Detection method and application of ezetimibe intermediate related substances

Abstract

The invention discloses a detection method and application of ezetimibe intermediate related substances, and belongs to the technical field of drug impurity detection. The method specifically comprises the following steps: dissolving a test article YZ1 by using acetonitrile-water as a solvent to obtain a test article solution; acetonitrile-water is used as a solvent to dissolve the standard substance YZ1 and the impurity standard substance YZ1-1, and a system applicability solution is obtained; an octadecyl silane bonded silica gel column is used as a chromatographic column, acetonitrile-water is used as a mobile phase, and the content of the related substances in the test solution is detected by adopting high performance liquid chromatography. According to the technical scheme provided by the invention, the impurities in the ezetimibe intermediate can be well separated from one another, and the content of the impurities in the ezetimibe intermediate can be accurately calculated. The method has the beneficial effects of accurate qualification, convenience, reliability and good repeatability.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of drug impurity detection, and particularly relates to a detection method and application of an ezetimibe intermediate related substance. BACKGROUND

[0002] Ezetimibe is a selective cholesterol inhibitor and has a wide range of clinical applications. YZ1 is a key intermediate in the synthesis of ezetimibe, and its chemical name is (4S)-3-[(5S)-5-(4-fluorophenyl)-5-hydroxyvaleryl]-4-phenyl-1,3-oxazolidin-2-one.

[0003] As a key intermediate in the synthesis of ezetimibe, the impurity YZ1-1 contained in YZ1 directly affects the quality of ezetimibe. In the prior art, the synthesis method of YZ1 is shown in formula 3:

[0004]

[0005] Since the production process of the ezetimibe intermediate YZ1 is chemical synthesis, process impurities are easily introduced, and these impurities are difficult to separate due to their similar polarity to the main component. Therefore, the related substances of the intermediate need to be detected and controlled to ensure the product quality of ezetimibe.

[0006] In summary, it is an urgent problem for those skilled in the art to establish a method for accurately determining the content of related substances in the ezetimibe intermediate YZ1. SUMMARY

[0007] The purpose of the present application is to provide a detection method and application of ezetimibe intermediate related substances, which can separate each impurity in the ezetimibe intermediate and accurately calculate the content of impurities in the ezetimibe intermediate. The method has the beneficial effects of accurate qualitative analysis, convenience, reliability and good repeatability.

[0008] To achieve the above purpose, the present application provides a detection method of ezetimibe intermediate related substances, which specifically comprises the following steps:

[0009] (1) dissolving the test sample YZ1 in an acetonitrile-water mixed solution to obtain a test sample solution;

[0010] (2) dissolving the control sample YZ1 and the impurity control sample YZ1-1 in an acetonitrile-water mixed solution to obtain a system suitability solution;

[0011] (3) taking octadecylsilane bonded silica gel column as a chromatographic column, taking acetonitrile-water mixed solution as a mobile phase, using high performance liquid chromatography, injecting blank solvent, system suitability solution and test sample solution into a liquid chromatograph respectively, recording chromatograms, and calculating the content of impurity YZ1-1 in the test sample solution according to the peak area of the chromatogram.

[0012] In a preferred embodiment, the YZ1 has a structural formula of formula 1:

[0013]

[0014] In a preferred embodiment, the YZ1-1 has a structural formula of formula 2:

[0015]

[0016] In a preferred embodiment, the concentration of YZ1 in the test sample solution is 0.8-1.2 mg / mL.

[0017] In a preferred embodiment, the column length of the chromatographic column is 250 mm, the inner diameter is 4.6 mm, and the packing particle size is 5 μm.

[0018] In a preferred embodiment, the volume ratio of acetonitrile to water in the blank solvent, the mobile phase acetonitrile-water mixed solution and the solvents of step (1) and step (2) of the method is (45-55):(55-45).

[0019] In a preferred embodiment, the flow rate of the mobile phase during the detection process of the high performance liquid chromatography is 0.8-1.2 mL / min, and the column temperature of the chromatographic column is 25-35 ℃.

[0020] In a preferred embodiment, the ultraviolet detection wavelength during the detection process of the high performance liquid chromatography is 255-265 nm.

[0021] Another object of the present application is to provide an application of any one of the above methods in detecting impurities in ezetimibe intermediate YZ1.

