Separation and detection method for related substances of initial material of rucotinib

Abstract

The invention discloses a method for separating and detecting related substances of a starting material 3-cyclopentyl-3-hydrazinopropionitrile-tosilate of rukotinib, which comprises the following steps of: adopting a mixed type chromatographic column in which a long carbon chain ligand is embedded into an acidic ion pair group as a filler, taking a mixed solution of an acidic additive, water and acetonitrile as a mobile phase, and adopting an electrical conductivity detector to detect the related substances of the starting material 3-cyclopentyl-3-hydrazinopropionitrile-tosilate. According to the method, the analysis of the related substances of the starting material 3-cyclopentyl-3-hydrazinopropionitrile-tosilate of the rukotinib is realized. The method disclosed by the invention is simple to operate, low in cost and short in analysis time, and a good guarantee is provided for quality control of the starting material 3-cyclopentyl-3-hydrazinopropionitrile-tosilate of the rukotinib.

Description

Technical Field

[0001] The invention belongs to the field of drug analysis methods, and specifically relates to a method for separating and detecting related substances of 3-cyclopentyl-3-hydrazinopropionitrile-p-toluenesulfonate, a starting material of ruxolitinib, by using high performance liquid chromatography. Background Art

[0002] Ruxolitinib is a targeted drug for the JAK-signaling and transcription activator protein signaling pathway. It was first used to treat myelofibrosis, significantly improving patients' symptoms, shrinking spleens and prolonging survival. Ruxolitinib inhibits cell proliferation, induces apoptosis of malignant cells and reduces the production of proinflammatory cytokines by inhibiting JAK-induced signal transduction and transcription activator phosphorylation. It has been widely used in basic and clinical research on a variety of blood system diseases, such as myeloproliferative neoplasms, acute lymphoblastic leukemia, graft-versus-host disease, hemophagocytic syndrome, aplastic anemia, multiple myeloma, etc.

[0003] The chemical name of ruxolitinib is (3R)-3-cyclopentyl-3-4-[(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazolyl]propionitrile {(R)-3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrile}, with a molecular formula of C17H18N6 and a molecular weight of 306.37. The structural formula is shown in Table 1.

[0004] 3-Cyclopentyl-3-hydrazineylpropanenitrile 4-methylbenzenesulfonic acid salt dihydrate has a molecular formula of C15H23O3N3S and a molecular weight of 325.43. The structural formula is shown in Table 1. 3-Cyclopentyl-3-hydrazineylpropanenitrile can be used to construct the main structure of ruxolitinib, but its stability in the free state is poor. Therefore, it is salified with p-toluenesulfonic acid and used as the starting material for the synthesis of ruxolitinib.

[0005] Table 1 Related structural formulas

[0006] At present, there is no literature report on the analysis method of related substances in 3-cyclopentyl-3-hydrazinopropionitrile-p-toluenesulfonate. Therefore, it is very necessary to develop an analysis method for related substances in 3-cyclopentyl-3-hydrazinopropionitrile-p-toluenesulfonate for the quality control of 3-cyclopentyl-3-hydrazinopropionitrile-p-toluenesulfonate and ruxolitinib products. Summary of the invention

[0007] The object of the present invention is to provide a method for separating and detecting related substances of 3-cyclopentyl-3-hydrazinopropionitrile-p-toluenesulfonate, a starting material of ruxolitinib, which can effectively separate the main component and its impurity I, and effectively control the quality of the starting material.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions.

[0009] The invention provides a method for separating and detecting related substances of 3-cyclopentyl-3-hydrazinopropionitrile-p-toluenesulfonate, a starting material of ruxolitinib. The method is a high performance liquid chromatography method, adopts a mixed chromatographic column with a long carbon chain ligand embedded in an acidic ion pair group as a filler, and takes a mixed solution of an acidic aqueous solution and acetonitrile as a mobile phase.

[0010] The chromatographic column described in this method is SiELC Primesep 100 (250 mm*4.6 mm, 3 μm).

[0011] Furthermore, in the above method of the present invention, the high performance liquid chromatography detector is a conductivity detector.

[0012] Furthermore, in the above method of the present invention, the acidic aqueous solution is a trifluoroacetic acid aqueous solution or a perchloric acid aqueous solution, preferably a perchloric acid aqueous solution.

