Method for separating and determining rucotinib mesylate intermediate Z1 and related impurities thereof

Abstract

The invention belongs to the technical field of chemical analysis, and particularly relates to a method for separating and determining a rucotinib mesylate intermediate Z1 and related impurities thereof. The impurities comprise any one or more of an impurity SM2a, an impurity SM1d, an impurity Z1c, an impurity Z1b, an impurity Z1a, triphenylphosphine oxide, an impurity SM2, an impurity SM1 and an impurity Z1d. Octadecylsilane chemically bonded silica is adopted as a chromatographic column filling agent; a monopotassium phosphate buffer solution is used as a mobile phase A, a mixed solution of methanol and acetonitrile is used as a mobile phase B, and main components are separated from impurities through gradient elution; and detecting in a detector to obtain a chromatogram. The method is high in specificity, short in separation time, high in sensitivity and good in durability, and has important significance on quality control of rukotinib mesylate bulk drugs and preparation products.

Description

Technical Field

[0001] This invention belongs to the field of chemical analysis technology, specifically relating to a method for separating and determining ruxolitinib mesylate intermediate Z1 and its related impurities. Background Technology

[0002] Ruxolitinib is a Janus kinase inhibitor, mainly used clinically to treat intermediate- or high-risk primary myelofibrosis, myelofibrosis secondary to polycythemia vera, myelofibrosis secondary to essential thrombocythemia, and other related diseases.

[0003] Ruxolitinib mesylate intermediate Z1 is a key intermediate of ruxolitinib mesylate. The structural formulas of ruxolitinib mesylate intermediate Z1 and ruxolitinib mesylate are shown in Formula 1 and Formula 11, respectively. Studies have found that ruxolitinib mesylate intermediate Z1 may contain the following nine impurities: Impurity SM 2a Impurities SM 1d Impurity Z 1c Impurity Z 1b Impurity Z 1a Triphenylphosphine oxide, impurity SM2, impurity SM1 and impurity Z 1d Its structural formula is shown in Formula 2-10. These impurities are difficult to remove in subsequent reactions, thus affecting the quality of ruxolitinib mesylate raw material and finished products.

[0004]

[0005] Currently, there is a lack of methods to control the relevant impurities in ruxolitinib mesylate intermediate Z1, and no patents or literature have reported on this. Therefore, a method is needed to separate and determine the relevant impurities in ruxolitinib mesylate intermediate Z1, in order to better control the quality of subsequent ruxolitinib mesylate API and related formulations. Summary of the Invention

[0006] In view of this, one of the objectives of the present invention is to provide a method for separating ruxolitinib mesylate intermediate Z1 and its related impurities using high performance liquid chromatography. This method can separate nine impurities in ruxolitinib mesylate intermediate Z1 within 75 minutes or more, which is of great significance for the subsequent qualitative and quantitative analysis of each component.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A method for separating ruxolitinib mesylate intermediate Z1 and its related impurities using high performance liquid chromatography, wherein the related impurities include impurity SM. 2a Impurities SM 1d Impurity Z 1c Impurity Z1b Impurity Z 1a Triphenylphosphine oxide, impurity SM2, impurity SM1 and impurity Z 1d Any one or more of the following; the high-performance liquid chromatography method includes: using octadecylsilane-bonded silica gel as the column packing material, using potassium dihydrogen phosphate buffer as mobile phase A, and a mixed solution of methanol and acetonitrile as mobile phase B, and sequentially removing impurities SM by gradient elution. 2a Impurities SM 1d Impurity Z 1c Impurity Z 1b Impurity Z 1a Triphenylphosphine oxide, impurity SM2, impurity SM1, ruxolitinib mesylate intermediate Z1 and / or impurity Z 1d Separation is performed; in the mobile phase B, the volume ratio of methanol to acetonitrile is 40-60:60-40;

[0009] The structural formula of the ruxolitinib mesylate intermediate Z1 is shown in Formula 1, and the impurity SM 2a The structural formula is shown in Formula 2, and the impurity SM 1d The structural formula is shown in Formula 3, wherein the impurity Z 1c The structural formula is shown in Formula 4, and the impurity Z 1b The structural formula is shown in Formula 5, wherein the impurity Z 1a The structural formula of the impurity is shown in Formula 6, the structural formula of the triphenylphosphine oxide is shown in Formula 7, the structural formula of the impurity SM2 is shown in Formula 8, the structural formula of the impurity SM1 is shown in Formula 9, and the structural formula of the impurity Z... 1d The structural formula is shown in Equation 10;

[0010]

[0011]

[0012] Furthermore, the relevant impurities include an impurity composition and / or other impurities; the impurity composition includes impurity Z. 1b Impurity Z 1a Impurity SM2, Impurity Z 1d Any one or more of the following; the other impurities include impurity SM. 2a Impurities SM 1d Impurity Z 1c Any one or more of the following: triphenylphosphine oxide, impurity SM1.

[0013] Furthermore, the separated components are as follows: 1) or 2):

[0014] 1) Impurity Z 1b Impurity Z 1a Impurity SM2, Impurity Z 1dAny one or more of the following;

[0015] 2) Impurity Z 1b Impurity Z 1a Impurity SM2, Impurity Z 1d Any one or more of the impurities SM 2a Impurities SM 1d Impurity Z 1c Any one or more of the following: triphenylphosphine oxide, impurity SM1, etc.

[0016] Preferably, in the mobile phase B, the volume ratio of methanol to acetonitrile is 50:50.

[0017] Furthermore, the concentration of the potassium dihydrogen phosphate buffer solution is 25-35 mmol / L, and the pH is 3-5.

[0018] Preferably, the concentration of the potassium dihydrogen phosphate buffer solution is 28-32 mmol / L, more preferably 0.03 mol / L.

[0019] Preferably, the pH of the potassium dihydrogen phosphate buffer solution is 3.8-4.2, more preferably 4.0.

[0020] Preferably, phosphoric acid is used to adjust the pH of mobile phase A.

[0021] Furthermore, the gradient elution procedure is as follows:

[0022] 0 minutes, set the volume ratio of mobile phase A to mobile phase B to 60-80:40-20;

[0023] Set the volume ratio of mobile phase A to mobile phase B to 60-80:40-20 for 3 minutes.

