Method for detecting enantiomer impurities in ixazomib citrate raw material medicine

Abstract

The invention provides a method for determining S-configuration enantiomer impurities in citric acid ixazomib, which adopts tetrahydrofuran as a dissolving solvent of a citric acid ixazomib sample, and adopts a polysaccharide bonding type normal-phase chiral column with cellulose-tri (3, 5-dichlorophenyl carbamate) covalently bonded on the surface of silica gel for separation and detection. The method has the advantages that the sample is not required to be pretreated, the operation is simple and convenient, the ixazomib citrate and the S-configuration enantiomer of the ixazomib citrate can be effectively separated and detected, the peak shape is good, the separation degree is qualified, the detection sensitivity is high, the detection limit concentration is about 0.285 mu g / ml, the quantitation limit concentration is about 0.571 mu g / ml, and the method is good in specificity, accurate and reliable. The invention provides a simpler and more effective method for the quality control of the ixazomib citrate raw material medicine, and provides guarantee for the medication safety of the ixazomib citrate medicine.

Description

Technical Field

[0001] The invention belongs to the field of pharmaceutical analytical chemistry, and particularly relates to a method for detecting enantiomeric impurities in an ixazomib citrate raw material drug. Background Art

[0002] Multiple myeloma is a malignant tumor with abnormal proliferation of plasma cells. Ixazomib is the first oral second-generation proteasome inhibitor developed by Takeda Pharmaceuticals of Japan. Compared with the first-generation proteasome inhibitor bortezomib, ixazomib has a shorter dissociation half-life from the 20S proteasome and a dissociation rate from the proteasome that is about 6 times faster than bortezomib. The longer-lasting binding will cause the drug to remain in the circulation, affecting its anti-tumor activity. In November 2015, ixazomib was approved for marketing by the US Food and Drug Administration (FDA) as a third-generation proteasome inhibitor for the treatment of relapsed or refractory multiple myeloma. It was approved for marketing by the European Union in November 2016 and in Japan in March 2017. In April 2018, ixazomib was approved for marketing by the China Food and Drug Administration (CFDA). The "Guidelines for the Diagnosis and Treatment of Multiple Myeloma in China" has recommended it as a second-line treatment for myeloma patients.

[0003] The chemical name of ixazomib citrate is 2-[(1R)-1-[[2-[(2,5-dichlorobenzoyl)amino]acetyl]amino]-3-methylbutyl]-5-oxo-1,3,2-dioxaborolane-4,4-diacetic acid, and the structural formula is as follows:

[0004]

[0005] Documents CN111065641A and CN102066386A disclose the synthetic route of ixazomib citrate:

[0006]

[0007] Ixazomib citrate contains a chiral center in its structure, which is of R configuration. It is introduced by the starting material (TPLM) containing a chiral structure during the amide condensation reaction. If the starting material (TPLM) contains an impurity of S configuration, it may lead to the introduction of an optical enantiomer of Ixazomib citrate of S configuration into the final product. The chemical name of the S-configuration enantiomer of Ixazomib citrate is 2,2'-[2-[(1S)-1-([[(2,5-dichlorobenzoyl)amino]acetyl]amino)-3-methylbutyl]-5-oxo-1,3,2-dioxaborolane-4,4-diyl]-diacetic acid, and the structural formula is as follows:

[0008]

[0009] In the process of API synthesis process development, isomer control of starting materials, intermediates and finished products is one of the important contents of quality research. According to the quality control requirements of ixazomib citrate API by the European Medicines Agency (EMA), the control limit of isomer impurities in the active ingredient shall not exceed 0.5%.

[0010] Chinese patent CN 115166073A records a method for detecting isomers of ixazomib or its salts. The patent specification records that when the method uses a polysaccharide-coated normal phase chiral column to directly detect enantiomers in ixazomib citrate, the chromatographic peak shape of the obtained ixazomib citrate is not good. Therefore, when the method is used to determine the enantiomers in ixazomib citrate, ixazomib citrate needs to be first hydrolyzed into ixazomib, extracted and concentrated with ethyl acetate, and then dissolved in a mobile phase to prepare a test sample, and the enantiomer content in ixazomib is determined. When the method is used to determine the enantiomers in ixazomib citrate, the sample preparation process is complicated, and the operation process is prone to loss, which causes errors in the detection results.

