A method for determining the optical isomers of eplerenone by high performance liquid chromatography
By optimizing chromatographic conditions in liquid chromatography, the problem of insufficient separation of impurity B of eplerenone optical isomer is solved, and more accurate impurity analysis and eplerenone quality control are achieved.
Patent Information
- Application Number
- CN202411834828.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-13
- Publication Date
- 2025-04-01
AI Technical Summary
The prior art is difficult to effectively separate the optical isomer impurity B of eplerenone, resulting in insufficient resolution and the inability to accurately analyze the impurity situation.
High performance liquid chromatography was used to optimize the chromatographic conditions, including the use of octadecylsilane bonded silica gel as a filler, Inertsil ODS-3 chromatography column, column temperature was 40°C, flow rate was 1.0mL/min, the detector was an ultraviolet detector, the detection wavelength was 240nm, the mobile phase was methanol: acetonitrile: ion-paired solution, and linear gradient elution was performed.
The separation of eprinione and its optical isomer impurity B is significantly improved, so that the impurity situation can be analyzed more accurately, thereby effectively controlling the isomers of the raw materials, which is of great significance to the quality control of eprinione.
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Abstract
Description
Technical Field
[0001] The invention relates to the technical field of drug analysis, and in particular to a method for determining the optical isomers of eplerenone by high performance liquid chromatography. Background Art
[0002] Eplerenone is a new type of selective aldosterone receptor antagonist. It was approved for clinical use by the State Food and Drug Administration in 2002. The pure product is white or off-white crystals. It has a stronger aldosterone antagonist effect than spironolactone, and has extremely low affinity for androgen and progesterone receptors, few adverse reactions, and has definite therapeutic effects on the treatment of hypertension, heart failure and myocardial infarction. It has fewer adverse reactions and good tolerance, and is a good alternative to spironolactone.
[0003] For severe hypertension that has not been controlled by multiple antihypertensive drugs, the addition of eplerenone can significantly reduce blood pressure, especially systolic blood pressure. Combination therapy with angiotensin-converting enzyme inhibitors (ACEI) and beta-blockers can improve the quality of life and reduce mortality in patients with severe heart failure and myocardial infarction.
[0004] Eplerenone is a steroidal drug with multiple isomers. The European Pharmacopoeia standard does not require a separation degree according to the usual liquid chromatography method for the most difficult optical isomer of this product, impurity B, but only requires a relative retention time of 1.2.
[0005] Therefore, the present invention provides a method for determining the optical isomers of eplerenone by high performance liquid chromatography to solve the above problems. Summary of the invention
[0006] The technical problem to be solved by the present invention is to provide a method for determining the optical isomers of eplerenone by high performance liquid chromatography, thereby increasing the separation degree of the optical isomer impurity B and analyzing the impurity situation more accurately.
[0007] In order to solve the above technical problems, the technical solution of the present invention is: a method for determining the optical isomers of eplerenone by high performance liquid chromatography, the innovation of which is: the chromatographic conditions are: octadecylsilane bonded silica gel is used as a filler, an Inertsil ODS-3 chromatographic column is used, the specification is 150mm×4.6mm, the filler particle size is 3μm, the column temperature is 40°C, the flow rate is 1.0mL / min, the detector is an ultraviolet detector, the detection wavelength is 240nm, the elution is performed according to a linear gradient, and the mobile phase is methanol: acetonitrile: ion pair solution.
[0008] Furthermore, in the ion pair solution, the ion pair includes one or more of sodium pentane sulfonate, sodium hexane sulfonate, sodium heptane sulfonate, and sodium octane sulfonate, and the purity thereof is chromatographically pure.
[0009] Furthermore, in the mobile phase, the ion pair solution is the mobile phase A, and acetonitrile and methanol are the mobile phase B, wherein the volume ratio of acetonitrile to methanol is 40:60.
[0010] Furthermore, the mobile phase A is a 100 mmol / L sodium octane sulfonate solution.
[0011] Further, the linear gradient elution is performed according to the following table: Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 40 60 30 40 60 40 30 70 50 30 70 53 40 60 60 40 60
[0012] Furthermore, the method comprises the following steps: S1. Preparation of test solution: Take 25 mg of eplerenone product, place it in a 50 mL volumetric flask, add mixed solvent to dissolve and dilute to the scale as the test solution; S2. System suitability solution preparation: Take 5 mg of the eplerenone system suitability reference substance containing impurity B, place it in a 10 mL volumetric flask, add the mixed solvent to dissolve and dilute to the scale, as the system suitability solution; S3, control solution preparation: accurately measure 1 mL of the test solution, place it in a 100 mL volumetric flask, add the mixed solvent to dilute to the scale, shake well to obtain the dilution, accurately measure 1 mL of the dilution, place it in a 10 mL volumetric flask, add the mixed solvent to dilute to the scale, shake well, as the control solution; S4. Liquid chromatography detection: Accurately measure 20 μL each of the test solution, system suitability solution, and control solution, inject them into the liquid chromatograph respectively, and record the chromatogram.
