Methods for the isolation, identification and content determination of utpatinib intermediate UPA-Z3 and its impurities

Abstract

The application belongs to the technical field of pharmaceutical analysis, and particularly relates to a method for separating and detecting an upatin intermediate UPA-Z3 and impurities thereof. 1b , impurity Z 2d , impurity Z 3a , impurity Z 3b , impurity Z 3c , impurity Z 3d , impurity Z 3e and / or impurity Z 3f . The mobile phase is composed of a mobile phase A and a mobile phase B, the mobile phase A is a 5mmol / L-50mmol / L phosphate buffer, the mobile phase B is methanol and / or acetonitrile, the chromatographic column adopts octadecylsilane pentafluorophenyl alternating bond silica gel as the filler, linear gradient elution of high performance liquid chromatography is carried out, and then detection is carried out in a detector, and then the content of each impurity is calculated according to the chromatogram by using the principal component self-control method multiplied by a correction factor. The method has the characteristics of good resolution, 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 utpatinib intermediate UPA-Z3 and its impurities. Background Technology

[0002] Upatinib, chemical name: (3S,4R)-3-ethyl-4-(3H-imidazo[1,2-a]pyrrolo[2,3-e]pyrazin-8-yl)-N-(2,2,2-trifluoroethyl)pyrrolidine-1-carboxamide, has the following chemical structural formula:

[0003]

[0004] Upadacitinib, developed by AbbVie Pharmaceuticals, is a Janus kinase (JAK) inhibitor used to treat moderate to severe atopic dermatitis, moderate to severe active rheumatoid arthritis, and psoriatic arthritis in adults. It was approved by the FDA in 2019 under the brand name RINVOQ. Atopic dermatitis is a common, chronic, relapsing, inflammatory skin disease, also known as atopic eczema. Its main clinical symptoms include dry skin, eczematous rashes, intense itching, redness, and scaling. Rheumatoid arthritis is a chronic autoimmune disease that can cause joint pain, swelling, stiffness, and dysfunction, and may lead to joint destruction and deformities, severely impacting quality of life. Symptoms of atopic dermatitis can last from several years to over a decade, and its pathogenesis remains unclear. Currently, treatment options for patients with atopic dermatitis and rheumatoid arthritis are extremely limited, making addressing the needs of these patients crucial for both the medical and pharmaceutical industries.

[0005] According to the original patent CN 108368121B, 8-((3R,4S)-4-ethylpyrrolidine-3-yl)-3H-imidazo[1,2-a]pyrrolo[2,3-e]pyrazine hydrochloride is a key intermediate in the preparation of utpatinib, which reacts with trifluoroethylamine in one step to generate utpatinib.

[0006]

[0007] The impurities related to utpatinib disclosed in patent CN 117805268A include impurities 6, 7, 8, 18, 31, F, and G, with the following structural formulas:

[0008]

[0009] In this invention, impurities Z3b and Z3c are the process sources of impurities 6 and G in the finished product of utpatinib in patent CN 117805268A. Therefore, the composition and content of impurity 8-((3R,4S)-4-ethylpyrrolidine-3-yl)-3H-imidazo[1,2-a]pyrrolo[2,3-e]pyrazine hydrochloride have a significant impact on the impurities of utpatinib and greatly affect drug safety. Therefore, by directionally preparing the target impurity of 8-((3R,4S)-4-ethylpyrrolidine-3-yl)-3H-imidazo[1,2-a]pyrrolo[2,3-e]pyrazine hydrochloride and establishing corresponding analytical methods, the quality of the active pharmaceutical ingredient intermediate can be controlled, thus ensuring the quality of utpatinib raw materials and preparations, and thereby ensuring drug safety. Summary of the Invention

[0010] One of the objectives of this invention is to provide a method for separating utpatinib intermediate UPA-Z3 and its impurities, which can complete the separation of multiple substances in a short time.

