Method for detecting genotoxic impurity acryloyl chloride in ibrutinib
Through the HPLC-UV method combined with the derivatization treatment of aniline compounds, the sensitivity and accuracy of trace acryloyl chloride detection in ibrutinib was solved, efficient and economical detection results were achieved, and the safety of product quality was ensured.
Patent Information
- Application Number
- CN202411793228.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-06
- Publication Date
- 2025-03-11
Smart Images
Figure CN119595790A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of drug analysis, and in particular to a method for detecting genotoxic impurity acryloyl chloride in ibrutinib. Background Art
[0002] Ibrutinib, also known as Ibrutinib, is a Bruton's tyrosine kinase inhibitor that can inhibit the proliferation of tumor cells by regulating apoptosis and cell cycle. It was first developed by Celera Genomics in the United States in 2007, and then transferred to Pharmacyclics in California for development. In 2011, Janssen Pharmaceuticals, a subsidiary of Johnson & Johnson, participated in the co-development. It is commonly used in clinical treatment of chronic lymphocytic leukemia and small cell lymphocytic lymphoma.
[0003] The chemical name of ibrutinib is 1-[(3R)-3-[4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]-1-piperidinyl]-2-propene-1-one, which mainly uses 3-(4-phenoxyphenyl)-1H-[3,4-d]pyrazolopyrimidin-4-aminepiperidine and (S)-1-tert-butyloxycarbonyl-3-hydroxypiperidine as starting materials to generate intermediate 1, intermediate 1 is de-Boc-ed to generate intermediate 2, and intermediate 2 is further reacted with acryloyl chloride to generate ibrutinib. The reaction process is shown below.
[0004]
[0005] Acyl halide compounds have warning structures and are potential genotoxic impurities that may be genotoxic and carcinogenic. Regulatory agencies in various countries attach great importance to the detection of residual acyl halide compounds in drugs. Therefore, it is necessary to establish a highly specific and sensitive method to control this type of impurity.
[0006] Acyl chloride compounds are active and highly reactive. Direct detection by HPLC or GC often results in poor reproducibility and inaccurate results. They are also corrosive to instruments and chromatographic columns. Through previous literature review, it was found that acyl chloride compounds are mostly detected by GC or GC-MS after chemical derivatization. The HPLC method is relatively rare, and there is no report on the detection of low-limit acryloyl chloride. According to the instructions, the maximum daily dose of ibrutinib is 560 mg. Based on the TTC calculation in ICH M7 (R1), the maximum acceptable daily intake of a single mutagenic impurity is 1.5 μg. The limit of genotoxic impurity acryloyl chloride is calculated to be 2.68 ppm. This limit is relatively low and often requires the use of more sensitive instruments such as mass spectrometry to complete the detection. Therefore, how to effectively determine the trace amount of acryloyl chloride in ibrutinib is an urgent problem to be solved. Summary of the invention
[0007] The purpose of the present invention is to provide a method for detecting acryloyl chloride, a genotoxic impurity in ibrutinib, to solve the problems of low sensitivity and inaccurate detection results of existing detection methods.
[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:
[0009] The present invention provides a method for detecting genotoxic impurity acryloyl chloride in ibrutinib, comprising the following steps:
[0010] Prepare blank solution, test solution and reference solution respectively, detect the blank solution, test solution and reference solution respectively by HPLC-UV, record the chromatogram, and calculate the content of acryloyl chloride in the test sample according to the external standard method;
[0011] The preparation method of the test solution is as follows: mixing the test sample with a solvent, adding a derivatization reagent for derivatization, and then adding a diluent to fix the volume to obtain a test solution;
[0012] The test sample is ibrutinib raw material or preparation.
[0013] Preferably, in the above-mentioned method for detecting the genotoxic impurity acryloyl chloride in ibrutinib, the derivatization reagent is one or more of aniline, benzylamine, and N-ethylaniline.
[0014] Preferably, in the above-mentioned method for detecting genotoxic impurity acryloyl chloride in ibrutinib, the dosage ratio of the test sample and the derivatization reagent is 0.1-2 g: 10-100 μL.
[0015] Preferably, in the above-mentioned method for detecting the genotoxic impurity acryloyl chloride in ibrutinib, the concentration of the test solution is 10 to 100 mg / mL.
