Detection method of ixazomib citrate optical isomer

Abstract

The invention discloses a method for detecting isomers of ixazomib citrate, and relates to the technical field of pharmaceutical analysis. According to the method, high performance liquid chromatography is adopted for detection, a chromatographic column is a chromatographic column with a silica gel surface covalently bonded with cellulose-tri (3, 5-dichlorophenyl carbamate) filler, and a mobile phase is a normal hexane-tetrahydrofuran-acetonitrile-trifluoroacetic acid mixed solution. The method disclosed by the invention can be used for well separating the citric acid ixazomib and the isomer thereof, so that the content of the isomer of the citric acid ixazomib can be accurately determined, and the method is stable, reliable and high in sensitivity.

Description

Technical Field

[0001] The present invention relates to the technical field of drug analysis, and in particular to a method for detecting isomers of ixazomib citrate. Background Art

[0002] Ixazomib citrate, English name Ixazomib citrate, chemical name: 2-[(1R)-1-[[2-[(2,5-dichlorobenzoyl)amino]acetyl]amino]-3-methylbutyl]-5-oxo-1,3,2-dioxocyclopentane-4,4-diacetic acid, the structural formula is as follows:

[0003]

[0004] Ixazomib citrate is an orally administered protease inhibitor that was first approved by the US FDA in November 2015, in combination with lenalidomide and dexamethasone, for the treatment of patients with multiple myeloma who have previously received at least one therapy.

[0005] The structure of ixazomib contains a chiral center, which is an R structure, which is introduced by the starting material containing the chiral structure during the amide condensation reaction. Therefore, isomeric impurities of the S configuration of ixazomib may also exist in R-ixazomib. According to the requirements of the European Medicines Agency for ixazomib citrate: the control limit of isomeric impurities in the active ingredient shall not exceed 0.5%.

[0006] Chinese patent CN115166073A discloses a method for determining the optical isomers of ixazomib or its salts, which adopts high performance liquid chromatography, uses a polysaccharide derivative coated normal phase and a supercritical SFC liquid phase as a chiral column, uses amylose tris (halogenated methylphenyl carbamate) as a stationary phase chromatographic column, and uses a mixture of n-hexane, ethanol, and tert-butyl alcohol as a mobile phase to determine the isomers of ixazomib or its salts. The method is stable and reliable, with high sensitivity, and can be used for quantitative analysis of enantiomers in the development process of ixazomib raw materials and quality control of products in the production process. However, the sample pretreatment of this method requires steps such as dissolution, stirring, extraction, reduced pressure concentration, and re-formulation, which is complicated to operate, time-consuming, and has the risk of incomplete extraction conversion, resulting in inaccurate test results, and cannot guarantee the safety, effectiveness, and quality control of drugs.

[0007] At present, there are few studies on the detection of isomers of ixazomib citrate, and there is still a lack of simpler and more accurate determination methods. Providing a simple, accurate and rapid method for detecting isomers of ixazomib citrate is of great significance to the quality control of ixazomib citrate and the safety of drugs. Summary of the invention

[0008] In view of this, the object of the present invention is to provide a simple method for determining the content of isomers in ixazomib citrate in the ixazomib citrate development process. The method of the present invention can well separate ixazomib citrate, thereby accurately determining the content of ixazomib citrate, and the method is stable, reliable and highly sensitive.

[0009] To achieve the above-mentioned purpose of the invention, the technical solution of the present invention is as follows:

[0010] A method for detecting isomers of ixazomib citrate adopts high performance liquid chromatography for detection, the chromatographic column is a chromatographic column with cellulose-tris(3,5-dichlorophenylcarbamate) filler covalently bonded to the surface of silica gel, and the mobile phase is a mixed solution of n-hexane-tetrahydrofuran-acetonitrile-trifluoroacetic acid.

[0011] Preferably, in the mobile phase, the volume ratio of n-hexane, tetrahydrofuran, acetonitrile and trifluoroacetic acid is 75-85:15-25:4-6:0.3, and more preferably 80:20:4:0.3.

[0012] Preferably, the flow rate of the mobile phase is 0.8-1.2 mL / min, more preferably 1.0 mL / min.

