CN113702514A - Method for determining atorvastatin calcium related impurity I - Google Patents
Method for determining atorvastatin calcium related impurity I Download PDFInfo
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- CN113702514A CN113702514A CN202010428943.XA CN202010428943A CN113702514A CN 113702514 A CN113702514 A CN 113702514A CN 202010428943 A CN202010428943 A CN 202010428943A CN 113702514 A CN113702514 A CN 113702514A
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- 239000012535 impurity Substances 0.000 title claims abstract description 193
- OJRHUICOVVSGSY-RXMQYKEDSA-N (2s)-2-chloro-3-methylbutan-1-ol Chemical compound CC(C)[C@H](Cl)CO OJRHUICOVVSGSY-RXMQYKEDSA-N 0.000 title claims abstract description 79
- 229960001770 atorvastatin calcium Drugs 0.000 title claims abstract description 79
- 238000000034 method Methods 0.000 title claims abstract description 25
- 239000013558 reference substance Substances 0.000 claims abstract description 104
- 239000000243 solution Substances 0.000 claims abstract description 64
- 239000002904 solvent Substances 0.000 claims abstract description 26
- 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 claims abstract description 25
- 238000005303 weighing Methods 0.000 claims abstract description 25
- XUKUURHRXDUEBC-SXOMAYOGSA-N (3s,5r)-7-[2-(4-fluorophenyl)-3-phenyl-4-(phenylcarbamoyl)-5-propan-2-ylpyrrol-1-yl]-3,5-dihydroxyheptanoic acid Chemical compound C=1C=CC=CC=1C1=C(C=2C=CC(F)=CC=2)N(CC[C@@H](O)C[C@H](O)CC(O)=O)C(C(C)C)=C1C(=O)NC1=CC=CC=C1 XUKUURHRXDUEBC-SXOMAYOGSA-N 0.000 claims abstract description 23
- 238000001514 detection method Methods 0.000 claims abstract description 23
- 239000012085 test solution Substances 0.000 claims abstract description 12
- 238000007865 diluting Methods 0.000 claims abstract description 11
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 9
- 239000012088 reference solution Substances 0.000 claims abstract description 8
- 239000000126 substance Substances 0.000 claims abstract description 6
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 45
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 43
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 30
- 229960000583 acetic acid Drugs 0.000 claims description 16
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 claims description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 15
- 239000008351 acetate buffer Substances 0.000 claims description 14
- 238000010828 elution Methods 0.000 claims description 13
- 239000007788 liquid Substances 0.000 claims description 12
- 238000002360 preparation method Methods 0.000 claims description 12
- 239000007853 buffer solution Substances 0.000 claims description 11
- BOGUJKRZZPTPOK-UHFFFAOYSA-N acetonitrile;2-(oxolan-2-yl)acetic acid Chemical compound CC#N.OC(=O)CC1CCCO1 BOGUJKRZZPTPOK-UHFFFAOYSA-N 0.000 claims description 10
- NSXFCODMXBYASQ-UHFFFAOYSA-N acetonitrile;oxolane;hydrate Chemical group O.CC#N.C1CCOC1 NSXFCODMXBYASQ-UHFFFAOYSA-N 0.000 claims description 10
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 claims description 5
- 239000005695 Ammonium acetate Substances 0.000 claims description 5
- 229940043376 ammonium acetate Drugs 0.000 claims description 5
- 235000019257 ammonium acetate Nutrition 0.000 claims description 5
- 239000012362 glacial acetic acid Substances 0.000 claims description 5
- -1 4-fluorobenzoyl Chemical group 0.000 claims 1
- 239000004305 biphenyl Substances 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 3
- 238000012360 testing method Methods 0.000 description 16
- 238000004090 dissolution Methods 0.000 description 10
- 239000000523 sample Substances 0.000 description 10
- 239000012488 sample solution Substances 0.000 description 10
- 238000000926 separation method Methods 0.000 description 7
- XUKUURHRXDUEBC-KAYWLYCHSA-N Atorvastatin Chemical compound C=1C=CC=CC=1C1=C(C=2C=CC(F)=CC=2)N(CC[C@@H](O)C[C@@H](O)CC(O)=O)C(C(C)C)=C1C(=O)NC1=CC=CC=C1 XUKUURHRXDUEBC-KAYWLYCHSA-N 0.000 description 6
- XUKUURHRXDUEBC-UHFFFAOYSA-N Atorvastatin Natural products C=1C=CC=CC=1C1=C(C=2C=CC(F)=CC=2)N(CCC(O)CC(O)CC(O)=O)C(C(C)C)=C1C(=O)NC1=CC=CC=C1 XUKUURHRXDUEBC-UHFFFAOYSA-N 0.000 description 6
