CN113311097A - Method for determining content of impurities in sofosbuvir enantiomer - Google Patents
Method for determining content of impurities in sofosbuvir enantiomer Download PDFInfo
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- 239000012535 impurity Substances 0.000 title claims abstract description 81
- TTZHDVOVKQGIBA-IQWMDFIBSA-N sofosbuvir Chemical class N1([C@@H]2O[C@@H]([C@H]([C@]2(F)C)O)CO[P@@](=O)(N[C@@H](C)C(=O)OC(C)C)OC=2C=CC=CC=2)C=CC(=O)NC1=O TTZHDVOVKQGIBA-IQWMDFIBSA-N 0.000 title claims abstract description 73
- 238000000034 method Methods 0.000 title claims abstract description 27
- 229960002063 sofosbuvir Drugs 0.000 claims abstract description 59
- 239000012074 organic phase Substances 0.000 claims abstract description 15
- 238000010828 elution Methods 0.000 claims abstract description 13
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 9
- 239000000945 filler Substances 0.000 claims abstract description 9
- 239000000741 silica gel Substances 0.000 claims abstract description 9
- 229910002027 silica gel Inorganic materials 0.000 claims abstract description 9
- 239000012071 phase Substances 0.000 claims abstract description 7
- 230000015572 biosynthetic process Effects 0.000 claims abstract description 4
- 238000003786 synthesis reaction Methods 0.000 claims abstract description 4
- SBTVLCPCSXMWIQ-UHFFFAOYSA-N (3,5-dimethylphenyl) carbamate Chemical compound CC1=CC(C)=CC(OC(N)=O)=C1 SBTVLCPCSXMWIQ-UHFFFAOYSA-N 0.000 claims abstract description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 72
- 239000000243 solution Substances 0.000 claims description 51
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 28
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 20
- 239000012085 test solution Substances 0.000 claims description 16
- 238000000926 separation method Methods 0.000 claims description 15
- 238000002360 preparation method Methods 0.000 claims description 9
- 238000001514 detection method Methods 0.000 claims description 7
- 239000002904 solvent Substances 0.000 claims description 6
- 239000003085 diluting agent Substances 0.000 claims description 3
- 239000011259 mixed solution Substances 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 2
- 238000002347 injection Methods 0.000 claims 1
- 239000007924 injection Substances 0.000 claims 1
- 238000007689 inspection Methods 0.000 abstract 1
- 238000007865 diluting Methods 0.000 description 15
- 238000005303 weighing Methods 0.000 description 15
- 238000004128 high performance liquid chromatography Methods 0.000 description 12
- 239000013558 reference substance Substances 0.000 description 10
- KPCOLEDDUNYSQA-UHFFFAOYSA-N (3,5-dimethylphenyl)carbamic acid Chemical compound CC1=CC(C)=CC(NC(O)=O)=C1 KPCOLEDDUNYSQA-UHFFFAOYSA-N 0.000 description 6
- 239000007983 Tris buffer Substances 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 230000014759 maintenance of location Effects 0.000 description 5
- 238000010606 normalization Methods 0.000 description 5
- 238000011003 system suitability test Methods 0.000 description 5
- HBAQYPYDRFILMT-UHFFFAOYSA-N 8-[3-(1-cyclopropylpyrazol-4-yl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl]-3-methyl-3,8-diazabicyclo[3.2.1]octan-2-one Chemical class C1(CC1)N1N=CC(=C1)C1=NNC2=C1N=C(N=C2)N1C2C(N(CC1CC2)C)=O HBAQYPYDRFILMT-UHFFFAOYSA-N 0.000 description 3
- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 3
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 description 3
- -1 sofosbuvir compound Chemical class 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 239000003814 drug Substances 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000003908 quality control method Methods 0.000 description 2
- 208000006154 Chronic hepatitis C Diseases 0.000 description 1
- 208000030453 Drug-Related Side Effects and Adverse reaction Diseases 0.000 description 1
- 208000005176 Hepatitis C Diseases 0.000 description 1
- 102000014150 Interferons Human genes 0.000 description 1
- 108010050904 Interferons Proteins 0.000 description 1
- 229940123066 Polymerase inhibitor Drugs 0.000 description 1
- XBDQKXXYIPTUBI-UHFFFAOYSA-N Propionic acid Chemical compound CCC(O)=O XBDQKXXYIPTUBI-UHFFFAOYSA-N 0.000 description 1
- 101800001554 RNA-directed RNA polymerase Proteins 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000001647 drug administration Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 208000010710 hepatitis C virus infection Diseases 0.000 description 1
- 230000008676 import Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 229940079322 interferon Drugs 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 229940127073 nucleoside analogue Drugs 0.000 description 1
- 230000003285 pharmacodynamic effect Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 229940076563 sovaldi Drugs 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
- G01N2030/8872—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample impurities
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- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
The invention provides a method for measuring the content of an enantiomer impurity of Sofosbuvir, which is characterized in that a chromatographic column using amylose-tri (3, 5-xylyl carbamate) coated on the surface of silica gel as a filler is adopted, an organic phase is used as a mobile phase for gradient elution, the enantiomer impurity in the synthesis process of Sofosbuvir is measured, and the inspection method is scientific, reasonable, accurate and objective, so that the quality of the Sofosbuvir enantiomer can be better controlled.
