CN115436492A - Liquid chromatography for detecting related substances in Sofosbuvir - Google Patents
Liquid chromatography for detecting related substances in Sofosbuvir Download PDFInfo
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- 229960002063 sofosbuvir Drugs 0.000 title claims abstract description 25
- TTZHDVOVKQGIBA-IQWMDFIBSA-N sofosbuvir Chemical compound 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 25
- 238000004811 liquid chromatography Methods 0.000 title claims abstract description 24
- 239000000126 substance Substances 0.000 title claims abstract description 17
- 239000012535 impurity Substances 0.000 claims abstract description 103
- 239000000243 solution Substances 0.000 claims abstract description 39
- 238000000034 method Methods 0.000 claims abstract description 25
- 239000012085 test solution Substances 0.000 claims abstract description 21
- 239000002904 solvent Substances 0.000 claims abstract description 16
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 13
- 239000013558 reference substance Substances 0.000 claims abstract description 13
- 238000001514 detection method Methods 0.000 claims description 27
- OAYLNYINCPYISS-UHFFFAOYSA-N ethyl acetate;hexane Chemical group CCCCCC.CCOC(C)=O OAYLNYINCPYISS-UHFFFAOYSA-N 0.000 claims description 26
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 25
- 239000000741 silica gel Substances 0.000 claims description 21
- 229910002027 silica gel Inorganic materials 0.000 claims description 21
- 238000010828 elution Methods 0.000 claims description 19
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 12
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical group CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims description 10
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 8
- 229910004298 SiO 2 Inorganic materials 0.000 claims description 2
- 239000000523 sample Substances 0.000 description 22
- 238000012360 testing method Methods 0.000 description 21
- 239000003814 drug Substances 0.000 description 12
- 238000011084 recovery Methods 0.000 description 10
- 239000012088 reference solution Substances 0.000 description 8
- 239000007788 liquid Substances 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 5
- 239000000945 filler Substances 0.000 description 5
- 208000005176 Hepatitis C Diseases 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- 238000002347 injection Methods 0.000 description 4
- 239000007924 injection Substances 0.000 description 4
- 239000011259 mixed solution Substances 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- IWUCXVSUMQZMFG-AFCXAGJDSA-N Ribavirin Chemical compound N1=C(C(=O)N)N=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 IWUCXVSUMQZMFG-AFCXAGJDSA-N 0.000 description 3
- 238000010812 external standard method Methods 0.000 description 3
- 229960000329 ribavirin Drugs 0.000 description 3
- HZCAHMRRMINHDJ-DBRKOABJSA-N ribavirin Natural products O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1N=CN=C1 HZCAHMRRMINHDJ-DBRKOABJSA-N 0.000 description 3
- 239000012488 sample solution Substances 0.000 description 3
- 238000013112 stability test Methods 0.000 description 3
- 238000005303 weighing Methods 0.000 description 3
- 206010067484 Adverse reaction Diseases 0.000 description 2
- 208000006154 Chronic hepatitis C Diseases 0.000 description 2
- 241000711549 Hepacivirus C Species 0.000 description 2
- 102000014150 Interferons Human genes 0.000 description 2
- 108010050904 Interferons Proteins 0.000 description 2
- 230000006838 adverse reaction Effects 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 208000010710 hepatitis C virus infection Diseases 0.000 description 2
- 229940079322 interferon Drugs 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 239000012452 mother liquor Substances 0.000 description 2
- 238000004305 normal phase HPLC Methods 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
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- 238000009210 therapy by ultrasound Methods 0.000 description 2
- 241001251200 Agelas Species 0.000 description 1
- 229940126656 GS-4224 Drugs 0.000 description 1
- 229940123066 Polymerase inhibitor Drugs 0.000 description 1
- 101800001554 RNA-directed RNA polymerase Proteins 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- -1 chloro (phenoxy) phosphoryl Chemical group 0.000 description 1
- 208000020403 chronic hepatitis C virus infection Diseases 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- TXFOLHZMICYNRM-UHFFFAOYSA-N dichlorophosphoryloxybenzene Chemical compound ClP(Cl)(=O)OC1=CC=CC=C1 TXFOLHZMICYNRM-UHFFFAOYSA-N 0.000 description 1
- 238000003821 enantio-separation Methods 0.000 description 1
- 208000006454 hepatitis Diseases 0.000 description 1
- 231100000283 hepatitis Toxicity 0.000 description 1
- 239000000543 intermediate Substances 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- RTHYZGURHQYQSH-UHFFFAOYSA-L potassium dihydrogen phosphate tetrabutylazanium bromide hydrate Chemical compound CCCC[N+](CCCC)(CCCC)CCCC.O.OP(=O)(O)[O-].[K+].[Br-] RTHYZGURHQYQSH-UHFFFAOYSA-L 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000007086 side reaction Methods 0.000 description 1
