CN110646550B - Method for detecting related substances in atorvastatin calcium - Google Patents
Method for detecting related substances in atorvastatin calcium Download PDFInfo
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Abstract
The invention relates to a method for detecting related substances in atorvastatin calcium, wherein the related substances are atorvastatin calcium An Naide tert-butyl ester (impurity I), the method comprises the step of detecting by adopting high performance liquid chromatography, a chromatographic column adopted by the liquid chromatography is a reverse phase chromatographic column, gradient elution is carried out by using a mobile phase A and a mobile phase B, the mobile phase A is a mixed solution of a solution A and a buffer solution, the volume ratio of the solution A to the buffer solution is 35-45-55-65, the solution A is an acetonitrile-tetrahydrofuran solution, the volume ratio of the solution A to the buffer solution is 800-950-50-200, the mobile phase B is a mixed solution of a buffer solution, a solution A and methanol, the volume ratio of the buffer solution to the solution A is 60-20.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a method for detecting a related substance (atorvastatin calcium An Naide tert-butyl ester) in atorvastatin calcium.
Background
Atorvastatin calcium is a third generation statin lipid regulating drug, is a selective and competitive inhibitor of HMG-CoA reductase, reduces cholesterol and lipoprotein levels in plasma by inhibiting the synthesis of HMG-CoA reductase (3-hydroxy-3-methylglutaryl coenzyme A reductase) and cholesterol in the liver, and enhances the uptake and metabolism of LDL by increasing hepatic LDL receptors on the cell surface.
The related substances of the atorvastatin calcium can cause untoward reactions such as myalgia and the like, and the analysis method improves the detection and identification capacity of impurities and improves the quality control level, thereby more effectively ensuring the safety of products. Several methods for measuring atorvastatin calcium related substances have been reported in the prior art, such as a method for detecting the related substances in a 201110197172.9 atorvastatin calcium capsule, a method for measuring the dissolution rate of a 201310239929.5 atorvastatin calcium preparation, a method for measuring the related substances in atorvastatin calcium by using high performance liquid chromatography in a 200910087797.2, a method for measuring the impurity E in atorvastatin calcium in a 201510178001.X, and a method for detecting the related substances in atorvastatin calcium by using an acetonitrile-tetrahydrofuran-ammonium acetate buffer solution in a medicine analysis impurity Chin J Pharm Anal 2014,34 (8) by using an HPLC method. In reference to quality standards: in the USP41 quality standard, high performance liquid chromatography is adopted for separation, a single mobile phase system, namely acetonitrile, tetrahydrofuran without a stabilizer and a buffer solution are adopted for a mobile phase, wherein the buffer solution is an ammonium dihydrogen phosphate buffer solution, the mobile phase system can be used for separating and detecting substances such as atorvastatin calcium, impurities H and the like, but for the impurities I (atorvastatin calcium An Naide tert-butyl ester), the system cannot realize effective separation, and the accuracy of a detection result cannot be guaranteed.
Disclosure of Invention
Problems to be solved by the invention
In order to solve the problem that the impurity I (atorvastatin calcium An Naide tert-butyl ester) cannot be effectively separated and detected in the prior art, the invention aims to provide a method for separating and detecting atorvastatin calcium An Naide tert-butyl ester in atorvastatin calcium.
Means for solving the problems
The technical scheme provided by the invention is as follows:
the method for detecting related substances in atorvastatin calcium comprises the step of detecting by using high performance liquid chromatography, wherein a chromatographic column used in the liquid chromatography is a reverse phase chromatographic column, and gradient elution is carried out by using a mobile phase A and a mobile phase B, the mobile phase A is a mixed solution of a solution A and a buffer solution in a volume ratio of 35-45.
Further, the related substances comprise atorvastatin calcium An Naide tert-butyl ester, and the reverse phase chromatographic column is a C18 chromatographic column, a C8 chromatographic column, a phenyl chromatographic column or an amino chromatographic column.
Further, the buffer solution is acetate solution or phosphate solution.
Furthermore, the buffer solution is 10-100mmol/L acetate solution, and the pH value is 3.0-4.0.
Further, the solution a is an acetonitrile-tetrahydrofuran solution with a volume ratio of 900 to 100, the mobile phase a is a mixed solution of the solution a and the buffer solution with a volume ratio of 41 to 59, and the mobile phase B is a mixed solution of the buffer solution-the solution a-methanol with a volume ratio of 60.
