Method for separating and detecting crizotinib starting material SM1a and impurities thereof

Abstract

The invention belongs to the technical field of pharmaceutical analysis, and particularly relates to a method for separating and detecting a crizotinib starting material SM1a and impurities thereof. The impurities comprise any one or more of an impurity SM < 1a-b >, an impurity SM < 1a-c >, an impurity SM < 1a-e >, an impurity SM < 1a-f >, an impurity SM < 1a-g >, an impurity SM < 1a-h >, an impurity SM < 1a-i >, an impurity SM < 1a-j > and an impurity SM < 1a-k >. According to the method, high performance liquid chromatography is adopted for detection, specifically, water is used as a mobile phase A, and acetonitrile is used as a mobile phase B; octadecylsilane chemically bonded silica is adopted as a chromatographic column filler, and linear gradient elution is carried out; and then calculating the content of each impurity by adopting a limit method according to a chromatogram. The method has the characteristics of good separation degree, good durability, high sensitivity and good reproducibility.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical analysis technology, and specifically relates to a method for separating and detecting crizotinib starting material SM 1a and its impurities. Background Art

[0002] Crizotinib is an oral small molecule tyrosine kinase inhibitor that inhibits tumor growth by suppressing abnormal protein kinase activity within tumor cells. It is primarily used to treat advanced ALK-positive and ROS1-positive non-small cell lung cancer (NSCLC). It is the world's first approved targeted drug for both ALK and ROS1 targets and has demonstrated significant efficacy in treating patients with ALK fusion genotype NSCLC.

[0003] SM 1a It is the key starting material for the synthesis of crizotinib, generally made from SM 1a-b It is obtained by dehydrogenase catalysis, and its synthetic route is as follows:

[0004]

[0005] The study found that SM 1a The following impurities are easily introduced during the synthesis process: Impurity SM 1a-b 、Impurity SM 1a-c 、Impurity SM 1a-e 、Impurity SM 1a-f 、Impurity SM 1a-g 、Impurity SM 1a-h 、Impurity SM 1a-i 、Impurity SM 1a-j 、Impurity SM 1a-k Among them, impurity SM 1a-b Is the residual raw material; impurity SM 1a-c It's SM 1a-b phenyl ring position isomers; impurity SM 1a-j 、Impurity SM 1a-k It is a halogen position isomer; impurity SM 1a-e 、Impurity SM 1a-f 、Impurity SM 1a-g 、Impurity SM 1a-h 、Impurity SM 1a-i It is a dehalogenated impurity.

[0006] The dosage of crizotinib needs to be strictly controlled during treatment. Impurities introduced into the starting materials and generated during the production process will inevitably affect the content of the active ingredient of the main drug and even cause adverse reactions. Therefore, the quality control of the starting materials and the detection of impurities during the production process are particularly important. However, these impurities are very similar in structure and difficult to separate. Currently, there is no method that can simultaneously separate and determine the crizotinib starting material SM.1a And the specific methods for the above 9 impurities.

[0007] Some existing technologies report analytical methods for crizotinib and its related substances. For example, patent CN111007158A discloses a method for separating and determining related substances in the preparation of crizotinib. The method employs reverse-phase high-performance liquid chromatography with gradient elution using the following mobile phase conditions: mobile phase A is a 0.1% trifluoroacetic acid solution by volume, mobile phase B is methanol or acetonitrile, and the mobile phase flow rate is 0.9-1.1 mL / min; the column temperature is 20-30°C; a diode array or ultraviolet detector is used at a detection wavelength of 254 nm; the injection volume is 10 μL; and the relevant impurities include CT-02 and CT-03.

[0008]

[0009] For example, patent CN117571843A discloses a method for detecting and analyzing 2-amino-3-hydroxypyridine in crizotinib starting materials. The method is performed on a high-performance liquid chromatograph using an UltraAQ C18 column with a buffered saline solution and an organic solvent as the mobile phase. Gradient elution detection using a liquid-phase diode array detector (DAD) or ultraviolet (UV) detector effectively detects the residual amount of 2-amino-3-hydroxypyridine.

[0010] However, the above methods are unable to simultaneously separate and determine the crizotinib starting material SM. 1a and its 9 related impurities. 1a For quality control of crizotinib, an efficient, robust, and sensitive method is needed for the determination of crizotinib starting material SM 1a And separation and detection of related impurities. Summary of the Invention

[0011] In view of this, one of the objects of the present invention is to provide a method for separating the crizotinib starting material SM 1a The method can complete the separation of multiple substances in a shorter time.

