An analytical method for determining related substances of pitavastatin tert-butyl ester using HPLC

Abstract

The invention discloses a method for analyzing related substances of pitavastatin tert-butyl ester by HPLC, and specifically relates to a method for analyzing tert-butyl ester of pitavastatin and four impurities thereof by HPLC. The chromatographic column type and specification, detection wavelength, mobile phase type, mobile phase ratio, running time, etc. described in the method of the present invention can be quickly, simply, accurately, and efficiently eluted, separated, and quantitatively detected for each impurity in pitavastatin tert-butyl ester, and complete separation between impurity peak and main peak, impurity and impurity peak can be achieved, and the best detection result is achieved. The analytical method is easy to operate, has a short running time, is highly specific, has high sensitivity, is highly stable, has a good linear curve in a low concentration range, and has strong repeatability and accuracy, is not affected by personnel and instruments, is stable and reliable, and provides a basis for research and development and quality detection for such compounds, and the method also has certain adjustable denaturation and is highly universal.

Description

Technical Field

[0001] The invention belongs to the field of chemical drug analysis methods, and particularly relates to an analysis method for determining pitavastatin tert-butyl ester-related substances by utilizing HPLC. Background Art

[0002] Pitavastatin calcium is a third-generation statin jointly developed by Nippon Chemical Industry Co., Ltd. and Kowa Co., Ltd. It was launched in Japan in September 2003. On March 27, 2009, the generic drug pitavastatin calcium tablets (trade name: Guanshuang) launched by Beijing Shuanghe Pharmaceutical Co., Ltd. were launched in my country. According to the existing clinical trial results and the comparison with similar products on the market abroad, pitavastatin calcium is the most effective lipid-lowering drug so far. It is called "super statin" by the pharmaceutical industry because of its small dosage and excellent efficacy. It has been listed as one of the 18 new drugs with the greatest sales potential in the world, and its development prospects are very broad.

[0003] (3R,5S,6E)-7-[2-cyclopropyl-4-(4-fluorophenyl)-3-quinolinyl]-3,5-dihydroxy-6-heptanoic acid tert-butyl ester, molecular formula: C 29 H 32 FNO4 molecular weight: 477.57, referred to as pitavastatin tert-butyl ester, is a key intermediate of pitavastatin calcium. Most of the existing pitavastatin calcium synthesis routes require the synthesis of this intermediate. The structural formula of pitavastatin tert-butyl ester is as follows:

[0004]

[0005] In order to ensure the safety and effectiveness of drugs, it is necessary to comprehensively examine the quality of drugs and study, test and monitor the materials in each step of the drug. As a key intermediate, it is of great significance to study the chromatographic conditions of the analysis method of its related substances for pitavastatin tert-butyl ester. At present, there is no discussion on the analysis method of related substances of pitavastatin tert-butyl ester. There is an urgent need for a method to detect its related substances to ensure accurate and effective monitoring of its quality level. Summary of the invention

[0006] Based on the technical problems existing in the background technology, the present invention proposes an analysis method for pitavastatin tert-butyl ester related substances. The present invention has a simple mobile phase system, high impurity separation, more detected impurities, high sensitivity, low detection concentration range, good linear range, and strong adjustable denaturation, and can accurately and effectively monitor the quality level of pitavastatin tert-butyl ester and its impurities.

[0007] In the present invention, the term "pitavastatin tert-butyl ester" means "(3R,5S,6E)-7-[2-cyclopropyl-4-(4-fluorophenyl)-3-quinolin-yl]-3,5-dihydroxy-6-heptanoic acid tert-butyl ester".

