Separation and determination method of luseovitine intermediate Z2 and its impurities

Abstract

The application belongs to the technical field of pharmaceutical analysis, and particularly relates to a separation and determination method of alosiltib mesylate intermediate Z2 and impurities thereof. 1d The impurities include one or more of trithiocyanic acid, triphenyl phosphine oxide, impurity SM1, impurity SM 2d 2, impurity SM 1a 2, impurity Z1, impurity Z 1b 2, impurity Z 1d 2, impurity B, impurity C, impurity D, impurity F, impurity SM 1b 2 and triphenyl phosphine. The method comprises the following steps: adopting octadecylsilane bonded silica gel as chromatographic column filler, taking phosphate buffer as mobile phase A, taking a mixed solution of methanol and acetonitrile as mobile phase B, performing linear gradient elution, and entering a detector for detection. Finally, whether the content of impurity SM1 is qualified is detected by limit method, and the content of other impurities is calculated by principal component self-control method with a correction factor. 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 technical field of drug analysis, and in particular relates to a method for separating and determining a ruxolitinib mesylate intermediate Z2 and its impurities. Background Art

[0002] Ruxolitinib mesylate is an effective drug for the treatment of myelofibrosis, polycythemia vera, and corticosteroid-refractory acute graft-versus-host disease. It selectively inhibits the activity of JAK1 and JAK2 protein kinases, interfering with the inflammatory response and cell proliferation that depend on JAK-STAT signaling, thereby effectively improving the symptoms of related diseases.

[0003] The ruxolitinib mesylate intermediate Z2 is a D-DMTA (resolving agent) salt of ruxolitinib, and its structural formula is shown in Formula 1.

[0004]

[0005] The study found that the intermediate Z2 of ruxolitinib mesylate may contain the following impurities: trithiocyanate, triphenylphosphine oxide, impurity SM1, impurity SM 1d , impurity SM2, impurity SM 2d , impurity Z1, impurity Z 1a , impurity Z 1b , impurity Z 1d , impurity B, impurity C, impurity D, impurity F, impurity SM 1b 、triphenylphosphine. Among them, impurity SM 1b To ensure the quality of ruxolitinib mesylate API and formulations, quality control of ruxolitinib mesylate intermediate Z2 is necessary. However, there is currently a lack of existing technology for separating and determining the relevant impurities in ruxolitinib mesylate intermediate Z2.

[0006] Some existing technologies report methods for separating and detecting ruxolitinib-related substances, but they are unable to achieve the separation and detection of ruxolitinib mesylate intermediate Z2 and its related impurities. For example, patent CN117517528A discloses a method for detecting related substances in ruxolitinib intermediates using high-performance liquid chromatography (HPLC). The chromatographic conditions include: chromatographic column: octadecylsilane bonded silica gel column; mobile phase: mobile phase A: dihydrogen phosphate solution with a pH of 2.0-4.0; mobile phase B: acetonitrile; gradient elution; and ultraviolet detection at a wavelength of 222 nm. This method can simultaneously detect 4-chloropyrrolopyrimidine, (4-chloro-7H-pyrrolo[2,3-D]pyrimidin-7-yl)methyl pivalate, 1-(1-ethoxyethyl)-4-iodo-1H-pyrazole, 1-(1-ethoxyethyl)-4-pyrazoleboronic acid pinacol ester, [(4-(1-ethoxyethyl)-1H-pyrazol-4-yl)-7H-pyrrolo[2,3-D]pyrimidin-7-yl]methyl pivalate, and triphenylphosphine oxide.

[0007] Therefore, it is necessary to establish a simple, efficient and sensitive method for the separation and determination of related impurities in ruxolitinib mesylate intermediate Z2. Summary of the Invention

[0008] In view of this, one of the objects of the present invention is to provide a method for separating ruxolitinib mesylate intermediate Z2 and its impurities based on high performance liquid chromatography, which can complete the separation of multiple substances in a relatively short time.

[0009] To achieve the above object, the technical solution of the present invention is:

[0010] A method for separating a mesylate ruxolitinib intermediate Z2 and its impurities based on high performance liquid chromatography, wherein the mesylate ruxolitinib intermediate Z2 and the impurities together form a composition, and the impurities include trithiocyanate, triphenylphosphine oxide, impurity SM1, impurity SM2, impurity SM3, impurity SM4, impurity SM5, impurity SM6, impurity SM7, impurity SM8, impurity SM9, impurity SM10, impurity SM11, impurity SM12, impurity SM13, impurity SM14, impurity SM15, impurity SM16, impurity SM17, impurity SM18, impurity SM19, impurity SM20, impurity SM21, impurity SM22, impurity SM23, impurity SM24, impurity SM3 1d , impurity SM2, impurity SM 2d , impurity Z1, impurity Z 1a , impurity Z 1b , impurity Z 1d , impurity B, impurity C, impurity D, impurity F, impurity SM 1b and any one or more of triphenylphosphine; the ruxolitinib mesylate intermediate Z2 is dissolved to generate ruxolitinib and D-DMTA; the structural formula of the above compound is as follows:

[0011]

[0012]

[0013] In the high performance liquid chromatography method, the stationary phase: the chromatographic column uses octadecylsilane bonded silica gel as the filler; the mobile phase: phosphate buffer is used as mobile phase A, and a mixed solution of methanol and acetonitrile is used as mobile phase B, and linear gradient elution is performed.

[0014] After separation and testing, it is used for the next production.

[0015] The above impurities can be arranged and combined in various ways. For example, combination 1: trithiocyanate, triphenylphosphine oxide. Another example is combination 2: triphenylphosphine oxide, impurity SM1, impurity SM 1d For example, combination 3: impurity SM 1d , impurity SM2, impurity SM 2d .

[0016] 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.

[0017] Further, the procedure of the linear gradient elution is as follows:

[0018] Time - minutes Mobile phase A - volume parts Mobile phase B-volume parts 0 70±10 30±10 4±1 70±10 30±10 32±1 20±10 80±10 41±1 20±10 80±10 42±1 70±10 30±10 50±1 70±10 30±10 .

