CN113419004B - Method for detecting impurities in methyl 3-amino-4-methylbenzoate - Google Patents
Method for detecting impurities in methyl 3-amino-4-methylbenzoate Download PDFInfo
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- 239000012535 impurity Substances 0.000 title claims abstract description 131
- FDCOHGHEADZEGF-OCEACIFDSA-N Glycerol 1,3-dihexadecanoate 2-(9Z-octadecenoate) Chemical compound CCCCCCCCCCCCCCCC(=O)OCC(COC(=O)CCCCCCCCCCCCCCC)OC(=O)CCCCCCC\C=C\CCCCCCCC FDCOHGHEADZEGF-OCEACIFDSA-N 0.000 title claims abstract description 28
- 238000000034 method Methods 0.000 title claims abstract description 14
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- 239000011550 stock solution Substances 0.000 claims abstract description 50
- 238000001514 detection method Methods 0.000 claims abstract description 30
- 239000007864 aqueous solution Substances 0.000 claims abstract description 25
- 239000012085 test solution Substances 0.000 claims abstract description 21
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- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims abstract description 20
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- 239000013558 reference substance Substances 0.000 claims abstract description 12
- 238000010828 elution Methods 0.000 claims abstract description 7
- 230000005526 G1 to G0 transition Effects 0.000 claims abstract description 6
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims abstract description 6
- 239000000377 silicon dioxide Substances 0.000 claims abstract description 6
- 239000002245 particle Substances 0.000 claims description 4
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- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 abstract description 6
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- 239000012088 reference solution Substances 0.000 abstract description 4
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- 229960001346 nilotinib Drugs 0.000 description 12
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- -1 4-methyl-1H-imidazol-1-yl Chemical group 0.000 description 2
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- AAEQXEDPVFIFDK-UHFFFAOYSA-N 3-(4-fluorobenzoyl)-2-(2-methylpropanoyl)-n,3-diphenyloxirane-2-carboxamide Chemical compound C=1C=CC=CC=1NC(=O)C1(C(=O)C(C)C)OC1(C=1C=CC=CC=1)C(=O)C1=CC=C(F)C=C1 AAEQXEDPVFIFDK-UHFFFAOYSA-N 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
-
- 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
-
- 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
- G01N30/86—Signal analysis
- G01N30/8624—Detection of slopes or peaks; baseline correction
- G01N30/8631—Peaks
- G01N30/8634—Peak quality criteria
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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
- G01N2030/042—Standards
- G01N2030/047—Standards external
Abstract
The invention relates to the technical field of medical chemistry, in particular to a method for detecting impurities in 3-amino-4-methyl benzoate, which comprises the following steps: 1) Mixing methyl 3-amino-4-methylbenzoate and acetonitrile aqueous solution to obtain a test solution a; respectively mixing the impurities to be detected with acetonitrile to obtain impurity stock solutions; mixing appropriate amount of each impurity stock solution with acetonitrile water solution to obtain each impurity reference solution; mixing 3-amino-4-methyl benzoate, a proper amount of impurity stock solutions and an acetonitrile aqueous solution to obtain a system applicability solution b; 2) Respectively detecting acetonitrile water solution, impurity reference substance solution, test solution a and system applicability solution b by adopting HPLC, wherein the detection conditions are as follows: octadecylsilane chemically bonded silica is used as a stationary phase; performing equal gradient elution by using a mixed solution of potassium dihydrogen phosphate solution and acetonitrile as a mobile phase; 3) And obtaining the impurity detection result through calculation. The method can accurately detect each impurity.
Description
Technical Field
The invention relates to the technical field of medical chemistry, in particular to a method for detecting impurities in 3-amino-4-methyl benzoate.