- 230000002459 sustained effect Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
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- 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
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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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
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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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
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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
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- G01N30/36—Control of physical parameters of the fluid carrier in high pressure liquid systems
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- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
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Abstract
The invention discloses a liquid chromatography method for detecting related substances in Sofosbuvir, which comprises the following steps: preparing a test solution, namely preparing the test solution by using a mobile phase as a solvent; preparing a reference substance solution, namely preparing the reference substance solution by using a mobile phase as a solvent; preparing a system applicability solution, namely preparing the system applicability solution by using a mobile phase as a solvent; detecting by high performance liquid chromatography, wherein the related substances are selected from one or two of impurities 1 and 2.
Description
Technical Field
The invention relates to the field of drug analysis, in particular to a liquid chromatography method for detecting related substances of fosbuvir.
Background
Sofosbuvir (Sofosbuvir, also known as Sofosbuvir or Sofosbuvir) is a Hepatitis C Virus (HCV) nucleotide analog NS5B polymerase inhibitor developed by Gillede (Gilead Sciences) for use in the treatment of chronic hepatitis C virus infections. Clinical trials demonstrated that the overall Sustained Virological Response rate (SVR) of sofosbuvir in combination with peginterferon and ribavirin is up to 90% for hepatitis c types 1 and 4; aiming at type 2 hepatitis C, the SVR of the medicine and ribavirin is 89-95 percent; aiming at type 3 hepatitis C, the SVR of the medicine and ribavirin is 61-63%; when the sofosbuvir is used for treating the specific genotype type chronic hepatitis C, the requirement of the traditional injection medicine Interferon (IFN) can be eliminated, the adverse reaction of a patient is reduced, the compliance of the patient is improved, and the sofosbuvir is widely considered as a breakthrough treatment medicine for the chronic hepatitis C. The raw research drug of Sofosbuvir has been approved for the market in the United states, european Union, japan, china and other countries since 2013.
The related substances are initial materials, intermediates, side reaction products, degradation impurities and the like brought in the process of synthesizing the medicament, influence the product quality of the medicament and even cause serious adverse reaction. The development of a detection method of related substances is an important part in the medicine quality research, and the safety, effectiveness and quality controllability of the medicine can be improved.
Regarding liquid chromatography for detecting sofosbuvir and related substances thereof, CN107402267A discloses a method for determining the diastereoisomers and impurity content of sofosbuvir bulk drugs by normal phase high performance liquid chromatography, wherein the impurities comprise:
CN112213418A discloses a method for detecting related substances in Sofosbuvir, which adopts a mobile phase A: potassium dihydrogen phosphate-tetrabutylammonium bromide-water, mobile phase B: and performing gradient elution on acetonitrile and octadecylsilane chemically bonded silica gel. The related substances comprise:
CN107402267A and CN112213418A disclose chiral impurities. In addition to chiral impurities, certain achiral impurities are involved in the synthesis process of the sofosbuvir. Therefore, there is an urgent need for liquid chromatography for detecting chiral and achiral impurities in fosbuvir by using an achiral column.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a liquid chromatography method for detecting chiral impurities and achiral impurities in Sofosbuvir by adopting a reversed-phase achiral column. The flow rate of the mobile phase in the liquid chromatography of the present invention is gradient-changed.
The invention provides a liquid chromatography method for detecting related substances in Sofosbuvir, which comprises the following steps:
preparing a test solution, namely preparing the test solution by using a mobile phase as a solvent;
preparing a reference substance solution, namely preparing the reference substance solution by using a mobile phase as a solvent;
preparing a system applicability solution, namely preparing the system applicability solution by using a mobile phase as a solvent;
detecting by high performance liquid chromatography;
wherein the related substance is selected from one or two of impurity 1 and impurity 2.
