Method for separating and determining SM1, the starting material of utpatinib, and its impurities

Abstract

This invention belongs to the field of pharmaceutical analysis technology, specifically relating to a method for separating and determining SM1, the starting material of utpatinib, and its impurities. The impurities include SM1. 1a SM 1b SM 1d SM 1f SM 1h SM 1i SM 1j SM 1m SM 1n and SM 1o One or more of the following. This invention provides a self-developed high-performance liquid chromatography (HPLC) method for the separation and determination of utpatinib starting material SM1 and its impurities, comprising using phosphoric acid solution as mobile phase A and an organic solvent as mobile phase B; using octadecylsilane-pentafluorophenyl alternating bonded silica gel as the packing material, performing linear gradient elution, and then entering a detector for detection; based on the measured chromatogram, the content of each impurity is calculated using the principal component self-comparison method with correction factors and / or the limit method. This method features good resolution, robustness, 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 determining umpatinib starting material SM1 and its impurities. Background Technology

[0002] Upadacitinib, chemically named (3S,4R)-3-ethyl-4-(3H-imidazo[1,2-a]pyrrolo[2,3-e]pyrazin-8-yl)-N-(2,2,2-trifluoroethyl)pyrrolidin-1-carboxamide, has the chemical structural formula shown in Formula 1.

[0003]

[0004] Upatinib is a Janus kinase (JAK) inhibitor developed by AbbVie Pharmaceuticals. It was approved by the FDA in 2019 for the treatment of moderate to severe atopic dermatitis, moderate to severe active rheumatoid arthritis, and psoriatic arthritis in adults. Upatinib works by precisely inhibiting JAK1, blocking the transmission of inflammatory signals, thereby effectively controlling inflammation-related disease symptoms.

[0005] The original patent CN 108368121B describes utpatinib as primarily prepared from compounds IV and III. Our company made adjustments based on this, selecting ethyl (5-(phenylsulfonyl)-5H-pyrrolo[2,3-b]pyrazin-2-yl)carbamate (also known as utpatinib starting material SM1 or UPA-SM1) with different protecting groups as the starting material to prepare utpatinib.

[0006]

[0007] The current mature process routes for umpatinib starting material SM1 are as follows:

[0008]

[0009] During the synthesis of utpatinib starting material SM1, the following impurities are easily introduced: starting material SM 1a and intermediate SM 1b SM 1d Impurities SM generated during degradation 1f SM 1h SM 1i (Highly polar degradation impurities), impurity SM 1d Dehalogenation byproduct SM 1j The oxidation product of the ligand Xantphos, SM 1m (Small polar oxidized ligand), impurity SM 1hReaction with CDI, impurity SM 1h With SM 1d Impurities SM that may be generated during the reaction 1n and SM 1o Among them, impurity SM 1a and impurities SM 1i It has a warning structure and is a basic toxic impurity.

[0010] As a key starting material for the synthesis of utpatinib, the composition and content of impurities in the utpatinib starting material SM1 have a significant impact on the impurity content of the drug and, consequently, on medication safety. Therefore, by directionally preparing the target impurities in the utpatinib starting material SM1 and establishing corresponding analytical methods to control the quality of the starting material, the quality of the utpatinib raw material and its formulation can be guaranteed, thereby ensuring medication safety. However, no relevant patents or literature have been found to report analytical methods for the aforementioned 10 impurities in the utpatinib starting material SM1.

[0011] Some existing patent literature has explored analytical methods for other related substances in utpatinib. For example, patent CN118311186A discloses a method for determining utpatinib intermediates and their enantiomers using liquid chromatography. This method employs a normal-phase high-performance liquid chromatograph (HPLC) with a column packed with linear starch-tris(3,5-dimethylphenylcarbamate)-coated silica gel, and a mobile phase consisting of a mixture of monohydric alcohol, ethylenediamine, a monobasic acid, and liquid alkanes with C6-C7 carbon chains. Another example is patent CN117805268A, which discloses a method for detecting related substances in utpatinib using HPLC. The chromatographic conditions are: a stationary phase of octadecylsilane-bonded silica gel column, mobile phase A consisting of a buffer solution of formic acid and ammonium formate, and mobile phase B consisting of methanol, with gradient elution. However, none of these existing technologies address the separation and detection of the aforementioned 10 impurities. Summary of the Invention

[0012] In view of this, one of the objectives of the present invention is to provide a method for separating umpatinib starting material SM1 and its impurities based on high performance liquid chromatography, which can complete the separation of multiple substances in a short time.