[0011] Therefore, finding an effective analytical detection method that can directly determine the enantiomers of ixazomib citrate and obtain more accurate and reliable test results is of great significance for the quality analysis and quality control of the drug and for improving the safety and effectiveness of the drug. Summary of the invention

[0012] The invention provides a method for detecting an S-configuration enantiomer impurity in ixazomib citrate. The method is simple to operate, has good separation between an ixazomib citrate main peak and an enantiomer peak, can accurately and effectively determine the content of the ixazomib citrate enantiomer, and is suitable for quality control of the ixazomib citrate enantiomer.

[0013] The invention provides a method for detecting S-configuration enantiomeric impurities in ixazomib citrate, which is characterized in that the method adopts normal high performance liquid chromatography, and the chromatographic conditions include using a polysaccharide bonded normal phase chiral column with cellulose-tris(3,5-dichlorophenylcarbamate) covalently bonded to the surface of silica gel, and using n-hexane-tetrahydrofuran-trifluoroacetic acid as the mobile phase.

[0014] Because ixazomib citrate will hydrolyze to form ixazomib when it comes into contact with water, which will lead to deviations in the test results, the mobile phase cannot contain water, and normal phase liquid chromatography must be used for analysis and detection. The solvents suitable for the chromatographic column of normal phase liquid chromatography are usually alkanes and alcohols, and ixazomib citrate can only be dissolved in methanol among these two types of solvents. The commonly used ethanol, isopropanol, and n-hexane cannot dissolve completely. Therefore, methanol is used as the sample dissolving solvent, and different types of polysaccharide-coated normal phase chiral columns and polysaccharide-bonded normal phase chiral columns are used for investigation. The results show that no good separation effect can be obtained. The results are shown in the table below:

[0015]

[0016]

[0017] Further research was conducted using tetrahydrofuran, a solvent that has good solubility for ixazomib citrate but is not commonly used in chromatography. After investigating multiple different types of chromatographic columns, it was found that only a polysaccharide-bonded normal phase chiral column with cellulose-tris(3,5-dichlorophenylcarbamate) covalently bonded to the silica gel surface can achieve effective separation of ixazomib citrate and its S-configuration enantiomer with good peak shape. Therefore, the sample dissolving solvent of the detection method of the present invention is tetrahydrofuran, and the preferred type of chromatographic column used is CHIRALPAK IC-3 or a chromatographic column with equivalent performance.

[0018] The volume ratio of the mobile phase n-hexane-tetrahydrofuran-trifluoroacetic acid is 4-8:2-6:0.01, preferably 6:4:0.01.

[0019] The chromatographic conditions of the present invention also include a flow rate of 0.9 to 1.1 ml / min, preferably 1.0 ml / min; a UV detector with a detection wavelength of 220 to 230 nm, preferably 225 nm; and a column temperature of 30 to 40° C., preferably 35° C.

[0020] The chromatographic conditions also include that the concentration of the prepared ixazomib citrate sample is 0.5-1.0 mg / ml, preferably 0.5 mg / mL.

[0021] The present invention provides a method for detecting the S-configuration enantiomer in ixazomib citrate, which specifically comprises the following steps:

[0022] 1) Preparation of test solution: Take ixazomib citrate sample, add tetrahydrofuran and shake to dissolve, dilute with mobile phase and shake well to prepare a solution with a sample concentration of 0.5 mg / mL as the test solution;

[0023] 2) Sample injection detection: Inject the test solution obtained in step 1) into a liquid chromatograph for detection and analysis, and record the chromatogram.

[0024] The present invention provides a method for determining the S-configuration enantiomer in ixazomib citrate. The method uses tetrahydrofuran as a dissolving solvent for the ixazomib citrate sample, and uses a polysaccharide bonded normal phase chiral column with cellulose-tris(3,5-dichlorophenylcarbamate) covalently bonded to the silica gel surface for separation and detection. No sample pretreatment is required, the operation is simple, and the ixazomib citrate and its S-configuration enantiomer can be effectively separated and detected. The peak shape is good, the separation degree is qualified, the detection sensitivity is high, the detection limit concentration is about 0.285 μg / ml, the quantitative limit concentration is about 0.571 μg / ml, the method has good specificity, and is accurate and reliable. The present invention provides a simpler and more effective method for the quality control of the ixazomib citrate raw material drug, and provides a guarantee for the drug safety of the ixazomib citrate drug.

