Separation, identification and content determination method for starting material SM3 of ibrutinib and impurities thereof

Abstract

The invention belongs to the technical field of pharmaceutical analysis, and particularly relates to a method for separating, detecting and determining an initial raw material SM3 and impurities thereof of ibrutinib. The impurities comprise one or more of propionyl chloride, 3-chloropropionyl chloride and benzoyl chloride. A mobile phase is a phosphate buffer solution as a mobile phase A, acetonitrile is a mobile phase B, a chromatographic column adopts octadecylsilane chemically bonded silica as a filler, and linear gradient elution of high performance liquid chromatography is carried out after pre-column derivatization. And then, according to a chromatogram, an external standard method is adopted to calculate the impurities according to a peak area. The method has the characteristics of good separation degree, good durability, high sensitivity and good reproducibility.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical analysis technology, specifically relating to a method for separating and detecting the starting material SM3 of ibrutinib and its impurities. Background Technology

[0002] Ibrutinib, also known as ibrutinib, is a Bruton's tyrosine kinase (BTK) inhibitor used to treat chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL). The synthesis of ibrutinib often involves seven impurities: acrylic anhydride, trichlorotoluene, acrylic acid, benzoic acid, propionyl chloride, 3-chloropropionyl chloride, and benzoyl chloride. Among these, propionyl chloride, 3-chloropropionyl chloride, and benzoyl chloride are genotoxic impurities. According to ICH M7(R1) Method 3, a standard higher than the acceptable limit for these impurities in the starting material IBR-SM3 must be established.

[0003] However, there is currently no method for detecting the three gene impurities.

[0004] For example, gas chromatography (GC) methods cannot be implemented. This is because propionyl chloride, 3-chloropropionyl chloride, benzoyl chloride, the starting material IBR-SM3, and any potential impurities have low melting and boiling points. While GC could be considered for determination, these impurities are highly reactive and unstable, easily reacting with the stationary phase of the GC column, compromising sample accuracy. Furthermore, they can corrode equipment, meaning propionyl chloride, 3-chloropropionyl chloride, and benzoyl chloride cannot be quantitatively determined by GC, compromising sample accuracy and potentially damaging GC column injectors and other GC equipment. Additionally, propionyl chloride, 3-chloropropionyl chloride, benzoyl chloride, the starting material IBR-SM3, and any potential impurities are highly irritating and toxic. Direct GC exposure exposes the sample to air, and the volatilized acyl chloride solvent gases pose significant health risks, making the experiment inherently dangerous.

[0005] Currently, there are no literature reports on using high-performance liquid chromatography (HPLC) as a detection method. For example, CN111007157A discloses a purity detection method in the preparation process of ibrutinib. Before the intermediate 003 reacts with acryloyl chloride, the optical purity of the ibrutinib intermediate is quantitatively analyzed by HPLC, and the content of the R-isomer is determined by HPLC to determine the purity of ibrutinib obtained by the reaction of the R-isomer and the acryloyl chloride. However, this document does not disclose specific impurities and differs significantly from the chromatographic conditions of this invention. As another example, CN104407067A discloses a HPLC separation and detection method for ibrutinib and its enantiomer impurities, belonging to the field of separation and detection technology. The detection method includes: using an AD-H, 4.6mm x 250mm, 5μm chiral column as the separation column; using a mixed solvent of alkane and alcohol as the mobile phase, with a volume ratio of alkane to alcohol of 35:65. This method can effectively separate and detect ibrutinib and its enantiomer impurities. The impurities of this invention are not disclosed and differ significantly from the chromatographic conditions of this invention.

[0006] Currently, there is an urgent need to develop a method for detecting three types of gene impurities. Summary of the Invention

[0007] One of the objectives of this invention is to provide a method for separating and determining the starting material SM3 and its impurities for ibrutinib, which can complete the separation of multiple substances in a short time.

[0008] To achieve the above objectives, the technical solution of the present invention is as follows:

[0009] A method for separating and determining the starting material SM3 and its impurities for ibrutinib using high-performance liquid chromatography (HPLC), wherein the starting material SM3 and the impurities together constitute a composition, and the impurities include any one or more of propionyl chloride, 3-chloropropionyl chloride, and benzoyl chloride; the composition is derivatized before column chromatography to generate derivatives of the starting material SM3, propionyl chloride derivatives, 3-chloropropionyl chloride derivatives, and benzoyl chloride derivatives, wherein the derivatizing reagent is 1-(4-nitrophenyl)piperazine; the derivatized compounds are separated by HPLC; the structural formulas of the above compounds are shown in Table A.

[0010] The separated material can be used in the next stage of production.

[0011] The aforementioned impurities can be arranged and combined in various ways. For example, combination 1: the starting material SM3 and propionyl chloride. Another example is combination 2: the starting material SM3 and 3-chloropropionyl chloride. Yet another example is combination 3: the starting material SM3 and benzoyl chloride.

