumarate, only the target configuration (RRS configuration) is the active ingredient. In the pharmaceutical field, the control of impurities in chemical drugs is related to the quality and safety of drugs. Therefore, it is very meaningful to accurately analyze and detect the relevant substances of chemical drugs, especially isomers, and control their content.

[0004] Patent CN01813161.1 Nucleotide phosphate analog prodrugs and screening and preparation methods thereof discloses an analytical method using reverse phase HPLC to separate and detect diastereoisomers of tenofovir alafenamide fumarate, with a single detection structure.

[0005] Patent CN111189947A discloses an analytical method for separating and detecting isomers of tenofovir alafenamide fumarate, using a daicel bonded chiral chromatographic column CHIRALPAK IC column, the mobile phase is a mixed solution of one or more of n-hexane, isopropanol, methanol, ethanol, methyl tert-butyl ether, tetrahydrofuran, dichloromethane, chloroform, ethyl acetate and acetonitrile, the additive is diethylamine or ethylenediamine, and the separated isomeric impurities are diastereoisomers SSS, diastereoisomers RSR, enantiomers SRR, phosphorus isomers RRS, diastereoisomers RRR, diastereoisomers SRS, diastereoisomers SSR, and its Example 2 and attached Figure 5 It shows that the combination and proportion of mobile phase and the choice of column cannot meet the requirements of baseline separation in method detection. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a method for detecting the isomers of tenofovir alafenamide fumarate, which is a detection method for effectively separating multiple isomeric impurities. The detection method has high sensitivity and good specificity, and can accurately evaluate the isomer content of the raw material drug, which is of great significance to the safety of clinical drug use.

[0007] The target compound of the tenofovir alafenamide isomer of the present invention is of RRS configuration. Toxicological data of each isomer impurity has not been obtained yet. The quality of the raw material can only be determined according to the detection of the original preparation and shall not be lower than that of the original preparation. Therefore, it is necessary to establish its isomer method to control the isomer content. The TAF-6-A reference substance used in the present invention is a racemate of TAF-6-A and TAF (free state). The configuration and isomer configuration of the target compound of tenofovir alafenamide fumarate, as well as the limit conditions in the raw material are as described in Table 1:

[0008] Table 1 Configuration and limit of tenofovir alafenamide fumarate isomers

[0009]

[0010]

[0011] The method for detecting tenofovir alafenamide fumarate isomers of the present invention comprises:

[0012] (1) Chromatographic column: chiral chromatographic column;

[0013] (2) Mobile phase: n-hexane, anhydrous ethanol, methanol, diethylamine;

[0014] (3) Detection wavelength: 258nm~262nm;

[0015] (4) Column temperature: 25°C to 35°C;

[0016] (5) Flow rate: 0.9 ml / min to 1.1 ml / min;

[0017] (6) Preparation of solution;

[0018] (7) High performance liquid chromatography detection;

[0019] The chiral chromatographic column is a normal phase chromatographic column with a derivative of amylose as the stationary phase;

[0020] The volume ratio of n-hexane, anhydrous ethanol and methanol in the mobile phase is 65:25:10, and the volume of diethylamine is 0.1% of the total volume of n-hexane, anhydrous ethanol and methanol.

[0021] The chromatographic column is a CHIRALPAK AD-H column, which has a particle size of 5 μm, an inner diameter of 4.6 mm, and a length of 250 mm.

[0022] The detection wavelength was 260 nm, the column temperature was 30 °C, the flow rate was 1.0 ml / min, the injection volume was 20 μl, and the running time was 60 min.

[0023] The detection concentration of tenofovir alafenamide fumarate isomers is 0.04μg / ml~2.5μg / ml.

[0024] The preparation of the solution of the present invention comprises:

[0025] (1) Preparation of isomer reference solution: Take each isomer reference (all racemic) and weigh accurately, place in a volumetric flask, add diluent to dilute, and prepare to obtain the required concentration;

[0026] (2) Preparation of system suitability solution: Take the tenofovir alafenamide fumarate raw material, accurately weigh it, place it in a volumetric bottle, add the isomer reference solution, dilute it, and prepare it to obtain the required concentration;

[0027] (3) Preparation of test solution: Take the tenofovir alafenamide fumarate raw material, accurately weigh it, place it in a volumetric flask, dilute it, and prepare it to the required concentration.