[0013] To achieve the above objectives, the technical solution of the present invention is as follows:

[0014] A method for separating utpatinib starting material SM1 and its impurities using high-performance liquid chromatography, wherein the utpatinib starting material SM1 and the impurities together constitute a composition, and the impurities include impurity SM1. 1a Impurities SM 1b Impurities SM 1d Impurities SM 1f Impurities SM 1h Impurities SM1i Impurities SM 1j Impurities SM 1m Impurities SM 1n and impurities SM 1o Any one or more of the following; the structural formula of each component in the composition is as follows:

[0015]

[0016] In the high-performance liquid chromatography method, the mobile phase is: phosphoric acid solution as mobile phase A and organic solvent as mobile phase B; the stationary phase is: the chromatographic column uses octadecylsilane pentafluorophenyl alternating bonded silica gel as the packing material, and linear gradient elution is performed.

[0017] After separation and testing, it is used for the next step of production.

[0018] The above impurities can be arranged and combined in various ways. For example, combination 1: impurity SM 1a Impurities SM 1b For example, combination method 2: impurity SM 1a Impurities SM 1b Impurities SM 1d For example, combination method 3: impurity SM 1h Impurities SM 1i Impurities SM 1j Impurities SM 1m .

[0019] All possible permutations and combinations are not exhaustive here. Theoretically, when the upper limit of the substances that can be separated, identified, and / or detected by this method is n (where n is the number of substances), it can naturally detect 1 to n substances.

[0020] As a preferred embodiment, the linear gradient elution procedure is as follows:

[0021] Time - minutes Mobile phase A - volume fraction Mobile phase B - volume fraction 0 70±10 30±10 5±1 70±10 30±10 35±1 35±10 65±10 40±1 35±10 65±10 41±1 70±10 30±10 60±1 70±10 30±10

[0022] Preferably, the linear gradient elution procedure is as follows:

[0023] Time - minutes Mobile phase A - volume fraction Mobile phase B - volume fraction 0 70±1 30±1 5 70±1 30±1 35 35 65 40 35 65 41 70±1 30±1 60 70±1 30±1

[0024] For example, mobile phase A accounts for 69 parts, and mobile phase B accounts for 31 parts. Figure 11 ); or mobile phase A accounts for 70 parts and mobile phase B accounts for 30 parts. Figure 2 ); or mobile phase A accounts for 71 parts and mobile phase B accounts for 29 parts. Figure 12 ).

[0025] Furthermore, the organic solvent is methanol and / or acetonitrile; the phosphoric acid content in the phosphoric acid solution is 0.05% to 0.2%.

[0026] As the most preferred embodiment, the mobile phase A is a phosphoric acid solution with a phosphoric acid content of 0.1%; and the mobile phase B is acetonitrile.

[0027] Furthermore, the flow rate was 0.7–1.3 mL / min; the column temperature was 25–35 °C.

[0028] As a preferred embodiment, the flow rate is 0.9–1.1 mL / min; the column temperature is 28–32 °C. For example, a flow rate of 0.9 mL / min ( Figure 13 ); or a flow rate of 1.0 mL / min ( Figure 2 ); or a flow rate of 1.1 mL / min ( Figure 14 For example, the column temperature is 28℃. Figure 15 ); or column temperature of 29℃; or column temperature of 30℃. Figure 2 ); or column temperature of 31℃; or column temperature of 32℃. Figure 16 ).

[0029] The optimal flow rate is 1.0 mL / min, and the column temperature is 30 °C.

[0030] Preferably, the chromatographic column has dimensions of 4.6 mm × 250 mm and a diameter of 5 μm.

[0031] As the most preferred option, the chromatographic column is a Hungpu XBT C18 / PFP, 4.6 mm × 250 mm, 5 μm or a column with equivalent performance.

[0032] Preferably, the injection volume is 5–30 μl.

[0033] As a preferred option, the running time is 60 minutes.

[0034] Preferably, the solvent for sample preparation is acetonitrile or an aqueous solution of acetonitrile, with acetonitrile being the most preferred.

[0035] Preferably, the sample preparation solvent is used to prepare system suitability solutions, sample solutions, and / or control solutions.

[0036] Preferably, the concentration of the sample solution is 0.1–1.0 mg / ml, more preferably 0.2–0.5 mg / ml, and even more preferably 0.25 mg / ml.

[0037] Preferably, the self-control solution is prepared by diluting the sample solution by 100 to 1000 times, preferably by 200 times or 500 times.

[0038] As a preferred method, the sample solution is prepared by taking an appropriate amount of utpatinib starting material SM1, dissolving the sample in acetonitrile, and preparing a sample solution containing 0.25 mg of utpatinib starting material SM1 per 1 ml.

[0039] As a preferred method, the system suitability solution is prepared by taking impurity SM separately. 1a Impurities SM 1b Impurities SM 1d Impurities SM 1f Impurities SM 1h Impurities SM 1i Impurities SM 1j Impurities SM 1m Impurities SM 1n Impurities SM 1o The reference standard was diluted with a diluent to prepare an impurity stock solution. Then, the sample solution and the impurity stock solution were diluted with acetonitrile to prepare a system suitability solution. In the system suitability solution, the concentration of UPA-SM1 was 0.25 mg / ml, and the impurity SM... 1a Impurities SM 1b Impurities SM 1d Impurities SM 1f Impurities SM 1h Impurities SM 1i Impurities SM 1j Impurities SM 1m Impurities SM 1n Impurities SM 1o The concentrations were all 0.375 μg / ml.

