Method for separating and determining ibrutinib SM3 and impurities thereof based on gas chromatography

Abstract

The invention belongs to the technical field of pharmaceutical analysis, and particularly relates to a method for separating and determining ibrutinib SM3 and impurities thereof based on gas chromatography. The impurities comprise any one or more of acrylic anhydride, trichlorotoluene, propionyl chloride, 3-chloropropionyl chloride, benzoyl chloride, acrylic acid and benzoic acid; the ibrutinib SM3 and impurities jointly form a composition to be detected. According to the method, (5% phenyl)-diphenylmethylsiloxane is adopted as a chromatographic column of a stationary liquid, or other stationary liquids with the same polarity are adopted; the ibrutinib SM3 and impurities of the ibrutinib SM3 are separated by adopting temperature programming; the temperature programming setting is as follows: the initial temperature is 35 + / -5 DEG C and is maintained for 5 + / -1 minutes, and the temperature is raised to 210 + / -10 DEG C at the rate of 30 + / -5 DEG C / min and is maintained for 8 + / -2 minutes. The method provided by the invention can effectively separate, identify and quantify various related substances in ibrutinib SM3, and has the advantages of short separation time, strong specificity, high sensitivity and good durability.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical analysis technology, specifically relating to a method for separating and determining ibrutinib SM3 and its impurities based on gas chromatography. Background Technology

[0002] Ibrutinib is an innovative anti-tumor drug primarily used to treat mantle cell lymphoma, chronic lymphocytic leukemia, small lymphocytic lymphoma, and Waldenström macroglobulinemia. As an inhibitor of Bruton's tyrosine kinase (BTK), it irreversibly inhibits BTK enzymatic activity by forming a covalent bond with cysteine ​​residues at the BTK active site. BTK is a key molecule in the B cell surface receptor activation signaling pathway, crucial for B cell migration, chemotaxis, and adhesion. Ibrutinib inhibits BTK, thereby suppressing the in vivo proliferation and survival of malignant B cells, as well as their migration and basal adhesion in vitro. This unique BTK inhibition mechanism makes ibrutinib play a vital role in the treatment of malignant B cell-related diseases.

[0003] Ibrutinib SM3 (IBR-SM3) is a key starting material for ibrutinib. Studies have found that ibrutinib SM3 may contain eight impurities: acrylic anhydride, trichlorotoluene, propionyl chloride, 3-chloropropionyl chloride, benzoyl chloride, acetyl chloride, acrylic acid, and benzoic acid. Among these, trichlorotoluene is a crucial raw material used in the preparation of ibrutinib SM3, and acrylic anhydride is a hydrolysis byproduct of ibrutinib SM3; both impurities contain warning structures. To ensure the quality of subsequent products, the content of acrylic anhydride and trichlorotoluene needs to be controlled.

[0004] In the prior art, patent CN112557571A reports a method for the quantitative detection of acryloyl chloride in the preparation of ibrutinib raw material. This method is gas chromatography, and the chromatographic conditions include: injection port temperature of 170-200℃; split ratio of 60-100:1; inert carrier gas; detector temperature of 180-250℃; column flow rate of 1.0-2.0 mL / min; column temperature: programmed temperature ramp, initial temperature 70-100℃, held for 5-8 min, then ramped to 270℃ at a rate of 65-70℃ / min, held for 4-10 min; headspace sampler: heating chamber temperature 80-100℃, equilibration time 15-25 min, injection valve temperature 100-130℃, transfer line temperature 120-155℃. Finally, the residual amount of acryloyl chloride in the sample is calculated using the internal standard method. This method cannot effectively separate and detect ibrutinib SM3, acrylic anhydride, and / or trichlorotoluene.

[0005] Currently, there are no relevant literature or methods that can simultaneously separate and detect acrylic anhydride and trichlorotoluene, two impurities in ibrutinib SM3, without interference from other impurities such as propionyl chloride, 3-chloropropionyl chloride, benzoyl chloride, acetyl chloride, acrylic acid, and benzoic acid. Therefore, it is necessary to establish an efficient, simple, sensitive, and reproducible method to achieve the effective separation and detection of acrylic anhydride and trichlorotoluene in ibrutinib SM3. Summary of the Invention

[0006] In view of this, one of the objectives of the present invention is to provide a method for separating ibrutinib SM3 and its impurities based on gas chromatography, which can complete the separation of multiple substances in a short time.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A method for separating ibrutinib SM3 and its impurities based on gas chromatography, wherein the gas chromatography method includes: using a chromatographic column with (5% phenyl)-diphenylmethylsiloxane as the stationary phase, or other stationary phases of equivalent polarity; separating ibrutinib SM3 and its impurities using a temperature programmed process; wherein the temperature programmed process is set as follows: initial temperature of 35±5℃, maintained for 5±1 minutes, increasing to 210±10℃ at a rate of 30±5℃ / min, and maintained for 8±2 minutes;

[0009] The impurities include any one or more of acrylic anhydride, trichlorotoluene, propionyl chloride, 3-chloropropionyl chloride, benzoyl chloride, acrylic acid, and benzoic acid; the ibrutinib SM3 and the impurities together constitute the test composition; the structural formula of the ibrutinib SM3 is shown in Formula I.