[0008] A qualitative and / or quantitative analysis method for pitavastatin tert-butyl ester and its related substances, wherein the related substances are:

[0009] Impurity A: (3R,5S,E)-7-[2-cyclopropyl-4-(4-fluorophenyl)quinolin-3-yl]-3,5-dihydroxyhept-6-enoic acid methyl ester,

[0010] Impurity B: (4R,6S,E)-6-[2-cyclopropyl-4-(4-fluorophenyl)-3-quinolyl-vinyl]-4-hydroxy-3,4,5,6-tetrahydro-2H-pyran-2-one,

[0011] Impurity C: (3R,5S,E)-7-[2-cyclopropyl-4-(4-fluorophenyl)quinolin-3-yl]-3,5-dihydroxyhept-6-enoic acid ethyl ester,

[0012] Impurity D: (4R,6S)-6-[[(1E)-2-cyclopropyl-4-(4-fluorophenyl)-3-quinolyl]vinyl]-2,2-dimethyl-1,3-dioxane-4-acetic acid tert-butyl ester,

[0013] The method comprises the following steps:

[0014] (1) Preparation of test sample: Take the test sample of pitavastatin tert-butyl ester and dilute the sample with acetonitrile to prepare a test sample solution of 0.1-0.5 mg / ml;

[0015] (2) Preparation of reference substance: Take pitavastatin tert-butyl ester reference substance, dilute the sample with acetonitrile, and prepare a 0.1-0.5 mg / ml test solution;

[0016] (3) Chromatographic conditions:

[0017] Chromatographic column: a chromatographic column that is chemically bonded as a stationary phase;

[0018] Detection wavelength: 220~280nm;

[0019] Column temperature: 20~45℃;

[0020] Injection volume: 5-20 μl;

[0021] Flow rate: 0.4~1.2ml / min;

[0022] Mobile phase: acetic acid aqueous solution is mobile phase A; acetonitrile is mobile phase B;

[0023] The mobile phase was gradient eluted in the following manner:

[0024] In the time range of 0 to 8 minutes, the proportion of mobile phase A is maintained at any value in the range of 30% to 50%, the proportion of mobile phase B is maintained at any value in the range of 50% to 70%, and the sum of mobile phase A and mobile phase B is 100%;

[0025] Within the time range of 8.01 to 13 minutes, the proportion of mobile phase A is maintained at any value within the range of 0% to 20%, the proportion of mobile phase B is maintained at any value within the range of 80% to 100%, and the sum of mobile phase A and mobile phase B is 100%;

[0026] 13. Within the time range of 01 to 20 minutes, the proportion of mobile phase A is maintained at any value within the proportion range of 0% to 20%, the proportion of mobile phase B is maintained at any value within the proportion range of 80% to 100%, and the sum of mobile phase A and mobile phase B is 100%;

[0027] In the time range of 22.01 to 30 minutes, the proportion of mobile phase A is maintained at any value within the range of 30% to 50%, the proportion of mobile phase B is maintained at any value within the range of 50% to 70%, and the sum of mobile phase A and mobile phase B is 100%.

[0028] Furthermore, the length of the chromatographic column is 150-250 mm, the diameter is 4.6 mm, and the filler particle size is 2-5 μm;

[0029] Preferably, the length of the chromatographic column is 150 mm and the filler particle size is 2.7 μm;

[0030] More preferably, the chromatographic column is an Agilent Phenyl Hexyl chromatographic column (150 mm*4.6 mm, 2.7 μm).

[0031] Furthermore, the detection wavelength is 245 nm.

[0032] Furthermore, the column temperature was 40°C.

[0033] Furthermore, the injection volume was 10 μl.

[0034] Further, the flow rate was 1.0 ml / min.

[0035] Furthermore, the mobile phase A is an acetic acid aqueous solution with a pH of 3.0 to 5.0;

[0036] Preferably, mobile phase A is an aqueous solution of acetic acid with a pH of 3.8.

[0037] Furthermore, the pH of mobile phase A was adjusted with sodium acetate solution.

[0038] Furthermore, the volume percentage of acetic acid in the mobile phase A is 0.01% to 0.5%. Preferably, the volume percentage of acetic acid in the mobile phase A is 0.06%.

[0039] Furthermore, during gradient elution from 0 to 8 min, the proportion of mobile phase A was 40%; during 8.01 to 13 min, the proportion of mobile phase A was maintained at 15%; during 13.01 to 20 min, the proportion of mobile phase A was maintained at 15%; during 20.01 to 30 min, the proportion of mobile phase A was maintained at 40%.