[0028] The diluent is anhydrous ethanol.

[0029] The calculation formula for the isomer content of tenofovir alafenamide fumarate is:

[0030]

[0031] in:

[0032] A 样 is the peak area of ​​the isomer in the test sample;

[0033] A 对 is the peak area of ​​the isomer in the reference substance;

[0034] C 样 is the concentration of the test article;

[0035] C 对 is the concentration of the reference substance.

[0036] Specifically, the method for detecting tenofovir alafenamide fumarate isomers comprises:

[0037] (1) Preparation of blank solvent: Use diluent as blank solvent and anhydrous ethanol as blank solvent;

[0038] (2) Preparation of isomer stock solutions: accurately weigh about 20 mg each of the isomers TAF-6-A, TAF-6-A1 and TAF-6-A3 and about 10 mg of TAF-6-A2, place them in four different 20 ml volumetric flasks, add diluent to dissolve and dilute to the mark, and shake well;

[0039] (3) Preparation of system suitability solution: Take about 10 mg of tenofovir alafenamide fumarate API, accurately weigh, place in a 20 ml volumetric flask, add 0.2 ml of each isomer stock solution in step (2), add diluent to dissolve and dilute to scale, and shake well;

[0040] (4) Preparation of isomer reference solution: accurately measure 1 ml of each isomer stock solution in step (2) and place it in a 100 ml volumetric flask, dilute to the mark with a diluent, and shake well; accurately measure 2 ml of each isomer stock solution and place it in a 20 ml volumetric flask, dilute to the mark with a diluent, and shake well;

[0041] (5) Prepare the test solution: Take about 10 mg of tenofovir alafenamide fumarate raw material, accurately weigh it, place it in a 20 ml volumetric flask, add diluent to dissolve and dilute to the scale, and shake well;

[0042] (6) Detection was performed using high performance liquid chromatography.

[0043] Preferably, the HPLC conditions are preferably:

[0044] Chromatographic column: CHIRALPAK AD-H chiral chromatographic column, with a particle size of 5 μm, an inner diameter of 4.6 mm, and a length of 250 mm;

[0045] Column temperature: 30°C;

[0046] Detection wavelength: 260nm;

[0047] Flow rate: 1.0ml / min;

[0048] Injection volume: 20 μl.

[0049] The present invention adopts diluent to prepare reference solution, system suitability solution and test solution. The raw material drug is easily soluble in anhydrous ethanol, so anhydrous ethanol is preferably used as the diluent and blank solvent.

[0050] The invention adopts a normal phase chiral chromatographic column with a derivative of amylose as a stationary phase, and a mobile phase of n-hexane, anhydrous ethanol and methanol, with a specific volume ratio of 65:25:10. The mobile phase has a strong elution ability, especially methanol, so that the separation degree between various isomers is improved and isocratic elution is performed; diethylamine is selected as a sweeping agent to improve the isomer peak shape, and the added volume of diethylamine is 0.1% of the total volume of n-hexane, anhydrous ethanol and methanol; in order to make the separation degree between various isomers meet the requirements and the running time is appropriate, the flow rate is preferably 1.0 ml / min.

[0051] The selection of chiral chromatographic column and mobile phase is particularly important in the normal phase chromatography method. The mobile phases of n-hexane, anhydrous ethanol, diethylamine and methanol are different from the conventional normal phase system.

[0052] Compared with the prior art, the present invention has the following beneficial effects:

[0053] (1) The present invention uses high performance liquid chromatography to detect tenofovir alafenamide fumarate isomers, with high detection sensitivity, a minimum detection limit of 0.01 μg / ml (S / N of 3.9), and a minimum quantification limit of 0.04 μg / ml (S / N of 10.7), which meets the requirements for low-concentration isomer detection;

[0054] (2) The linear range of the method of the present invention is wide. Within the concentration range of quantification limit to 500% of the limit concentration (0.04 μg / ml to 2.5 μg / ml), the peak area and concentration of each isomer impurity show a good linear relationship, and the correlation coefficient R 2 All greater than 0.999;