[0010]

[0011] Furthermore, the test composition also contains acetyl chloride.

[0012] The separated material can be used in the next stage of production.

[0013] The aforementioned impurities can be arranged and combined in various ways.

[0014] For example, combination 1: acrylic anhydride and trichlorotoluene; combination 2: acrylic anhydride, propionyl chloride and 3-chloropropionyl chloride; combination 3: acrylic anhydride, trichlorotoluene, benzoyl chloride, acrylic acid and benzoic acid.

[0015] All possible permutations and combinations are not exhaustive. Theoretically, when the upper limit for the separation, identification, and / or content detection of this method is 7 substances, it can naturally detect any 1 to 7 substances.

[0016] Preferably, the programmed temperature rise is set as follows: the initial temperature is 35±2℃, maintained for 5 minutes, and then the temperature is increased to 210℃ at a rate of 30±2℃ / min, and maintained for 8 minutes.

[0017] As the optimal setting, the initial temperature is set to 35°C and maintained for 5 minutes, then the temperature is increased to 210°C at a rate of 30°C / min and maintained for 8 minutes.

[0018] Furthermore, the carrier gas is nitrogen; the injection port temperature is 250±20℃; the detector temperature is 250±20℃; and the column flow rate is 1.0±0.3 ml / min.

[0019] Preferably, the injection port temperature is 250℃.

[0020] Preferably, the detector temperature is 250°C.

[0021] Preferably, the column flow rate is 1.0 ± 0.1 ml / min, and most preferably 1.0 ml / min.

[0022] Furthermore, the injection volume was 3.0 μL.

[0023] Furthermore, the separation time is preferably 18.8 minutes.

[0024] Furthermore, the test solution was prepared using dichloromethane as a solvent.

[0025] Furthermore, the detection limit concentration of ibrutinib SM3 is 1.0592 μg / ml and the quantitation limit concentration is 3.0263 μg / ml, the detection limit concentration of acrylic anhydride is 1.0763 μg / ml and the quantitation limit concentration is 3.0750 μg / ml, and the detection limit concentration of trichlorotoluene is 1.1718 μg / ml and the quantitation limit concentration is 3.0132 μg / ml.

[0026] The second objective of this invention is to provide a method for identifying ibrutinib SM3 and its impurities, which can complete the identification of multiple substances in a short time.

[0027] To achieve the above objectives, the present invention adopts the following technical solution:

[0028] The method for identifying ibrutinib SM3 and its impurities involves separating the test composition using the aforementioned separation method and detecting it in a detector to obtain a chromatogram; by comparing the chromatographic characteristics of the test sample and the reference sample, it is determined whether the test sample contains ibrutinib SM3 and its impurities.

[0029] The components of the test composition can be identified according to their relative retention times. The components of the test composition are in ascending order as follows: ibrutinib SM3, propionyl chloride, acrylic acid, 3-chloropropionyl chloride, acrylic anhydride, benzoyl chloride, benzoic acid, and trichlorotoluene.

[0030] Relative retention time (R0) typically describes the relative retention of different components in a mixture on a chromatographic column. It is the ratio of the retention time of a particular component to the retention time of a reference component (usually the main peak or a known component). This ratio reflects the relative retention performance of different components on the column and is an important parameter used for localization, qualitative, and quantitative analysis in chromatographic analysis. R0 is calculated by dividing the retention time of the target component (tR) by the retention time of the reference component (tR0).

[0031] As a preferred method, using ibrutinib SM3 as a reference peak, the following relative retention times were identified: 1.04 for propionyl chloride; 1.47 for acrylic acid; 1.96 for 3-chloropropionyl chloride; 2.17 for acrylic anhydride; 2.50 for benzoyl chloride; 2.60 for benzoic acid; and 2.70 for trichlorotoluene. The relative retention times of each component fluctuated within the range of 5%.

[0032] Besides relative retention time, retention time can also be used to identify components. Retention time refers to the time required for a sample to travel from entering the chromatographic column to being detected by the detector. This time is calculated based on the migration speed of the component on the chromatographic column, that is, the time interval from the start of injection to the chromatographic peak (maximum concentration) of a certain component. It is mainly used to determine the elution order and position of each component in the sample and is one of the basic data in chromatographic analysis. In quality control, changes in retention time can reflect factors such as the state of the chromatographic column, the stability of the mobile phase, and the performance of the instrument.