[0013] Furthermore, in the above method of the present invention, in the acidic aqueous solution, the volume ratio of acid to water is 0.1% to 0.3%, preferably 0.3%.

[0014] Furthermore, in the above method of the present invention, the volume ratio of the acidic aqueous solution to acetonitrile in the mobile phase is (58-62):(42-38), preferably 58:42.

[0015] Furthermore, in the above method of the present invention, the temperature of the chromatographic column is 28-32°C, preferably 30°C.

[0016] Furthermore, in the above method of the present invention, the flow rate of the mobile phase is 0.9-1.1 ml / min, preferably 1.0 / min.

[0017] In a specific embodiment, the method of the present invention comprises the following steps: Preparation of system suitability solution: Take an appropriate amount of ruxolitinib starting material 3-cyclopentyl-3-hydrazinopropionitrile-p-toluenesulfonate and impurity I reference substance, add mobile phase to dissolve and dilute to prepare a system suitability solution; Preparation of test solution: Take an appropriate amount of ruxolitinib starting material 3-cyclopentyl-3-hydrazinopropionitrile-p-toluenesulfonate, add mobile phase to dissolve and dilute to prepare a test solution; Take the system suitability solution and the test solution, inject them into the high performance liquid chromatograph for detection, and record the chromatogram.

[0018] The method of the present invention uses a conductivity detector, a mixed chromatographic column with a long carbon chain ligand embedded in an acidic ion pair group as a filler, and a system with a mixed solution of an acidic aqueous solution and acetonitrile as a mobile phase, to achieve separation and detection of 3-cyclopentyl-3-hydrazine propionitrile-p-toluenesulfonate, a starting material of ruxolitinib, and impurity I. The method of the present invention does not require derivatization, has a simple operation process, a short analysis time, low cost, and good accuracy, and can more conveniently and accurately achieve quality control of the production process of 3-cyclopentyl-3-hydrazine propionitrile-p-toluenesulfonate and ruxolitinib products, thereby improving the quality of the final product. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the HPLC chromatogram of the blank solution of Example 2; Figure 2 This is the HPLC chromatogram of the system suitability solution of Example 2; Figure 3 HPLC chromatogram of the solution for locating impurity I in Example 2; Figure 4 This is the HPLC chromatogram of the blank test solution of Example 2; Figure 5 This is the principal component linear investigation diagram of Example 3; Figure 6 This is the linear investigation diagram of impurity I in Example 3; Figure 7 The HPLC chromatogram of the quantitative limit of the main component and impurity I in Example 4 is shown; Figure 8 The HPLC chromatogram of the test solution of Comparative Example 1 is shown. DETAILED DESCRIPTION

[0020] The following examples are used to further illustrate and understand the spirit of the present invention, but are not intended to limit the scope of protection of the present invention in any way.

[0021] In the following examples, the instruments and reagents used are as follows: Instrument: Shimadzu LC-20AD high performance liquid chromatograph (equipped with conductivity detector) Reagents: Perchloric acid was AR grade (70%-72%), formic acid was LC-MS grade, trifluoroacetic acid and acetonitrile were chromatography grade, and water was ultrapure water.

[0022] 3-Cyclopentyl-3-hydrazinopropionitrile-p-toluenesulfonate and impurity I reference substances were both produced by Chongqing Yaoyou Pharmaceutical Co., Ltd. and calibrated by Chongqing Southwest University.

[0023] Example 1 Screening of mobile phase

[0024] 1.1 Detection effect test of different types of acidic aqueous solutions

[0025] Chromatographic conditions: Mobile phase 1: 0.2% trifluoroacetic acid aqueous solution-acetonitrile (50:50, v / v) Mobile phase 2: 0.5% formic acid aqueous solution-acetonitrile (50:50, v / v) Mobile phase 3: 0.2% perchloric acid aqueous solution-acetonitrile (50:50, v / v) Chromatographic instrument: Shimadzu LC-20AD high performance liquid chromatograph Chromatographic column: SiELC Primesep 100 (250 mm*4.6 mm, 3 μm) Detector: Conductivity detector (Polarity: -; Gain: 1) Flow rate: 1.0ml / min Injection volume: 25 μl Column temperature: 30°C Solvent: Mobile phase.