[0011] To achieve the above objectives, the technical solution of the present invention is as follows:

[0012] A method for separating utpatinib intermediate UPA-Z3 and its impurities using high-performance liquid chromatography (HPLC), wherein the HPLC method is reversed-phase HPLC, and the utpatinib intermediate UPA-Z3 and the impurities together form a composition. The impurities include impurity Z1 (chemical name: (3R,4S)-3-(N-(ethoxycarbonyl)-N-(5-(phenylsulfonyl)-5H-pyrrolo[2,3-b]pyrazin-2-yl)glycyl)-4-ethylpyrrolidine-1-carboxylic acid benzyl ester), impurity Z... 1b (Chemical name: benzyl(3R,4S)-3-(N-(ethoxycarbonyl)-N-(5H-pyrrolo[2,3-b]pyrazin-2-yl)glycyl)-4-ethylpyrrolidin-1-carboxylate), Impurity Z2 (Chemical name: benzyl(3S,4R)-3-ethyl-4-(3H-imidazo[1,2-a]pyrrolo[2,3-e]pyrazin-8-yl)pyrrolidin-1-carboxylate), Impurity Z 2d (Chemical name: Benzyl(3S,4R)-3-ethyl-4-(3-(phenylsulfonyl)-3H-imidazo[1,2-a]pyrrolo[2,3-e]pyrazin-8-yl)pyrrolidin-1-carboxylate), Impurity Z 3a (Chemical name: 8-((3R,4S)-1,4-diethylpyrrolidine-3-yl)-3H-imidazo[1,2-a]pyrrolo[2,3-e]pyrazine), Impurity Z 3b (Chemical name: 3-ethyl-8-((3R,4S)-4-ethylpyrrolidine-3-yl)-3H-imidazo[1,2-a]pyrrolo[2,3-e]pyrazine), Impurity Z3c (Chemical name: 6-ethyl-8-((3R,4S)-4-ethylpyrrolidine-3-yl)-6H-imidazo[1,2-a]pyrrolo[2,3-e]pyrazine), Impurity Z 3d (Chemical name: 8-((3S,4S)-4-ethylpyrrolidine-3-yl)-3H-imidazo[1,2-a]pyrrolo[2,3-e]pyrazine), Impurity Z 3e (Chemical name: 8-((3R,4S)-4-ethylpyrrolidine-3-yl)-3-(phenylsulfonyl)-3H-imidazo[1,2-a]pyrrolo[2,3-e]pyrazine) and / or impurity Z 3f (Chemical name: (3S,4R)-3-ethyl-4-(3H-imidazo[1,2-a]pyrrolo[2,3-e]pyrazin-8-yl)pyrrolidine-1-carboxylic acid ethyl ester); the structural formula of each component in the composition is shown in Table A. Mobile phase: composed of mobile phase A and mobile phase B, wherein mobile phase A is 5 mmol / L-50 mmol / L phosphate buffer and mobile phase B is methanol and / or acetonitrile; stationary phase: the chromatographic column is packed with octadecylsilane-pentafluorophenyl alternating bonded silica gel as the packing material, and linear gradient elution is performed.

[0013] After separation and testing, it is used for the next step of production.

[0014] The aforementioned impurities can be arranged and combined in various ways. For example, combination 1: impurity Z1, impurity Z 1b Impurity Z2, Impurity Z 3b Impurity Z 3c Impurity Z 3d Impurity Z 3e and impurity Z 3f For example, combination 2: impurity Z1, impurity Z 1b Impurity Z2, Impurity Z 2d Impurity Z 3a Impurity Z 3b Impurity Z 3c Impurity Z 3d For example, combination 3: impurity Z1, impurity Z 1b Impurity Z2, Impurity Z 2d Impurity Z 3a Impurity Z 3b Impurity Z 3c Impurity Z 3d and impurities Z 3f .

[0015] All possible permutations and combinations are not exhaustive 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.

[0016] Regarding mobile phase A, as a preferred embodiment, the concentration of the phosphate buffer is preferably 10 mmol / L to 30 mmol / L, more preferably 20 mmol / L. Regarding mobile phase B, it can be methanol and / or acetonitrile, preferably acetonitrile.

[0017] When it comes to sample solvents, acetonitrile or an aqueous solution of acetonitrile or a phosphate buffer-acetonitrile solution is used to dissolve the sample, with phosphate buffer-acetonitrile solution being preferred.