[0033] The third objective of this invention is to provide a method for quantitative detection of ibrutinib SM3 and its impurities.

[0034] To achieve the above objectives, the present invention adopts the following technical solution:

[0035] A method for quantitative detection of ibrutinib SM3 and its impurities was proposed, wherein ibrutinib SM3 and its impurities were separated and identified using the aforementioned identification method, and chromatograms were obtained; based on the obtained chromatograms, the content of acrylic anhydride, trichlorotoluene and other individual impurities in ibrutinib SM3 was calculated using the peak area normalization method.

[0036] Preferably, the detection was performed under the following chromatographic conditions, and chromatograms were obtained: a chromatographic column with (5% phenyl)-diphenylmethylsiloxane as the stationary phase, the column size being 30m × 0.32mm and 0.25μm; nitrogen as the carrier gas; and the following temperature program settings: initial temperature of 35±2℃, maintained for 5 minutes, then increased to 210℃ at a rate of 30±2℃ / min, and maintained for 8 minutes; injection port temperature of 250℃; detector temperature of 250℃; and column flow rate of 1.0±0.1ml / min.

[0037] As a preferred technical solution:

[0038] Step 1: Accurately weigh 60 mg of this product (ibrutinib SM3), place it in a 10 ml volumetric flask containing approximately 5 ml of dichloromethane, dissolve and dilute to the mark with dichloromethane to obtain the sample solution.

[0039] Step 2: Take appropriate amounts of propionyl chloride, 3-chloropropionyl chloride, benzoyl chloride, acetyl chloride, acrylic acid, and benzoic acid, weigh them accurately, place them in a volumetric flask containing dichloromethane, and dilute with dichloromethane to prepare a solution containing 30 μg of each impurity per 1 ml, which is used as the impurity localization solution.

[0040] Step 3: Measure 50 μl each of acrylic anhydride and trichlorotoluene and place them in the same 50 ml volumetric flask containing approximately 10 ml of dichloromethane. Dissolve and dilute to the mark with dichloromethane, and shake well. Measure 0.5 ml of the solution and place it in a 10 ml volumetric flask containing approximately 5 ml of dichloromethane. Weigh approximately 60 mg of the test sample and place it in the same volumetric flask. Dilute to the mark with dichloromethane and shake well. This solution is used as the system suitability solution.

[0041] Step 4: Accurately measure 3.0 μl each of the system suitability solution, impurity localization solution, and test solution, and inject them into the gas chromatograph, recording the chromatograms; calculate the content of impurities acrylic anhydride, trichlorotoluene, and other individual impurities using the peak area normalization method.

[0042] The beneficial effects of this invention are as follows:

[0043] 1. This invention provides a self-developed gas chromatography method for the separation and detection of related impurities in ibrutinib SM3. This method is highly efficient and robust, enabling the simultaneous and accurate quantification of acrylic anhydride, trichlorotoluene, and other individual impurities in ibrutinib SM3 within a single procedure. This invention solves the problem of separating and determining acrylic anhydride and trichlorotoluene in ibrutinib SM3 that has not been addressed by existing technologies, and features high sensitivity, strong specificity, good reproducibility, and simple and feasible operation.

[0044] 2. The method of the present invention can also effectively separate five other known impurities (propionyl chloride, 3-chloropropionyl chloride, benzoyl chloride, acrylic acid and benzoic acid) that may be present in ibrutinib SM3. The separation and detection of acrylic anhydride and trichlorotoluene in ibrutinib SM3 are not affected by these five known impurities, solvents and acetyl chloride.

[0045] 3. The method of the present invention can simultaneously separate acrylic anhydride, trichlorotoluene, propionyl chloride, 3-chloropropionyl chloride, benzoyl chloride, acrylic acid and benzoic acid in ibrutinib SM3 within 18.8 minutes, with short separation time and good separation effect.

[0046] 4. Using the method of this invention to identify acrylic anhydride and trichlorotoluene in ibrutinib SM3, the detection limit concentration (LOC) for acrylic anhydride is 1.0763 μg / ml, which is 0.018% as expressed in the sample, with a mean signal-to-noise ratio (SNR) of 14.1; the LOC for trichlorotoluene is 1.1718 μg / ml, which is 0.020% as expressed in the sample, with a mean SNR of 25.3; and the LOC for ibrutinib SM3 is 1.0592 μg / ml, which is 0.018% as expressed in the sample, with a mean SNR of 6.8. That is, the method of this invention can simultaneously identify ibrutinib SM3, acrylic anhydride, and trichlorotoluene at concentrations as low as approximately 1.17 μg / ml in the sample, exhibiting a low LOC.