Description
Technical Field
The invention relates to the field of pharmaceutical chemicals, and particularly relates to a method for determining the content of impurities in an enantiomer of sofosbuvir.
Background
The chemical name of the sofosbuvir is as follows: (S) -isopropyl 2- ((S) - ((((2R, 3R, 4R, 5R) -5- (2, 2-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl) methoxy) - (phenoxy) phosphinylamino) propanoate. The structural formula is as follows:
is a nucleoside analogue (NS5B) polymerase inhibitor of Jilided, is approved by the national drug administration (NMPA) to be on the market at 20 days 9 and 9 months 2017, has the trade name of Sovaldi, and is clinically used for treating chronic hepatitis C. The product has no need of using interferon for HCV infection of some genotypes, can reduce side effect, and has good therapeutic effect.
Chinese patent CN101918425A discloses a sofosbuvir compound and a preparation method thereof, so sofosbuvir is a well-known compound.
In the prior art, the method for analyzing related substances of Sofosbuvir tablets is controlled by Sofosbuvir tablet import registration standard (standard number: JX20160007), the enantiomers of Sofosbuvir are not controlled, some process impurities are easily introduced into the Sofosbuvir during the production process, the content of the impurities affects the pharmacodynamic activity on one hand, adverse drug reactions can be increased on the other hand, and the enantiomers need to be controlled in the production process of the Sofosbuvir. One impurity in the sofosbuvir is isopropyl ((R) - ((2S, 3S, 4S, 5S) -5- (2, 4-dioxy-3, 4-dihydropyrimidin-1 (2H) -yl) -4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl) methoxy) (phenoxy) phosphoryl) -D-alanine (impurity one, enantiomer), the enantiomer impurity is similar to the sofosbuvir in structure and polarity, and the content of the impurity is difficult to monitor. The quality monitoring of the medicine is very important in the process of medicine preparation and use, so that the development of the quality control capable of effectively separating and determining the enantiomers in the raw material or the preparation of the fosbuvir has practical significance.
Disclosure of Invention
The invention aims to provide a method for measuring the content of enantiomer impurities in Sofosbuvir, which can separate isopropyl ((R) - ((2S, 3S, 4S, 5S) -5- (2, 4-dioxy-3, 4-dihydropyrimidine-1 (2H) -yl) -4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl) methoxy) (phenoxy) phosphoryl) -D-alanine from a main peak and monitor the impurities, so that the content of the enantiomer impurities in Sofosbuvir can be accurately measured, the quality of the enantiomer and a preparation in Sofosbuvir can be well evaluated, the content of the enantiomer impurities in the produced Sofosbuvir is ensured to be in a safe range, realize the quality control of the sofosbuvir enantiomer and the preparation.
In order to achieve the purpose, the invention adopts the technical scheme that:
a method for measuring the content of impurities in Sofosbuvir enantiomers comprises the steps of dissolving Sofosbuvir, adopting a chromatographic column which takes amylose-tri (3, 5-xylyl carbamate coated on the surface of silica gel as a filler, and taking an organic phase as a mobile phase for gradient elution to measure the impurities in the synthesis process of the Sofosbuvir enantiomers, wherein the mobile phase is a mixed solution of ethanol solution of trifluoroacetic acid and n-hexane, and the gradient elution conditions are as follows:
| time (minutes) | N-hexane (%) | Trifluoroacetic acid in ethanol (%) |
| 0→25 | 67 | 33 |
| 25→35 | 67→15 | 33→85 |
| 35→40 | 15 | 85 |
| 40→41 | 15→67 | 85→33 |
| 41→55 | 67 | 33 |
The said diluent is composed of n-hexane or ethanol and trifluoroacetic acid mixed according to a certain proportion, the proportion of ethanol is 80% -100%, V/V.
The trifluoroacetic acid solution has the volume proportion concentration of 0.05-0.15% and V/V.
