A method for analyzing related substances of tenofovir alafenamide hemifumarate

Abstract

The present invention relates to the field of drug analysis technology, and in particular to a high performance liquid phase detection method for tenofovir alafenamide hemifumarate related substances. The present invention discloses a simultaneous separation degree analysis method for 10 related impurities of tenofovir alafenamide. The present invention can effectively and accurately determine the related substances and contents in tenofovir alafenamide, and ensure product quality.

Description

Technical Field

[0001] The present invention relates to the technical field of chemical drug analysis methods, and in particular to an analysis method for determining tenofovir alafenamide hemifumarate-related substances. Background Art

[0002] Tenofovir Alafenamide Fumarate, chemical name 9-[(R)-2-[((S)-[[(S)-1-(isopropoxycarbonyl)ethyl]amino]phenoxyphosphinyl)methoxy]propyl]adenine hemifumarate, molecular formula C 21 H 29 O 5 N 6 P·1 / 2(C 4 H 4 O 4 ), molecular weight is 534.51, and its structural formula is as follows:

[0003]

[0004] Hepatitis B virus infection is a worldwide infectious disease. According to the World Health Organization, about 2 billion people have been infected with hepatitis B virus, and about one million people die from cirrhosis or liver failure caused by hepatitis B infection every year. At present, the only nucleotide drugs used in my country for anti-hepatitis B virus are lamivudine, adefovir dipivoxil, telbivudine, entecavir and tenofovir disoproxil. In the process of anti-nucleotide treatment of hepatitis B, drug resistance is a more difficult problem, and the development of tenofovir alafenamide provides a new option for the clinical treatment of hepatitis B infection. In the production process of tenofovir alafenamide API, related substances such as starting materials, intermediates, by-products and chiral isomers may be introduced. At the same time, by-products or other degradation products may be produced during the production, storage and transportation of APIs and preparations. Therefore, the determination of related substances is extremely important for the control of drug quality. Currently, there is no literature reporting the specific determination of related substances in tenofovir alafenamide hemifumarate raw materials and preparations, so it is necessary to explore suitable chromatographic conditions, adjust corresponding chromatographic parameters, and control corresponding impurities. Summary of the invention

[0005] The present invention provides an analytical method for determining tenofovir alafenamide hemifumarate-related substances, and the present invention can solve the problem that the number of tenofovir alafenamide-related substances is large and the separation is difficult.

[0006] The technical solution of the present invention includes:

[0007] The high performance liquid chromatography method for the determination of related substances of tenofovir alafenamide hemifumarate comprises the following steps:

[0008] (1) preparing a tenofovir alafenamide hemifumarate analytical solution;

[0009] (2) High performance liquid chromatography was used, and the chromatographic conditions included: the chromatographic column was an octadecyl bonded silica gel column, a solution of phosphate buffer and methanol in a volume ratio of 92:8 was used as mobile phase A, and a solution of phosphate buffer and methanol in a volume ratio of 30:70 was used as mobile phase B. The gradient elution process was: the ratio of phase A and phase B in the mobile phase was 90-95%, 40-45%, 30-35%, 0%, 90-95%, 90-95% at the time points of 0, 15-20, 35-38, 40-45, 50, and 60 minutes, and the ratio of phase A was 90-95%, 40-45%, 30-35%, 0%, 90-95%, and 90-95%, and the detection wavelength was 260 nm.

[0010] (3) Sample determination: inject the analytical solution prepared in step (1) into a liquid chromatograph, record the chromatogram, and separate the following related substances:

[0011]

[0012] The preferred embodiment, the method for analyzing related substances of tenofovir alafenamide hemifumarate, mainly comprises the following steps:

[0013] (1) Preparation of analytical solution

[0014] The sample is dissolved in a single solvent of methanol or acetonitrile or a mixed solution of methanol-water, acetonitrile-water, methanol-phosphate aqueous solution and acetonitrile-phosphate aqueous solution to prepare an analytical solution.