[0022] Compared with the prior art, the technical scheme of the present application has the following advantages:

[0023] The present application provides a simple and feasible way of separating the main component from the related substances by using HPLC method and accurately calculating the content of YZ1 in the test sample solution, so that the content of impurities in ezetimibe intermediate YZ1 can be quickly, accurately and simply detected. BRIEF DESCRIPTION OF DRAWINGS

[0024] These and / or other aspects and advantages of the present application will become more apparent from the following detailed description, taken in conjunction with the accompanying drawings, illustrating by way of example the principles of the application.

[0025] Figure 1 Chromatogram of the system suitability solution for the system of Example 1 of the present application;

[0026] Figure 2 Chromatogram of the YZ1-1 positioning solution in Example 2 of the present application. DETAILED DESCRIPTION

[0027] In order to make the skilled in the art better understand the present application, the present application is further described in detail below in conjunction with the drawings and specific embodiments, but it should be understood that the protection scope of the present application is not limited by the specific embodiments.

[0028] The embodiment of the present application provides a detection method for impurities of ezetimibe intermediate YZ1, solves the technical problem that there is no quality control standard for ezetimibe intermediate YZ1 in the prior art, and impurities of ezetimibe intermediate cannot be separated and accurately calculated.

[0029] The technical solution in the present application is to solve the above problems, and the general idea is as follows:

[0030] The purpose of the present application is to provide a detection method for impurities of ezetimibe intermediate YZ1, which specifically comprises the following steps:

[0031] (1) Dissolving the test product YZ1 in an acetonitrile-water mixed solution as a solvent to obtain a test product solution;

[0032] (2) Dissolving the control product YZ1 and the impurity control product YZ1-1 in an acetonitrile-water mixed solution as a solvent to obtain a system suitability solution;

[0033] (3) Using a high-performance liquid chromatography method, injecting the blank solvent, the system suitability solution and the test product solution into a liquid chromatograph respectively, recording chromatograms, and calculating the content of the impurity YZ1-1 in the test product according to the peak area of the chromatogram, with an octadecylsilane bonded silica gel column as a chromatographic column and an acetonitrile-water mixed solution as a mobile phase.

[0034] In a preferred embodiment, the YZ1 has a structural formula of formula 1:

[0035]

[0036] In a preferred embodiment, the YZ1-1 has a structural formula of formula 2:

[0037]

[0038]

[0039] In a preferred embodiment, the concentration of YZ1 in the test sample solution is 0.8-1.2 mg / mL.

[0040] Preferably, the concentration of YZ1 in the test sample solution is 1.0 mg / mL.

[0041] In a preferred embodiment, the column length of the chromatographic column is 250 mm, the inner diameter is 4.6 mm, and the packing particle size is 5 μm.

[0042] Preferably, the chromatographic column is Agilent Eclipse XDB-C18 with a specification of 4.6 x 250 mm and 5 μm.

[0043] In a preferred embodiment, the mobile phase in the HPLC detection process is acetonitrile-water (45-55):(55-45).

[0044] Preferably, the mobile phase in the HPLC detection process is acetonitrile-water 50:50.

[0045] In a preferred embodiment, the flow rate of the mobile phase in the HPLC detection process is 0.9-1.1 mL / min, and preferably, the flow rate of the mobile phase is 1.0 mL / min.

[0046] In a preferred embodiment, the column temperature of the chromatographic column in the HPLC detection process is 25-35 °C, and preferably, the column temperature is 30 °C.

[0047] In a preferred embodiment, the UV detection wavelength in the HPLC detection process is 255-265 nm, and preferably, the UV detection wavelength is 260 nm.

[0048] After the test sample is subjected to HPLC detection according to the above conditions, an HPLC chromatogram is obtained, and the content of each substance is calculated according to the chromatogram. In the specific embodiments of the present application, preferably, a standard solution of each substance is first prepared, and then subjected to HPLC detection according to the above detection conditions. The separated substances are qualitatively determined according to the retention time of the standard in the HPLC chromatogram and the retention time of the test sample in the HPLC chromatogram. The test sample is quantitatively determined according to the peak area of the standard in the HPLC chromatogram, the concentration of the standard, and the peak area of the test sample in the HPLC chromatogram. The present application does not have special requirements for the specific calculation method, and the calculation can be performed according to the methods well known to those skilled in the art. Preferably, the calculation method is the area normalization method.

[0049] Another object of the present application is to provide an application of any of the above methods in detecting ezetimibe intermediate YZ1.