[0040] As a preferred method, the control solution is prepared as follows: accurately measure 0.5 ml of the sample solution, place it in a 100 ml volumetric flask, dilute quantitatively to the mark with diluent, and shake well.

[0041] The second objective of this invention is to provide a method for identifying umpatinib starting material SM1 and its impurities, which can complete the identification of multiple substances in a short time.

[0042] To achieve the above objectives, the technical solution of the present invention is as follows:

[0043] A method for identifying utpatinib starting material SM1 and its impurities involves separating the 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 utpatinib starting material SM1 and its impurities.

[0044] Preferably, the detector is an ultraviolet (VWD) detector.

[0045] As a preferred embodiment, the detection wavelength of the detector is 230 ± 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.

[0046] As the most preferred option, the detection wavelength of the detector is 230 nm.

[0047] As a preferred embodiment, the components in the composition can be identified according to their relative retention times, wherein the components of the composition, in ascending order, are: impurities SM. 1i Impurities SM 1f Impurities SM 1b Impurities SM 1h Impurities SM 1j Upatinib starting material SM1, impurities SM 1d Impurities SM 1a Impurities SM 1m Impurities SM 1n Impurities SM 1o .

[0048] 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).

[0049] As a preferred method, SM1, the starting material of utpatinib, was used as the reference peak; the relative retention time was 0.11, and it was determined to be impurity SM. 1i The relative retention time was 0.26, indicating it was classified as impurity SM. 1f The relative retention time was 0.39, indicating it was classified as impurity SM. 1b The relative retention time was 0.51, indicating it was classified as impurity SM. 1h The relative retention time was 0.74, indicating it was classified as impurity SM. 1j The relative retention time was 1.00, which was determined to be the starting material SM1 for utpatinib; the relative retention time was 1.24, which was determined to be the impurity SM. 1d The relative retention time was 1.39 seconds, indicating it was classified as impurity SM. 1a The relative retention time was 1.44, indicating it was classified as impurity SM. 1m The relative retention time was 1.51, indicating it was classified as impurity SM. 1nThe relative retention time was 1.58, indicating it was classified as impurity SM. 1o The relative retention times of each component fluctuated within ±5%.

[0050] 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.

[0051] The third objective of this invention is to provide a method for determining the content of umpatinib starting material SM1 and its impurities, which can complete the identification and content determination of multiple substances in a short time.

[0052] To achieve the above objectives, the technical solution of the present invention is as follows:

[0053] The method for determining the content of umpatinib starting material SM1 and its impurities involves separating and identifying umpatinib starting material SM1 and its impurities using the aforementioned identification method to obtain a chromatogram; and calculating the content of each impurity based on the obtained chromatogram using the principal component self-comparison method with correction factor and / or the limit method.

[0054] Furthermore, the limit method was used to determine the SM content in the umpatinib starting material SM1. 1m Whether the content is within the acceptable range; calculate the impurity SM using the principal component self-comparison method with correction factors. 1a Impurities SM 1b Impurities SM 1d Impurities SM 1f Impurities SM 1h Impurities SM 1i Impurities SM 1j Impurities SM 1n and impurities SM 1o The content of any one or more impurities in the sample.

[0055] Content determination can further be used to determine whether the content of umpatinib starting material SM1 and its impurities is within acceptable limits. If impurity SM1... 1a Impurities SM 1b Impurities SM 1d Impurities SM 1f Impurities SM 1h Impurities SM 1i Impurities SM 1j Impurities SM 1m Impurities SM1n and impurities SM 1o If the peak area of ​​any one or more impurities in the solution is larger than the peak area of ​​the corresponding impurity in the control solution, it indicates that the impurity content is unqualified; conversely, if the impurity SM 1a Impurities SM 1b Impurities SM 1d Impurities SM 1f Impurities SM 1h Impurities SM 1i Impurities SM 1j Impurities SM 1m Impurities SM 1n and impurities SM 1o 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 control solution, it indicates that the impurity content is qualified.

[0056] 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.

[0057] Furthermore, the impurity SM 1a The correction factor is 1.6; the impurity SM 1b The correction factor is 0.79; the impurity SM 1f The correction factor is 0.55; the impurity SM 1h The correction factor is 1.2; the impurity SM 1i The correction factor is 1.6; the impurity SM 1n The correction factor is 1.1; the impurity SM 1o The correction factor is 1.2; the impurity SM 1j and the impurities SM 1d The correction factor is 1.0.

