Method for detecting pitavastatin calcium intermediate and related substances thereof
The detection of Pivastatin calcium intermediate YPF-SM-A and its impurities by liquid chromatography solved the problem of impurity detection during the synthesis process and achieved efficient control of finished product quality.
Patent Information
- Application Number
- CN202311108020.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-08-30
- Publication Date
- 2025-02-28
AI Technical Summary
During the synthesis of the Pivastatin calcium intermediate YPF-SM-A, it is difficult to effectively detect and control the impurity compounds I, II, III and IV, which affects the quality of the finished product.
The detection is carried out by liquid chromatography, and through the combination of specific chromatographic conditions and mobile phases, effective separation and quantitative detection of YPF-SM-A and its related impurities are achieved.
The accurate and quantitative detection of the Pivastatin calcium intermediate YPF-SM-A and its related substances was achieved, and the accuracy and efficiency of finished product quality control was improved.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of drug analysis, and in particular relates to a method for detecting a pitavastatin calcium intermediate and related substances thereof, and is particularly suitable for detecting mutagenic impurities in the pitavastatin calcium intermediate. Background Art
[0002] Pitavastatin calcium, chemical name: (+)-bis{(3R,5S,6E)-7-[2-cyclopropyl-4-(fluorophenyl)quinoline-3-phenyl]-3,5-dihydroxy-6-heptenoic acid} calcium salt (2:1), molecular formula: C 50 H 46 CaF2N2O8, relative molecular mass: 880.98. Coronary heart disease is a common and frequently occurring disease that threatens human health and life. Hypercholesterolemia, characterized by elevated low-density lipoprotein (LDL), is one of the main risk factors for coronary heart disease. The efficacy of commonly used lipid-lowering drugs such as niacin, resins or fibrates is unsatisfactory. The best lipid-lowering drugs are called statins. The common mechanism of action of statins is that they all belong to 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase inhibitors. Currently, there are 6 main types widely used in the world: lovastatin, pravastatin, simvastatin, cerivastatin, fluvastatin and atorvastatin. The new statin, pitavastatin calcium, recently developed by Kowa, is called a "superstatin" for its good cholesterol-lowering effect. Tests have shown that pitavastatin calcium (NK-104) has a significant effect on lowering low-density lipoprotein cholesterol (LDL-C), similar to atorvastatin, but stronger than the other five statins. Pitavastatin calcium has good safety and tolerance and has been approved for marketing in Japan. Experts predict that this drug and another super statin from AstraZeneca, rosuvastan, will become the two main drugs leading the statin market in the next few years.
[0003] The synthetic route of pitavastatin calcium is as follows:
[0004]
[0005] The quality of the key intermediate YPF-SM-A seriously affects the quality of the finished product of pitavastatin calcium. Therefore, the quality of YPF-SM-A needs to be strictly controlled to ensure the quality of the finished product.
[0006] The synthetic route of YPF-SM-A is as follows:
[0007]
[0008] The intermediate compounds I and II and the by-product compounds III and IV in the synthesis process of YPF-SM-A are all impurities, so analytical methods must be established to strictly control them. The molecular structure of compound II also contains a halogenated alkane warning structure. It is necessary to establish a corresponding quality control method and perform quality control on it as a specific impurity in order to further improve the quality of the raw material drug. Summary of the invention
[0009] The purpose of the present invention is to provide a method for detecting a pitavastatin calcium intermediate (YPF-SM-A) and related substances thereof.
[0010] The method for detecting pitavastatin calcium intermediates and related substances provided by the present invention adopts liquid chromatography for detection, and comprises the following steps:
[0011] 1) Chromatographic conditions:
[0012] Chromatographic column: a chromatographic column with octadecylsilane bonded silica gel as filler;
[0013] Detector: UV detector;
[0014] Detection wavelength: 240nm; column temperature: 25℃~35℃; flow rate: 0.8-1.2ml / min;
[0015] The mobile phases are the following mobile phase A and mobile phase B, and the mobile phase A and mobile phase B are used for linear gradient elution; the mobile phase A is a mixed solution of water: trifluoroacetic acid: ammonia water according to a volume ratio of 1000:1:0.5; the mobile phase B is acetonitrile;
[0016] The linear gradient elution procedure is as follows:
[0017]
[0018] 2) Sample preparation:
[0019] Preparation of test solution: Accurately weigh an appropriate amount of pitavastatin calcium intermediate YPF-SM-A, dissolve it in acetonitrile-water (4:1, v / v), and quantitatively dilute it to make a solution containing about 0.4 mg of YPF-SM-A per 1 ml, as the test solution;
[0020] Preparation of control solution: Accurately measure an appropriate amount of the test solution and quantitatively dilute it with acetonitrile-water (4:1, v / v) to make a solution containing about 0.6 μg of YPF-SM-A per 1 ml as the control solution;
[0021] System suitability solution: Take appropriate amounts of YPF-SM-A reference substance, impurity I, impurity II, impurity III, and impurity IV reference substances, dissolve them in acetonitrile-water (4:1, v / v), and quantitatively dilute them to make a mixed solution containing approximately 0.4 mg of YPF-SM-A, 0.6 μg each of impurity I, impurity III, and impurity IV, and 0.15 μg of impurity II per 1 ml, as the system suitability solution.