[0050] The technical solutions of the present application are described in detail below through specific examples:

[0051] If not specifically indicated, the technical means used in the present application are conventional means familiar to those skilled in the art, and various raw materials, reagents, instruments and equipment used in the present application can be purchased from the market or prepared by existing methods. The reagents used in the present application are analytical pure unless otherwise specified. Specifically, the instruments and reagents used in the examples of the present application are as follows:

[0052] Shimadzu LC-2030C HPLC system, electronic analytical balance (Shimadzu, one ten-thousandth);

[0053] Ezetimibe intermediate YZ1 reference substance (purchased, purity ≥ 95%), ezetimibe intermediate test substance YZ1 (laboratory self-made), impurity reference substance YZ1-1 (purchased, purity ≥ 95%), acetonitrile is chromatographically pure.

[0054] Example 1

[0055] Step 1, preparation of mobile phase:

[0056] Mobile phase: acetonitrile: water = 50:50 (volume ratio)

[0057] Step 2, solution preparation:

[0058] Blank solvent: acetonitrile and water were each measured 500 mL, mixed, and filtered.

[0059] Test sample solution: 20 mg of ezetimibe intermediate YZ1 test sample was accurately weighed and placed in a 20 mL volumetric flask. The solvent was dissolved and diluted to constant volume, shaken well, and used as the test sample solution.

[0060] System suitability solution: appropriate amounts of YZ1 and YZ1-1 reference substances were accurately weighed and placed in a volumetric flask. Solvent was added to prepare a YZ1-1 solution with a concentration of 20 μg / mL and a YZ1 reference substance with a concentration of 1 mg / mL.

[0061] Step 3, using high performance liquid chromatography, the blank solvent, system suitability solution and test sample solution were injected into the liquid chromatograph, and the chromatogram was recorded. The chromatogram of the system suitability solution is shown in Figure 1 According to the peak area of the chromatogram, the content of each impurity was calculated.

[0062] In this example, the chromatographic conditions were screened and determined by changing the chromatographic conditions, such as the chromatographic column, the proportion of the mobile phase, etc.

[0063] Example 1-a

[0064] Chromatographic conditions:

[0065] Column: Agilent SB-C8 column (4.6 x 250 mm, particle size 5 μm)

[0066] Mobile phase: Acetonitrile: water = 50:50 (volume ratio)

[0067] Solvent: Acetonitrile: water = 50:50 (volume ratio)

[0068] Flow rate: 1 mL / min; detection wavelength: 260 nm; column temperature: 30 °C; injection volume: 20 μL;

[0069] The chromatogram of the system suitability solution of Example 1-a shows a tailing phenomenon for the impurity peak, and the tailing factor is greater than 1, indicating that the chromatographic column is not suitable.

[0070] Example 1-b

[0071] Chromatographic conditions

[0072] Column: Agilent Eclipse XDB-C18 (4.6 x 250 mm, particle size 5 μm)

[0073] Mobile phase: Acetonitrile: water = 40:60 (volume ratio)

[0074] Solvent: Acetonitrile: water = 50:50 (volume ratio)

[0075] Flow rate: 1 mL / min; detection wavelength: 260 nm; column temperature: 30 °C; injection volume: 20 μL;

[0076] The chromatogram of the system suitability solution of Example 1-b shows only one peak, indicating that the proportion of the mobile phase is not suitable.

[0077] Example 1-c

[0078] Chromatographic conditions

[0079] Column: Agilent Eclipse XDB-C18 (4.6 x 250 mm, particle size 5 μm)

[0080] Mobile phase: Acetonitrile: water = 50:50 (volume ratio)

[0081] Solvent: Acetonitrile: water = 50:50 (volume ratio)

[0082] Flow rate: 1 mL / min; detection wavelength: 260 nm; column temperature: 30 °C; injection volume: 20 μL;

[0083] The chromatogram of the system suitability solution of Example 1-c is shown in Figure 1and Table 1. It can be seen from Figure 1 and Table 1. It can be seen from

[0084] Table 1. It can be seen from

[0085]

[0086] Example 2 Method Specificity

[0087] The specificity test was performed according to the method of Example 1-c.

[0088] Step 1, Preparation of mobile phase:

[0089] Mobile phase: acetonitrile-water = 50:50 (volume ratio)

[0090] Solvent: acetonitrile-water = 50:50 (volume ratio)

[0091] Step 2, Preparation of solution:

[0092] Blank solvent: acetonitrile and water were each measured 500 mL, mixed and filtered.

[0093] Test solution: 20 mg of ezetimibe intermediate YZ1 test sample was accurately weighed into a 20 mL volumetric flask, dissolved with solvent to constant volume, shaken well, and used as the test solution.

