Separation and determination method of rukotinib mesylate intermediate Z2 and impurities thereof

Abstract

The invention belongs to the technical field of pharmaceutical analysis, and particularly relates to a method for separating and determining a rukotinib mesylate intermediate Z2 and impurities thereof. The impurities comprise one or more of trithiocyanuric acid, triphenylphosphine oxide, an impurity SM1, an impurity SM1d, an impurity SM2, an impurity SM2d, an impurity Z1, an impurity Z1a, an impurity Z1b, an impurity Z1d, an impurity B, an impurity C, an impurity D, an impurity F, an impurity SM1b and triphenylphosphine. The method comprises the following steps: carrying out linear gradient elution by using octadecylsilane chemically bonded silica as a chromatographic column filler, a phosphate buffer as a mobile phase A and a mixed solution of methanol and acetonitrile as a mobile phase B, and detecting in a detector. And finally, detecting whether the content of the impurity SM1 is qualified by adopting a limit method, and calculating the content of other impurities by adopting a main component self-contrast method with correction factors. The method has the characteristics of good separation degree, good durability, high sensitivity and good reproducibility.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and specifically 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 the 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 preparation products, it is necessary to control the quality of ruxolitinib mesylate intermediate Z2. However, there is currently a lack of existing technology that can separate and determine the relevant impurities in ruxolitinib mesylate intermediate Z2.

[0006] Some prior art reports on separation and determination methods of ruxolitinib related substances, but cannot achieve 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, which uses high performance liquid chromatography for detection; chromatographic conditions include: chromatographic column: octadecylsilane bonded silica gel column; mobile phase: mobile phase A is a dihydrogen phosphate solution with a pH of 2.0-4.0, and mobile phase B is acetonitrile; gradient elution is used; ultraviolet detection, and the detection wavelength is 222nm. 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 method can complete the separation of multiple substances in a shorter time.

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

[0010] A method for separating a ruxolitinib mesylate intermediate Z2 and its impurities based on high performance liquid chromatography, wherein the ruxolitinib mesylate intermediate Z2 and the impurities together form a composition, wherein the impurities include 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 and any one or more of triphenylphosphine; the mesylate ruxolitinib 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 a 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 step.

[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 to be separated, identified and / or detected by the method is n (number), 1-n substances can be detected naturally.

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

[0018] Time - Minutes Mobile phase A - volume Mobile phase B - volume 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 Mobile phase B - volume 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 ( Fig.21 ); or mobile phase A accounts for 69 parts and mobile phase B accounts for 31 parts; or mobile phase A accounts for 70 parts and 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 ( Fig. 20 ).

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

[0023] Time - Minutes Mobile phase A - volume Mobile phase B - volume 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; 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; in the mobile phase B, the volume ratio of methanol to acetonitrile 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 adjuster 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 ( Fig.12 ); or flow rate of 1.0 mL / min ( Figure 3 ); or flow rate of 1.1 mL / min ( Fig.13 ). For example, the column temperature is 33°C ( Fig.14 ); or column temperature is 34°C; or column temperature is 35°C ( Figure 3 ); or column temperature is 36°C; or column temperature is 37°C ( Fig.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 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, methanol was used as a diluent to prepare the test solution.

[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 by taking an appropriate amount of the product, weighing it accurately, adding a diluent to dissolve it and quantitatively diluting it to make a solution containing about 2 mg per 1 ml 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 about 3 μg per 1 ml 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 , trithiocyanate) in appropriate amount, dissolve in diluent and dilute to make a solution containing about 2 mg per 1 ml as the system suitability solution.

[0045] The 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 intermediate Z2 of ruxolitinib mesylate 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 intermediate Z2 of ruxolitinib mesylate and its impurities by comparing the chromatogram characteristics of the test sample with those of the reference sample.