- 229960005370 atorvastatin Drugs 0.000 description 6
- 239000011550 stock solution Substances 0.000 description 6
- 238000011084 recovery Methods 0.000 description 5
- 238000010521 absorption reaction Methods 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 239000002994 raw material Substances 0.000 description 3
- 238000010998 test method Methods 0.000 description 3
- PAYRUJLWNCNPSJ-UHFFFAOYSA-N Aniline Chemical compound NC1=CC=CC=C1 PAYRUJLWNCNPSJ-UHFFFAOYSA-N 0.000 description 2
- 229910052791 calcium Inorganic materials 0.000 description 2
- 239000011575 calcium Substances 0.000 description 2
- 125000004427 diamine group Chemical group 0.000 description 2
- 230000010354 integration Effects 0.000 description 2
- UNKLVVGSJZVOBR-KAYWLYCHSA-N (3r,5r)-7-[2,3-bis(4-fluorophenyl)-4-(phenylcarbamoyl)-5-propan-2-ylpyrrol-1-yl]-3,5-dihydroxyheptanoic acid Chemical compound C=1C=C(F)C=CC=1C1=C(C=2C=CC(F)=CC=2)N(CC[C@@H](O)C[C@@H](O)CC(O)=O)C(C(C)C)=C1C(=O)NC1=CC=CC=C1 UNKLVVGSJZVOBR-KAYWLYCHSA-N 0.000 description 1
- 125000001255 4-fluorophenyl group Chemical group [H]C1=C([H])C(*)=C([H])C([H])=C1F 0.000 description 1
- OUCSEDFVYPBLLF-KAYWLYCHSA-N 5-(4-fluorophenyl)-1-[2-[(2r,4r)-4-hydroxy-6-oxooxan-2-yl]ethyl]-n,4-diphenyl-2-propan-2-ylpyrrole-3-carboxamide Chemical compound C=1C=CC=CC=1C1=C(C=2C=CC(F)=CC=2)N(CC[C@H]2OC(=O)C[C@H](O)C2)C(C(C)C)=C1C(=O)NC1=CC=CC=C1 OUCSEDFVYPBLLF-KAYWLYCHSA-N 0.000 description 1
- 206010067484 Adverse reaction Diseases 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- HNNNBAPVNTVXBM-UHFFFAOYSA-N O.O.O.C(CCCCCC)(=O)O Chemical compound O.O.O.C(CCCCCC)(=O)O HNNNBAPVNTVXBM-UHFFFAOYSA-N 0.000 description 1
- 230000006838 adverse reaction Effects 0.000 description 1
- 239000003524 antilipemic agent Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 150000004985 diamines Chemical class 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000010812 external standard method 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
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000013112 stability test Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 150000004684 trihydrates Chemical class 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
Images
Classifications
-
- 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
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
-
- 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 discloses a method for measuring atorvastatin calcium related impurities I, which belongs to the field of detection of pharmaceutical impurities and adopts the technical scheme that the method comprises the following steps: (a) preparing a test solution: weighing atorvastatin calcium, adding a solvent to dissolve the atorvastatin calcium, and quantitatively diluting the atorvastatin calcium to prepare a solution containing 0.8-1.2mg of atorvastatin calcium in every 1mL of atorvastatin calcium as a test solution; (b) preparing a mixed reference substance solution: weighing an impurity A reference substance, an impurity B reference substance, an impurity C reference substance, an impurity D reference substance, an impurity E reference substance, an impurity I reference substance and an atorvastatin calcium reference substance, adding the above solvents to dissolve and quantitatively dilute the above substances to prepare a solution containing 54-66 mu g of impurity B, 5-15 mu g of each of impurity A, impurity C, impurity D, impurity E and impurity I and 45-55 mu g of atorvastatin calcium in each 1mL of solution as a mixed reference substance solution; (C) and analyzing the test solution and the mixed reference solution by adopting high performance liquid chromatography. The effect of detecting other impurities in the atorvastatin calcium is achieved.
Description
Technical Field
The invention relates to the field of detection of pharmaceutical impurities, and particularly relates to a method for determining atorvastatin calcium related impurity I.
Background
Atorvastatin calcium usually exists in the form of trihydrate, with the chemical name: [ R- (R,) 2- (4-fluorophenyl) -beta, delta-dihydroxy-5- (1-methylethyl) -3-phenyl-4- [ (aniline) carbonyl ] -1-hydro-pyrrole-1-calcium heptanate trihydrate.