[0018] As a preferred embodiment, the linear gradient elution procedure is as follows:

[0019]

[0020] As a more preferred method, the linear gradient elution procedure is as follows: at 0 minutes, the volume ratio of mobile phase A to mobile phase B is set to 85:15; at 3 minutes, the volume ratio of mobile phase A to mobile phase B is set to 85:15; at 45 minutes, the volume ratio of mobile phase A to mobile phase B is set to 30:70; at 50 minutes, the volume ratio of mobile phase A to mobile phase B is set to 30:70; at 50.01 minutes, the volume ratio of mobile phase A to mobile phase B is set to 85:15; and at 60 minutes, the volume ratio of mobile phase A to mobile phase B is set to 85:15.

[0021] As a preferred method, the flow rate is 0.5-1.5 ml / min; the column temperature is 20-40°C. More preferably, the flow rate is 0.9-1.1 ml / min, and most preferably, 1 ml / min. More preferably, the column temperature is 25-35°C, and most preferably, the column temperature is 30°C.

[0022] The second objective of this invention is to provide a method for identifying utpatinib intermediate UPA-Z3 and its impurities, which can complete the identification of multiple substances in a short time.

[0023] To achieve the above objectives, the technical solution of the present invention is as follows:

[0024] A method for identifying utpatinib intermediate UPA-Z3 and its impurities, characterized in that the composition is separated using the method described above and detected by a detector to obtain a chromatogram; the presence of utpatinib intermediate UPA-Z3 and its impurities in the test sample is determined by comparing the chromatographic characteristics of the test sample and the reference sample.

[0025] As a preferred method, the detection wavelength of the detector is 190-240 nm.

[0026] The set detection wavelength range is based on a comprehensive consideration of factors such as error tolerance, methodological superiority, and practical application requirements. This set range helps ensure the reliability of detection results, improve measurement repeatability and flexibility, and meet the requirements of specific experiments.

[0027] As a preferred embodiment, the components in the composition can be identified according to their relative retention times, wherein the components in the composition, in ascending order, are: UPA-Z3, impurity Z. 3d Impurity Z 3a Impurity Z 3c Impurity Z 3b Impurity Z 3f Impurity Z 3e Impurity Z2, Impurity Z 1b Impurity Z 2d Impurity Z1.

[0028] Furthermore, using the utpatinib intermediate UPA-Z3 as a reference peak, the relative retention time was 1.10, which was determined to be impurity Z. 3d The relative retention time was 1.24, which was determined to be impurity Z. 3a The relative retention time was 1.27, which was determined to be impurity Z. 3c The relative retention time was 1.60, which was determined to be impurity Z. 3b The relative retention time was 1.88, indicating it was impurity Z3. f The relative retention time was 2.06, which was determined to be impurity Z. 3e The relative retention time was 2.48, and it was determined to be impurity Z2; the relative retention time was 2.97, and it was determined to be impurity Z. 1b The relative retention time was 3.55 seconds, which was determined to be impurity Z. 2d The relative retention time was 3.81, which was determined to be impurity Z1.

[0029] 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).

[0030] 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.

[0031] The third objective of this invention is to provide a method for determining the content of utpatinib intermediate UPA-Z3 and its impurities, which can complete the identification and content determination of multiple substances in a short time.

[0032] To achieve the above objectives, the technical solution of the present invention is as follows:

[0033] A method for determining the content of utpatinib intermediate UPA-Z3 and its impurities is described, wherein the method is used to separate and identify utpatinib intermediate UPA-Z3 and its impurities, and chromatograms are obtained; based on the obtained chromatograms, the content of each impurity is calculated by the principal component self-comparison method multiplied by a correction factor.

[0034] More specifically, the formula for calculating the content of each impurity is as follows:

[0035] Individual impurity % = A impurity * F / A content * self-control content / 100 * 100%

[0036] Other impurities (total) % = (Aimpurity1 + Aimpurity2 + ... + Aimpurityn) / Acomponent * content of its own control

[0037] / 100*100%

[0038] Total impurities % = Impurities 1% + Impurities 2% + ... + Impurities n% + Other total impurities %

[0039] In the formula: Aimpurity: peak area of ​​a single impurity; F: correction factor; Apair: peak area of ​​its own control.