[0047] 5. The method of this invention was used to quantitatively detect acrylic anhydride and trichlorotoluene in ibrutinib SM3. The limit of quantification (LOQ) for acrylic anhydride was 3.0750 μg / ml, expressed as 0.05% in the sample, with a 6-shot peak area RSD of 2.3% and a mean signal-to-noise ratio (SNR) of 45.6. The LQ for trichlorotoluene was 3.0132 μg / ml, expressed as 0.05% in the sample, with a 6-shot peak area RSD of 3.1% and a mean SNR of 77.2. The LQ for ibrutinib SM3 was 3.0263 μg / ml, expressed as 0.05% in the sample, with a 6-shot peak area RSD of 2.7% and a mean SNR of 20.2. In other words, the method of this invention can simultaneously quantify ibrutinib SM3, acrylic anhydride, and trichlorotoluene at concentrations as low as approximately 3.08 μg / ml in the sample, exhibiting a low LQ. Attached Figure Description

[0048] Figure 1 The gas chromatogram is for a blank solution.

[0049] Figure 2 The gas chromatogram of the acrylic anhydride positioning solution;

[0050] Figure 3 The gas chromatogram of the trichlorotoluene positioning solution;

[0051] Figure 4 The gas chromatogram of the acetyl chloride positioning solution;

[0052] Figure 5 The gas chromatogram of the propionyl chloride positioning solution is shown.

[0053] Figure 6 The gas chromatogram of the 3-chloropropionyl chloride positioning solution;

[0054] Figure 7 The gas chromatogram of the benzoyl chloride positioning solution;

[0055] Figure 8 Gas chromatogram of acrylic acid positioning solution;

[0056] Figure 9 This is the gas chromatogram of the benzoic acid positioning solution;

[0057] Figure 10 The gas chromatogram of the test solution;

[0058] Figure 11 The image shows a gas chromatogram of the mixed solution.

[0059] Figure 12 Gas chromatogram of the solution at the limit of quantitation;

[0060] Figure 13 Gas chromatogram of the solution at the detection limit;

[0061] Figure 14 The gas chromatogram of the mixed solution at an initial column temperature of 33°C during the durability test;

[0062] Figure 15 This is a gas chromatogram of the mixed solution at an initial column temperature of 37°C during the durability test.

[0063] Figure 16 This is a gas chromatogram of the mixed solution under a heating rate of 28 °C / min during the durability test;

[0064] Figure 17 This is a gas chromatogram of the mixed solution under a heating rate of 32℃ / min during the durability test;

[0065] Figure 18 This is a gas chromatogram of the mixed solution at a flow rate of 0.9 ml / min during the durability test;

[0066] Figure 19 This is a gas chromatogram of the mixed solution at a flow rate of 1.1 ml / min during the durability test. Detailed Implementation

[0067] The technical solution of the present invention will be described more clearly and completely below with reference to specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention.

[0068] This patent contains Figures 1 to 19 Visual aids are provided for understanding and interpretation. Some figures overlap with each other, or with column peaks. To address this, this patent provides supplementary tables (Tables 1-19) to the accompanying figures to facilitate better review of experimental results by those skilled in the art. In case of any ambiguity, users should refer to the corresponding numbered tables (Tables 1-9) for more detailed information. Conversely, if any potentially misleading or ambiguous information is found while reviewing Tables 1-9, the content of the corresponding numbered figure should be considered definitive. The above guidelines aim to ensure the correct interpretation of this document and the consistency of its information.

[0069] Table 1. Figure 1 Integral Results Table

[0070]

[0071] Table 2. Figure 2 Integral Results Table

[0072]

[0073] In Table 2, the peak name for number 1 is acrylic anhydride.

[0074] Table 3. Figure 3 Integral Results Table

[0075]

[0076] In Table 3, the peak name for number 1 is trichlorotoluene.

[0077] Table 4. Figure 4 Integral Results Table

[0078]

[0079] Table 5. Figure 5 Integral Results Table

[0080]

[0081] In Table 5, the peak name for number 1 is propionyl chloride.

[0082] Table 6. Figure 6 Integral Results Table

[0083]

[0084] In Table 6, the peak name for number 1 is 3-chloropropionyl chloride.

[0085] Table 7. Figure 7 Integral Results Table

[0086]

[0087] In Table 7, the peak name for number 1 is benzoyl chloride.

[0088] Table 8. Figure 8 Integral Results Table

[0089]

[0090] In Table 8, the peak name for number 1 is acrylic acid.

[0091] Table 9. Figure 9 Integral Results Table

[0092]

[0093] In Table 9, the peak name for number 1 is benzoic acid.

[0094] Table 10. Figure 10 Integral Results Table

[0095]

[0096] In Table 10, number 1 is ibrutinib SM3; number 2 is acrylic acid; and numbers 4, 6, 7, and 8 are all unknown impurities.

[0097] Table 11. Figure 11 Integral Results Table

[0098]

[0099] In Table 11, serial numbers 4, 6, 8, 10, and 13-16 are all unknown impurities.

