Separation and detection method of tenofovir and enantiomer

Abstract

The invention provides a tenofovir and enantiomer separation and detection method, and belongs to the field of pharmaceutical analysis. Octadecylsilane chemically bonded silica is adopted as a filler chromatographic column, a mobile phase is a mixed solution of a mixed aqueous solution (D-phenylalanine, copper sulfate pentahydrate and ammonium dihydrogen phosphate) and methanol, and tenofovir and enantiomers can be effectively separated and accurately determined. The method disclosed by the invention is simple, convenient, high in sensitivity, good in separation degree and reproducibility and accurate and reliable in result, and can be used for detecting tenofovir diastereoisomers and enantiomers.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis and relates to a high performance liquid chromatography separation and detection method for tenofovir and its enantiomers. Background Art

[0002] Tenofovir is a nucleotide reverse transcriptase inhibitor that has an inhibitory effect on human immunodeficiency virus (HIV) and hepatitis B virus (HBV). The drug can inhibit viral polymerase by directly competing with natural deoxyribose substrates and terminate the DNA chain by inserting into DNA. In vitro, this product can effectively fight a variety of viruses, including those strains that are resistant to nucleoside reverse transcriptase inhibitors. The chemical name is: R-9-(2-phosphomethoxypropyl) adenine, and its chemical structure is as follows:

[0003]

[0004] Tenofovir has a chiral center. According to the synthesis process of tenofovir and the starting materials used in the synthesis, it can be known that enantiomers exist in the synthesis route of this product.

[0005] Since enantiomers are difficult to separate on ordinary HPLC, the development of a method for detecting the enantiomers of tenofovir is necessary to detect and separate the enantiomers of tenofovir, which can ensure the effectiveness and safety of tenofovir products.

[0006] Isomers produced during the preparation of tenofovir need to be strictly controlled, whether in raw materials or preparations. Currently, there is no enantiomer detection method in the pharmacopoeias of various countries. The enantiomer detection methods of tenofovir in the literature all use chiral chromatographic columns, which are expensive, have certain restrictions on the use of mobile phases, and are more troublesome to maintain during use.

[0007] Therefore, it is necessary to develop a simple, feasible, sensitive and specific method for analyzing and detecting tenofovir and its enantiomers for the quality control of tenofovir, which is of great significance for ensuring the safety of tenofovir. Summary of the invention

[0008] The present invention provides a high performance liquid chromatography separation and detection method for tenofovir and its enantiomers. The method has the advantages of simplicity, high efficiency and high accuracy, and can accurately reflect the content of isomers in tenofovir raw materials, thereby better controlling and mastering product quality and improving the safety of clinical medication.

[0009] In view of the deficiencies in the background technology, the present invention provides a method for detecting tenofovir enantiomers, which has the advantages of simplicity, high efficiency and high accuracy, and can accurately reflect the content of enantiomers in tenofovir raw materials, thereby better controlling and mastering product quality and improving the safety of clinical medication.

[0010] The present invention adopts the following technical solutions:

[0011] The present invention provides a high performance liquid chromatography for effectively separating tenofovir and its enantiomers.

[0012] Analysis method. The method comprises: determining according to high performance liquid chromatography, using an octadecylsilane bonded silica gel chromatography column, using phosphate buffer (take 0.16g of D-phenylalanine, 0.76g of copper sulfate pentahydrate and 2.31g of ammonium dihydrogen phosphate, add 1000ml of water to dissolve, and adjust the pH value to 4.0 with ammonia water)-methanol as the mobile phase, and performing isocratic elution under certain conditions. It comprises the following steps:

[0013] Detection on high performance liquid chromatography

[0014] Chromatographic column filler: octadecylsilane bonded silica gel

[0015] Column temperature: 33℃-37℃

[0016] Flow rate: 0.8mL / min-1.2mL / min

[0017] Detection wavelength: 262nm

[0018] Mobile phase: phosphate buffer (take 0.16g D-phenylalanine, 0.76g copper sulfate pentahydrate and 2.31g ammonium dihydrogen phosphate, add 1000ml water to dissolve, and adjust the pH value to 4.0 with ammonia water) - methanol

[0019] Diluent: Water

[0020] Preparation of system suitability solution: Take appropriate amount of enantiomer reference substance and tenofovir reference substance, dissolve in water and dilute to a mixed solution containing about 1 mg of tenofovir and 5 μg of enantiomer per 1 ml, as the system suitability solution;

[0021] Sample solution preparation: Take the tenofovir sample to be tested and use the diluent to prepare a solution with a concentration of 0.8mg-1.2mg / ml as the test solution;

[0022] The chromatographic column uses octadecylsilane bonded silica gel as a filler, preferably an Inertsil ODS-SP (250 mm×4.6 mm, 5 μm) chromatographic column.

[0023] The volume ratio of the mobile phase phosphate buffer (take 0.16g of D-phenylalanine, 0.76g of copper sulfate pentahydrate and 2.31g of ammonium dihydrogen phosphate, add 1000ml of water to dissolve, and adjust the pH value to 4.0 with ammonia water)-methanol is 80-95:20-5, preferably 85-95:15-5.