[0040] The correction factors for each impurity are as follows: impurity Z1: 2.2; impurity Z1b: 1.7; impurity Z3a: 0.8; impurity Z3c: 1.2.

[0041] Content determination can further be used to determine whether the content of utpatinib intermediate UPA-Z3 and its impurities is within acceptable limits. If impurities Z1 and Z... 1b Impurity Z2, Impurity Z 2d Impurity Z 3a Impurity Z 3b Impurity Z 3c Impurity Z3d Impurity Z 3e and / or impurity Z 3f If the peak area of ​​any one or more impurities in the solution is greater than the peak area of ​​the corresponding impurity in the reference solution, it indicates that the impurity content is unqualified; conversely, if the peak area of ​​impurity Z1, impurity Z... 1b Impurity Z2, Impurity Z 2d Impurity Z 3a Impurity Z 3b Impurity Z 3c Impurity Z 3d Impurity Z 3e and / or impurity Z 3f If the peak area of ​​any one or more impurities in the solution is not greater than the peak area of ​​the corresponding impurity in the reference solution, it indicates that the impurity content is qualified.

[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] As a preferred method, the sample is prepared using a mixed solution of phosphate buffer and acetonitrile. For example, 0.02 mol / L phosphate buffer and acetonitrile are prepared in a 50:50 ratio.

[0044] As a preferred embodiment, the concentration of the test sample solution is 0.1-1.0 mg / ml, preferably 0.2-0.5 mg / ml, and more preferably 0.25 mg / ml. As a preferred embodiment, the concentration of the self-control solution can be achieved by diluting the test sample solution 100 times or 1000 times, preferably 200 times or 500 times.

[0045] The beneficial effects of this invention are: the method of this invention is simple and effective, has good reproducibility and high sensitivity, and can simultaneously detect utpatinib intermediate and 10 related impurities, including positional isomer impurity Z. 3a Impurity Z 3b Impurity Z 3c Research and control can be carried out in the same system, and impurity Z 3b Z 3c This method identifies the process sources of impurities 6 and G in the finished utpatinib product, and can quantitatively detect impurity Z. 3b Impurity Z 3cThe content of [the substance] is equivalent to 0.03% of the concentration of the test sample, which can effectively control the content of impurities 6 and G in the finished product. This invention solves the problem of separating and determining 10 process impurities in utpatinib intermediates, thereby ensuring the quality control of utpatinib intermediates and ultimately determining the safety and efficacy of the product. Attached Figure Description

[0046] Figure 1 High-performance liquid chromatogram for blank solvent;

[0047] Figure 2 High-performance liquid chromatogram of a mixed solution of utpatinib intermediates and all impurities;

[0048] Figure 3 The high-performance liquid chromatogram of the test solution;

[0049] Figure 4 This is the high-performance liquid chromatogram of the control solution;

[0050] Figure 5 It consists of utpatinib intermediates and impurities Z1 and Z. 1b Impurity Z2, Impurity Z 2d Impurity Z 3a Impurity Z 3b Impurity Z 3c Impurity Z 3d Impurity Z 3e and impurity Z 3f High-performance liquid chromatogram for determination of the limit of quantitation;

[0051] Figure 6 It consists of utpatinib intermediates and impurities Z1 and Z. 1b Impurity Z2, Impurity Z 2d Impurity Z 3a Impurity Z 3b Impurity Z 3c Impurity Z 3d Impurity Z 3e and impurity Z 3f High-performance liquid chromatogram for detection limit determination;

[0052] Figure 7 The high-performance liquid chromatogram is for a mixed solution with an initial mobile phase ratio of 84%-16%.

[0053] Figure 8 The high-performance liquid chromatogram is for a mixed solution with an initial mobile phase ratio of 86%-14%.

[0054] Figure 9 The image shows a high-performance liquid chromatogram of the mixed solution at a column temperature of 25°C.

[0055] Figure 10 The image shows a high-performance liquid chromatogram of a mixed solution at a column temperature of 35°C.

[0056] Figure 11 The high-performance liquid chromatogram of the mixed solution at a flow rate of 0.9 ml / min is shown.