[0015] (2) Chromatographic conditions

[0016] High performance liquid chromatography was used, and the chromatographic conditions included: an octadecyl bonded silica gel column, a solution with a volume ratio of phosphate buffer to methanol of 92:8 as mobile phase A, a solution with a volume ratio of phosphate buffer to methanol of 30:70 as mobile phase B, a detection wavelength of 260 nm, and gradient elution.

[0017] According to the present invention, the length of the chromatographic column is 150 mm, the diameter is 4.6 mm, and the filler particle size is 5 μm.

[0018] According to the present invention, the chromatographic column filler is octadecyl bonded silica gel, and preferably, the chromatographic column model is ACE ExcelC18.

[0019] According to the present invention, in the phosphate buffer, the concentration of sodium dihydrogen phosphate is 15-25 mmol / L; preferably, the concentration of sodium dihydrogen phosphate is 20 mmol / L.

[0020] According to the present invention, the concentration of the sodium hydroxide solution for adjusting the pH value is 0.1-1.0 mol / L; preferably, the concentration of the sodium hydroxide solution is 0.5 mol / L.

[0021] According to the present invention, in a phosphate buffer, a 0.5 mol / L sodium hydroxide solution is used to adjust the pH value to 4-7, preferably, the pH value is 6.0.

[0022] According to the present invention, the flow rate is 0.8-1.5 ml / min; preferably, the flow rate is 1.0 ml / min.

[0023] According to the present invention, the column temperature is 10-30°C; preferably, the column temperature is 20°C.

[0024] According to the present invention, the gradient elution process is as follows: the ratio of phase A and phase B in the mobile phase is 0, 15-20, 35-38, 40-45, 50, 60 minutes, and the ratio of phase A is 90-95%, 40-45%, 30-35%, 0%, 90-95%, 90-95%

[0025] The sample is tenofovir alafenamide hemifumarate raw material or its preparation, and the preparation is tablets, capsules, granules, suppositories, pills, ointments, creams, pastes, inhalation preparations, sprays, aerosols, gels, powders, syrups, liniments, coatings, film coatings, tinctures, patches, oral solutions, implants, films, lotions, rinses, decoctions, pastes, dews, and teas.

[0026] Beneficial effects:

[0027] The present inventors have effectively developed a method for determining related substances of tenofovir alafenamide hemifumarate with reference to the present invention, which can achieve simultaneous separation of 10 impurities in one system, and provide an effective means for quality control of raw materials and preparations. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 is the mixed solution chromatogram of Example 1;

[0029] Figure 2 is the mixed solution chromatogram of Example 2;

[0030] Figure 3 is the mixed solution chromatogram of Example 3;

[0031] Figure 4 It is the chromatogram of the mixed solution of Example 4. DETAILED DESCRIPTION

[0032] The preferred embodiments of the present invention are described in detail below. The experimental methods without specific conditions in the preferred embodiments are usually carried out under conventional conditions. The examples are provided to better illustrate the contents of the present invention, but the contents of the present invention are not limited to the examples. Those skilled in the art can make non-essential improvements and adjustments to the implementation scheme according to the above invention content, which still fall within the scope of protection of the present invention.

[0033] Example 1

[0034] (1) Instruments and HPLC conditions

[0035] High performance liquid chromatography: Thermo U3000 high performance liquid chromatography system and workstation;

[0036] Chromatographic column: Welch XB-phenyl (4.6×150mm, 5μm) phenylsilane bonded silica gel column

[0037] 20mmo / L sodium dihydrogen phosphate buffer (pH6.0) was used as mobile phase A, and 20mmo / L sodium dihydrogen phosphate buffer (pH4.0)-methanol (35:65) was used as mobile phase B. The ratio of phase A to phase B was 90%, 40%, 30%, 0%, 90%, and 90% at 0, 20, 38, 45, 50, and 60 minutes. The detection wavelength was 260nm, the flow rate was 1.0ml / min, and the column temperature was 30℃.