Further, the chromatographic condition is that the detection wavelength is 240-250nm, preferably 244nm; the column temperature is 25-40 deg.C, preferably 30 deg.C, and the flow rate of mobile phase is 1-2ml/min, preferably 1.4-1.8ml/min.
Further, the atorvastatin calcium exists in the form of a bulk drug, a solid preparation, a semi-solid preparation or a liquid preparation.
Further, the mobile phase a and the mobile phase B were subjected to gradient elution using the following elution procedure:
| time (min) | Mobile phase A (%) | Mobile phase B (%) | Flow rate (ml/min) |
| 0 | 100～90 | 0～10 | 1.8 |
| 30 | 100～90 | 0～10 | 1.8 |
| 45 | 20～30 | 80～70 | 1.4～1.7 |
| 50 | 20～30 | 80～70 | 1.4～1.7 |
| 55 | 15～25 | 85～75 | 1.4～1.7 |
| 70 | 15～25 | 85～75 | 1.4～1.7 |
| 73 | 100～90 | 0～10 | 1.8 |
| 80 | 100～90 | 0～10 | 1.8 |
Wherein the percentages of mobile phases a and B are volume percentages.
Further, the flow rate is 1.5-1.7ml/min when the time is 45-55 min in the elution procedure.
Further, before the step of detecting by adopting high performance liquid chromatography, the atorvastatin calcium is added into a solvent to prepare a solution to be treated.
ADVANTAGEOUS EFFECTS OF INVENTION
The detection method provided by the invention can realize sufficient separation of all components of atorvastatin calcium related substances, and simultaneously, impurities I which cannot be detected in a reference method can also be accurately and effectively detected, so that the medicine quality and safety of atorvastatin calcium can be effectively improved.
Drawings
FIG. 1 chromatogram of a blank adjuvant solution;
FIG. 2 chromatogram of blank solvent;
FIG. 3 is a chromatogram of a sample solvent;
FIGS. 4-10 chromatograms of mixed solution separations under different chromatographic conditions;
FIG. 11 chromatogram of the mixed solution with reference to the method.
Detailed Description
Firstly, the invention provides a method for detecting related substances in atorvastatin calcium, wherein the related substances are atorvastatin calcium An Naide tert-butyl ester, the method comprises the step of detecting by using high performance liquid chromatography, a chromatographic column used by the liquid chromatography is a reverse phase chromatographic column, gradient elution is carried out by using a mobile phase A and a mobile phase B, the mobile phase A is a mixed solution of a solution A and a buffer solution, the volume ratio of the solution A to the buffer solution is 35-45-55-65, the solution A is an acetonitrile-tetrahydrofuran solution, the volume ratio of the solution A to the buffer solution is 800-950-200-50, and the mobile phase B is a mixed solution of a buffer solution-solution A-methanol, the volume ratio of the buffer solution to the solution is 60-20.
In the context of the present invention, the term "atorvastatin calcium" has the formula C 66 H 68 CaF 2 N 4 O 10 In the specific scheme of the invention, the Atorvastatin Calcium exists in the form of bulk drug, solid preparation, semi-solid preparation or liquid preparation. Preferably, the solid preparation comprises tablets, capsules, suppositories, granules, films, pastes or pills, the semisolid preparation comprises gels and ointments, and the liquid preparation comprises injections, mixtures and solutions.
In the context of the present invention, the term "atorvastatin calcium An Naide tert-butyl ester" is an impurity I in atorvastatin calcium, which is an impurity of atorvastatin calcium.
In the context of the present invention, the term "mobile phase" refers to a substance that moves forward carrying the component to be measured during chromatography.
In the context of the present invention, the term "reverse phase chromatography column" refers to a chromatography column in which the mobile phase is more polar than the stationary phase. The present invention may use reverse phase chromatography columns conventional in the art. For example, in the specific embodiment, a column including a C18 column, a C8 column, a phenyl column, an amino column, or the like is used. The C18 chromatographic column refers to a chromatographic column using octadecylsilane chemically bonded silica as a filler.
In a preferred embodiment, the buffer solution is a 10-100mmol/L acetate solution with a pH of 3.0-4.0, and the acid for adjusting the pH of the buffer solution is phosphoric acid, glacial acetic acid or a mixture of acid and water.