Background
Nilotinib (Nilotinib), chemical name 4-methyl-N- [3- (4-methyl-1H-imidazol-1-yl) -5- (trifluoromethyl) phenyl]-3- [ [4- (3-pyridinyl) -2-pyrimidinyl]Amino group]-benzamide of the formula C 28 H 22 F 3 N 7 O, molecular weight 529.51600, developed by norwalk, switzerland in 2007. Nilotinib has stronger selectivity on BCR-ABL kinase activity, has 30 times stronger inhibition effect on tyrosine kinase than imatinib, can inhibit kinase activity of BCR-ABL mutant resistant to imatinib, and is mainly used for treating chronic granulocytic leukemia resistant to gleevec (imatinib) clinically. Nilotinib has the chemical formula:
the methyl 3-amino-4-methylbenzoate is used as a main starting reaction material for synthesis of nilotinib, potential impurities existing in the methyl 3-amino-4-methylbenzoate can be introduced into API, and in order to reduce potential risks of the API, the quality of the methyl 3-amino-4-methylbenzoate needs to be monitored.
Disclosure of Invention
In view of this, the technical problem to be solved by the present invention is to provide a method for detecting impurities in methyl 3-amino-4-methylbenzoate, which can effectively detect impurities in methyl 3-amino-4-methylbenzoate, and the separation degree between the impurities and methyl 3-amino-4-methylbenzoate is high.
The invention provides a method for detecting impurities in 3-amino-4-methyl benzoate, which comprises the following steps:
1) Mixing 3-amino-4-methyl benzoate and acetonitrile aqueous solution to obtain a test solution a;
respectively mixing impurities to be detected with acetonitrile to obtain impurity stock solutions;
mixing appropriate amount of each impurity stock solution with acetonitrile water solution to obtain each impurity reference solution;
mixing 3-amino-4-methyl benzoate, a proper amount of impurity stock solutions and an acetonitrile aqueous solution to obtain a system applicability solution b;
2) Respectively detecting acetonitrile aqueous solution, impurity reference substance solution, test solution a and system applicability solution b by adopting HPLC, wherein the detection conditions are as follows:
octadecylsilane chemically bonded silica is used as a stationary phase;
performing equal gradient elution by using a mixed solution of potassium dihydrogen phosphate solution and acetonitrile as a mobile phase;
3) And (3) obtaining the detection result of the impurity by calculation according to the HPLC map obtained in the step 2).
Preferably, in step 3), the mobile phase comprises mobile phase a and mobile phase B;
the mobile phase A comprises:
the volume ratio is 20:80 of 0.005-0.1 mol/L potassium dihydrogen phosphate solution and acetonitrile;
the mobile phase B comprises:
the volume ratio is 80:20 of 0.005-0.1 mol/L potassium dihydrogen phosphate solution and acetonitrile;
the pH value of the potassium dihydrogen phosphate solution is 3.0-5.5.
Preferably, the volume ratio of the mobile phase A to the mobile phase B is 15-85: 85 to 15.
Preferably, in the step 3), the flow rate of the mobile phase is 0.8-1.2 mL/min;
the column temperature of the chromatographic column to be detected is 20-40 ℃.
Preferably, in the step 3), the flow rate of the mobile phase is 1mL/min;
the column temperature of the detected chromatographic column is 30 ℃;
the chromatographic column for detection is YMC-Triart C18, the column length is 250mm, the inner diameter is 4.6mm, and the particle size of the filler is 5 mu m;
the wavelength of the detection is 225nm.
Preferably, in step 3), during the isocratic elution, the volume ratio of the mobile phase a to the mobile phase B is:
preferably, each impurity stock solution comprises a stock solution of the impurity of formula (1), a stock solution of the impurity of formula (2), a stock solution of the impurity of formula (3), a stock solution of the impurity of formula (4), a stock solution of the impurity of formula (5), and a stock solution of the impurity of formula (6);
preferably, the concentration of each impurity in the stock solution is 0.0005mg to 1mg:1mL;
in each impurity reference substance solution, the concentration of the impurity is 0.0001-0.01 mg:1mL.
Preferably, in the test solution a, the concentration of methyl 3-amino-4-methylbenzoate is 0.1-5 mg:1mL.