[0013] Preferably, the column temperature of the chromatographic column is 25-35°C, more preferably 30°C.

[0014] Preferably, the chromatographic column is a CHIRALPAK IC chromatographic column, and as an example, it is a DAICEL CHIRALPAK IC chromatographic column.

[0015] Preferably, in the detection method, an ultraviolet detector or a diode array detector is used for detection, preferably an ultraviolet detector.

[0016] Preferably, the detection wavelength is 220-230 nm, more preferably 225 nm.

[0017] Preferably, the detection method further comprises the step of preparing a test solution, comprising:

[0018] Take ixazomib citrate, dissolve it in a solvent, and dilute it with n-hexane to obtain a test solution.

[0019] More preferably, the solvent is a tetrahydrofuran citrate solution, and even more preferably, the concentration of citric acid in the tetrahydrofuran citrate solution is 0.5-2 mg / mL, most preferably 1 mg / mL.

[0020] Further preferably, in the test solution, the concentration of ixazomib citrate is 0.5-1.5 mg / mL, most preferably 1.0 mg / mL.

[0021] As a specific example of the present invention, the step of preparing a test solution includes: taking ixazomib citrate, dissolving it in 1 mg / mL tetrahydrofuran citrate solution, and then diluting it with n-hexane to form a solution containing 1.0 mg of ixazomib citrate per 1 mL as the test solution.

[0022] Preferably, the detection method further comprises the step of preparing a control solution, comprising:

[0023] Dilute the test solution with diluent to obtain the control solution.

[0024] Further preferably, the diluent is a mixed solution of tetrahydrofuran citrate solution and n-hexane. Further preferably, the volume ratio of the tetrahydrofuran citrate solution to n-hexane is 45-55:45-55, most preferably 50:50; further preferably, the concentration of citric acid in the tetrahydrofuran citrate solution is 0.5-2 mg / mL, most preferably 1 mg / mL.

[0025] Further preferably, in the control solution, the concentration of ixazomib citrate is 3-7 μg / mL, most preferably 5 μg / mL.

[0026] Preferably, the detection method further comprises the step of preparing a system suitability solution, comprising:

[0027] Take ixazomib citrate and ixazomib citrate isomer reference substances, dissolve them in a solvent, and dilute them with n-hexane to prepare a mixed solution containing ixazomib citrate and ixazomib citrate isomers.

[0028] More preferably, the solvent is a tetrahydrofuran citrate solution, and even more preferably, the concentration of citric acid in the tetrahydrofuran citrate solution is 0.5-2 mg / mL, most preferably 1 mg / mL.

[0029] Further preferably, in the system suitability solution, the concentration of the ixazomib citrate isomer is 3-7 μg / mL, most preferably 5 μg / mL; the concentration of ixazomib citrate is 0.5-1.5 mg / mL, most preferably 1 mg / mL.

[0030] As a specific example of the present invention, it includes:

[0031] Take the ixazomib citrate reference substance, dissolve it in 1 mg / mL citrate tetrahydrofuran solution, add the reference substance stock solution, and dilute it with n-hexane.

[0032] Preferably, the detection method comprises the following steps:

[0033] (1) Prepare test solution, control solution and system suitability solution;

[0034] (2) Inject the system suitability solution into the liquid chromatograph and record the chromatogram;

[0035] (3) Inject the test solution and the control solution into a liquid chromatograph, record the chromatogram, and calculate the isomer content of ixazomib citrate.

[0036] As a specific example of the present invention, step (2) is specifically as follows: accurately measure 25 μl of the system suitability solution, inject it into a liquid chromatograph, record the chromatogram, and the peak order is ixazomib and ixazomib isomers, and the separation degree of the two chromatographic peaks should be no less than 1.5.

[0037] As a specific example of the present invention, step (3) specifically comprises: accurately measuring 25 μl of the test solution and the control solution, injecting them into a liquid chromatograph, recording the chromatogram, and calculating the content of the isomers of ixazomib citrate by the self-control method.