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

[0020] Time - minutes Mobile phase A - volume parts Mobile phase B-volume parts 0 70±2 30±2 4 70±2 30±2 32 20 80 41 20 80 42 70±2 30±2 50 70±2 30±2 .

[0021] For example, at 0 minutes, 4 minutes, 42 minutes and / or 50 minutes, mobile phase A accounts for 68 parts and mobile phase B accounts for 32 parts ( Figure 21 ); 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 3 ); or mobile phase A accounts for 71 parts and mobile phase B accounts for 29 parts; or mobile phase A accounts for 72 parts and mobile phase B accounts for 28 parts ( Figure 20 ).

[0022] Most preferably, the procedure of the linear gradient elution is as follows:

[0023] Time - minutes Mobile phase A - volume parts Mobile phase B-volume parts 0 70 30 4 70 30 32 20 80 41 20 80 42 70 30 50 70 30 .

[0024] Furthermore, the mobile phase A is a potassium dihydrogen phosphate solution with a concentration of 20-50 mmol / L and a pH of 3-5; and in the mobile phase B, the volume ratio of methanol to acetonitrile is 40-60:60-40.

[0025] Preferably, the mobile phase A is a potassium dihydrogen phosphate solution with a concentration of 28-32 mmol / L and a pH of 3.9-4.1; and the volume ratio of methanol to acetonitrile in the mobile phase B is 50:50.

[0026] Most preferably, the mobile phase A is a potassium dihydrogen phosphate solution with a concentration of 30 mmol / L and a pH of 4.0.

[0027] Preferably, the pH regulator of the mobile phase A is phosphoric acid.

[0028] Furthermore, the flow rate is 0.7-1.3 mL / min; the column temperature is 30-40°C; and the injector temperature is 3-10°C.

[0029] As a preferred solution, the flow rate is 0.9-1.1 mL / min; the column temperature is 33-37°C. For example, the flow rate is 0.9 mL / min ( Figure 12 ); or a flow rate of 1.0 mL / min ( Figure 3 ); or a flow rate of 1.1 mL / min ( Figure 13 For example, the column temperature is 33°C ( Figure 14 ); or column temperature is 34℃; or column temperature is 35℃( Figure 3 ); or column temperature is 36℃; or column temperature is 37℃( Figure 15 ).

[0030] Most preferably, the flow rate is 1.0 mL / min and the column temperature is 35°C.

[0031] Preferably, the injector temperature is 5°C.

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

[0033] Preferably, the running time is 50 minutes.

[0034] Preferably, the chromatographic column has a specification of 4.6 mm × 150 mm and a diameter of 3 μm.

[0035] As the most preferred chromatography column, GL Inersil ODS-3, 4.6 mm×150 mm, 3 μm is selected.

[0036] Preferably, the chromatographic conditions further include: adding a mobile phase impurity trapping column, wherein the specification of the impurity trapping column is 4.6 mm×50 mm.

[0037] Most preferably, the impurity trapping column is a Ghost-Sniper Column, 4.6 mm × 50 mm.

[0038] Furthermore, before separation, the test solution was prepared using methanol as a diluent.

[0039] Preferably, the solution to be tested includes a sample solution, a control solution and / or a system suitability solution.

[0040] Preferably, the concentration of the sample solution is 2.0 mg / ml.

[0041] Preferably, the concentration of the control solution is 3 μg / ml.

[0042] As a preferred solution, the sample solution is prepared as follows: take an appropriate amount of the product, accurately weigh it, dissolve it in a diluent, and quantitatively dilute it to make a solution containing about 2 mg per 1 ml, which is used as the sample solution.

[0043] As a preferred solution, the control solution is prepared by accurately measuring an appropriate amount of the sample solution and quantitatively diluting it with a diluent to prepare a solution containing approximately 3 μg per 1 ml, which serves as the control solution.

[0044] As a preferred solution, the system suitability solution is prepared by taking the system suitability reference substance of ruxolitinib mesylate intermediate Z2 (containing ruxolitinib mesylate intermediate Z2, impurity B, impurity C, impurity D, impurity F, impurity Z1, impurity SM 2d An appropriate amount of thiocyanate (Citric Acid, Polythiocyanate) was dissolved in a diluent and diluted to prepare a solution containing about 2 mg per ml, which was used as the system suitability solution.

[0045] A second object of the present invention is to provide a method for identifying ruxolitinib mesylate intermediate Z2 and its impurities, which can complete the identification of multiple substances in a relatively short time.

[0046] To achieve the above object, the technical solution of the present invention is:

[0047] The method for identifying the ruxolitinib mesylate intermediate Z2 and its impurities comprises separating the composition by the aforementioned separation method, and entering a detector for detection to obtain a chromatogram; and determining whether the test sample contains the ruxolitinib mesylate intermediate Z2 and its impurities by comparing the chromatogram characteristics of the test sample with those of the reference sample.

[0048] As a preferred solution, the detector's detection wavelength is 220 ± 10 nm. This ± 10 nm range is based on a comprehensive consideration of multiple factors, including error tolerance, method superiority, and practical application requirements. This range helps ensure the reliability of test results, improves measurement repeatability and flexibility, and meets specific experimental requirements.

[0049] Most preferably, the detection wavelength of the detector is preferably 220 nm.

[0050] 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 in ascending order: impurity C, impurity SM 2d , thiocyanate, D-DMTA, impurity SM 1d , impurity D, impurity F, impurity Z 1b , Ruxolitinib, Impurity Z 1a , impurity B, impurity SM 1b , triphenylphosphine oxide, impurity SM2, impurity SM1, impurity Z1, impurity Z 1d , triphenylphosphine.

[0051] 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 (tR) by the retention time of the reference component (tR0).