Preferably, in the system suitability solution b, the concentration of the methyl 3-amino-4-methylbenzoate is 0.1 to 5mg:1mL, the concentration of impurities is 0.0001-0.01 mg:1mL.
The invention provides a method for detecting impurities in 3-amino-4-methyl benzoate, which comprises the following steps: 1) Mixing 3-amino-4-methyl benzoate and acetonitrile aqueous solution to obtain a test solution a; respectively mixing impurities to be detected with acetonitrile to obtain impurity stock solutions; mixing appropriate amount of each impurity stock solution with acetonitrile water solution to obtain each impurity reference solution; mixing 3-amino-4-methyl benzoate, a proper amount of impurity stock solutions and an acetonitrile aqueous solution to obtain a system applicability solution b; 2) Respectively detecting acetonitrile aqueous solution, impurity reference substance solution, test solution a and system applicability solution b by adopting HPLC, wherein the detection conditions are as follows: octadecylsilane chemically bonded silica is used as a stationary phase; performing equal gradient elution by using a mixed solution of potassium dihydrogen phosphate solution and acetonitrile as a mobile phase; 3) And (3) obtaining the detection result of the impurity by calculation according to the HPLC map obtained in the step 2). The method for detecting the nilotinib starting material methyl 3-amino-4-methylbenzoate is an external standard method, can accurately detect each impurity in the nilotinib starting material methyl 3-amino-4-methylbenzoate, has high separation degree between the impurities and the methyl 3-amino-4-methylbenzoate, has high separation degree between the impurities, meets all indexes, is simple and convenient to operate, has low cost, and is suitable for wide application.
Drawings
FIG. 1 is an HPLC chromatogram of an aqueous solution of acetonitrile at a volume concentration of 50%;
FIG. 2 is an HPLC chromatogram of a system suitability solution b of example 1 of the present invention;
FIG. 3 is an HPLC chromatogram of the test solution a in example 1 of the present invention.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The invention provides a method for detecting impurities in 3-amino-4-methyl benzoate, which comprises the following steps:
1) Mixing methyl 3-amino-4-methylbenzoate and acetonitrile aqueous solution to obtain a test solution a;
respectively mixing impurities to be detected with acetonitrile to obtain impurity stock solutions;
mixing a proper amount of each impurity stock solution with an acetonitrile aqueous solution respectively to obtain each impurity reference substance solution;
mixing 3-amino-4-methyl benzoate, a proper amount of impurity stock solutions and an acetonitrile aqueous solution to obtain a system applicability solution b;
2) Respectively detecting acetonitrile aqueous solution, impurity reference substance solution, test solution a and system applicability solution b by adopting HPLC, wherein the detection conditions are as follows:
octadecylsilane chemically bonded silica is used as a stationary phase;
performing equal gradient elution by using a mixed solution of potassium dihydrogen phosphate solution and acetonitrile as a mobile phase;
3) And (3) obtaining the detection result of the impurity by calculation according to the HPLC map obtained in the step 2).
According to the invention, methyl 3-amino-4-methylbenzoate and acetonitrile are mixed to obtain a test solution a.
In certain embodiments of the invention, the aqueous solution of acetonitrile has a concentration of 50% by volume.
In some embodiments of the present invention, the concentration of methyl 3-amino-4-methylbenzoate in the test solution a is 0.1 to 5mg:1mL. In certain embodiments, the concentration of methyl 3-amino-4-methylbenzoate in the test solution a is 0.2mg:1mL.
The invention mixes the impurities to be detected with acetonitrile respectively to obtain the impurity stock solutions.
In certain embodiments of the invention, each of the impurity stock solutions comprises a stock solution of the impurity of formula (1), a stock solution of the impurity of formula (2), a stock solution of the impurity of formula (3), a stock solution of the impurity of formula (4), a stock solution of the impurity of formula (5), and a stock solution of the impurity of formula (6);
in certain embodiments of the invention, the concentration of each impurity in the stock solution of impurities is from 0.0005mg to 1mg:1mL. In certain embodiments, the concentration of each impurity in the stock solution is 0.05mg:1mL.