[0012] To achieve the above object, the technical solution of the present invention is:

[0013] Separation of crizotinib starting material SM based on HPLC 1a and its impurities, the crizotinib starting material SM 1a and the impurities together form a composition, wherein the impurities include impurity SM 1a-b 、Impurity SM 1a-c 、Impurity SM 1a-e 、Impurity SM 1a-f、Impurity SM 1a-g 、Impurity SM 1a-h 、Impurity SM 1a-i 、Impurity SM 1a-j 、Impurity SM 1a-k Any one or more of; the structural formula of each component in the composition is as follows:

[0014]

[0015] In the high performance liquid chromatography method, the mobile phase is water as mobile phase A and acetonitrile as mobile phase B; the stationary phase is octadecylsilane bonded silica gel as the chromatographic column filler, and linear gradient elution is performed.

[0016] After separation and testing, it is used for the next production.

[0017] The above impurities can be arranged and combined in many ways. For example, combination 1: impurity SM 1a-b 、Impurity SM 1a-c For example, combination 2: impurity SM 1a-e 、Impurity SM 1a-f 、Impurity SM 1a-g For example, combination 3: impurity SM 1a-b 、Impurity SM 1a-c 、Impurity SM 1a-e 、Impurity SM 1a-f 、Impurity SM 1a-g 、Impurity SM 1a-h 、Impurity SM 1a-i 、Impurity SM 1a-j 、Impurity SM 1a-k .

[0018] Theoretically, when the upper limit of the substances that can be separated, identified and / or detected by this method is n (number), 1-n substances can be detected.

[0019] Further, the procedure of the linear gradient elution is as follows:

[0020] Time - minutes Mobile phase A-% Mobile phase B-% 0 70±10 30±10 4±1 70±10 30±10 27±1 35±10 65±10 30±1 35±10 65±10 31±1 70±10 30±10 40±1 70±10 30±10 .

[0021] Preferably, the procedure of the linear gradient elution is as follows:

[0022] Time - minutes Mobile phase A-% Mobile phase B-% 0 70±2 30±2 4 70±2 30±2 27 35 65 30 35 65 31 70±2 30±2 40 70±2 30±2 .

[0023] For example, mobile phase A accounts for 68 parts and mobile phase B accounts for 32 parts ( Figure 12 ); or mobile phase A accounts for 69 parts, mobile phase B accounts for 31 parts; or mobile phase A accounts for 70 parts, mobile phase B accounts for 30 parts ( Figure 4 ); or mobile phase A accounts for 71 parts, mobile phase B accounts for 29 parts; or mobile phase A accounts for 72 parts, mobile phase B accounts for 28 parts ( Figure 11 ).

[0024] More preferably, the linear gradient elution procedure is as follows:

[0025] Time - minutes Mobile phase A-% Mobile phase B-% 0 70 30 4 70 30 27 35 65 30 35 65 31 70 30 40 70 30 .

[0026] Furthermore, the flow rate was 0.5-1.5 mL / min and the column temperature was 30-50°C.

[0027] Preferably, the flow rate is 0.9-1.1 mL / min; and the column temperature is 38-42°C.

[0028] For example, the flow rate is 0.9 mL / min ( Figure 7 ); or a flow rate of 1.0 mL / min ( Figure 4 ); or a flow rate of 1.1 mL / min ( Figure 8 For example, the column temperature is 38°C ( Figure 9 ); or column temperature is 39℃; or column temperature is 40℃( Figure 4 ); or column temperature is 41°C; or column temperature is 42°C ( Figure 10 ).

[0029] More preferably, the flow rate is 1.0 mL / min and the column temperature is 40°C.

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

[0031] More preferably, the chromatographic column is SHIMADZU Shim-pack Scepter C18, 4.6 mm×250 mm 5 μm.

[0032] Preferably, the injection volume is 10 μl.

[0033] Preferably, the running time is 40 minutes.

[0034] The second object of the present invention is to provide a method for identifying the crizotinib starting material SM 1a The method can complete the identification of multiple substances in a shorter time.

[0035] To achieve the above object, the technical solution of the present invention is:

[0036] Identification of crizotinib starting material SM 1aThe method for separating the composition and its impurities is as follows: the composition is separated by the aforementioned separation method, and the composition is detected by a detector with a detection wavelength of 210±10 nm to obtain a chromatogram; by comparing the chromatogram characteristics of the test sample with those of the reference sample, it is determined whether the test sample contains the crizotinib starting material SM 1a and its impurities.

[0037] As a preferred approach, the detection wavelength range of ±10 nm is based on a comprehensive consideration of factors including error tolerance, method superiority, and practical application requirements. This range helps ensure the reliability of test results, improve measurement repeatability and flexibility, and meet specific experimental requirements.