[0012] All possible permutations and combinations will not be listed here. Theoretically, when the upper limit of the substances that this method can separate, identify and / or detect is n (where n is the number of substances), it can naturally detect 1 to n substances.

[0013] The compositions with structural formulas as described in I-IV involve four components, which are typically mixed together with four other components (due to the synthetic process route). This method requires not only separating the composition from the other four components, but also separating each component within the composition independently. The other four components are acrylic anhydride, trichlorotoluene, acrylic acid, and benzoic acid.

[0014] As a preferred embodiment, the linear gradient elution procedure is as follows:

[0015] Time - minutes Mobile phase A - (volume parts) Mobile phase B - (volume parts) 0 70±7 30±3 5±0.5 70±7 30±3 25±0.5 45±4.5 55±5.5 30±0.5 20±2 80±8 35±0.5 20±2 80±8 36±0.5 70±7 30±3 45±0.5 70±7 30±3

[0016] Preferably, the linear gradient elution procedure is as follows:

[0017] Time - minutes Mobile phase A - (volume parts) Mobile phase B - (volume parts) 0 70 30 5 70 30 25 45 55 30 20 80 35 20 80 36 70 30 45 70 30

[0018] As a preferred embodiment, an example of the preparation method for the phosphate buffer in the mobile phase is as follows: dissolve 1.36g of potassium dihydrogen phosphate in 1000ml of water, and adjust the pH to 3.0 with phosphoric acid.

[0019] The pH value, except for the standard value of 3.0, can be in the range of 2.8-3.2. For example, when the pH value is 2.8 ( Figure 14 ); or when the pH value is 3.2 ( Figure 15 ).

[0020] As a preferred embodiment, the pre-column derivatization specifically involves heating propionyl chloride, 3-chloropropionyl chloride, benzoyl chloride, and 1-(4-nitrophenyl)piperazine at 40-60°C for at least 20 minutes to generate derivatives of the starting material SM3, propionyl chloride derivatives, 3-chloropropionyl chloride derivatives, and benzoyl chloride derivatives.

[0021] As a preferred option, the heating method can be a water bath. The heating temperature can be 45℃. Figure 4 ); or the heating temperature can be 50℃ ( Figure 3 , Figure 6 and Figure 7 The heating temperature can be 55℃. Figure 5 Heating time should be 25-35 minutes.

[0022] As a preferred embodiment, in the method described, the initial volume ratio of the mobile phase at 0 minutes, the volume ratio of mobile phase A to mobile phase B, is 68-72:32-28. For example, the volume ratio of mobile phase A to mobile phase B is 68:32. Figure 12For example, the volume ratio of mobile phase A to mobile phase B is 72:28. Figure 13 ).

[0023] As a preferred method, the flow rate is 1.0-1.2 mL / min; the column temperature is 20-40 °C. For example, the flow rate is 1.0 mL / min ( Figure 10 ); or a flow rate of 1.2 mL / min ( Figure 11 For example, the column temperature is 28℃. Figure 8 ); or column temperature is 32℃ ( Figure 9 ).

[0024] The second objective of this invention is to provide a method for identifying and determining the starting material SM3 and its impurities in ibrutinib, which can complete the identification of multiple substances in a short time.

[0025] To achieve the above objectives, the technical solution of the present invention is as follows:

[0026] A method for identifying and determining the starting material SM3 and its impurities for ibrutinib involves separating the composition using the method described above 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 the starting material SM3 and its impurities for ibrutinib determination.

[0027] As a preferred embodiment, the detection wavelength of the detector is 228 ± 10 nm. The ± 10 nm setting range is based on a comprehensive consideration of factors such as error tolerance, methodological superiority, and practical application requirements. This setting range helps ensure the reliability of the detection results, improves the repeatability and flexibility of the measurement, and meets the requirements of specific experiments.

[0028] As a preferred embodiment, the components and / or their corresponding derivatives in the composition can be identified according to the order of retention time. The derivatives corresponding to each component, in ascending order, are: the derivative of the starting material SM3, the propionyl chloride derivative, the 3-chloropropionyl chloride derivative, and the benzoyl chloride derivative. As a preferred embodiment, the specific retention time is:

[0029]

[0030] Retention time refers to the time required for a sample to travel from the point of entry into the chromatographic column to its detection by the detector. This time is calculated based on the migration velocity of the components on the column, specifically the time interval from the start of injection to the chromatographic peak (maximum concentration) of a particular component. It is primarily used to determine the elution order and position of each component in a sample and is one of the fundamental data points 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.

[0031] Besides retention time, relative retention time can also be used to determine impurities. Relative retention time describes the relative retention degree 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 chromatographic column and is an important parameter used for localization, qualitative, and quantitative analysis in chromatographic analysis. The relative retention time is calculated by dividing the retention time of the target component (tR) by the retention time of the reference component (tR0).