[0040] That is, use the mobile phase for gradient elution as shown in the table below:

[0041] time Mobile phase A Mobile phase B 0min 40％ 60％ 8min 40％ 60％ 13min 15％ 85％ 20min 15％ 85％ 20.01min 40％ 60％ 30min 40％ 60％

[0042] In one embodiment, the analytical method is subjected to a specificity test according to the above-mentioned chromatographic conditions. The specific steps are: prepare diluent, pitavastatin tert-butyl ester test solution, pitavastatin tert-butyl ester positioning solution, impurity A positioning solution, impurity B positioning solution, impurity C positioning solution, impurity D positioning solution, test mixed solution and system suitability solution for injection respectively, wherein the system suitability solution is continuously injected 5 times. Record the peak time of pitavastatin tert-butyl ester and the separation between its adjacent impurity peaks, and calculate the RSD of the peak area of ​​hydroxylamine hydrochloride peak, impurity A peak, impurity B peak, impurity C peak and impurity D peak of 5 consecutive injections.

[0043] In one embodiment, the analysis method is subjected to detection limit and quantification limit tests, and the specific steps of elution according to the above chromatographic conditions are: preparing detection limit solutions and quantification limit solutions of pitavastatin tert-butyl ester, impurity A, impurity B, impurity C and impurity D, respectively, and injecting them separately, and calculating the detection limit concentration and quantification limit concentration of each component, respectively.

[0044] In one embodiment, the linearity and range test of the analytical method is carried out, and the specific steps of eluting according to the above chromatographic conditions are: linear solutions of different concentrations of pitavastatin tert-butyl ester, impurity A, impurity B, impurity C and impurity D (quantitative limit concentration to 120% of the limit concentration) are prepared respectively, and the linear equation and linear range are calculated.

[0045] In one embodiment, the analytical method is subjected to repeatability and intermediate precision tests, and the specific steps of eluting according to the above-mentioned chromatographic conditions are: preparing a repeatability solution and an intermediate precision solution for injection respectively, and calculating the RSD of pitavastatin tert-butyl ester, impurity A, impurity B, impurity C and impurity D in each solution.

[0046] In one embodiment, the analysis method is subjected to an accuracy test, and the specific steps of eluting according to the above-mentioned chromatographic conditions are: prepare 20% spiked recovery solution, 40% spiked recovery solution and 120% spiked recovery solution of impurity A, impurity B, impurity C and impurity D respectively, inject them separately, and calculate the recovery rate of each solution.

[0047] In one embodiment, the analytical method is subjected to a solution stability test, and the specific steps of eluting according to the above-mentioned chromatographic conditions are: preparing a system suitability solution of pitavastatin tert-butyl ester and a test mixed solution, and injecting the samples at 0, 12, 24, 36, and 48 hours after preparation, recording the amount of pitavastatin tert-butyl ester and each impurity, and calculating the change value compared with 0 hours.

[0048] The technical solution of the present invention has the following beneficial effects compared with the prior art:

[0049] The inventors screened the chromatographic column type, mobile phase type, elution condition, injector temperature, column temperature, and injection volume, detected pitavastatin tert-butyl ester and its impurity content, determined the chromatographic analysis method of the present invention, and verified the method for specificity, quantitative limit, detection limit, linearity, repeatability, intermediate precision, accuracy, solution stability, and method durability. It is finally proved that the high-performance liquid chromatography analysis method provided by the present invention, the quantitative limit can reach 0.0101 μg / mL, the detection limit can reach 0.0031 μg / mL, and the sensitivity is high. It can accurately and effectively detect the content of pitavastatin tert-butyl ester and its impurities, and the analysis method has strong specificity, has a good linear curve in the low concentration range, and is not affected by personnel and instruments, and has strong repeatability and accuracy; and the detection method has a strong elution ability to pitavastatin tert-butyl ester and its impurities. Within 20 minutes, the main peak and impurity peaks are all eluted, and the separation is good. At the same time, the method is made to have a certain adjustable denaturation, and universality is strong.