[0024] The column temperature is 33°C-37°C, preferably 35°C.

[0025] The flow rate is 0.8 mL / min-1.2 mL / min, preferably 1.0 mL / min.

[0026] The wavelength is 262 nm.

[0027] The peaks in the system suitability solution can achieve baseline separation, with a resolution of ≥1.5.

[0028] The detection method provided by the present invention can effectively separate tenofovir from its enantiomers, ensure the effectiveness of the detection, and accurately and strictly control the quality of tenofovir. The method has strong specificity, high sensitivity, good accuracy and durability. DETAILED DESCRIPTION

[0029] In order to explain the present invention in detail, the following is further described in conjunction with specific embodiments. The following embodiments are only used to illustrate the present invention and do not limit the protection scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The HPLC chromatogram of the solvent in Example 1 is

[0031] Figure 2 4 is the HPLC chromatogram of the system suitability solution in Example 1.

[0032] Figure 3 It is the HPLC chromatogram of the tenofovir test solution in Example 1.

[0033] Example 1

[0034] Specificity test

[0035] Adopt the method in the present invention.

[0036] Chromatographic column: Inertsil ODS-SP (250mm×4.6mm, 5μm)

[0037] Mobile phase: phosphate buffer (take 0.16g D-phenylalanine, 0.76g copper sulfate pentahydrate and 2.31g ammonium dihydrogen phosphate, add 1000ml water to dissolve, and adjust the pH value to 4.0 with ammonia water) - methanol, the volume ratio is 90:10

[0038] Flow rate: 1.0ml / min

[0039] Column temperature: 35°C

[0040] Detection wavelength: 262nm

[0041] Injection volume: 20 μl

[0042] Experimental steps:

[0043] Blank solvent: water

[0044] Preparation of system suitability solution: Take appropriate amount of enantiomer reference substance and tenofovir reference substance, dissolve in water and dilute to a mixed solution containing about 1 mg of tenofovir and 5 μg of enantiomer per 1 ml, as the system suitability solution;

[0045] Preparation of sample solution: Take an appropriate amount of this product, weigh it accurately, place it in a volumetric flask, add water to dissolve and dilute it to make a 1 mg / ml solution.

[0046] Preparation of control solution: Take an appropriate amount of sample solution and dilute it quantitatively with water to make a solution containing about 5 μg / ml.

[0047] Take each of the above solutions, inject them into a high performance liquid chromatograph respectively, and record the chromatogram.

[0048] The results of blank solvent are shown in the attached Figure 1 .

[0049] The results of the system suitability solution are attached. Figure 2 .

[0050] The results of the sample solution are shown in the attached Figure 3 .

[0051] Results: The blank solvent did not interfere with the detection of tenofovir and its enantiomers, and the method had good specificity. Figure 2 The peaks from left to right are enantiomers and tenofovir, with a separation degree of 2.28, which is greater than 1.5 and can achieve baseline separation.

[0052] Example 2

[0053] Limit of detection and limit of quantitation tests.

[0054] The chromatographic method in Example 1 was used.

[0055] Experimental steps:

[0056] Blank solvent: water

[0057] Detection limit and quantification limit solution: Take appropriate amount of tenofovir reference substance and enantiomer reference substance, add anhydrous ethanol to prepare a series of concentration solutions, and measure according to the law. The quantification limit and detection limit of the substance are determined by the signal-to-noise ratio. The results are shown in the table below:

[0058] Limit of Quantitation Results

[0059] name Limit of quantification concentration (μg / mL) S / N Equivalent to sample concentration (1mg / ml) (%) Enantiomers 0.1100 21.2 0.01％ Tenofovir 0.1005 18.4 0.01％

[0060] Detection limit results

[0061] name Limit of quantification concentration (μg / mL) S / N Equivalent to sample concentration (1mg / ml) (%) Enantiomers 0.03300 3.6 0.003％ Tenofovir 0.03015 3.3 0.003％

[0062] Example 3

[0063] Linear Relationship and Range

[0064] The chromatographic method in Example 1 was used.

[0065] Experimental steps:

[0066] Blank solvent: water

[0067] Linear solution: Take appropriate amount of tenofovir reference substance and enantiomer reference substance, add water to prepare solutions with a series of concentrations, and measure according to the method.

[0068] Results: The linear equation of tenofovir was y=0.4490x+0.0014, r=0.9998 (n=5) in the concentration range of 0.1005-2.0100 μg / ml; the linear equation of enantiomer was y=0.4536x-0.0006, r=0.9993 (n=5) in the concentration range of 0.1100-2.2000 μg / ml; the results showed that the linear range was good.

[0069] Example 3

[0070] Recovery test

[0071] The chromatographic method in Example 1 was used.