[0100] Table 12. Figure 12 Integral Results Table

[0101]

[0102] Table 13. Figure 13 Integral Results Table

[0103]

[0104] Table 14. Figure 14 Integral Results Table

[0105]

[0106] In Table 14, number 1 is ibrutinib SM3; number 2 is propionyl chloride; number 3 is acrylic acid; number 5 is 3-chloropropionyl chloride; number 7 is acrylic anhydride; number 9 is benzoyl chloride; number 11 is benzoic acid; number 12 is trichlorotoluene; and numbers 4, 6, 8, 10, 13, and 14 are all unknown impurities.

[0107] Table 15. Figure 15 Integral Results Table

[0108]

[0109] In Table 15, number 1 is ibrutinib SM3; number 2 is propionyl chloride; number 3 is acrylic acid; number 5 is 3-chloropropionyl chloride; number 7 is acrylic anhydride; number 8 is benzoyl chloride; number 10 is benzoic acid; number 11 is trichlorotoluene; and numbers 4, 6, 9, 12, and 13 are all unknown impurities.

[0110] Table 16. Figure 16 Integral Results Table

[0111]

[0112] In Table 16, number 1 is ibrutinib SM3; number 2 is propionyl chloride; number 3 is acrylic acid; number 5 is 3-chloropropionyl chloride; number 7 is acrylic anhydride; number 9 is benzoyl chloride; number 11 is benzoic acid; number 12 is trichlorotoluene; and numbers 4, 6, 8, 10, 13, and 14 are all unknown impurities.

[0113] Table 17. Figure 17 Integral Results Table

[0114]

[0115] In Table 17, number 1 is ibrutinib SM3; number 2 is propionyl chloride; number 3 is acrylic acid; number 5 is 3-chloropropionyl chloride; number 7 is acrylic anhydride; number 8 is benzoyl chloride; number 10 is benzoic acid; number 11 is trichlorotoluene; and numbers 4, 6, 9, 12, 13, and 14 are all unknown impurities.

[0116] Table 18. Figure 18 Integral Results Table

[0117]

[0118] In Table 18, number 1 is ibrutinib SM3; number 2 is propionyl chloride; number 3 is acrylic acid; number 5 is 3-chloropropionyl chloride; number 7 is acrylic anhydride; number 8 is benzoyl chloride; number 10 is benzoic acid; number 11 is trichlorotoluene; and numbers 4, 6, 9, 12, 13, and 14 are all unknown impurities.

[0119] Table 19. Figure 19 Integral Results Table

[0120]

[0121] In Table 19, number 1 is ibrutinib SM3; number 2 is propionyl chloride; number 3 is acrylic acid; number 5 is 3-chloropropionyl chloride; number 6 is acrylic anhydride; number 7 is benzoyl chloride; number 9 is benzoic acid; number 10 is trichlorotoluene; and numbers 4, 8, 11, and 12 are all unknown impurities.

[0122] To enhance understanding of the present invention, certain key technologies and scientific terms will be clearly defined below. Unless specifically defined herein, all other technical and scientific terms shall follow their generally accepted and understood meanings within the art to which this invention pertains. It should be emphasized that the scope of the present invention is not limited to the specific methods, reagents, compounds, compositions, reference standards, and test items described, but allows for reasonable variations and adjustments in these aspects. Furthermore, please understand that the terminology used herein is intended to illustrate specific embodiments and not to impose a limiting interpretation.

[0123] Furthermore, all references cited in this document, including but not limited to patents, patent applications, academic papers, textbooks, and further citations therein, are considered to be incorporated into this document in their entirety through citation, unless directly cited, as a reference. If there are any inconsistencies or conflicts between the content of these cited references or similar materials and this application, particularly regarding terminology definitions, usage, or technical descriptions, the content of this application shall prevail.

[0124] the term

[0125] The limit of quantitation (LOQ) is the lowest amount of an analyte in a sample that can be quantitatively determined. The determination result should possess a certain degree of accuracy and precision. In other words, the LOQ is the lowest level at which an analytical method can accurately and reliably determine the concentration of the analyte in a sample. In gas chromatography, the determination of the LOQ typically relies on the signal-to-noise ratio (S / N) method, where the analyte concentration at which the signal-to-noise ratio reaches a certain level is taken as the LOQ. Determining the LOQ is crucial for ensuring the accuracy and reliability of analytical results.

[0126] The limit of detection (LOD) is the lowest concentration or amount of an analyte in a sample that can be detected. It reflects the sensitivity and noise level of the analytical method and instrument, and also indicates the level of the blank (background) value after sample processing. The LOD is an important indicator for evaluating the sensitivity of instruments and analytical methods.