[0038] As a preferred example of the present invention, the detection method comprises the following steps:

[0039] (1) Preparation of test solution: Dissolve the product in 1 mg / mL tetrahydrofuran citrate solution and dilute with n-hexane;

[0040] Preparation of control solution: dilute the test solution with 1 mg / mL citrate tetrahydrofuran-n-hexane (50:50);

[0041] Reference substance stock solution: Take ixazomib citrate isomer reference substance, dissolve it in 1 mg / mL tetrahydrofuran citrate solution, and dilute it with n-hexane;

[0042] Reference solution: dilute the reference stock solution with 1 mg / mL citrate tetrahydrofuran-n-hexane (50:50);

[0043] System suitability solution: Take ixazomib citrate reference substance, dissolve it in 1 mg / mL tetrahydrofuran citrate solution, add reference substance stock solution, and dilute with n-hexane;

[0044] (2) Inject system suitability solution, control solution and test solution, record chromatograms and calculate content. The chromatographic conditions are:

[0045] Column: CHIRALPAK IC

[0046] Column temperature: 30°C

[0047] UV detection wavelength: 225nm

[0048] Mobile phase: n-hexane-tetrahydrofuran-acetonitrile-trifluoroacetic acid in a volume ratio of 80:20:4:0.3

[0049] Flow rate: 1.0mL / min

[0050] Injection volume: 25 μl.

[0051] The beneficial effects of the present invention are:

[0052] (1) The detection method of the present invention is simple to operate, highly practical, highly sensitive, and highly accurate, and can effectively control the optical isomers in ixazomib citrate.

[0053] (2) The HPLC separation method of ixazomib citrate isomers provided by the present invention can effectively separate and determine the isomers of ixazomib citrate. According to the test results, the blank solution does not interfere with the determination of ixazomib citrate isomers. The quantitative limit concentration of ixazomib citrate isomers is equivalent to 0.026% of the test sample concentration, and the detection limit is equivalent to 0.008% of the test sample concentration. When the concentration of ixazomib citrate isomers is in the range of 0.259-10.340 μg / mL, the peak area and the concentration show a good linear relationship, and the linear equation is y =34.9859x+11.4891, the linear correlation coefficient r is 0.9990, the method has high sensitivity, 6 samples were repeatedly prepared, the RSD of the measured content of ixazomib citrate isomers was 1.9%, the repeatability was good, the recovery rates of 9 samples at three concentrations (0.25%, 0.5%, 0.75%) were all between 80% and 120%, the accuracy was good, the separation degree between ixazomib citrate and ixazomib citrate isomers was good, the results were accurate and reliable, the operation was simple, and the isomers in ixazomib citrate raw materials could be effectively determined. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 This is the blank solution chromatogram of Example 1;

[0055] Figure 2 This is the chromatogram of the control solution of Example 1;

[0056] Figure 3 This is the chromatogram of the reference substance solution in Example 1;

[0057] Figure 4 is the chromatogram of the system suitability solution of Example 1;

[0058] Figure 5 This is the chromatogram of the 1% spiked test solution of Comparative Example 1;

[0059] Figure 6 This is the chromatogram of the 1% spiked test solution of Comparative Example 2. DETAILED DESCRIPTION

[0060] The following non-limiting examples can enable those skilled in the art to more fully understand the present invention, but do not limit the present invention in any way. The following content is merely an exemplary description of the scope of the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, which should also fall within the scope of the present application.

[0061] The present invention is further described below by way of specific examples. The various chemical reagents used in the examples of the present invention are obtained through conventional commercial channels unless otherwise specified. Unless otherwise specified, the contents described below are all weight contents. Unless otherwise specified, it is understood that the process is carried out at room temperature.

[0062] A method for detecting isomers of ixazomib citrate, the specific detection method is as follows:

[0063] Preparation of test solution: Take about 10 mg of the product, accurately weigh it, put it in a 10 mL volumetric flask, add 5 mL of solvent to dissolve it, dilute it to the scale with n-hexane, shake well, and you have it.

[0064] Preparation of control solution: Accurately measure 0.5 mL of the test solution, place it in a 100 mL volumetric flask, dilute to the scale with diluent, and shake well.

[0065] Stock solution of reference substance: accurately weigh 10 mg of ixazomib citrate isomer reference substance, place in a 20 mL volumetric flask, add 10 mL of solvent to dissolve, dilute to the mark with n-hexane, shake well, accurately measure 1 mL and place in a 10 mL volumetric flask, dilute to the mark with diluent, shake well, and obtain.