[0072] Experimental steps:

[0073] Accurately weigh an appropriate amount of tenofovir, place it in a volumetric flask, accurately add an appropriate amount of tenofovir enantiomer stock solution, dissolve it with water and dilute it to the scale, prepare solutions with relative concentrations of 50%, 100%, and 150%, shake well, and use them as test solutions. Prepare 3 copies of each concentration in parallel. Accurately weigh an appropriate amount of tenofovir, dissolve it with ethanol and dilute it to the scale, shake well, and use it as a blank sample solution. Accurately measure an appropriate amount of tenofovir enantiomer stock solution, quantitatively dilute it with water to make a solution of appropriate concentration, and use it as a reference solution. Accurately measure 20μl of each of the above solutions, inject them into the liquid chromatograph, and record the chromatogram and peak area. Calculate the recovery rate.

[0074] Results: The recovery rates of tenofovir enantiomers at each concentration were in line with the requirements of the pharmacopoeia. The accuracy of this method was good. The specific data are shown in the table below:

[0075] Tenofovir enantiomer recovery results

[0076]

[0077] The recoveries of tenofovir enantiomers at the limit values ​​of 50%, 100% and 150% were 90.78% to 110.66%, respectively. The results showed that the method had good accuracy.

[0078] Example 4 Durability test of the method of the present invention

[0079] The tolerance of the test method was investigated by changing parameters such as flow rate, column temperature, and mobile phase ratio. Only one parameter was changed in each test, and the results were compared with the results when the parameters were not changed. The separation of enantiomers and tenofovir was not less than 1.5, which met the requirements. The specific results are shown in the table below:

[0080]

[0081] Example 5 Sample detection test

[0082] 1. Instruments and conditions:

[0083] High performance liquid chromatograph: Thermofisher Dixonex U3000, UV detector

[0084] Chromatographic column: Inertsil ODS-SP (250mm×4.6mm, 5μm)

[0085] Mobile phase: phosphate buffer (take 0.16g D-phenylalanine, 0.76g copper sulfate pentahydrate and 2.31g ammonium dihydrogen phosphate, add 1000ml water to dissolve, and adjust the pH value to 4.0 with ammonia water) - methanol, the volume ratio is 90:10

[0086] Flow rate: 1.0ml / min

[0087] Column temperature: 35°C

[0088] Injection volume: 20 μl

[0089] Detection wavelength: 262nm

[0090] 2. Experimental methods and results

[0091] Diluent: Water

[0092] Preparation of system suitability solution: Take appropriate amount of enantiomer reference substance and tenofovir reference substance, dissolve in water and dilute to a mixed solution containing about 1 mg of tenofovir and 5 μg of enantiomer per 1 ml, as the system suitability solution;

[0093] Sample solution preparation: Take the tenofovir sample to be tested and prepare it into a solution with a concentration of 1.0 mg / ml with water as the test solution; take 20 μl of each of the above solutions, inject them into the liquid chromatograph in sequence, and record the chromatogram.

[0094] Table 4 Tenofovir isomer detection experimental results

[0095] batch number Enantiomers GS3-201611001 Not detected GS3-201701001 Not detected GS3-201701002 Not detected

Claims

1. A detection and analysis method for tenofovir and its enantiomers, characterized in that: octadecylsilane bonded silica gel is used as filler, the mobile phase is a mixed solution of a mixed aqueous solution (D-phenylalanine, copper sulfate pentahydrate and ammonium dihydrogen phosphate) and methanol, and the volume ratio of the mixed aqueous solution to methanol is 85-95:15-5.

2. The method for detecting tenofovir enantiomers as claimed in claim 1, characterized in that: The volume ratio of the mixed aqueous solution to methanol is 90:

10.

3. The method for detecting tenofovir and its enantiomers as claimed in claim 2, characterized in that: The HPLC detection adopts the following optional chromatographic conditions: the mobile phase flow rate is 0.8 to 1.2 ml / min; the chromatographic column temperature is 33 to 37°C; The detector is an ultraviolet detector with a detection wavelength of 262 nm.

4. The method for detecting tenofovir enantiomers as claimed in claim 3, characterized in that: The flow rate of the mobile phase is 1.0 ml / min; the temperature of the chromatographic column is 35°C.

5. The method for detecting tenofovir enantiomers according to any one of claims 1 to 4, characterized in that: The detection method includes three steps: preparation of the test solution, preparation of the system suitability solution and determination: The preparation of the test solution comprises: taking a tenofovir sample to be tested, and preparing a solution with a concentration of 0.8 mg to 1.2 mg / ml with a diluent as the test solution; The preparation of the system suitability solution comprises: accurately weighing a tenofovir reference substance, a diastereomer and an enantiomer reference substance, and diluting them with a diluent to prepare a system suitability solution containing about 1 mg of tenofovir and 5 μg of enantiomer per 1 ml; The determination steps include: injecting 20 μl of the system suitability solution into a high performance liquid chromatograph, and the separation degree between the tenofovir peak and the enantiomer peak must not be less than 1.5; then injecting 20 μl of the test solution into the liquid chromatograph, and recording the chromatogram.

6. The separation and detection method according to any one of claims 1 to 5, characterized in that The separation degree between tenofovir and the enantiomer peak in the method is not less than 1.5, and the repeatability, accuracy and sensitivity of the method can meet the detection requirements.

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