[0026] Experimental steps: (1) Preparation of mobile phase 0.2% trifluoroacetic acid aqueous solution-acetonitrile (50:50, v / v): Take 1000ml of ultrapure water, add 2ml of trifluoroacetic acid, mix well to obtain 0.2% trifluoroacetic acid aqueous solution; take 500ml of 0.2% trifluoroacetic acid aqueous solution, add 500ml of acetonitrile, mix well to obtain; 0.5% formic acid aqueous solution-acetonitrile (50:50, v / v): Take 1000ml of ultrapure water, add 5ml of formic acid, mix thoroughly, and obtain 0.5% formic acid aqueous solution: Take 500ml of 0.5% formic acid aqueous solution, add 500ml of acetonitrile, mix well, and obtain; 0.2% perchloric acid aqueous solution-acetonitrile (50:50, v / v): Take 1000 ml of ultrapure water, add 2 ml of perchloric acid, mix thoroughly to obtain 0.2% perchloric acid aqueous solution; take 500 ml of 0.2% perchloric acid aqueous solution, add 500 ml of acetonitrile, mix thoroughly to obtain.

[0027] (2) Preparation of the test solution: Take an appropriate amount of ruxolitinib starting material 3-cyclopentyl-3-hydrazinopropionitrile-p-toluenesulfonate, add mobile phase to dissolve and quantitatively dilute to make a solution containing 2.5 mg of 3-cyclopentyl-3-hydrazinopropionitrile-p-toluenesulfonate per 1 ml, which is used as the test solution.

[0028] (3) Detection: Inject the above test sample solution into a high performance liquid chromatograph and perform the measurement under three different mobile phase conditions, and record the chromatograms.

[0029] Table 2 Detection of different types of acidic additives

[0030] Conclusion: In order to keep the pH of the acidic aqueous solutions under investigation similar, 0.2% trifluoroacetic acid aqueous solution, 0.5% formic acid aqueous solution and 0.2% perchloric acid aqueous solution were selected for the experiment. The above results show that no peaks appear in the formic acid aqueous solution system, and the perchloric acid aqueous solution system is better than the trifluoroacetic acid aqueous solution system in terms of response and tailing, so the perchloric acid aqueous solution is used.

[0031] 1.2 Detection effect test of different acidic aqueous solution ratios

[0032] Chromatographic conditions: Mobile phase 1: 0.1% perchloric acid aqueous solution-acetonitrile (60:40, v / v) Mobile phase 2: 0.3% perchloric acid aqueous solution-acetonitrile (60:40, v / v) Chromatographic instrument: Shimadzu LC-20AD high performance liquid chromatograph Chromatographic column: SiELC Primesep 100 (250 mm*4.6 mm, 3 μm) Detector: Conductivity detector (Polarity: -; Gain: 1) Flow rate: 1.0ml / min Injection volume: 25 μl Column temperature: 30°C Solvent: Mobile phase.

[0033] Experimental steps: (1) Preparation of mobile phase 0.1% perchloric acid aqueous solution-acetonitrile (60:40, v / v): Take 1000ml of ultrapure water, add 1ml of perchloric acid, mix thoroughly to obtain 0.1% perchloric acid aqueous solution; take 600ml of 0.1% perchloric acid aqueous solution, add 400ml of acetonitrile, mix thoroughly to obtain; 0.3% perchloric acid aqueous solution-acetonitrile (60:40, v / v): Take 1000 ml of ultrapure water, add 3 ml of perchloric acid, mix thoroughly to obtain 0.3% perchloric acid aqueous solution; take 600 ml of 0.3% perchloric acid aqueous solution, add 400 ml of acetonitrile, mix thoroughly to obtain.

[0034] (2) Preparation of system suitability solution: Take appropriate amount of ruxolitinib starting material 3-cyclopentyl-3-hydrazinopropionitrile-p-toluenesulfonate and impurity I reference substance, add mobile phase to dissolve and quantitatively dilute to make a solution containing 20 mg of 3-cyclopentyl-3-hydrazinopropionitrile-p-toluenesulfonate and 0.1 mg of impurity I per 1 ml, which is used as the system suitability solution.

[0035] (3) Detection: Inject the above system suitability solution into a high performance liquid chromatograph and perform the measurement under two different mobile phase conditions, and record the chromatograms.