[0052] As a preferred solution, the main component of ruxolitinib in the intermediate Z2 of ruxolitinib mesylate is used as the reference peak, and the relative retention time is 0.15, which is determined to be impurity C; the relative retention time is 0.19, which is determined to be impurity SM 2d The relative retention time was 0.20, which was determined to be thiocyanate; the relative retention time was 0.39, which was determined to be D-DMTA; the relative retention time was 0.56, which was determined to be impurity SM 1d The relative retention time is 0.77, which is determined to be impurity D; the relative retention time is 0.89, which is determined to be impurity F; the relative retention time is 0.96, which is determined to be impurity Z 1b The relative retention time was 1.00, which was determined to be ruxolitinib; the relative retention time was 1.13, which was determined to be impurity Z 1a ; The relative retention time is 1.17, which is determined to be impurity B; the relative retention time is 1.21 and / or 1.25, which is determined to be impurity SM 1b The relative retention time was 1.29, which was determined to be triphenylphosphine oxide; the relative retention time was 1.54, which was determined to be impurity SM2; the relative retention time was 1.56, which was determined to be impurity SM1; the relative retention time was 1.76, which was determined to be impurity Z1; the relative retention time was 1.81, which was determined to be impurity Z 1d The relative retention time was 2.24, which was determined to be triphenylphosphine. The relative retention time of each component fluctuated within the range of ±10%.

[0053] 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.

[0054] A third object of the present invention is to provide a method for determining the content of ruxolitinib mesylate intermediate Z2 and its impurities, which can complete the identification and content determination of multiple substances in a relatively short time.

[0055] To achieve the above object, the technical solution of the present invention is:

[0056] The method for determining the content of ruxolitinib mesylate intermediate Z2 and its impurities comprises separating and identifying ruxolitinib mesylate intermediate Z2 and its impurities by the aforementioned identification method to obtain a chromatogram; according to the obtained chromatogram, the limit method is used to detect whether the content of impurity SM1 is qualified, and / or the main component self-control method with a correction factor is used to calculate the content of the thiocyanate, the triphenylphosphine oxide, the impurity SM 1d , the impurity SM2, the impurity SM 2d , the impurity Z1, the impurity Z 1a , the impurity Z 1b , the impurity Z 1d , the content of any one or more impurities among the impurity B, the impurity C, the impurity D, and the impurity F.

[0057] Through the content determination, we can further determine whether the content of ruxolitinib mesylate intermediate Z2 and its impurities are qualified. 1d , impurity SM2, impurity SM 2d , impurity Z1, impurity Z 1a , impurity Z 1b , impurity Z 1d , impurity B, impurity C, impurity D, impurity F, any one or more of the impurity peak area is greater than the peak area of ​​the corresponding impurity in the control solution, indicating that the impurity content is unqualified; on the contrary, if trithiocyanate, triphenylphosphine oxide, impurity SM1, impurity SM2 1d , impurity SM2, impurity SM 2d , impurity Z1, impurity Z 1a , impurity Z 1b , impurity Z 1dIf the peak area of any one or more of impurity A, impurity B, impurity C, impurity D, impurity F in the test solution is not more than the peak area of the corresponding impurity in the control solution, it indicates that the impurity content is qualified.

[0058] The above determination method can be used as a drug quality determination model and further as an indispensable key module in an intelligent production process. The model realizes accurate separation and quantitative analysis of active ingredients, impurities and degradation products in drugs by precisely controlling parameters such as mobile phase composition, flow rate and column temperature, thereby providing a scientific basis for comprehensive evaluation of drug quality. In an intelligent production system, this model is seamlessly integrated, can receive raw data from the production line in real time, automatically perform analysis tasks, and quickly feed back the determination results based on the preset quality standards.

[0059] The beneficial effects of the present application are:

[0060] The present application establishes a high performance liquid chromatography method for separating and determining the intermediate Z2 of lucitanib mesylate and impurities thereof. The method can realize effective separation of multiple impurities in the intermediate Z2 of lucitanib mesylate within 50 minutes, and the impurities include any one or more of trithiocyanic acid, triphenyl phosphine oxide, impurity SM1, impurity SM2, impurity SM3, impurity Z1, impurity Z2, impurity Z3, impurity Z4, impurity B, impurity C, impurity D, impurity F, impurity SM3 and triphenyl phosphine. 1d 2d 1a 1b 1d 1b The method verification experiment results show that the method has strong specificity, good separation degree, high sensitivity, good durability, strong practicability, accurate and reliable detection results, and is of great significance for realizing quality control of the intermediate Z2 of lucitanib mesylate and the finished product of lucitanib mesylate. BRIEF DESCRIPTION OF DRAWINGS

[0061] Figure 1 The chromatogram is for a blank solvent;

[0062] Figure 2 The chromatogram is for a test solution;

[0063] Figure 3 The chromatogram is for a test solution;

[0064] Figure 4 The chromatogram is for a limit of quantification solution 1#;

[0065] Figure 5 The chromatogram is for a limit of quantification solution 2#;

[0066] Figure 6 The chromatogram is for a limit of quantification solution 3#;

[0067] ​​​​​ Figure 7 This is the chromatogram of the detection limit solution 1#;

[0068] Figure 8 This is the chromatogram of the detection limit solution 2#;

[0069] Figure 9 This is the chromatogram of the detection limit solution 3#;

[0070] Figure 10 This is the chromatogram of the detection limit solution 4#;

[0071] Figure 11 Chromatograms of different column batches for the durability experiment;

[0072] Figure 12 This is the chromatogram at a flow rate of 0.9 ml / min in the durability experiment;

[0073] Figure 13 This is the chromatogram at a flow rate of 1.1 ml / min in the durability experiment;

[0074] Figure 14 This is the chromatogram when the column temperature is 33°C in the durability experiment;

[0075] Figure 15 This is the chromatogram when the column temperature is 37°C in the durability experiment;

[0076] Figure 16 This is the chromatogram when the buffer pH is 3.9 in the durability experiment;

[0077] Figure 17 This is the chromatogram when the buffer pH is 4.1 in the durability experiment;

[0078] Figure 18 This is the chromatogram at a salt concentration of 28 mmol / L in the durability experiment;

[0079] Figure 19 This is the chromatogram at a salt concentration of 32 mmol / L in the durability experiment;

[0080] Figure 20 This is the chromatogram of the initial mobile phase ratio A:B = 72:28 in the durability experiment;

[0081] Figure 21 This is the chromatogram of the initial mobile phase ratio A:B = 68:32 in the durability experiment. DETAILED DESCRIPTION

[0082] 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.