In certain embodiments of the invention, the concentration of impurities in each impurity stock solution is the same.
In the invention, a proper amount of each impurity stock solution is taken and respectively mixed with an acetonitrile water solution to obtain each impurity reference substance solution.
The amount of each impurity stock solution used is not particularly limited, and the required amount can be used according to actual conditions.
In certain embodiments of the invention, the concentration of the impurity in each impurity control solution is from 0.0001 to 0.01mg:1mL. In certain embodiments, the concentration of impurities in each impurity control solution is 0.001mg:1mL.
In certain embodiments of the invention, the aqueous solution of acetonitrile has a concentration of 50% by volume.
In certain embodiments of the invention, the concentration of the impurity in each of the impurity control solutions is the same.
The invention mixes 3-amino-4-methyl benzoate, a proper amount of each impurity stock solution and acetonitrile aqueous solution to obtain a system applicability solution b.
The amount of each impurity stock solution mentioned herein is not particularly limited, and may be selected as needed in accordance with the actual circumstances.
In certain embodiments of the invention, the aqueous solution of acetonitrile has a concentration of 50% by volume.
In certain embodiments of the present invention, the concentration of the methyl 3-amino-4-methylbenzoate in the system suitability solution b is 0.1 to 5mg:1mL, and the concentration of impurities is 0.0001-0.01 mg:1mL. In certain embodiments, the system suitability solution b, the concentration of the methyl 3-amino-4-methylbenzoate is 0.2mg:1mL, concentration of impurities 0.001mg:1mL.
After obtaining each impurity stock solution, each impurity reference substance solution, the test solution a and the system applicability solution b, the acetonitrile aqueous solution, the impurity reference substance solution, the test solution a and the system applicability solution b are respectively detected by HPLC.
In certain embodiments of the invention, the aqueous solution of acetonitrile has a concentration of 50% by volume.
The detection conditions are as follows:
octadecylsilane chemically bonded silica is used as a stationary phase;
and (3) taking a mixed solution of potassium dihydrogen phosphate solution and acetonitrile as a mobile phase for carrying out equal gradient elution.
In certain embodiments of the invention, the mobile phase comprises mobile phase a and mobile phase B;
the mobile phase A comprises:
the volume ratio is 15-25: 75 to 85 of 0.005 to 0.1mol/L potassium dihydrogen phosphate solution and acetonitrile;
the mobile phase B comprises:
the volume ratio is 75-85: 15 to 25 portions of 0.005 to 0.1mol/L potassium dihydrogen phosphate solution and acetonitrile.
In some embodiments of the invention, in the mobile phase a, the volume ratio of 0.005-0.1 mol/L potassium dihydrogen phosphate solution to acetonitrile is 20:80.
in some embodiments of the invention, in the mobile phase B, the volume ratio of 0.005-0.1 mol/L potassium dihydrogen phosphate solution to acetonitrile is 80:20.
in certain embodiments of the invention, the pH of the potassium dihydrogen phosphate solution is 3.0 to 5.5. In certain embodiments, the potassium dihydrogen phosphate solution has a pH of 4.3.
In certain embodiments, the potassium dihydrogen phosphate solution has a concentration of 0.02mol/L.
In certain embodiments of the invention, the volume ratio of mobile phase a to mobile phase B is 15 to 85:85 to 15.
In certain embodiments of the present invention, during the gradient elution, the volume ratio of mobile phase a to mobile phase B is:
in certain embodiments of the invention, the flow rate of the mobile phase is 0.8 to 1.2mL/min. In certain embodiments, the flow rate of the mobile phase is 1mL/min.
In certain embodiments of the invention, the column temperature of the chromatographic column is measured at 20-40 ℃. In certain embodiments, the column temperature of the chromatographic column being tested is 30 ℃.