[0055] (3) The accuracy of the method of the present invention is good. The recoveries of the isomeric impurities in the test solution spiked at three levels of 50%, 100% and 150% are all between 90.0% and 110.0%, and the results are reliable. The solution has good stability. The system suitability solution, reference solution and test solution are injected continuously for 24 hours. The RSD of the peak area % of each isomeric impurity in the system suitability solution is between 0.02% and 1.21%. The difference in the detected amount of isomeric impurities in the test solution is small, and the accurate quantification of each isomeric impurity in this product can be achieved. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 Typical chromatograms of the chromatographic column screening in Example 1 Figure 1 ;

[0057] Figure 2 Typical chromatograms of the chromatographic column screening in Example 1 Figure 2 ;

[0058] Figure 3 Typical chromatogram of mobile phase screening in Example 1 Figure 1 ;

[0059] Figure 4 Typical chromatogram of mobile phase screening in Example 1 Figure 2 ;

[0060] Figure 5 It is the specific overlay diagram of Example 2;

[0061] Figure 6 It is the overlay of detection limit and quantitation limit of Example 3;

[0062] Figure 7 is the linear graph of peak 1 isomer impurity of TAF-6-A1 of Example 4;

[0063] Figure 8 This is the linear graph of the peak 2 isomer impurity of TAF-6-A1 in Example 4;

[0064] Fig. 9 is the linear impurity diagram of TAF-6-A isomers in Example 4;

[0065] Fig.10 is the TAF linear graph of Example 4;

[0066] Fig.11 is the linear graph of peak 1 isomer impurity of TAF-6-A3 of Example 4;

[0067] Fig.12 This is the linear graph of the peak 2 isomer impurity of TAF-6-A3 in Example 4;

[0068] Fig.13 is the linear impurity diagram of TAF-6-A2 isomers in Example 4;

[0069] Fig.14 is the HPLC chromatogram of the system suitability solution in Example 7;

[0070] Fig.15 is the HPLC chromatogram of the reference substance solution in Example 7;

[0071] Fig.16 is the HPLC chromatogram of the test solution in Example 7;

[0072] Fig.17 is the HPLC chromatogram of the system suitability solution in Comparative Example 1;

[0073] Fig.18 is the HPLC chromatogram of the system suitability solution in Comparative Example 2;

[0074] Fig.19 It is the HPLC graph of the system suitability solution in Comparative Example 3. DETAILED DESCRIPTION

[0075] The present invention is further described below with reference to the embodiments.

[0076] The raw materials and reagents used in the examples are shown in Table 2:

[0077] Table 2 Raw materials and reagents

[0078] Reagents level factory purity n-Hexane AR Tianjin Fuchen Chemical Reagent Factory / Anhydrous ethanol AR Tianjin Guangfu Technology Development Co., Ltd. / Methanol HPLC Fisher / Diethylamine AR Tianjin Fuchen Chemical Reagent Factory / TAF-6-A control Reference substance (racemate) Beijing Zhongshuo Pharmaceutical Technology Development Co., Ltd. 96.4％ TAF-6-A1 control Reference substance (racemate) Beijing Zhongshuo Pharmaceutical Technology Development Co., Ltd. 96.4％ TAF-6-A2 Reference Reference substance (racemate) Beijing Zhongshuo Pharmaceutical Technology Development Co., Ltd. 95.9％ TAF-6-A3 reference Reference substance (racemate) Beijing Zhongshuo Pharmaceutical Technology Development Co., Ltd. 96.2％ Tenofovir Alafenamide Fumarate sample Beijing New Leading Pharmaceutical Technology Development Co., Ltd. 100.1％

[0079] The instruments and equipment used in the examples are shown in Table 3:

[0080] Table 3 Instruments and Equipment

[0081]

[0082]

[0083] Example 1

[0084] Establishment of detection method and optimization of conditions:

[0085] Limit regulations:

[0086] Isomer impurities are controlled as general impurities, and according to the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) guidelines, the limit is set at 0.10% as a reference for the development of detection methods.

[0087] Each isomeric impurity has a maximum absorption wavelength at around 260nm, and the detection wavelength can be selected from 258nm to 262nm, and 260nm is preferred in this embodiment; when the injection volume is 20μl, the minimum peak height of each isomeric impurity is 0.5mV, which can meet the sensitivity requirement of the method, so the test sample concentration is determined to be 0.5mg / ml, the isomeric reference concentration is 0.5μg / ml, and the system suitability solution and impurity location solution are prepared for each isomeric impurity at a concentration of 1% of the test sample.