In a more preferred embodiment, the buffer solution is an ammonium acetate solution with a pH of 3.5.
In a preferred embodiment, the solution a is an acetonitrile-tetrahydrofuran solution with a volume ratio of 900.
In a preferred embodiment, the mobile phase B is a buffer solution-solution a-methanol mixed solution in a volume ratio of 60.
In a preferred embodiment, the chromatographic conditions are a detection wavelength of 240 to 250nm, preferably 244nm, a column temperature of 25 to 40 ℃, preferably 30 ℃, and a flow rate of the mobile phase of 1 to 2ml/min, preferably 1.4 to 1.8ml/min.
In a preferred embodiment, the mobile phase a and mobile phase B are subjected to gradient elution using the elution procedure of the following table:
| time (min) | Mobile phase A (%) | Mobile phase B (%) | Flow rate (ml/min) |
| 0 | 100～90 | 0～10 | 1.8 |
| 30 | 100～90 | 0～10 | 1.8 |
| 45 | 20～30 | 80～70 | 1.4～1.7 |
| 50 | 20～30 | 80～70 | 1.4～1.7 |
| 55 | 15～25 | 85～75 | 1.4～1.7 |
| 70 | 15～25 | 85～75 | 1.4～1.7 |
| 73 | 100～90 | 0～10 | 1.8 |
| 80 | 100～90 | 0～10 | 1.8 |
Wherein the percentages of mobile phases a and B are volume percentages.
In a preferred embodiment, the flow rate during the elution procedure is from 1.5 to 1.7ml/min for a time of from 45 to 55 min.
In a preferred embodiment, the atorvastatin calcium is formulated as a solution to be treated prior to the step of performing detection using high performance liquid chromatography.
In a preferred embodiment, a process for preparing a test sample solution is also included.
Solvent: DMF (dimethyl formamide)
Preparing a blank auxiliary material solution: taking a proper amount of blank auxiliary materials (about 10mg of atorvastatin calcium according to the formula proportion), precisely weighing, putting into a 20ml measuring flask, adding about 80% of solvent, shaking for 10min, adding the solvent to dilute to a scale, shaking uniformly, filtering, and taking the subsequent filtrate as a blank auxiliary material solution.
Preparing a reference substance stock solution (1) of each impurity: accurately weighing proper amounts of an impurity A, PD140728, an impurity B, an impurity C, PD130694, PD148996, PD153824, atorvastatin calcium methyl ester, atorvastatin calcium epoxy tetrahydrofuran, PD139884, atorvastatin calcium tert-butyl ester and an impurity I reference substance respectively, adding a solvent to dissolve and dilute the mixture to prepare a solution containing about 0.1mg of the corresponding impurity in each 1 ml.
Preparing a mixed solution: about 10mg of atorvastatin calcium control was weighed out precisely, placed in a 20ml measuring flask, dissolved by adding a proper amount of solvent, 0.2ml each of the aforementioned impurity A, B, C, atorvastatin calcium methyl ester, atorvastatin calcium t-butyl ester, impurity I control stock solution (1), PD140728, PD148996, PD153824 control stock solution (1), 0.5ml each, 1.0ml of PD130694 control stock solution (1), atorvastatin calcium epoxy tetrahydrofuran control stock solution (1): 0.25ml, D139884 control stock solution (1): 0.35ml, diluted to the scale with solvent, and shaken.
The following table is a comparison of the abbreviations and full names of the above impurities:
preparing a test solution: taking a proper amount of fine powder (about 10mg equivalent to atorvastatin calcium) of the product, precisely weighing, placing in a 20ml measuring flask, adding about 80% solvent, shaking for 10min, diluting to scale with the solvent, shaking uniformly, filtering, and taking the subsequent filtrate as a test solution.
Preparation of a control solution:
precisely measuring 1ml of the test solution, placing the test solution in a 50ml measuring flask, diluting the test solution to the scale with a solvent, shaking up, precisely measuring 1ml of the test solution, placing the test solution in a 10ml measuring flask, diluting the test solution to the scale with the solvent, and shaking up.
The present invention will be described in further detail with reference to specific examples, but the present invention is not limited thereto.
Example 1
Preparation of test sample solution
Preparing a blank auxiliary material solution: taking a proper amount of blank auxiliary materials (about 10mg of atorvastatin calcium according to the formula proportion), precisely weighing, putting into a 20ml measuring flask, adding about 80 percent of DMF, shaking for 10min, adding a solvent to dilute to a scale, shaking uniformly, filtering, and taking a subsequent filtrate as a blank auxiliary material solution.