[0058] The beneficial effects of this invention are as follows:

[0059] 1. This invention proposes a method for separating and determining utpatinib starting material SM1 and its impurities. This method is a reversed-phase high-performance liquid chromatography (RP-HPLC) method, which can effectively separate and determine multiple impurities in utpatinib starting material SM1 within a single chromatographic system. These impurities include impurity SM. 1a Impurities SM 1b Impurities SM 1d Impurities SM 1fImpurities SM 1h Impurities SM 1i Impurities SM 1j Impurities SM 1m Impurities SM 1n and impurities SM 1o Any one or more of the following. This detection method is highly sensitive, robust, practical, and provides accurate and reliable results, which is of great significance for achieving quality control of utpatinib SM1 and utpatinib finished products.

[0060] 2. The method of this invention can completely separate 10 process impurities from the utpatinib starting material simultaneously within 60 minutes, including the highly polar degradation impurity SM. 1i and small polar oxidized ligand impurities SM 1m The research and control can be carried out within the same system. This invention solves the problem of separating and determining 10 process impurities in utpatinib starting materials, thereby ensuring the quality control of utpatinib starting materials and ultimately determining the safety and efficacy of the product. This method has the advantages of being simple and feasible, having a short separation time, good reproducibility, strong specificity, and high accuracy.

[0061] 3. The method of this invention has a low limit of quantitation: the limit of quantitation concentration of utpatinib starting material SM1 is 0.0761 μg / ml; impurity SM 1i The limit of quantitation (LOQ) was 0.0723 μg / ml; impurity SM 1f The limit of quantitation (LOQ) is 0.0724 μg / ml; impurity SM 1b The limit of quantitation was 0.0757 μg / ml; impurity SM 1h The limit of quantitation (LOQ) is 0.0728 μg / ml; impurity SM 1j The limit of quantitation (LOQ) is 0.0745 μg / ml; impurity SM 1d The limit of quantitation was 0.0757 μg / ml; impurity SM 1a The limit of quantitation (LOQ) was 0.0736 μg / ml; impurity SM 1m The limit of quantitation (LOQ) was 0.0751 μg / ml; impurity SM 1n The limit of quantitation (LOQ) is 0.0753 μg / ml; impurity SM 1o The limit of quantitation is 0.0735 μg / ml.

[0062] 4. The method of this invention has a low detection limit: the detection limit concentration of utpatinib starting material SM1 is 0.0266 μg / ml; impurity SM 1i The detection limit concentration is 0.0253 μg / ml; impurity SM 1f The detection limit concentration is 0.0253 μg / ml; impurity SM 1bThe detection limit concentration is 0.0265 μg / ml; impurity SM 1h The detection limit concentration is 0.0255 μg / ml; impurity SM 1j The detection limit concentration was 0.0261 μg / ml; impurity SM 1d The detection limit concentration is 0.0265 μg / ml; impurity SM 1a The detection limit concentration is 0.0258 μg / ml; impurity SM 1m The detection limit concentration is 0.0263 μg / ml; impurity SM 1n The detection limit concentration is 0.0264 μg / ml; impurity SM 1o The detection limit concentration is 0.0257 μg / ml. Attached Figure Description

[0063] Figure 1 High-performance liquid chromatogram of blank solvent;

[0064] Figure 2 The high-performance liquid chromatogram of a mixed solution of utpatinib starting material and all impurities;

[0065] Figure 3 The high-performance liquid chromatogram of the test solution;

[0066] Figure 4 This is the high-performance liquid chromatogram of the control solution;

[0067] Figure 5 The starting material SM1 and impurity SM for utpatinib 1f Impurities SM 1h Impurities SM 1i Impurities SM 1j and impurities SM 1m High-performance liquid chromatogram for determination of the limit of quantitation;

[0068] Figure 6 SM is an impurity 1a Impurities SM 1b and impurities SM 1d High-performance liquid chromatogram for determination of the limit of quantitation;

[0069] Figure 7 SM is an impurity 1n and impurities SM 1o High-performance liquid chromatogram for determination of the limit of quantitation;

[0070] Figure 8 Starting material and impurities of utpatinib (SM) 1f Impurities SM 1h Impurities SM 1i Impurities SM 1j and impurities SM1m High-performance liquid chromatogram for detection limit determination;

[0071] Figure 9 SM is an impurity 1a Impurities SM 1b and impurities SM 1d High-performance liquid chromatogram for detection limit determination;

[0072] Figure 10 SM is an impurity 1n and impurities SM 1o High-performance liquid chromatogram for detection limit determination;

[0073] Figure 11 The high-performance liquid chromatograms are for the mixed solution in the durability test when the initial proportion of the mobile phase is 69%-31%.

[0074] Figure 12 The high-performance liquid chromatograms of the mixed solution in the mobile phase with an initial ratio of 71% to 29% are shown in the durability test.