[0088] Column screening:

[0089] The concentration of the test sample was 0.5 mg / ml, and the system suitability solution and impurity location solution were prepared according to the concentration of each isomer impurity of 1% of the test sample. The chromatographic column was screened using a high performance liquid chromatograph. The screening conditions and screening results are shown in Table 4:

[0090] Table 4 Screening conditions and results of chromatographic columns

[0091]

[0092] Screening of mobile phase:

[0093] The concentration of the test sample was 0.5 mg / ml, and the system suitability solution of each isomer impurity was prepared according to the concentration of 1% of the test sample. The mobile phase was screened using a high performance liquid chromatograph. The screening type, screening volume ratio and screening results are shown in Table 5:

[0094] Table 5 Screening types and screening volume ratios of mobile phases and screening results

[0095]

[0096] The conditions for selecting the validation analysis method and the impurity calculation formula are as follows:

[0097] Chromatographic column: CHIRALPAK AD-H (4.6mm×250mm, 5μm)

[0098] Mobile phase: n-hexane: anhydrous ethanol: methanol: diethylamine, volume ratio 65:25:10:0.1

[0099] Column temperature: 30°C

[0100] Flow rate: 1.0ml / min

[0101] Wavelength: 260nm

[0102] Injection volume: 20 μl

[0103] Running time: 60 minutes

[0104] Test sample concentration: 0.5mg / ml

[0105] Diluent: anhydrous ethanol

[0106]

[0107] A 样 is the peak area of ​​the isomer in the test sample;

[0108] A 对 is the peak area of ​​the isomer in the reference substance;

[0109] C 样 is the concentration of the test article;

[0110] C 对 is the concentration of the reference substance.

[0111] Example 2

[0112] Verify system suitability and specificity:

[0113] Solution preparation:

[0114] Blank solvent: anhydrous ethanol, i.e. diluent;

[0115] Isomer stock solution: respectively eliminate about 20 mg of the isomers TAF-6-A, TAF-6-A1 and TAF-6-A3, and about 10 mg of TAF-6-A2, accurately weigh, and place in four different 20 ml volumetric flasks, add diluent to dissolve and dilute to the scale to obtain isomer stock solution, wherein the concentrations of TAF-6-A, TAF (free state), TAF-6-A1 peak 1, TAF-6-A1 peak 2, TAF-6-A3 peak 1, TAF-6-A3 peak 2 and TAF-6-A2 peak are about 0.5 mg / ml, respectively;

[0116] System suitability solution: take about 10 mg of tenofovir alafenamide fumarate bulk drug, accurately weigh, place in a 20 ml volumetric flask, add 0.2 ml of each of the above isomer stock solutions, add diluent to dissolve and dilute to the scale to obtain a system suitability solution, wherein the concentration of tenofovir alafenamide fumarate is about 0.5 mg / ml, and the concentrations of TAF-6-A, TAF-6-A1 peak 1, TAF-6-A1 peak 2, TAF-6-A3 peak 1, TAF-6-A3 peak 2 and TAF-6-A2 peak are about 5 μg / ml respectively;

[0117] Isomer reference solution: accurately measure 1 ml of each of the above isomer stock solutions and place them in a 100 ml volumetric flask, dilute to the mark with a diluent, and shake well; accurately measure 2 ml of the above solution and place it in a 20 ml volumetric flask, dilute to the mark with a diluent, and shake well to obtain a reference solution, wherein the concentrations of TAF-6-A, TAF (free state), TAF-6-A1 peak 1, TAF-6-A1 peak 2, TAF-6-A3 peak 1, TAF-6-A3 peak 2, and TAF-6-A2 peak are approximately 0.5 μg / ml, respectively;

[0118] Test solution: Take about 10 mg of tenofovir alafenamide fumarate raw material, accurately weigh, place in a 20 ml volumetric flask, add diluent to dissolve and dilute to the scale to obtain the test solution, the concentration of tenofovir alafenamide fumarate is about 0.5 mg / ml.

[0119] The injection procedure is as shown in Table 6.

[0120] Table 6 System suitability and specificity injection procedure

[0121] serial number Solution name Injection times 1 Blank solvent ≥1 2 System Suitability Solutions 1 3 Reference solution 1 1 4 Reference solution 2 1 5 Test solution 1

[0122] The measurement results are shown in Table 7.