[0013] Furthermore, the mixed solvent is a mixture of acetonitrile, methanol and water, wherein the volume ratio of acetonitrile, methanol and water is 25:25:50.
[0014] Furthermore, if there is a chromatographic peak in the chromatogram of the test solution with the same retention time as impurity B in the chromatogram of the system suitability solution, the impurity content is calculated by peak area according to the self-reference method.
[0015] The advantages of the present invention are: (1) The present invention is based on the principle of ion-pair liquid chromatography and uses ion-pair reagents to optimize the mobile phase, thereby effectively increasing the separation degree between eplerenone and its optical isomer impurity B. The method of the present invention can better separate the optical isomer impurity B and more accurately analyze the impurity situation, so that the present invention can effectively control the isomers of the raw material drug, which is of indispensable significance for the quality control of eplerenone.
[0016] (2) The present invention uses methanol: acetonitrile: ion pair solution as the mobile phase, adjusts and optimizes the mobile phase ratio and gradient, and finally establishes a high performance liquid chromatography system and conducts methodological verification. All indicators of the method meet the requirements of the current Chinese Pharmacopoeia. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments.
[0018] Figure 1 It is a chromatogram of a comparative example of the present invention.
[0019] Figure 2 It is the chromatogram of Example 1 of the present invention. DETAILED DESCRIPTION
[0020] In order to further explain the technical means and effects adopted by the present invention to achieve the predetermined invention purpose, the specific implementation mode, structure, characteristics and effects of the present invention are described in detail below in combination with the accompanying drawings and preferred embodiments.
[0021] The compound structure of eplerenone is as follows: ; Among them, the compound structure of impurity B is as follows: .
[0022] Eplerenone is a steroidal drug with multiple isomers. The European Pharmacopoeia standard does not require a separation degree according to the usual liquid chromatography method for the most difficult optical isomer of this product, impurity B, but only requires a relative retention time of 1.2.
[0023] Comparative Example This comparative example determines the optical isomer impurity B of eplerenone according to the European Pharmacopoeia chromatographic conditions, and the European Pharmacopoeia chromatographic conditions are: Octadecylsilane bonded silica gel was used as filler, and the filler used was Inertsil ODS-3 chromatographic column, model 4.6mm×150mm, filler particle size 3μm; Mobile phase A was 0.1% phosphoric acid; Mobile phase B was phosphoric acid: acetonitrile: methanol = 0.1:40:60 (volume ratio); Flow rate: 1.0 ml / min; Detection wavelength: 240nm; Injection volume 20 μl; Perform linear gradient elution according to Table 1.
[0024] Table 1 Linear gradient elution process table of European Pharmacopoeia Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 54 46 25 54 46 32 40 60 45 40 60 46 54 46 56 54 46 The chromatogram of the results is shown in Figure 1 , Figure 1 The number 3 is the chromatographic peak of eplerenone, and the number 4 is the chromatographic peak of impurity B.
[0025] from Figure 1 It can be seen that the separation degree between the optical isomer impurity B and eplerenone is small, and the chromatographic conditions according to the European Pharmacopoeia in the comparative example are not suitable for the separation of the optical isomer impurity B.
[0026] Therefore, the present invention creatively explored the mobile phase system, adopted a system of methanol: acetonitrile: ion pair solution, adjusted the mobile phase ratio and gradient, and ultimately established the high performance liquid chromatography method of the present invention and conducted methodological verification.
[0027] The method for determining the optical isomers of eplerenone by high performance liquid chromatography of the present invention comprises the following steps: S1. Preparation of test solution: Take 25 mg of eplerenone product, place it in a 50 mL volumetric flask, add mixed solvent to dissolve and dilute to scale as the test solution, wherein the mixed solvent is a mixture of acetonitrile, methanol and water, wherein the volume ratio of acetonitrile, methanol and water is 25:25:50; S2. System suitability solution preparation: Take 5 mg of the eplerenone system suitability reference substance containing impurity B, place it in a 10 mL volumetric flask, add the mixed solvent to dissolve and dilute to the scale, as the system suitability solution; S3, control solution preparation: accurately measure 1 mL of the test solution, place it in a 100 mL volumetric flask, add the mixed solvent to dilute to the scale, shake well to obtain the dilution, accurately measure 1 mL of the dilution, place it in a 10 mL volumetric flask, add the mixed solvent to dilute to the scale, shake well, as the control solution; S4. Liquid chromatography detection: Accurately measure 20 μL each of the test solution, system suitability solution, and control solution, and inject them into the liquid chromatograph respectively, and record the chromatograms; if the chromatogram of the test solution contains a chromatographic peak with the same retention time as the impurity B in the chromatogram of the system suitability solution, calculate the impurity content by peak area using the self-reference method.