[0016] Preferably, in the above-mentioned method for detecting genotoxic impurity acryloyl chloride in ibrutinib, the blank solution is prepared by mixing the solvent with the derivatization reagent, and then adding a diluent to make up the volume to obtain a blank solution.
[0017] Preferably, in the above-mentioned method for detecting the genotoxic impurity acryloyl chloride in ibrutinib, the preparation method of the reference solution is: mixing the acryloyl chloride reference substance with a solvent, adding a derivatization reagent for derivatization treatment, and then adding a diluent to make up the volume to obtain a reference solution.
[0018] Preferably, in the above-mentioned method for detecting the genotoxic impurity acryloyl chloride in ibrutinib, the concentration of the reference solution is 0.1-1 μg / mL.
[0019] Preferably, in the above-mentioned method for detecting the genotoxic impurity acryloyl chloride in ibrutinib, the detection conditions of the HPLC-UV include: the chromatographic column is one of Waters XBridge C18, Waters XBridge Shield RP18, and Waters Symmetry C18; the column temperature is 20-40°C; the detection wavelength is 240-260nm; the injection volume is 5-20μL; the flow rate is 0.5-1mL / min; the mobile phase A is triethylamine with a volume concentration of 0.05-0.2%; the mobile phase B is acetonitrile; and the elution method is gradient elution.
[0020] Preferably, in the above-mentioned method for detecting genotoxic impurity acryloyl chloride in ibrutinib, the procedure of the gradient elution is as shown in the following table:
[0021]
[0022] Wherein, mobile phase A / % is the volume fraction of mobile phase A, and mobile phase B / % is the volume fraction of mobile phase B.
[0023] It can be seen from the above technical solution that compared with the prior art, the present invention has the following beneficial effects:
[0024] The present invention uses aniline compounds to derivatize acryloyl chloride, the treatment method is simple to operate, and the trace amount of acryloyl chloride in the sample can be accurately quantified by ordinary high performance liquid chromatography, which greatly reduces the detection cost. The detection method of the present invention is highly sensitive, accurate and reliable, achieves effective control of impurities, strictly controls the quality of ibrutinib products, and ensures product safety and effectiveness. BRIEF DESCRIPTION OF THE DRAWINGS
[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the description of the embodiments or the prior art are briefly introduced below.
[0026] Figure 1 The blank solution test chromatogram of Example 1;
[0027] Figure 2 The chromatogram of the reference solution of Example 1 is detected;
[0028] Figure 3 The test solution of Example 1 is detected chromatogram;
[0029] Figure 4 The specific solution detection chromatogram of Example 1. DETAILED DESCRIPTION
[0030] The present invention provides a method for detecting genotoxic impurity acryloyl chloride in ibrutinib, comprising the following steps:
[0031] Prepare blank solution, test solution and reference solution respectively, detect the blank solution, test solution and reference solution respectively by HPLC-UV, record the chromatograms, and calculate the content of acryloyl chloride in the test sample according to the external standard method.
[0032] In the present invention, the test sample is the ibrutinib bulk drug or preparation. The test samples used in the embodiments of the present invention are homemade, with batch numbers 2402001, 2402002, and 2402003.
[0033] In the present invention, the reference substance is acryloyl chloride reference substance. The reference substance information used in the examples of the present invention is shown in Table 1.
[0034] Table 1 Reference material information
[0035]
[0036] In the present invention, the preparation method of the test solution is: mixing the test sample with a solvent, adding a derivatization reagent for derivatization treatment, and then adding a diluent to fix the volume to obtain the test solution.
[0037] In the present invention, the blank solution is prepared by mixing a solvent with a derivatization reagent, and then adding a diluent to a fixed volume to obtain a blank solution.
[0038] In the present invention, the preparation method of the reference substance solution is: mixing the acryloyl chloride reference substance with a solvent, adding a derivatization reagent for derivatization treatment, and then adding a diluent to fix the volume to obtain a reference substance solution.
[0039] In the present invention, the derivatization agent is preferably one or more of aniline, benzylamine, and N-ethylaniline, more preferably aniline or benzylamine, and more preferably aniline.