Compared with the prior art, the invention has the following advantages:
the method adopts liquid chromatography for gradient elution, and under the conditions of experiment and changing color spectrum, the sofosbuvir and enantiomer impurities can be completely separated and better detected quantitatively.
Drawings
FIG. 1 HPLC chromatogram of Sofosbuvir and various impurities
FIG. 2 HPLC chromatogram of sofosbuvir and enantiomeric impurity
Detailed Description
The present invention is further illustrated by the following detailed description, which is to be construed as merely illustrative and not limitative of the remainder of the disclosure, and modifications and variations such as those ordinarily skilled in the art are intended to be included within the scope of the present invention as defined in the appended claims.
Dissolving Sofosbuvir in normal hexane or ethanol or a mixture of the two, coating a chromatographic column with amylose-tri (3, 5-xylylcarbamate as a filler on the surface of silica gel, performing gradient elution by taking an organic phase as a mobile phase, and determining the enantiomer impurities in the synthesis process of the Sofosbuvir, wherein the mobile phase is a mixed solution of ethanol solution of trifluoroacetic acid and normal hexane, the organic phase is ethanol solution of normal hexane and trifluoroacetic acid, the volume ratio concentration of the trifluoroacetic acid is 0.05% -0.15%, and the V/V is carried out under the gradient elution conditions:
the sofosbuvir enantiomer contains six main impurities as follows:
by adopting the method, the sofosbuvir enantiomer or the preparation can be completely separated from the six impurities; the problem of separation of the enantiomer in the Sofosbuvir and impurities thereof is solved, so that the quality of the enantiomer in the Sofosbuvir and the preparation is controllable; the impurity I (isomer) and the sofosbuvir enantiomer have similar structures and similar polarities, and the method can well separate the impurity I and the impurity II from the main peak so as to ensure the accuracy of impurity content detection.
[ example 1 ]
High performance liquid chromatography for determining enantiomer impurities in Sofosbuvir
According to high performance liquid chromatography (Chinese pharmacopoeia 2020 edition four-part general rules 0512)
Chromatographic conditions and System suitability test
A chromatographic column: the surface of the silica gel is coated with amylose-tris (3, 5-xylylcarbamate as a filler (e.g., CHIRALPAK AD-H (4.6X 250mm, 5 μm))
Organic phase A: n-hexane
And (3) organic phase B: 0.1% trifluoroacetic acid in ethanol
The detection wavelength was 260nm, the column temperature was 30 ℃ and the flow rate was 0.5 ml/min.
Solvent: ethanol
Taking a proper amount of the sofosbuvir and the impurity I (enantiomer), precisely weighing, adding ethanol for dissolving and diluting to prepare a solution containing about 1mg of the sofosbuvir and 1.5 mu g of the impurity I (enantiomer) in each 1ml, and taking the solution as a system applicability solution. Precisely measuring 5 mu l of system applicability solution, injecting into a high performance liquid chromatograph, and recording a chromatogram, wherein the separation degree of a main peak and adjacent impurities in the system applicability solution is more than 1.5.
Gradient elution
The determination method comprises the following steps: taking the product, precisely weighing, adding ethanol for dissolving, and diluting to prepare a solution containing about 1mg of Sofosbuvir in 1ml as a test solution; taking a proper amount of the Sofosbuvir reference substance and each impurity reference substance, precisely weighing, adding ethanol for dissolving and diluting to prepare a solution containing about 1mg of the Sofosbuvir, 1mg of the impurity I (enantiomer) to 1.5 mu g of the impurity six in each 1ml, and taking the solution as a system applicability solution. If a chromatographic peak which is consistent with the retention time of the impurity-I (enantiomer) peak exists in the chromatogram of the test solution, the test solution is obtained by an area normalization method. The results are shown in Table 1.
Results for the main peaks and respective impurities listed in Table 1
Example 2: high performance liquid chromatography for determining enantiomer impurities in Sofosbuvir
According to high performance liquid chromatography (Chinese pharmacopoeia 2020 edition four-part general rules 0512)
Chromatographic conditions and System suitability test
A chromatographic column: the surface of the silica gel is coated with amylose-tris (3, 5-xylylcarbamate as a filler (e.g., CHIRALPAK AD-H (4.6X 250mm, 5 μm))
Organic phase A: n-hexane
And (3) organic phase B: 0.1% trifluoroacetic acid in ethanol
The detection wavelength was 260nm, the column temperature was 35 ℃ and the flow rate was 0.4 ml/min.