[0036] Table 3 Detection results of different acid additive ratios

[0037] Conclusion: Considering that too high a concentration of perchloric acid will damage the chromatographic column, only the ratio of perchloric acid aqueous solution in the range of 0.1%-0.3% was investigated. The results showed that in the 0.3% perchloric acid aqueous solution system, the peak-to-valley ratio of the main peak to impurity I was 2.9, which was better than the 0.1% level, so the 0.3% perchloric acid aqueous solution was selected.

[0038] 1.3 Detection effect test of different organic phase ratios

[0039] Mobile phase 1: 0.3% perchloric acid aqueous solution-acetonitrile (62:38, v / v) Mobile phase 2: 0.3% perchloric acid aqueous solution-acetonitrile (60:40, v / v) Mobile phase 3: 0.3% perchloric acid aqueous solution-acetonitrile (58:42, v / v) Chromatographic instrument: Shimadzu LC-20AD high performance liquid chromatograph Chromatographic column: SiELC Primesep 100 (250 mm*4.6 mm, 3 μm) Detector: Conductivity detector (Polarity: -; Gain: 1) Flow rate: 1.0ml / min Injection volume: 25 μl Column temperature: 30°C Solvent: Mobile phase.

[0040] Experimental steps: (1) Preparation of mobile phase 0.3% perchloric acid aqueous solution-acetonitrile (62:38, v / v): Take 1000ml of ultrapure water, add 3ml of perchloric acid, mix thoroughly to obtain 0.3% perchloric acid aqueous solution; Take 620ml of 0.3% perchloric acid aqueous solution, add 380ml of acetonitrile, mix thoroughly to obtain; 0.3% perchloric acid aqueous solution-acetonitrile (60:40, v / v): Take 1000ml of ultrapure water, add 3ml of perchloric acid, mix thoroughly to obtain 0.3% perchloric acid aqueous solution; Take 600ml of 0.3% perchloric acid aqueous solution, add 400ml of acetonitrile, mix thoroughly to obtain; 0.3% perchloric acid aqueous solution-acetonitrile (58:42, v / v): Take 1000 ml of ultrapure water, add 3 ml of perchloric acid, mix thoroughly to obtain 0.3% perchloric acid aqueous solution; take 580 ml of 0.3% perchloric acid aqueous solution, add 420 ml of acetonitrile, mix thoroughly to obtain.

[0041] (2) Preparation of system suitability solution: Take appropriate amount of ruxolitinib starting material 3-cyclopentyl-3-hydrazinopropionitrile-p-toluenesulfonate and impurity I reference substance, add mobile phase to dissolve and quantitatively dilute to make a solution containing 20 mg of 3-cyclopentyl-3-hydrazinopropionitrile-p-toluenesulfonate and 0.1 mg of impurity I per 1 ml, which is used as the system suitability solution.

[0042] (3) Detection: Inject the above system suitability solution into a high performance liquid chromatograph and perform the measurement under three different mobile phase conditions. Record the chromatograms.

[0043] Table 4 Detection results of different organic phase ratios

[0044] Conclusion: The above results show that under the conditions of 0.3% perchloric acid aqueous solution-acetonitrile (58:42, v / v), the peak-to-valley ratio of the main peak to impurity I is the largest.

[0045] Example 2 System Applicability and Specificity Experiment

[0046] Chromatographic conditions: Mobile phase: 0.3% perchloric acid aqueous solution-acetonitrile (60:40, v / v) Chromatographic instrument: Shimadzu LC-20AD high performance liquid chromatograph Chromatographic column: SiELC Primesep 100 (250 mm*4.6 mm, 3 μm) Detector: Conductivity detector (Polarity: -; Gain: 1) Flow rate: 1.0ml / min Injection volume: 25 μl Column temperature: 30°C Solvent: Mobile phase.