[0048] As a preferred solution, the detection wavelength of the detector is 220±10nm. The setting range of ±10nm is a comprehensive consideration based on multiple factors such as error considerations, method superiority, and practical application requirements. This setting range helps to ensure the reliability of the detection results, improve the repeatability and flexibility of the measurement, and meet the requirements of specific experiments.

[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 according to 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] Generally, relative retention time is used to describe the relative retention of different components in a mixture on a chromatographic column. It refers to 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 a chromatographic column and is an important parameter for positioning, qualitative and quantitative analysis in chromatographic analysis. The relative retention time is calculated by dividing the retention time of the target component (tR) by the retention time of the reference component (tR0).

[0052] As a preferred solution, the main component ruxolitinib in the mesylate ruxolitinib intermediate Z2 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 is 0.20, which is determined to be thiocyanate; the relative retention time is 0.39, which is determined to be D-DMTA; the relative retention time is 0.56, which is 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 is 1.00, which is determined to be ruxolitinib; the relative retention time is 1.13, which is 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 is 1.29, which is determined to be triphenylphosphine oxide; the relative retention time is 1.54, which is determined to be impurity SM2; the relative retention time is 1.56, which is determined to be impurity SM1; the relative retention time is 1.76, which is determined to be impurity Z1; the relative retention time is 1.81, which is 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 determine components. Retention time refers to the time required for a sample to enter the chromatographic column and be detected by the detector. This time is calculated based on the movement speed of the component on the chromatographic column, that is, the time interval from the start of injection to the chromatographic apex (maximum concentration) of a certain component. It is mainly used to determine the peak order and position of each component in the sample and is one of the basic data in chromatographic analysis. In quality control, changes in retention time can reflect factors such as the state of the chromatographic column, the stability of the mobile phase, and the performance of the instrument.

[0054] The 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 the ruxolitinib mesylate intermediate Z2 and its impurities by the aforementioned identification method to obtain a chromatogram; according to the obtained chromatogram, using the limit method to detect whether the content of impurity SM1 is qualified, and / or using the main component self-control method with a correction factor to calculate the content of thiocyanate, triphenylphosphine oxide, and 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 impurity content is 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 areas are greater than the peak areas of the corresponding impurities in the control solution, indicating that the impurity content is unqualified; on the contrary, if trithiocyanate, triphenylphosphine oxide, impurity SM1, impurity SM 1d , impurity SM2, impurity SM 2d , impurity Z1, impurity Z 1a 、Impurity Z 1b 、Impurity Z 1dThe peak area of ​​any one or more impurities among impurity A, impurity B, impurity C, impurity D, and impurity F is not greater than the peak area of ​​the corresponding impurity in the control solution, indicating 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 the intelligent production process. This model can achieve accurate separation and quantitative analysis of active ingredients, impurities and degradation products in drugs by accurately controlling parameters such as mobile phase composition, flow rate, column temperature, etc., providing a scientific basis for the comprehensive evaluation of drug quality. In the intelligent production system, this model is seamlessly integrated, able to receive raw data from the production line in real time, automatically perform analysis tasks, and quickly feedback the determination results based on preset quality standards.

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

[0060] The present invention establishes a high performance liquid chromatography method for separating and determining the ruxolitinib mesylate intermediate Z2 and its impurities. The method can effectively separate multiple impurities in the ruxolitinib mesylate intermediate Z2 within 50 minutes. The impurities include 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 and triphenylphosphine. The method validation experimental results show that the method has strong specificity, good separation, high sensitivity, good durability, strong practicality, accurate and reliable detection results, and is of great significance for achieving quality control of ruxolitinib mesylate intermediate Z2 and ruxolitinib mesylate finished product. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 is the chromatogram of blank solvent;

[0062] Figure 2 is the chromatogram of the test solution;

[0063] Figure 3 is the chromatogram of the test sample mixed solution;

[0064] Figure 4 This is the chromatogram of the quantitative limit solution 1#;

[0065] Figure 5 This is the chromatogram of the quantitative limit solution 2#;

[0066] Figure 6 This is the chromatogram of the quantitative limit solution 3#;

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

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

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

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

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

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

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

[0074] Fig.14 This is the chromatogram with a column temperature of 33°C in the durability experiment;

[0075] Fig.15 This is the chromatogram with a column temperature of 37°C in the durability experiment;

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

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

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

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

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

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

[0082] The technical solution of the present invention will be further described clearly and completely in conjunction with specific embodiments below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work belong to the protection scope of the present invention.