[0022] Reference solution: Accurately weigh an appropriate amount of impurity II reference substance, add acetonitrile-water (4:1, v / v) to dissolve and quantitatively dilute to make a solution containing about 0.15 μg of impurity II per 1 ml, as the reference solution.
[0023] The structural formula of the YPF-SM-A is shown below:
[0024]
[0025] The YPF-SM-A related substances are selected from the impurities shown in the following formulas I to IV:
[0026]
[0027] 3) Assay method: Take 10 μl of each of the test solution, control solution, system suitability solution and reference solution and inject them into the liquid chromatograph, and record the chromatogram;
[0028] If there is a chromatographic peak with the same retention time as impurity II in the chromatogram of the test solution, the content of impurity II in the test sample is calculated by peak area according to the external standard method; if there is no chromatographic peak with the same retention time as impurity II, it is judged that the test sample does not contain impurity II;
[0029] If there is a chromatographic peak with the same retention time as impurity I, impurity III and / or impurity IV in the chromatogram of the test sample solution, the content of impurity I, impurity III and impurity IV in the test sample is calculated by the self-control method; if there is no chromatographic peak with the same retention time as impurity I, impurity III and / or impurity IV, it is judged that the test sample does not contain impurity I, impurity III and / or impurity IV.
[0030] Further, the chromatographic column may be YMC-Pack ODS-AQ 250 mm×4.6 mm, 5 μm;
[0031] Furthermore, the UV detector may specifically be a PDA detector;
[0032] Furthermore, in the test, a trapping column is installed after the HPLC pump (ie, the trapping column is installed after the mixer and before the injector).
[0033] Furthermore, in the chromatogram corresponding to the system suitability solution, the order of peaks is the impurity shown in formula IV (impurity 4), the impurity shown in formula I (impurity 1), the impurity shown in formula III (impurity 3), YPF-SM-A, and the impurity shown in formula II (impurity 2), and the separation degree of each peak should meet the requirements.
[0034] Furthermore, the relative retention time of impurity IV is 0.3; the relative retention time of impurity I is 0.4; the relative retention time of impurity III is 0.7; and the relative retention time of impurity II is 1.3.
[0035] Relative retention time (RRT): the ratio of the calibrated retention time of a component to the calibrated retention time of the corresponding standard.
[0036] The invention can realize effective separation and quantitative detection of multiple related substances of pitavastatin calcium intermediate YPF-SM-A, realize effective separation between chromatographic peaks (separation degree is greater than 1.5), can accurately determine the quality and quantity of the intermediate and its related substances, the method is simple and easy to implement, the instrument is a general high performance liquid chromatograph, and the detection cost is low; the specificity, linearity and range, precision, durability and other tests are all good, and the method can be used for conventional analysis and quality control of pitavastatin calcium intermediate YPF-SM-A and related substances, meeting the needs of research and development and production. BRIEF DESCRIPTION OF THE DRAWINGS
[0037] Figure 1 This is the adaptability map of the YPF-SM-A related substance detection system. DETAILED DESCRIPTION
[0038] The present invention is further described below in conjunction with specific examples, but the present invention is not limited to the following examples. The methods described are conventional methods unless otherwise specified. The raw materials described can be obtained from public commercial channels unless otherwise specified.
[0039] Impurity 1 described in the following embodiments is an impurity represented by the following formula I, impurity 2 is an impurity represented by the following formula II, impurity 3 is an impurity represented by the following formula III, and impurity 4 is an impurity represented by the following formula IV.
[0040]
[0041] The standard product of impurity 1 used in the following examples was purchased from Ark Pharm, Inc.
[0042] The standard product of impurity 2 used in the following examples was purchased from Jiangsu Alpha Pharmaceutical Co., Ltd.
[0043] The standard product of impurity 3 used in the following examples was purchased from SIGMA.