In the present invention, impurity 1 is: phenyl dichlorophosphate; impurity 2 is: isopropyl N- (chloro (phenoxy) phosphoryl) -L-alaninate.
In a preferred embodiment of the present invention, the related substance is selected from either or both of impurity 1 and impurity 2.
In a preferred embodiment of the invention, the related substances consist of impurity 1 and impurity 2.
In a preferred embodiment of the present invention, the concentration of the test solution is 1mg/ml to 50mg/ml, and the concentration of the control solution may be 0.1. Mu.g/ml to 10. Mu.g/ml.
In a preferred embodiment of the present invention, the concentration of the test solution is 5mg/ml to 30mg/ml, and the concentration of the control solution may be 0.2. Mu.g/ml to 5. Mu.g/ml.
In a preferred embodiment of the present invention, the concentration of the test solution is 20mg/ml, and the concentration of the control solution may be 1. Mu.g/ml.
In a preferred embodiment of the invention, the packed column is a silica gel column.
In the present invention, the octadecylsilane bonded silica gel column is commercially available. For example, the octadecylsilane-bonded silica gel column may be selected from the octadecylsilane-bonded silica gel columns manufactured by YMC, phenomenex, ES, merck, agilent, kromasil, agela, or Techmate.
In a preferred embodiment of the invention, the octadecylsilane-bonded silica gel column is selected from any one of YMC Triat C18, YMC ODS C18, YMC-Pack C18, phenomenex kinetex C18, titank C18, ES-C18, epic C18, ZORBAX SB-C18, poroshell 120EC-C18, ZORBAX 300SB-C18, XDB-C18, eclipse Plus C18, TC-C18, extended-C18, bonshell C18, venusil C18 Plus, bonshell ASB C18, venusil HLP C18, venusil MP C18, innoval Neo XD C18, venusil XBP C18 (A), venusil XBP C18 (B), techmate C18-ST, tachmate C18-ST II, etnussil XB C18-10-C18, kruserl 5-10 Etmesil C18, kruserl C18-C10-C18, krusemi C10-C10, or Krusemi C10-C10.
In a preferred embodiment of the invention, the packed column is a carbo-octadecylsilicon column using silica gel as the packing agent, preferably Ultimate SiO 2 4.6mm×250mm，5μm。
In a preferred embodiment of the invention, the mobile phase is n-hexane-ethyl acetate. The volume ratio of n-hexane-ethyl acetate is 70-95, preferably 80-10, more preferably 85.
In a preferred embodiment of the invention, the flow rate is between 0.7ml and 1.6ml per minute.
In a preferred embodiment of the invention, the flow rate is varied during the elution by the following procedure:
in a preferred embodiment of the invention, the elution procedure is:
in a preferred embodiment of the invention, the elution procedure is:
in the invention, when preparing a test solution or a reference solution, the solvent is n-hexane-ethyl acetate. The volume ratio of n-hexane to ethyl acetate is 70:30-95, preferably 80-90, more preferably 85.
In a preferred embodiment of the invention, the column temperature is from 30 ℃ to 60 ℃, preferably from 35 ℃ to 55 ℃, more preferably 45 ℃.
In a preferred embodiment of the invention, the detection wavelength is from 240nm to 280nm, preferably from 250nm to 270nm, more preferably 262nm.