[0038] More preferably, the detection wavelength is 210 nm.

[0039] As a preferred solution, the components in the composition can be identified in the order of relative retention time. The components of the composition are as follows in ascending order: impurity SM 1a-g 、Impurity SM 1a-f 、Impurity SM 1a-e 、Impurity SM 1a-i 、Impurity SM 1a-h , Crizotinib starting material SM 1a 、Impurity SM 1a-k 、Impurity SM 1a-j 、Impurity SM 1a-b 、Impurity SM 1a-c .

[0040] Relative retention time is typically used to describe the relative retention of different components in a mixture on a chromatographic column. It is the ratio of the retention time of a 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 positioning, qualitative, and quantitative analysis in chromatographic analysis. Relative retention time is calculated by dividing the retention time of the target component by the retention time of the reference component.

[0041] As a preferred solution, the crizotinib starting material SM 1a It is a reference peak with a relative retention time of 0.48, and is determined to be impurity SM 1a-g ; The relative retention time is 0.59, which is determined to be impurity SM 1a-f ; The relative retention time is 0.87, which is determined to be impurity SM 1a-e ; The relative retention time is 0.92, which is determined to be impurity SM 1a-i ; The relative retention time is 0.95, which is determined to be impurity SM 1a-h ; The relative retention time is 1.04, which is determined to be impurity SM 1a-k; The relative retention time is 1.22, which is determined to be impurity SM 1a-j ; The relative retention time is 1.29, which is determined to be impurity SM 1a-b ; The relative retention time is 1.32, which is determined to be impurity SM 1a-c The relative retention time range of each impurity fluctuates in sequence within the range of ±0.05 of its respective relative retention time.

[0042] In addition to relative retention time, retention time can also be used to identify components. Retention time is the time it takes for a sample to enter the chromatographic column and be detected by the detector. This time is calculated based on the migration speed of the component along the column, that is, the interval from the start of injection to the chromatographic apex (maximum concentration) of a component. It is primarily used to determine the order and location of peaks of each component in a sample and is one of the essential data in chromatographic analysis. In quality control, changes in retention time can reflect factors such as the condition of the chromatographic column, the stability of the mobile phase, and the performance of the instrument.

[0043] The third object of the present invention is to provide a method for detecting the starting material SM of crizotinib 1a This method can complete the content determination of multiple substances in a relatively short time.

[0044] To achieve the above object, the technical solution of the present invention is:

[0045] Detection of crizotinib starting material SM 1a The method for determining whether the impurity content in the crizotinib is qualified is to separate and identify the crizotinib starting material SM using the above-mentioned identification method. 1a and its impurities to obtain a chromatogram; based on the obtained chromatogram, the limit method is used to determine whether the impurity content in the test product is qualified.

[0046] As a preferred solution, if the impurity SM in the test sample 1a-b 、Impurity SM 1a-c 、Impurity SM 1a-e 、Impurity SM 1a-f 、Impurity SM 1a-g 、Impurity SM 1a-h 、Impurity SM 1a-i 、Impurity SM 1a-j 、Impurity SM 1a-k If the peak area of ​​any one or more impurities in the test sample is greater than the peak area of ​​the corresponding impurities in the reference solution, it indicates that the impurity content is unqualified; on the contrary, if the impurity SM in the test sample is 1a-b 、Impurity SM 1a-c 、Impurity SM 1a-e 、Impurity SM 1a-f 、Impurity SM 1a-g 、Impurity SM 1a-h 、Impurity SM 1a-i、Impurity SM 1a-j 、Impurity SM 1a-k The peak area of ​​any one or more impurities in the sample is not greater than the peak area of ​​the corresponding impurities in the reference solution, indicating that the impurity content is qualified.

[0047] The above-mentioned determination method can be used as a drug quality determination model and further as an indispensable key module in the intelligent production process. By precisely controlling parameters such as mobile phase composition, flow rate, and column temperature, this model achieves accurate separation and quantitative analysis of active ingredients, impurities, and degradation products in drugs, providing a scientific basis for comprehensive assessment of drug quality. In the intelligent production system, this model is seamlessly integrated, capable of receiving raw data from the production line in real time, automatically performing analysis tasks, and quickly providing feedback on determination results based on preset quality standards.

[0048] As a preferred solution, the sample preparation solvent is methanol.

[0049] As a preferred solution, the test solution is prepared as follows:

[0050] Sample solution: Take an appropriate amount of this product, weigh accurately, dissolve it in methanol and quantitatively dilute it to make a solution with a concentration of 1 mg / ml.