[0025] The present invention will be further described below in conjunction with specific implementation examples and accompanying drawings.

[0026] Instruction Manual

[0027] Attached Figure 1 Spectrum of blank solution for specificity test

[0028] Attached Figure 2 Spectrum of sample solution for specificity test

[0029] Attached Figure 3 Specificity test Ixazomib citrate enantiomer solution spectrum

[0030] Attached Figure 4 Specificity test system suitability solution map

[0031] Attached Figure 5 Example 2 Ixazomib citrate sample determination spectrum DETAILED DESCRIPTION

[0032] Example 1 Method Verification Test

[0033] Instrument: Waters e2695 liquid chromatograph

[0034] Reagents: n-hexane (chromatographic grade), tetrahydrofuran (chromatographic grade), trifluoroacetic acid (analytical grade)

[0035] Blank solution: Use the mobile phase as the blank solution.

[0036] Test solution: Accurately weigh 50 mg of ixazomib citrate, place in a 100 ml volumetric flask, add 5 ml of tetrahydrofuran and shake to dissolve, dilute to the scale with mobile phase, and shake well.

[0037] Control solution: Accurately measure 1 ml of the test solution, place it in a 100 ml volumetric flask, dilute to the scale with mobile phase, and shake well.

[0038] Ixazomib citrate enantiomer stock solution: Take 25 mg of ixazomib citrate enantiomer, place it in a 100 ml volumetric flask, add 5 ml of tetrahydrofuran and shake to dissolve, dilute to the scale with mobile phase, and shake well.

[0039] System suitability solution: Take appropriate amounts of ixazomib citrate and ixazomib citrate enantiomers, add tetrahydrofuran to dissolve them, and then dilute with mobile phase to prepare a mixed solution containing approximately 0.5 mg and 0.0025 mg per ml, respectively, as the system suitability solution.

[0040] Chromatographic conditions: Use a polysaccharide bonded normal phase chiral column (CHIRALPAK IC-3, 4.6×250mm, 3μm or a chromatographic column with equivalent performance) with n-hexane-tetrahydrofuran-trifluoroacetic acid (6:4:0.01) as the mobile phase; flow rate is 1.0 ml per minute; detection wavelength is 225nm; column temperature is 35℃; sample chamber temperature is 10℃; injection volume is 20μl.

[0041] (1) Specificity test

[0042] Determination method: Using the above chromatographic conditions, inject the blank solution, test solution, ixazomib citrate enantiomer stock solution, and system suitability solution into the chromatograph respectively and record the chromatogram. See attached for the spectrum. Figures 1 to 4 .

[0043] Specificity test results

[0044]

[0045] Conclusion: The blank solution should have no interference with the determination results, and the separation degree of ixazomib citrate and its enantiomers should be greater than 1.5.

[0046] (2) Repeatability test

[0047] Determination method: Prepare 6 test sample solutions in parallel according to the above method, use the above chromatographic conditions, inject the 6 test sample solutions into the chromatograph, and record the chromatogram.

[0048] Repeatability test results

[0049]

[0050] Conclusion: None of the enantiomers of ixazomib citrate were detected in the 6 test solutions, indicating that this method has good reproducibility.

[0051] (3) Linear test

[0052] Ixazomib citrate stock solution: Take 25 mg of ixazomib citrate reference substance, place it in a 100 ml volumetric flask, add 5 ml of tetrahydrofuran and shake to dissolve, dilute to the scale with mobile phase, and shake well.

[0053] Linear stock solution: Accurately measure 5 ml of ixazomib citrate stock solution and 5 ml of ixazomib citrate enantiomer stock solution under the "Specificity" item, place them in a 20 ml volumetric flask, dilute to the scale with mobile phase, and shake well.