[0028] Table 2 Linear gradient elution process of the high performance liquid chromatography of the present invention Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 40 60 30 40 60 40 30 70 50 30 70 53 40 60 60 40 60 Example 1 The chromatographic conditions of this embodiment are: Octadecylsilane bonded silica was used as filler, using an Inertsil ODS-3 chromatographic column with specifications of 150 mm × 4.6 mm and a filler particle size of 3 μm; Column temperature: 40°C; Flow rate: 1.0 mL / min; The detector is an ultraviolet detector; The detection wavelength is 240nm; Perform linear gradient elution according to Table 2; Mobile phase A is 100 mmol / L sodium octane sulfonate solution, the purity is chromatographic grade; The mobile phase B was acetonitrile:methanol = 40:60 (volume ratio).
[0029] The chromatogram of the results is shown in Figure 2 , Figure 2 The chromatographic peak of eplerenone is at 19 minutes, and the chromatographic peak of impurity B is at 23 minutes. It can be seen that the separation degree of eplerenone and impurity B is increased. Compared with the chromatographic conditions of the European Pharmacopoeia, the chromatographic conditions of this embodiment are more suitable for the separation of impurity B.
[0030] Example 2 The chromatographic conditions of this embodiment are: Octadecylsilane bonded silica was used as filler, using an Inertsil ODS-3 chromatographic column with specifications of 150 mm × 4.6 mm and a filler particle size of 3 μm; Column temperature: 40°C; Flow rate: 1.0 mL / min; The detector is an ultraviolet detector; The detection wavelength is 240nm; Perform linear gradient elution according to Table 2; Mobile phase A is 100 mmol / L sodium pentane sulfonate solution, the purity is chromatographic grade; The mobile phase B was acetonitrile:methanol = 40:60 (volume ratio).
[0031] Example 3 The chromatographic conditions of this embodiment are: Octadecylsilane bonded silica was used as filler, using an Inertsil ODS-3 chromatographic column with specifications of 150 mm × 4.6 mm and a filler particle size of 3 μm; Column temperature: 40°C; Flow rate: 1.0 mL / min; The detector is an ultraviolet detector; The detection wavelength is 240nm; Perform linear gradient elution according to Table 2; Mobile phase A is 100 mmol / L sodium hexane sulfonate solution, the purity is chromatographic grade; The mobile phase B was acetonitrile:methanol = 40:60 (volume ratio).
[0032] Example 4 The chromatographic conditions of this embodiment are: Octadecylsilane bonded silica was used as filler, using an Inertsil ODS-3 chromatographic column with specifications of 150 mm × 4.6 mm and a filler particle size of 3 μm; Column temperature: 40°C; Flow rate: 1.0 mL / min; The detector is an ultraviolet detector; The detection wavelength is 240nm; Perform linear gradient elution according to Table 2; Mobile phase A is 100 mmol / L sodium heptane sulfonate solution, the purity is chromatographic grade; The mobile phase B was acetonitrile:methanol = 40:60 (volume ratio).
[0033] Example 5 The chromatographic conditions of this embodiment are: Octadecylsilane bonded silica was used as filler, using an Inertsil ODS-3 chromatographic column with specifications of 150 mm × 4.6 mm and a filler particle size of 3 μm; Column temperature: 40°C; Flow rate: 1.0 mL / min; The detector is an ultraviolet detector; The detection wavelength is 240nm; Perform linear gradient elution according to Table 2; Mobile phase A is a 100 mmol / L ion pair solution, the ion pair in the solution is sodium pentane sulfonate: sodium hexane sulfonate = 1:1 (mass ratio), and the purity is chromatographic grade; The mobile phase B was acetonitrile:methanol = 40:60 (volume ratio).