[0047] (1) Solution preparation: Blank solution: Take an appropriate amount of p-toluenesulfonic acid, add mobile phase to dissolve and quantitatively dilute to make a solution containing 10 mg of p-toluenesulfonic acid per 1 ml as the blank solution; System suitability solution: Take an appropriate amount of ruxolitinib starting material 3-cyclopentyl-3-hydrazinopropionitrile-p-toluenesulfonate and impurity I reference substance, add mobile phase to dissolve and quantitatively dilute to prepare a solution containing 20 mg of 3-cyclopentyl-3-hydrazinopropionitrile-p-toluenesulfonate and 0.1 mg of impurity I per 1 ml, as the system suitability solution; Impurity I localization solution: Take an appropriate amount of impurity I reference substance, add mobile phase to dissolve and quantitatively dilute to make a solution containing 0.1 mg of impurity I per 1 ml, as the impurity I localization solution. Blank test solution: Take an appropriate amount of ruxolitinib starting material 3-cyclopentyl-3-hydrazinopropionitrile-p-toluenesulfonate, add mobile phase to dissolve and quantitatively dilute to make a solution containing 20 mg of 3-cyclopentyl-3-hydrazinopropionitrile-p-toluenesulfonate per 1 ml, as the blank test solution.

[0048] (2) Detection: Take 25 μl of each of the blank solution, system suitability solution, impurity I location solution and blank test solution and inject them into the HPLC for determination. Record the chromatogram.

[0049] Conclusion: From Figure 1~Figure 4 The results show that in the system suitability solution, the peak-to-valley ratio of the main peak to impurity I is 2.9, and both the blank solution and the blank test sample have no obvious interference with the detection.

[0050] Example 3 Linearity experiment

[0051] Chromatographic conditions: Same as Example 2

[0052] (1) Solution preparation: Impurity I reference substance stock solution: Take about 25 mg of impurity I reference substance, weigh accurately, place in a 25 ml volumetric bottle, add solvent to dissolve and dilute to scale, shake well to obtain. (Contains impurity I concentration of about 1 mg / ml) Standard series solutions: According to the preparation method in Table 5, prepare a series of main component standard solutions with concentrations of approximately 0.02, 0.05, 0.1, 0.2, 0.4, 1, 20, and 24 mg / ml, and a series of impurity I standard solutions with concentrations of approximately 20, 25, 50, 100, 200, and 400 μg / ml.

[0053] Table 5 Linear solution preparation method

[0054] (2) Detection: Take the above-mentioned standard series solutions of various concentrations and inject them into high performance liquid chromatography for measurement, and record the chromatogram.

[0055] According to the above chromatographic conditions, the peak area was analyzed and recorded, and the peak area was linearly regressed against the concentration to obtain the regression curve. The results are shown in Table 6 and Table 7. The regression curve is shown in Figure 5 , Figure 6The principal component regression equation is Y=571.2X+12729 (Y is the peak area, X is the concentration in μg / ml), the correlation coefficient r is 0.9990, and the linearity is good in the concentration range of 19.77μg / ml~23738μg / ml; the impurity I regression equation is Y=598.7X-4569 (Y is the peak area, X is the concentration in μg / ml), the correlation coefficient r is 0.9995, and the linearity is good in the concentration range of 20.38μg / ml~407.7μg / ml.

[0056] Table 6 Principal component standard curve

[0057] Table 7 Standard curve of impurity I

[0058] Example 4 Quantitation Limit and Detection Limit Experiment

[0059] Chromatographic conditions: Same as Example 2

[0060] (1) Solution preparation: Quantitative limit solution: Take appropriate amount of 3-cyclopentyl-3-hydrazinopropionitrile-p-toluenesulfonate reference substance and impurity I reference substance, add mobile phase to dissolve and quantitatively dilute to make a solution containing 0.02 mg of 3-cyclopentyl-3-hydrazinopropionitrile-p-toluenesulfonate and impurity I per 1 ml, as the quantitative limit solution, prepare 6 quantitative limit solutions in parallel for detection; Detection limit solution: Take appropriate amount of 3-cyclopentyl-3-hydrazinopropionitrile-p-toluenesulfonate reference substance and impurity I reference substance, add mobile phase to dissolve and quantitatively dilute to make a solution containing 0.01 mg of 3-cyclopentyl-3-hydrazinopropionitrile-p-toluenesulfonate and impurity I per 1 ml, as the detection limit solution;

[0061] (2) Detection: Take the above-mentioned quantitative limit solution and detection limit solution and inject them into the high performance liquid chromatograph for measurement, and record the chromatogram.