[0038] (2) Experimental procedures

[0039] Take appropriate amounts of impurity A to impurity J reference substances and tenofovir alafenamide hemifumarate respectively, and prepare a mixed solution containing 1 mg of tenofovir alafenamide and 10 μg of the remaining impurity reference substances per 1 ml with 80% methanol solution;

[0040] Take 5 μl of the above analytical solution, inject it into the liquid chromatograph, and record the chromatogram. The results are shown in the attached Figure 1 Impurity H and impurity I completely overlap with API, and the peak shape of impurity B is poor under this condition, so the chromatographic conditions still need to be adjusted and optimized.

[0041] Example 2

[0042] (1) Instruments and HPLC conditions

[0043] High performance liquid chromatography: Thermo U3000 high performance liquid chromatography system and workstation;

[0044] Chromatographic column: Intersil ODS-3 (4.6×150mm, 5μm) octadecylsilane bonded silica gel chromatographic column

[0045] 20mmo / L sodium dihydrogen phosphate buffer (pH6.0)-methanol (92:8) was used as mobile phase A, and 20mmo / L sodium dihydrogen phosphate buffer (pH5.0)-methanol (35:65) was used as mobile phase B. The ratio of phase A to phase B was 92%, 45%, 35%, 0%, 92%, and 92% at 0, 15, 35, 40, 50, and 60 minutes. The detection wavelength was 260nm, the flow rate was 1.0ml / min, and the column temperature was 15°C.

[0046] (3) Experimental procedures

[0047] Take appropriate amounts of impurity A to impurity J reference substances and tenofovir alafenamide hemifumarate respectively, and prepare a mixed solution containing 1 mg of tenofovir alafenamide and 10 μg of the remaining impurity reference substances per 1 ml with 80% methanol solution;

[0048] Take 5 μl of the above analytical solution, inject it into the liquid chromatograph, and record the chromatogram. The results are shown in the attached Figure 2 Tenofovir alafenamide and all impurities can be completely separated.

[0049] Example 3

[0050] (1) Instruments and HPLC conditions

[0051] High performance liquid chromatography: Thermo U3000 high performance liquid chromatography system and workstation;

[0052] Chromatographic column: ACE Excel C18 (4.6×150mm, 5μm) octadecylsilane bonded silica gel column

[0053] 20mmo / L sodium dihydrogen phosphate buffer (pH6.0)-methanol (92:8) was used as mobile phase A, and 20mmo / L sodium dihydrogen phosphate buffer (pH5.0)-methanol (35:65) was used as mobile phase B. The ratio of phase A to phase B was 90%, 40%, 30%, 0%, 90%, and 90% at 0, 15, 35, 40, 50, and 60 minutes. The detection wavelength was 260nm, the flow rate was 1.5ml / min, and the column temperature was 30℃.

[0054] (2) Experimental procedures

[0055] Take appropriate amounts of impurity A to impurity J reference substances and tenofovir alafenamide hemifumarate respectively, and prepare a mixed solution containing 1 mg of tenofovir alafenamide and 10 μg of each of the other impurity reference substances per 1 ml with methanol;

[0056] Take 5 μl of the above analytical solution, inject it into the liquid chromatograph, and record the chromatogram. The results are shown in the attached Figure 3Tenofovir alafenamide and all impurities can be completely separated.

[0057] Example 4

[0058] (1) Instruments and HPLC conditions

[0059] High performance liquid chromatography: Thermo U3000 high performance liquid chromatography system and workstation;

[0060] Chromatographic column: ACE Excel C18 (4.6×150mm, 5μm) octadecylsilane bonded silica gel column

[0061] 20mmo / L sodium dihydrogen phosphate buffer (pH6.0)-methanol (92:8) was used as mobile phase A, and 20mmo / L sodium dihydrogen phosphate buffer (pH6.0)-methanol (35:65) was used as mobile phase B. The ratio of phase A to phase B was 95%, 45%, 35%, 0%, 95%, and 95% at 0, 15, 35, 40, 50, and 60 minutes. The detection wavelength was 260nm, the flow rate was 1.0ml / min, and the column temperature was 20℃.