[0024] Set the volume ratio of mobile phase A to mobile phase B to 35-50:65-50 for 15 minutes.

[0025] 55 minutes, set the volume ratio of mobile phase A to mobile phase B to be 20-30:80-70;

[0026] For 65 minutes, set the volume ratio of mobile phase A to mobile phase B to be 20-30:80-70;

[0027] 66 minutes, with the volume ratio of mobile phase A to mobile phase B set at 60-80:40-20;

[0028] Set the volume ratio of mobile phase A to mobile phase B to 60-80:40-20 for 75 minutes.

[0029] Preferably, the gradient elution procedure is as follows:

[0030] 0 minutes, set the volume ratio of mobile phase A to mobile phase B to 67-71:33-29;

[0031] For 3 minutes, set the volume ratio of mobile phase A to mobile phase B to 67-71:33-29;

[0032] After 15 minutes, the volume ratio of mobile phase A to mobile phase B was set to 44:56.

[0033] 55 minutes, with the volume ratio of mobile phase A to mobile phase B set at 25:75;

[0034] 65 minutes, with the volume ratio of mobile phase A to mobile phase B set at 25:75;

[0035] 66 minutes, with the volume ratio of mobile phase A to mobile phase B set at 67-71:33-29;

[0036] For 75 minutes, the volume ratio of mobile phase A to mobile phase B was set to 67-71:33-29.

[0037] As the optimal choice, the gradient elution procedure is as follows:

[0038] 0 minutes, set the volume ratio of mobile phase A to mobile phase B to 69:31;

[0039] For 3 minutes, set the volume ratio of mobile phase A to mobile phase B to 69:31;

[0040] After 15 minutes, the volume ratio of mobile phase A to mobile phase B was set to 44:56.

[0041] 55 minutes, with the volume ratio of mobile phase A to mobile phase B set at 25:75;

[0042] 65 minutes, with the volume ratio of mobile phase A to mobile phase B set at 25:75;

[0043] 66 minutes, with the volume ratio of mobile phase A to mobile phase B set at 69:31;

[0044] For 75 minutes, the volume ratio of mobile phase A to mobile phase B was set to 69:31.

[0045] Furthermore, the separation time is 75 minutes.

[0046] Furthermore, the flow rate was 1.0-1.5 ml / min; the column temperature was 35-45℃.

[0047] Preferably, the flow rate is 1.0-1.2 ml / min, more preferably 1.1 ml / min.

[0048] Preferably, the column temperature is 38-42℃, more preferably 40℃.

[0049] Furthermore, the injector temperature is 4-8℃, preferably 5℃.

[0050] Furthermore, the injection volume was 10 μl.

[0051] Preferably, the chromatographic column used is the Phenomen SuperLu C18.

[0052] Preferably, the chromatographic column has a size of 4.6 mm × 150 mm and a diameter of 3 μm.

[0053] The second objective of this invention is to provide a method for identifying ruxolitinib mesylate intermediate Z1 and its related impurities. This method can identify impurities in ruxolitinib mesylate intermediate Z1 with a content as low as 0.003%-0.015%, with a low detection limit and high method sensitivity.

[0054] To achieve the above objectives, the present invention adopts the following technical solution:

[0055] A method for identifying ruxolitinib mesylate intermediate Z1 and its related impurities involves separating ruxolitinib mesylate intermediate Z1 and any one or more of the related impurities using the aforementioned separation method; detecting the separated ruxolitinib mesylate intermediate Z1 using a detector with a detection wavelength of 210-230 nm; and determining whether the ruxolitinib mesylate intermediate Z1 sample contains related impurities by comparing the chromatographic behavior of the test sample and the reference sample.

[0056] Preferably, the detection wavelength is 220 nm.

[0057] Furthermore, the retention times of each component, from shortest to longest, are as follows: impurity SM 2a Impurities SM 1d Impurity Z 1c Impurity Z 1b Impurity Z 1a Triphenylphosphine oxide, impurity SM2, impurity SM1, ruxolitinib mesylate intermediate Z1, impurity Z 1d .

[0058] Based on the retention time, the components in ruxolitinib mesylate intermediate Z1 can be qualitatively identified.

[0059] Furthermore, using ruxolitinib mesylate intermediate Z1 as a reference peak, when the relative retention time was 0.20 ± 0.05, it was identified as impurity SM. 2a When the relative retention time is 0.25 ± 0.05, it is determined to be impurity SM. 1d When the relative retention time is 0.35 ± 0.05, it is determined to be impurity Z. 1c When the relative retention time is 0.44 ± 0.05, it is determined to be impurity Z. 1bWhen the relative retention time is 0.52 ± 0.05, it is determined to be impurity Z. 1a When the relative retention time is 0.61±0.05, it is identified as triphenylphosphine oxide; when the relative retention time is 0.73±0.05, it is identified as impurity SM2; when the relative retention time is 0.78±0.05, it is identified as impurity SM1; when the relative retention time is 1.03±0.05, it is identified as impurity Z. 1d The retention time of the ruxolitinib mesylate intermediate Z1 was 29.3 ± 0.5 min.

[0060] Preferably, a retention time of 5.7 ± 0.5 min is considered to be impurity SM. 2a When the retention time is 7.2 ± 0.5 min, it is identified as impurity SM. 1d When the retention time is 10.4 ± 0.5 min, it is determined to be impurity Z. 1c When the retention time is 12.7 ± 0.5 min, it is determined to be impurity Z. 1b When the retention time is 15.4 ± 0.5 min, it is determined to be impurity Z. 1a The following substances were identified: a retention time of 17.7 ± 0.5 min, which was determined to be triphenylphosphine oxide; a retention time of 21.4 ± 0.5 min, which was determined to be impurity SM2; a retention time of 22.8 ± 0.5 min, which was determined to be impurity SM1; a retention time of 29.3 ± 0.5 min, which was determined to be ruxolitinib mesylate intermediate Z1; and a retention time of 30.1 ± 0.5 min, which was determined to be impurity Z. 1d .

[0061] The third objective of this invention is to provide a method for detecting the content of relevant impurities in ruxolitinib mesylate intermediate Z1. This method can achieve quantitative detection of impurities in ruxolitinib mesylate intermediate Z1 with a content as low as 0.01%-0.05%, and has a low limit of quantification.