[0044] The standard product of impurity 4 used in the following examples was purchased from Anaiji Chemical.
[0045] The standard product of YPF-SM-A used in the following examples was purchased from Jiangsu Alpha Pharmaceutical Co., Ltd.
[0046] The YPF-SM-A samples used in the following examples were purchased from Jiangsu Alpha Pharmaceutical Co., Ltd.
[0047] Embodiment 1,
[0048] (1) Chromatographic conditions
[0049] Use octadecylsilane bonded silica gel as filler (YMC-Pack ODS-AQ 250mm×4.6mm, 5μm or equivalent column is applicable), and install a capture column after the pump.
[0050] Water: trifluoroacetic acid: ammonia water (1000:1:0.5, v / v / v) was used as mobile phase A, and acetonitrile was used as mobile phase B; linear gradient elution was performed according to Table 1 below.
[0051] Detector: PDA detector.
[0052] The detection wavelength was 240 nm, the column temperature was 30°C, the flow rate was 1.0 ml / min, and the diluent was acetonitrile: water (4:1, v / v). (2) Sample preparation
[0053] System suitability solution: Take appropriate amounts of YPF-SM-A reference substance, impurity I, impurity II, impurity III, and impurity IV reference substances, dissolve them in acetonitrile-water (4:1, v / v), and quantitatively dilute them to make a mixed solution containing approximately 0.4 mg of YPF-SM-A, 0.6 μg each of impurity I, impurity III, and impurity IV, and 0.15 μg of impurity II per 1 ml, as the system suitability solution.
[0054] Preparation of reference solution: Accurately weigh an appropriate amount of impurity II, dissolve it in acetonitrile-water (4:1, v / v), and quantitatively dilute it to make a solution containing about 0.15 μg per 1 ml as the reference solution;
[0055] Preparation of test solution: Take an appropriate amount of YPF-SM-A, weigh it accurately, dissolve it in acetonitrile-water (4:1, v / v), and quantitatively dilute it to make a solution containing about 0.4 mg per 1 ml as the test solution.
[0056] Preparation of control solution: Accurately measure an appropriate amount of the test solution and quantitatively dilute it with acetonitrile-water (4:1, v / v) to make a solution containing about 0.6 μg per 1 ml as the control solution;
[0057] (3) Determination method:
[0058] Accurately measure 10 μl of system suitability solution, reference solution, control solution and test solution respectively and inject them into the liquid chromatograph to record the chromatogram. In the system suitability solution, the peak order is impurity 4, impurity 1, impurity 3, YPF-SM-A, impurity 2, and the separation degree of each peak should meet the requirements. Calculate the content of impurity 2 in the test sample by peak area according to the external standard method; calculate the content of impurity 1, impurity 3 and impurity 4 by the self-control method.
[0059] Table 1. Gradient elution program
[0060]
[0061] (4) Methodological validation of YPF-SM-A related substances
[0062] The method validation items and results are shown in Table 2.
[0063] Table 2. Summary of Methodological Validation of Related Substances of YPF-SM-A
[0064]
[0065]
[0066]
[0067] The experimental process of the above verification project is as follows:
[0068] 1. Exclusivity
[0069] System suitability solution: Take appropriate amounts of YPF-SM-A reference substance, impurity I, impurity II, impurity III, and impurity IV reference substances, dissolve them in acetonitrile-water (4:1, v / v), and quantitatively dilute them to make a mixed solution containing approximately 0.4 mg of YPF-SM-A, 0.6 μg each of impurity I, impurity III, and impurity IV, and 0.15 μg of impurity II per 1 ml, as the system suitability solution.
[0070] Data collection was performed according to the proposed method, and the results are shown in Table 3.
[0071] Table 3
[0072]
[0073] 2. Forced degradation experiment
[0074] The solution preparation is shown in Table 4 below.
[0075] Table 4
[0076]
[0077]
[0078] Data collection was performed according to the proposed method, and the results are shown in Table 5.