In a preferred embodiment of the present invention, the detection conditions of the high performance liquid chromatography are as follows: bonding a silica gel column by using octadecylsilane; using n-hexane-ethyl acetate (80-90); the column temperature is 35-55 ℃; the detection wavelength is 250nm-270nm; elution was carried out with the following gradient:
in a preferred embodiment of the present invention, the detection conditions of the high performance liquid chromatography are as follows: bonding a silica gel column by using octadecylsilane; using n-hexane-ethyl acetate (80-90); the column temperature is 35-55 ℃; the detection wavelength is 250nm-270nm; elution was carried out with the following gradient:
in a preferred embodiment of the present invention, the detection conditions of the high performance liquid chromatography are as follows: bonding a silica gel column by using octadecylsilane; using n-hexane-ethyl acetate (80-90); the column temperature is 35-55 ℃; the detection wavelength is 250nm-270nm; elution was carried out with the following gradient:
in a preferred embodiment of the present invention, the detection conditions of the high performance liquid chromatography are: bonding a silica gel column by using octadecylsilane; using n-hexane-ethyl acetate (70-95); the column temperature is 30-60 ℃; the detection wavelength is 250nm-270nm; elution was carried out with the following gradient:
in a preferred embodiment of the present invention, the detection conditions of the high performance liquid chromatography are as follows: bonding a silica gel column by using octadecylsilane; using n-hexane-ethyl acetate (70; the column temperature is 30-60 ℃; the detection wavelength is 250nm-270nm; elution was carried out with the following gradient:
in a preferred embodiment of the present invention, the detection conditions of the high performance liquid chromatography are: bonding a silica gel column by using octadecylsilane; using n-hexane-ethyl acetate (70; the column temperature is 30-60 ℃; the detection wavelength is 250nm-270nm; elution was carried out with the following gradient:
in a preferred embodiment of the present invention, the detection conditions of the high performance liquid chromatography are as follows: ultimate SiO using silica gel as filler 2 4.6 mm. Times.250mm, 5 μm; n-hexane-ethyl acetate (85); the column temperature is 45 ℃; the detection wavelength is 262nm; elution was carried out with the following gradient:
in a preferred embodiment of the present inventionThe detection conditions of the high performance liquid chromatography are as follows: ultimate SiO using silica gel as filler 2 4.6 mm. Times.250mm, 5 μm; n-hexane-ethyl acetate (85); the column temperature is 45 ℃; the detection wavelength is 262nm; elution was carried out with the following gradient:
in a preferred embodiment of the present invention, the detection conditions of the high performance liquid chromatography are: ultimate SiO using silica gel as filler 2 4.6 mm. Times.250mm, 5 μm; n-hexane-ethyl acetate (85); the column temperature is 45 ℃; the detection wavelength is 262nm; elution was carried out with the following gradient:
in the present invention, impurity 1 is not more than 50ppm; impurity 2 is not more than 50ppm; the sum of impurity 1 and impurity 2 does not exceed 75ppm.
In a preferred embodiment of the present invention, the liquid chromatography comprises:
placing a sample in a measuring flask, precisely adding ethyl acetate, performing ultrasonic treatment to dissolve, precisely adding n-hexane, shaking, filtering, and collecting filtrate as sample solution;
taking an impurity 1 and an impurity 2 reference substance, and preparing a solution containing about 1 mu g of each of the impurity 1 and the impurity 2 in 1ml by using a mobile phase as a reference solution;
silica gel as filler (Ultimate SiO) 2 4.6 mm. Times.250mm, 5 μm); n-hexane-ethyl acetate (85); the column temperature was 45 ℃; the detection wavelength is 262nm; gradient elution was as follows;
and (4) respectively injecting 100 mu l of the reference solution and the test solution into a liquid chromatograph, and recording the chromatogram. And (4) calculating the contents of the impurity 1 and the impurity 2 by peak areas according to an external standard method to obtain the product. Impurity 1 does not exceed 50ppm; impurity 2 is not more than 50ppm; the sum of impurity 1 and impurity 2 does not exceed 75ppm.
In the present invention, when referring to the ratio between the mobile phases, it refers to the volume ratio between the mobile phases. In the present invention, references to concentrations refer to the weight/volume ratio of the substance in the solvent. In the present invention, ppm refers to a concentration expressed in terms of parts per million of the mass of solute to the mass of the entire solution, and is also referred to as a part per million concentration.
The method of the invention has the following beneficial effects: the invention adopts a reversed-phase achiral column to detect chiral impurities and achiral impurities in the fosbuvir. The flow rate of the mobile phase in the liquid chromatography of the present invention is gradient-changed. The method has strong specificity, the Sofosbuvir and the blank solvent do not interfere with impurities, and the separation degree is good; the invention has high sensitivity of detecting impurities under chromatographic conditions; the correlation coefficients of impurities are all larger than 0.99, and the linearity is good; the sample injection precision is good; under the item of a repeatability test, the measurement results of different analysts on different instruments at different times are consistent; the solution stability test result shows that the solution is stable when the test solution and the reference solution are placed at room temperature for 8 hours; the average recovery rate of the impurities is between 90 and 110 percent.