[0062] To achieve the above objectives, the present invention adopts the following technical solution:

[0063] A method for detecting the content of relevant impurities in ruxolitinib mesylate intermediate Z1 includes the following steps:

[0064] (1) The intermediate Z1 of ruxolitinib mesylate and its related impurities were separated and detected using the aforementioned method, and a chromatogram was obtained;

[0065] (2) Based on the chromatogram obtained in step (1), calculate the content of each impurity using the external standard method or the principal component self-comparison method with correction factor.

[0066] As a preferred method, the principal component self-comparison method with correction factors is used to calculate the impurities SM in the sample. 2aImpurities SM 1d Impurity Z 1c Impurity Z 1b Impurity Z 1a Triphenylphosphine oxide, impurity SM2, impurity SM1 and impurity Z 1d The content of any one or more of the following. Further, before separation, the test solution is prepared using a mixed solution of methanol and acetonitrile as a solvent; the volume ratio of methanol to acetonitrile is 50:50.

[0067] Furthermore, the impurity Z 1a The correction factor is 0.8; the impurity Z 1b The correction factor is 1.8; the impurity Z 1c The correction factor is 0.7; the impurity Z 1d The correction factor for impurity SM1 is 0.5; the correction factor for impurity SM2 is 3.0; the correction factor for impurity SM3 is 0.7; the correction factor for impurity SM4 is 0.5. 1d The correction factor is 2.1; the impurity SM 2a The correction factor is 0.3; the correction factor for triphenylphosphine oxide is 0.5.

[0068] As a preferred technical solution, the method includes the following steps:

[0069] 1. Take an appropriate amount of this product, dissolve it in a solvent, and quantitatively dilute it to prepare a solution with a concentration of 1 mg / ml, which will be used as the sample solution;

[0070] 2. Take an appropriate amount of the test solution and dilute it quantitatively with a solvent to prepare a solution with a concentration of 10 μg / ml, which will serve as the control solution;

[0071] 3. Take intermediate Z1 system suitability reference standard (containing ruxolitinib mesylate intermediate Z1, impurity SM1, impurity SM2, and impurity SM). 1d Impurities SM 2a Impurity Z 1a Impurity Z 1b Impurity Z 1c Impurity Z 1d Add an appropriate amount of triphenylphosphine oxide, dissolve and dilute with solvent to prepare a solution containing approximately 1 mg per 1 ml, which is used as a system suitability solution;

[0072] 4. Take 10 μl of the above system suitability solution, sample solution, and control solution, respectively, and inject them into the liquid chromatograph. Perform the detection according to the aforementioned chromatographic conditions and record the chromatograms.

[0073] 5. Based on the chromatogram obtained in step 4, calculate the content of each impurity using the principal component self-comparison method with correction factors.

[0074] The beneficial effects of this invention are as follows:

[0075] 1. The method for separating and determining ruxolitinib mesylate intermediate Z1 and its related impurities provided by the present invention can simultaneously separate ruxolitinib mesylate intermediate Z1 and its nine impurities within 75 minutes, with good separation between impurities, and features short analysis time and high specificity.

[0076] 2. The analytical method of the present invention can effectively detect impurities with a concentration of 0.003%-0.015% in ruxolitinib mesylate intermediate Z1, and accurately quantify impurities with a concentration of 0.01%-0.05% in ruxolitinib mesylate intermediate Z1, and has the characteristics of high sensitivity.

[0077] 3. The analytical method of the present invention has high accuracy, and the recovery rate of each impurity is between 90% and 108%.

[0078] 4. The analytical method of the present invention has good robustness. When the flow rate, column temperature and mobile phase ratio fluctuate, the elution order of each impurity and the main component remains unchanged. Under each condition, the relative retention time of each impurity changes within the range of ±0.05. The resolution between the peaks of each component is greater than 1.5. Attached Figure Description

[0079] Figure 1 The HPLC chromatogram is for the blank solution.

[0080] Figure 2 The HPLC chromatogram of mixed solution 1 is shown below.

[0081] Figure 3 SM is an impurity 2a HPLC chromatogram of the positioning solution;

[0082] Figure 4 SM is an impurity 1d HPLC chromatogram of the positioning solution;

[0083] Figure 5 Impurity Z 1c HPLC chromatogram of the positioning solution;

[0084] Figure 6 Impurity Z 1b HPLC chromatogram of the positioning solution; Figure 7 Impurity Z 1a HPLC chromatogram of the positioning solution;

[0085] Figure 8 The HPLC chromatogram shows the solution containing the impurity triphenylphosphine oxide.

[0086] Figure 9 The HPLC chromatogram of the solution containing impurity SM2 is shown.

[0087] Figure 10The HPLC chromatogram of the solution containing impurity SM1 is shown.

[0088] Figure 11 Impurity Z 1d HPLC chromatogram of the positioning solution;

[0089] Figure 12 The HPLC chromatogram of the localization solution of intermediate Z1;

[0090] Figure 13 HPLC chromatogram of the mixed solution under the chromatographic conditions of "mobile phase A is water, mobile phase B is methanol, mobile phase C is acetonitrile, and chromatographic column is SHIMADZU Shim-pack VP-ODS" in the exploratory experiment for the method establishment stage;

[0091] Figure 14 HPLC chromatogram of the mixed solution under the chromatographic conditions of "mobile phase A: 0.1% phosphoric acid solution, mobile phase B: methanol, mobile phase C: acetonitrile, and chromatographic column: SHIMADZU Shim-pack VP-ODS" in the exploratory experiment for the method establishment stage;

[0092] Figure 15 HPLC chromatogram of the mixed solution under the chromatographic conditions of "mobile phase A: 0.03 mol / L potassium dihydrogen phosphate buffer, mobile phase B: methanol-acetonitrile, chromatographic column: Phenomen SuperLu C18, elution time: 45 min" in the exploratory experiment for the method establishment stage;

[0093] Figure 16 HPLC chromatogram of the mixed solution under the chromatographic conditions of "mobile phase A: 0.03 mol / L potassium dihydrogen phosphate buffer, mobile phase B: methanol-acetonitrile, chromatographic column: Phenomen SuperLu C18, elution time: 70 min" in the exploratory experiment for the method establishment stage;

[0094] Figure 17 The HPLC chromatogram of the test solution is shown below.