[0050] The method of the present invention optimizes the detection result and the separation between the components by comprehensively considering the comprehensive influence of the chromatographic column type and specification, detection wavelength, mobile phase type, mobile phase ratio, running time, sample preparation and injection volume on separation and detection, and has the advantages of fast, simple, accurate and efficient elution, separation and quantitative detection. The analytical method is easy to operate, has a short running time, strong specificity, high sensitivity and strong stability, and provides a basis for research and development and quality detection for the detection of pitavastatin tert-butyl ester and its impurities. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 This is the chromatogram of the system suitability solution of pitavastatin tert-butyl ester;

[0052] Figure 2 This is the chromatogram of the test mixed solution of pitavastatin tert-butyl ester;

[0053] Figure 3 This is the chromatogram of the quantification limit solution of pitavastatin tert-butyl ester;

[0054] Figure 4 This is the detection limit solution chromatogram of pitavastatin tert-butyl ester; DETAILED DESCRIPTION

[0055] The technical scheme of the present invention is described in detail below through specific examples. The experimental methods in the following examples without specifying specific conditions are generally based on the known means in the art. The reagents not specifically specified are all reagents commonly purchased in the art.

[0056] Example 1 Comparison of different chromatographic columns and different mobile phases

[0057] HPLC conditions:

[0058] This embodiment is divided into three groups, and the Welch Ultimate XB-C18 chromatographic column (250*4.6mm, 5μm), Agilent ZORBAX SB-C18 chromatographic column (250*4.6mm, 3.5μm) and Agilent Phenyl Hexyl chromatographic column (150*4.6mm, 2.7μm) are tested using methods A, B and C respectively (because the stationary phases of the three groups of chromatographic columns are different, different chromatographic conditions are selected considering their characteristics). Methods A, B and C are shown in Table 1, and the gradient conditions are the same as the gradient conditions in the content of the invention.

[0059] Table 1 Comparison of mobile phase screening methods

[0060]

[0061] Sample preparation:

[0062] Pitavastatin tert-butyl ester system suitability solution: Weigh appropriate amounts of pitavastatin tert-butyl ester reference substance and each impurity reference substance to prepare a mixed solution containing approximately 0.3 mg of pitavastatin tert-butyl ester reference substance, 0.6 μg of impurity A, 0.6 μg of impurity B, 0.6 μg of impurity C and 0.6 μg of impurity D per 1 ml.

[0063] Test operation: Take 10 μl of the system suitability solution and inject it separately, and record the chromatogram. The system suitability solution was tested using these three chromatographic conditions respectively, and the results are shown in Table 2.

[0064] Table 2 Summary of chromatographic condition screening results

[0065]

[0066] As can be seen from Table 2, method C is significantly better than method A and method B in terms of separation between the main peak and adjacent peaks and component retention time. Method C has better separation and shorter running time.

[0067] Example 2 Specificity Verification

[0068] HPLC conditions:

[0069] Agilent Phenyl Hexyl chromatographic column (150*4.6mm, 2.7μm) was used, with 0.06% acetic acid aqueous solution adjusted to pH 3.8 by sodium acetate solution as mobile phase A, acetonitrile as mobile phase B, detection wavelength of 245nm, flow rate of 1.0ml / min, column temperature of 40℃, injection volume of 10μl, gradient elution, elution gradient:

[0070] time Mobile phase A Mobile phase B 0min 40％ 60％ 8min 40％ 60％ 13min 15％ 85％ 20min 15％ 85％ 20.1min 40％ 60％ 30min 40％ 60％

[0071] Sample preparation:

[0072] Pitavastatin tert-butyl ester reference solution: Weigh the reference substance and dilute to the mark with acetonitrile to obtain a reference solution containing approximately 0.3 mg of pitavastatin tert-butyl ester per 1 ml.

[0073] Pitavastatin tert-butyl ester test solution: Weigh the test sample and dilute to the mark with acetonitrile to obtain a test solution containing approximately 0.3 mg of pitavastatin tert-butyl ester per 1 ml.

[0074] Component localization solution: Weigh appropriate amount of each component reference substance to prepare impurity A localization solution containing approximately 0.6 μg per 1 ml, impurity B localization solution containing approximately 0.6 μg per 1 ml, impurity C localization solution containing approximately 0.6 μg per 1 ml, impurity D localization solution containing approximately 0.6 μg per 1 ml and pitavastatin tert-butyl ester localization solution containing approximately 0.6 μg per 1 ml.