Atorvastatin calcium is used as a global blood lipid lowering drug, the content of impurities must be strictly controlled, so that the medication safety is ensured, and impurities, harmful substances and the like generated in the preparation process can cause adverse reactions, so that the impurities of atorvastatin calcium are necessary to be researched to hopefully obtain higher-quality atorvastatin calcium.
The existing chinese patent with reference to publication No. CN104931599A discloses a method for determining atorvastatin calcium related substances, wherein an impurity a: defluorinated atorvastatin calcium, impurity B: atorvastatin diastereomer calcium, impurity C: difluoro atorvastatin calcium, impurity D: atorvastatin lactone, impurity E: atorvastatin condensate.
However, atorvastatin calcium also contains other impurities, and detection of other impurities is not mentioned in related impurity detection standards of atorvastatin calcium, so that continuous research on other impurities is needed, and the quality of atorvastatin calcium is improved.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide a method for measuring atorvastatin calcium related impurity I, so as to achieve the effect of detecting the impurity in atorvastatin calcium.
The technical purpose of the invention is realized by the following technical scheme:
a method for determining atorvastatin calcium related impurity I comprises the following steps:
(a) preparing a test solution:
weighing atorvastatin calcium, adding a solvent to dissolve the atorvastatin calcium, and quantitatively diluting the atorvastatin calcium to prepare a solution containing 0.8-1.2mg of atorvastatin calcium in every 1mL of atorvastatin calcium as a test solution;
(b) preparing a mixed reference substance solution:
weighing an impurity A reference substance, an impurity B reference substance, an impurity C reference substance, an impurity D reference substance, an impurity E reference substance, an impurity I reference substance and an atorvastatin calcium reference substance, adding the above solvents to dissolve and quantitatively dilute the above substances to prepare a solution containing 54-66 mu g of impurity B, 5-15 mu g of each of impurity A, impurity C, impurity D, impurity E and impurity I and 45-55 mu g of atorvastatin calcium in each 1mL of solution as a mixed reference substance solution;
(c) analyzing the test solution and the mixed reference solution by adopting high performance liquid chromatography, and calculating the content of impurities according to the peak area of a chromatogram;
wherein the mobile phase: the mobile phase A is acetonitrile-tetrahydrofuran-acetate buffer solution, and acetonitrile: tetrahydrofuran: the volume ratio of the acetate buffer solution is 35:3: 62-45: 7: 48; the mobile phase B is acetonitrile-tetrahydrofuran-acetate buffer solution, and acetonitrile: tetrahydrofuran: the volume ratio of the acetate buffer solution is 82:3: 15-87: 7:6.
By adopting the technical scheme, when the impurity I is detected, the maximum absorption is found to be 244nm, and the maximum absorption is the same as the detection wavelength of the impurity A, B, C, D, E, so that the wavelength is used as the detection wavelength of the impurity I, and then when the impurity A, B, C, D, E, I and the atorvastatin calcium reference substance are detected simultaneously, the separation degree of the impurity I from other impurities can be effectively seen, so that the content of the impurity I in the atorvastatin calcium can be effectively calculated, the content of the impurity I in the atorvastatin calcium can be effectively determined, the impurity I can be conveniently removed from the atorvastatin calcium at the later stage, and the purity of the atorvastatin calcium is improved.
The invention further provides that the solvent is acetonitrile-tetrahydrofuran-water, wherein the ratio of acetonitrile: tetrahydrofuran: the volume ratio of water is 1:1: 2.
By adopting the technical scheme, the acetonitrile-tetrahydrofuran-water can be well compatible with atorvastatin calcium, so that the dissolution of impurities and a solvent in the atorvastatin calcium is facilitated, the elution efficiency is improved, and the detection of the impurity I is facilitated.
The invention further provides that the impurity I is named 3- (4-fluorobenzoyl) -2-isobutyryl-N, 3-diphenyloxirane-2-amide.
The invention is further configured that the preparation method of the acetic acid buffer solution comprises the steps of taking 1.50-1.58g of ammonium acetate, adding 700-900 mL of water for dissolution, adjusting the pH value to 4.0 by glacial acetic acid after dissolution, and adding water to 1000 mL.