[0123] Table 7 System suitability and specificity results

[0124]

[0125] According to Table 6, we can obtain the specificity overlay diagram, such as Figure 5From the overlay of the specificity, it can be seen that there is no interfering peak in the blank solvent at the elution point of each isomer impurity; and there is no impurity in the test solution that interferes with the isomer detection, and the specificity of this method is good; the system suitability solution has good separation between each isomer impurity and between the isomer impurity and the main peak, the theoretical plate number of each isomer chromatographic peak is greater than 5000, and the tailing factor is less than 1.5, and the system suitability is good.

[0126] Embodiment 3:

[0127] Verify the detection limit and quantification limit:

[0128] Solution preparation:

[0129] Blank solvent: anhydrous ethanol, i.e. diluent;

[0130] Isomer stock solution: respectively eliminate about 20 mg of the isomers TAF-6-A, TAF-6-A1 and TAF-6-A3, and about 10 mg of TAF-6-A2, accurately weigh, and place in four different 20 ml volumetric flasks, add diluent to dissolve and dilute to the scale to obtain isomer stock solution, wherein the concentrations of TAF-6-A, TAF (free state), TAF-6-A1 peak 1, TAF-6-A1 peak 2, TAF-6-A3 peak 1, TAF-6-A3 peak 2 and TAF-6-A2 peak are about 0.5 mg / ml, respectively;

[0131] Isomer localization solution: take 1 ml of each isomer stock solution and place it in four different 50 ml volumetric flasks, dilute to the scale with diluent, shake well to obtain isomer localization solution, in which the concentrations of TAF-6-A, TAF (free state), TAF-6-A1 peak 1, TAF-6-A1 peak 2, TAF-6-A3 peak 1, TAF-6-A3 peak 2 and TAF-6-A2 peak are about 10 μg / ml respectively;

[0132] Quantitative limit solution: Take 0.1 ml of TAF-6-A and TAF-6-A1 isomer positioning solutions, 0.3 ml of TAF-6-A2 isomer positioning solution and 0.2 ml of TAF-6-A3 isomer positioning solution and put them into the same 20 ml volumetric flask, dilute to the mark with diluent, shake well to obtain the quantitative limit solution, in which the concentrations of TAF-6-A, TAF (free state), TAF-6-A1 peak 1 and TAF-6-A1 peak 2 are all 0.05 μg / ml; the concentrations of TAF-6-A3 peak 1 and TAF-6-A3 peak 2 are all 0.1 μg / ml; the concentration of TAF-6-A2 peak is 0.15 μg / ml;

[0133] Detection limit solution: Take 3 ml of the quantitative limit solution and place it in a 10 ml volumetric flask, dilute it to the scale with diluent, shake well to obtain the detection limit solution, in which the concentrations of TAF-6-A, TAF (free state), TAF-6-A1 peak 1, and TAF-6-A1 peak 2 are all 0.015 μg / ml; the concentrations of TAF-6-A3 peak 1 and TAF-6-A3 peak 2 are both 0.03 μg / ml; the concentration of TAF-6-A2 peak is 0.045 μg / ml.

[0134] The injection procedure is as shown in Table 8:

[0135] Table 8 Detection limit and quantification limit injection procedure of the detection method

[0136] serial number Solution name Injection times 1 Blank solvent ≥1 2 Quantitative limit solution 1 3 Detection limit solution 1

[0137] The quantitative limit and detection limit were tested according to the injection procedure in Table 8. The test spectrum was as follows: Figure 6 The detection limit and quantification limit are shown in the overlay graph, and the test results are shown in Table 9:

[0138] Table 9 Quantitation limit and detection limit test results

[0139]

[0140]

[0141] It can be seen from the data in Table 9 above that the signal-to-noise ratio of the quantitative limit solution of each isomer impurity is about 10, and the concentration is far less than the reporting limit of 0.05%. The signal-to-noise ratio of the detection limit is about 3, and the concentration is one-third of the quantitative limit, indicating that the method has a high sensitivity.