[0051] Reference solution: take impurity SM 1a-c , SM 1a-e , SM 1a-f , SM 1a-g , SM 1a-h , SM 1a-i , SM 1a-j and SM 1a-k An appropriate amount of each reference substance was accurately weighed, dissolved in methanol and quantitatively diluted to prepare a solution with a concentration of 1 μg / ml.

[0052] The beneficial effects of the present invention are:

[0053] 1) The present invention provides a high performance liquid chromatography method for separating and detecting the starting material SM of crizotinib 1a The method for separating and analyzing crizotinib starting material SM can be used to separate and analyze crizotinib starting material SM 1a and its related impurities SM 1a-b , SM 1a-c , SM 1a-e , SM 1a-f , SM 1a-g , SM 1a-h , SM 1a-i , SM 1a-j , SM 1a-k The separation degree between each impurity is greater than 1.5. The method of the present invention has the advantages of simplicity, rapidity, strong specificity and high accuracy.

[0054] 2) This method is highly specific and is not affected by blank solvents and other unknown impurities.

[0055] 3) This method is a self-developed high-performance liquid chromatography method, which fills the gap in this detection field.

[0056] 4) This method can be used to make the crizotinib starting material SM 1a The quality of the product is effectively controlled, and the crizotinib raw material that meets the quality standards is obtained.

[0057] 5) The present invention screens different chromatographic columns, adjusts the gradient elution procedure, etc. to make the crizotinib starting material SM 1a The method can be used to separate the impurities and perform qualitative and quantitative analysis. This method has a short detection time, detects many impurities, and has a simple detection process, effectively reducing the time and labor costs for production inspection work.

[0058] 6) The mobile phase preparation process of this method is simple and easy to operate.

[0059] 7) This method has the characteristics of high sensitivity and low detection limit and quantification limit. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 The chromatogram is of the blank solution.

[0061] Figure 2 The chromatogram is that of the reference solution.

[0062] Figure 3 is the chromatogram of the test solution.

[0063] Figure 4 The chromatogram of the mixed solution.

[0064] Figure 5 The chromatogram is the quantification limit solution.

[0065] Figure 6 The chromatogram is the detection limit solution.

[0066] Figure 7 This is the chromatogram of the mixed solution at a flow rate of 0.9 ml / min in the durability experiment.

[0067] Figure 8 This is the chromatogram of the mixed solution at a flow rate of 1.1 ml / min in the durability experiment.

[0068] Figure 9 This is the chromatogram of the mixed solution when the column temperature is 38°C in the durability experiment.

[0069] Figure 10 This is the chromatogram of the mixed solution when the column temperature is 42°C in the durability experiment.

[0070] Figure 11 This is the chromatogram of the mixed solution under the initial ratio of the mobile phase (water-acetonitrile = 72:28) in the durability experiment.

[0071] Figure 12 This is the chromatogram of the mixed solution under the initial ratio of the mobile phase (water-acetonitrile = 68:32) in the durability experiment. DETAILED DESCRIPTION

[0072] The technical solutions of the present invention will be further described in detail below with reference to specific embodiments. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Therefore, all other embodiments derived by those skilled in the art based on the embodiments of the present invention without inventive effort are intended to fall within the scope of protection of the present invention.

[0073] Supplementary tables to the drawings in the specification. Figures 1-12 Visual aids are provided for the purpose of understanding and interpretation. If there is any ambiguity, the user should refer to the corresponding numbered tables (Tables 1-12) and data for more detailed information. Conversely, if any information that may cause misunderstanding or ambiguity is found in the process of reviewing Tables 1-12, the content in the corresponding numbered figures shall prevail. The above guidelines are intended to ensure the correct interpretation of this document and the consistency of its information. Although some of the characters in the spectrum of this application document overlap, they can still be viewed clearly, and the integration results of each figure are recorded in detail in the specification. In addition, the numbers in the spectrum have no effect on the full disclosure of the technical solutions in the claims and the specification.

[0074] Table 1

[0075]

[0076] Table 2

[0077]

[0078] Table 3

[0079]

[0080] Table 4

[0081]

[0082] Table 5

[0083]

[0084] Table 6

[0085]

[0086] Table 7

[0087]

[0088] Table 8

[0089]

[0090] Table 9

[0091]

[0092] Table 10

[0093]

[0094] Table 11

[0095]

[0096] Table 12

[0097]

[0098] In order to enhance understanding of the present invention, certain key technical and scientific terms will be clearly defined below. Unless otherwise specifically defined herein, all other technical and scientific terms follow the meanings generally accepted and understood in the field to which the present invention belongs. It should be emphasized that the scope of the present invention is not limited to the specific methods, reagents, compounds, compositions, reference substances, and test products described, but allows reasonable flexibility and adjustment in these aspects. At the same time, please understand that the terms used herein are intended to illustrate specific embodiments and are not to be interpreted restrictively.