The invention is further configured such that the impurity I control is prepared as follows: weighing 5mg of an impurity I reference substance, dissolving the impurity I reference substance by adding a solvent, then placing the impurity I reference substance in a 20mL volumetric flask and diluting the impurity I reference substance to a scale, shaking up, measuring 5-7mL again, measuring the impurity I reference substance in a 100mL volumetric flask, diluting the impurity I reference substance to the scale by adding the solvent, shaking up, measuring 0.5mL, 1.0mL, 2.0mL, 3.0mL and 5.0mL again, respectively placing the impurity I reference substance in the 20mL volumetric flask, diluting the impurity I reference substance to the scale by adding the solvent, shaking up, measuring 20 mu L again, and injecting the obtained product into a liquid chromatograph for linear detection.
By adopting the technical scheme, the selection of the concentration in the impurity detection is crucial to the final linear detection, and when the concentration of the impurity I reference substance is 0.375-3.75 mug/mL, the final linear relation is concentrated, so that the detection of the impurity I is more accurate, and the accuracy of the detection of the impurity I is effectively improved.
The invention is further configured that, in the mobile phase gradient elution procedure: the mobile phase A accounts for 60 to 85 percent; the mobile phase B accounts for 15-40%.
Through adopting above-mentioned technical scheme, mobile phase gradient elution procedure can make impurity A, B, C, D, E, I have good peak area, plays certain boosting effect to the appearance of peak order of impurity simultaneously to help the volume of the impurity I who contains in the rapid detection extremely atorvastatin calcium more, thereby get rid of impurity I for the later stage and play certain additional role.
In conclusion, the invention has the following beneficial effects: when the impurity I is detected, the maximum absorption of the impurity I is found to be 244nm, the maximum absorption is the same as the detection wavelength of the impurity A, B, C, D, E, so that the wavelength is used as the detection wavelength of the impurity I, and then when the impurity A, B, C, D, E is used as a reference substance to detect the impurity I and atorvastatin calcium, the separation degree of the impurity I from other impurities can be effectively seen, so that the content of the impurity I in the atorvastatin calcium can be effectively calculated, the content of the impurity I in the atorvastatin calcium can be effectively determined, the impurity I can be conveniently removed from the atorvastatin calcium at a later stage, and the purity of the atorvastatin calcium is improved.
Drawings
FIG. 1 is a chromatogram in example 1;
FIG. 2 is a chromatogram in example 2;
FIG. 3 is a chromatogram in example 3.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings.
In this example, impurity I is named 3- (4-fluorobenzoyl) -2-isobutyryl-N, 3-diphenyloxirane-2-amide.
Example 1
1. Chromatographic condition chromatographic column of high performance liquid chromatograph: wondasil C18(4.6 mm. times.250 mm, 5 μm);
mobile phase: the mobile phase A is acetonitrile-tetrahydrofuran-acetate buffer solution, wherein the ratio of acetonitrile: tetrahydrofuran: the volume ratio of the acetic acid buffer solution is 35:3: 62;
the mobile phase B is acetonitrile-tetrahydrofuran-acetate buffer solution, wherein the acetonitrile: tetrahydrofuran: the volume ratio of the acetic acid buffer solution is 82:3: 15;
the preparation method of the acetic acid buffer solution comprises the following steps: 1.50g of ammonium acetate is taken and added with 700mL of water for dissolution, after dissolution, glacial acetic acid is used for adjusting the pH value to 4.0, and then water is added to 1000 mL;
flow rate: 1.0 ml/min;
detection wavelength: 244nm, and the column temperature is 30 ℃;
sample introduction amount: 20 mu L of the solution;
the mobile phase gradient elution conditions are shown in table 1.1.
TABLE 1.1 gradient elution conditions for mobile phase
| Time/min | Mobile phase A/%) | Mobile phase B/%) |
| 0 | 60 | 40 |
| 25 | 60 | 40 |
| 40 | 40 | 60 |
| 50 | 40 | 60 |
| 51 | 60 | 40 |
| 60 | 60 | 40 |
2. The method for measuring the impurity I by high performance liquid chromatography comprises the following steps:
2.1 preparation of a test sample solution: weighing a proper amount of atorvastatin calcium, and adding acetonitrile-tetrahydrofuran-hydrosolvent for dissolving, wherein the acetonitrile: tetrahydrofuran: the volume ratio of water is 1:1:2, and after dissolution, the solution is quantitatively diluted into a solution containing 0.8mg of atorvastatin calcium in every 1mL and is used as a test solution;
2.2 preparation of mixed control solution: accurately weighing a proper amount of an impurity A reference substance, an impurity B reference substance, an impurity C reference substance, an impurity D reference substance, an impurity E reference substance, an impurity I reference substance and an atorvastatin calcium reference substance, adding acetonitrile-tetrahydrofuran-water solvent to dissolve and quantitatively dilute into a solution containing 54 micrograms of impurity B, 5 micrograms of impurity A, 5 micrograms of impurity C, 5 micrograms of impurity D, 5 micrograms of impurity E and 45 micrograms of atorvastatin calcium in each 1mL of solution to serve as a mixed reference substance solution;
2.3 HPLC analysis, precisely measuring 20 μ L of each of the test sample solution and the mixed reference solution, respectively injecting into a high performance liquid chromatograph, and recording the chromatogram until the gradient elution process is finished.