[0032] The third objective of this invention is to provide a method for determining the content of SM3, the starting material of ibrutinib, and its impurities. This method can complete the identification and content determination of multiple substances in a short time.

[0033] To achieve the above objectives, the technical solution of the present invention is as follows:

[0034] The method described above was used to separate and identify the starting material SM3 and its impurities for ibrutinib, and chromatograms were obtained. Based on the obtained chromatograms, impurities were calculated by peak area using the external standard method.

[0035] Known

[0036] In the formula: C S —The concentration of known impurities in the reference solution, in μg / ml;

[0037] Cx—Concentration of the test solution, μg / ml;

[0038] Ax—Peak area of ​​known impurities in the test solution;

[0039] Ps—The content of known impurity reference standards in the reference standard solution;

[0040] As—the peak area of ​​known impurities in the reference solution.

[0041] The content determination method can be used to further determine whether the content of the starting material SM3 and its impurities in ibrutinib is up to standard. If the peak area of ​​one or more impurities among propionyl chloride, 3-chloropropionyl chloride, and benzoyl chloride is greater than the peak area of ​​the corresponding impurity in the reference solution, it indicates that the impurity content is not up to standard; conversely, if the peak area of ​​one or more impurities among propionyl chloride, 3-chloropropionyl chloride, and benzoyl chloride is not greater than the peak area of ​​the corresponding impurity in the reference solution, it indicates that the impurity content is up to standard.

[0042] The aforementioned judgment method can serve as a drug quality assessment model and further as an indispensable key module in intelligent production processes. This model, through precise control of parameters such as mobile phase composition, flow rate, and column temperature, achieves accurate separation and quantitative analysis of active ingredients, impurities, and degradation products in drugs, providing a scientific basis for comprehensive drug quality assessment. In intelligent production systems, this model is seamlessly integrated, capable of receiving raw data from the production line in real time, automatically executing analysis tasks, and rapidly providing judgment results based on preset quality standards.

[0043] Furthermore, a fourth objective of the present invention is to provide a novel application of 1-(4-nitrophenyl)piperazine that can enhance the UV response of the three impurities.

[0044] To achieve the above objectives, the technical solution of the present invention is as follows:

[0045] The 1-(4-nitrophenyl)piperazine is used as a pre-column derivatization reagent in the composition, which contains three components: propionyl chloride, 3-chloropropionyl chloride, and benzoyl chloride.

[0046] The beneficial effects of this invention are as follows: It innovates the high-performance liquid chromatography (HPLC) method. By using a pre-column derivatization method with heating in a 50°C water bath for 30 minutes, propionyl chloride, 3-chloropropionyl chloride, and benzoyl chloride, which have very weak UV responses, are derivatized with 1-(4-nitrophenyl)piperazine to produce derivatives with stronger UV responses, thus achieving high sensitivity in the analytical method. Simultaneously, within 45 minutes, the propionyl chloride derivative, 3-chloropropionyl chloride derivative, and benzoyl chloride derivative are effectively separated from IBR-SM3 and any possible impurities or their derivatives. This solves the problem of separating and determining propionyl chloride, 3-chloropropionyl chloride, and benzoyl chloride impurities in the starting material IBR-SM3, which has not been addressed by existing technologies. The derivatization of the sample (starting material IBR-SM3 and any possible impurities) inactivates and devolatiles the sample, reducing human harm and ensuring experimental safety. This analytical method is short in time, highly sensitive, highly specific, reproducible, simple and feasible to operate, and safe. Attached Figure Description

[0047] Figure 1 The chromatogram is of the mixed solution;

[0048] Figure 2 Chromatogram of the solution at the detection limit;

[0049] Figure 3 Chromatogram for derivatization condition robustness (heated at -50°C for 30 min);

[0050] Figure 4 Chromatogram for derivatization condition robustness after heating at -45°C for 30 min;

[0051] Figure 5 Chromatogram for derivatization condition robustness (heated at -55°C for 30 min);

[0052] Figure 6 Chromatogram for derivatization condition robustness after heating at -50°C for 25 min;

[0053] Figure 7 Chromatogram for derivatization condition robustness at -50°C for 35 min;

[0054] Figure 8 The chromatogram is for a mixed-robust column at a temperature of 28°C.

[0055] Figure 9 The chromatogram is for a mixed-durability column at 32°C.

[0056] Figure 10 Chromatogram for a mixed-durability flow rate of 1.0 / min;

[0057] Figure 11 Chromatogram for a mixed-durability flow rate of 1.2 / min;

[0058] Figure 12 Chromatogram for a mixed-durability starting volume ratio of 68:32;

[0059] Figure 13 Chromatogram for a mixed-durability starting volume ratio of 72:28;

[0060] Figure 14 Chromatogram for mixed-durability pH 2.8;

[0061] Figure 15 Chromatogram for mixed-durability pH 3.2;

[0062] Figure 16 This is the chromatogram of the solution at the limit of quantitation.