[0040] In the present invention, the solvent is preferably dichloromethane.
[0041] In the present invention, the diluent is preferably acetonitrile.
[0042] In the present invention, the usage ratio of the test sample and the derivatization reagent is preferably 0.1-2 g:10-100 μL, more preferably 0.3-1 g:20-60 μL, and more preferably 0.5 g:50 μL.
[0043] In the present invention, the concentration of the test solution is preferably 10 to 100 mg / mL, more preferably 40 to 70 mg / mL, and even more preferably 50 mg / mL.
[0044] In the present invention, the concentration of the reference solution is preferably 0.1 to 1 μg / mL, more preferably 0.12 to 0.4 μg / mL, and more preferably 0.134 μg / mL.
[0045] In the present invention, the detection conditions of the HPLC-UV include: the chromatographic column is preferably one of Waters XBridge C18, Waters XBridge Shield RP18, and Waters Symmetry C18, more preferably Waters XBridge Shield RP18 or Waters Symmetry C18, and more preferably Waters XBridge Shield RP18; the column temperature is preferably 20-40°C, more preferably 25-35°C, and more preferably 30°C; the detection wavelength is preferably 240-260nm, more preferably 250-255nm, and more preferably 254nm; the injection volume is preferably 5-20μL, more preferably 8-12μL, and more preferably 10μL; the flow rate is preferably 0.5-1mL / min, more preferably 0.5-0.7mL / min, and more preferably 0.6mL / min; the mobile phase A is preferably triethylamine with a volume concentration of 0.05-0.2%, more preferably triethylamine with a volume concentration of 0.05-0.2% adjusted to pH 6.0-8.0 using phosphoric acid, and more preferably triethylamine with a volume concentration of 0.1% adjusted to pH 7.0 using phosphoric acid; the mobile phase B is preferably acetonitrile; the elution method is preferably gradient elution.
[0046] In the present invention, the procedure of the gradient elution is shown in the following table:
[0047]
[0048] Wherein, mobile phase A / % is the volume fraction of mobile phase A, and mobile phase B / % is the volume fraction of mobile phase B.
[0049] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0050] Example 1
[0051] 1.1 Chromatographic conditions
[0052] Chromatographic column: XBridge Shield RP18 (4.6*250mm, 5μm);
[0053] Column temperature: 30°C;
[0054] Detection wavelength: 254nm;
[0055] Flow rate: 0.6 mL / min;
[0056] Injection volume: 10 μL;
[0057] Running time: 35min;
[0058] Mobile phase A: 0.1% triethylamine aqueous solution (pH adjusted to 7.0 with phosphoric acid);
[0059] Mobile phase B: acetonitrile;
[0060] The gradient elution program is as follows:
[0061]
[0062] Wherein, mobile phase A / % is the volume fraction of mobile phase A, and mobile phase B / % is the volume fraction of mobile phase B.
[0063] 1.2 Solution preparation
[0064] Blank solution: Accurately measure 1.0 mL of dichloromethane and 50 μL of aniline and place them in a 10 mL volumetric flask. Shake for 10 min, then dilute to the mark with acetonitrile and shake well.
[0065] Reference solution: Accurately weigh 53.6 mg of acryloyl chloride reference substance and place it in a 50 mL volumetric flask with an appropriate amount of acetonitrile, dilute to the mark with acetonitrile, and shake well to serve as reference stock solution 1. Accurately measure 1.0 mL of reference stock solution 1 and place it in a 20 mL volumetric flask with an appropriate amount of acetonitrile, dilute to the mark with acetonitrile, and shake well to serve as reference stock solution 2. Accurately measure 1.25 mL of reference stock solution 2 and place it in a 50 mL volumetric flask with an appropriate amount of acetonitrile, dilute to the mark with acetonitrile, and shake well to serve as reference stock solution 3. Accurately measure 1.0 mL of dichloromethane and 50 μL of aniline in a 10 mL volumetric flask, accurately add 1.0 mL of reference stock solution 3 to the volumetric flask, shake for 10 minutes, dilute to the mark with acetonitrile, and shake well.
[0066] Test solution: Accurately weigh 0.5 g of ibrutinib test sample and place it in a 10 mL volumetric flask. Add 1.0 mL of dichloromethane and ultrasonically dissolve until clear. Accurately add 50 μL of aniline, shake for 10 min, and then dilute to the mark with acetonitrile and shake well.