3. Specificity experiments
Accurately weighing a proper amount of an impurity A reference substance, an impurity B reference substance, an impurity C reference substance, an impurity D reference substance, an impurity E reference substance, an impurity I reference substance and an atorvastatin calcium reference substance, adding acetonitrile-tetrahydrofuran-water solvent to dissolve and dilute the mixture to prepare a solution containing 45 mu g of atorvastatin calcium, 5 mu g of each of the impurity A, the impurity B, the impurity C, the impurity D, the impurity E and the impurity I in every 1 mL. The chromatogram is shown in FIG. 1, and the integral result of the mixed control solution is shown in Table 1.2;
TABLE 1.2 Integrated results of Mixed control
The peak appearance sequence is diamine without peak, impurity A without peak, impurity B (5.283min), atorvastatin (5.560min), impurity C without peak, impurity D (8.420min), impurity I without peak and impurity E (11.198 min).
The results show that various impurities cannot be detected well under this condition, so the test condition is not selected as the detection condition.
Example 2
1. Chromatographic condition chromatographic column of high performance liquid chromatograph: wondasil C18(4.6 mm. times.250 mm, 5 μm);
mobile phase: the mobile phase A is acetonitrile-tetrahydrofuran-acetate buffer solution, wherein the ratio of acetonitrile: tetrahydrofuran: the volume ratio of the acetic acid buffer solution is 40:5: 55;
the mobile phase B is acetonitrile-tetrahydrofuran-acetate buffer solution, wherein the acetonitrile: tetrahydrofuran: the volume ratio of the acetic acid buffer solution is 85:5: 10;
the preparation method of the acetic acid buffer solution comprises the following steps: 1.55g of ammonium acetate is taken and added with 800mL of water for dissolution, after dissolution, glacial acetic acid is used for adjusting the pH value to 4.0, and then water is added to 1000 mL;
flow rate: 1.0 ml/min;
detection wavelength: 244nm, and the column temperature is 30 ℃;
sample introduction amount: 20 mu L of the solution;
the mobile phase gradient elution conditions are shown in table 2.1.
TABLE 2.1 gradient elution conditions for mobile phase
| Time/min | Mobile phase A/%) | Mobile phase B/%) |
| 0 | 85 | 15 |
| 25 | 85 | 15 |
| 40 | 15 | 85 |
| 50 | 15 | 85 |
| 51 | 85 | 15 |
| 60 | 85 | 15 |
2. The method for measuring the impurity I by high performance liquid chromatography comprises the following steps:
2.1 preparation of a test sample solution: weighing a proper amount of atorvastatin calcium, and adding acetonitrile-tetrahydrofuran-hydrosolvent for dissolving, wherein the acetonitrile: tetrahydrofuran: the volume ratio of water is 1:1:2, and after dissolution, the solution is quantitatively diluted into a solution containing 1.0mg of atorvastatin calcium in every 1mL and is used as a test solution;
2.2 preparation of mixed control solution: accurately weighing a proper amount of an impurity A reference substance, an impurity B reference substance, an impurity C reference substance, an impurity D reference substance, an impurity E reference substance, an impurity I reference substance and an atorvastatin calcium reference substance, adding acetonitrile-tetrahydrofuran-water solvent to dissolve and quantitatively dilute into a solution containing 60 micrograms of impurity B, 10 micrograms of impurity A, 10 micrograms of impurity C, 10 micrograms of impurity D, 10 micrograms of impurity E and 50 micrograms of atorvastatin calcium in every 1mL, and taking the solution as a mixed reference substance solution;
2.3 HPLC analysis, precisely measuring 20 μ L of each of the test sample solution and the mixed reference solution, respectively injecting into a high performance liquid chromatograph, and recording the chromatogram until the gradient elution process is finished.