[0142] Example 4

[0143] Verify linearity and range:

[0144] Solution preparation:

[0145] Blank solvent: anhydrous ethanol, i.e. diluent;

[0146] Isomer stock solution: respectively eliminate about 20 mg of the isomers TAF-6-A, TAF-6-A1 and TAF-6-A3, and about 10 mg of TAF-6-A2, accurately weigh, and place in four different 20 ml volumetric flasks, add diluent to dissolve and dilute to the scale to obtain isomer stock solution, wherein the concentrations of TAF-6-A, TAF (free state), TAF-6-A1 peak 1, TAF-6-A1 peak 2, TAF-6-A3 peak 1, TAF-6-A3 peak 2 and TAF-6-A2 peak are about 0.5 mg / ml, respectively;

[0147] Linear stock solution: Take 5 ml of each of the above isomer stock solutions and place them in the same 50 ml volumetric flask, dilute to the mark with diluent, shake well to obtain linear stock solution, wherein the concentrations of TAF-6-A, TAF (free state), TAF-6-A1 peak 1, TAF-6-A1 peak 2, TAF-6-A3 peak 1, TAF-6-A3 peak 2 and TAF-6-A2 peak are approximately 0.05 mg / ml respectively;

[0148] Quantitation limit solution: Take 0.1 ml of the above TAF-6-A and TAF-6-A1 isomer positioning solutions, 0.3 ml of the TAF-6-A2 isomer positioning solution, and 0.2 ml of the TAF-6-A3 isomer positioning solution, respectively, and place them in the same 20 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the quantitation limit solution;

[0149] Linear 50% (limit concentration) solution: Take 5 ml of the linear stock solution and place it in a 100 ml volumetric flask, dilute it to the mark with a diluent, and shake well; then accurately measure 1 ml of the above solution and place it in a 10 ml volumetric flask, dilute it to the mark with a diluent, and shake well;

[0150] Linear 100% (limiting concentration) solution: Take 1 ml of the linear stock solution and place it in a 100 ml volumetric flask, dilute to the mark with diluent, and shake well;

[0151] Linear 200% (limit concentration) solution: Take 5 ml of the linear stock solution and place it in a 25 ml volumetric flask, dilute it to the mark with a diluent, and shake well; then accurately measure 1 ml of the above solution and place it in a 10 ml volumetric flask, dilute it to the mark with a diluent, and shake well;

[0152] Linear 500% (limiting concentration) solution: Take 5 ml of the linear stock solution and place it in a 100 ml volumetric flask, dilute to the scale with diluent, and shake well.

[0153] Perform the linearity test according to the injection procedure in Table 10:

[0154] Table 10 Linearity test injection procedure

[0155] serial number Solution name Injection times 1 Blank solvent ≥1 2 Quantitative limit solution 1 3 Linear 50% (limit concentration) solution 1 4 Linear 100% (limit concentration) solution 1 5 Linear 200% (limit concentration) solution 6 Linear 500% (limit concentration) solution 1

[0156] According to the above solution, the linear test results of high performance liquid phase analysis are shown in Table 11:

[0157] Table 11 Linearity test results

[0158]

[0159] Within the concentration range of the quantitative limit to 500% of each isomer impurity, a linear regression line is drawn with the isomer concentration as the horizontal axis and the peak area as the vertical axis. The linear regression coefficient r 2All are greater than 0.999, with good linearity. Figure 7-13 Linear graph shown.

[0160] Example 5

[0161] Verify the accuracy of the system:

[0162] Solution preparation: The preparation of blank solvent, isomer stock solution, isomer reference solution, and test solution is the same as in Example 2;

[0163] Accuracy stock solution: Take 1 ml of each isomer stock solution and place it in the same 100 ml volumetric flask, dilute to the mark with diluent, and shake well;

[0164] Spiked test solution (50% limit concentration): Take about 10 mg of tenofovir alafenamide fumarate raw material, accurately weigh, place in a 20 ml volumetric flask, add 1 ml of accuracy stock solution, add diluent to dissolve and dilute to the scale, shake well; prepare 3 copies in the same way;

[0165] Spiked test solution (100% limit concentration): Take about 10 mg of tenofovir alafenamide fumarate raw material, accurately weigh, place in a 20 ml volumetric flask, add 2 ml of accuracy stock solution, add diluent to dissolve and dilute to the scale, shake well; prepare 3 copies in the same way;

[0166] Spiked test solution (150% limit concentration): Take about 10 mg of tenofovir alafenamide fumarate raw material, accurately weigh, place in a 20 ml volumetric flask, add 3 ml of accuracy stock solution, add diluent to dissolve and dilute to the scale, shake well; prepare 3 portions in the same way.