[0066] Reference substance solution: Accurately measure 1 mL of reference substance stock solution and place it in a 20 mL volumetric flask, add diluent to dilute to the mark, and shake well.

[0067] System suitability solution: Take 10 mg of ixazomib citrate reference substance, accurately weigh it, put it in a 10 mL volumetric flask, add 4.5 mL of solvent to dissolve it, accurately measure and add 1 mL of reference substance stock solution, dilute to the scale with n-hexane, shake well, and obtain.

[0068] Diluent: n-hexane-1 mg / mL citrate tetrahydrofuran solution (50:50).

[0069] Solvent: 1 mg / mL citrate tetrahydrofuran solution.

[0070] Use high performance liquid chromatography and ultraviolet detector to detect blank solution, system suitability solution, control solution and test solution, record the chromatogram, the peak order in the system suitability chromatogram is ixazomib and ixazomib isomers, and the separation degree of the two chromatographic peaks should be not less than 1.5. If there is an ixazomib isomer peak in the test solution, calculate the content by the self-control method.

[0071] In the detection method, cellulose-tris(3,5-dichlorophenylcarbamate) covalently bonded to the surface of silica gel is used as a filler chromatographic column (CHIRALPAK IC, 4.6mm×250mm, 5μm), and the detector is an ultraviolet detector or a diode array detector, with an ultraviolet detector being the best. n-hexane-tetrahydrofuran-acetonitrile-trifluoroacetic acid (75-85:15-25:4-6:0.3) is used as the mobile phase, with n-hexane-tetrahydrofuran-acetonitrile-trifluoroacetic acid (80:20:4:0.3) being the best. The flow rate is 0.8-1.2mL / min, with 1.0mL / min being the best. The column temperature is 25-35°C, with 30°C being the best. The detection wavelength is 225nm.

[0072] Example 1

[0073] 1. Instruments and chromatographic conditions

[0074] Instrument: Agilent 1260 High Performance Liquid Chromatograph

[0075] Chromatographic column: CHIRALPAK IC (4.6mm×250mm, 5μm)

[0076] Column temperature: 30°C

[0077] UV detection wavelength: 225nm

[0078] Mobile phase: n-hexane-tetrahydrofuran-acetonitrile-trifluoroacetic acid (80:20:4:0.3)

[0079] Flow rate: 1.0mL / min

[0080] 2. Solution preparation:

[0081] Diluent (blank solution): n-hexane-1 mg / mL citrate tetrahydrofuran solution (50:50).

[0082] Solvent: 1 mg / mL citrate tetrahydrofuran solution.

[0083] Test solution: Take about 10 mg of ixazomib citrate, weigh accurately, place in a 10 mL volumetric flask, add 5 mL of solvent to dissolve, and dilute to the scale with n-hexane.

[0084] Stock solution of reference substance: Take 10 mg of ixazomib citrate isomer reference substance, weigh accurately, place in a 20 mL volumetric flask, add 10 mL of solvent to dissolve and dilute to the scale with n-hexane, then accurately transfer 1 mL to a 10 mL volumetric flask, add diluent to dilute to the scale, shake well, and obtain.

[0085] System suitability solution: Accurately weigh 10 mg of ixazomib citrate reference substance, place in a 10 mL volumetric flask, add 4.5 mL of solvent to dissolve, accurately add 1 mL of reference substance stock solution, dilute to the mark with n-hexane, and shake well.

[0086] Reference substance solution: Accurately measure 1 mL of reference substance stock solution and place it in a 20 mL volumetric flask, add diluent to dilute to the mark, and shake well.

[0087] Preparation of control solution: Accurately measure 0.5 mL of the test solution, place it in a 100 mL volumetric flask, dilute to the scale with diluent, and shake well.

[0088] (1) Specificity test:

[0089] Accurately measure 25 μl of blank solution, reference solution, test solution and system suitability solution and inject them, and record the chromatogram. The measured results are shown in Table 1.