In certain embodiments of the invention, the chromatographic column tested is YMC-Triart C18, the column length is 250mm, the inner diameter is 4.6mm, and the packing particle size is 5 μm.
In certain embodiments of the invention, the wavelength of detection is 225nm.
And after the detection is finished, obtaining the detection result of the impurity by calculation according to the HPLC map obtained in the step.
In certain embodiments of the present invention, the method of calculation is area normalization.
The source of the above-mentioned raw materials is not particularly limited in the present invention, and may be generally commercially available.
The method for detecting the nilotinib starting material methyl 3-amino-4-methylbenzoate is an external standard method, can accurately detect each impurity in the nilotinib starting material methyl 3-amino-4-methylbenzoate, has high separation degree of the impurity from the methyl 3-amino-4-methylbenzoate and high separation degree among the impurities, meets the requirements of each index, and is simple and convenient to operate, low in cost and suitable for wide application.
In order to further illustrate the present invention, the following examples are provided to describe the method for detecting impurities in methyl 3-amino-4-methylbenzoate in detail, but should not be construed as limiting the scope of the present invention.
Example 1
Instruments and conditions:
a detector: a UV detector;
a chromatographic column: YMC-Triart C18, with a column length of 250mm, an inner diameter of 4.6mm, and a filler particle size of 5 μm;
detection wavelength: 225nm;
mobile phase:
a0.02 mol/L potassium dihydrogen phosphate solution (pH 4.3) -acetonitrile (volume ratio 20
B0.02 mol/L potassium dihydrogen phosphate solution (pH 4.3) -acetonitrile (volume ratio 80;
column temperature: 30 ℃;
flow rate: 1.0mL/mim;
time program:
solvent: an aqueous solution of acetonitrile, the volume concentration being 50%;
sample injection amount: 10 μ L.
The experimental steps are as follows:
taking 10mg of nilotinib starting material 3-amino-4-methyl benzoate, precisely weighing, adding a solvent to dissolve and dilute into a test solution a containing 0.2mg of nilotinib per 1mL;
taking 5mg of each impurity of the formula (1), the formula (2), the formula (3), the formula (4), the formula (5) and the formula (6), precisely weighing, and respectively adding acetonitrile to dissolve and dilute into an impurity stock solution containing 50 mu g of impurities per 1mL; precisely measuring 1.0mL of each impurity stock solution, and diluting with a solvent to obtain a reference solution containing 1 microgram of each impurity in 1mL; taking 10mg of nilotinib starting material 3-amino-4-methyl benzoate, precisely weighing, adding a proper amount of solvent for dissolving, precisely weighing 1.0mL of each impurity stock solution respectively, adding, and diluting with the solvent to obtain a system applicability solution b containing 0.2mg of each 1 mu g of 3-amino-4-methyl benzoate, impurities of formula (1), formula (2), formula (3), formula (4), formula (5) and formula (6) in each 1mL.
Precisely sucking 10 μ L acetonitrile water solution with volume concentration of 50%, injecting into high performance liquid chromatograph for detection, and recording map. Precisely absorbing 10 mu L of each impurity reference substance solution, injecting into a high performance liquid chromatograph for detection, and recording the map. Precisely sucking 10 μ L of the sample solution, injecting into high performance liquid chromatograph, detecting, and recording the chromatogram. Precisely sucking 10 μ L of the system applicability solution b, injecting into a high performance liquid chromatograph for detection, and recording the map. Wherein, impurity formula (1) corresponds impurity A, impurity formula (2) corresponds impurity B, impurity formula (3) corresponds impurity C, impurity formula (4) corresponds impurity D, impurity formula (5) corresponds impurity E, and impurity formula (6) corresponds impurity F.
And (4) obtaining the detection result of the impurity by calculation according to the HPLC map obtained in the step.
FIG. 1 is an HPLC chromatogram of a 50% acetonitrile-containing aqueous solution.