[0054] Preparation of linear 1 solution: Accurately measure 3 ml of linear stock solution, place in a 25 ml volumetric flask, dilute to scale with mobile phase, and shake well. (equivalent to 1.5% concentration of the test sample)

[0055] Preparation of linear 2 solution: Accurately measure 2 ml of linear stock solution, place in a 25 ml volumetric flask, dilute to scale with mobile phase, and shake well. (equivalent to 1.0% concentration of the test sample)

[0056] Preparation of linear 3 solution: Accurately measure 3 ml of linear stock solution, place in a 50 ml volumetric flask, dilute to scale with mobile phase, and shake well. (equivalent to 0.75% concentration of the test sample)

[0057] Preparation of linear 4 solution: Accurately measure 1 ml of linear stock solution, place in a 25 ml volumetric flask, dilute to scale with mobile phase, and shake well. (equivalent to 0.5% concentration of the test sample)

[0058] Preparation of linear 5 solution: Accurately measure 1 ml of linear stock solution, place in a 50 ml volumetric flask, dilute to scale with mobile phase, and shake well. (equivalent to 0.25% test sample concentration)

[0059] Preparation of linear 6 solution: Accurately measure 1 ml of linear stock solution, place in a 100 ml volumetric flask, dilute to scale with mobile phase, and shake well. (equivalent to a test sample concentration of 0.125%)

[0060] Accurately measure each of the above linear solutions, inject them into the liquid chromatograph, and record the chromatogram. Perform linear regression analysis with concentration as the horizontal axis (X) and peak area as the vertical axis (Y). The correlation coefficient r should be no less than 0.99.

[0061] Results of linearity test of ixazomib citrate

[0062]

[0063] Results of linearity test of enantiomers of ixazomib citrate

[0064]

[0065]

[0066] Conclusion: The main peak area of ​​ixazomib citrate and its enantiomers has a good linear relationship with the concentration.

[0067] (4) Accuracy test

[0068] Test solution: Accurately weigh 50 mg of ixazomib citrate, place in a 100 ml volumetric flask, add 5 ml of tetrahydrofuran and shake to dissolve, dilute to the scale with mobile phase, and shake well.

[0069] Recovery rate stock solution: Accurately measure 25 ml of the ixazomib citrate enantiomer stock solution under the "Specificity" item, place it in a 100 ml volumetric flask, dilute to the scale with mobile phase, and shake well.

[0070] 50% recovery solution: accurately weigh 50 mg of ixazomib citrate, place in a 100 ml volumetric flask, add 5 ml of tetrahydrofuran and shake to dissolve, accurately add 2 ml of recovery stock solution, dilute to scale with mobile phase, shake well. Prepare 3 copies in parallel.

[0071] 100% recovery solution: accurately weigh 50 mg of ixazomib citrate, place in a 100 ml volumetric flask, add 5 ml of tetrahydrofuran and shake to dissolve, accurately add 4 ml of recovery stock solution, dilute to scale with mobile phase, shake well. Prepare 3 copies in parallel.

[0072] 150% recovery solution: accurately weigh 50 mg of ixazomib citrate, place in a 100 ml volumetric flask, add 5 ml of tetrahydrofuran and shake to dissolve, accurately add 6 ml of recovery stock solution, add mobile phase to dilute to scale, shake well. Prepare 3 copies in parallel.

[0073] Accurately measure the above blank solution, test solution and recovery solutions of each concentration, inject them into the liquid chromatograph respectively, record the chromatogram, and substitute the results into the linear equation to calculate the recovery rate.

[0074]

[0075]

[0076] Conclusion: In the concentration range of 50% to 150%, the recovery of ixazomib citrate enantiomers was between 98.8% and 105.4%, with an average recovery of 103.1% and an RSD of 2.12%, indicating that this method has high accuracy in determining enantiomers.

[0077] (5) Detection limit and quantification limit test

[0078] Refer to the low-concentration linear solution under the "Linearity and Range" item to prepare appropriate concentrations and inject them separately. When the signal-to-noise ratio is about 3 or above and the solution concentration is not higher than the respective limit concentration, it is taken as the detection limit; when the signal-to-noise ratio is about 10 or above and the solution concentration is not higher than the respective limit concentration, it is taken as the quantification limit. Accurately measure the quantification limit solution and inject it continuously for 6 times.