[0083] Supplementary table to the drawings in the specification. Figure 1-Figure 21 Visual aids are provided for the purpose of understanding and interpretation. In the event of 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 during the review of 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 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 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 the understanding of the present invention, some key technologies and scientific terms are clearly defined below. Unless 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 substances described, but reasonable flexibility and adjustment in these aspects are allowed. At the same time, please understand that the terms used in this article are intended to illustrate specific embodiments rather than 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 as a whole to the extent not directly cited. If there is any inconsistency or conflict between the contents of these cited documents or similar materials and this application, especially in terms of term definitions, term usage, technical descriptions, etc., the contents of this application shall prevail.

[0128] If there are any unmentioned conditions in the chromatographic conditions, they can be determined by referring to the high performance liquid chromatography method (Chinese Pharmacopoeia 2020 Edition Part 4 0512).

[0129] the term

[0130] The limit of quantitation refers to the lowest amount of the substance to be measured in the sample, and the measurement result should have a certain degree of accuracy and precision. In other words, the limit of quantitation is the lowest level at which the analytical method can accurately and reliably measure the concentration of the substance to be measured in the sample. In HPLC, the determination of the limit of quantitation usually relies on the signal-to-noise ratio (S / N) method, that is, the concentration of the substance to be measured corresponding to a certain level of signal-to-noise ratio is used as the limit of quantitation. The determination of the limit of quantitation is crucial to ensure the accuracy and reliability of the analysis results.

[0131] The durability of chromatographic conditions refers to the ability of the chromatographic analysis system to maintain stable analytical performance and not significantly affect the measurement results when there is a slight change in the measurement conditions. This durability is crucial to ensure the reliability, repeatability and stability of the analysis results.

[0132] The limit of detection (LOD) refers to the lowest concentration or amount of the substance in the sample that can be detected. It reflects the sensitivity and noise of the analytical method and instrument, and also indicates the level of the blank (background) value after the sample is processed.

[0133] The correction factor is a coefficient or parameter used to correct the results during the analysis process. It is intended to improve data accuracy and reliability. In HPLC analysis, since the same detector has different response values ​​for different substances, when different substances of the same mass pass through the detector, the peak areas generated are not necessarily equal. In order to make the peak area accurately reflect the content of the component to be measured, standard substances are used for correction, the correction factor is calculated, and it is applied to the measurement results of the sample to be measured.

[0134] The ratio of peak height to noise (S / N, i.e. signal-to-noise ratio) is used in HPLC to evaluate the detection sensitivity and resolution of the instrument and is an important indicator for measuring instrument performance. Among them: peak height refers to the signal value output by the detector when the component to be tested is eluted from the column; and noise refers to the fluctuation of the baseline signal, that is, the signal value measured by the blank sample. The signal-to-noise ratio is the ratio of the signal measured by a sample of known concentration to the signal measured by a blank sample. The larger the signal-to-noise ratio, the more accurately the instrument can separate and identify the target component when detecting the sample, while also reducing the interference of background noise.

[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 table

[0137]

[0138]

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

[0140] Quantitation limit (expressed as the 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 Table

[0146]

[0147]

[0148] Example 1. Separation and determination method of ruxolitinib mesylate intermediate Z2 and its impurities

[0149] (1) Prepare the test solution

[0150] Diluent: methanol.