Preparation of each impurity reference stock solution (1): accurately weighing appropriate amounts of A, PD140728 impurities, B impurities, C, PD130694 impurities, 148996 PD153824, atorvastatin calcium methyl ester, atorvastatin calcium epoxy tetrahydrofuran, PD139884 impurities, atorvastatin calcium tert-butyl ester and I reference substances, adding DMF solvent for dissolving and diluting to prepare a solution containing about 0.1mg of corresponding impurities in each 1 ml.
Preparing a mixed solution: about 10mg of atorvastatin calcium control was precisely weighed, placed in a 20ml measuring flask, dissolved by adding a proper amount of DMF solvent, added with 0.2ml each of the aforementioned impurity A, B, C, atorvastatin calcium methyl ester, atorvastatin calcium tert-butyl ester, impurity I control stock solution (1), PD140728, PD148996, PD153824 control stock solution (1), 0.5ml each, PD130694 control stock solution (1), 1.0ml, atorvastatin calcium epoxy tetrahydrofuran control stock solution (1), 0.25ml, D139884 control stock solution (1): 0.35ml, diluted to the scale with DMF, and shaken.
Preparing a test solution: taking a proper amount of fine powder (about 10mg of atorvastatin calcium) of the product, accurately weighing, putting the product in a 20ml measuring flask, adding about 80 percent of DMF solution, shaking for 10min, diluting to scale with the DMF solution, shaking uniformly, filtering, and taking the subsequent filtrate as a test solution.
Preparation of a control solution:
precisely measuring 1ml of the test solution, placing the test solution in a 50ml measuring flask, diluting the test solution to the scale with a DMF solution, shaking up, precisely measuring 1ml of the test solution, placing the test solution in a 10ml measuring flask, diluting the test solution to the scale with the DMF solution, and shaking up.
Detection apparatus and chromatographic conditions
A detection instrument: high Performance Liquid Chromatograph (HPLC)
Chromatographic conditions are as follows: a column (C18 column) using octadecylsilane chemically bonded silica as a packing material was selected. Acetonitrile-tetrahydrofuran (90: 10, v/v) as solution A,50mmol/L ammonium acetate (pH adjusted to 3.5. + -. 0.1 with glacial acetic acid) -solution A (59.
TABLE 1
| Time (min) | Mobile phase A (%) | Mobile phase B (%) | Flow rate (ml/min) |
| 0 | 98 | 2 | 1.8 |
| 30 | 98 | 2 | 1.8 |
| 45 | 22 | 78 | 1.6 |
| 50 | 22 | 78 | 1.6 |
| 55 | 18 | 82 | 1.6 |
| 70 | 18 | 82 | 1.6 |
| 73 | 98 | 2 | 1.8 |
| 80 | 98 | 2 | 1.8 |
1. Interference test
Respectively sampling 20 μ l of blank solvent, blank adjuvant solution and sample solution, and analyzing with figure 1-figure 3. The result shows that the blank solvent and the blank auxiliary materials do not interfere the detection of the related substances of the atorvastatin calcium tablet, and the impurity I contained in the test sample can be definitely detected from the chromatogram of the test sample shown in the attached figure 3.
2. Experiment for mixing and separating impurities
Taking 20 microliter of mixed solution, and observing the number of theoretical plates, the separation degree and the tailing factor of each component by feeding samples (see attached figure 4). The results are shown in Table 2.
TABLE 2
| Components | Retention time (min) | Degree of separation | Number of theoretical plate | Tailing factor |
| Impurity A | 17.38 | -- | 13400 | 0.95 |
| PD140728 | 18.29 | 1.32 | 8800 | 0.98 |
| Impurity B | 19.46 | 1.59 | 14900 | 0.93 |
| Atorvastatin calcium | 20.65 | 1.73 | 12800 | 1.00 |
| Impurity C | 22.55 | 2.60 | 13200 | 1.00 |
| PD130694 | 40.51 | 22.41 | 39300 | 0.97 |
| PD148996 | 43.80 | 4.33 | 61600 | 1.03 |
| Methyl ester | 45.12 | 2.08 | 104900 | 0.96 |
| PD153824 | 46.36 | 2.23 | 112300 | 0.98 |
| Epoxy tetrahydrofuran | 47.97 | 3.52 | 279200 | 0.96 |
| PD139884 | 50.56 | 7.37 | 358200 | 0.98 |
| Tert-butyl ester | 58.98 | 19.83 | 212600 | 1.00 |
| Impurity I | 71.60 | 23.59 | 198900 | 0.98 |
3. Precision of the instrument
Preparing a test solution: taking a proper amount of fine powder (about 10mg equivalent to atorvastatin calcium) of the product, accurately weighing, placing in a 20ml measuring flask, adding about 80% solvent, shaking for 10min, diluting to scale with the solvent, shaking uniformly, filtering, and taking the subsequent filtrate as a test solution.