[0063] in, Figure 1-16 The terms "SM3, propionyl chloride, 3-chloropropionyl chloride, and benzoyl chloride" mentioned in Table 1-16 actually refer to "SM3 derivatives, propionyl chloride derivatives, 3-chloropropionyl chloride derivatives, and benzoyl chloride derivatives." The word "derivatives" is omitted to minimize the overlap between the text and the chromatographic peaks. Detailed Implementation

[0064] 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.

[0065] Supplementary tables to the accompanying drawings in the specification. Included in this patent. Figure 1-16 Visual aids are provided for understanding and interpretation. In case of any ambiguity, users should refer to the corresponding numbered tables (Tables 1-16) for more detailed information. Conversely, if any potentially misleading or ambiguous information is found during the review of Tables 1-16, the content of the corresponding numbered figures should be considered authoritative. The above guidelines aim to ensure the correct interpretation of this document and the consistency of its information. Although some text overlaps in the spectra of this application, it is still clearly legible, and the specification details the integration results of each figure. Furthermore, the numbers in the spectra do not affect the scope of protection of the claims or the full disclosure of the technical solutions in the specification.

[0066] Table 1

[0067]

[0068] Table 2

[0069]

[0070] Table 3

[0071]

[0072] Table 4

[0073]

[0074] Table 5

[0075]

[0076] Table 6

[0077]

[0078] Table 7

[0079]

[0080] Table 8

[0081]

[0082] Table 9

[0083]

[0084] Table 10

[0085]

[0086] Table 11

[0087]

[0088] Table 12

[0089]

[0090] Table 13

[0091]

[0092] Table 14

[0093]

[0094] Table 15

[0095]

[0096] Table 16

[0097]

[0098] 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.

[0099] 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.

[0100] If any chromatographic conditions are not mentioned, please refer to the high performance liquid chromatography method (Chinese Pharmacopoeia 2020 Edition, Part IV, 0512 for determination).

[0101] the term

[0102] The limit of quantitation (LOQ) is the lowest amount of an analyte in a sample that can be quantitatively determined, and the measurement result should have a certain degree of accuracy and precision. In other words, the LQ is the lowest level at which an analytical method can accurately and reliably determine the concentration of the analyte in a sample. In HPLC, the determination of the LQ usually relies on the signal-to-noise ratio (S / N) method, that is, the concentration of the analyte corresponding to a certain level of signal-to-noise ratio is taken as the LQ. Determining the LQ is crucial for ensuring the accuracy and reliability of analytical results.

[0103] 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.

[0104] 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.

[0105] The peak height to noise ratio (S / N, or signal-to-noise ratio) is used in high-performance liquid chromatography (HPLC) to evaluate the detection sensitivity and resolution of an instrument, and is an important indicator of instrument performance. Peak height refers to the signal value output by the detector when the analyte elutes from the column; noise refers to the fluctuation of the baseline signal, i.e., the signal value measured for a blank sample. The signal-to-noise ratio is the ratio of the signal measured for a sample of known concentration to the signal measured for a blank sample. A higher signal-to-noise ratio means that the instrument can more accurately separate and identify the target component when detecting samples, while also reducing interference from background noise.

[0106] Derivatization peaks refer to the analyte (usually a compound without direct UV absorption or with low detection sensitivity) reacting chemically with the derivatizing reagent during pre-column derivatization in high-performance liquid chromatography (HPLC), generating compounds with UV absorption, fluorescence, or other detectable properties. When these newly formed compounds are separated by the chromatographic column, they produce corresponding chromatographic peaks on the detector; these peaks are called derivatization peaks.

[0107] In this embodiment of the invention, because the related substances analysis method needs to separate a large number of impurities, and the impurities have significant differences in polarity and response, as well as significant differences in solubility between the impurities and the sample, it is necessary to elute from a low-proportion organic phase to a high-proportion organic phase. To ensure accurate quantification of each impurity, the external standard method using impurity reference standards is employed to quantitatively calculate each known impurity.

[0108] In this patent, IBR-SM3 is also referred to as the starting material SM3 or SM3 of ibrutinib. The absence of superscript or subscript does not affect the definition of the patent. For example, SM3 is equivalent to SM3.

[0109] In this embodiment of the invention, information about SM3 and its related impurities is shown in Table A.

[0110] Table A: Compound Information Table

[0111]

[0112]

[0113] Table B Gradient Elution Procedure

[0114] Time - minutes Mobile phase A - (volume parts) Mobile phase B - (volume parts) 0 70 30 5 70 30 25 45 55 30 20 80 35 20 80 36 70 30 45 70 30

[0115] Example 1

[0116] Stock solution of test sample: Weigh 50 mg of the test sample accurately and place it in a 10 ml volumetric flask containing about 2 ml of acetonitrile. Dissolve and dilute to the mark with acetonitrile and shake well.