[0075] Figure 13 The image shows the high-performance liquid chromatogram of the mixed solution at a flow rate of 0.9 ml / min during the durability test.

[0076] Figure 14 The image shows the high-performance liquid chromatogram of the mixed solution at a flow rate of 1.1 ml / min during the durability test.

[0077] Figure 15 The image shows the high-performance liquid chromatogram of the mixed solution at a column temperature of 28°C during the durability test.

[0078] Figure 16 This is a high-performance liquid chromatogram of the mixed solution at a column temperature of 32°C during the durability test. Detailed Implementation

[0079] 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.

[0080] Supplementary tables to the accompanying drawings in the specification. Included in this patent. Figures 1-16Visual 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 information that may cause misunderstanding or ambiguity is found during the review of Tables 1-16, the content of the corresponding numbered figures should be taken as the standard. 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.

[0081] Table 1

[0082]

[0083] Table 2

[0084]

[0085] Table 3

[0086]

[0087] Table 4

[0088]

[0089] Table 5

[0090]

[0091] Table 6

[0092]

[0093] Table 7

[0094]

[0095] Table 8

[0096]

[0097] Table 9

[0098]

[0099] Table 10

[0100]

[0101] Table 11

[0102]

[0103] Table 12

[0104]

[0105] Table 13

[0106]

[0107] Table 14

[0108]

[0109] Table 15

[0110]

[0111] Table 16

[0112]

[0113] In Tables 1 to 16 above, any peaks not specified are unknown impurities.

[0114] 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.

[0115] 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.

[0116] If any chromatographic conditions are not mentioned, refer to the high performance liquid chromatography method (Chinese Pharmacopoeia 2020 Edition, Part IV, 0512) for determination.

[0117] the term

[0118] 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.

[0119] 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.

[0120] 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.

[0121] 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.

[0122] 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.

[0123] In this embodiment of the invention, information on the utpatinib starting material SM1 and its related impurities is shown in Table A.

[0124] Table A. Compound Information Table

[0125]

[0126]

[0127]

[0128] In this embodiment of the invention, the preparation method of mobile phase A (phosphoric acid solution) is as follows: 1 ml of phosphoric acid is measured into a 1000 ml volumetric flask, dissolved in water and diluted to the mark.

[0129] In this embodiment of the invention, the diluent (the solvent for dissolving the reference standard and the sample to be tested) is acetonitrile.

[0130] In this embodiment of the invention, "——" in the table indicates none.

[0131] Example 1. Method for separating and determining umpatinib starting material SM1 and its impurities

[0132] (1) Preparation of the test solution

[0133] System suitability solution: Take impurity SM separately 1a Impurities SM 1b Impurities SM 1d Impurities SM 1f Impurities SM 1h Impurities SM 1i Impurities SM 1j Impurities SM 1m Impurities SM 1n Impurities SM 1o The reference standard was diluted to prepare an impurity stock solution. Then, the test solution and the impurity stock solution were diluted with acetonitrile to prepare a system suitability solution. The concentration of UPA-SM1 in the system suitability solution was 0.25 mg / ml, and the impurity SM... 1a Impurities SM 1b Impurities SM 1d Impurities SM 1f Impurities SM 1h Impurities SM 1i Impurities SM 1j Impurities SM 1m Impurities SM 1n Impurities SM 1o The concentrations were all 0.375 μg / ml.

[0134] Test solution: Take an appropriate amount of utpatinib starting material SM1, dissolve the sample in acetonitrile, and prepare a sample solution containing 0.25 mg of utpatinib starting material SM1 per 1 ml (operate in the dark).

[0135] Control solution: Accurately measure 0.5 ml of the test solution and place it in a 100 ml volumetric flask. Dilute quantitatively to the mark with diluent and shake well.

[0136] (2) Chromatographic conditions

[0137] The high-performance liquid chromatograph (HPLC) used was a Shimadzu 2050; the column was packed with octadecylsilane-pentafluorophenyl alternating bonded silica gel (Hungpu XBT C18 / PFP, 250×4.6 mm, 5 μm or equivalent column); mobile phase A was 0.1% phosphoric acid solution, and mobile phase B was acetonitrile; the detector was a UV detector (VWD); the detection wavelength was 230 nm; the mobile phase flow rate was 1.0 ml / min; the column oven temperature was 30 ℃; the injection volume was 10 μl; the elution method was linear gradient elution, and the gradient elution program is shown in Table B.