Precisely measuring 1ml of the test solution, placing the test solution in a 50ml measuring flask, diluting the test solution to the scale with a solvent, shaking up, precisely measuring 1ml of the test solution, placing the test solution in a 10ml measuring flask, diluting the test solution to the scale with the solvent, and shaking up to obtain the control solution.
Precisely measuring 20 μ l of control solution, injecting into liquid chromatograph, and continuously feeding sample for 6 times to examine instrument precision results shown in Table 3
TABLE 3
| Needle insertion sequence number | Area of self-control peak |
| 1 | 28186 |
| 2 | 28035 |
| 3 | 28401 |
| 4 | 28165 |
| 5 | 28433 |
| 6 | 28248 |
| Mean value of | 28245 |
| RSD(％) | 0.5 |
And (4) conclusion: the Relative Standard Deviation (RSD) of the chromatographic peaks of each reference substance is less than 2 percent, and the repeatability meets the requirement.
4. Destructive test
An acid-breaking solution, a base-breaking solution, an oxidative-breaking solution, a high-temperature-breaking solution and a light-breaking solution of atorvastatin calcium were prepared.
Precisely absorbing 20 microliters of acid destruction solution, alkali destruction solution, oxidation destruction solution, high-temperature destruction solution and illumination destruction solution respectively, observing the number and content of each related substance peak by sample injection, and detecting the peak purity. The atorvastatin calcium peak and the related substance peak can be well separated, and the atorvastatin calcium tablet damages the basic balance of the materials before and after the sample is damaged; and (3) analyzing the peak purity of the atorvastatin calcium chromatographic peak as the main peak in the acid, alkali, oxidation, high temperature and illumination damage test of the atorvastatin calcium tablet by using a diode array detector, wherein all peaks are single chromatographic peaks, the peak purity is more than 99%, and the result is shown in a table 4. The detection method can be used for detecting related substances of the atorvastatin calcium capsules.
TABLE 4
And (4) conclusion:
(1) the atorvastatin calcium is not obviously degraded under the conditions of oxidation and strong alkali degradation. Under the condition of illumination, the known degradation product of atorvastatin calcium is PD148996; under the high-temperature condition, the known main degradation products of atorvastatin calcium are PD130694; the known main degradation products of the bulk drug and atorvastatin calcium under acid conditions are all PD130694. Each degradation product can be completely separated from the main peak.
(2) According to peak purity inspection, no impurity is detected under all conditions, the peak purity is qualified, and the specificity of chromatographic conditions is verified.
(3) The undamaged content is 100%, the balance condition of the main component and the materials under each damaged condition is calculated according to an external standard method, the materials are all conserved, and the chromatographic condition is further verified to have good specificity.
5. Minimum limit of detection
Taking a proper amount of each impurity reference substance, precisely weighing, adding a solvent to gradually dilute to a proper concentration, and taking the amount injected into an instrument when the signal-to-noise ratio is 3:1 as a detection limit under the chromatographic condition. The results are given in Table 5 below:
TABLE 5
And (4) conclusion: the detection concentration of the atorvastatin calcium is 0.5mg/ml, the detection limit is 0.2ng, and the impurities can be detected at the concentration of more than 0.002 percent, so that the method shows that the product has high chromatographic condition sensitivity and is suitable for impurity control.
Example 2
The mixed solution was prepared in the same manner as in example 1.
Detection apparatus and chromatographic conditions
A detection instrument: high performance liquid chromatograph
Chromatographic conditions are as follows: chromatographic column using octadecylsilane chemically bonded silica as filler. Acetonitrile-tetrahydrofuran (90: 10, v/v) as solution A,50mmol/L ammonium acetate (pH adjusted to 3.5. + -. 0.1 with glacial acetic acid) -solution A (59.