[0062] Calculate the relative standard deviation of the peak area. The results are shown in Table 8. The chromatogram of the quantitative limit is shown in Figure 7 .

[0063] Table 8 Results of quantitation limit and detection limit

[0064] Example 5 Accuracy Experiment

[0065] Chromatographic conditions: Same as Example 2

[0066] Quantitative method: Peak area normalization method with correction factor

[0067] (1) Solution preparation: Accuracy solution: Take an appropriate amount of ruxolitinib starting material 3-cyclopentyl-3-hydrazinopropionitrile-p-toluenesulfonate and impurity I reference substance, add mobile phase to dissolve and quantitatively dilute to make a solution containing 20 mg of 3-cyclopentyl-3-hydrazinopropionitrile-p-toluenesulfonate and 0.1 mg of impurity I per 1 ml, as the accuracy solution, prepare 6 copies in parallel; Blank test solution: Take an appropriate amount of ruxolitinib starting material 3-cyclopentyl-3-hydrazinopropionitrile-p-toluenesulfonate, add mobile phase to dissolve and quantitatively dilute to make a solution containing 20 mg of 3-cyclopentyl-3-hydrazinopropionitrile-p-toluenesulfonate per 1 ml as the blank test solution, and prepare 3 copies in parallel.

[0068] (2) Detection: Take the above accuracy solutions and inject them into high performance liquid chromatography for measurement, and record the chromatogram.

[0069] The contents of impurity I in the starting material of ruxolitinib, 3-cyclopentyl-3-hydrazinopropionitrile-p-toluenesulfonate, were calculated respectively. The results are shown in Table 9.

[0070] It can be seen from Table 9 that the average recovery rate of 6 accuracy solutions of impurity I is 86%, and the recovery RSD is 4.0%. The accuracy of this method is good.

[0071] Table 9 Accuracy test results

[0072] Example 6 Repeatability experiment.

[0073] Chromatographic conditions: same as in Example 2; Quantitative method: Peak area normalization method with correction factor

[0074] (1) Preparation of reproducible solution: Take an appropriate amount of ruxolitinib starting material 3-cyclopentyl-3-hydrazinopropionitrile-p-toluenesulfonate, add mobile phase to dissolve and quantitatively dilute to make a solution containing 20 mg of 3-cyclopentyl-3-hydrazinopropionitrile-p-toluenesulfonate per 1 ml. Prepare 6 replicates as the reproducible solution.

[0075] (2) Detection: Take the above reproducible solution and inject it into the high performance liquid chromatograph for measurement and record the chromatogram.

[0076] The impurity I, the maximum unknown single impurity, the total impurity content and the chromatographic purity in the starting material of ruxolitinib 3-cyclopentyl-3-hydrazinopropionitrile-p-toluenesulfonate were calculated respectively. The results are shown in Table 10.

[0077] It can be seen from Table 10 that the detection method provided by the present invention has high precision.

[0078] Table 10 Repeatability test results

[0079] Example 7 Intermediate Precision Experiment

[0080] Chromatographic conditions: same as in Example 2; Quantitative method: Peak area normalization method with correction factor added.

[0081] (1) Preparation of reproducible solution: Take an appropriate amount of ruxolitinib starting material 3-cyclopentyl-3-hydrazinopropionitrile-p-toluenesulfonate, add mobile phase to dissolve and quantitatively dilute to make a solution containing 20 mg of 3-cyclopentyl-3-hydrazinopropionitrile-p-toluenesulfonate per 1 ml. Prepare 6 replicates as the reproducible solution.

[0082] (2) Detection: Take the above reproducible solution and inject it into the high performance liquid chromatograph for measurement and record the chromatogram.

[0083] Different experimenters used different instruments on different dates to measure the impurity I, the maximum unknown single impurity, the total impurity content and the chromatographic purity in 6 replicate solutions in parallel, and calculated the results 12 times. The results are shown in Table 11. The results show that the intermediate precision of this method is good.

[0084] Table 11 Intermediate precision test results

[0085] Example 8 Durability Test

[0086] The robustness of the method was investigated by changing parameters such as column temperature and chromatographic column batches. Only one parameter was changed each time, with a parameter range of column temperature ±2°C and two batches of chromatographic columns. The other chromatographic conditions were the same as in Example 2.