[0099] In addition, all literature cited herein, including but not limited to patents, patent applications, academic papers, textbooks, and further citations therein, are deemed to be incorporated herein by reference in their entirety, to the extent not directly cited. In the event of any inconsistency or conflict between the contents of such cited literature or similar materials and this application, particularly with respect to term definitions, term usage, or technical descriptions, the contents of this application shall prevail.

[0100] If there are any unmentioned conditions in the chromatographic conditions, the determination can be referred to the high performance liquid chromatography method (Chinese Pharmacopoeia 2020 Edition Part 4 0512).

[0101] the term

[0102] The limit of quantification (LOQ) is the lowest amount of an analyte in a sample that can be quantitatively determined with a certain degree of accuracy and precision. In other words, the LOQ is the lowest level at which an analytical method can accurately and reliably determine the concentration of the analyte in a sample. In HPLC, the determination of the LOQ typically relies on the signal-to-noise (S / N) ratio, where the analyte concentration corresponding to a certain signal-to-noise ratio is used as the LOQ. Determining the LOQ is crucial to 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 unsignificantly affect results even when minor changes in measurement conditions occur. 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 the substance 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 the sample is processed.

[0105] 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, the peak areas produced by the same mass of different substances passing through the detector may not be equal. To ensure that the peak area accurately reflects the content of the component being measured, calibration is performed using a standard substance. The correction factor is calculated and applied to the measurement results of the sample being measured.

[0106] The peak height to noise ratio (S / N, or signal-to-noise ratio) is used in HPLC to assess instrument sensitivity and resolution and is a key performance metric. Peak height refers to the detector signal output when the analyte elutes from the post-column column, while noise refers to the fluctuation of the baseline signal, i.e., the signal value measured with a blank sample. The S / N ratio is the ratio of the signal measured with a sample of known concentration to the signal measured with a blank sample. A higher S / N ratio indicates a more accurate separation and identification of the target component while also minimizing background noise interference.

[0107] In the present invention, the crizotinib starting material SM 1a The information of the impurities is shown in Table A.

[0108] Table A Compound Information

[0109]

[0110] Example 1. Separation and detection of crizotinib starting material SM 1a and impurities thereof

[0111] (1) Prepare the test solution

[0112] Test solution: Take an appropriate amount of the product, weigh accurately, dissolve it in methanol and quantitatively dilute it to make a solution containing approximately 1 mg per 1 ml.

[0113] Reference solution: take impurity SM 1a-c , SM 1a-e , SM 1a-f , SM 1a-g , SM 1a-h , SM 1a-i , SM 1a-j and SM 1a-k An appropriate amount of each reference substance was accurately weighed, dissolved in methanol and quantitatively diluted to prepare a solution containing approximately 1 μg per 1 ml.

[0114] (2) Chromatographic conditions

[0115] The main instruments and chromatographic conditions are shown in Table B and Table C.

[0116] Table B Main instruments and chromatographic conditions information

[0117]

[0118] Table C Gradient elution program

[0119] Time - minutes Mobile phase A-% Mobile phase B-% 0 70 30 4 70 30 27 35 65 30 35 65 31 70 30 40 70 30

[0120] (3) Detection

[0121] System suitability: In the reference solution, the RSD of each impurity peak was less than 5.0%, the number of theoretical plates was greater than 5000, and the tailing factor was within the range of 0.8 to 1.8, which met the requirements.

[0122] Determination method: Accurately measure 10 μl of the test solution and the reference solution, inject them separately for detection, and record the chromatogram. The detection results of the reference solution are as follows: Figure 2 , as shown in Table 2.

[0123] Example 2. Specificity

[0124] (1) Prepare the test solution

[0125] Blank solvent (diluent): methanol.

[0126] Impurity SM 1a-b Stock solution: Accurately weigh SM 1a-b Place 50.48 mg of the reference substance in a 100 ml volumetric flask, add methanol to dissolve and dilute to the scale, shake well, and obtain the product.

[0127] Impurity SM 1a-cStock solution: Accurately weigh SM 1a-c Place 19.55 mg of the reference substance in a 100 ml volumetric flask, add methanol to dissolve and dilute to the scale, shake well, and obtain the product.

[0128] Impurity SM 1a-e Stock solution: Accurately weigh SM 1a-e Place 19.45 mg of the reference substance in a 100 ml volumetric flask, add methanol to dissolve and dilute to the scale, shake well, and obtain the product.

[0129] Impurity SM 1a-f Stock solution: Accurately weigh SM 1a-f Place 19.49 mg of the reference substance in a 100 ml volumetric flask, add methanol to dissolve and dilute to the scale, shake well, and obtain the product.