[0075] System suitability solution: Weigh appropriate amounts of pitavastatin tert-butyl ester reference substance and each impurity reference substance to prepare a mixed solution containing approximately 0.3 mg of pitavastatin tert-butyl ester reference substance, 0.6 μg of impurity A, 0.6 μg of impurity B, 0.6 μg of impurity C and 0.6 μg of impurity D per 1 ml.

[0076] Test mixed solution: Weigh appropriate amounts of pitavastatin tert-butyl ester test sample and each impurity reference substance to prepare a mixed solution containing approximately 0.3 mg of pitavastatin tert-butyl ester test sample, 0.6 μg of impurity A, 0.6 μg of impurity B, 0.6 μg of impurity C and 0.6 μg of impurity D per 1 ml.

[0077] Experimental operation: Take 10 μl of each component positioning solution, system suitability solution and test mixed solution and inject them separately, and record the chromatogram for 30 minutes.

[0078] Typical chromatograms are shown in Figure 1 System suitability solution chromatogram, Figure 2 Chromatogram of the test mixed solution.

[0079] The specificity of the present invention was verified by using the system suitability solution, the test mixed solution, and each impurity location solution. The results are shown in Table 3, Table 4 and Figure 1 , Figure 2 .

[0080] Table 3 Specificity test results 1

[0081]

[0082] Table 4 Specificity test results 2

[0083]

[0084] From Table 3 and Figure 1 , Figure 2 It can be seen that under the chromatographic conditions described in the present invention, pitavastatin tert-butyl ester and each impurity can be effectively detected. The retention time (t R ) and the main peak retention time (t R ) are consistent.

[0085] From Table 4 and Figure 1 It can be seen that in the systematic solution spectrum, the peak order is impurity A, impurity B, impurity C, pitavastatin tert-butyl ester and impurity D. The separation between the main peak pitavastatin tert-butyl ester and the impurity C peak is greater than 1.5, and the separation between impurities is greater than 1.5. And the RSD of the 5-shot system suitability solution is ≤1.0%.

[0086] Example 3 Detection limit and quantification limit verification

[0087] The HPLC conditions were the same as those in Example 2.

[0088] Sample preparation:

[0089] The pitavastatin tert-butyl ester and each impurity location solution were diluted with acetonitrile as solvent, and the diluted solution with a corresponding concentration with a signal-to-noise ratio of about 3:1 was used as the detection limit solution.

[0090] The location solutions of pitavastatin tert-butyl ester and each impurity were diluted with acetonitrile as solvent, and the diluted solution with a corresponding concentration having a signal-to-noise ratio of about 10:1 was used as the quantitative limit solution.

[0091] Experimental operation: Take 10 μl of the detection limit solution and the quantitative limit solution and inject them separately. Each sample is injected 5 times in parallel. Record the chromatogram until 30 minutes. Calculate the RSD of the peak area of ​​each component in each group of samples. The results are shown in Table 5.

[0092] Typical chromatograms are shown in Figure 3 This is the chromatogram of the quantitative limit solution of pitavastatin tert-butyl ester. Figure 4 This is the chromatogram of the quantification limit solution of pitavastatin tert-butyl ester.

[0093] Table 5 Detection limit and quantification limit test results

[0094]

[0095] The inventors have verified the detection limit and quantitative limit of pitavastatin tert-butyl ester and its impurities, and the detection limit and quantitative limit of pitavastatin tert-butyl ester are the detection limit and quantitative limit of a single impurity. It can be seen that the detection limit and quantitative limit of pitavastatin tert-butyl ester and its impurities are very low, proving that the present invention has a higher and stable detection sensitivity.

[0096] Example 4 Linearity and range verification

[0097] The HPLC conditions were the same as those in Example 2.

[0098] Sample preparation:

[0099] Solution 1: Prepare solutions of pitavastatin tert-butyl ester and its impurity limit concentration of 120% respectively.