[0127] Chromatographic robustness refers to the ability of a chromatographic analysis system to maintain stable analytical performance and unaffected analytical results when measurement conditions are slightly changed. This robustness is crucial for ensuring the reliability, repeatability, and stability of analytical results.

[0128] The signal-to-noise ratio (S / N) in gas chromatography refers to the ratio of the peak height of the main component in a gas chromatogram to the baseline noise. It is an important indicator of the quality of the measuring electrical system, the instrument's detection sensitivity, and the reliability of the analytical method. The peak height of the main component refers to the signal value output by the detector when the analyte elutes to its maximum concentration after passing through the column; it is usually used to represent the relative content or concentration of the component. Noise, on the other hand, refers to the fluctuation of the baseline signal, i.e., the signal value output by the detector when no sample passes through the column. A higher S / N means that the instrument can more accurately separate and identify the target component when detecting a sample, while reducing interference from background noise.

[0129] If any part of the chromatographic conditions in this invention is not mentioned, the gas chromatography method (Chinese Pharmacopoeia 2015 Edition, Part IV, General Chapter 0521) can be used for testing.

[0130] The structural formulas of the compounds involved in this invention are shown in Table A.

[0131] Table A. Compound Information Table

[0132]

[0133]

[0134] Example 1. Method for separating and determining impurities in ibrutinib SM3

[0135] (1) Preparation of the test solution

[0136] Test solution: Weigh 60 mg of this product accurately and place it in a 10 ml volumetric flask containing about 5 ml of dichloromethane. Dissolve and dilute to the mark with dichloromethane to obtain the test solution.

[0137] Impurity localization solution: Weigh appropriate amounts of propionyl chloride, 3-chloropropionyl chloride, benzoyl chloride, acetyl chloride, acrylic acid, and benzoic acid accurately, place them in a volumetric flask containing dichloromethane, and dilute with dichloromethane to prepare a solution containing 30 μg of each impurity per 1 ml, which is used as the impurity localization solution.

[0138] System suitability solution: Measure 50 μl each of acrylic anhydride and trichlorotoluene into the same 50 ml volumetric flask containing approximately 10 ml of dichloromethane. Dissolve and dilute to the mark with dichloromethane, and shake well. Measure 0.5 ml of the solution into a 10 ml volumetric flask containing approximately 5 ml of dichloromethane. Weigh approximately 60 mg of the test sample into the same volumetric flask, dilute to the mark with dichloromethane, and shake well. This is the system suitability solution.

[0139] (2) Chromatographic conditions

[0140] A chromatographic column (HP-5, 30m × 0.32mm, 0.25μm) with (5% phenyl)-diphenylmethylsiloxane (or similar polarity) as the stationary phase was used; nitrogen was used as the carrier gas; the initial temperature was 35℃, maintained for 5 minutes, and then increased to 210℃ at a rate of 30℃ / min, maintained for 8 minutes; the injection port temperature was 250℃; the detector temperature was 250℃; the column flow rate was 1.0 ml per minute; and the injection volume was 3.0 μL.

[0141] (3) Measurement

[0142] Accurately measure 3.0 μl each of the system suitability solution, impurity localization solution, and test solution, and inject them separately into the gas chromatograph, recording the chromatograms. The resolution between the main component and each impurity peak in the system suitability solution should meet the requirements. Calculate the content of acrylic anhydride, trichlorotoluene, and other individual impurities using the peak area normalization method. If there are chromatographic peaks of impurities propionyl chloride, 3-chloropropionyl chloride, and benzoyl chloride, do not integrate them. The content of each impurity should conform to the specifications in Table B below.

[0143] Table B. Impurity Limits

[0144] Impurity Name limit acrylic anhydride 0.5％ Trichlorotoluene 0.1％ Single impurity 0.5％

[0145] Example 2. Specificity

[0146] Potential impurities in ibrutinib SM3 include: acrylic anhydride, trichlorotoluene, propionyl chloride, 3-chloropropionyl chloride, benzoyl chloride, acetyl chloride, acrylic acid, and benzoic acid, totaling eight impurities. This example investigates acrylic anhydride, trichlorotoluene, and other individual impurities. The specific methods are as follows:

[0147] (1) Preparation of the test solution

[0148] Blank solution: solvent.

[0149] Acrylic anhydride stock solution: Accurately weigh about 300 mg and place it in a 50 ml volumetric flask containing about 20 ml of dichloromethane. Dissolve and dilute to the mark with dichloromethane and shake well.

[0150] Trichlorotoluene stock solution: Accurately weigh about 100 mg and place it in a 50 ml volumetric flask containing about 20 ml of dichloromethane. Dissolve and dilute to the mark with dichloromethane and shake well.

[0151] Propionyl chloride, 3-chloropropionyl chloride, benzoyl chloride, acetyl chloride, and acrylic acid stock solutions: Take about 50 μl of each of the above impurities and place them in a 25 ml volumetric flask containing about 10 ml of dichloromethane. Dissolve and dilute to the mark with dichloromethane and shake well.