[0094] System suitability solution: an appropriate amount of YZ1 and YZ1-1 control sample was accurately weighed into a volumetric flask, solvent was added to prepare a YZ1-1 control sample with a concentration of 20 μg / mL and a YZ1 control sample with a concentration of 1 mg / mL.

[0095] Component positioning solution: an appropriate amount of YZ1-1 control sample was accurately weighed into a volumetric flask, dissolved with solvent to constant volume to prepare a YZ1-1 control sample with a concentration of 20 μg / mL as the positioning solution.

[0096] Step 3, using high performance liquid chromatography, each component positioning solution and system suitability solution was injected, and the chromatogram was recorded. The chromatogram of the component positioning solution is shown in Figure 2 Table 1. The specific chromatographic conditions are as follows:

[0097] Chromatographic column: Agilent Eclipse XDB-C18 (4.6 x 250 mm, filler particle size 5 μm)

[0098] Mobile phase: acetonitrile: water = 50:50 (volume ratio)

[0099] Flow rate: 1 mL / min; detection wavelength: 260 nm; column temperature: 30°C; injection volume: 20 μL.

[0100] Example 3 Precision experiment

[0101] According to the method of Example 2, the system suitability solution was continuously injected for 6 times, the chromatograms were recorded, and the results were evaluated, and the results are shown in Table 2.

[0102] Table 2 Results of injection precision experiment

[0103]

[0104] The results of the injection precision test showed that the RSD% of the peak area of the components in the injection precision solution was ≤2%, indicating that this method was suitable for detecting the impurity content in YZ1, and the instrument injection precision was good.

[0105] Example 4 Durability experiment

[0106] According to the method of Example 2, by fine-tuning the relevant parameters, the flow rate, mobile phase ratio, column temperature and other conditions of the chromatographic conditions were adjusted respectively, and the determination results were not affected. The durability results are shown in Tables 3 to 7.

[0107] Table 3 Separation degree at different flow rates

[0108]

[0109] The test results of the durability of different flow rates showed that the change of the test method between 0.9 mL / min and 1.1 mL / min had little effect on the determination results, indicating that the durability of the method was good.

[0110] Table 4 Separation degree at different mobile phase ratios

[0111]

[0112] The test results of the durability of different mobile phase ratios showed that the change of the mobile phase ratio of the test method between 9:11 and 11:9 (45:55 and 55:45) had little effect on the determination results, indicating that the durability of the method was good.

[0113] Table 5 Separation degree at different column temperatures

[0114]

[0115] The test results of the durability of different column temperatures showed that the change of the column temperature of the test method between 25°C and 35°C had little effect on the determination results, indicating that the durability of the method was good.

[0116] Table 6 Separation degree at different wavelengths

[0117]

[0118] The test results of different wavelengths show that the wavelength of the test method varies between 255 nm and 265 nm, which has little effect on the determination results, indicating that the method has good durability.

[0119] Separation of different chromatographic columns

[0120]

[0121] The test results of different columns show that the change of different columns in the test method has little effect on the determination results, indicating that the method has good durability.

[0122] Limit of detection and limit of quantitation experiment of example 5

[0123] The limit of detection and limit of quantitation experiment was carried out according to the method of example 2.

[0124] Quantitation limit solution configuration: gradually dilute the component positioning solution to the signal-to-noise ratio ≥ 10;

[0125] Detection line solution configuration: gradually dilute the component positioning solution to the signal-to-noise ratio ≥ 3;

[0126] The test results of the limit of detection and limit of quantitation experiment show that:

[0127] The limit of detection of YZ1-1 is 0.07 μg / mL, and the limit of quantitation is 0.3 μg / mL. The limit of detection of YZ1-1 is 0.07 μg / mL, and the limit of quantitation is 0.3 μg / mL. Divide the limit of detection and the limit of quantitation by the concentration of the test solution 1 mg / mL, and the limit of detection of YZ1-1 in the test sample is 0.07 μg / mg, and the limit of quantitation is 0.3 μg / mg. The component quantitation limit is much lower than its standard limit, indicating that the method has strong detection ability for each component.

[0128] The component quantitation limit is much lower than its standard limit, indicating that the method has strong detection ability for each component.