[0057] Figure 12 The image shows a high-performance liquid chromatogram of a mixed solution at a flow rate of 1.1 ml / min. Detailed Implementation

[0058] 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.

[0059] Supplementary tables to the accompanying drawings in the specification. Included in this patent. Figure 1-12 Visual aids are provided for understanding and interpretation. In case of any ambiguity, users should refer to the corresponding numbered tables (Tables 1-12) for more detailed information. Conversely, if any potentially misleading or ambiguous information is found during the review of Tables 1-12, 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 full disclosure of the technical solutions in the claims and specification.

[0060] Table 1

[0061]

[0062] Table 2

[0063]

[0064] Table 3

[0065]

[0066] Table 4

[0067]

[0068] Table 5

[0069]

[0070] Table 6

[0071]

[0072] Table 7

[0073]

[0074] Table 8

[0075]

[0076] Table 9

[0077]

[0078] Table 10

[0079]

[0080] Table 11

[0081]

[0082] Table 12

[0083]

[0084] 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.

[0085] 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.

[0086] If any chromatographic conditions are not mentioned, refer to the high performance liquid chromatography method (Chinese Pharmacopoeia 2020 Edition, Part IV, 0512) for determination.

[0087] the term

[0088] 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.

[0089] Chromatographic robustness refers to the ability of a chromatographic analysis system to maintain stable analytical performance and ensure that the results are not significantly affected by minor changes in measurement conditions. This robustness is crucial for ensuring the reliability, repeatability, and stability of analytical results.

[0090] 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.

[0091] A correction factor is a coefficient or parameter used to correct analytical results. It aims to improve data accuracy and reliability. In HPLC analysis, because the same detector responds differently to different substances, peak areas produced when the same mass of different substances passes through the detector may not be equal. To ensure that the peak area accurately reflects the content of the analyte, standard substances are used for correction, a correction factor is calculated, and this factor is applied to the measurement results of the sample.

[0092] 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.

[0093] In this embodiment of the invention, information on the utpatinib intermediate UPA-Z3 and its related impurities is shown in Table A.

[0094] Table A: Compound Information Table

[0095]

[0096]

[0097] In the table above, the three impurities numbered XI, XII, and IX are those added to the mixed solution; they are potential impurities and were added here to assess separation. SM 1g It is a degradation product of Z3, Z 1d It's SM. 1g The byproduct of the reaction with ethanol, pyridine, is a reaction reagent.

[0098] Table B Gradient Elution Procedure

[0099]

[0100] The following four examples all used the following instruments and chromatographic conditions: High-performance liquid chromatograph: Shimadzu LC-20A; Column: Hungpu XBT C18 / PFP (250×4.6mm, 5μm). Mobile phase: Mobile phase A: Phosphate buffer: Dissolve 3.68g of potassium hexafluorophosphate in 1000ml of water, and adjust the pH to 3.0 with phosphoric acid; Mobile phase B: Acetonitrile. Detector wavelength: 230nm. Mobile phase flow rate: 1.0ml / min. Column oven temperature: 30℃. Injection volume: 10μl. Diluent (solvent for dissolving the reference standard and the test sample): 0.02mol / L phosphate buffer and acetonitrile were mixed at a volume ratio of 50:50.

[0101] Example 1: Specificity study of uropatinib intermediates and 10 process impurities using a chromatographic system.

[0102] Preparation of blank solvent: mobile phase A - acetonitrile (1:1).

[0103] Preparation of a mixed stock solution of various impurities: Take impurities Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z1 ...1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z1, Z2, Z3, Z4, 1b Z 3f Dissolve and dilute with acetonitrile to prepare a 0.47 mg / ml solution. Take impurities Z2 and Z... 2d Z 3a Z 3b Z 3d Z 3e Dissolve and dilute with acetonitrile aqueous solution to prepare a 0.47 mg / ml solution. Dissolve and dilute impurity Z3c with acetonitrile aqueous solution to prepare a 3.1 mg / ml solution. Accurately transfer 1.0 ml of each impurity stock solution into the same 25 ml volumetric flask and dilute with diluent to prepare a mixed stock solution of each impurity.