[0083] Supplementary tables to the drawings in the specification. Figures 1-21 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 1-21) and data for more detailed information. Conversely, if any information that may cause misunderstanding or ambiguity is found in the process of reviewing Tables 1-21, 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 handwriting in the spectra of this application document overlaps, it 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 spectra have no effect on the scope of protection of the claims and the full disclosure of the technical solutions in the specification.

[0084] Table 1

[0085]

[0086] Table 2

[0087]

[0088] Table 3

[0089]

[0090] Table 4

[0091]

[0092] Table 5

[0093]

[0094] Table 6

[0095]

[0096] Table 7

[0097]

[0098] Table 8

[0099]

[0100] Table 9

[0101]

[0102] Table 10

[0103]

[0104] Table 11

[0105]

[0106] Table 12

[0107]

[0108] Table 13

[0109]

[0110] Table 14

[0111]

[0112] Table 15

[0113]

[0114] Table 16

[0115]

[0116] Table 17

[0117]

[0118] Table 18

[0119]

[0120] Table 19

[0121]

[0122] Table 20

[0123]

[0124] Table 21

[0125]

[0126] 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.

[0127] 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.

[0128] 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).

[0129] the term

[0130] 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.

[0131] 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.

[0132] 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.

[0133] 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.

[0134] 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.

[0135] In the examples of the present invention, information on the intermediate Z2 of ruxolitinib mesylate and its related impurities is shown in Table A.

[0136] Table A. Compound Information

[0137]

[0138]

[0139] In the embodiment of the present invention, the calculation formula of the quantitative limit of impurities is as follows:

[0140] Quantitation limit (expressed as concentration in the sample) % = (quantitation limit concentration ÷ sample concentration) × 100%.

[0141] In the embodiment of the present invention, the impurity detection limit calculation formula is as follows:

[0142] Detection limit (expressed as the concentration in the sample) % = (detection limit concentration ÷ sample concentration) × 100%.

[0143] In the embodiment of the present invention, the main instrument is a high performance liquid chromatograph, and its manufacturer / model is Shimadzu LC-20A (LC3004) and Thermo Fisher Ultimate 3000 (LC3011).

[0144] In the embodiments of the present invention, the reagent information is shown in Table B.

[0145] Table B. Reagent Information

[0146]

[0147]

[0148] Example 1. Separation and determination method of luspaterin intermediate Z2 in methanesulfonic acid and its impurities

[0149] (1) Preparation of the test solution

[0150] Diluent: methanol.

[0151] Test solution (2.0 mg / ml): An appropriate amount of the product (luspaterin intermediate Z2 in methanesulfonic acid) was accurately weighed, dissolved and quantitatively diluted with the diluent to prepare a solution containing about 2 mg per 1 ml.

[0152] Control solution (3 μg / ml): An appropriate amount of the test solution was accurately measured and quantitatively diluted with the diluent to prepare a solution containing about 3 μg per 1 ml.

[0153] System suitability solution: An appropriate amount of luspaterin intermediate Z2 system suitability control (containing luspaterin intermediate Z2, impurity B, impurity C, impurity D, impurity F, impurity Z1, impurity SM 2d , trithiocyanic acid) was dissolved and diluted with the diluent to prepare a solution containing about 2 mg per 1 ml.

[0154] (2) Chromatographic conditions

[0155] Octadecylsilane-bonded silica gel as the filler (GL Inersil ODS-3, 4.6 mm x 150 mm, 3 μm or a chromatographic column with equivalent performance); 0.03 mol / L potassium dihydrogen phosphate solution (pH value adjusted to 4.0 with phosphoric acid) as mobile phase A, methanol-acetonitrile (50:50) as mobile phase B, linear gradient elution according to Table C, with a mobile phase impurity trapping column (Chromrex Ghost-Sniper Column, 4.6 mm x 50 mm or an equivalent trapping column); detection wavelength was 220 nm; flow rate was 1.0 ml per minute; column temperature was 35°C; injector temperature was 5°C; injection volume was 10 μl.

[0156] Table C. Gradient elution program table

[0157] Time - minutes Mobile phase A - volume parts Mobile phase B-volume parts 0 70 30 4 70 30 32 20 80 41 20 80 42 70 30 50 70 30

[0158] (3) Determination

[0159] The system suitability solution, test solution and control solution were accurately measured and injected into the liquid chromatograph, and the chromatogram was recorded. Except that impurity SM1 was determined by the limit method, the remaining impurities were calculated for the content of each impurity by the self-control method with a correction factor.

[0160] System suitability requirements: In the system suitability solution chromatogram, according to impurity C, impurity SM 2d , thiocyanate, D-DMTA, impurity D, impurity F, ruxolitinib, impurity B and impurity Z1 appear in sequence, and the separation between each impurity peak should meet the requirements.

[0161] Limit: If there are impurity peaks in the chromatogram of the test solution, except for the blank solvent peak and D-DMTA peak, the impurity B calculated by the corrected peak area (multiplied by the correction factor 0.43) shall not be greater than 2 times (0.3%) the main peak area of ​​the control solution, and the impurities C, D, F, SM and SM 2d The peak areas of impurities Z1, thiocyanate, and thiocyanate calculated after correction (multiplied by correction factors of 0.29, 0.44, 0.52, 0.48, 0.63, and 2.0, respectively) shall not be greater than the main peak area of ​​the control solution (0.15%). The peak areas of other individual impurities shall not be greater than the main peak area of ​​the control solution (0.15%). The sum of the peak areas of all impurities calculated after correction shall not be greater than 6.66 times the main peak area of ​​the control solution (1.0%). Any peak in the chromatogram of the test solution that is less than 0.33 times the main peak area of ​​the control solution can be ignored (0.05%).