[0100] Solution 2: Prepare solutions of pitavastatin tert-butyl ester and its impurity limit concentration of 100% respectively.

[0101] Solution 3: prepare solutions of pitavastatin tert-butyl ester and its impurity limit concentration of 80% respectively.

[0102] Solution 4: Prepare solutions of pitavastatin tert-butyl ester and its impurity limit concentration of 40% respectively.

[0103] Solution 5: Prepare solutions of pitavastatin tert-butyl ester and its impurity quantitative limit concentration respectively.

[0104] Experimental operation: Take 10 μl of each of the five linear solutions of pitavastatin tert-butyl ester and each impurity, inject them separately, and record the chromatogram for 30 minutes.

[0105] The inventors verified the linearity and range of pitavastatin tert-butyl ester and each impurity. The results are shown in Table 6.

[0106] Table 6 Linearity and range test results

[0107]

[0108] It can be seen from Table 6 that the main peak of pitavastatin tert-butyl ester and its impurities of the present invention have a good linear relationship between the quantitative limit concentration and 120% of the limit concentration.

[0109] Example 5 Repeatability and Intermediate Precision Verification

[0110] The HPLC conditions were the same as those in Example 2.

[0111] Sample preparation:

[0112] Pitavastatin tert-butyl ester test solution: Weigh the test sample and dilute to the mark with acetonitrile to obtain a test solution containing approximately 0.3 mg of pitavastatin tert-butyl ester per 1 ml.

[0113] Test mixed solution: Weigh appropriate amounts of pitavastatin tert-butyl ester test sample and each impurity reference substance to prepare a mixed solution containing approximately 0.3 mg of pitavastatin tert-butyl ester test sample, 0.6 μg of impurity A, 0.6 μg of impurity B, 0.6 μg of impurity C and 0.6 μg of impurity D per 1 ml.

[0114] Repeatability is that one experimenter prepares 6 test mixed solutions;

[0115] The intermediate precision is that two experimenters prepare 6 portions of the test mixed solution respectively, for a total of 12 portions.

[0116] Two of the researchers were required to perform six independent tests on different days and different instrument models. The chromatograms were recorded. Repeatability and intermediate precision were verified, and the results are shown in Table 7.

[0117] Table 7 Repeatability and intermediate precision test results

[0118]

[0119] It can be concluded from Table 7 that the RSDs of the repeatability and intermediate precision of pitavastatin tert-butyl ester and its impurities all meet the acceptance criteria. It can be seen that the invention is not affected by personnel and instruments and has good repeatability and intermediate precision.

[0120] Example 6 Accuracy Verification

[0121] The HPLC conditions were the same as those in Example 2.

[0122] Sample preparation:

[0123] Pitavastatin tert-butyl ester test solution: Weigh the test sample and dilute to the mark with acetonitrile to obtain a test solution containing approximately 0.3 mg of pitavastatin tert-butyl ester per 1 ml.

[0124] Weigh appropriate amounts of pitavastatin tert-butyl ester test sample and each impurity reference substance to prepare 20% limit concentration spiked recovery solution, 50% limit concentration spiked recovery solution and 120% limit concentration spiked recovery solution.

[0125] Experimental operation: 10 μl of each of 20% spiked recovery solution, 80% spiked recovery solution, and 120% spiked recovery solution were injected respectively, and the chromatogram was recorded for 30 minutes. The inventor calculated the concentration of each impurity in each spiked recovery solution by reading the content of each impurity peak and the content of each impurity in the test solution on 9 spiked recovery solution spectra, and compared it with the actual theoretical concentration added, and calculated the sample recovery rate of each impurity and the relative standard deviation (RSD) between the recovery rates, n=9. The accuracy of the method was verified, and the results are shown in Table 8.

[0126] Table 8 Accuracy test results

[0127]

[0128] It can be seen from Table 8 that the spiked recoveries of impurities A, B, C and D meet the acceptable standards, indicating that the present invention can accurately and stably detect related substances of pitavastatin tert-butyl ester.

[0129] Example 7 Solution Stability Verification

[0130] The HPLC conditions were the same as those in Example 2.