[0138] Table B Gradient Elution Procedure

[0139] Time - minutes Mobile phase A - volume fraction Mobile phase B - volume fraction 0 70 30 5 70 30 35 35 65 40 35 65 41 70 30 60 70 30

[0140] (3) Measurement

[0141] Accurately measure the system suitability solution, test solution, and self-control solution, and inject them separately into the liquid chromatograph. Record the chromatograms and extract impurity information, such as impurity quantity, impurity type, resolution between impurity peaks, and peak area. Based on the measured impurity information, determine the impurity SM in the test solution using the limit method. 1m Whether the content is qualified or not, the content of other impurities is calculated according to the self-comparison method with correction factors. Among them, impurity SM 1a The correction factor is 1.6; impurity SM 1b The correction factor is 0.79; impurity SM 1f The correction factor is 0.55; impurity SM 1h The correction factor is 1.2; impurity SM 1i The correction factor is 1.6; impurity SM 1n The correction factor is 1.1; impurity SM 1o The correction factor is 1.2; impurity SM 1j and impurities SM 1d The correction factor is 1.0.

[0142] The formulas for calculating the content of each impurity are as follows:

[0143] Individual impurity % = A impurity * F / A content * self-control content / 100 * 100%;

[0144] Other impurities (total) % = (A impurity 1 + A impurity 2 + ... + A impurity n) / A content * self-control / 100 * 100%;

[0145] Total impurities % = Impurities 1% + Impurities 2% + ... + Impurities n% + Other total impurities %;

[0146] In the formula, Aimpurity: peak area of ​​a single impurity; F: correction factor; Apair: peak area of ​​its own control.

[0147] Example 2. Specificity Experiment

[0148] (1) Preparation of the test solution

[0149] Blank solvent / diluent: acetonitrile.

[0150] Preparation of stock solutions for each impurity: Take impurity SM 1h Dissolve in 10% (v / v) tetrahydrofuran, then dissolve and dilute in acetonitrile to prepare a 20 μg / ml solution. Take the impurity SM. 1i Dissolve and dilute with 50% acetonitrile to prepare a 20 μg / ml solution. Take appropriate amounts of the remaining reference standards, dissolve and dilute with diluent to prepare a 20 μg / ml solution, and use these as stock solutions for each impurity.

[0151] Preparation of 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 each impurity stock solution to the same volumetric flask, add diluent to dissolve and dilute to the mark, and shake well to obtain the solution.

[0152] Preparation of the test solution: Weigh about 12.5 mg of the test sample, place it in a 50 ml volumetric flask, add diluent to dissolve and dilute to the mark, shake well, and the solution is ready.

[0153] Preparation of the control solution: Accurately measure 0.5 ml of the test solution and place it in a 100 ml volumetric flask. Dilute quantitatively to the mark with diluent and shake well.

[0154] (2) Detection

[0155] Take blank solvent, test solution, control solution and mixed solution respectively, inject them into the chromatographic conditions described above and record the chromatograms.

[0156] The measurement results are shown in Table C. Figures 1-4 As shown in Tables 1 to 4. The blank solvent does not interfere with the detection of related substances in this product. The resolution between the main peak and adjacent impurity peaks is greater than 1.5, and the resolution between all other known impurity peaks is also greater than 1.5, indicating that they do not interfere with each other's determination. The test solution does not interfere with the detection of known impurities. These experimental results demonstrate that the main peak and impurity peaks are well separated and have strong specificity.

[0157] Table C. Test Results

[0158]

[0159] Example 3. Limit of Quantitation Experiment

[0160] (1) Preparation of the test solution

[0161] Limit of Quantification Solution 1: Accurately weigh impurities SM1 and SM2. 1fSM 1h SM 1i SM 1j SM 1m Prepare a solution by diluting an appropriate amount of reference standard to contain approximately 0.075 μg of each impurity per ml.

[0162] Limit of Quantification Solution 2: Accurately weigh impurity SM 1a SM 1b SM 1d Prepare a solution by diluting an appropriate amount of reference standard to contain approximately 0.075 μg of each impurity per ml.

[0163] Limit of Quantification Solution 3: Accurately weigh impurity SM 1n SM 1o Prepare a solution by diluting an appropriate amount of reference standard to contain approximately 0.075 μg of each impurity per ml.

[0164] (2) Measurement

[0165] Take the above-mentioned limit of quantitation solution 1, limit of quantitation solution 2, and limit of quantitation solution 3, and inject them 6 times consecutively. Calculate the ratio of the main peak height to the noise (signal-to-noise ratio) and record the chromatogram.