[0162] Triphenylphosphine oxide, impurity SM 1d , impurity SM2, impurity Z 1a , impurity Z 1b , impurity Z 1d For research purposes only, the limits, calculated based on the corrected peak area (multiplied by the correction factors 1.3, 0.44, 0.49, 1.2, 0.33, and 0.31, respectively), must not be greater than 0.66 times (0.10%) the main peak area of ​​the control solution.

[0163] Impurity SM1 has poor stability and needs to be prepared fresh before use. Therefore, the limit method is used for research. In the chromatogram of the test solution, the peak area of ​​impurity SM1 shall not be greater than the peak area of ​​impurity SM1 in the reference solution (0.10%).

[0164] Example 2. Specificity

[0165] (1) Prepare the test solution

[0166] Diluent (blank solvent): methanol.

[0167] D-DMTA stock solution: Accurately weigh 20.47 mg of D-DMTA, place it in a 50 ml volumetric flask, add methanol to dissolve and dilute to the scale, shake well, and obtain.

[0168] Triphenylphosphine stock solution: Accurately weigh 50.78 mg of triphenylphosphine reference substance, place it in a 50 ml volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well, and obtain.

[0169] Triphenylphosphine oxide stock solution: Accurately weigh 20.27 mg of triphenylphosphine oxide reference substance, place it in a 50 ml volumetric flask, add methanol to dissolve and dilute to the scale, shake well, and obtain.

[0170] Thiocyanate stock solution: Accurately weigh 20.50 mg of thiocyanate reference substance into a 50 ml volumetric flask, add methanol to dissolve and dilute to the scale, shake well, and obtain.

[0171] Impurity SM1 stock solution: Accurately weigh 49.96 mg of impurity SM1 reference substance, place it in a 50 ml volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well, and obtain.

[0172] Impurity SM 1b Stock solution: Accurately weigh impurity SM 1b Place 13.50 mg of the reference substance in a 25 ml volumetric flask, add methanol to dissolve and dilute to the scale, shake well, and obtain the product.

[0173] Impurity SM 1d Stock solution: Accurately weigh impurity SM 1d Place 20.11 mg of the reference substance in a 50 ml volumetric flask, add methanol to dissolve and dilute to the scale, shake well, and obtain the product.

[0174] Impurity SM2 stock solution: Accurately weigh 20.18 mg of impurity SM2 reference substance, place it in a 50 ml volumetric flask, add methanol to dissolve and dilute to the scale, shake well, and obtain.

[0175] Impurity SM 2d Stock solution: Accurately weigh impurity SM 2d Place 21.00 mg of the reference substance in a 50 ml volumetric flask, add methanol to dissolve and dilute to the scale, shake well, and obtain the product.

[0176] Impurity Z1 stock solution: Accurately weigh 20.13 mg of impurity Z1 reference substance, place it in a 50 ml volumetric flask, add methanol to dissolve and dilute to the scale, shake well, and obtain.

[0177] Impurity Z 1a Stock solution: Accurately weigh impurity Z 1a Place 20.04 mg of the reference substance in a 100 ml volumetric flask, add diluent [methanol-acetonitrile (1:2)] to dissolve and dilute to the scale, shake well, and obtain the product.

[0178] Impurity Z 1b Stock solution: Accurately weigh impurity Z 1b Place 21.12 mg of the reference substance in a 50 ml volumetric flask, add methanol to dissolve and dilute to the scale, shake well, and obtain the product.

[0179] Impurity Z 1d Stock solution: Accurately weigh impurity Z 1dPlace 20.36 mg of the reference substance in a 50 ml volumetric flask, add methanol to dissolve and dilute to the scale, shake well, and obtain the product.

[0180] Impurity B stock solution: Accurately weigh 20.11 mg of impurity B reference substance, place it in a 50 ml volumetric flask, add methanol to dissolve and dilute to the scale, shake well, and obtain.

[0181] Impurity C stock solution: Accurately weigh 20.14 mg of impurity C reference substance, place it in a 50 ml volumetric flask, add methanol to dissolve and dilute to the scale, shake well, and obtain.

[0182] Impurity D stock solution: Accurately weigh 20.06 mg of impurity D reference substance, place it in a 50 ml volumetric flask, add methanol to dissolve and dilute to the scale, shake well, and obtain.

[0183] Impurity F stock solution: Accurately weigh 20.63 mg of impurity F reference substance, place it in a 50 ml volumetric flask, add methanol to dissolve and dilute to the scale, shake well, and obtain.

[0184] Z2 stock solution: Accurately weigh 40.55 mg of impurity Z2 reference substance, place it in a 100 ml volumetric flask, add methanol to dissolve and dilute to the scale, shake well, and obtain.

[0185] Triphenylphosphine positioning solution: Accurately pipette 2 ml of triphenylphosphine stock solution into a 100 ml volumetric flask, dilute to the mark with acetonitrile, and shake well.

[0186] Impurity SM1 positioning solution: Accurately pipette 2 ml of impurity SM1 stock solution into a 100 ml volumetric flask, dilute to the scale with acetonitrile, and shake well to obtain.

[0187] Impurity Z 1a Positioning solution: Precise removal of impurities Z 1a Place 5 ml of the stock solution in a 50 ml volumetric flask, dilute to the mark with methanol, and shake well.

[0188] The remaining impurity location solutions: accurately pipette D-DMTA, triphenylphosphine oxide, trithiocyanate, impurity SM 1b 、Impurity SM 1d , impurity SM2, impurity SM 2d , impurity Z1, impurity Z 1b , impurity Z 1d Place 5 ml each of the stock solutions of impurity B, impurity C, impurity D, impurity F, and Z2 in different 100 ml volumetric flasks, dilute to the scale with methanol, and shake well to obtain.

[0189] Test solution: Accurately weigh 0.17 mg of ruxolitinib mesylate intermediate Z25, place it in a 25 ml volumetric flask, add methanol to dissolve and dilute to the scale, shake well, and obtain the solution.