[0095] Figure 18 The HPLC chromatogram of the mixed solution under normal conditions in the durability test;

[0096] Figure 19 The HPLC chromatogram is shown for a mixed solution with an initial mobile phase ratio of 71:29, used in the durability test.

[0097] Figure 20 The HPLC chromatogram is shown for a mixed solution with an initial mobile phase ratio of 67:33, used in the durability test.

[0098] Figure 21 This is the HPLC chromatogram of the mixed solution at a flow rate of 1.0 ml / min during the durability test;

[0099] Figure 22 The HPLC chromatogram is for a mixed solution with a flow rate of 1.2 ml / min during the durability test.

[0100] Figure 23 This is the HPLC chromatogram of the mixed solution at a column temperature of 38°C during the durability test;

[0101] Figure 24 This is the HPLC chromatogram of the mixed solution at a column temperature of 42℃ during the durability test;

[0102] Figure 25 This is an HPLC chromatogram of a mixed solution with a buffer salt pH of 3.8 used in the durability test.

[0103] Figure 26 This is an HPLC chromatogram of a mixed solution with a buffer salt pH of 4.2 used in the durability test.

[0104] Figure 27 The image shows the HPLC chromatogram of the mixed solution under the condition of a buffer salt concentration of 28 mmol / L during the durability test.

[0105] Figure 28 The image shows the HPLC chromatogram of the mixed solution under the condition of a buffer salt concentration of 32 mmol / L during the durability test.

[0106] Figure 29 The HPLC chromatogram is for the limit of quantitation solution.

[0107] Figure 30 This is the HPLC chromatogram of the solution at the detection limit. Detailed Implementation

[0108] The technical solution of the present invention will be described more clearly and completely below with reference to specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention.

[0109] In this embodiment of the invention, the determination was performed according to high performance liquid chromatography (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0512).

[0110] In this embodiment of the invention, all experimental solvents and detection solutions are freshly prepared before use.

[0111] Example 1

[0112] (1) Preparation of solution

[0113] Solvent: A mixed solution of methanol and acetonitrile in a volume ratio of 50:50.

[0114] Test solution: Take an appropriate amount of this product (Ruxolitinib mesylate intermediate Z1), accurately weigh it, dissolve it in solvent and dilute it quantitatively to prepare a solution containing about 1 mg per 1 ml.

[0115] Control solution: Accurately measure an appropriate amount of the test solution and dilute it quantitatively with a solvent to prepare a solution containing approximately 10 μg per ml.

[0116] System suitability solution is taken from intermediate Z1 system suitability reference standard (containing ruxolitinib mesylate intermediate Z1, impurity SM1, impurity SM2, and impurity SM). 1d Impurities SM 2a Impurity Z 1a Impurity Z 1b Impurity Z 1c Impurity Z 1d Add an appropriate amount of triphenylphosphine oxide, dissolve and dilute with a solvent to prepare a solution containing approximately 1 mg per 1 ml.

[0117] (2) Chromatographic conditions

[0118] The column was packed with octadecylsilane-bonded silica gel (Pheromen SuperLu C18, 4.6 mm × 150 mm, 3 μm or equivalent column); mobile phase A was 0.03 mol / L potassium dihydrogen phosphate buffer (pH adjusted to 4.0 with phosphoric acid), and mobile phase B was a mixture of methanol and acetonitrile (50:50 v / v). Gradient elution was performed according to Table 1. The detection wavelength was 220 nm; the flow rate was 1.1 mL / min; the column temperature was 40 °C; the injection volume was 10 μL; and the injector temperature was 5 °C.

[0119] Table 1. Gradient elution program table

[0120] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 69 31 3 69 31 15 44 56 55 25 75 65 25 75 66 69 31 75 69 31

[0121] (3) Detection

[0122] System suitability requirements: In the system suitability solution chromatogram, according to impurities SM 2a Impurities SM 1d Impurity Z 1c Impurity Z 1b Impurity Z 1a Triphenylphosphine oxide, impurity SM2, impurity SM1, Z1, impurity Z 1d Peaks should be extracted sequentially, and the separation between peaks of each component should meet the requirements.

[0123] Assay: Accurately measure the test solution and the control solution, inject them separately into the liquid chromatograph, and record the chromatograms.

[0124] Limit: If impurity peaks are present in the chromatogram of the test solution, impurity SM 2aImpurities SM 1d Impurity Z 1c Impurity Z 1b Impurity Z 1a Triphenylphosphine oxide, impurity SM2, impurity SM1, impurity Z 1d The content of each impurity should be calculated according to the principal component self-comparison method with correction factors, and should meet the limits specified in Table 2. The total amount of impurities should not exceed 15.0%. Table 2 lists the correction factors and limits for each impurity peak. Any peak in the chromatogram of the test solution that is less than 0.05 times the area of ​​the main peak in the control solution can be ignored (0.05%).

[0125] Table 2. Limitation Regulations

[0126]

[0127]

[0128] Example 2. Specificity

[0129] (1) Solution preparation

[0130] 1) Blank solvent interference experiment

[0131] Blank solvent (diluent): a mixed solution of acetonitrile and methanol, wherein the volume ratio of acetonitrile to methanol is 50:50.

[0132] 2) Separation of potential impurities by the chromatographic system

[0133] Impurity SM1 stock solution: Accurately weigh 51.17 mg of impurity SM1, place it in a 25 ml volumetric flask, dissolve and dilute to the mark with acetonitrile, and shake well to obtain the solution.

[0134] Impurity SM2 stock solution: Accurately weigh 25.75 mg of impurity SM2, place it in a 25 ml volumetric flask, dissolve and dilute to the mark with acetonitrile, and shake well to obtain the solution.

[0135] Impurities SM 1d Stock solution: Accurately weigh impurity SM 1d 26.17 mg was placed in a 25 ml volumetric flask, dissolved and diluted to the mark with acetonitrile, and shaken well to obtain the final product.

[0136] Impurities SM 2a Stock solution: Accurately weigh impurity SM 2a 25.66 mg, placed in a 25 ml volumetric flask, dissolved and diluted to the mark with diluent, shaken well, and the product is obtained.