[0034] Example 6 The chromatographic conditions of this embodiment are: Octadecylsilane bonded silica was used as filler, using an Inertsil ODS-3 chromatographic column with specifications of 150 mm × 4.6 mm and a filler particle size of 3 μm; Column temperature: 40°C; Flow rate: 1.0 mL / min; The detector is an ultraviolet detector; The detection wavelength is 240nm; Perform linear gradient elution according to Table 2; Mobile phase A is a 100 mmol / L ion pair solution, the ion pair in the solution is sodium heptane sulfonate: sodium octane sulfonate = 1:1 (mass ratio), and the purity is chromatographic grade; The mobile phase B was acetonitrile:methanol = 40:60 (volume ratio).
[0035] Example 7 The chromatographic conditions of this embodiment are: Octadecylsilane bonded silica was used as filler, using an Inertsil ODS-3 chromatographic column with specifications of 150 mm × 4.6 mm and a filler particle size of 3 μm; Column temperature: 40°C; Flow rate: 1.0 mL / min; The detector is an ultraviolet detector; The detection wavelength is 240nm; Perform linear gradient elution according to Table 2; Mobile phase A is a 100 mmol / L ion pair solution, the ion pair in the solution is sodium hexane sulfonate: sodium octane sulfonate = 1:2 (mass ratio), and the purity is chromatographic grade; The mobile phase B was acetonitrile:methanol = 40:60 (volume ratio).
[0036] Example 8 The chromatographic conditions of this embodiment are: Octadecylsilane bonded silica was used as filler, using an Inertsil ODS-3 chromatographic column with specifications of 150 mm × 4.6 mm and a filler particle size of 3 μm; Column temperature: 40°C; Flow rate: 1.0 mL / min; The detector is an ultraviolet detector; The detection wavelength is 240nm; Perform linear gradient elution according to Table 2; Mobile phase A is a 100 mmol / L ion pair solution, the ion pair in the solution is sodium pentane sulfonate: sodium hexane sulfonate: sodium octane sulfonate = 1:1:1 (mass ratio), and the purity is chromatographic grade; The mobile phase B was acetonitrile:methanol = 40:60 (volume ratio).
[0037] Example 9 The chromatographic conditions of this embodiment are: Octadecylsilane bonded silica was used as filler, using an Inertsil ODS-3 chromatographic column with specifications of 150 mm × 4.6 mm and a filler particle size of 3 μm; Column temperature: 40°C; Flow rate: 1.0 mL / min; The detector is an ultraviolet detector; The detection wavelength is 240nm; Perform linear gradient elution according to Table 2; Mobile phase A is a 100 mmol / L ion pair solution, the ion pair in the solution is sodium hexane sulfonate: sodium heptane sulfonate: sodium octane sulfonate = 1:1:1 (mass ratio), and the purity is chromatographic grade; The mobile phase B was acetonitrile:methanol = 40:60 (volume ratio).
[0038] Example 10 The chromatographic conditions of this embodiment are: Octadecylsilane bonded silica was used as filler, using an Inertsil ODS-3 chromatographic column with specifications of 150 mm × 4.6 mm and a filler particle size of 3 μm; Column temperature: 40°C; Flow rate: 1.0 mL / min; The detector is an ultraviolet detector; The detection wavelength is 240nm; Perform linear gradient elution according to Table 2; Mobile phase A is a 100 mmol / L ion pair solution, the ion pair in the solution is sodium pentane sulfonate: sodium hexane sulfonate: sodium heptane sulfonate: sodium octane sulfonate = 1:1:1:1 (mass ratio), and the purity is chromatographic grade; The mobile phase B was acetonitrile:methanol = 40:60 (volume ratio).
[0039] Methodological validation According to the requirements of the 2020 edition of the Chinese Pharmacopoeia, the method proposed in the present invention was methodologically validated. The results of the methodological validation of the relevant substances are as follows: (1) Exclusivity Acceptable criteria: blank solvent should be free of interference; Verification result: The solvent does not interfere with the determination of content.
[0040] (2) Injection precision Acceptable criteria: RSD of peak area and retention time after 6 repeated injections is no more than 2%; Verification results: The test solution and the control solution were injected 6 times continuously, and the RSD of retention time and peak area were both less than 2.0%. This method has good injection precision.
[0041] (3) Detection limit Acceptable criteria: When the signal-to-noise ratio (S / N) is about 3, the corresponding concentration is the detection limit, and when the signal-to-noise ratio (S / N) is about 10, the corresponding concentration is the quantification limit; Verification results: The detection limit is about 0.5μg / ml (0.1% percentage), and the quantification limit is 1μg / ml (0.1% percentage). This method has good sensitivity and meets the detection needs.