[0067] Specific solution: Accurately weigh 0.5 g of ibrutinib test sample and place it in a 10 mL volumetric flask. Add 1.0 mL of dichloromethane and ultrasonically dissolve until clear. Accurately add 50 μL of aniline and 1.0 mL of reference stock solution 3. Shake for 10 min, then dilute to the mark with acetonitrile and shake well.
[0068] 1.3 Determination method
[0069] Take 10 μL of blank solution, reference solution and test solution and inject them into liquid chromatograph respectively, record the chromatograms, the chromatograms are as follows: Figure 1 to Figure 3 As shown, the content of acryloyl chloride was calculated by external standard method.
[0070] 1.4 Measurement results
[0071] The contents of acryloyl chloride in three batches of ibrutinib test samples (batch numbers were 2402001, 2402002, and 2402003, respectively) were detected according to the above method. The results are shown in Table 2.
[0072] Table 2 Test results of three batches of test products
[0073]
[0074] From the results in Table 2, it can be seen that acryloyl chloride was not detected in the three batches of test samples, which met the limit requirements (≤2.68ppm).
[0075] 1.5 Methodological Validation:
[0076] (1) Specificity and system applicability investigation
[0077] Take blank solution, specific solution, reference solution and test solution and test them according to the chromatographic conditions in 1.1. The reference solution is injected 5 times continuously and the others are injected 1 time each. The chromatogram of the specific solution is as follows: Figure 4 The test results are shown in Table 3.
[0078] Table 3 Specificity and system suitability test results
[0079]
[0080] From the results in Table 3, it can be seen that in this method, the blank solution and the test solution have no interference with the detection of acryloyl chloride, and the RSD of the peak area of acryloyl chloride in the 5-pronged reference solution is less than 10%, indicating that the method has good specificity and the instrument system has good precision.
[0081] (2) Investigation of limit of quantification and limit of detection
[0082] The reference stock solution 3 was diluted to a certain concentration for derivatization treatment. The concentration when the impurity peak signal-to-noise ratio was about 10:1 was taken as the quantitative limit concentration, and 6 injections of the quantitative limit solution were injected continuously. The concentration when the impurity peak signal-to-noise ratio was about 3:1 was taken as the detection limit concentration, and 2 injections of the detection limit solution were injected continuously. The test results are shown in Table 4.
[0083] Table 4 Quantitation limit and detection limit test results
[0084]
[0085] It can be seen from the results in Table 4 that the detection limit and quantification limit of acryloyl chloride in the method of the present invention are low, indicating that the detection sensitivity of the method is high and can fully meet the determination requirements.
[0086] (3) Linearity and range investigation
[0087] The reference substance stock solution 3 was diluted to prepare linear solutions of different concentrations and then subjected to derivatization treatment. Samples were injected separately and the standard curve was drawn with concentration as the horizontal axis and peak area as the vertical axis. The linearity and range results are shown in Table 5.
[0088] Table 5 Linearity and range test results
[0089] name Linear equations Correlation coefficient scope Acryloyl chloride y=70.978x+0.42 0.9990 0.0142 μg / mL~0.1838 μg / mL
[0090] It can be seen from the results in Table 5 that acryloyl chloride has a good linear relationship in the method of the present invention.
[0091] (4) Precision inspection
[0092] According to the test solution preparation method in 1.2, 6 samples were prepared in parallel and tested separately. The repeatability results are shown in Table 6.
[0093] Table 6 Repeatability test results
[0094]
[0095] Another experimenter prepared 6 test solutions in parallel at different times according to the test solution preparation method in 1.2, and tested them on another instrument according to the chromatographic conditions in 1.1. The intermediate precision results are shown in Table 7.
[0096] Table 7 Intermediate precision test results
[0097]
[0098] It can be seen from the results in Tables 6 and 7 that acryloyl chloride was not detected in any of the 12 test solutions, and the method had good precision.
[0099] (5) Accuracy test
[0100] Weigh the test sample accurately, add the reference stock solution 3 quantitatively, and further derivatize to prepare the recovery solution. Prepare 3 copies of each of the high, medium and low concentrations in parallel. The accuracy results are shown in Table 8.