[0152] Benzoic acid stock solution: Accurately weigh about 15 mg, place it in a 25 ml volumetric flask, dissolve and dilute with dichloromethane to the mark, and shake well.

[0153] For each impurity localization solution: accurately measure 0.5 ml of each of the above stock solutions, place them in 10 ml volumetric flasks, dilute to the mark with dichloromethane, and shake well.

[0154] Test solution: Take the test sample and add dichloromethane to prepare a solution containing approximately 6 mg per 1 ml.

[0155] Reference stock solution: Accurately measure 5 ml of acrylic anhydride stock solution and 3 ml of trichlorotoluene stock solution into a 100 ml volumetric flask, dilute to the mark with solvent, and shake well to obtain the reference solution.

[0156] Mixed solution: Take 0.5 ml of each stock solution of acyl chloride impurities, stock solution of acrylic acid and stock solution of benzoic acid, and 2.5 ml of stock solution of reference standard, put them into a 25 ml volumetric flask, then take the test sample into the same volumetric flask, add solvent to dissolve and dilute to prepare a solution containing 36 mg of ibrutinib SM per ml.

[0157] (2) Measurement

[0158] Take 3.0 μl each of the blank solution, the localization solution for each impurity, the test solution, and the mixed solution, and inject them sequentially, recording the chromatograms.

[0159] The measurement results are shown in Table C. Figures 1 to 11 Tables 1 to 11 show that the blank solvent and acetyl chloride did not interfere with the detection of impurities in the test sample. The resolution between the main component, acrylic anhydride, trichlorotoluene, and adjacent component peaks was not less than 3.51, indicating that the method specificity met the requirements.

[0160] Table C. Results of Specificity Test

[0161]

[0162] Note: As Figure 4 As shown in Table 4, acetyl chloride did not produce a peak in this system. This peak was located here to demonstrate that acetyl chloride does not interfere with the detection of benzoyl chloride, trichlorotoluene, and other individual impurities in this method.

[0163] Example 3. Limit of Quantification

[0164] Impurity Quantitative Limit Stock Solution: Accurately measure 1 ml of acrylic anhydride stock solution and 3 ml of trichlorotoluene stock solution, place them in the same 100 ml volumetric flask, dilute to the mark with dichloromethane, and shake well.

[0165] Stock solution of main component quantitation limit: Weigh an appropriate amount of ibrutinib SM3 accurately, place it in a volumetric flask containing dichloromethane, dilute with dichloromethane to prepare a solution containing 60 μg per ml, and shake well.

[0166] Limit of Quantification Solution: Take 2.5 ml each of the impurity limit of quantification stock solution and the main component limit of quantification stock solution, place them in a 50 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0167] The limit of quantitation solution was injected six times consecutively to obtain a chromatogram, and the ratio of the main peak height to the noise (signal-to-noise ratio) was calculated.

[0168] The test results are shown in Table D. Figure 12 See Table 12. Data shows that the limit of quantitation (LOQ) concentration for acrylic anhydride was 3.0750 μg / ml, expressed as 0.05% in the sample, with a 6-needle peak area RSD of 2.3% and a mean signal-to-noise ratio of 45.6; the LQ concentration for trichlorotoluene was 3.0132 μg / ml, expressed as 0.05% in the sample, with a 6-needle peak area RSD of 3.1% and a mean signal-to-noise ratio of 77.2; and the LQ concentration for ibrutinib SM3 was 3.0263 μg / ml, expressed as 0.05% in the sample, with a 6-needle peak area RSD of 2.7% and a mean signal-to-noise ratio of 20.2. All these results met the requirements for the LQ test.

[0169] Table D. Results of Limit of Quantitation Test

[0170]

[0171] Example 4. Detection Limit

[0172] Accurately measure 3.5 ml of the limit of quantitation solution and place it in a 10 ml volumetric flask. Dilute to the mark with solvent and mix well to obtain the limit of detection solution. Inject the limit of detection solution three times consecutively and calculate the ratio of the main peak height to the noise level (signal-to-noise ratio).

[0173] The test results are shown in Table E. Figure 13See Table 13. Data shows that the detection limit concentration (LOD) for acrylic anhydride was 1.0763 μg / ml, expressed as 0.018% of the concentration present in the sample, with a mean signal-to-noise ratio (SNR) of 14.1; the LOD concentration for trichlorotoluene was 1.1718 μg / ml, expressed as 0.020% of the concentration present in the sample, with a mean SNR of 25.3; and the LOD concentration for ibrutinib SM3 was 1.0592 μg / ml, expressed as 0.018% of the concentration present in the sample, with a mean SNR of 6.8. All of these met the requirements for the detection limit test.