[0137] Impurity Z 1a Stock solution: Accurately weigh impurity Z 1a25.60 mg was placed in a 100 ml volumetric flask, dissolved and diluted to the mark with the solvent [methanol-acetonitrile (1:2)], and shaken well to obtain the final product.

[0138] Impurity Z 1b Stock solution: Accurately weigh impurity Z 1b 51.05 mg, placed in a 25 ml volumetric flask, dissolved and diluted to the mark with diluent, shaken well, and the product is obtained.

[0139] Impurity Z 1c Stock solution: Accurately weigh impurity Z 1c 25.96 mg was placed in a 25 ml volumetric flask, dissolved and diluted to the mark with methanol, and shaken well to obtain the final product.

[0140] Impurity Z 1d (Triphenylphosphine sulfide) stock solution: Accurately weigh impurity Z 1d 200.91 mg, placed in a 100 ml volumetric flask, dissolved and diluted to the mark with diluent, shaken well, and the product is obtained.

[0141] Triphenylphosphine oxide stock solution: Accurately weigh 51.57 mg of triphenylphosphine oxide, place it in a 25 ml volumetric flask, dissolve and dilute to the mark with diluent, and shake well to obtain the solution.

[0142] Impurity localization solution: Take impurity SM1, impurity SM2, and impurity SM 1d Impurities SM 2a Impurity Z 1a Impurity Z 1b Impurity Z 1c Impurity Z 1d 1.0 ml each of triphenylphosphine stock solution and dilute to the mark with diluent in separate 10 ml volumetric flasks and shake well to obtain the final product.

[0143] Test solution: Accurately weigh 51.51 mg of intermediate Z1, place it in a 50 ml volumetric flask, dissolve and dilute to the mark with diluent, shake well, and the solution is ready.

[0144] Mixed Solution 1: Accurately weigh 101.83 mg of intermediate Z1 and place it in a 100 ml volumetric flask. Then accurately measure impurity Z. 1c Impurities SM 2a 0.15 ml of each stock solution, SM for impurities. 1d Impurity SM2 stock solution 0.5 ml each, triphenylphosphine oxide stock solution 0.75 ml, impurity Z 1b 1.5 ml each of impurity SM1 stock solution and impurity Z 1a Impurity Z 1d Place 2.0 ml of each stock solution into the same 100 ml volumetric flask, dissolve and dilute to the mark with diluent, and shake well to obtain the final product.

[0145] (2) Detection

[0146] Precisely measure 10 μl of each of the above-mentioned blank solvent, impurity positioning solution, test solution, and mixed solution 1, and inject them into the chromatographic conditions described in Example 1. Record the chromatograms.

[0147] Results: As shown in Table 3. Figures 1-12 , Figure 17 As shown, the blank solvent does not interfere with the detection of the test sample; the integral results of mixed solution 1 are shown in Table 4; impurity Z 1d The resolution between the peak and the Z1 peak was 2.14, and the resolution between each impurity peak was greater than 1.5. This result indicates that the method has good specificity.

[0148] Table 3. Results of specificity test

[0149]

[0150] Table 4. Integral Results of Mixed Solution 1

[0151]

[0152] Example 3. Limit of Quantification and Limit of Detection

[0153] (1) Limit of quantitation

[0154] 1) Preparation of the test solution

[0155] Impurities SM 1d Limit of quantity stock solution: Accurately measure impurity SM 1d Place 0.5 ml of the stock solution into a 100 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the final product.

[0156] Impurity Z 1a Limit of quantity stock solution: Accurately measure impurity Z 1a Place 2 ml of the stock solution into a 100 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the final product.

[0157] Impurity SM2 Quantitative Limit Stock Solution: Accurately measure 0.5 ml of impurity SM2 stock solution, place it in a 100 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.

[0158] Impurity Z 1b Limit of quantity stock solution: Accurately measure impurity Z 1b Place 1.5 ml of the stock solution into a 100 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the final product.

[0159] Impurity SM1 Quantitative Limit Stock Solution: Accurately measure 1.5 ml of impurity SM1 stock solution, place it in a 100 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.

[0160] Impurity Z 1c Limit of quantity stock solution: Accurately measure impurity Z 1c Place 1.5 ml of the stock solution into a 10 ml volumetric flask, dilute to the mark with diluent, and shake well. Then accurately measure 1 ml into a 100 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the final product.

[0161] Impurities SM 2a Limit of quantity stock solution: Accurately measure impurity SM 2a Place 1.5 ml of the stock solution into a 10 ml volumetric flask, dilute to the mark with diluent, and shake well. Then accurately measure 1 ml into a 100 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the final product.

[0162] Triphenylphosphine oxide stock solution: Accurately measure 0.75 ml of triphenylphosphine oxide stock solution, place it in a 100 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.

[0163] Impurity Z 1d Limit of quantity stock solution: Accurately measure impurity Z 1d Place 2 ml of the stock solution into a 100 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the final product.

[0164] Impurity Z1 Quantitative Limit Stock Solution: Accurately measure 0.5 ml of the impurity Z1 reference stock solution under the "Linearity" section, place it in a 10 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.

[0165] Limit of Quantification Solution: Accurately measure impurity SM 1d Z 1c 10 ml of each stock solution at the limit of quantitation, and impurity Z. 1a Quantitative limit stock solution 3 ml, impurity SM2 Quantitative limit stock solution 5 ml, impurity Z 1b 1 ml each of Z1 limit of quantitation stock solution and impurity SM1 limit of quantitation stock solution, 1.5 ml of impurity SM 2a 7.5 ml of limit-of-quantity stock solution, 0.75 ml of triphenylphosphine oxide limit-of-quantity stock solution, and impurity Z. 1d Take 0.5 ml of the limit of quantitation stock solution and place it in the same 100 ml volumetric flask. Dilute to the mark with diluent and shake well.

[0166] 2) Inject the above-mentioned limit of quantitation solution 6 times consecutively, and perform detection under the chromatographic conditions of Example 1. Calculate the ratio of the main peak height to the noise (signal-to-noise ratio) and the peak area RSD, and record the chromatogram.