[0087] (1) Solution preparation: Blank solution: Take an appropriate amount of p-toluenesulfonic acid, add mobile phase to dissolve and quantitatively dilute to make a solution containing 10 mg of p-toluenesulfonic acid per 1 ml as the blank solution; System suitability solution: Take an appropriate amount of ruxolitinib starting material 3-cyclopentyl-3-hydrazinopropionitrile-p-toluenesulfonate and impurity I reference substance, add mobile phase to dissolve and quantitatively dilute to make a solution containing 20 mg of 3-cyclopentyl-3-hydrazinopropionitrile-p-toluenesulfonate and 0.1 mg of impurity I per 1 ml as the system suitability solution.

[0088] (2) Detection: Take the blank solution and system suitability solution mentioned above and inject them into the high performance liquid chromatograph for measurement and record the chromatogram.

[0089] The test results show that the blank solution does not interfere with the determination, and the peak-to-valley ratios of the main peak and impurity I are both greater than 1.5. The results are shown in Table 12.

[0090] Table 12 Durability test results

[0091] Comparative Example 1 Comparison of different types of chromatographic columns Mobile phase: 0.1% trifluoroacetic acid aqueous solution-acetonitrile (60:40, v / v) Chromatographic instrument: Shimadzu LC-20AD high performance liquid chromatograph Chromatographic column: BioBasic SCX (150mm*4.6mm, 5μm) Detector: Conductivity detector (Polarity: -; Gain: 1) Flow rate: 0.8ml / min Injection volume: 25 μl Column temperature: 40°C Solvent: Mobile phase

[0092] (1) Preparation of test solution: Take an appropriate amount of ruxolitinib starting material 3-cyclopentyl-3-hydrazinopropionitrile-p-toluenesulfonate, add mobile phase to dissolve and quantitatively dilute to make a solution containing 20 mg of 3-cyclopentyl-3-hydrazinopropionitrile-p-toluenesulfonate per 1 ml.

[0093] (2) Detection: Take 25 μl of the test solution and inject it for measurement.

[0094] Typical spectra Figure 8 ,The results show that there are impurities behind the main peak and the method is not applicable.

Claims

1. A method for separating and detecting related substances of 3-cyclopentyl-3-hydrazinopropionitrile-p-toluenesulfonate, a starting material of ruxolitinib, the method being a high performance liquid chromatography method, characterized in that A mixed chromatographic column with a long carbon chain ligand embedded in an acidic ion pair group as filler is used, and a mixed solution of an acidic aqueous solution and acetonitrile is used as a mobile phase. The related substance is an impurity 1 shown in the following formula 。 2. The separation and detection method according to claim 1, wherein the chromatographic column is SiELC Primesep 100.

3. The separation and detection method according to claim 1, wherein the detector is a conductivity detector.

4. The separation and detection method according to claim 1, wherein the acidic aqueous solution is a trifluoroacetic acid aqueous solution or a perchloric acid aqueous solution, preferably a perchloric acid aqueous solution.

5. The separation and detection method according to claim 1, wherein in the acidic aqueous solution, the volume ratio of acid to water is 0.1% to 0.3%, preferably 0.3%.

6. The separation and detection method according to claim 1, wherein the volume ratio of the acidic aqueous solution to acetonitrile in the mobile phase is (58-62): (42-38), preferably 58:

42.

7. The separation and detection method according to claim 1, wherein the temperature of the chromatographic column is 28-32°C, preferably 30°C.

8. The separation and detection method according to claim 1, wherein the flow rate of the mobile phase is 0.9-1.1 ml / min, preferably 1.0 ml / min.

9. The method according to any one of claims 1 to 8, characterized in that: The method comprises the following steps: 1) Preparation of system suitability solution: Take appropriate amount of ruxolitinib starting material 3-cyclopentyl-3-hydrazinopropionitrile-p-toluenesulfonate and impurity I reference substance, add mobile phase to dissolve and dilute to prepare system suitability solution; 2) Preparation of test solution: Take an appropriate amount of ruxolitinib starting material 3-cyclopentyl-3-hydrazinopropionitrile-p-toluenesulfonate, add mobile phase to dissolve and dilute to prepare a test solution; 3) Take the system suitability solution and the test sample solution, inject them into the high performance liquid chromatograph for detection, and record the chromatogram.

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