[0104] Mixed solution: Weigh about 12.5 mg of the test sample and place it in a 50 ml volumetric flask. Accurately transfer 1.0 ml of the mixed stock solution of each impurity to the same volumetric flask, add diluent to dissolve and dilute to the mark, and shake well to obtain the solution.

[0105] Test solution: Weigh approximately 12.5 mg of the test sample and place it in a 50 ml volumetric flask. Dissolve and dilute to the mark with diluent, and shake well to obtain the solution.

[0106] Control solution: Accurately transfer 0.5 ml of the test solution into a 100 ml volumetric flask, dilute quantitatively to the mark with diluent, and shake well to obtain the control solution.

[0107] Blank solvent, test solution, control solution, and mixed solution were injected separately under the chromatographic conditions described above, and the chromatograms were recorded. The results are shown in Table C. Figures 1-4 .

[0108] Table C shows the test results.

[0109]

[0110]

[0111] Conclusion: The blank solvent does not interfere with the detection of related substances in this product. The resolution between the main peak and the adjacent impurity peak is greater than 1.5, and the resolution between all other known impurity peaks is also greater than 1.5, without interfering with each other's determination. The test solution does not interfere with the detection of known impurities. The above tests demonstrate that the main peak and impurity peaks are well separated and have strong specificity.

[0112] Example 2: Basic study on the quantitation limits of uropatinib intermediates and 10 process impurities using a chromatographic system.

[0113] Limit of Quantitation Solution: Accurately weigh impurities Z1 and Z2. 1b Z 2d Z2, Z 3a Z 3b Z 3c Z 3d Z 3e Z 3f Add an appropriate amount of Z3 reference standard and dilute to prepare a solution containing approximately 0.075 μg of each impurity per ml.

[0114] Determination method: Inject the above-mentioned limit-of-quantity solution six times consecutively, and calculate the ratio of the main peak height to the noise (signal-to-noise ratio). Record the chromatogram, as follows. Figure 5 As shown, the experimental results are shown in Table D.

[0115]

[0116] Table D Results of Limit of Quantitation Determination

[0117]

[0118]

[0119]

[0120] Conclusion: Impurity Z 3d The limit of quantitation (LOQ) was 0.0726 μg / ml, the concentration present in the sample was 0.029%, the average signal-to-noise ratio was 71.8, and the peak area RSD was 0.1%; impurity Z 3a The limit of quantitation (LOQ) was 0.0711 μg / ml, the concentration present in the sample was 0.028%, the average signal-to-noise ratio was 80.4, and the peak area RSD was 1.6%; impurity Z 3c The limit of quantitation (LOQ) was 0.0742 μg / ml, the concentration present in the sample was 0.030%, the average signal-to-noise ratio was 57.5, and the peak area RSD was 1.2%; impurity Z 3b The limit of quantitation (LOQ) was 0.0726 μg / ml, the concentration present in the sample was 0.029%, the average signal-to-noise ratio was 81.1, and the peak area RSD was 1.2%; impurity Z 3f The limit of quantitation (LOQ) was 0.0701 μg / ml, the concentration present in the sample was 0.028%, the average signal-to-noise ratio was 66.9, and the peak area RSD was 1.0%; impurity Z 3e The limit of quantitation (LOQ) for impurity Z1 was 0.0765 μg / ml, with a concentration of 0.031% in the sample, an average signal-to-noise ratio (SNR) of 90.7, and a peak area RSD of 1.4%. The LQ for impurity Z2 was 0.0728 μg / ml, with a concentration of 0.029% in the sample, an average SNR of 65.4, and a peak area RSD of 1.9%. 1b The limit of quantitation (LOQ) was 0.0706 μg / ml, the concentration present in the sample was 0.028%, the average signal-to-noise ratio was 38.3, and the peak area RSD was 2.6%; impurity Z 3e The limit of quantitation (LOQ) for impurity Z1 was 0.0756 μg / ml, with a concentration of 0.030% in the sample. The average signal-to-noise ratio (SNR) was 62.2, and the peak area RSD was 2.3%. The LOQ for impurity Z1 was 0.0770 μg / ml, with a concentration of 0.031% in the sample. The average SNR was 33.7, and the peak area RSD was 3.5%. The LOQ for intermediate Z3 was 0.0743 μg / ml, with a concentration of 0.030% in the sample. The average SNR was 72.6, and the peak area RSD was 0.9%. At their respective LOQ concentrations, all impurities exhibited a SNR greater than 10 and a peak area RSD less than 10%, meeting the requirements for related substance detection. This indicates that the impurities can be accurately quantified at these levels.