[0090] Table 1. Specificity results

[0091]

[0092] (2) Limit of quantitation and limit of detection test:

[0093] The reference stock solution was diluted, and the concentration when S / N ≥ 10 was used as the quantitative limit solution, and the concentration when S / N ≥ 3 was used as the detection limit solution. The measured results are shown in Table 2.

[0094] Table 2. Results of determination of limit of quantitation and limit of detection

[0095]

[0096] (3) Linear test:

[0097] Linearity 1: Limit of Quantitation Solution

[0098] Linearity 2 to Linearity 5: Accurately measure 1 mL, 2 mL, 3 mL, and 4 mL of the reference stock solution and place them in different 20 mL volumetric flasks. Add solvent to dilute to the scale and shake well.

[0099] Linear regression was performed with the peak area as the ordinate and the linear solution concentration as the abscissa. The measured results are shown in Table 3.

[0100] Table 3. Isomer linearity determination results

[0101]

[0102] (4) Repeatability test:

[0103] Weigh 10 mg of this product accurately, place it in a 10 mL volumetric flask, add 4.5 mL of 1 mg / mL tetrahydrofuran citrate solution to dissolve, accurately add 1 mL of reference substance stock solution, dilute to scale with n-hexane, and shake well. Prepare 6 portions in parallel. The results are shown in Table 4.

[0104] Table 4. Isomer repeatability test results

[0105]

[0106] (5) Recovery test:

[0107] 0.25% spiked test solution: Accurately weigh 10 mg of the product and place it in a 10 mL volumetric flask. Add 4.5 mL of 1 mg / mL citrate tetrahydrofuran solution to dissolve it. Accurately add 0.5 mL of reference stock solution, dilute to the mark with n-hexane, and shake well.

[0108] 0.5% spiked test solution: Accurately weigh 10 mg of the product and place it in a 10 mL volumetric flask. Add 4.5 mL of 1 mg / mL citrate tetrahydrofuran solution to dissolve it. Accurately add 1 mL of the reference stock solution, then dilute to the scale with n-hexane and shake well.

[0109] 0.75% spiked test solution: Accurately weigh 10 mg of the product and place it in a 10 mL volumetric flask. Add 4.5 mL of 1 mg / mL citrate tetrahydrofuran solution to dissolve it. Accurately add 1.5 mL of reference substance stock solution, dilute to the mark with n-hexane, and shake well.

[0110] The recovery results are shown in Table 5, and the 100% spike recovery graph is shown in Figure 4 .

[0111] Table 5. Isomer recovery determination results

[0112]

[0113] Example 2

[0114] The difference from Example 1 is that the volume ratios of the mobile phases n-hexane-tetrahydrofuran-acetonitrile-trifluoroacetic acid are (80:20:4:0.3) and (80:20:6:0.3), and the rest are the same. The results are shown in Table 6.

[0115] Table 6. Isomer determination results at different mobile phase ratios

[0116]

[0117] Example 3

[0118] Experiments with different column temperatures:

[0119] The difference from Example 1 is that column temperatures (25°C, 35°C) are used for injection, and the rest are the same. The test results are shown in Table 7.

[0120] Example 4

[0121] Experiments with different flow rates:

[0122] The difference from Example 1 is that the flow rates (0.8 ml / min, 1.2 ml / min) are used for injection, and the rest are the same. The test results are shown in Table 7.

[0123] Table 7. Isomer determination results at different flow rates and column temperatures

[0124]

[0125] Example 5

[0126] The difference from Example 1 is that the volume ratios of the mobile phase n-hexane-tetrahydrofuran-acetonitrile-trifluoroacetic acid are (75:25:4:0.3) and (85:15:4:0.3), and the rest are the same. The results are shown in Table 8.

[0127] Table 8. Isomer determination results at different mobile phase ratios

[0128]

[0129] Comparative Example 1

[0130] The difference from Example 1 is that the mobile phase is: n-hexane-ethanol-diethylamine-trifluoroacetic acid, the volume ratio is (70:30:0.1:0.05), and the rest are the same. The results are shown in Figure 5 .

[0131] Conclusion: The isomers were wrapped in the main peak and could not be well detected.

[0132] Comparative Example 2

[0133] The difference from Example 1 is that the chromatographic column is DAICEL CHIRALPAK IA (4.6×250 mm, 5 um), and the rest is the same. The results are shown in Figure 6 .