[0167] The injection procedure for the accuracy experiment is shown in Table 12:

[0168] Table 12 Injection procedure for accuracy experiment

[0169] serial number Solution name Injection times 1 Blank solvent ≥1 2 Reference solution 1 1 3 Reference solution 2 1 4 Test solution 1 5 Spiked test solution (50% limit concentration -1) 1 6 Spiked test solution (50% limit concentration - 2) 1 7 Spiked test solution (50% limit concentration -3) 1 8 Spiked test solution (100% limit concentration - 1) 1 9 Spiked test solution (100% limit concentration - 2) 1 10 Spiked test solution (100% limit concentration - 3) 1 11 Spiked test solution (150% limit concentration - 1) 1 12 Spiked test solution (150% limit concentration - 2) 1 13 Spiked test solution (150% limit concentration - 3) 1

[0170] By measuring the recovery of spiked samples of different concentrations ((actual measured amount - target content in sample solution) / added amount × 100%), the recovery of each isomer at three concentration levels was between 90.0% and 110.0%, and the recovery of each concentration level and the recovery RSD of all levels were less than 10.0%, which proved that the method had good sample recovery and high accuracy. The test results are shown in Table 13.

[0171] Table 13 Results of spike recovery experiments for each isomer

[0172]

[0173]

[0174] Example 6

[0175] Verify the stability of the solution:

[0176] Preparation of solutions: The preparation of blank solvent, isomer stock solution, isomer reference solution, test solution, and system suitability solution is the same as in Example 2.

[0177] At room temperature, the experimental injection procedure for solution stability is shown in Table 14:

[0178] Table 14 Stability test injection procedure

[0179]

[0180]

[0181] After being placed at room temperature for 5h, 8h, 12h, and 24h, the RSD of the peak area % of each isomer impurity in the system suitability solution was measured to be between 0.02% and 1.21%, and there was basically no difference in the amount of isomer impurities detected in the test solution, indicating that the solution was stable after being placed at room temperature for 24h. The stability test results of the test solution and the system suitability solution at 5h, 8h, 12h, and 24h are shown in Table 15.

[0182] Table 15 Stability test results

[0183]

[0184] Example 7

[0185] The detection method of the present invention is used to detect isomers in the tenofovir alafenamide fumarate raw material drug.

[0186] Diluent: anhydrous ethanol.

[0187] Isomer stock solution: Dissolve approximately 20 mg each of the isomers TAF-6-A, TAF-6-A1 and TAF-6-A3 and approximately 10 mg of TAF-6-A2, accurately weigh and place in four different 20 ml volumetric flasks, add diluent to dissolve and dilute to the scale, and shake well.

[0188] System suitability solution: Take about 10 mg of tenofovir alafenamide fumarate raw material, accurately weigh it, put it in a 20 ml volumetric flask, add 0.2 ml of each of the above isomer stock solutions, add diluent to dissolve and dilute to the scale, and shake well.

[0189] Isomer reference solution: Accurately measure 1 ml of each of the above isomer stock solutions and place them in a 100 ml volumetric flask, dilute to the mark with diluent, and shake well; then accurately measure 2 ml of the above solution and place it in a 20 ml volumetric flask, dilute to the mark with diluent, and shake well.

[0190] Test solution: Take 10 mg of tenofovir alafenamide fumarate raw material, accurately weigh it, put it in a 20 ml volumetric flask, add diluent to dissolve and dilute to the scale, and shake well.

[0191] High performance liquid chromatography was used for detection, and the specific chromatographic conditions are shown in Table 16:

[0192] Table 16 Chromatographic conditions

[0193]

[0194]

[0195] After testing, the HPLC chromatograms of the system suitability solution, reference solution and test solution are as follows: Fig.14 , Fig.15 and Fig.16 As shown in the analytical spectrum, the peaks of each isomer impurity have good shape and high separation.

[0196] According to the formula Calculate the isomer content in the sample;

[0197] Among them, the peak area of ​​each isomer impurity in the test solution is 0, so the isomers in the test sample are calculated to be undetectable.