Drawings
FIG. 1 is a linear plot of impurities 1, wherein diamond-solid represents 0.302. Mu.g/ml, \9632; represents 0.503. Mu.g/ml,. Tangle-solidup represents 0.805. Mu.g/ml,. Gamma represents 1.006. Mu.g/ml,. Gamma.represents 1.509. Mu.g/ml,. 9679; represents 2.012. Mu.g/ml;
FIG. 2 is a linear plot of impurity 2, where diamond-solid represents 0.261. Mu.g/ml, \9632; represents 0.434. Mu.g/ml, tangle-solidup represents 0.695 μ g/ml, gamma represents 0.869 μ g/ml, x represents 1.303 μ g/ml, \9679; represents 1.737 μ g/ml.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, embodiments of the present invention will be described in detail below with reference to the accompanying drawings. It should be noted that the embodiments and features of the embodiments of the present invention may be arbitrarily combined with each other without conflict.
Examples
Example 1: HPLC method
1.1 analytical methods
Table 1 instruments and reagents used in example 1
Chromatographic conditions are as follows: silica gel as filler (Ultimate SiO) 2 4.6 mm. Times.250mm, 5 μm); n-hexane-ethyl acetate (85); the column temperature was 45 ℃; the detection wavelength is 262nm; elution was carried out with a gradient as follows.
The determination method comprises the following steps: taking about 100mg of a test sample, placing the test sample in a 5ml measuring flask, precisely adding 1.5ml of ethyl acetate, carrying out ultrasonic treatment to dissolve the test sample, precisely adding 3.5ml of n-hexane, shaking up, filtering, and taking a subsequent filtrate as a test sample solution;
taking an impurity 1 and an impurity 2 reference substance, and preparing a solution containing about 1 mu g of each of the impurity 1 and the impurity 2 in 1ml by using a mobile phase as a reference solution;
detecting according to the chromatographic conditions;
and (4) respectively injecting 100 mu l of the reference solution and the test solution into a liquid chromatograph, and recording the chromatogram. And calculating the contents of the impurity 1 and the impurity 2 by peak areas according to an external standard method to obtain the product. Impurity 1 does not exceed 50ppm; impurity 2 is not more than 50ppm; the sum of impurity 1 and impurity 2 does not exceed 75ppm.
1.2 methodological validation
1.2.1, specificity
Taking Sofosbuvir (SFB), impurity 1 and impurity 2 reference substances respectively, and preparing a solution containing each component of 1 mu g/ml by using a mobile phase to serve as a single impurity positioning solution; and preparing a mixed solution of the reference substances of the impurities 1 and 2 by using a mobile phase.
A blank solvent, a single impurity-localized solution, and a mixed solution were taken 100. Mu.l each and injected into a chromatograph, and the results are shown in Table 2.
TABLE 2 results of the specificity test
The blank solvent and the sofosbuvir do not interfere with the determination of the impurity 1 and the impurity 2.
1.2.2, limit of quantitation, limit of detection
The reference samples of impurity 1 and impurity 2 were dissolved in a mobile phase and diluted to give a mixed solution containing about 1. Mu.g of each of impurity 1 and impurity 2 per 1ml, as a mother liquor. The mother liquor was diluted stepwise with the mobile phase as the test solution. Precisely measuring 100 mu l of each level of test solution, injecting the solution into a liquid chromatograph, and determining a quantitative limit according to a signal-to-noise ratio (S/N is about 10; the detection limit is determined in terms of signal-to-noise ratio (S/N is about 3. The results are shown in Table 3.
TABLE 3 detection limit and quantitation limit test results
Test results show that the method has good sensitivity for measuring each impurity.