[0131] Sample preparation:

[0132] Prepare the system suitability solution of pitavastatin tert-butyl ester and the test mixed solution. The sample preparation process is the same as the sample preparation in Example 2. After preparation, store them in a refrigerator temporarily.

[0133] Experimental operation: take 10 μl of the applicable solution of pitavastatin tert-butyl ester system and the mixed solution for testing at 0, 12, 24, 36, 48h, respectively, sample respectively, and record the chromatogram to 30min. Record each pitavastatin tert-butyl ester and its impurity content, and calculate the amount of each component after storing 12h, 24h, 36h and 48h in a cold place and the change value of 0h. The inventor verifies the solution stability of pitavastatin tert-butyl ester and its impurity, and the results are shown in Table 9.

[0134] Table 9 Accuracy test results

[0135]

[0136] During the solution stability experiment period, the changes in the contents of pitavastatin tert-butyl ester and its impurities at 12h, 24h, 36h, 48h and 0h were all less than the acceptable standards.

[0137] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, and they should be covered by the protection scope of the present invention.

Claims

1. A qualitative and / or quantitative analysis method for pitavastatin tert-butyl ester and its related substances, characterized in that: The related substances are: Impurity A: (3R,5S,E)-7-[2-cyclopropyl-4-(4-fluorophenyl)quinolin-3-yl]-3,5-dihydroxyhept-6-enoic acid methyl ester, Impurity B: (4R,6S,E)-6-[2-cyclopropyl-4-(4-fluorophenyl)-3-quinolyl-vinyl]-4-hydroxy-3,4,5,6-tetrahydro-2H-pyran-2-one, Impurity C: (3R,5S,E)-7-[2-cyclopropyl-4-(4-fluorophenyl)quinolin-3-yl]-3,5-dihydroxyhept-6-enoic acid ethyl ester, Impurity D: (4R,6S)-6-[[(1E)-2-cyclopropyl-4-(4-fluorophenyl)-3-quinolyl]vinyl]-2,2-dimethyl-1,3-dioxane-4-acetic acid tert-butyl ester, The method comprises the following steps: (1) Preparation of test sample: Take the test sample of pitavastatin tert-butyl ester and dilute the sample with acetonitrile to prepare a test sample solution of 0.1-0.5 mg / ml; (2) Preparation of reference substance: Take pitavastatin tert-butyl ester reference substance, dilute the sample with acetonitrile, and prepare a 0.1-0.5 mg / ml test solution; (3) Chromatographic conditions: Chromatographic column: Agilent Phenyl Hexyl chromatographic column; Detection wavelength: 220~280nm; Column temperature: 20~45℃; Injection volume: 5-20 μl; Flow rate: 0.4~1.2ml / min; Mobile phase: acetic acid aqueous solution is mobile phase A; acetonitrile is mobile phase B; the mobile phase A is acetic acid aqueous solution with a pH of 3.0 to 5.0; the volume percentage of acetic acid in mobile phase A is 0.01% to 0.5%; The mobile phase was gradient eluted in the following manner: 。 2. The analysis method according to claim 1, characterized in that The length of the chromatographic column is 150-250 mm, the diameter is 4.6 mm, and the filler particle size is 2-5 μm.

3. The analysis method according to claim 2, characterized in that The length of the chromatographic column is 150 mm, and the filler particle size is 2.7 μm.

4. The analysis method according to claim 3, characterized in that The length*inner diameter of the chromatographic column is 150 mm*4.6 mm, and the filler particle size is 2.7 μm.

5. The analysis method according to claim 1, characterized in that The detection wavelength is 245 nm.

6. The analysis method according to claim 1, characterized in that The column temperature was 40°C.

7. The analysis method according to claim 1, characterized in that The injection volume was 10 μl.

8. The analysis method according to claim 1, characterized in that The flow rate is 1.0 ml / min.

9. The analysis method according to claim 1, characterized in that Mobile phase A was acetic acid in water with a pH of 3.

8.

10. The analysis method according to claim 1, characterized in that The pH of mobile phase A was adjusted with sodium acetate solution.

11. The analysis method according to claim 1, characterized in that The volume percentage of acetic acid in mobile phase A is 0.06%.

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