[0121] Example 3: Basic Study on the Detection Limits of the Chromatographic System for Upatinib Intermediates and 10 Process Impurities

[0122] Detection limit solution: Accurately transfer 3.5 ml of the quantitation limit solution into a 10 ml volumetric flask, and dilute with diluent to prepare a solution containing approximately 0.025 μg of each impurity per ml.

[0123] Measurement method:

[0124] Inject the above-mentioned detection limit solution three times consecutively, and calculate the ratio of the main peak height to the noise (signal-to-noise ratio).

[0125] Record the chromatogram, such as Figure 6 As shown, the experimental results are shown in Table E.

[0126]

[0127] Table E shows the results of the detection limit determination.

[0128]

[0129]

[0130] Conclusion: Impurity Z 3d The detection limit was 0.0254 μg / ml, the concentration in the sample was 0.010%, and the average signal-to-noise ratio was 22.9.

[0131] Impurity Z 3a The detection limit was 0.0249 μg / ml, the concentration in the sample was 0.010%, and the average signal-to-noise ratio was 25.2.

[0132] Impurity Z 3c The detection limit was 0.0260 μg / ml, the concentration in the sample was 0.010%, and the average signal-to-noise ratio was 15.6.

[0133] Impurity Z 3b The detection limit was 0.0254 μg / ml, the concentration in the sample was 0.010%, and the average signal-to-noise ratio was 27.1.

[0134] Impurity Z 3f The detection limit was 0.0245 μg / ml, the concentration in the sample was 0.010%, and the average signal-to-noise ratio was 22.1.

[0135] Impurity Z 3e The detection limit was 0.0268 μg / ml, the concentration in the sample was 0.011%, and the average signal-to-noise ratio was 30.8.

[0136] The detection limit for impurity Z2 was 0.0255 μg / ml, its concentration in the sample was 0.010%, and the average signal-to-noise ratio was 21.8.

[0137] Impurity Z1b The detection limit was 0.0247 μg / ml, the concentration in the sample was 0.010%, and the average signal-to-noise ratio was 13.4.

[0138] Impurity Z 2d The detection limit was 0.0265 μg / ml, the concentration in the sample was 0.011%, and the average signal-to-noise ratio was 20.7.

[0139] The detection limit for impurity Z1 was 0.0270 μg / ml, its concentration in the sample was 0.011%, and the average signal-to-noise ratio was 11.7.

[0140] The detection limit of intermediate Z3 was 0.0260 μg / ml, the concentration in the sample was 0.010%, and the average signal-to-noise ratio was 22.7.

[0141] At the detection limit concentration, the signal-to-noise ratio of each impurity was greater than 3, which meets the requirements for related substance detection, indicating that the impurities can be effectively detected at this level.

[0142] Example 4: Robustness study of the chromatographic system against utpatinib intermediates and 10 process impurities.

[0143] Mixed stock solution of various impurities: Take the mixed stock solution of impurities from Example 1.

[0144] Other impurity stock solution: Take impurity SM 1g Z 1d Pyridine was dissolved and diluted with a diluent to prepare a solution of approximately 62.5 μg / ml.

[0145] Mixed solution: Weigh about 12.5 mg of the test sample and place it in a 50 ml volumetric flask. Accurately transfer 1.0 ml each of the mixed stock solution of each impurity and the stock solution of other impurities into the same volumetric flask. Add diluent to dissolve and dilute to the mark, and shake well to obtain the solution.

[0146] Measurement method:

[0147] Accurately measure 10 μl of the mixed solution, and adjust each experimental condition (initial mobile phase ratio ±1%, column flow rate ±0.1 ml / min, column temperature ±5℃). After the instrument system stabilizes, conduct the test. Examine the resolution between each peak. The experimental results are shown in Table F.