Solvent: ethanol
Taking a proper amount of the sofosbuvir and the impurity I (enantiomer), precisely weighing, adding ethanol for dissolving and diluting to prepare a solution containing about 1mg of the sofosbuvir and 1.5 mu g of the impurity I (enantiomer) in each 1ml, and taking the solution as a system applicability solution. Precisely measuring 5 mu l of system applicability solution, injecting into a high performance liquid chromatograph, and recording a chromatogram, wherein the separation degree of a main peak and adjacent impurities in the system applicability solution is more than 1.5.
Gradient elution
The determination method comprises the following steps: taking the product, precisely weighing, adding ethanol for dissolving, and diluting to prepare a solution containing about 1mg of Sofosbuvir in 1ml as a test solution; taking a proper amount of the Sofosbuvir reference substance and an appropriate amount of each impurity reference substance, precisely weighing, adding ethanol for dissolving and diluting to prepare a solution containing about 1mg of the Sofosbuvir and 1.5 mu g of the impurity I (enantiomer) in each 1ml, and taking the solution as a system applicability solution. If a chromatographic peak which is consistent with the retention time of the impurity-I (enantiomer) peak exists in the chromatogram of the test solution, the test solution is obtained by an area normalization method. The results are shown in Table 2.
The degrees of separation of the main peaks from impurity one (enantiomer) as listed in Table 2
| Sample (I) | Degree of separation from post impurity peak |
| Main peak | 3.1 |
| Impurities | —— |
Example 3: high performance liquid chromatography for determining enantiomer impurities in Sofosbuvir
According to high performance liquid chromatography (Chinese pharmacopoeia 2020 edition four-part general rules 0512)
Chromatographic conditions and System suitability test
A chromatographic column: the surface of the silica gel is coated with amylose-tris (3, 5-xylylcarbamate as a filler (e.g., CHIRALPAK AD-H (4.6X 250mm, 5 μm))
Organic phase A: n-hexane
And (3) organic phase B: 0.1% trifluoroacetic acid in ethanol
The detection wavelength was 265nm, the column temperature was 35 ℃ and the flow rate was 0.6 ml/min.
Solvent: ethanol
Taking a proper amount of the sofosbuvir and the impurity I (enantiomer), precisely weighing, adding ethanol for dissolving and diluting to prepare a solution containing about 1mg of the sofosbuvir and 1.5 mu g of the impurity I (enantiomer) in each 1ml, and taking the solution as a system applicability solution. Precisely measuring 5 mu l of system applicability solution, injecting into a high performance liquid chromatograph, and recording a chromatogram, wherein the separation degree of a main peak and adjacent impurities in the system applicability solution is more than 1.5.
Gradient elution
The determination method comprises the following steps: taking the product, precisely weighing, adding ethanol for dissolving, and diluting to prepare a solution containing about 1mg of Sofosbuvir in 1ml as a test solution; taking a proper amount of the Sofosbuvir reference substance and an appropriate amount of each impurity reference substance, precisely weighing, adding ethanol for dissolving and diluting to prepare a solution containing about 1mg of the Sofosbuvir and 1.5 mu g of the impurity I (enantiomer) in each 1ml, and taking the solution as a system applicability solution. If a chromatographic peak which is consistent with the retention time of the impurity-I (enantiomer) peak exists in the chromatogram of the test solution, the test solution is obtained by an area normalization method. The results are shown in Table 3.
Degree of separation of the main peaks from impurity one (enantiomer) as listed in Table 3
| Sample (I) | Degree of separation from post impurity peak |
| Main peak | 2.7 |
| Impurities | —— |
Example 4: high performance liquid chromatography for determining enantiomer impurities in Sofosbuvir
According to high performance liquid chromatography (Chinese pharmacopoeia 2020 edition four-part general rules 0512)
Chromatographic conditions and System suitability test
A chromatographic column: the surface of the silica gel is coated with amylose-tris (3, 5-xylylcarbamate as a filler (e.g., CHIRALPAK AD-H (4.6X 250mm, 5 μm))
Organic phase A: n-hexane
And (3) organic phase B: 0.05% trifluoroacetic acid in ethanol
The detection wavelength was 265nm, the column temperature was 35 ℃ and the flow rate was 0.6 ml/min.
Solvent: ethanol
Taking a proper amount of the sofosbuvir and the impurity I (enantiomer), precisely weighing, adding ethanol for dissolving and diluting to prepare a solution containing about 1mg of the sofosbuvir and 1.5 mu g of the impurity I (enantiomer) in each 1ml, and taking the solution as a system applicability solution. Precisely measuring 5 mu l of system applicability solution, injecting into a high performance liquid chromatograph, and recording a chromatogram, wherein the separation degree of a main peak and adjacent impurities in the system applicability solution is more than 1.5.