[0117] Test solution: Weigh 25 mg of 1-(4-nitrophenyl)piperazine and place it in a 10 ml volumetric flask. Add 0.5 ml of acetonitrile and sonicate for 1 minute. Accurately measure 1 ml of the test stock solution and place it in the same 10 ml volumetric flask. Add 2 ml of acetonitrile and shake. Add 25 μl of triethylamine and shake. Place in a 50 °C water bath for 30 minutes. Remove and cool. Dissolve and dilute to the mark with acetonitrile and shake well.

[0118] Reference stock solution: Accurately weigh 200 mg of 3-chloropropionyl chloride, 100 mg of propionyl chloride, and 100 mg of benzoyl chloride, and place them in a 50 ml volumetric flask containing about 10 ml of acetonitrile. Dissolve and dilute to the mark with acetonitrile, and shake well. Accurately measure 5 ml of the solution and place it in a 50 ml volumetric flask. Dissolve and dilute to the mark with acetonitrile, and shake well. Accurately measure 2.5 ml of the solution and place it in a 100 ml volumetric flask. Dissolve and dilute to the mark with acetonitrile, and shake well.

[0119] Reference solution: Weigh 25 mg of 1-(4-nitrophenyl)piperazine and place it in a 10 ml volumetric flask. Add 0.5 ml of acetonitrile and sonicate for 1 minute. Accurately measure 1 ml of the reference stock solution and place it in the same 10 ml volumetric flask. From the point “add 2 ml of acetonitrile and shake”, the preparation method is the same as that for the test solution.

[0120] System suitability solution: Measure 1 ml of the test solution and 1 ml of the reference solution and mix well.

[0121] Chromatographic conditions: Octadecylsilane-bonded silica gel was used as the stationary phase (Kromasil 100-5-C18, 4.6 mm × 250 mm, 5 μm or equivalent column). Phosphate buffer (1.36 g potassium dihydrogen phosphate dissolved in 1000 ml of water, pH adjusted to 3.0 with phosphoric acid) was used as mobile phase A, and acetonitrile was used as mobile phase B. Linear gradient elution was performed according to Table B. The flow rate was 1.1 ml / min; the column temperature was 30 °C; the detection wavelength was 228 nm; and the injection volume was 10 μl.

[0122] System suitability requirements: In the system suitability solution chromatogram, the resolution between the main component derivative peak and the propionyl chloride derivative peak should meet the requirements.

[0123] Assay: Accurately measure the test solution and the reference solution, inject them separately into the liquid chromatograph, and record the chromatograms.

[0124] Limits: In the chromatogram of the test solution, the content of impurities propionyl chloride, 3-chloropropionyl chloride, and benzoyl chloride is calculated by peak area using the external standard method.

[0125] Calculation formula:

[0126] Known

[0127] Where: CS—concentration of known impurities in the reference solution, μg / ml;

[0128] Cx—Concentration of the test solution, μg / ml;

[0129] Ax—Peak area of ​​known impurities in the test solution;

[0130] Ps—The content of known impurity reference standards in the reference standard solution;

[0131] As—the peak area of ​​known impurities in the reference solution.

[0132] Example 2 Specificity

[0133] Other potential impurities in the starting material IBR-SM3 include propionyl chloride, 3-chloropropionyl chloride, benzoyl chloride, acrylic anhydride, trichlorotoluene, benzoic acid, and acrylic acid, totaling seven impurities. We investigated the separation of propionyl chloride, 3-chloropropionyl chloride, benzoyl chloride derivatives, derivatizing reagents, IBR-SM3 and the other four impurities, or derivatives of IBR-SM3 and the other four impurities.

[0134] Solvent: Acetonitrile

[0135] Stock solutions of acyl chloride impurities (propionyl chloride, 3-chloropropionyl chloride, benzoyl chloride): Accurately weigh an appropriate amount of the acyl chloride impurity, dissolve it in acetonitrile, and quantitatively dilute it to prepare a solution containing approximately 2 mg per milliliter. (Prepare stock solutions of propionyl chloride, 3-chloropropionyl chloride, and benzoyl chloride separately.)

[0136] Positioning solutions for acyl chloride impurities (propionyl chloride, 3-chloropropionyl chloride, benzoyl chloride) derivatives: Weigh 25 mg of 1-(4-nitrophenyl)piperazine and place it in a 10 ml volumetric flask. Add 0.5 ml of acetonitrile and sonicate for 1 minute. Measure 30 μl of the acyl chloride impurity stock solution and place it in the same 10 ml volumetric flask. Add 2 ml of acetonitrile and shake. Add 25 μl of triethylamine and shake. Incubate at 50°C for 30 minutes. Remove, cool, dissolve in acetonitrile, and dilute to the mark. Shake well. (Prepare positioning solutions for propionyl chloride, 3-chloropropionyl chloride, and benzoyl chloride derivatives separately.)

[0137] Other impurity mixed solution: Take appropriate amounts of benzoic acid, acrylic acid, acrylic anhydride and trichlorotoluene, dissolve and dilute with acetonitrile to prepare a solution containing approximately 40 μg of benzoic acid and 80 μl each of acrylic acid, acrylic anhydride and trichlorotoluene per milliliter.