[0166] The formula for calculating the limit of quantitation is as follows:

[0167] Limit of quantitation (expressed as concentration present in the sample) % = (Limit of quantitation concentration ÷ Sample concentration) × 100%

[0168] The test results are shown in Table D. Figures 5-7 Tables 5-7 show the results. The data indicate that the limit of quantitation (LOQ) concentration of the starting material SM1 for utpatinib was 0.0761 μg / ml, the concentration present in the sample was 0.03%, the average signal-to-noise ratio was 32.0, and the peak area RSD was 1.7%. Impurity SM... 1i The limit of quantitation (LOQ) was 0.0723 μg / ml, the concentration present in the sample was 0.03%, the average signal-to-noise ratio was 34.3, and the RSD of the peak area was 1.8%; impurity SM 1f The limit of quantitation (LOQ) was 0.0724 μg / ml, the concentration present in the sample was 0.03%, the average signal-to-noise ratio was 46.7, and the RSD of the peak area was 5.5%; impurity SM 1b The limit of quantitation (LOQ) was 0.0757 μg / ml, the concentration present in the sample was 0.03%, the average signal-to-noise ratio was 32.5, and the RSD of the peak area was 3.9%; impurity SM 1h The limit of quantitation (LOQ) was 0.0728 μg / ml, the concentration of which was present in the sample was 0.03%, the average signal-to-noise ratio was 20.1, and the RSD of the peak area was 4.7%; impurity SM 1jThe limit of quantitation (LOQ) was 0.0745 μg / ml, the concentration present in the sample was 0.03%, the average signal-to-noise ratio was 26.1, and the RSD of the peak area was 5.4%; impurity SM 1d The limit of quantitation (LOQ) was 0.0757 μg / ml, the concentration present in the sample was 0.03%, the average signal-to-noise ratio was 28.2, and the RSD of the peak area was 2.0%; impurity SM 1a The limit of quantitation (LOQ) was 0.0736 μg / ml, the concentration present in the sample was 0.03%, the average signal-to-noise ratio was 16.0, and the RSD of the peak area was 4.6%; impurity SM 1m The limit of quantitation (LOQ) was 0.0751 μg / ml, the concentration present in the sample was 0.03%, the average signal-to-noise ratio was 15.3, and the RSD of the peak area was 5.6%; impurity SM 1n The limit of quantitation (LOQ) was 0.0753 μg / ml, the concentration present in the sample was 0.03%, the average signal-to-noise ratio was 27.2, and the RSD of the peak area was 3.9%; impurity SM 1o The limit of quantitation (LOQ) concentration was 0.0735 μg / ml, the concentration present in the sample was 0.03%, the average signal-to-noise ratio (SNR) was 25.5, and the RSD of the peak area was 1.6%. At the LOQ concentration, the SNR of each impurity was greater than 10, and the RSD of the peak area was less than 10%, meeting the requirements for related substance detection and indicating that the impurities could be accurately quantified at these levels.

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

[0170]

[0171]

[0172]

[0173] Example 4. Detection Limit Experiment

[0174] (1) Preparation of detection limit solution

[0175] Detection Limit Solution 1: Accurately transfer 13.5 ml of the Quantification Limit Solution into a 10 ml volumetric flask, and dilute with diluent to prepare a solution containing approximately SM1 and SM2 impurities per ml. 1f SM 1h SM 1i SM 1j SM 1m A solution of 0.025 μg each.

[0176] Detection limit solution 2: Accurately transfer 23.5 ml of the quantitation limit solution into a 10 ml volumetric flask, and dilute with diluent to prepare a solution containing approximately SM impurities per ml. 1a SM 1b SM1d A solution of 0.025 μg each.

[0177] Detection limit solution 3: Accurately transfer 33.5 ml of the quantitation limit solution into a 10 ml volumetric flask, and dilute with diluent to prepare a solution containing approximately SM impurities per ml. 1n SM 1o A solution of 0.025 μg each.

[0178] (2) Measurement

[0179] Take the above detection limit solution 1, detection limit solution 2 and detection limit solution 3, and inject them three times consecutively. Calculate the ratio of the main peak height to the noise (signal-to-noise ratio) and record the chromatogram.

[0180] The formula for calculating the detection limit is as follows:

[0181] Limit of detection (expressed as concentration present in the sample) % = (Limit of detection concentration ÷ Sample concentration) × 100%

[0182] The test results are shown in Table E. Figures 8-10 As shown in Tables 8-10, the detection limit concentration of umpatinib starting material SM1 was 0.0266 μg / ml, the concentration present in the sample was 0.01%, and the average signal-to-noise ratio was 18.5; impurity SM 1i The detection limit was 0.0253 μg / ml, the concentration in the sample was 0.01%, and the average signal-to-noise ratio was 14.6; impurity SM 1f The detection limit was 0.0253 μg / ml, the concentration of which was present in the sample was 0.01%, and the average signal-to-noise ratio was 20.2; impurity SM 1b The detection limit was 0.0265 μg / ml, the concentration of which was present in the sample was 0.01%, and the average signal-to-noise ratio was 12.2; impurity SM 1h The detection limit was 0.0255 μg / ml, the concentration present in the sample was 0.01%, and the average signal-to-noise ratio was 8.5; impurity SM 1j The detection limit was 0.0261 μg / ml, the concentration in the sample was 0.01%, and the average signal-to-noise ratio was 10.3; impurity SM 1d The detection limit was 0.0265 μg / ml, the concentration in the sample was 0.01%, and the average signal-to-noise ratio was 10.5; impurity SM 1a The detection limit was 0.0258 μg / ml, the concentration present in the sample was 0.01%, and the average signal-to-noise ratio was 6.2; impurity SM 1m The detection limit was 0.0263 μg / ml, the concentration present in the sample was 0.01%, and the average signal-to-noise ratio was 5.0; impurity SM 1nThe detection limit was 0.0264 μg / ml, the concentration of which was present in the sample was 0.01%, and the average signal-to-noise ratio was 10.3; impurity SM 1o The detection limit concentration was 0.0257 μg / ml, the concentration present in the sample was 0.01%, and the average signal-to-noise ratio was 8.8. At the detection limit concentration, the signal-to-noise ratio for each impurity was greater than 3, meeting the requirements for related substance detection, indicating that the impurities could be effectively detected at this level.