[0198] Comparative Example 1

[0199] Comparative Example 1 and Example 7, the sample preparation, chromatographic conditions, detection steps are all the same, only the mobile phase is changed to: n-hexane: anhydrous ethanol: diethylamine (75:25:0.1), the detection results are as follows Fig.17 HPLC chromatogram of system suitability solution, Fig.17 It can be seen that after removing methanol, the isomer peaks overlap with the main peak, the separation does not meet the requirements, and cannot be used to detect isomers.

[0200] Comparative Example 2

[0201] Comparative Example 1 and Example 7, the sample preparation, chromatographic conditions, and detection steps are all the same, only the chromatographic column is replaced with: CHIRALPAK AY-H (4.6mm×250mm, 5μm), and the detection results are as follows: Fig.18 HPLC chromatogram of system suitability solution, Fig.18It can be seen that after changing to CHIRALPAK AY-H chromatographic column, the peak shapes of the isomer impurities and main components are poor, which does not meet the system suitability requirements and cannot be used to detect isomers.

[0202] Comparative Example 3

[0203] Comparative Example 1 and Example 7, the sample preparation, chromatographic conditions, and detection steps are all the same, only the chromatographic column is replaced with: CHIRALPAK OD-H (4.6mm×250mm, 5μm), and the detection results are as follows: Fig.19 HPLC chromatogram of system suitability solution, Fig.19 It can be seen that after changing to the CHIRALPAK OD-H column, the peak shapes of the isomer impurities and main components are poor, which does not meet the system suitability requirements and cannot be used to detect isomers.

Claims

1. A method for detecting isomers of tenofovir alafenamide fumarate, characterized in that: include: (1) Chromatographic column: chiral chromatographic column; (2) Mobile phase: n-hexane, anhydrous ethanol, methanol, diethylamine; (3) Detection wavelength: 258nm~262nm; (4) Column temperature: 25℃~35℃; (5) Flow rate: 0.9 ml / min~1.1 ml / min; (6) Preparation of solution; (7) High performance liquid chromatography detection; The chiral chromatographic column is a CHIRALPAK AD-H column; The volume ratio of the mobile phase n-hexane, anhydrous ethanol and methanol is 65:25:10, the volume of diethylamine is 0.1% of the total volume of n-hexane, anhydrous ethanol and methanol; the mobile phase elution mode is isocratic elution; The name and structural formula of the tenofovir alafenamide fumarate isomer are as follows: TAF-6-A: ， TAF-6-A1: ， TAF-6-A2: ， TAF-6-A3: 。 2. The method for detecting tenofovir alafenamide fumarate isomers according to claim 1, characterized in that: The chromatographic column is a CHIRALPAK AD-H column with a particle size of 5 µm, an inner diameter of 4.6 mm, and a length of 250 mm.

3. The method for detecting tenofovir alafenamide fumarate isomers according to claim 1, characterized in that: The detection wavelength was 260 nm, the column temperature was 30 °C, the flow rate was 1.0 ml / min, the injection volume was 20 µl, and the running time was 60 min.

4. The method for detecting tenofovir alafenamide fumarate isomers according to claim 1, characterized in that: The solution preparation includes: (1) Preparation of isomer reference solution: Take each isomer reference, which is a racemic isomer, accurately weigh it, place it in a volumetric bottle, add a diluent to dilute it, and prepare it to obtain the required concentration; (2) Preparation of system suitability solution: Take the tenofovir alafenamide fumarate API, accurately weigh it, place it in a volumetric flask, add the isomer reference solution, dilute it, and prepare the required concentration; (3) Preparation of test solution: Take the tenofovir alafenamide fumarate raw material, accurately weigh it, place it in a volumetric flask, dilute it, and prepare it to the required concentration.

5. The method for detecting tenofovir alafenamide fumarate isomers according to claim 4, characterized in that: The diluent is anhydrous ethanol.

6. The method for detecting tenofovir alafenamide fumarate isomers according to claim 1, characterized in that: The calculation formula for the isomer content of tenofovir alafenamide fumarate is: Isomer percentage = in: A 样 is the peak area of ​​the isomer in the test sample; A 对 is the peak area of ​​the isomer in the reference substance; C 样 is the concentration of the test article; C 对 is the concentration of the reference substance.

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