3. Specificity test
Accurately weighing appropriate amounts of an impurity A reference substance, an impurity B reference substance, an impurity C reference substance, an impurity D reference substance, an impurity E reference substance, an impurity I reference substance and an atorvastatin calcium reference substance, adding acetonitrile-tetrahydrofuran-water solvent to dissolve and dilute the appropriate amounts to prepare a solution containing 50 micrograms of atorvastatin calcium, 10 micrograms of each of the impurity A, the impurity B, the impurity C, the impurity D, the impurity E and the impurity I in each 1 mL. The chromatogram is shown in FIG. 2, and the integral result of the mixed control solution is shown in Table 2.2;
TABLE 2.2 integration of the mixed control solutions
The appearance sequence of the peak is diamine (10.205min), impurity A (18.420min), impurity B (20.123min), atorvastatin (21.183min), impurity C (23.068min), impurity D (32.607min), impurity I (37.548min) and impurity E (43.593 min); the degrees of separation of atorvastatin from adjacent impurities B and C were 1.53 and 2.51, respectively, the degree of separation of impurity a from impurity B was 2.59, the degree of separation of impurity C from impurity D was 17.41, the degree of separation of impurity I from its preceding adjacent peak was 7.40, and the degree of separation of impurity I from impurity E was 32.78.
The chromatographic conditions in example 2 were effective to separate the individual impurities.
3.1 detection and quantitation limits
Accurately weighing a proper amount of the impurity I reference substance, dissolving the impurity I reference substance by using acetonitrile-tetrahydrofuran-hydrosolvent and gradually diluting the impurity I reference substance, wherein when the concentration is 0.143ug/mL and the sample amount is 20 mu L, the S/N of the impurity is 10, so that the quantitative limit is 2.86 mu g; when the concentration is 0.098ug/mL and the sample size is 20. mu.L, the S/N ratio of the impurity is 3, so that the detection limit is 1.96. mu.g.
4. Verification of determination method of impurity I in atorvastatin calcium
4.1 Linear relationship
4.11 precisely weighing 5mg of an impurity I reference substance, placing the reference substance in a 20mL measuring flask, adding a solvent to dissolve and dilute the reference substance to a scale, shaking up, precisely weighing 6mL to a 100mL measuring flask, adding a solvent to dilute the reference substance to a scale, shaking up, precisely weighing 0.5mL, 1.0mL, 2.0mL, 3.0mL and 5.0mL, respectively placing the reference substance in a 20mL measuring flask, adding a solvent to dilute the reference substance to a scale, shaking up to prepare the reference substance with the concentrations of 0.375 mu g/mL, 0.75 mu g/mL, 1.5 mu g/mL, 2.25 mu g/mL and 3.75 mu g/mL, precisely weighing 20 mu L, injecting the reference substance into a liquid chromatograph to perform linear detection, performing linear regression by taking the concentration as X and the peak area as Y, wherein the regression equation of the impurity I is as follows: y is 0.6399X-0.0058r is 0.9999.
4.12 test article testing
And (3) measuring the prepared test sample solution according to the chromatographic conditions, and calculating the content of the impurity I in the test sample through a standard curve.
4.2 sample recovery test for known impurities
The test method comprises the following steps: precisely weighing 5mg of impurity I pair photograph, placing the impurity I pair photograph in a 20mL volumetric flask, adding a solvent to dissolve and dilute the impurity I pair photograph to a scale, shaking up, precisely weighing 6mL, placing the impurity I pair photograph in a 100mL volumetric flask, adding acetonitrile-tetrahydrofuran-hydrosolvent to dissolve and dilute the impurity I pair photograph to the scale, shaking up to serve as a stock solution; accurately weighing 20mg (9 parts in total) of atorvastatin calcium raw material medicine, respectively placing the atorvastatin calcium raw material medicine into a 20mL measuring flask, respectively and accurately adding 1.0mL, 2.0mL and 3.0mL of stock solution 3 parts respectively, adding acetonitrile-tetrahydrofuran-hydrosolvent to dissolve and dilute the atorvastatin calcium raw material medicine to a scale, and shaking up to be used as a sample background solution; precisely measuring 2mL of stock solution, placing the stock solution in a 20mL volumetric flask, adding acetonitrile-tetrahydrofuran-hydrosolvent to dissolve and dilute the stock solution to a scale, and shaking up the stock solution to serve as a reference solution; precisely measuring 20 μ L of each of the reference solution, the sample solution and the sample background solution, respectively adding into a liquid chromatograph, recording chromatogram, and calculating the recovery rate of each impurity by peak area according to an external standard method, wherein the result is shown in Table 2.3;
the calculation formula is as follows: recovery (%) - (C-a)/B × 100%, wherein: a is the measured component content of the test sample; b is the amount of the added reference substance; c is an observed value.