FIG. 2 is an HPLC chromatogram of a system suitability solution b of example 1 of the present invention.
FIG. 3 is an HPLC chromatogram of the test solution a in example 1 of the present invention.
The results of the experiments are summarized in tables 1 and 2.
TABLE 1 HPLC DETECTION AND CALCULATION OF SYSTEM APPLICABILITY SOLUTION B OF EXAMPLE 1
TABLE 2 HPLC DETECTION AND CALCULATION OF TEST SOLUTION A IN EXAMPLE 1
As can be seen from Table 1, the tailing factor of the main peak Nil-SM1 is 1.027 < 2.0, the separation degrees of the main peak and adjacent impurities are both greater than 1.5, the separation degrees of the adjacent impurity peaks are both greater than 1.2, the theoretical plate numbers of the peaks are both high, the base line is stable, and the requirements are met.
As can be seen from table 2, the purity of the batch of samples was 99.94% and the total impurity content was 0.06% by mass, calculated by area normalization.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (7)
1. A method for detecting impurities in methyl 3-amino-4-methylbenzoate comprises the following steps:
1) Mixing methyl 3-amino-4-methylbenzoate and acetonitrile aqueous solution to obtain a test solution a;
respectively mixing impurities to be detected with acetonitrile to obtain impurity stock solutions;
each impurity stock solution comprises a stock solution of the impurity of formula (1), a stock solution of the impurity of formula (2), a stock solution of the impurity of formula (3), a stock solution of the impurity of formula (4), a stock solution of the impurity of formula (5) and a stock solution of the impurity of formula (6);
mixing a proper amount of each impurity stock solution with an acetonitrile aqueous solution respectively to obtain each impurity reference substance solution;
mixing 3-amino-4-methyl benzoate, a proper amount of impurity stock solutions and an acetonitrile aqueous solution to obtain a system applicability solution b;
2) Respectively detecting acetonitrile aqueous solution, impurity reference substance solution, test solution a and system applicability solution b by adopting HPLC, wherein the detection conditions are as follows:
octadecylsilane chemically bonded silica is used as a stationary phase;
the mobile phase comprises a mobile phase A and a mobile phase B;
the mobile phase A is as follows:
the volume ratio is 20:80 of 0.005 to 0.1mol/L potassium dihydrogen phosphate solution and acetonitrile;
the mobile phase B is as follows:
the volume ratio is 80:20 of a potassium dihydrogen phosphate solution of 0.005 to 0.1mol/L and acetonitrile;
during gradient elution, the volume ratio of mobile phase a to mobile phase B is:
3) And (3) obtaining the detection result of the impurity by calculation according to the HPLC map obtained in the step 2).
2. The detection method according to claim 1, wherein in step 2),
the pH value of the potassium dihydrogen phosphate solution is 3.0 to 5.5.
3. The detection method according to claim 1, wherein in the step 2), the flow rate of the mobile phase is 0.8 to 1.2mL/min;
the column temperature of the chromatographic column to be detected is 20 to 40 ℃.
4. The detection method according to claim 1, wherein in step 2), the flow rate of the mobile phase is 1mL/min;
the column temperature of the detected chromatographic column is 30 ℃;
the detected chromatographic column is YMC-Triart C18, the column length is 250mm, the inner diameter is 4.6mm, and the filler particle size is 5 mu m;
the wavelength of the detection is 225nm.
5. The detection method according to claim 1, wherein the concentration of the impurity in the stock solution of each impurity is 0.0005mg to 1mg:1mL;
in each impurity control solution, the concentration of impurities is 0.0001 to 0.01mg:1mL.
6. The detection method according to claim 1, wherein the concentration of methyl 3-amino-4-methylbenzoate in the test solution a is 0.1 to 5mg:1mL.
7. The detection method according to claim 1, wherein the concentration of the methyl 3-amino-4-methylbenzoate in the system-compatible solution b is 0.1 to 5mg:1mL, and the concentration of impurities is 0.0001 to 0.01mg:1mL.
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