1.2.3, linearity and Range
Respectively taking reference substances of the impurity 1 and the impurity 2, precisely weighing, and preparing into solution containing the impurity 1 and the impurity 2 with concentration in a range of 16.1-16.2 by using a flowing phase. The preparation method comprises preparing solutions with the solvent at limit of 200%, 150%, 100%, 80%, 50% and 30%. Precisely measuring 100 μ l of the solution with each series of concentrations, injecting into a chromatograph, recording chromatogram, and performing linear regression with the concentration as abscissa (μ g/ml) and peak area as ordinate, wherein the test results are shown in tables 4.1-4.2.
TABLE 4.1 Linear test results for impurity 1
TABLE 4.2 Linear test results for impurity 2
Test results show that the correlation coefficients of all impurities are more than 0.999 within the concentration range from the limit of 30% to the limit of 200%, and the linear relation is good.
1.2.4, precision
(1) Sample introduction precision: precisely weighing reference substances of the impurity 1 and the impurity 2, and preparing a mixed solution of the impurity 1 and the impurity 2 with each concentration of 1 mu g/ml by using a mobile phase as a sample injection precision test solution; the samples were continuously injected 6 times, and the peak area was examined for changes, the results are shown in Table 5.
TABLE 5 sample introduction precision test results
| Peak area | Sample 1 | Sample 2 | Sample 3 | Sample 4 | Sample No. 5 | Sample 6 | RSD(％) |
| Impurity 1 | 4451 | 4422 | 4473 | 4547 | 4508 | 4492 | 0.98 |
| Impurity 2 | 2279 | 2037 | 2288 | 2201 | 2048 | 2227 | 5.11 |
Test results show that the sample injection precision solution is continuously injected for 6 times, the RSD value of the peak area value of the impurity 1 is 0.98 percent, and the RSD value of the peak area value of the impurity 2 is 5.11 percent.
(2) Repeatability: a total of 6 parts of this product were collected and measured by the analytical method of example 1.1. The results are shown in Table 6. TABLE 6 results of the repeatability tests
| Name(s) | Sample 1 | Sample 2 | Sample 3 | Sample 4 | Sample No. 5 | Sample 6 | Mean value of | RSD |
| Impurity 1 | Undetected | Undetected | Not detected out | Not detected out | Undetected | Undetected | Undetected | -- |
| Impurity 2 | Not detected out | Not detected out | Undetected | Not detected out | Undetected | Undetected | Undetected | -- |
The test result shows that the detected number of the known impurities of 6 samples under the repeatability test item is consistent with the detected amount, the detected amount of the total impurities is consistent, and the repeatability of the analysis method is good. The results of 12 measurements made by different analysts at different times and on different instruments are consistent.
1.2.5 solution stability
The test solution and the reference solution were taken and left at room temperature for 0, 5, 6, 7, and 8 hours, respectively, and 100. Mu.l of each solution was injected into a liquid chromatograph, and the test results are shown in Table 7 below.
TABLE 7 solution stability test results
| Name(s) | Sample-impurity 1 | Reference substance-impurity 1 | Test article-impurity 2 | Reference substance-impurity 2 |
| 0 hour | Undetected | 4379 | Undetected | 2360 |
| 5 hours | Undetected | 4528 | Undetected | 2394 |
| 6 hours | Not detected out | 4698 | Undetected | 2118 |
| 7 hours | Not detected out | 4693 | Undetected | 2427 |
| 8 hours | Undetected | 4669 | Undetected | 2270 |
| Difference by% | --- | 6.8 | --- | 10.3 |
Note: the difference% means the ratio of the absolute value of the difference between the peak area at the observation point where the difference between the area and the 0-hour peak area is largest at 0 hour after 0 hour to the area at 0 hour.
Calculating the formula: difference% = |. 0 hour peak area-peak area at point of investigation with maximum difference |/0 hour peak area × 100%
The solution stability test result shows that when the reference solution is placed at room temperature for 8 hours, the maximum difference of the peak areas of the impurity 1 is 6.8 percent, and the maximum difference of the peak areas of the impurity 2 is 10.3 percent, so that the detection requirements are basically met; the sample solution is placed at room temperature for 8 hours, no impurity 1 or impurity 2 is detected, and the solution is stable when the sample is placed at room temperature for 8 hours. In conclusion, the test solution and the control solution were stable when left at room temperature for 8 hours.
1.2.6, accuracy
The recovery rates of impurity 1 and impurity 2 were measured.