[0151] Test solution (2.0 mg / ml): Take an appropriate amount of the product (ruxolitinib mesylate intermediate Z2), accurately weigh it, add diluent to dissolve it and quantitatively dilute it to make a solution containing about 2 mg per 1 ml.

[0152] Control solution (3 μg / ml): Accurately measure an appropriate amount of the test solution and quantitatively dilute it with a diluent to make a solution containing approximately 3 μg per 1 ml.

[0153] System suitability solution: Take 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 , trithiocyanate) in appropriate amount, dissolve in diluent and dilute to make a solution containing about 2 mg per 1 ml.

[0154] (2) Chromatographic conditions

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

[0156] Table C. Gradient elution schedule

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

[0158] (3) Determination

[0159] Accurately measure the system suitability solution, test solution and reference solution, inject them into the liquid chromatograph respectively, and record the chromatogram. Except for impurity SM1, which is determined by the limit method, the other impurities are calculated by the self-control method with correction factors.

[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 degree 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 twice the main peak area of ​​the control solution (0.3%), and the impurities C, D, F, SM 2d The peak areas of impurities Z1 and thiocyanate after correction (multiplied by correction factors 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 after correction shall not be greater than 6.66 times (1.0%) of the main peak area of ​​the control solution. 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 limit, 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, so 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 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 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] Thiocyanuric acid stock solution: Accurately weigh 20.50 mg of thiocyanuric acid reference substance, place it in 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 the 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 the solution is ready.

[0173] Impurity SM 1d Stock solution: Accurately weigh the 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 the solution is ready.

[0174] Impurity SM2 stock solution: Accurately weigh 20.18 mg of impurity SM2 reference substance, place 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 the 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 the solution is ready.

[0176] Impurity Z1 stock solution: Accurately weigh 20.13 mg of impurity Z1 reference substance, place 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 the solution is ready.

[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 the solution is ready.

[0180] Impurity B stock solution: Accurately weigh 20.11 mg of impurity B reference substance, place 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 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 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 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 in a 100 ml volumetric flask, add methanol to dissolve and dilute to the scale, shake well, and obtain.

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

[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, shake well, and 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, shake well, and obtain.

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

[0190] Mixed solution of test sample: accurately weigh 20.19 mg of ruxolitinib mesylate intermediate Z2, place it in a 10 ml volumetric flask, add impurity Z 1a 0.5 ml of stock solution, add 0.2 ml each of triphenylphosphine and impurity SM1 stock solution, add D-DMTA, triphenylphosphine oxide, trithiocyanate, 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 0.25 ml each, add methanol to dissolve and dilute to the scale, shake well, and you have it.

[0191] (2) Detection

[0192] Take 10 μl of blank solvent, positioning solution, test solution and test mixed solution respectively, inject and detect them, and record the chromatogram to examine the separation between the 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 between the peaks of each component meets the requirements. This method has good specificity.

[0194] Table D. Specificity test results

[0195]

[0196]

[0197] Note: Ruxolitinib mesylate intermediate Z2 is the D-DMTA (resolving agent) salt of ruxolitinib (main component). After dissolution, the main peaks of ruxolitinib and D-DMTA will appear; impurity SM 1b (Impurity SM 1b There are cis-trans isomers at the double bond) and triphenylphosphine are impurities in the impurity spectrum of ruxolitinib mesylate intermediate Z2, which are only used to locate and see whether they interfere with the detection of other impurities.

[0198] Example 3. Limit of Quantitation

[0199] (1) Ruxolitinib mesylate intermediate Z2

[0200] Quantitative limit solution 1#: Accurately pipette 3.75 ml of the stock solution of ruxolitinib mesylate intermediate Z2 under the "Specificity" item, place it in a 50 ml volumetric flask, dilute it to the scale with methanol, shake well, accurately pipette 5 ml, place it in a 50 ml volumetric flask, dilute it to the scale with methanol, shake well, accurately pipette 3.5 ml, place it in a 50 ml volumetric flask, dilute it to the scale with methanol, shake well, and obtain.