[0174] Table E. Results of Limit of Quantitation Test

[0175]

[0176]

[0177] Example 5. Durability of chromatographic conditions

[0178] Take the mixed solution under “Example 2. Specificity” and test it under normal chromatographic conditions, different initial column temperatures (±2℃), column flow rates (±0.1ml / min), and heating rates (±2℃ / min) as planned. After the instrument system stabilizes, test and record the chromatograms to examine the changes in the resolution between the main component and each known impurity and adjacent components, as well as the changes in the normalized content of acrylic anhydride, trichlorotoluene, and other individual impurities.

[0179] The test results are shown in Tables F and G. Figures 14-19 Tables 14 to 19. When chromatographic conditions fluctuate slightly, the resolution between the test sample and each known impurity and adjacent components is not less than 3.16. The contents of acrylic anhydride, trichlorotoluene and the largest other single impurity are basically consistent by normalization, and the method robustness meets the requirements.

[0180] Table F. Summary of resolution between components in the chromatographic condition robustness test

[0181]

[0182] Table G. Results of Normalized Content Determination of Each Component in Chromatographic Condition Variation Test

[0183]

Claims

1. A method for separating ibrutinib SM3 and its impurities based on gas chromatography, characterized in that, The gas chromatography method includes: using a chromatographic column with (5% phenyl)-diphenylmethylsiloxane as the stationary phase, or other stationary phases of equivalent polarity; separating ibrutinib SM3 and its impurities using a temperature programmed process; the temperature programmed process is set as follows: initial temperature of 35±5℃, maintained for 5±1 minutes, increasing to 210±10℃ at a rate of 30±5℃ / min, and maintained for 8±2 minutes; The impurities include any one or more of acrylic anhydride, trichlorotoluene, propionyl chloride, 3-chloropropionyl chloride, benzoyl chloride, acrylic acid, and benzoic acid; the ibrutinib SM3 and the impurities together constitute the test composition; the structural formula of the ibrutinib SM3 is shown in Formula I.

2. The method according to claim 1, characterized in that, The test composition also contains acetyl chloride.

3. The method according to claim 1, characterized in that, The carrier gas was nitrogen; the injection port temperature was 250±20℃; the detector temperature was 250±20℃; and the column flow rate was 1.0±0.3 ml / min.

4. The method according to claim 1, characterized in that, The test solution was prepared using dichloromethane as a solvent.

5. The method according to claim 1, characterized in that, The detection limit (LOD) of ibrutinib SM3 is 1.0592 μg / ml and the quantitation limit (LOQ) is 3.0263 μg / ml; the LOD of acrylic anhydride is 1.0763 μg / ml and the LOQ is 3.0750 μg / ml; and the LOD of trichlorotoluene is 1.1718 μg / ml and the LOQ is 3.0132 μg / ml.

6. A method for identifying ibrutinib SM3 and its impurities, characterized in that, The test composition is separated by the method according to any one of claims 1-5 and detected by a detector to obtain a chromatogram; by comparing the chromatogram characteristics of the test sample and the reference sample, it is determined whether the test sample contains ibrutinib SM3 and its impurities.

7. The method according to claim 6, characterized in that, The components of the test composition can be identified according to their relative retention times. The components of the test composition are in ascending order as follows: ibrutinib SM3, propionyl chloride, acrylic acid, 3-chloropropionyl chloride, acrylic anhydride, benzoyl chloride, benzoic acid, and trichlorotoluene.

8. The method according to claim 6, characterized in that, Using ibrutinib SM3 as a reference peak, the relative retention time was 1.04, identified as propionyl chloride; 1.47, identified as acrylic acid; 1.96, identified as 3-chloropropionyl chloride; 2.17, identified as acrylic anhydride; 2.50, identified as benzoyl chloride; 2.60, identified as benzoic acid; and 2.70, identified as trichlorotoluene. The relative retention times of each component fluctuated within 5%.

9. A method for quantitative detection of ibrutinib SM3 and its impurities, characterized in that, The method described in any one of claims 6-8 is used to separate and identify ibrutinib SM3 and its impurities, and a chromatogram is obtained. Based on the obtained chromatogram, the content of acrylic anhydride, trichlorotoluene and other individual impurities in ibrutinib SM3 is calculated by peak area normalization method.

10. The method according to claim 9, characterized in that, The following chromatographic conditions were used for detection, and chromatograms were obtained: a chromatographic column with (5% phenyl)-diphenylmethylsiloxane as the stationary phase (30m × 0.32mm, 0.25μm) was used; nitrogen was used as the carrier gas; the temperature program was set as follows: initial temperature 35±2℃, held for 5 minutes, then increased to 210℃ at a rate of 30±2℃ / min, held for 8 minutes; the injection port temperature was 250℃; the detector temperature was 250℃. The column flow rate was 1.0 ± 0.1 ml / min.

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