[0079] Table 5
[0080]
[0081] 3. Accuracy experiment
[0082] Solution preparation:
[0083] Impurity I stock solution (3 portions): Take an appropriate amount of this product, add diluent, dissolve by ultrasonication and quantitatively dilute to make a solution containing about 0.5 mg per 1 ml, shake well, and obtain. (Respectively Impurity I stock solution ①②③)
[0084] Impurity II stock solution (3 portions): Take an appropriate amount of this product, add a diluent, dissolve by ultrasonication, and quantitatively dilute to make a solution containing about 0.5 mg per 1 ml, shake well, and obtain. (Respectively, Impurity II stock solution ①②③)
[0085] Impurity III stock solution (3 portions): Take an appropriate amount of this product, add diluent to dissolve and quantitatively dilute to make a solution containing about 0.5 mg per 1 ml and shake well. (Respectively Impurity III stock solution ①②③)
[0086] Impurity IV stock solution (3 portions): Take an appropriate amount of this product, add a diluent, dissolve by ultrasonication, and quantitatively dilute to make a solution containing about 0.5 mg per 1 ml, shake well, and obtain. (Respectively, Impurity IV stock solution ①②③)
[0087] Mixed impurity stock solution ①: Accurately measure 3 ml of impurity I stock solution ①, 0.75 ml of impurity II stock solution ①, 3 ml of impurity III stock solution ①, and 3 ml of impurity IV stock solution ① and place them in the same 100 ml volumetric flask, add diluent to make up to volume, shake well, and obtain.
[0088] Mixed impurity stock solution ②: Accurately measure 3 ml of impurity I stock solution ②, 0.75 ml of impurity II stock solution ②, 3 ml of impurity III stock solution ②, and 3 ml of impurity IV stock solution ② and place them in the same 100 ml volumetric flask, add diluent to make up to volume, shake well, and obtain.
[0089] Mixed impurity stock solution ③: Accurately measure 3 ml of impurity I stock solution ③, 0.75 ml of impurity II stock solution ③, 3 ml of impurity III stock solution ③, and 3 ml of impurity IV stock solution ③ and place them in the same 100 ml volumetric flask, add diluent to make up to volume, shake well, and obtain.
[0090] Reference solution: Accurately weigh 1 ml of mixed impurity stock solution① and place it in a 25 ml volumetric flask, add diluent to make up the volume, shake well, and the solution is ready. (Prepare two in parallel)
[0091] The preparation method of the accuracy test solution is shown in Table 6.
[0092] Table 6
[0093]
[0094] Data collection was performed according to the method proposed in Table 6 above, and the results are shown in Table 7 below.
[0095] Table 7 Accuracy determination experimental results
[0096] name Impurity I Impurity II Impurity III Impurity IV Recovery - 50% concentration 101.4％ 104.3％ 99.22％ 98.06％ Recovery - 100% concentration 99.97％ 102.0％ 97.26％ 97.81％ Recovery - 150% concentration 100.2％ 103.7％ 99.22％ 98.47％
[0097] From the results in Table 2, it can be seen that the HPLC method is used to determine the related substances of YPF-SM-A. The methodological validation includes system suitability, linearity, specificity, detection limit, quantification limit, precision (injection precision, repeatability, intermediate precision), accuracy and durability. The method has good linearity, sensitivity and specificity, good method precision and high accuracy. The durability test results show that slight changes in the flow rate and column temperature in the chromatographic conditions of this product have no effect on the determination of related substances of this product. This method is suitable for the detection of related substances of this product.
[0098] (5) YPF-SM-A sample measurement
[0099] Preparation of test solution: Accurately weigh an appropriate amount of pitavastatin calcium intermediate YPF-SM-A, dissolve it in acetonitrile-water (4:1, v / v), and quantitatively dilute it to make a solution containing about 0.4 mg of YPF-SM-A per 1 ml, as the test solution;
[0100] Preparation of control solution: Accurately measure an appropriate amount of the test solution and quantitatively dilute it with acetonitrile-water (4:1, v / v) to make a solution containing about 0.6 μg of YPF-SM-A per 1 ml as the control solution;
[0101] System suitability solution: Take appropriate amounts of YPF-SM-A reference substance, impurity I, impurity II, impurity III, and impurity IV reference substances, dissolve them in acetonitrile-water (4:1, v / v), and quantitatively dilute them to make a mixed solution containing approximately 0.4 mg of YPF-SM-A, 0.6 μg each of impurity I, impurity III, and impurity IV, and 0.15 μg of impurity II per 1 ml, as the system suitability solution.
[0102] Reference solution: Take an appropriate amount of impurity II reference substance, add acetonitrile-water (4:1, v / v) to dissolve and quantitatively dilute to make a solution containing about 0.15 μg of impurity II per 1 ml, as the reference solution.
[0103] Determination method: Take 10 μl of the test solution, control solution, system suitability solution and reference solution mentioned above and inject them into liquid chromatograph, record the chromatogram, calculate the content of impurity II in the test sample by peak area according to the external standard method, and calculate the contents of impurity I, impurity III and impurity IV by the self-control method.