[0201] Take the above 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 the peak area RSD.

[0202] The test results are shown in Table E. Figure 4 and Table 4. The quantification limit of intermediate Z2 was 0.2103 μg / ml, which was expressed as 0.011% 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) Thiocyanate, impurity SM 2d , impurity Z1, impurity B, impurity C, impurity D and impurity F

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

[0207] Take the above 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 the 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% in the test sample, with a peak area RSD of 2.2% and a signal-to-noise ratio of 28.7; impurity SM 2dThe quantitative limit concentration is 0.1129μg / ml, which is expressed as 0.006% of the concentration in the test sample, the peak area RSD is 1.8%, and the average signal-to-noise ratio is 43.3; the quantitative limit concentration of impurity Z1 is 0.2739μg / ml, which is expressed as 0.014% of the concentration in the test sample, the peak area RSD is 1.6%, and the average signal-to-noise ratio is 63.1; the quantitative limit concentration of impurity B is 0.1180μg / ml, which is expressed as 0.006% of the concentration in the test sample, the peak area RSD is 1.3%, and the average signal-to-noise ratio is 39.6; The quantitative limit concentration of impurity C is 0.0390μg / ml, expressed as 0.002% in the test sample, the peak area RSD is 2.5%, and the average signal-to-noise ratio is 26.9; the quantitative limit concentration of impurity D is 0.1196μg / ml, expressed as 0.006% in the test sample, the peak area RSD is 1.1%, and the average signal-to-noise ratio is 40.7; the quantitative limit concentration of impurity F is 0.1176μg / ml, expressed as 0.006% in the test sample, the peak area RSD is 2.1%, and the average signal-to-noise ratio is 32.7. The average signal-to-noise ratios of the peak quantitative limits of the above impurities are all greater than 10, and the peak area RSDs are all less than 10.0%, which meets the requirements.

[0209] Table F. Quantitation limit 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 0.4 ml of triphenylphosphine oxide positioning solution and impurity SM under the "Specificity" item 1d 2ml of positioning solution, 0.4ml of impurity SM2 positioning solution, and impurity Z 1a Positioning solution 0.6ml, impurity Z 1b 1.6 ml of localization solution and impurity Z 1d Place 0.3 ml of the positioning solution in a 100 ml volumetric flask, dilute to the mark 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 the peak area RSD.

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

[0216] Table G. Quantitation limit 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 in a 25 ml volumetric flask, dilute to the scale with methanol, shake well, and obtain.

[0222] Take the above detection limit solution 1# and measure it continuously for 3 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 and Table 7. The detection limit concentration of ruxolitinib mesylate intermediate Z2 is 0.0673 μg / ml, expressed as 0.003% in the test sample, and the average signal-to-noise ratio is 8.6. The average signal-to-noise ratio of the detection limit of ruxolitinib mesylate intermediate Z2 is greater than 3, which meets the requirements.

[0224] Table H. Detection limit test results

[0225]

[0226]

[0227] (2) Thiocyanate, 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 in a 25 ml volumetric flask, dilute to the scale with methanol, shake well, and obtain.