[0130] Impurity SM 1a-g Stock solution: Accurately weigh SM 1a-g Place 19.46 mg of the reference substance in a 100 ml volumetric flask, add methanol to dissolve and dilute to the scale, shake well, and obtain the product.

[0131] Impurity SM 1a-h Stock solution: Accurately weigh SM 1a-h Place 19.91 mg of the reference substance in a 100 ml volumetric flask, add methanol to dissolve and dilute to the scale, shake well, and obtain the product.

[0132] Impurity SM 1a-i Stock solution: Accurately weigh SM 1a-i Place 19.96 mg of the reference substance in a 100 ml volumetric flask, add methanol to dissolve and dilute to the scale, shake well, and obtain the product.

[0133] Impurity SM 1a-j Stock solution: Accurately weigh SM 1a-j Place 19.63 mg of the reference substance in a 100 ml volumetric flask, add methanol to dissolve and dilute to the scale, shake well, and obtain the product.

[0134] Impurity SM 1a-k Stock solution: Accurately weigh SM 1a-k Place 19.81 mg of the reference substance in a 100 ml volumetric flask, add methanol to dissolve and dilute to the scale, shake well, and obtain the product.

[0135] Impurity SM 1a-b Positioning solution: Precisely measure impurities SM 1a-b Place 2.5 ml of the stock solution in a 100 ml volumetric flask, dilute to the mark with methanol, and shake well.

[0136] Other impurity location solutions: Precisely measure impurity SM 1a-c 、Impurity SM 1a-e 、Impurity SM 1a-f 、Impurity SM1a-g 、Impurity SM 1a-h 、Impurity SM 1a-i 、Impurity SM 1a-j and impurity SM 1a-k Place 5 ml of each stock solution in different 100 ml volumetric flasks, dilute to the scale with methanol, and shake well.

[0137] Reference stock solution: Accurately measure the impurity SM 1a-c 、Impurity SM 1a-e 、Impurity SM 1a-f 、Impurity SM 1a-g 、Impurity SM 1a-h 、Impurity SM 1a-i 、Impurity SM 1a-j and impurity SM 1a-k Place 5 ml of each stock solution in the same 100 ml volumetric flask, dilute to the scale with methanol, and shake well.

[0138] Test solution: Accurately weigh 10.11 mg of this product (batch number: HJ008-20221101), place it in a 10 ml volumetric flask, add methanol to dissolve and dilute to the scale, shake well, and obtain.

[0139] Mixed solution: Accurately weigh 10.73 mg of this product (batch number: HJ008-20221101) and place it in a 10 ml volumetric flask. Accurately add the reference stock solution and impurity SM 1a-b Dissolve 1 ml of each positioning solution in methanol and dilute to the scale, shake well, and you are done.

[0140] (2) Detection

[0141] Accurately measure 10 μl of blank solvent, each positioning solution, test solution, and mixed solution, inject them into the liquid chromatograph, and record the chromatogram. Inspect the separation between impurities and adjacent peaks.

[0142] The test results are shown in Table D. Figure 1 、 Figure 3 、 Figure 4 , Table 1, Table 3 and Table 4. Blank solvent, other impurities and other components in the test sample did not interfere with the extraction of impurity SM 1a-g , SM 1a-f , SM 1a-e , SM 1a-i , SM 1a-h , SM 1a-k , SM 1a-j , SM 1a-c Detection of impurities SM 1a-g , SM 1a-f , SM 1a-e , SM 1a-i , SM 1a-h , SM1a-k , SM 1a-j , SM 1a-c The separation between adjacent peaks meets the requirements, and this method has good specificity.

[0143] Table D Specificity test results

[0144]

[0145] In addition, the results of the standard addition test showed that the recovery rates of each impurity were between 90.0% and 108.0%, meeting the requirements. The results of the solution stability test showed that the reference solution and the spiked test solution were stable for 34.5 hours at room temperature.

[0146] Example 3. Limit of Quantitation

[0147] Quantitation limit solution: Accurately measure 5 ml of the reference substance stock solution under the "Specificity" item, place it in a 50 ml volumetric flask, dilute it to the scale with methanol, shake well, then accurately measure 4 ml, place it in a 25 ml volumetric flask, dilute it to the scale with methanol, shake well, and the solution is obtained.

[0148] Accurately measure 10 μl of the quantitation limit solution and inject it into the liquid chromatograph. Repeat the test six times and record the chromatogram. Calculate the peak height to noise ratio (SNR) and the peak area RSD to assess the sensitivity of the chromatographic system.