[0062] (2) Experimental procedures

[0063] Take appropriate amounts of impurity A to impurity J reference substances and tenofovir alafenamide hemifumarate respectively, and prepare a mixed solution containing 1 mg of tenofovir alafenamide and 10 μg of each of the other impurity reference substances per 1 ml with methanol;

[0064] Take 5 μl of the above analytical solution, inject it into the liquid chromatograph, and record the chromatogram. The results are shown in the attached Figure 4 Tenofovir alafenamide and all impurities can be completely separated.

[0065] As shown in the table below, the separation degree of the method in Example 1 is poor, and impurities H and I are encapsulated by the API. Examples 2 to 4 have good separation and can achieve complete separation of 10 impurities in the same system.

[0066] Table 1 Comparison of impurity separation degree in Examples 1 to 4

[0067]

[0068]

[0069] Note: Impurity G has two isomers, so it appears as two peaks in the chromatogram.

Claims

1. An analytical method for determining related substances of tenofovir alafenamide hemifumarate, characterized in that: (1) preparing a tenofovir alafenamide hemifumarate analytical solution; (2) High performance liquid chromatography was used, and the chromatographic conditions included: an octadecyl bonded silica gel column, a solution of phosphate buffer with a pH value of 6.0 and methanol in a volume ratio of 92:8 as mobile phase A, and a solution of phosphate buffer with a pH value of 6.0 and methanol in a volume ratio of 30:70 as mobile phase B; a gradient elution process: the ratio of phase A to phase B in the mobile phase was 90-95%, 40-45%, 30-35%, 0%, 90-95%, 90-95% at 0, 15-20, 35-38, 40-45, 50, and 60 minutes, and the detection wavelength was 260 nm; (3) Sample determination: inject the analytical solution prepared in step (1) into a liquid chromatograph, record the chromatogram, and separate the following related substances:

2. The analytical method for determining related substances of tenofovir alafenamide hemifumarate according to claim 1, characterized in that: Step (1) Preparation of analytical solution: Specific steps: dissolving the sample with a single solvent of methanol or acetonitrile or a mixed solution of methanol-water, acetonitrile-water, methanol-phosphate aqueous solution, or acetonitrile-phosphate aqueous solution to prepare an analytical solution.

3. The analytical method for determining related substances of tenofovir alafenamide hemifumarate according to claim 1, characterized in that: The length of the chromatographic column is 150 mm, the diameter is 4.6 mm, and the filler particle size is 5 μm.

4. The analytical method for determining related substances of tenofovir alafenamide hemifumarate according to claim 1, characterized in that: The model of the chromatographic column is ACE Excel C18 chromatographic column.

5. The analytical method for determining related substances of tenofovir alafenamide hemifumarate according to claim 1, characterized in that: In phosphate buffer, the concentration of sodium dihydrogen phosphate is 15-25 mmol / L.

6. The analytical method for determining related substances of tenofovir alafenamide hemifumarate according to claim 1, characterized in that: The concentration of the sodium hydroxide solution for adjusting the pH value is 0.1 to 1.0 mol / L.

7. The analytical method for determining related substances of tenofovir alafenamide hemifumarate according to claim 1, characterized in that: In phosphate buffer, the pH value was adjusted to 6.0 with 0.5 mol / L sodium hydroxide solution.

8. The analytical method for determining related substances of tenofovir alafenamide hemifumarate according to claim 1, characterized in that: The flow rate is 1.2~1.8ml / min.

9. The analytical method for determining related substances of tenofovir alafenamide hemifumarate according to claim 1, characterized in that: The column temperature is 15-40°C.

10. The analytical method for determining related substances of tenofovir alafenamide hemifumarate according to claim 2, characterized in that: The sample is the raw material of tenofovir alafenamide hemifumarate or its preparation, and the preparation is tablets, capsules, granules, suppositories, pills, ointments, creams, pastes, inhalation preparations, sprays, aerosols, gels, powders, syrups, liniments, coatings, films, tinctures, patches, oral solutions, implants, films, lotions, rinses, ointments, dews, and teas.

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