[0129] Linear range experiment of example 6

[0130] The linear relationship experiment was carried out according to the method of example 1-c. Reference stock solution: take YZ1-1 reference substance, accurately weigh, put into a capacity bottle, add solvent to make a solution containing about 35 μg / mL of YZ1-1;

[0131] Linear 1: the reference stock solution as linear 1 solution;

[0132] Linear 2: accurately take 8.0 mL of reference stock solution, put it into a 10 mL capacity bottle, dilute to the mark line with solvent, shake well;

[0133] Linearity 3: precisely pipet 6.0 mL of the control stock solution into a 10 mL volumetric flask, dilute to the mark with solvent, and shake to mix;

[0134] Linearity 4: precisely pipet 4.0 mL of the control stock solution into a 10 mL volumetric flask, dilute to the mark with solvent, and shake to mix;

[0135] Linearity 5: precisely pipet 2.0 mL of the control stock solution into a 10 mL volumetric flask, dilute to the mark with solvent, and shake to mix;

[0136] Precisely pipet 20 μL of each of the above solutions into the liquid chromatograph, record the chromatogram, and plot the standard curve with the peak area as the ordinate and the concentration of the control (μg / mL) as the abscissa. The results are shown in Table 8.

[0137] Table 8

[0138]

[0139] The test results of the linearity experiment show that YZ1-1 has good linearity in a certain range above the quantitation limit, indicating that the method has stable detection ability for each component and can be used for quantitative analysis of each component.

[0140] Example 7

[0141] The experiment was performed according to the method of Example 1-c.

[0142] Test sample solution: take 20 mg of ezetimibe intermediate YZ1 test sample, precisely weigh, place in a 20 mL volumetric flask, dissolve with solvent, dilute to volume, shake to mix, and use as the test sample solution. Continuously inject 6 times, record the chromatogram, and bring the peak area into the standard curve equation to obtain the concentration of YZ1-1. The results are shown in Table 9.

[0143] Table 9

[0144]

[0145]

[0146] The results show that the RSD of the content of YZ1-1 is 0.2% when the test sample solution is continuously injected, indicating that the method can be used for actual experimental application. The content of YZ1-1 in the test sample is obtained by dividing the concentration of YZ1-1 in Table 9 by the concentration of the test sample.

[0147] The foregoing description of specific exemplary embodiments of the invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A method for detecting related substances of an ezetimibe intermediate, characterized by, Specifically comprising the following steps: (1) dissolving the test sample YZ1 in acetonitrile-water mixed solution to obtain a test sample solution; (2) dissolving the reference substance YZ1 and the impurity reference substance YZ1-1 in acetonitrile-water mixed solution to obtain a system suitability solution; (3) using a high performance liquid chromatography method, injecting the blank solvent, the system suitability solution and the test sample solution into a liquid chromatograph respectively, recording the chromatogram, and calculating the content of the impurity YZ1-1 in the test sample according to the peak area of the chromatogram.

2. The method for detecting impurities of an ezetimibe intermediate according to claim 1, wherein, The YZ1 has a structural formula of formula 1: Formula 1.

3. The method for detecting related substances in ezetimibe intermediates as described in claim 1, characterized in that, The YZ1-1 has a structural formula of formula 2: Formula 2.

4. The method for detecting related substances in ezetimibe intermediates as described in claim 1, characterized in that, In step (1), the concentration of YZ1 in the test sample solution is 0.8 mg / mL-1.2 mg / mL.

5. The method for detecting related substances in ezetimibe intermediates as described in claim 1, characterized in that, In step (2), the concentration of YZ1 in the system suitability solution is 0.8 mg / mL-1.2 mg / mL, and the concentration of YZ1-1 is 15-25 μg / mL.

6. The method of detecting impurities of ezetimibe intermediate YZ1 according to claim 1, wherein, The column length of the chromatographic column is 250 mm, the inner diameter is 4.6 mm, and the particle size of the filler is 5 μm.

7. The method for detecting related substances in ezetimibe intermediates as described in claim 1, characterized in that, The volume ratio of acetonitrile to water in the solvent of step (1), step (2), the blank solvent and the mobile phase acetonitrile-water mixed solution of step (3) is (45-55):(55-45).

8. The method for detecting related substances in ezetimibe intermediates as described in claim 1, characterized in that, In the process of high performance liquid chromatography detection, the flow rate of the mobile phase is 0.8-1.2 mL / min, and the column temperature of the chromatographic column is 25-35℃.

9. The method for detecting related substances in ezetimibe intermediates as described in claim 1, characterized in that, In the process of high performance liquid chromatography detection, the ultraviolet detection wavelength is 255-265 nm.

10. Use of the method of detection according to any one of claims 1 to 9 for the detection of related substances of an ezetimibe intermediate, characterized in that, The YZ1 has a structural formula of formula 1: Formula 1, The related substance has a structural formula of formula 2: Formula 2.

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