[0101] Table 8 Accuracy test results
[0102]
[0103] From the results in Table 8, it can be seen that the recoveries of acryloyl chloride measured by this method are all in the range of 70.0% to 130.0%, and the recovery RSD is less than 10%, indicating that the detection accuracy of this method is good.
[0104] (6) Durability test
[0105] The column temperature (±5°C), flow rate (±0.1mL / min), mobile phase pH (±0.2), initial mobile phase ratio (±5%), injection volume (±2μL), and chromatographic column number (1# and 2# are two chromatographic columns from different batches of XBridge ShieldRP18) in 1.1 chromatographic conditions were fine-tuned respectively. Other conditions remained unchanged and the tests were carried out in sequence. The durability results are shown in Table 9.
[0106] Table 9 Durability test results
[0107]
[0108]
[0109] From the results in Table 9, it can be seen that after fine-tuning the chromatographic conditions, the system applicability still meets the requirements, has no effect on the detection of impurities, and the method is highly durable.
[0110] (7) Solution stability
[0111] The reference solution and the test solution were placed at room temperature for about 80 hours and tested at different time points. The solution stability results are shown in Table 10.
[0112] Table 10 Solution stability test results
[0113]
[0114] From the results in Table 10, it can be seen that the reference solution and the test solution are placed at room temperature for about 80 hours without affecting the measurement results, and the stability is good.
[0115] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.
Claims
1. A method for detecting genotoxic impurity acryloyl chloride in ibrutinib, characterized in that: The following steps are involved: Prepare blank solution, test solution and reference solution respectively, detect the blank solution, test solution and reference solution respectively by HPLC-UV, record the chromatogram, and calculate the content of acryloyl chloride in the test sample according to the external standard method; The preparation method of the test solution is as follows: mixing the test sample with a solvent, adding a derivatization reagent for derivatization, and then adding a diluent to fix the volume to obtain a test solution; The test sample is ibrutinib raw material or preparation.
2. The method for detecting genotoxic impurity acryloyl chloride in ibrutinib according to claim 1, characterized in that: The derivatization reagent is one or more of aniline, benzylamine and N-ethylaniline.
3. A method for detecting genotoxic impurity acryloyl chloride in ibrutinib according to claim 1 or 2, characterized in that: The dosage ratio of the test sample and the derivatization reagent is 0.1-2 g: 10-100 μL.
4. The method for detecting genotoxic impurity acryloyl chloride in ibrutinib according to claim 3, characterized in that: The concentration of the test solution is 10-100 mg / mL.
5. The method for detecting genotoxic impurity acryloyl chloride in ibrutinib according to claim 1, characterized in that: The blank solution is prepared by mixing the solvent with the derivatization reagent, and then adding a diluent to make up the volume to obtain a blank solution.
6. The method for detecting genotoxic impurity acryloyl chloride in ibrutinib according to claim 5, characterized in that: The preparation method of the reference substance solution is: mixing the acryloyl chloride reference substance with a solvent, adding a derivatization reagent for derivatization treatment, and then adding a diluent to fix the volume to obtain the reference substance solution.
7. The method for detecting genotoxic impurity acryloyl chloride in ibrutinib according to claim 6, characterized in that: The concentration of the reference solution is 0.1-1 μg / mL.
8. The method for detecting genotoxic impurity acryloyl chloride in ibrutinib according to claim 3, characterized in that: The detection conditions of the HPLC-UV include: the chromatographic column is one of Waters XBridge C18, Waters XBridgeShield RP18, and Waters Symmetry C18; the column temperature is 20-40°C; the detection wavelength is 240-260nm; the injection volume is 5-20μL; the flow rate is 0.5-1mL / min; the mobile phase A is triethylamine with a volume concentration of 0.05-0.2%; the mobile phase B is acetonitrile; and the elution method is gradient elution.
9. The method for detecting genotoxic impurity acryloyl chloride in ibrutinib according to claim 8, characterized in that: The procedure of the gradient elution is shown in the following table: Wherein, mobile phase A / % is the volume fraction of mobile phase A, and mobile phase B / % is the volume fraction of mobile phase B.