[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 is 0.1019 μg / ml, expressed as 0.005% in the test sample, and the average signal-to-noise ratio is 8.6; impurity SM 2d The detection limit concentration is 0.0361μg / ml, which is expressed as 0.002% of the concentration in the test sample, and the average signal-to-noise ratio is 13.0; the detection limit concentration of impurity Z1 is 0.0877μg / ml, which is expressed as 0.004% of the concentration in the test sample, and the average signal-to-noise ratio is 19.6; the detection limit concentration of impurity B is 0.0378μg / ml, which is expressed as 0.002% of the concentration in the test sample, and the average signal-to-noise ratio is 12.0; The detection limit concentration of impurity C is 0.0125μg / ml, expressed as 0.001% in the test sample, and the average signal-to-noise ratio is 8.0; the detection limit concentration of impurity D is 0.0383μg / ml, expressed as 0.002% in the test sample, and the average signal-to-noise ratio is 12.6; the detection limit concentration of impurity F is 0.0376μg / ml, expressed as 0.002% in the test sample, and the average signal-to-noise ratio is 9.7. The average signal-to-noise ratios of the detection limits of the above impurities are all greater than 3, which meets 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 quantitation limit solution 3# under the “quantitation 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 for 3 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. Fig. 9 and Table 9. The detection limit of triphenylphosphine oxide is 0.0255 μg / ml, expressed as 0.001% of the concentration in the test sample, and the average signal-to-noise ratio is 10.8; impurity SM 1d The detection limit concentration is 0.1241μg / ml, which is expressed as 0.006% in the test sample, and the average signal-to-noise ratio is 10.4; the detection limit concentration of impurity SM2 is 0.0257μg / ml, which is expressed as 0.001% in the test sample, and the average signal-to-noise ratio is 8.1; impurity Z 1a The detection limit concentration is 0.0377μg / ml, which is expressed as 0.002% 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% 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% in the test sample, and the average signal-to-noise ratio is 6.0. The average signal-to-noise ratios of the detection limits of the above impurities are all 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, add acetonitrile to dissolve and dilute to scale, shake well, accurately transfer 2 ml, place in a 100 ml volumetric flask, dilute to scale with acetonitrile, shake well, accurately transfer 1 ml, place in a 100 ml volumetric flask, dilute to scale with methanol, shake well, and the solution is ready. (Prepare freshly before 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. Fig.10and Table 10. The detection limit concentration of impurity SM1 is 0.1982 μg / ml, expressed as 0.010% in the test sample, and the average signal-to-noise ratio is 8.9. The detection limit signal-to-noise ratios are all greater than 3, which meets the requirements.

[0243] Table K. Detection limit test results

[0244]

[0245] Example 5. Durability

[0246] Use the mixed solution of the test sample 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 of 1.0 ml / min; column temperature of 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 Tables L to O. Figure 3 , Figure 11 to Figure 21 , Table 3, Table 11 to Table 21. When there were slight fluctuations in flow rate, column temperature, buffer pH value, buffer concentration, and initial ratio of mobile phase, and when different batches of chromatographic columns from the same manufacturer were replaced, the resolution between peaks was greater than 1.5, and the relative retention time changes of each known impurity were within the range of ±0.12. The change of chromatographic conditions had no significant effect on the detection, and the method had good durability.

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

[0251]

[0252] Table M. Chromatographic Conditions 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 (Degree of 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 mesylate ruxolitinib intermediate Z2 and the impurities together form a composition, and the impurities include 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 and any one or more of triphenylphosphine; the mesylate ruxolitinib 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 stationary phase: the chromatographic column uses octadecylsilane bonded silica gel as a 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.

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

5. The method according to claim 1, characterized in that 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.

6. A method for identifying ruxolitinib mesylate intermediate Z2 and its impurities, characterized in that: The composition is separated by the method described in any one of claims 1 to 5, 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.

7. The method according to claim 6, characterized in that The detection wavelength of the detector is 220±10 nm.

8. The method according to claim 6, 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 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.

9. The method according to claim 7, characterized in that: Taking ruxolitinib, the main component of ruxolitinib mesylate intermediate Z2, as the reference peak, the relative retention time was 0.15, and it was 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 is 0.56, which is 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 is 1.13, which is 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 is 1.76, which is determined to be impurity Z1; The relative retention time is 1.81, which is 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%.

10. A method for determining the content of ruxolitinib mesylate intermediate Z2 and its impurities, characterized in that: The method according to any one of claims 6 to 9 is used to separate and identify the mesylate ruxolitinib intermediate Z2 and its impurities 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.

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