[0183] Table E. Results of Limit of Detection

[0184]

[0185]

[0186] Example 5. Durability Test

[0187] Mixed solution: The same as the mixed solution in Example 2.

[0188] 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 ±2℃). After the instrument system stabilizes, conduct the test to examine the resolution between each peak.

[0189] The test results are shown in Table F. Figures 11-16 As shown in Tables 11 to 16, when there are fluctuations in the chromatographic system, 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.

[0190] Table F. Results of Resolution Determination in Chromatographic Condition Variation Robustness Test

[0191]

Claims

1. A method for separating umpatinib starting material SM1 and its impurities based on high performance liquid chromatography, characterized in that, The utpatinib starting material SM1 and the impurities together constitute the composition, the impurities including impurity SM. 1a Impurities SM 1b Impurities SM 1d Impurities SM 1f Impurities SM 1h Impurities SM 1i Impurities SM 1j Impurities SM 1m Impurities SM 1n and impurities SM 1o Any one or more of the following; the structural formula of each component in the composition is as follows: In the high-performance liquid chromatography method, the mobile phase is: phosphoric acid solution as mobile phase A and organic solvent as mobile phase B; the stationary phase is: the chromatographic column uses octadecylsilane pentafluorophenyl alternating bonded silica gel as the packing material, 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 organic solvent is methanol and / or acetonitrile; the phosphoric acid content in the phosphoric acid solution is 0.05% to 0.2%.

4. The method according to claim 1, characterized in that, The flow rate was 0.7–1.3 mL / min; the column temperature was 25–35 °C.

5. A method for identifying umpatinib starting material SM1 and its impurities, characterized in that, The composition is separated using the method described in 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 starting material SM1 and its impurities.

6. The method according to claim 5, characterized in that, The detector has a detection wavelength of 230±10nm.

7. The method according to claim 5, characterized in that, The components of the composition can be identified according to their relative retention times. The components of the composition, in ascending order, are: impurities SM. 1i Impurities SM 1f Impurities SM 1b Impurities SM 1h Impurities SM 1j Upatinib starting material SM1, impurities SM 1d Impurities SM 1a Impurities SM 1m Impurities SM 1n Impurities SM 1o .

8. The method according to claim 7, characterized in that, Using SM1, the starting material for utpatinib, as a reference peak; with a relative retention time of 0.11, it was identified as impurity SM. 1i ; The relative retention time was 0.26, and it was determined to be impurity SM. 1f ; The relative retention time was 0.39, and it was determined to be impurity SM. 1b ; The relative retention time was 0.51, indicating it was classified as impurity SM. 1h ; The relative retention time was 0.74, and it was determined to be impurity SM. 1j ; The relative retention time was 1.00, which was determined to be the starting material SM1 for utpatinib; The relative retention time was 1.24, and it was determined to be impurity SM. 1d ; The relative retention time was 1.39 seconds, indicating it was classified as impurity SM. 1a ; The relative retention time was 1.44, and it was determined to be impurity SM. 1m ; The relative retention time was 1.51, indicating it was classified as impurity SM. 1n ; The relative retention time was 1.58, and it was determined to be impurity SM. 1o The relative retention times of each component fluctuated within ±5%.

9. A method for determining the content of umpatinib starting material SM1 and its impurities, characterized in that, The starting material SM1 of utpatinib and its impurities are separated and identified by the method described in any one of claims 6-8, and a chromatogram is obtained. Based on the obtained chromatogram, the content of each impurity is calculated by principal component self-comparison with correction factor and / or limit method.

10. The method according to claim 9, characterized in that, The impurity SM 1a The correction factor is 1.6; the impurity SM 1b The correction factor is 0.79; the impurity SM 1f The correction factor is 0.55; the impurity SM 1h The correction factor is 1.2; the impurity SM 1i The correction factor is 1.6; the impurity SM 1n The correction factor is 1.1; the impurity SM 1o The correction factor is 1.2.

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