TABLE 2.3 test results for known impurity recoveries
The recovery rate of the impurities is between 95 and 100.2 percent, and the RSD is less than 3 percent.
4.3 precision (repeatability and intermediate precision)
The test method comprises the following steps:
repeatability: the product is taken and added with acetonitrile-tetrahydrofuran-hydrosolvent to be dissolved and diluted to prepare 1mg solution in each 1mL, and 6 samples are measured in parallel. 20. mu.L of the suspension was measured precisely and injected into a liquid chromatograph to measure.
Intermediate precision: at different time, different personnel and different instruments take the product, add acetonitrile-tetrahydrofuran-hydrosolvent to dissolve and dilute the product to prepare 1mg solution in each 1mL, measure 6 samples in parallel, measure 20 muL precisely, inject into liquid chromatograph, measure.
The result shows that the repeatability of the sample solution is 7.8 percent, and the RSD is less than 9 percent; the intermediate precision was 8.7% and the RSD was less than 9%, indicating that the precision of the method was good.
4.4 solution stability test
4.4.1 test methods
Dissolving the product in acetonitrile-tetrahydrofuran-water solvent, diluting to obtain solution containing 1mg per 1mL, and measuring at 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 24 hr.
4.4.2 results
The deviation of the results for impurity I in the sample solution was within ± 0.02%, thus indicating that it was stable within 24 h.
Example 3
1. Chromatographic condition chromatographic column of high performance liquid chromatograph: wondasil C18(4.6 mm. times.250 mm, 5 μm);
mobile phase: the mobile phase A is acetonitrile-tetrahydrofuran-acetate buffer solution, wherein the ratio of acetonitrile: tetrahydrofuran: the volume ratio of the acetic acid buffer solution is 45:7: 48;
the mobile phase B is acetonitrile-tetrahydrofuran-acetate buffer solution, wherein the acetonitrile: tetrahydrofuran: the volume ratio of the acetic acid buffer solution is 87:7: 6; the preparation method of the acetic acid buffer solution comprises the following steps: adding 900mL of water into 1.58g of ammonium acetate for dissolving, adjusting the pH to 4.0 by using glacial acetic acid after dissolving, and adding water to 1000 mL;
flow rate: 1.0 ml/min;
detection wavelength: 244nm, and the column temperature is 30 ℃;
sample introduction amount: 20 mu L of the solution;
the mobile phase gradient elution conditions are shown in table 3.1.
TABLE 3.1 gradient elution conditions for mobile phase
| Time/min | Mobile phase A/%) | Mobile phase B/%) |
| 0 | 70 | 30 |
| 25 | 70 | 30 |
| 40 | 30 | 70 |
| 50 | 30 | 70 |
| 51 | 70 | 30 |
| 60 | 70 | 30 |
2. The method for measuring the impurity I by high performance liquid chromatography comprises the following steps:
2.1 preparation of a test sample solution: weighing a proper amount of atorvastatin calcium, and adding acetonitrile-tetrahydrofuran-hydrosolvent for dissolving, wherein the acetonitrile: tetrahydrofuran: the volume ratio of water is 1:1:2, and after dissolution, the solution is quantitatively diluted into a solution containing 1.2mg of atorvastatin calcium in every 1mL and is used as a test solution;
2.2 preparation of mixed control solution: accurately weighing a proper amount of an impurity A reference substance, an impurity B reference substance, an impurity C reference substance, an impurity D reference substance, an impurity E reference substance, an impurity I reference substance and an atorvastatin calcium reference substance, adding acetonitrile-tetrahydrofuran-water solvent to dissolve and quantitatively dilute into a solution containing 66 mu g of impurity B, 15 mu g of impurity A, 15 mu g of impurity C, 15 mu g of impurity D, 15 mu g of impurity E and 55 mu g of atorvastatin calcium in every 1mL of solution to serve as a mixed reference substance solution;
2.3 HPLC analysis, precisely measuring 20 μ L of each of the test sample solution and the mixed reference solution, respectively injecting into a high performance liquid chromatograph, and recording the chromatogram until the gradient elution process is finished.