Taking the reference substances of the sofosbuvir, the impurity 1 and the impurity 2, precisely weighing, and preparing the solution containing the sofosbuvir 20mg and the concentrations of the impurity 1 and the impurity 2 which are respectively 50%, 100% and 150% of the limit concentration according to the preparation method of the test solution, wherein 3 parts of each concentration solution are prepared. The analysis was carried out as in example 1.1. The recovery (%) and RSD (%) of impurity 1 and impurity 2 were calculated by peak area according to the external standard method, and the results of the recovery test are shown in Table 8.
TABLE 8 recovery test results
The recovery rate of the impurity 1 is in the range of 80.18-103.06%, the average recovery rate is 91.92%, and the RSD value is 7.59%; the recovery rate of the impurity 2 is in the range of 88.89-96.91%, the average recovery rate is 92.39%, the RSD value is 2.54%, the average recovery rates of the impurity 1 and the impurity 2 are both 90-110%, and the method has good accuracy.
Although the present invention has been described with reference to the above embodiments, the above description is only for illustrative purposes and is not intended to limit the scope of the present invention. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (10)
1. A liquid chromatography method for detecting related substances in Sofosbuvir comprises the following steps:
preparing a test solution, namely preparing the test solution by using a mobile phase as a solvent;
preparing a reference substance solution, namely preparing the reference substance solution by using a mobile phase as a solvent;
preparing a system applicability solution, namely preparing the system applicability solution by using a mobile phase as a solvent;
detecting by high performance liquid chromatography;
wherein the related substance is selected from one or two of impurity 1 and impurity 2
2. The liquid chromatography of claim 1, wherein the concentration of the test solution is 1mg/ml to 50mg/ml, preferably 5mg/ml to 30mg/ml, and more preferably 20mg/ml.
3. A liquid chromatography as claimed in claim 1, wherein the concentration of the control solution is from 0.1 μ g/ml to 10 μ g/ml, preferably from 0.2 μ g/ml to 5 μ g/ml, more preferably 1 μ g/ml.
4. Liquid chromatography according to claim 1, wherein the packed column of the liquid chromatography is a silica gel column, preferably an octadecylsilane bonded silica gel column.
5. A liquid chromatography as claimed in claim 4, the octadecylsilane bonded silica gel column is selected from the group consisting of YMC Triat C18, YMC ODS C18, YMC-Pack C18, phenomenex kinetex C18, titank C18, ES-C18, epic C18, ZORBAX SB-C18, poroshell 120EC-C18, ZORBAX 300SB-C18, XDB-C18, eclipse Plus C18, TC-C18, extended-C18, bonshell C18, venusil C18 Plus, bonshell ASB C18, and combinations thereof Venusil HLP C18, venusil MP C18, innoval Neo XD C18, venusil XBP C18 (A), venusil XBP C18 (B), techmate C18-ST II, techmate CI8 UG, kromasil Eternity-5-C18, kromasil EternitylXT-10-C18, kromasil100-5-C18, kromasil 300-5-C18, and Ulltice SiO mate 2 Any one or combination thereof, preferably Ultimate SiO 2 4.6mm×250mm，5μm。
6. The liquid chromatography according to claim 1, wherein the mobile phase of the liquid chromatography is n-hexane-ethyl acetate, the volume ratio of n-hexane-ethyl acetate being 70.
7. A liquid chromatography method according to claim 1, having a flow rate of 0.7-1.6 ml per minute,
preferably the flow rate of the liquid chromatography is varied during the elution procedure, the elution procedure being:
preferably the elution procedure is:
more preferably, the elution procedure is:
8. the liquid chromatography according to claim 1, wherein the solvent used in preparing the test solution or the control solution is n-hexane-ethyl acetate,
the volume ratio of n-hexane to ethyl acetate is 70:30-95, preferably 80-90, more preferably 85.
9. A liquid chromatography method according to claim 1 having a column temperature of 30 ℃ to 60 ℃, preferably 35 ℃ to 55 ℃, more preferably 45 ℃.
10. Liquid chromatography according to claim 1, having a detection wavelength of 240nm to 280nm, preferably 250nm to 270nm, more preferably 262nm.
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