[0138] Other impurities mixed derivatization localization solution: Weigh 25 mg of 1-(4-nitrophenyl)piperazine and place it in a 10 ml volumetric flask. Add 0.5 ml of acetonitrile and sonicate for 1 minute. Measure 0.25 ml of the other impurities mixed solution and place it in the same 10 ml volumetric flask. Add 2 ml of acetonitrile and shake. Add 25 μl of triethylamine and shake. Place in a 50 °C water bath for 30 minutes. Remove and cool. Dissolve and dilute to the mark with acetonitrile and shake well.

[0139] Stock solution of test sample: Take an appropriate amount of test sample, accurately weigh it, dissolve it in acetonitrile and dilute it quantitatively to prepare a solution containing about 5 mg per milliliter.

[0140] Test solution: Weigh 25 mg of 1-(4-nitrophenyl)piperazine and place it in a 10 ml volumetric flask. Add 0.5 ml of acetonitrile and sonicate for 1 minute. Accurately measure 1 ml of the test stock solution and place it in the same 10 ml volumetric flask. Add 2 ml of acetonitrile and shake. Add 25 μl of triethylamine and shake. Place in a 50 °C water bath for 30 minutes. Remove and cool. Dissolve and dilute to the mark with acetonitrile and shake well.

[0141] Reference stock solution: Accurately measure appropriate amounts of propionyl chloride, 3-chloropropionyl chloride and benzoyl chloride stock solutions, and quantitatively dilute with acetonitrile to prepare a solution containing approximately 5 μg each of propionyl chloride and benzoyl chloride and 7.5 μg of 3-chloropropionyl chloride per milliliter.

[0142] Mixed solution: Weigh 25 mg of 1-(4-nitrophenyl)piperazine and place it in a 10 ml volumetric flask. Add 0.5 ml of acetonitrile and sonicate for 1 minute. Take 1 ml of the test sample stock solution, 0.25 ml of the mixed solution of other impurities, and 1 ml of the reference stock solution and place them in the same 10 ml volumetric flask. Add 2 ml of acetonitrile and shake. Add 25 μl of triethylamine and shake. Place in a 50 °C water bath for 30 minutes. Remove and cool. Dissolve and dilute to the mark with acetonitrile and shake well.

[0143] Derivatizing reagent (blank solution): Weigh 25 mg of 1-(4-nitrophenyl)piperazine and place it in a 10 ml volumetric flask. Add 0.5 ml of acetonitrile, sonicate for 1 minute, add 2 ml of acetonitrile, shake, add 25 μl of triethylamine, shake, place in a 50 °C water bath for 30 minutes, remove, cool, dissolve and dilute to the mark with acetonitrile, and shake well.

[0144] Take 10 μl each of the solvent, derivatizing reagent solution, propionyl chloride positioning solution (derivative), 3-chloropropionyl chloride positioning solution (derivative), benzoyl chloride positioning solution (derivative), other impurity mixed solution (after derivatization), test solution (IBR-SM3 derivative), and mixed solution (after derivatization), inject them according to the method, record the chromatogram, and the determination results are shown in Table C or Figure 1 .

[0145] Table C: Results of Specificity Test

[0146]

[0147]

[0148] Conclusion: Solvents, derivatizing reagents (blank solution), main components, and other possible impurities do not interfere with the detection of each acyl chloride impurity. The peak-to-peak resolution between each acyl chloride impurity derivative and adjacent components is not less than 3.25, and the method specificity meets the requirements.

[0149] Example 3 Limit of Quantification

[0150] Propionyl chloride stock solution, 3-chloropropionyl chloride stock solution, benzoyl chloride stock solution: prepared according to the preparation method of each stock solution under the "Specificity of Example 2" section.

[0151] Limit of Quantitation (LOQ) Stock Solutions: Accurately measure appropriate amounts of propionyl chloride stock solution, 3-chloropropionyl chloride stock solution, and benzoyl chloride stock solution, and dilute with acetonitrile to prepare a solution containing approximately 1 μg of each per milliliter. (Relative concentration 0.2%)

[0152] Limit of Quantitation (LOQ) solution: Weigh 25 mg of 1-(4-nitrophenyl)piperazine into a 10 ml volumetric flask, add 0.5 ml of acetonitrile, and sonicate for 1 minute. Accurately measure 1 ml of the LOQ stock solution and place it in the same 10 ml volumetric flask. Add 2 ml of acetonitrile, shake, add 25 μl of triethylamine, shake, and incubate in a 50°C water bath for 30 minutes. Remove, cool, dissolve and dilute to the mark with acetonitrile, and mix well. (0.02%)

[0153] The above-mentioned limit of quantitation solution was injected six times consecutively, and the peak height to noise ratio (S / N) of each acyl chloride impurity derivative was calculated.