[0167] The formula for calculating the limit of quantitation is as follows:

[0168]

[0169] Results: As shown in Table 5. Figure 29 As shown, impurity SM 2a The limit of quantitation (LOQ) was 0.1134 μg / ml, which is expressed as 0.011% in Z1. The RSD of the peak area was 2.9%, and the mean signal-to-noise ratio was 27.9. Impurity SM 1d The limit of quantitation (LOQ) was 0.5046 μg / ml, expressed as 0.050% in Z1. The RSD of the peak area was 3.7%, and the mean signal-to-noise ratio was 17.2. Impurity Z 1c The limit of quantitation (LOQ) was 0.1548 μg / ml, expressed as 0.015% in Z1. The RSD of the peak area was 4.4%, and the mean signal-to-noise ratio was 33.5. Impurity Z 1b The limit of quantitation (LOQ) was 0.3048 μg / ml, expressed as 0.030% in Z1. The RSD of the peak area was 3.5%, and the mean signal-to-noise ratio was 20.2. Impurity Z 1a The limit of quantitation (LOQ) concentration was 0.1504 μg / ml, expressed as 0.015% in Z1, with an RSD of 2.0% for peak area and a mean signal-to-noise ratio of 12.0%; the limit of quantitation (LOQ) concentration of triphenylphosphine was 0.1139 μg / ml, expressed as 0.011% in Z1, with an RSD of 1.3% for peak area and a mean signal-to-noise ratio of 16.9%; the limit of quantitation (LOQ) concentration of impurity SM2 was 0.2562 μg / ml, expressed as 0.015% in Z1. The concentration of impurity Z1 was 0.026%, with an RSD of 1.9% for peak area and a mean SNR of 41.3; the limit of quantitation (LOQ) for impurity SM1 was 0.4568 μg / ml, which, expressed as 0.046% in Z1, had an RSD of 2.0% for peak area and a mean SNR of 26.1; the limit of quantitation (LOQ) for impurity Z1 was 0.4899 μg / ml, which, expressed as 0.049% in the test sample, had an RSD of 2.2% for peak area and a mean SNR of 43.0; impurity Z... 1d The limit of quantitation (LOQ) concentration was 0.2004 μg / ml, expressed as 0.020% in Z1. The RSD of the peak area was 1.5%, and the mean signal-to-noise ratio (SNR) was 22.3. At the LOQ concentrations of all impurities, the SNR was greater than 10, and the RSD of the peak area was less than 10%, meeting the requirements for the determination of related substances and indicating that each impurity could be accurately quantified at these levels.

[0170] Table 5. Results of Limit of Quantitation Test

[0171]

[0172]

[0173] (2) Detection limit

[0174] Accurately measure 3 ml of the quantitation limit solution under the "Limit of Quantitation" section, place it in the same 10 ml volumetric flask, dilute to the mark with diluent, shake well, and prepare the detection limit solution. Inject this detection limit solution three times consecutively, and perform detection according to the chromatographic conditions of Example 1. Calculate the ratio of the main peak height to the noise (signal-to-noise ratio); and record the chromatogram.

[0175] The formula for calculating the limit of detection is as follows:

[0176]

[0177] Results: As shown in Table 6. Figure 30 As shown, impurity SM 2a The detection limit was 0.0340 μg / ml, expressed as 0.003% in Z1, with a mean signal-to-noise ratio of 17.5; impurity SM 1d The detection limit was 0.1514 μg / ml, expressed as 0.015% in Z1, with a mean signal-to-noise ratio of 7.4; impurity Z 1c The detection limit was 0.0464 μg / ml, expressed as 0.005% in Z1, with a mean signal-to-noise ratio of 11.6; impurity Z 1b The detection limit was 0.0914 μg / ml, expressed as 0.009% in Z1, with a mean signal-to-noise ratio of 9.5; impurity Z 1a The detection limit concentration (LOC) was 0.0451 μg / ml, expressed as 0.005% in Z1, with a mean signal-to-noise ratio (SNR) of 10.2; the LOC of triphenylphosphine was 0.0342 μg / ml, expressed as 0.003% in Z1, with a mean SNR of 16.0; the LOC of impurity SM2 was 0.0769 μg / ml, expressed as 0.008% in Z1, with a mean SNR of 16.0; the LOC of impurity SM1 was 0.1371 μg / ml, expressed as 0.014% in Z1, with a mean SNR of 9.6; the LOC of Z1 was 0.1470 μg / ml, expressed as 0.015% in the test sample, with a mean SNR of 13.8; impurity Z... 1d The detection limit concentration was 0.0601 μg / ml, which is expressed as 0.006% in Z1, with a mean signal-to-noise ratio of 11.1. At the impurity detection limit concentration, the signal-to-noise ratio was greater than 3, meeting the requirements for related substance determination, indicating that impurities can be effectively detected at this level.

[0178] Table 6. Results of Limit of Detection

[0179]

[0180]

[0181] Example 4. Accuracy

[0182] (1) Accuracy ①

[0183] 1) Preparation of the test solution

[0184] Impurity Stock Solution ①: Accurately measure impurity Z under the "Specificity" section. 1c Impurities SM 2a 0.75 ml of each stock solution, impurity SM2, impurity SM 1d 2.5 ml of each stock solution, impurity Z 1a Impurity Z 1d 10 ml of each stock solution, 3.75 ml of triphenylphosphine oxide stock solution, impurity SM1, impurity Z 1b Place 7.5 ml of each stock solution into the same 50 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the final product.

[0185] Reference solution: Accurately measure 2.5 ml of impurity stock solution ①, place it in a 25 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.

[0186] Unspecified test solution: Accurately weigh 25.70 mg of this product, place it in a 25 ml volumetric flask, dissolve and dilute to the mark with diluent, and shake well to obtain the solution.

[0187] Self-control solution: Accurately measure 1.0 ml of the above unspecified test solution, place it in a 100 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.

[0188] Spiked test solution 1#: Accurately weigh 25.51 mg, 25.59 mg, and 25.16 mg of test sample Z1, and place them in separate 25 ml volumetric flasks. Add 2.0 ml of impurity stock solution ① to each flask, dissolve and dilute to the mark with diluent, and shake well. Prepare a self-control solution according to the same method.

[0189] Spiked test solution #2: Accurately weigh 25.80 mg, 25.37 mg, and 25.46 mg of test sample Z1, and place them in separate 25 ml volumetric flasks. Add 2.5 ml of impurity stock solution ① to each flask, then dissolve and dilute to the mark with diluent, and shake well. Prepare a self-control solution according to the same method.