3. Specificity test
Accurately weighing appropriate amounts of an impurity A reference substance, an impurity B reference substance, an impurity C reference substance, an impurity D reference substance, an impurity E reference substance, an impurity I reference substance and an atorvastatin calcium reference substance, adding acetonitrile-tetrahydrofuran-water solvent to dissolve and dilute the appropriate amounts to prepare a solution containing 50 micrograms of atorvastatin calcium, 10 micrograms of each of the impurity A, the impurity B, the impurity C, the impurity D, the impurity E and the impurity I in each 1 mL. The chromatogram is shown in FIG. 3, and the integral results of the mixed control solution are shown in Table 3.2;
TABLE 3.2 integration of the mixed control solutions
The appearance sequence of the peaks is diamine (3.088min), impurity A is not appeared, impurity B (6.237min), atorvastatin (6.697min), impurity C is not detected, impurity D is not detected, impurity I (10.92min) and impurity E is not appeared.
It is clear that under these conditions, various impurities could not be detected well.
Comparing example 2 with example 3, the chromatographic conditions of example 2 can separate each impurity more effectively, and the requirements of data validity and accuracy are met.
The present embodiment is only for explaining the present invention, and it is not limited to the present invention, and those skilled in the art can make modifications of the present embodiment without inventive contribution as needed after reading the present specification, but all of them are protected by patent law within the scope of the claims of the present invention.
Claims (6)
1. The method for determining atorvastatin calcium related impurity I is characterized by comprising the following steps:
(a) preparing a test solution:
weighing atorvastatin calcium, adding a solvent to dissolve the atorvastatin calcium, and quantitatively diluting the atorvastatin calcium to prepare a solution containing 0.8-1.2mg of atorvastatin calcium in every 1mL of atorvastatin calcium as a test solution;
(b) preparing a mixed reference substance solution:
weighing an impurity A reference substance, an impurity B reference substance, an impurity C reference substance, an impurity D reference substance, an impurity E reference substance, an impurity I reference substance and an atorvastatin calcium reference substance, adding the above solvents to dissolve and quantitatively dilute the above substances to prepare a solution containing 54-66 mu g of impurity B, 5-15 mu g of each of impurity A, impurity C, impurity D, impurity E and impurity I and 45-55 mu g of atorvastatin calcium in each 1mL of solution as a mixed reference substance solution;
(C) analyzing the test solution and the mixed reference solution by adopting high performance liquid chromatography, and calculating the content of impurities according to the peak area of a chromatogram;
wherein the mobile phase: the mobile phase A is acetonitrile-tetrahydrofuran-acetate buffer solution, and acetonitrile: tetrahydrofuran: the volume ratio of the acetate buffer solution is 35:3: 62-45: 7: 48; the mobile phase B is acetonitrile-tetrahydrofuran-acetate buffer solution, and acetonitrile: tetrahydrofuran: the volume ratio of the acetate buffer solution is 82:3: 15-87: 7:6.
2. The method for determining atorvastatin calcium related impurity I according to claim 1, wherein: the solvent is acetonitrile-tetrahydrofuran-water, wherein the acetonitrile: tetrahydrofuran: the volume ratio of water is 1:1: 2.
3. The method for determining atorvastatin calcium related impurity I according to claim 1, wherein: the impurity I is named as 3- (4-fluorobenzoyl) -2-isobutyryl-N, 3-diphenyl oxirane-2-amide.
4. The method for determining atorvastatin calcium related impurity I according to claim 1, wherein: the preparation method of the acetic acid buffer solution comprises the steps of taking 1.50-1.58g of ammonium acetate, adding 700-900 mL of water for dissolving, adjusting the pH value to 4.0 by glacial acetic acid after dissolving, and adding water to 1000 mL.
5. The method for determining atorvastatin calcium related impurity I according to claim 1, wherein: the impurity I control was prepared as follows: weighing 5mg of an impurity I reference substance, dissolving the impurity I reference substance by adding a solvent, placing the impurity I reference substance in a 20mL volumetric flask and diluting the impurity I reference substance to a scale, shaking up, measuring 6mL again, measuring the impurity I reference substance in a 100mL volumetric flask, diluting the impurity I reference substance to the scale by adding the solvent, shaking up, measuring 0.5mL, 1.0mL, 2.0mL, 3.0mL and 5.0mL again, placing the impurity I reference substance in the 20mL volumetric flask respectively, diluting the impurity I reference substance to the scale by adding the solvent, shaking up, measuring 20 mu L again, and injecting the impurity I reference substance into a liquid chromatograph for linear detection.
6. The method for determining atorvastatin calcium related impurity I according to claim 1, wherein: mobile phase gradient elution procedure: the mobile phase A accounts for 60 to 85 percent; the mobile phase B accounts for 15-40%.
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