[0154] Table D shows the results of the determination of the limit of quantitation. Figure 16 )

[0155]

[0156] The limit of quantitation (LOQ) concentration for propionyl chloride was 0.1037 μg / ml, expressed as 0.021% in the sample, with a derivative peak area RSD of 0.9% and a mean signal-to-noise ratio of 23.8; the LOQ concentration for 3-chloropropionyl chloride was 0.1044 μg / ml, expressed as 0.021% in the sample, with a derivative peak area RSD of 0.5% and a mean signal-to-noise ratio of 24.2; and the LOQ concentration for benzoyl chloride was 0.1179 μg / ml, expressed as 0.024% in the sample, with a derivative peak area RSD of 0.5% and a mean signal-to-noise ratio of 26.7. All of these met the requirements for the limit of quantitation test.

[0157] Example 4 Detection Limit

[0158] Propionyl chloride stock solution, 3-chloropropionyl chloride stock solution, benzoyl chloride stock solution: prepared according to the preparation method of each stock solution under the "Specificity of Example 2" section.

[0159] Detection limit stock solutions: Accurately measure appropriate amounts of propionyl chloride stock solution, 3-chloropropionyl chloride stock solution, and benzoyl chloride stock solution, and dilute with acetonitrile to prepare a solution containing approximately 1 μg of each per milliliter (relative concentration 0.2%).

[0160] Limit of detection solution: Weigh 25 mg of 1-(4-nitrophenyl)piperazine and place it in a 10 ml volumetric flask. Add 0.5 ml of acetonitrile and sonicate for 1 minute. Accurately measure 0.5 ml of the limit of detection stock solution and place it in the same 10 ml volumetric flask. Add 2 ml of acetonitrile and shake. Add 25 μl of triethylamine and shake. Incubate in a 50°C water bath for 30 minutes. Remove, cool, dissolve and dilute to the mark with acetonitrile, and mix well. (0.01%)

[0161] Inject the above-mentioned limit-of-detection solution three times consecutively, and calculate the peak height to noise ratio (S / N) of each acyl chloride impurity derivative. Record the chromatograms; the experimental results are shown in the table below.

[0162] Table E shows the results of the detection limit determination. Figure 2 (Atlas attached)

[0163]

[0164] The detection limit concentration of propionyl chloride was 0.0519 μg / ml, which is expressed as 0.010% in the sample, with a mean signal-to-noise ratio of 11.2; the detection limit concentration of 3-chloropropionyl chloride was 0.0522 μg / ml, which is expressed as 0.010% in the sample, with a mean signal-to-noise ratio of 13.3; and the detection limit concentration of benzoyl chloride was 0.0590 μg / ml, which is expressed as 0.012% in the sample, with a mean signal-to-noise ratio of 16.9. All of these concentrations meet the requirements for detection limit testing.

[0165] Changes in the derived conditions of Example 5

[0166] Test sample stock solution: Prepared according to the test sample stock solution preparation method under “Specificity in Example 2”.

[0167] Reference stock solution: Prepared according to the preparation method of reference stock solution under the "Specificity of Example 2" section.

[0168] Test solution 1: Weigh 25 mg of 1-(4-nitrophenyl)piperazine and place it in a 10 ml volumetric flask. Add 0.5 ml of acetonitrile and sonicate for 1 minute. Accurately measure 1 ml of the test sample stock solution and 1 ml of the reference stock solution and place them in the same 10 ml volumetric flask. Add 2 ml of acetonitrile and shake. Add 25 μl of triethylamine and shake. Place in a 50 °C water bath for 30 minutes. Remove and cool. Dissolve and dilute to the mark with acetonitrile and shake well.

[0169] Test solution 2: Weigh 25 mg of 1-(4-nitrophenyl)piperazine and place it in a 10 ml volumetric flask. Add 0.5 ml of acetonitrile and sonicate for 1 minute. Accurately measure 1 ml of the test sample stock solution and 1 ml of the reference stock solution and place them in the same 10 ml volumetric flask. Add 2 ml of acetonitrile and shake. Add 25 μl of triethylamine and shake. Place in a 45 °C water bath for 30 minutes. Remove and cool. Dissolve and dilute to the mark with acetonitrile and shake well.

[0170] Test solution 3: Weigh 25 mg of 1-(4-nitrophenyl)piperazine and place it in a 10 ml volumetric flask. Add 0.5 ml of acetonitrile and sonicate for 1 minute. Accurately measure 1 ml of the test sample stock solution and 1 ml of the reference stock solution and place them in the same 10 ml volumetric flask. Add 2 ml of acetonitrile and shake. Add 25 μl of triethylamine and shake. Place in a 55 °C water bath for 30 minutes. Remove and cool. Dissolve and dilute to the mark with acetonitrile and shake well.