[0190] Spiked test solution #3: Accurately weigh 25.25 mg, 25.81 mg, and 25.31 mg of test sample Z1, and place them in separate 25 ml volumetric flasks. Add 3.0 ml of impurity stock solution ① to each flask, then dissolve and dilute to the mark with diluent, and shake well. Prepare a self-control solution according to the same method.

[0191] 2. Detection

[0192] Accurately measure 10 μl each of the blank solvent, reference solution, and spiked test solution, and inject them into the liquid chromatograph. Perform detection under the chromatographic conditions of Example 1 and record the chromatograms. Calculate the impurity content and recovery rate using the external standard method and the self-comparison method with correction factors.

[0193] Results: The recovery rates of each impurity were calculated using the external standard method. Impurity SM 2a Z 1c Z 1b Z 1a Triphenylphosphine oxide, SM2, SM1, and Z 1d The average recoveries obtained were 100.6%, 99.3%, 101.0%, 99.7%, 100.0%, 100.3%, 98.0%, and 101.8%, respectively. The recoveries of each impurity were calculated using a self-comparison method with correction factors. Impurity SM... 2a Z 1c Z 1b Z 1a Triphenylphosphine oxide, SM2, SM1, and Z 1d The average recoveries obtained were 94.6%, 95.1%, 95.2%, 100.0%, 96.1%, 97.0%, 98.8%, and 97.8%, respectively. The recoveries of each impurity obtained by the two calculation methods were all between 90% and 108%, indicating that the accuracy of the determination of each impurity by the two methods met the requirements.

[0194] (2) Accuracy ②

[0195] 1) Preparation of the test solution

[0196] Impurities SM 1d Accuracy stock solution: Accurately weigh impurity SM 1d 25.37 mg was placed in a 25 ml volumetric flask, dissolved and diluted to the mark with acetonitrile, and shaken well to obtain the final product.

[0197] Impurity Stock Solution ②: Accurately measure impurity SM 1d Place 2.5 ml of the accuracy stock solution into a 50 ml volumetric flask, dilute to the mark with diluent, and shake well.

[0198] Reference solution: Accurately measure 2.5 ml of impurity stock solution ②, place it in a 25 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.

[0199] Unspecified test solution: Accurately weigh 25.32 mg of this product, place it in a 25 ml volumetric flask, dissolve and dilute to the mark with diluent, and shake well to obtain the solution.

[0200] Self-control solution: Accurately measure 1.0 ml of the above unspecified test solution, place it in a 100 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.

[0201] Test solution: Accurately weigh 5.32 mg of test sample Z1, place it in a 25 ml volumetric flask, dissolve and dilute to the mark with diluent, shake well, and the solution is ready.

[0202] Spiked test solution 1#: Accurately weigh 25.33 mg, 25.56 mg, and 25.66 mg of test sample Z1, and place them in separate 25 ml volumetric flasks. Add 2.0 ml of impurity stock solution ② to each flask, and then dilute to the mark with diluent. Shake well to obtain the solution. Prepare a self-control solution according to the same method.

[0203] Spiked test solution 2#: Accurately weigh 25.79 mg, 25.46 mg, and 25.56 mg of test sample Z1, and place them in separate 25 ml volumetric flasks. Add 2.5 ml of impurity stock solution ② to each flask, and then dilute to the mark with diluent. Shake well to obtain the solution. Prepare a self-control solution according to the same method.

[0204] Spiked test solution #3: Accurately weigh 25.49 mg, 25.45 mg, and 25.56 mg of test sample Z1, and place them in separate 25 ml volumetric flasks. Add 3.0 ml of impurity stock solution ② to each flask, and then dilute to the mark with diluent. Shake well to obtain the solution. Prepare a self-control solution according to the same method.

[0205] 2) Detection

[0206] Accurately measure 10 μl each of the blank solvent, reference solution, and spiked test solution, and inject them into the liquid chromatograph. Perform detection under the chromatographic conditions of Example 1 and record the chromatograms. Calculate the impurity content and recovery rate using the external standard method and the self-comparison method with correction factors.

[0207] Results: The recovery rate of impurities was calculated using the external standard method, and the impurity SM was... 1d The average recovery rate was 100.6%. The impurity recovery rate was calculated using a self-comparison method with correction factors. Impurity SM... 1d The average recovery rate was 102.1%. The recovery rates of impurities obtained by the two calculation methods were both between 90% and 108%, indicating that the accuracy of the determination of each impurity by the two methods met the requirements.

[0208] Example 5 Durability

[0209] Take the mixed solution 1 under the "Specificity" section, and test it under normal chromatographic conditions and the modified chromatographic conditions described below. After the instrument system stabilizes, record the resolution and relative retention time between each peak.

[0210] Normal conditions: flow rate (1.1 ml / min); column temperature (40 °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, initial mobile phase ratio: 69:31; the rest is the same as in Example 1.

[0211] The changes in chromatographic conditions included: (1) flow rate: 1.0 ml / min, 1.2 ml / min; (2) column temperature: 38℃, 42℃; (3) initial ratio of mobile phase: 71:29, 67:33; (4) pH of mobile phase: 3.8, 4.2; (5) salt concentration of mobile phase: 0.028 mol / L, 0.032 mol / L.

[0212] Results: See Tables 7 and 8. Figures 18-28 As shown, when the flow rate, column temperature, and mobile phase ratio fluctuate, the elution order of each impurity and the main component remains unchanged, and the relative retention time of each impurity varies within ±0.05 under all conditions; the inter-peak resolution of each component is greater than 1.5, indicating good robustness of the method.

[0213] Table 7. Results of Chromatographic Condition Robustness Test (Resolution)

[0214]

[0215] Table 8. Results of Chromatographic Condition Robustness Test (Retention Time / min)

[0216]

[0217] Example 6. Exploratory Experiment in the Method Establishment Phase

[0218] (1) Optimization of chromatographic conditions

[0219] The detection effects of different mobile phases were compared. The experimental conditions and detection results are shown in Table 9. It can be seen that with a three-phase mobile phase, multiple impurities could not be effectively separated.