[0190] Tested product mixed solution: 0.19 g of luspaterin intermediate Z2 methanesulfonic acid was accurately weighed into a 10 ml volumetric flask, and impurity Z was added 1a Stock solution 0.5 ml, add triphenylphosphine, impurity SM1 stock solution each 0.2 ml, add D-DMTA, triphenylphosphine, trithiocyanic acid, impurity SM 1b , impurity SM 1d , impurity SM2, impurity SM 2d , impurity Z1, impurity Z 1b , impurity Z 1d , impurity B, impurity C, impurity D, impurity F stock solution each 0.25 ml, add methanol to dissolve and dilute to the mark, shake well, and it is obtained.

[0191] (2) Detection

[0192] Take 10 μl of blank solvent, positioning solution, test sample solution and test sample mixed solution respectively, and detect and record the chromatogram, and investigate the separation between peaks.

[0193] The test results are shown in Table D and Figure 1-Figure 3 , Table 1 to Table 3. The blank solvent does not interfere with the detection; the separation degree between each component peak meets the requirements. The method has good specificity.

[0194] Table D. Specificity test results

[0195]

[0196]

[0197] Note: Luspaterin intermediate Z2 methanesulfonic acid is the D-DMTA (resolving agent) salt of luspaterin (main component), which will produce main peak luspaterin and D-DMTA peak after dissolution; impurity SM 1b (impurity SM 1b with cis-trans isomers at double bond) and triphenylphosphine are impurities in luspaterin intermediate Z2 methanesulfonic acid impurity spectrum, which are only used for positioning and looking at whether they interfere with the detection of other impurities.

[0198] Example 3. Quantitative limit

[0199] (1) Luspaterin intermediate Z2 methanesulfonic acid

[0200] Quantitative limit solution 1#: 3.75 ml of luspaterin intermediate Z2 methanesulfonic acid stock solution under "specificity" was accurately transferred into a 50 ml volumetric flask, diluted to the mark with methanol, shaken well, 5 ml was accurately transferred into a 50 ml volumetric flask, diluted to the mark with methanol, shaken well, 3.5 ml was accurately transferred into a 50 ml volumetric flask, diluted to the mark with methanol, shaken well, and it was obtained.

[0201] Take the above-mentioned quantitative limit solution 1# and measure it continuously for 6 times, record the chromatogram, and calculate the ratio of each peak to noise (signal-to-noise ratio) and peak area RSD.

[0202] The test results are shown in Table E. Figure 4 and Table 4. The limit of quantification concentration of intermediate Z2 was 0.2103 μg / ml, which was expressed as 0.011% of the concentration in the test sample. The RSD of the peak area of ​​ruxolitinib was 1.7%, the average signal-to-noise ratio was 25.4, the average signal-to-noise ratio was greater than 10, and the peak area RSD was less than 10.0%, which met the requirements.

[0203] Table E. Quantitation limit test results

[0204]

[0205] (2) Thiocyanic acid, impurity SM 2d , impurity Z1, impurity B, impurity C, impurity D and impurity F

[0206] Quantitative limit solution 2#: Accurately pipette 1.6ml of thiocyanate fixation solution under the "Specificity" item and impurity SM 2d Place 0.6 ml of the localizing solution, 1.4 ml of the impurity Z1 localizing solution, 0.6 ml of the impurity B localizing solution, 0.2 ml of the impurity C localizing solution, 0.6 ml of the impurity D localizing solution and 0.6 ml of the impurity F localizing solution in a 100 ml volumetric flask, dilute to the mark with methanol, and shake well to obtain the product.

[0207] Take the above-mentioned quantitative limit solution 2# and measure it continuously for 6 times, record the chromatogram, and calculate the ratio of each peak to noise (signal-to-noise ratio) and peak area RSD.

[0208] The test results are shown in Table F. Figure 5 and Table 5. The limit of quantification of thiocyanate was 0.3183 μg / ml, which was expressed as 0.016% of the concentration in the test sample, with an RSD of 2.2% for the peak area and an average signal-to-noise ratio of 28.7; the impurity SM 2dThe quantitative limit concentration was 0.1129 μg / ml, which was expressed as 0.006% of the concentration in the test sample, with a peak area RSD of 1.8% and an average signal-to-noise ratio of 43.3; the quantitative limit concentration of impurity Z1 was 0.2739 μg / ml, which was expressed as 0.014% of the concentration in the test sample, with a peak area RSD of 1.6% and an average signal-to-noise ratio of 63.1; the quantitative limit concentration of impurity B was 0.1180 μg / ml, which was expressed as 0.006% of the concentration in the test sample, with a peak area RSD of 1.3% and an average signal-to-noise ratio of 39.6; The limit of quantification for impurity C was 0.0390 μg / ml, expressed as 0.002% of the test sample, with a peak area RSD of 2.5% and an average signal-to-noise ratio of 26.9. The limit of quantification for impurity D was 0.1196 μg / ml, expressed as 0.006% of the test sample, with a peak area RSD of 1.1% and an average signal-to-noise ratio of 40.7. The limit of quantification for impurity F was 0.1176 μg / ml, expressed as 0.006% of the test sample, with a peak area RSD of 2.1% and an average signal-to-noise ratio of 32.7. The average signal-to-noise ratios of the peak quantification limits for each of the above impurities were all greater than 10, and the peak area RSDs were all less than 10.0%, meeting the requirements.

[0209] Table F. Limit of Quantitation Test Results

[0210]

[0211]

[0212] (3) Triphenylphosphine oxide, impurity SM 1d , impurity SM2, impurity Z 1a , impurity Z 1b and impurity Z 1d

[0213] Quantitative limit solution 3#: Accurately pipette 0.4ml of triphenylphosphine oxide positioning solution under the "Specificity" item and impurity SM 1d Positioning solution 2ml, impurity SM2 positioning solution 0.4ml, impurity Z 1a Positioning solution 0.6ml, impurity Z 1b 1.6 ml of positioning solution and impurity Z 1d Place 0.3 ml of the positioning solution in a 100 ml volumetric flask, dilute to the scale with methanol, and shake well.