TABLE 6
| Time (min) | Mobile phase A (%) | Mobile phase B (%) | Flow rate (ml/min) |
| 0 | 98 | 2 | 1.8 |
| 30 | 98 | 2 | 1.8 |
| 45 | 25 | 75 | 1.6 |
| 50 | 25 | 75 | 1.6 |
| 55 | 20 | 80 | 1.6 |
| 70 | 20 | 80 | 1.6 |
| 73 | 98 | 2 | 1.8 |
| 80 | 98 | 2 | 1.8 |
The determination method comprises the following steps: measuring 20 mu l of the mixed solution, injecting the mixed solution into a liquid chromatograph, and recording a chromatogram to finish measurement; the resulting chromatogram is shown in FIG. 5.
The results show that: the atorvastatin calcium related substance can be well separated from each component, the impurity I can be effectively detected, and the method is suitable for detecting the atorvastatin calcium related substance.
Example 3
The mixed solution was prepared in the same manner as in example 1.
A detection instrument: high performance liquid chromatograph
Chromatographic conditions are as follows: octadecylsilane chemically bonded silica is used as a chromatographic column of a filling agent. Acetonitrile-tetrahydrofuran (90: 10, v/v) as solution A,50mmol/L ammonium acetate (pH adjusted to 3.5. + -. 0.1 with glacial acetic acid) -solution A (59.
TABLE 7
| Time (min) | Mobile phase A (%) | Mobile phase B (%) | Flow rate (ml/min) |
| 0 | 98 | 2 | 1.8 |
| 30 | 98 | 2 | 1.8 |
| 45 | 21 | 79 | 1.6 |
| 50 | 21 | 79 | 1.6 |
| 55 | 16 | 84 | 1.6 |
| 70 | 16 | 84 | 1.6 |
| 73 | 98 | 2 | 1.8 |
| 80 | 98 | 2 | 1.8 |
The determination method comprises the following steps: measuring 20 mu l of the mixed solution, injecting the mixed solution into a liquid chromatograph, and recording a chromatogram to finish measurement; the resulting chromatogram is shown in FIG. 6.
The results show that: the results of the examples 2 and 3 are summarized in the table 8, each component of the atorvastatin calcium related substance can be well separated, the impurity I can be effectively detected, and the method is suitable for detecting the atorvastatin calcium related substance.
TABLE 8
The results show that: under the conditions of the two embodiments, all components of the atorvastatin calcium related substance can be well separated, and the impurity I can be effectively detected.
Example 4
The mixed solution was prepared in the same manner as in example 1.
Detection apparatus and chromatographic conditions
A detection instrument: high performance liquid chromatograph
Chromatographic conditions are as follows: octadecylsilane chemically bonded silica is used as a chromatographic column of a filling agent. Acetonitrile-tetrahydrofuran (90: 10, v/v) as solution A,50mmol/L ammonium acetate (pH adjusted to 3.5. + -. 0.1 with glacial acetic acid) -solution A (59.
TABLE 9
| Time (min) | Mobile phase A (%) | Mobile phase B (%) | Flow rate (ml/min) |
| 0 | 98 | 2 | 1.8 |
| 30 | 98 | 2 | 1.8 |
| 45 | 22 | 78 | 1.6 |
| 50 | 22 | 78 | 1.6 |
| 55 | 18 | 82 | 1.6 |
| 70 | 18 | 82 | 1.6 |
| 73 | 98 | 2 | 1.8 |
| 80 | 98 | 2 | 1.8 |
The determination method comprises the following steps: measuring 20 μ l of the mixed solution, injecting into a liquid chromatograph, recording chromatogram, and completing measurement; the resulting chromatogram is shown in FIG. 7.
The results show that: the atorvastatin calcium related substance can be well separated from each component, the impurity I can be effectively detected, and the method is suitable for detecting the atorvastatin calcium related substance.
Example 5
The mixed solution was prepared in the same manner as in example 1.