[0104] The calculation formula is as follows:
[0105] 1. The calculation formula of the self-control method is as follows:
[0106]
[0107] 2. Calculation formula of external standard method:
[0108]
[0109] Where: A1 is the impurity peak area in the test solution;
[0110] A2 is the impurity peak area in the reference solution;
[0111] C1 is the concentration of the test solution, μg / ml;
[0112] C2 is the concentration of the reference solution, μg / ml.
[0113] Table 3. Test results of test products
[0114] Batch No. / Content% Impurity I Impurity II Impurity III Impurity IV Sample 1 0.005 Not detected 0.007 Not detected Sample 2 0.007 Not detected 0.013 Not detected Sample 3 0.010 Not detected 0.011 Not detected
[0115] From the results in Table 3, it can be seen that the proposed method is used to detect the related substances of this product, and the test results of multiple batches are consistent, which proves that this method has high sensitivity and strong specificity and is suitable for the detection of related substances of this product.
[0116] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.
Claims
1. A method for detecting a pitavastatin calcium intermediate and its related substances, using liquid chromatography for detection, comprising the following steps: 1) Chromatographic conditions: Chromatographic column: a chromatographic column with octadecylsilane bonded silica gel as filler; Detector: UV detector; Detection wavelength: 240nm; column temperature: 25℃~35℃; flow rate: 0.8-1.2ml / min; The mobile phases are the following mobile phase A and mobile phase B, and the mobile phase A and mobile phase B are used for linear gradient elution; the mobile phase A is a mixed solution of water: trifluoroacetic acid: ammonia water according to a volume ratio of 1000:1:0.5; the mobile phase B is acetonitrile; The linear gradient elution procedure is as follows: 2) Sample preparation: Preparation of test solution: accurately weigh an appropriate amount of pitavastatin calcium intermediate YPF-SM-A, dissolve it in acetonitrile-water (4:1, v / v), and quantitatively dilute it to make a solution containing 0.4 mg of YPF-SM-A per 1 ml, as the test solution; Preparation of control solution: Accurately measure an appropriate amount of the test solution and quantitatively dilute it with acetonitrile-water (4:1, v / v) to make a solution containing 0.6 μg of YPF-SM-A per 1 ml as the control solution; System suitability solution: Take appropriate amount of YPF-SM-A reference substance, impurity I, impurity II, impurity III, and impurity IV reference substances, dissolve them in acetonitrile-water (4:1, v / v), and quantitatively dilute them to prepare a mixed solution containing 0.4 mg of YPF-SM-A, 0.6 μg each of impurity I, impurity III, and impurity IV, and 0.15 μg of impurity II per 1 ml, as the system suitability solution; Preparation of reference solution: Accurately weigh an appropriate amount of impurity II reference substance, add acetonitrile-water (4:1, v / v) to dissolve and quantitatively dilute to make a solution containing 0.15 μg of impurity II per 1 ml as the reference solution; The structural formula of the YPF-SM-A is shown below: The YPF-SM-A related substances are selected from the impurities shown in the following formulas I to IV: 3) Determination method: Take 10 μl of the test solution, control solution, system suitability solution and reference solution respectively and inject them into the liquid chromatograph to record the chromatogram; If there is a chromatographic peak with the same retention time as impurity II in the chromatogram of the test solution, the content of impurity II in the test sample is calculated by peak area according to the external standard method; if there is no chromatographic peak with the same retention time as impurity II, it is judged that the test sample does not contain impurity II; If there is a chromatographic peak with the same retention time as impurity I, impurity III and / or impurity IV in the chromatogram of the test sample solution, the content of impurity I, impurity III and impurity IV in the test sample is calculated by the self-control method; if there is no chromatographic peak with the same retention time as impurity I, impurity III and / or impurity IV, it is judged that the test sample does not contain impurity I, impurity III and / or impurity IV.
2. The method according to claim 1, characterized in that: The chromatographic column is YMC-Pack ODS-AQ 250*4.6mm, 5μm.
3. The method according to claim 1 or 2, characterized in that: In the method, a trapping column is installed after the HPLC pump.
4. The method according to any one of claims 1 to 3, characterized in that: In the chromatogram corresponding to the system suitability solution, the order of peaks is the impurity shown in Formula IV, the impurity shown in Formula I, the impurity shown in Formula III, YPF-SM-A, and the impurity shown in Formula II.
5. The method according to claim 4, characterized in that: The relative retention time of the impurity represented by formula IV is 0.3; the relative retention time of the impurity represented by formula I is 0.4; the relative retention time of impurity III is 0.7; and the relative retention time of impurity II is 1.3.