[0148] Table F shows the results of the resolution determination in the robustness test for changes in chromatographic conditions.

[0149]

[0150] Note: Impurities SM 1g No peak was observed at this wavelength, and no data statistics were performed.

[0151] Conclusion: When the chromatographic system is fluctuating, the resolution between all known impurities in the mixed solution is greater than 1.5, and the resolution between the main component and adjacent impurities is greater than 1.5, which meets the requirements.

[0152] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for separating utpatinib intermediate UPA-Z3 and its impurities based on high performance liquid chromatography, characterized in that, The high-performance liquid chromatography method is reversed-phase high-performance liquid chromatography. The utpatinib intermediate UPA-Z3 and the impurities together form a composition. The impurities include impurity Z1 and impurity Z2. 1b Impurity Z2, Impurity Z 2d Impurity Z 3a Impurity Z 3b Impurity Z 3c Impurity Z 3d Impurity Z 3e and / or impurity Z 3f Any one or more of the following; the structural formula of each component in the composition is as follows: Mobile phase: Composed of mobile phase A and mobile phase B, wherein mobile phase A is 5 mmol / L-50 mmol / L phosphate buffer and mobile phase B is methanol and / or acetonitrile; Stationary phase: The chromatographic column is packed with octadecylsilane-pentafluorophenyl alternating bonded silica gel and linear gradient elution is performed.

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 linear gradient elution procedure is as follows: at 0 minutes, the volume ratio of mobile phase A to mobile phase B is set to 85:15; at 3 minutes, the volume ratio of mobile phase A to mobile phase B is set to 85:15; at 45 minutes, the volume ratio of mobile phase A to mobile phase B is set to 30:70; at 50 minutes, the volume ratio of mobile phase A to mobile phase B is set to 30:70; at 50.01 minutes, the volume ratio of mobile phase A to mobile phase B is set to 85:15; and at 60 minutes, the volume ratio of mobile phase A to mobile phase B is set to 85:

15.

4. The method according to claim 1, characterized in that, The flow rate is 0.5-1.5 ml / min; the column temperature is 20-40℃.

5. A method for identifying utpatinib intermediate UPA-Z3 and its impurities, characterized in that, The composition is separated by the method according to any one of claims 1-4 and detected by 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 utpatinib intermediate UPA-Z3 and its impurities.

6. The method according to claim 5, characterized in that, The detector has a detection wavelength of 190-240nm.

7. The method according to claim 5, characterized in that, The components in the composition can be identified according to their relative retention times. The components of the composition, in ascending order, are: UPA-Z3, impurity Z. 3d Impurity Z 3a Impurity Z 3c Impurity Z 3b Impurity Z 3f Impurity Z 3e Impurity Z2, Impurity Z 1b Impurity Z 2d Impurity Z1.

8. The method according to claim 7, characterized in that, Using utpatinib intermediate UPA-Z3 as a reference peak; the relative retention time was 1.10, which was determined to be impurity Z. 3d ; The relative retention time is 1.24, and it is identified as impurity Z. 3a ; The relative retention time was 1.27, and it was determined to be impurity Z. 3c ; The relative retention time is 1.60, and it is identified as impurity Z. 3b ; The relative retention time was 1.88, and it was determined to be impurity Z3. f ; The relative retention time was 2.06, and it was determined to be impurity Z. 3e ; The relative retention time was 2.48, and it was determined to be impurity Z2; The relative retention time was 2.97, and it was determined to be impurity Z. 1b ; The relative retention time is 3.55, and it is determined to be impurity Z. 2d ; The relative retention time was 3.81, which was determined to be impurity Z1.

9. A method for determining the content of utpatinib intermediate UPA-Z3 and its impurities, characterized in that, The intermediate UPA-Z3 of utpatinib and its impurities were separated and identified by the method described in any one of claims 5-8, and chromatograms were obtained. Based on the obtained chromatograms, the content of each impurity was calculated by the principal component self-comparison method multiplied by a correction factor.

10. The method according to claim 9, characterized in that, The sample preparation solvent is 0.01-0.03 mol / L phosphate buffer and acetonitrile at a volume ratio of 45-55:50.

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