[0134] Conclusion: The main peak of the 1% spiked test solution was severely tailed and could not be well detected.

[0135] The above embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention.

Claims

1. A method for detecting isomers of ixazomib citrate, characterized in that: High performance liquid chromatography was used for detection. The chromatographic column was a chromatographic column with cellulose-tris(3,5-dichlorophenylcarbamate) filler covalently bonded to the surface of silica gel, and the mobile phase was a mixture of n-hexane-tetrahydrofuran-acetonitrile-trifluoroacetic acid.

2. The detection method according to claim 1, characterized in that: In the mobile phase, the volume ratio of n-hexane, tetrahydrofuran, acetonitrile and trifluoroacetic acid is 75-85:15-25:4-6:0.3, preferably 80:20:4:0.

3.

3. The detection method according to claim 1, characterized in that: The flow rate of the mobile phase is 0.8-1.2 mL / min, preferably 1.0 mL / min.

4. The detection method according to claim 1, characterized in that: The column temperature of the chromatographic column is 25-35°C, preferably 30°C.

5. The detection method according to claim 1, characterized in that: The chromatographic column is a CHIRALPAK IC chromatographic column.

6. The detection method according to claim 1, characterized in that: The detection wavelength is 220-230 nm, preferably 225 nm.

7. The detection method according to claim 1, characterized in that: The detection method further includes the step of preparing a test solution, including: taking ixazomib citrate, adding a solvent to dissolve it, and diluting it with n-hexane to obtain a test solution; The method also includes the steps of preparing a control solution, including: diluting the test solution with a diluent to obtain a control solution; The method also includes the step of preparing a system suitability solution, including: taking ixazomib citrate and ixazomib citrate isomer reference substances, dissolving them with a solvent, and diluting them with n-hexane to prepare a mixed solution containing ixazomib citrate and ixazomib citrate isomers.

8. The detection method according to claim 7, characterized in that: The diluent is a mixed solution of tetrahydrofuran citrate solution and n-hexane; preferably, the volume ratio of the tetrahydrofuran citrate solution to n-hexane is 45-55:45-55, most preferably 50:50; preferably, the concentration of citric acid in the tetrahydrofuran citrate solution is 0.5-2 mg / mL, most preferably 1 mg / mL; The solvent is a tetrahydrofuran citrate solution. Preferably, the concentration of citric acid in the tetrahydrofuran citrate solution is 0.5-2 mg / mL, and most preferably is 1 mg / mL.

9. The detection method according to claim 7, characterized in that: In the test solution, the concentration of ixazomib citrate is 0.5-1.5 mg / mL, in the control solution, the concentration of ixazomib citrate is 3-7 μg / mL, in the system suitability solution, the concentration of ixazomib citrate isomers is 3-7 μg / mL, and the concentration of ixazomib citrate is 0.5-1.5 mg / mL.

10. The detection method according to claim 1, characterized in that: The detection method comprises the following steps: (1) Preparation of test solution: Dissolve the product in 1 mg / mL tetrahydrofuran citrate solution and dilute with n-hexane; Preparation of control solution: dilute the test solution with 1 mg / mL tetrahydrofuran citrate solution-n-hexane, 50:50; Reference substance stock solution: Take ixazomib citrate isomer reference substance, dissolve it in 1 mg / mL tetrahydrofuran citrate solution, and dilute it with n-hexane; Reference solution: Add 1 mg / mL tetrahydrofuran citrate solution-n-hexane to the reference stock solution, dilute 50:50; System suitability solution: Take ixazomib citrate reference substance, dissolve it in 1 mg / mL tetrahydrofuran citrate solution, add reference substance stock solution, and dilute with n-hexane; (2) Inject system suitability solution, control solution and test solution, record chromatograms and calculate content. The chromatographic conditions are: Column: CHIRALPAK IC Column temperature: 30°C UV detection wavelength: 225nm Mobile phase: n-hexane-tetrahydrofuran-acetonitrile-trifluoroacetic acid in a volume ratio of 80:20:4:0.3 Flow rate: 1.0 mL / min Injection volume: 25μl.

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