[0042] (4) Linearity and range Acceptable criteria: The correlation coefficient r of linear regression is not less than 0.995; Verification results: In the concentration range of 0.5-5 μg / ml, the concentration of eplerenone showed a good linear relationship with the peak area, r=0.9996.
[0043] (5) Solution stability Acceptable standard: The RSD of the main peak area before and after sample placement is no more than 2%; Verification results: The test solution, control solution and system suitability solution were stable after being placed at room temperature for 30 hours. The main peak area RSD of the test solution was 0.52%, the main peak area RSD of the control solution was 0.73%, and the main peak area RSD of the system suitability solution was 0.67%.
[0044] (6) Repeatability Acceptable standard: The RSD of the content and recovery results of 6 measurements of impurities near the limit concentration is no more than 2%.
[0045] Verification results: The same batch of samples was measured 6 times in parallel, and the RSD of the content results was no more than 2%. This method has good repeatability.
[0046] (7) Durability Acceptable criteria: After changing the testing conditions, the separation degree between the main peak and the isomer under each condition meets the requirements, and the detection content of the isomer does not change significantly; Verification results: When the relative value of flow rate changes by ±0.05ml / min, column temperature changes by ±5℃, and the proportion of mobile phase B in the mobile phase changes by ±2%, there is no significant change in system applicability and test results. This method has good durability.
[0047] The validation results show that all indicators of this method meet the requirements of the 2020 edition of the Chinese Pharmacopoeia and are suitable for testing.
[0048] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used to limit the present invention. Any technical personnel in this field can make some changes or modify the technical contents disclosed above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any brief modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.
Claims
1. A method for determining the optical isomers of eplerenone by high performance liquid chromatography, characterized in that: The chromatographic conditions were as follows: octadecylsilane bonded silica gel was used as the filler, an Inertsil ODS-3 chromatographic column was used, the specification was 150 mm × 4.6 mm, the filler particle size was 3 μm, the column temperature was 40°C, the flow rate was 1.0 mL / min, the detector was an ultraviolet detector, the detection wavelength was 240 nm, linear gradient elution was performed, and the mobile phase was methanol: acetonitrile: ion pair solution.
2. The method for determining the optical isomers of eplerenone by high performance liquid chromatography according to claim 1, characterized in that: In the ion pair solution, the ion pair includes one or more of sodium pentane sulfonate, sodium hexane sulfonate, sodium heptane sulfonate, and sodium octane sulfonate, and the purity thereof is chromatographically pure.
3. The method for determining the optical isomers of eplerenone by high performance liquid chromatography according to claim 2, characterized in that: In the mobile phase, the ion pair solution is the mobile phase A, and acetonitrile and methanol are the mobile phase B, wherein the volume ratio of acetonitrile to methanol is 40:
60.
4. The method for determining the optical isomers of eplerenone by high performance liquid chromatography according to claim 3, characterized in that: The mobile phase A is a 100 mmol / L sodium octane sulfonate solution.
5. The method for determining the optical isomers of eplerenone by high performance liquid chromatography according to claim 4, characterized in that: The linear gradient elution was performed as follows: 。 6. The method for determining the optical isomers of eplerenone by high performance liquid chromatography according to claim 1, characterized in that: The method comprises the following steps: S1. Preparation of test solution: Take 25 mg of eplerenone product, place it in a 50 mL volumetric flask, add mixed solvent to dissolve and dilute to the scale as the test solution; S2. System suitability solution preparation: Take 5 mg of the eplerenone system suitability reference substance containing impurity B, place it in a 10 mL volumetric flask, add the mixed solvent to dissolve and dilute to the scale, as the system suitability solution; S3, control solution preparation: accurately measure 1 mL of the test solution, place it in a 100 mL volumetric flask, add the mixed solvent to dilute to the scale, shake well to obtain the dilution, accurately measure 1 mL of the dilution, place it in a 10 mL volumetric flask, add the mixed solvent to dilute to the scale, shake well, as the control solution; S4. Liquid chromatography detection: Accurately measure 20 μL each of the test solution, system suitability solution, and control solution, inject them into the liquid chromatograph respectively, and record the chromatogram.
7. The method for determining the optical isomers of eplerenone by high performance liquid chromatography according to claim 6, characterized in that: The mixed solvent is a mixture of acetonitrile, methanol and water, wherein the volume ratio of acetonitrile, methanol and water is 25:25:
50.
8. The method for determining the optical isomers of eplerenone by high performance liquid chromatography according to claim 7, characterized in that: If the chromatogram of the test solution contains a chromatographic peak with the same retention time as that of impurity B in the chromatogram of the system suitability solution, the impurity content shall be calculated by peak area using the self-reference method.