[0220] Table 9

[0221]

[0222] (2) Optimization of chromatographic conditions

[0223] The chromatographic column, mobile phase, and gradient elution program were optimized, and the preliminary method was established. The specific conditions and detection results are shown in Table 10.

[0224] Table 10

[0225]

[0226]

[0227] (3) Optimization of chromatographic conditions

[0228] Further exploration of the phosphate:acetonitrile:methanol gradient elution method was conducted, and the established method was optimized. Specific conditions and detection results are shown in Table 11.

[0229] Table 11

[0230]

[0231]

Claims

1. A method for separating ruxolitinib mesylate intermediate Z1 and its related impurities using high performance liquid chromatography, characterized in that, The relevant impurities include impurity SM. 2a Impurities SM 1d Impurity Z 1c Impurity Z 1b Impurity Z 1a Triphenylphosphine oxide, impurity SM2, impurity SM1 and impurity Z 1d Any one or more of the following; the high-performance liquid chromatography method includes: using octadecylsilane-bonded silica gel as the column packing material, using potassium dihydrogen phosphate buffer as mobile phase A, and a mixed solution of methanol and acetonitrile as mobile phase B, and sequentially removing impurities SM by gradient elution. 2a Impurities SM 1d Impurity Z 1c Impurity Z 1b Impurity Z 1a Triphenylphosphine oxide, impurity SM2, impurity SM1, ruxolitinib mesylate intermediate Z1 and / or impurity Z 1d Separation is performed; in the mobile phase B, the volume ratio of methanol to acetonitrile is 40-60:60-40; the structural formula of the ruxolitinib mesylate intermediate Z1 is shown in Formula 1, and the impurity SM... 2a The structural formula is shown in Formula 2, and the impurity SM 1d The structural formula is shown in Formula 3, wherein the impurity Z 1c The structural formula is shown in Formula 4, and the impurity Z 1b The structural formula is shown in Formula 5, wherein the impurity Z 1a The structural formula of the impurity is shown in Formula 6, the structural formula of the triphenylphosphine oxide is shown in Formula 7, the structural formula of the impurity SM2 is shown in Formula 8, the structural formula of the impurity SM1 is shown in Formula 9, and the structural formula of the impurity Z... 1d The structural formula is shown in Equation 10; 2. The method according to claim 1, characterized in that, The concentration of the potassium dihydrogen phosphate buffer solution is 25-35 mmol / L, and the pH is 3-5.

3. The method according to claim 1, characterized in that, The gradient elution procedure is as follows: 0 minutes, set the volume ratio of mobile phase A to mobile phase B to 60-80:40-20; Set the volume ratio of mobile phase A to mobile phase B to 60-80:40-20 for 3 minutes. Set the volume ratio of mobile phase A to mobile phase B to 35-50:65-50 for 15 minutes. 55 minutes, set the volume ratio of mobile phase A to mobile phase B to be 20-30:80-70; For 65 minutes, set the volume ratio of mobile phase A to mobile phase B to be 20-30:80-70; 66 minutes, set the volume ratio of mobile phase A to mobile phase B to be 60-80:40-20; Set the volume ratio of mobile phase A to mobile phase B to 60-80:40-20 for 75 minutes.

4. The method according to claim 1, characterized in that, The flow rate is 1.0-1.5 ml / min; the column temperature is 35-45℃.

5. A method for identifying ruxolitinib mesylate intermediate Z1 and its related impurities, characterized in that, The intermediate Z1 of ruxolitinib mesylate and any one or more of the related impurities are separated by the method described in any one of claims 1-4; after separation, the sample is detected by a detector with a detection wavelength of 210-230 nm; by comparing the chromatographic behavior of the test sample and the reference sample, it is determined whether the sample of intermediate Z1 of ruxolitinib mesylate contains related impurities.

6. The method according to claim 5, characterized in that, The retention times of each component, from shortest to longest, are as follows: Impurity SM 2a Impurities SM 1d Impurity Z 1c Impurity Z 1b Impurity Z 1a Triphenylphosphine oxide, impurity SM2, impurity SM1, ruxolitinib mesylate intermediate Z1, impurity Z 1d .

7. The method according to claim 5, characterized in that, Using ruxolitinib mesylate intermediate Z1 as a reference peak, when the relative retention time is 0.20 ± 0.05, it is identified as impurity SM. 2a When the relative retention time is 0.25 ± 0.05, it is determined to be impurity SM. 1d When the relative retention time is 0.35 ± 0.05, it is determined to be impurity Z. 1c When the relative retention time is 0.44 ± 0.05, it is determined to be impurity Z. 1b When the relative retention time is 0.52 ± 0.05, it is determined to be impurity Z. 1a When the relative retention time is 0.61±0.05, it is identified as triphenylphosphine oxide. When the relative retention time is 0.73±0.05, it is identified as impurity SM2; when the relative retention time is 0.78±0.05, it is identified as impurity SM1; when the relative retention time is 1.03±0.05, it is identified as impurity Z. 1d The retention time of the ruxolitinib mesylate intermediate Z1 was 29.3 ± 0.5 min.

8. A method for detecting the content of relevant impurities in ruxolitinib mesylate intermediate Z1, characterized in that, Includes the following steps: (1) The intermediate Z1 of ruxolitinib mesylate and its related impurities are separated and detected by the method according to any one of claims 5-7, and a chromatogram is obtained; (2) Based on the chromatogram obtained in step (1), calculate the content of each impurity using the external standard method or the principal component self-comparison method with correction factor.

9. The method according to claim 8, characterized in that, Before separation, a mixed solution of methanol and acetonitrile was used as a solvent to prepare the test solution; the volume ratio of methanol to acetonitrile was 50:

50.

10. The method according to claim 8, characterized in that, The impurity Z 1a The correction factor is 0.8; the impurity Z 1b The correction factor is 1.8; the impurity Z 1c The correction factor is 0.7; the impurity Z 1d The correction factor for impurity SM1 is 0.5; the correction factor for impurity SM2 is 3.0; the correction factor for impurity SM3 is 0.7; the correction factor for impurity SM4 is 0.

5. 1d The correction factor is 2.1; the impurity SM 2a The correction factor is 0.3; the correction factor for triphenylphosphine oxide is 0.5.

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