Detection apparatus and chromatographic conditions
A detection instrument: high performance liquid chromatograph
Chromatographic conditions are as follows: octadecylsilane chemically bonded silica is used as a chromatographic column of a filling agent. Acetonitrile-tetrahydrofuran (90: 10, v/v) as solution A,50mmol/L ammonium acetate (pH adjusted to 3.5. + -. 0.1 with glacial acetic acid) -solution A (59.
| Time (min) | Mobile phase A (%) | Mobile phase B (%) | Flow rate (ml/min) |
| 0 | 98 | 2 | 1.8 |
| 30 | 98 | 2 | 1.8 |
| 45 | 22 | 78 | 1.6 |
| 50 | 22 | 78 | 1.6 |
| 55 | 18 | 82 | 1.6 |
| 70 | 18 | 82 | 1.6 |
| 73 | 98 | 2 | 1.8 |
| 80 | 98 | 2 | 1.8 |
The determination method comprises the following steps: measuring 20 mu l of the mixed solution, injecting the mixed solution into a liquid chromatograph, and recording a chromatogram to finish measurement; the resulting chromatogram is shown in FIG. 8.
The results show that: the results of the examples 4 and 5 are summarized in the table 11, the components of the atorvastatin calcium related substance can be well separated, the impurity I can be effectively detected, and the method is suitable for detecting the atorvastatin calcium related substance.
TABLE 11
The results show that: under the conditions of the two embodiments, all components of the atorvastatin calcium related substance can be well separated, and the impurity I can be effectively detected.
Example 6
The mixed solution was prepared in the same manner as in example 1.
Detection apparatus and chromatographic conditions
A detection instrument: high performance liquid chromatograph
Chromatographic conditions are as follows: octadecylsilane chemically bonded silica is used as a chromatographic column of a filling agent. Acetonitrile-tetrahydrofuran (88: 12, v/v) as solution A,50mmol/L ammonium acetate (pH adjusted to 3.5. + -. 0.1 with glacial acetic acid) solution A (59.
TABLE 12
| Time (min) | Mobile phase A (%) | Mobile phase B (%) | Flow rate (ml/min) |
| 0 | 98 | 2 | 1.8 |
| 30 | 98 | 2 | 1.8 |
| 45 | 22 | 78 | 1.6 |
| 50 | 22 | 78 | 1.6 |
| 55 | 18 | 82 | 1.6 |
| 70 | 18 | 82 | 1.6 |
| 73 | 98 | 2 | 1.8 |
| 80 | 98 | 2 | 1.8 |
The determination method comprises the following steps: measuring 20 μ l of the mixed solution, injecting into a liquid chromatograph, recording chromatogram, and completing measurement; the resulting chromatogram is shown in FIG. 9.
The results show that: the atorvastatin calcium related substance can be well separated from each component, the impurity I can be effectively detected, and the method is suitable for detecting the atorvastatin calcium related substance.
Example 7
The mixed solution was prepared in the same manner as in example 1.
Detection apparatus and chromatographic conditions
A detection instrument: high performance liquid chromatograph
Chromatographic conditions are as follows: octadecylsilane chemically bonded silica is used as a chromatographic column of a filling agent. Acetonitrile-tetrahydrofuran (92: 8,v/v) as solution a,50mmol/L ammonium acetate (pH adjusted to 3.5 ± 0.1 with glacial acetic acid) -solution a (59, 41, v/v) as mobile phase a,50mmol/L ammonium acetate (pH adjusted to 3.5 ± 0.1 with glacial acetic acid) -solution a-methanol (60: 15:25, v/v) as mobile phase B, gradient elution procedure see table 13 below, detection wavelength of 244nm, column temperature of 30 ℃.
Watch 13
| Time (min) | Mobile phase A (%) | Mobile phase B (%) | Flow rate (ml/min) |
| 0 | 98 | 2 | 1.8 |
| 30 | 98 | 2 | 1.8 |
| 45 | 22 | 78 | 1.6 |
| 50 | 22 | 78 | 1.6 |
| 55 | 18 | 82 | 1.6 |
| 70 | 18 | 82 | 1.6 |
| 73 | 98 | 2 | 1.8 |
| 80 | 98 | 2 | 1.8 |
The determination method comprises the following steps: measuring 20 mu l of the mixed solution, injecting the mixed solution into a liquid chromatograph, and recording a chromatogram to finish measurement; the resulting chromatogram is shown in FIG. 10.
The results show that: the results of the examples 6 and 7 are summarized in the table 14 that each component of the atorvastatin calcium related substance can be well separated, the impurity I can be effectively detected, and the method is suitable for detecting the atorvastatin calcium related substance.