[0171] Test solution 4: Weigh 25 mg of 1-(4-nitrophenyl)piperazine and place it in a 10 ml volumetric flask. Add 0.5 ml of acetonitrile and sonicate for 1 minute. Accurately measure 1 ml of the test sample stock solution and 1 ml of the reference stock solution and place them in the same 10 ml volumetric flask. Add 2 ml of acetonitrile and shake. Add 25 μl of triethylamine and shake. Place in a 50 °C water bath for 35 minutes. Remove and cool. Dissolve and dilute to the mark with acetonitrile and shake well.

[0172] Test solution 5: Weigh 25 mg of 1-(4-nitrophenyl)piperazine and place it in a 10 ml volumetric flask. Add 0.5 ml of acetonitrile and sonicate for 1 minute. Accurately measure 1 ml of the test sample stock solution and 1 ml of the reference stock solution and place them in the same 10 ml volumetric flask. Add 2 ml of acetonitrile and shake. Add 25 μl of triethylamine and shake. Place in a 50 °C water bath for 25 minutes. Remove and cool. Dissolve and dilute to the mark with acetonitrile and shake well.

[0173] Take samples 1–5 of the test solutions respectively and detect them under the aforementioned chromatographic conditions, then record the chromatograms. Calculate the RSD of the peak area for each known impurity derivative.

[0174] Table F shows the experimental data on changes in derived conditions. Figures 3-7 )

[0175]

[0176] Conclusion: When there are slight fluctuations in derivation temperature and derivation time, the RSD values ​​of the peak areas of each acyl chloride impurity derivative are all less than 5.0%, indicating good robustness of the derivation conditions.

[0177] Example 6: Chromatographic Condition Durability

[0178] Take the mixed solution under “Specificity of Example 2” and test it under normal chromatographic conditions, different column temperatures (±2℃), column flow rates (±0.1ml / min), initial mobile phase ratios (±1%), and buffer salt pH values ​​(±0.2). After the instrument system stabilizes, test the solutions and record the chromatograms to examine the changes in the resolution between the peaks of each component.

[0179] Table G shows the resolution between components in the chromatographic condition variation test. Figure 1 , Figures 8-15 )

[0180]

[0181] Conclusion: When chromatographic conditions fluctuate slightly, the peak resolution between each acyl chloride impurity derivative and its neighboring components is not less than 3.10, and the method robustness meets the requirements.

Claims

1. A method for separating and determining the starting material SM3 and its impurities of ibrutinib based on high performance liquid chromatography, characterized in that, The starting material SM3 for determining ibrutinib and the impurities together constitute a composition, wherein the impurities include any one or more of propionyl chloride, 3-chloropropionyl chloride, and benzoyl chloride; the composition is derivatized before a column to generate derivatives of the starting material SM3, propionyl chloride derivatives, 3-chloropropionyl chloride derivatives, and benzoyl chloride derivatives, wherein the derivatizing reagent is 1-(4-nitrophenyl)piperazine, and the derivatized compounds are separated by high performance liquid chromatography; the structural formulas of the above compounds are as follows: Mobile phase: phosphate buffer as mobile phase A, acetonitrile as mobile phase B; stationary phase: octadecylsilane-bonded silica gel as the packing material for linear gradient elution.

2. The method according to claim 1, characterized in that, The procedure for linear gradient elution is as follows:

3. The method according to claim 1, characterized in that, The pre-column derivatization specifically involves heating propionyl chloride, 3-chloropropionyl chloride, benzoyl chloride, and 1-(4-nitrophenyl)piperazine at 40-60°C for at least 20 minutes to generate derivatives of the starting material SM3, propionyl chloride derivatives, 3-chloropropionyl chloride derivatives, and benzoyl chloride derivatives.

4. The method according to claim 1, characterized in that, In the mobile phase, the initial volume ratio of mobile phase A to mobile phase B is 68-72:32-28.

5. The method according to claim 1, characterized in that, The flow rate is 1.0-1.2 mL / min; the column temperature is 20-40℃.

6. A method for identifying and determining the starting material SM3 and its impurities for ibrutinib, characterized in that, The composition is separated using the method described in 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 the starting material SM3 for the determination of ibrutinib and its impurities.

7. The method according to claim 6, characterized in that, The detector has a detection wavelength of 228±10nm.

8. The method according to claim 6, characterized in that, Based on the order of retention time, each component in the composition and / or its corresponding derivative can be identified. The derivatives corresponding to each component are, in ascending order, the derivatives of the starting material SM3, the propionyl chloride derivative, the 3-chloropropionyl chloride derivative, and the benzoyl chloride derivative.

9. A method for determining the content of SM3, the starting material for ibrutinib, and its impurities, characterized in that, The starting material SM3 and its impurities for ibrutinib are separated and identified using the method described in any one of claims 5-8, and a chromatogram is obtained; based on the obtained chromatogram, the impurities are calculated by peak area using the external standard method.

10. The use of the 1-(4-nitrophenyl)piperazine as a pre-column derivatization agent for the composition of claim 1.

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