[0214] Take the above-mentioned quantitative limit solution 3# and measure it continuously for 6 times, record the chromatogram, and calculate the ratio of each peak to noise (signal-to-noise ratio) and peak area RSD.

[0215] The test results are shown in Table G. Figure 6and Table 6. The limit of quantification of triphenylphosphine oxide was 0.0796 μg / ml, which was expressed as 0.004% of the concentration in the test sample, with an RSD of 2.4% for the peak area and an average signal-to-noise ratio of 26.8; the impurity SM 1d The quantitative limit concentration was 0.3877 μg / ml, which was expressed as 0.019% of the concentration in the test sample, with a peak area RSD of 2.8% and an average signal-to-noise ratio of 25.6. The quantitative limit concentration of impurity SM2 was 0.0803 μg / ml, which was expressed as 0.004% of the concentration in the test sample, with a peak area RSD of 3.1% and an average signal-to-noise ratio of 19.2. The quantitative limit concentration of impurity Z 1a The limit of quantification was 0.1177 μg / ml, which was expressed as 0.006% in the sample, with an RSD of 1.2% for the peak area and an average signal-to-noise ratio of 25.2. 1b The limit of quantification concentration was 0.3362 μg / ml, which was expressed as 0.017% of the concentration in the test sample, with a peak area RSD of 2.1% and an average signal-to-noise ratio of 29.2. 1d The limit of quantification (LOQ) was 0.0609 μg / mL, representing a concentration of 0.003% in the test sample. The RSD for the peak area was 3.5%, and the average signal-to-noise ratio was 20.2. The average signal-to-noise ratio for the peak quantification limits of each impurity was greater than 10, and the RSD for the peak area was less than 10.0%, meeting the requirements.

[0216] Table G. Limit of Quantitation Test Results

[0217]

[0218]

[0219] Example 4. Detection limit

[0220] (1) Ruxolitinib mesylate intermediate Z2

[0221] Detection limit solution 1#: Accurately pipette 8 ml of the quantification limit solution 1# under the "quantification limit" item, place it into a 25 ml volumetric flask, dilute to the scale with methanol, and shake well.

[0222] Take the above detection limit solution 1# and measure it continuously three times, record the chromatogram, and calculate the ratio of each peak to noise (signal-to-noise ratio).

[0223] The test results are shown in Table H. Figure 7 The detection limit of ruxolitinib mesylate intermediate Z2 was 0.0673 μg / mL, expressed as 0.003% of the test sample concentration, and the average signal-to-noise ratio was 8.6. The average signal-to-noise ratio of the detection limit of ruxolitinib mesylate intermediate Z2 was greater than 3, meeting the requirements.

[0224] Table H. Detection limit test results

[0225]

[0226]

[0227] (2) Thiocyanic acid, impurity SM 2d , impurity Z1, impurity B, impurity C, impurity D and impurity F

[0228] Detection limit solution 2#: Accurately pipette 8 ml of the quantification limit solution 2# under the "quantification limit" item, place it into a 25 ml volumetric flask, dilute to the scale with methanol, and shake well.

[0229] Take the above detection limit solution 2# and measure it continuously for 3 times, record the chromatogram, and calculate the ratio of each peak to noise (signal-to-noise ratio).

[0230] The test results are shown in Table 1. Figure 8 and Table 8. The detection limit of thiocyanate was 0.1019 μg / ml, which was expressed as 0.005% of the concentration in the test sample, and the average signal-to-noise ratio was 8.6; the impurity SM 2d The detection limit concentration was 0.0361 μg / ml, which was expressed as 0.002% of the concentration in the test sample, and the average signal-to-noise ratio was 13.0; the detection limit concentration of impurity Z1 was 0.0877 μg / ml, which was expressed as 0.004% of the concentration in the test sample, and the average signal-to-noise ratio was 19.6; the detection limit concentration of impurity B was 0.0378 μg / ml, which was expressed as 0.002% of the concentration in the test sample, and the average signal-to-noise ratio was 12.0; The detection limit of impurity C was 0.0125 μg / ml, expressed as 0.001% of the concentration in the test sample, with an average signal-to-noise ratio of 8.0. The detection limit of impurity D was 0.0383 μg / ml, expressed as 0.002% of the concentration in the test sample, with an average signal-to-noise ratio of 12.6. The detection limit of impurity F was 0.0376 μg / ml, expressed as 0.002% of the concentration in the test sample, with an average signal-to-noise ratio of 9.7. The average signal-to-noise ratios of the detection limits of the above impurities were all greater than 3, meeting the requirements.

[0231] Table I. Detection limit test results

[0232]

[0233] (3) Triphenylphosphine oxide, impurity SM 1d , impurity SM2, impurity Z 1a , impurity Z 1b and impurity Z 1d

[0234] Detection limit solution 3#: Accurately pipette 8 ml of the quantification limit solution 3# under the "quantification limit" item, place it into a 25 ml volumetric flask, dilute to the scale with methanol, shake well, and obtain.

[0235] Take the above detection limit solution 3# and measure it continuously three times, record the chromatogram, and calculate the ratio of each peak to noise (signal-to-noise ratio).

[0236] The test results are shown in Table J. Figure 9 and Table 9. The detection limit of triphenylphosphine oxide was 0.0255 μg / ml, which was expressed as 0.001% of the concentration in the test sample, and the average signal-to-noise ratio was 10.8; the impurity SM 1d The detection limit concentration was 0.1241 μg / ml, which was expressed as 0.006% of the concentration in the test sample, and the average signal-to-noise ratio was 10.4; the detection limit concentration of impurity SM2 was 0.0257 μg / ml, which was expressed as 0.001% of the concentration in the test sample, and the average signal-to-noise ratio was 8.1; the detection limit concentration of impurity Z 1a The detection limit concentration is 0.0377 μg / ml, which is expressed as 0.002% of the concentration in the test sample, and the average signal-to-noise ratio is 10.2; impurity Z 1b The detection limit concentration is 0.1076μg / ml, which is expressed as 0.005% of the concentration in the test sample, and the average signal-to-noise ratio is 11.6; impurity Z 1d The detection limit concentration is 0.0195 μg / ml, expressed as 0.001% of the concentration in the test sample, and the average signal-to-noise ratio is 6.0. The average signal-to-noise ratio of the detection limit of each impurity is greater than 3, which meets the requirements.