Gradient elution
The determination method comprises the following steps: taking the product, precisely weighing, adding ethanol for dissolving, and diluting to prepare a solution containing about 1mg of Sofosbuvir in 1ml as a test solution; taking a proper amount of the Sofosbuvir reference substance and an appropriate amount of each impurity reference substance, precisely weighing, adding ethanol for dissolving and diluting to prepare a solution containing about 1mg of the Sofosbuvir and 1.5 mu g of the impurity I (enantiomer) in each 1ml, and taking the solution as a system applicability solution. If a chromatographic peak which is consistent with the retention time of the impurity-I (enantiomer) peak exists in the chromatogram of the test solution, the test solution is obtained by an area normalization method. The results are shown in Table 4.
Degree of separation of the main peaks from impurity one (enantiomer) as listed in Table 4
| Sample (I) | Degree of separation from post impurity peak |
| Main peak | 2.2 |
| Impurities | —— |
Example 5: high performance liquid chromatography for determining enantiomer impurities in Sofosbuvir
According to high performance liquid chromatography (Chinese pharmacopoeia 2020 edition four-part general rules 0512)
Chromatographic conditions and System suitability test
A chromatographic column: the surface of the silica gel is coated with amylose-tris (3, 5-xylylcarbamate as a filler (e.g., CHIRALPAK AD-H (4.6X 250mm, 5 μm))
Organic phase A: n-hexane
And (3) organic phase B: 0.15% trifluoroacetic acid in ethanol
The detection wavelength was 265nm, the column temperature was 35 ℃ and the flow rate was 0.6 ml/min.
Solvent: ethanol
Taking a proper amount of the sofosbuvir and the impurity I (enantiomer), precisely weighing, adding ethanol for dissolving and diluting to prepare a solution containing about 1mg of the sofosbuvir and 1.5 mu g of the impurity I (enantiomer) in each 1ml, and taking the solution as a system applicability solution. Precisely measuring 5 mu l of system applicability solution, injecting into a high performance liquid chromatograph, and recording a chromatogram, wherein the separation degree of a main peak and adjacent impurities in the system applicability solution is more than 1.5.
Gradient elution
The determination method comprises the following steps: taking the product, precisely weighing, adding ethanol for dissolving, and diluting to prepare a solution containing about 1mg of Sofosbuvir in 1ml as a test solution; taking a proper amount of the Sofosbuvir reference substance and an appropriate amount of each impurity reference substance, precisely weighing, adding ethanol for dissolving and diluting to prepare a solution containing about 1mg of the Sofosbuvir and 1.5 mu g of the impurity I (enantiomer) in each 1ml, and taking the solution as a system applicability solution. If a chromatographic peak which is consistent with the retention time of the impurity-I (enantiomer) peak exists in the chromatogram of the test solution, the test solution is obtained by an area normalization method. The results are shown in Table 5.
Degree of separation of the main peaks from impurity one (enantiomer) as listed in Table 5
| Sample (I) | Degree of separation from post impurity peak |
| Main peak | 1.8 |
| Impurities | —— |
Claims (3)
1. A method for measuring the content of impurities in an enantiomer of sofosbuvir is characterized by comprising the following steps: adopting a chromatographic column which takes amylose-tri (3, 5-xylyl carbamate coated on the surface of silica gel as a filler, taking an organic phase as a mobile phase for gradient elution, and determining impurities in the synthesis process of the Sofosbuvir enantiomer, wherein the mobile phase is a mixed solution of ethanol solution of trifluoroacetic acid and n-hexane, and the gradient elution conditions are as follows:
the sofosbuvir enantiomer contains six main impurities as follows:
by adopting the method, the sofosbuvir enantiomer or the preparation can be completely separated from the six impurities; the problem of separation of the determined sofosbuvir enantiomer and impurities thereof is solved, so that the quality of the sofosbuvir enantiomer and the preparation is ensured to be controllable; the impurity I and the impurity II have similar structures and similar polarities to the isomer of the sofosbuvir, and the method can well separate the impurity I from a main peak so as to ensure the accuracy of impurity content detection.
2. The method as claimed in claim 1, wherein the test solution is prepared by taking a proper amount of sofosbuvir test sample, using a diluent as a solvent, and properly processing the mixture to prepare a solution containing sofosbuvir 0.5-1.5 mg/ml, wherein the diluent is composed of ethanol or ethanol and trifluoroacetic acid which are mixed according to a certain proportion, and the ethanol proportion is 80-100% and V/V.
3. The method of claim 2, wherein the detector: ultraviolet detector for detecting wavelength of 260-270 nm and sample injection volume of 5-20 μ l.
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