TABLE 14
The results show that: under the conditions of the two embodiments, all components of the atorvastatin calcium related substance can be well separated, and the impurity I can be effectively detected.
Example 8 (full reference USP41 method)
The mixed solution was prepared in the same manner as in example 1.
Detection apparatus and chromatographic conditions
A detection instrument: high performance liquid chromatograph
Chromatographic conditions are as follows: chromatographic column using octadecylsilane chemically bonded silica as filler. Acetonitrile-tetrahydrofuran (925: 75, v/v) was used as solution A,5.75g/L of an aqueous ammonium dihydrogen phosphate solution (adjusted to pH 4.3. + -. 0.05 with dilute 10% acetic acid or 10% aqueous ammonia) solution A (58.
| Time (min) | Mobile phase A (%) | Mobile phase B (%) | Flow rate (ml/min) |
| 0 | 100 | 0 | 1.8 |
| 30 | 100 | 0 | 1.8 |
| 45 | 25 | 75 | 1.5 |
| 50 | 25 | 75 | 1.5 |
| 55 | 20 | 80 | 1.5 |
| 58 | 100 | 0 | 1.8 |
| 65 | 100 | 0 | 1.8 |
The determination method comprises the following steps: measuring 20 mu l of the mixed solution, injecting the mixed solution into a liquid chromatograph, and recording a chromatogram to finish measurement; the resulting chromatogram is shown in FIG. 11.
The results show that: the results of example 8 are summarized in Table 16 that the components of atorvastatin calcium related substances can be well separated, impurity I cannot be detected, and the method is not suitable for detecting atorvastatin calcium related substances.
TABLE 16
From the implementation results, under the conditions of the invention, the components of the atorvastatin calcium related substance can be well separated, and the invention method can also accurately and effectively detect the impurity I which cannot be detected in the reference method. Therefore, the method is an effective method for detecting atorvastatin calcium related substances.
Claims (6)
1. A method for detecting related substances in atorvastatin calcium is characterized in that: comprises the step of detecting by adopting high performance liquid chromatography; the related substances include the following compounds:
the chromatographic column adopted by the liquid chromatogram is a reverse chromatographic column, and the reverse chromatographic column is a C18 chromatographic column; performing gradient elution by using a mobile phase A and a mobile phase B, wherein the mobile phase A is a mixed solution of a solution A and a buffer solution with a volume ratio of 41:
Wherein the percentages of mobile phases a and B are volume percentages.
2. The method for detecting related substances in atorvastatin calcium according to claim 1, wherein: the solution a is an acetonitrile-tetrahydrofuran solution with a volume ratio of 900 to 100, and the mobile phase B is a mixed solution of a buffer solution with a volume ratio of 60.
3. The method for detecting related substances in atorvastatin calcium according to claim 1, wherein: the chromatographic conditions include detection wavelength of 240-250nm and column temperature of 25-40 deg.c.
4. The method for detecting related substances in atorvastatin calcium according to claim 3, wherein: the chromatographic conditions are that the detection wavelength is 244nm and the column temperature is 30 ℃.
5. The method for detecting related substances in atorvastatin calcium according to claim 1, wherein: the atorvastatin calcium exists in the form of a bulk drug, a solid preparation, a semi-solid preparation or a liquid preparation.
6. The method for detecting related substances in atorvastatin calcium according to claim 1, wherein: before the step of detecting by adopting high performance liquid chromatography, adding the atorvastatin calcium into a solvent to prepare a solution to be treated.
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| CN112782333B (en) * | 2020-12-25 | 2022-07-12 | 石家庄四药有限公司 | HPLC detection method for pitavastatin isopropyl tert-butyl ester diastereoisomer |
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| CN114280181B (en) * | 2021-12-23 | 2024-06-18 | 浙江海翔川南药业有限公司 | Rosuvastatin intermediate and detection method of related substances thereof |
| CN114295746A (en) * | 2021-12-27 | 2022-04-08 | 卓和药业集团股份有限公司 | Analysis and detection method of pitavastatin calcium |
| CN114384178B (en) * | 2021-12-30 | 2023-10-20 | 苏州正济医药研究有限公司 | Method for detecting pitavastatin calcium intermediate and impurities |
| CN114295748B (en) * | 2021-12-30 | 2023-11-10 | 苏州正济药业有限公司 | Method for detecting pitavastatin calcium intermediate and impurities |
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