[0079] Six injection results when the concentration of ixazomib citrate enantiomers was 0.571 μg / ml (quantification limit concentration)

[0080] The concentration of ixazomib citrate enantiomers was 0.285 μg / ml (detection limit concentration)

[0081]

[0082] (6) Durability test

[0083] Change the chromatographic parameters according to the parameters specified in the table below, accurately measure the blank solution, system suitability solution, test solution and control solution and inject them into the liquid chromatograph respectively, and record the chromatogram.

[0084]

[0085] Durability test results

[0086]

[0087] Conclusion: Slight changes in column temperature and flow rate have no effect on the separation and content determination of enantiomers, indicating that this method has good durability.

[0088] Example 2 Determination of Ixazomib Citrate API Samples

[0089] Instrument: Waters e2695 liquid chromatograph

[0090] Reagents: n-hexane (chromatographic grade), tetrahydrofuran (chromatographic grade), trifluoroacetic acid (analytical grade)

[0091] Chromatographic conditions: a polysaccharide bonded normal phase chiral column (CHIRALPAK IC-3, 4.6×250 mm, 3 μm, with n-hexane-tetrahydrofuran-trifluoroacetic acid (6:4:0.01) as the mobile phase; a flow rate of 1.0 ml per minute; a detection wavelength of 225 nm; a column temperature of 35°C; a sample chamber temperature of 10°C; and an injection volume of 20 μl.

[0092] Preparation of test solution: Accurately weigh 50 mg of ixazomib citrate bulk drug sample, place in a 100 ml volumetric flask, add 5 ml of tetrahydrofuran and shake to dissolve, dilute to the scale with mobile phase, and shake well.

[0093] Determination method: Using the above chromatographic conditions, inject the test solution into the liquid chromatograph for detection and analysis, and record the chromatogram. Figure 5 .

[0094] Results of enantiomer content testing of three batches of ixazomib citrate

[0095]

Claims

1. A method for detecting the S-configuration enantiomer in ixazomib citrate, characterized in that: This method adopts normal high performance liquid chromatography, and its chromatographic conditions include using a polysaccharide bonded normal phase chiral column with cellulose-tris (3,5-dichlorophenyl carbamate) covalently bonded to the silica gel surface and using n-hexane-tetrahydrofuran-trifluoroacetic acid as the mobile phase.

2. The detection method according to claim 1, characterized in that: The polysaccharide bonded normal phase chiral column model is CHIRALPAK IC-3.

3. The detection method according to claim 1, characterized in that: The volume ratio of the mobile phase n-hexane-tetrahydrofuran-trifluoroacetic acid is 4-8:2-6:0.

01.

4. The detection method according to claim 3, characterized in that: The volume ratio of the mobile phase n-hexane-tetrahydrofuran-trifluoroacetic acid is 6:4:0.

01.

5. The detection method according to claim 1, characterized in that: The chromatographic conditions also include that the sample dissolving solvent is tetrahydrofuran.

6. The detection method according to claim 1, characterized in that: The chromatographic conditions also include a flow rate of 0.9 to 1.1 ml / min, a detector using an ultraviolet detector, a detection wavelength of 220 to 230 nm, and a column temperature of 30 to 40°C.

7. The detection method according to claim 6, characterized in that: The flow rate was 1.0 ml / min, the detection wavelength was 225 nm, and the column temperature was 35°C.

8. The detection method according to claim 1, characterized in that: The chromatographic conditions also include that the concentration of the prepared ixazomib citrate test sample is 0.5-1.0 mg / ml.

9. The detection method according to claim 1, characterized in that The detection method specifically comprises the following steps: 1) Preparation of test solution: Take ixazomib citrate sample, add tetrahydrofuran and shake to dissolve, dilute with mobile phase and shake well to prepare a solution with a sample concentration of 0.5 mg / mL as the test solution; 2) Sample injection detection: Inject the test solution obtained in step 1) into a liquid chromatograph for detection and analysis, and record the chromatogram.

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