[0237] Table J. Detection limit test results

[0238]

[0239] (4) Impurity SM1 (limit method)

[0240] Detection limit solution 4#: Accurately weigh 49.96 mg of SM1 reference substance, place in a 50 ml volumetric flask, dissolve in acetonitrile and dilute to the mark, shake well, accurately pipette 2 ml, place in a 100 ml volumetric flask, dilute to the mark with acetonitrile, shake well, accurately pipette 1 ml, place in a 100 ml volumetric flask, dilute to the mark with methanol, shake well, and the solution is ready. (Prepare fresh for use)

[0241] Take the above detection limit solution 4# and measure it continuously for 3 times, record the chromatogram, and calculate the ratio of each peak to noise (signal-to-noise ratio).

[0242] The test results are shown in Table K. Figure 10and Table 10. The detection limit concentration of impurity SM1 is 0.1982 μg / ml, which is expressed as 0.010% of the concentration in the test sample, and the average signal-to-noise ratio is 8.9. The detection limit signal-to-noise ratio is greater than 3, which meets the requirements.

[0243] Table K. Detection limit test results

[0244]

[0245] Example 5. Durability

[0246] Use the test sample mixed solution under the "Specificity" item, use normal chromatographic conditions and change the chromatographic conditions as follows, test them separately after the instrument system is stable, and record the relative retention time and separation between each peak.

[0247] Normal conditions: flow rate, 1.0 ml / min; column temperature, 35°C; mobile phase A: 0.03 mol / L potassium dihydrogen phosphate buffer (pH adjusted to 4.0 with phosphoric acid), mobile phase B: methanol-acetonitrile (50:50), gradient elution; chromatographic column (GLInersil ODS-3, 4.6 mm × 150 mm, 3 μm; S / N: 22H0349434).

[0248] The chromatographic conditions were changed as follows: (1) Chromatographic column: GL Inersil ODS-3, 4.6 mm × 150 mm; S / N: 0HI82436; (2) Flow rate: 0.9 ml / min, 1.1 ml / min; (3) Column temperature: 33 °C, 37 °C; (4) Buffer pH: 3.9, 4.1; (5) Buffer concentration: 28 mmol / L, 32 mmol / L; (6) Initial ratio of mobile phase: 72:28, 68:32.

[0249] The results are shown in Table L to Table O. Figure 3 、 Figures 11 to 21 , Table 3, and Tables 11–21. Peak resolution was consistently greater than 1.5 when subject to minor fluctuations in flow rate, column temperature, buffer pH, buffer concentration, and initial mobile phase ratio, as well as when using columns from different batches from the same manufacturer. The relative retention time of each known impurity varied within ±0.12. Changes in chromatographic conditions had no significant impact on detection, demonstrating the robustness of the method.

[0250] Table L. Chromatographic Conditions Durability Test Results (Relative Retention Time)

[0251]

[0252] Table M. Chromatographic Condition Durability Test Results (Relative Retention Time)

[0253]

[0254]

[0255] Table N. Chromatographic Conditions Durability Test Results (Separation)

[0256]

[0257] Table O. Chromatographic Conditions Durability Test Results (Separation)

[0258]

[0259]

Claims

1. A method for separating ruxolitinib mesylate intermediate Z2 and its impurities based on high performance liquid chromatography, characterized in that: The ruxolitinib mesylate intermediate Z2 and the impurities together form a composition, wherein the impurities include impurity B; the ruxolitinib mesylate intermediate Z2 is dissolved to generate ruxolitinib and D-DMTA; The structural formula of the above compound is as follows: ； In the high-performance liquid chromatography method, the chromatographic column uses octadecylsilane bonded silica gel as a filler, specifically GLInersil ODS-3, 4.6 mm × 150 mm, 3 μm; a potassium dihydrogen phosphate solution with a concentration of 28-32 mmol / L and a pH of 3.9-4.1 is used as mobile phase A, and a mixed solution of methanol and acetonitrile is used as mobile phase B, and linear gradient elution is performed; in the mobile phase B, the volume ratio of methanol to acetonitrile is 50:50; the flow rate is 0.9-1.1 mL / min, the column temperature is 33-37°C, and the injector temperature is 3-10°C; The procedure of the linear gradient elution is as follows: 。 2. A method for identifying ruxolitinib mesylate intermediate Z2 and its impurities, characterized in that: The composition is separated by the method described in claim 1, and enters a detector for detection to obtain a chromatogram; by comparing the chromatogram characteristics of the test sample and the reference sample, it is determined whether the test sample contains the ruxolitinib mesylate intermediate Z2 and its impurities.

3. The method according to claim 2, characterized in that The detection wavelength of the detector is 220±10 nm.

4. The method according to claim 2, characterized in that The components in the composition can be identified according to the order of relative retention time. The components of the composition are, in ascending order, D-DMTA, ruxolitinib, and impurity B.

5. The method according to claim 2, characterized in that The main component of ruxolitinib mesylate intermediate Z2 was used as the reference peak, and the relative retention time was 0.39, which was determined to be D-DMTA; The relative retention time was 1.00, which was determined to be ruxolitinib; the relative retention time was 1.17, which was determined to be impurity B; the relative retention time of each component fluctuated within the range of ±10%.

6. A method for determining the content of ruxolitinib mesylate intermediate Z2 and its impurity content, characterized in that: The method according to any one of claims 2 to 5 is used to separate and identify the ruxolitinib mesylate intermediate Z2 and its impurities to obtain a chromatogram; based on the obtained chromatogram, the content of the impurity B is calculated by the principal component self-reference method with a correction factor.

Images