[0149] The formula for calculating the limit of quantitation is as follows:

[0150]

[0151] The test results are shown in Table E. Figure 5 , Table 5. Impurity SM 1a-g The limit of quantification concentration was 0.1524 μg / ml, equivalent to 0.0152% of the test sample concentration, the average signal-to-noise ratio was 21.7, and the peak area RSD was 1.8%; the impurity SM 1a-f The limit of quantification concentration was 0.1529 μg / ml, equivalent to 0.0153% of the test sample concentration, the average signal-to-noise ratio was 14.9, and the peak area RSD was 5.7%; the impurity SM 1a-e The limit of quantification concentration was 0.1478 μg / ml, equivalent to 0.0148% of the test sample concentration, the average signal-to-noise ratio was 15.4, and the peak area RSD was 4.7%; the impurity SM 1a-i The limit of quantification concentration was 0.1554 μg / ml, equivalent to 0.0155% of the test sample concentration, the average signal-to-noise ratio was 14.8, and the peak area RSD was 4.7%; the impurity SM 1a-hThe limit of quantification concentration was 0.1545 μg / ml, equivalent to 0.0155% of the test sample concentration, the average signal-to-noise ratio was 30.7, and the peak area RSD was 2.7%; the impurity SM 1a-k The limit of quantification concentration was 0.1513 μg / ml, equivalent to 0.0151% of the test sample concentration, the average signal-to-noise ratio was 25.8, and the peak area RSD was 5.0%; the impurity SM 1a-j The limit of quantification concentration was 0.1547 μg / ml, equivalent to 0.0155% of the test sample concentration, the average signal-to-noise ratio was 19.0, and the peak area RSD was 6.1%; the impurity SM 1a-c The limit of quantification concentration was 0.1561 μg / ml, equivalent to 0.0156% of the test sample concentration, the average signal-to-noise ratio was 33.3, and the peak area RSD was 1.6%, indicating that the above impurities could be accurately quantified at this level.

[0152] Table E Quantitation Limit Test Results

[0153]

[0154]

[0155] Example 4. Detection limit

[0156] Detection limit solution: Accurately measure 5 ml of the quantification limit solution under the "quantification limit" item, place it in a 10 ml volumetric flask, dilute to the scale with methanol, and shake well.

[0157] Accurately measure 10 μl of the detection limit solution and inject it into the liquid chromatograph. Repeat the test three times and record the chromatogram. Calculate the ratio of peak height to noise (signal-to-noise ratio) to evaluate the sensitivity of the chromatographic system.

[0158] The detection limit is calculated as follows:

[0159]

[0160] The test results are shown in Table F. Figure 6 , Table 6. Impurity SM 1a-g The detection limit concentration is 0.0762μg / ml, which is equivalent to 0.0076% of the test sample concentration, and the average signal-to-noise ratio is 10.4; the impurity SM 1a-f The detection limit concentration is 0.0764μg / ml, which is equivalent to 0.0076% of the test sample concentration, and the average signal-to-noise ratio is 7.6; the impurity SM 1a-e The detection limit concentration is 0.0739μg / ml, which is equivalent to 0.0074% of the test sample concentration, and the average signal-to-noise ratio is 7.7; the impurity SM 1a-iThe detection limit concentration is 0.0777μg / ml, which is equivalent to 0.0078% of the test sample concentration, and the average signal-to-noise ratio is 7.5; the impurity SM 1a-h The detection limit concentration is 0.0773μg / ml, which is equivalent to 0.0077% of the test sample concentration, and the average signal-to-noise ratio is 14.5; the impurity SM 1a-k The detection limit concentration is 0.0756μg / ml, which is equivalent to 0.0076% of the test sample concentration, and the average signal-to-noise ratio is 12.3; the impurity SM 1a-j The detection limit concentration was 0.0773 μg / ml, equivalent to 0.0077% of the test sample concentration, and the average signal-to-noise ratio was 9.0; the impurity SM 1a-c The detection limit concentration is 0.0780μg / ml, which is equivalent to 0.0078% of the test sample concentration, and the average signal-to-noise ratio is 15.4, indicating that the above impurities can be effectively detected at this level.

[0161] Table F Detection limit test results

[0162]

[0163]

[0164] Example 5. Durability

[0165] Mixed solution: Use the mixed solution and its spectrum under the "Specificity" item.

[0166] Accurately measure 10 μl of the mixed solution and adjust the chromatographic conditions (flow rate ±0.1 ml / min, column temperature ±2°C, initial mobile phase ratio ±2%). After the instrument system stabilizes, perform sample injections and record the chromatograms. Inspect the separation of each impurity from adjacent peaks.

[0167] The test results are shown in Table G. Figures 7 to 12 , Table 7 to Table 12. When the chromatographic conditions fluctuate, the impurity SM 1a-g , SM 1a-f , SM 1a-e , SM 1a-i , SM 1a-h , SM 1a-k , SM 1a-j , SM 1a-c The separation between adjacent peaks was greater than 1.5, indicating that the method had good robustness.

[0168] Table G Durability test results

[0169]

Claims

1. Separation of crizotinib starting material SM based on high performance liquid chromatography 1a and impurities thereof, characterized in that, The crizotinib starting material SM 1a and the impurities together form a composition, wherein the impurities include impurity SM 1a-b 、Impurity SM 1a-c 、Impurity SM 1a-e 、Impurity SM 1a-f 、Impurity SM 1a-g 、Impurity SM 1a-h 、Impurity SM 1a-i 、Impurity SM 1a-j 、Impurity SM 1a-k Any one or more of; the structural formula of each component in the composition is as follows: In the high performance liquid chromatography method, the mobile phase is water as mobile phase A and acetonitrile as mobile phase B; the stationary phase is octadecylsilane bonded silica gel as the chromatographic column filler, and linear gradient elution is performed.

2. The method according to claim 1, characterized in that The procedure of the linear gradient elution is as follows: 。 3. The method according to claim 1, characterized in that The procedure of the linear gradient elution is as follows: 。 4. The method according to claim 1, wherein The flow rate was 0.5-1.5 mL / min; the column temperature was 30-50°C.

5. Identification of Crizotinib Starting Material SM 1a and impurities thereof, characterized in that, The composition is separated by the method according to any one of claims 1 to 4, and the detection is performed by a detector with a detection wavelength of 210 ± 10 nm to obtain a chromatogram; and the chromatogram characteristics of the test sample and the reference sample are compared to determine whether the test sample contains the crizotinib starting material SM. 1a and its impurities.

6. The method according to claim 5, characterized in that According to the order of relative retention time, the components in the composition can be identified. The components of the composition are as follows in ascending order: impurity SM 1a-g 、Impurity SM 1a-f 、Impurity SM 1a-e 、Impurity SM 1a-i 、Impurity SM 1a-h , Crizotinib starting material SM 1a 、Impurity SM 1a-k 、Impurity SM 1a-j 、Impurity SM 1a-b 、Impurity SM 1a-c .

7. The method according to claim 6, characterized in that Starting from crizotinib SM 1a It is a reference peak with a relative retention time of 0.48, and is determined to be impurity SM 1a-g ; The relative retention time is 0.59, which is determined to be impurity SM 1a-f ; The relative retention time is 0.87, which is determined to be impurity SM 1a-e ; The relative retention time is 0.92, which is determined to be impurity SM 1a-i ; The relative retention time is 0.95, which is determined to be impurity SM 1a-h ; The relative retention time is 1.04, which is determined to be impurity SM 1a-k ; The relative retention time is 1.22, which is determined to be impurity SM 1a-j ; The relative retention time is 1.29, which is determined to be impurity SM 1a-b ; The relative retention time is 1.32, which is determined to be impurity SM 1a-c The relative retention time ranges of the impurities fluctuate in sequence within the range of ±0.05 of their respective relative retention times.

8. Detection of Crizotinib Starting Material SM 1a The method for determining whether the impurity content in a liquid is qualified is characterized in that: Separate and identify the crizotinib starting material SM using the method described in any one of claims 5 to 7 1a and its impurities to obtain a chromatogram; based on the obtained chromatogram, the limit method is used to determine whether the impurity content in the test product is qualified.

9. The method according to claim 8, characterized in that If the impurity SM 1a-b 、Impurity SM 1a-c 、Impurity SM 1a-e 、Impurity SM 1a-f 、Impurity SM 1a-g 、Impurity SM 1a-h 、Impurity SM 1a-i 、Impurity SM 1a-j 、Impurity SM 1a-k If the peak area of ​​any one or more impurities in the test sample is greater than the peak area of ​​the corresponding impurities in the reference solution, it indicates that the impurity content is unqualified; on the contrary, if the impurity SM in the test sample is 1a-b 、Impurity SM 1a-c 、Impurity SM 1a-e 、Impurity SM 1a-f 、Impurity SM 1a-g 、Impurity SM 1a-h 、Impurity SM 1a-i 、Impurity SM 1a-j 、Impurity SM 1a-k The peak area of ​​any one or more impurities in the sample is not greater than the peak area of ​​the corresponding impurities in the reference solution, indicating that the impurity content is qualified.

10. The method according to claim 8, characterized in that The solvent for sample preparation was methanol.

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