Method for detecting position isomer of rucotinib phosphate
By using reverse phase chromatography in drug analysis and using specific chromatographic columns and mobile phase conditions, efficient separation and detection of position isomer impurity A in ructinib phosphate is achieved, solving the problem of difficult detection in the prior art and ensuring the control of drug quality.
Patent Information
- Application Number
- CN202510530689.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-25
- Publication Date
- 2025-06-13
AI Technical Summary
The prior art is difficult to effectively detect and isolate the position isomer impurity A in rucortinib phosphate, making it difficult to evaluate the impurity content and control its level during drug synthesis.
Reverse phase chromatography is used, and the specific conditions include the use of phenylhexyl bonded silica gel as the filler, perchloric acid solution and methanol as the mobile phase, gradient elution mode, combined with an ultraviolet detector, to achieve effective separation and detection of rucortinib phosphate and its position isomers.
Accurate detection of impurity A in rucotinib phosphate is achieved, with the detection limit reaching 0.02%, high sensitivity and good accuracy, and effective drug quality control to ensure product quality.
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Abstract
Description
Technical Field
[0001] The present invention relates to the field of chemical drug analysis methods, and particularly to a detection method for position isomers of ruxolitinib phosphate. Background Art
[0002] Ruxolitinib phosphate tablets are used for the treatment of acute graft-versus-host disease in patients 12 years of age and older who are refractory to corticosteroids or other systemic therapies, and its chemical formula is C 17 H 21 N 6 O 4 P, and its structural formula is as follows:
[0003] Impurity A is an important position isomer impurity of ruxolitinib, which may be generated during the preparation process of ruxolitinib phosphate, and its structural formula is as follows:
[0004] Ruxolitinib and its position isomer (hereinafter referred to as Impurity A) only differ in the substitution position of cyclopentylpropionitrile on ruxolitinib, and they have similar physical and chemical properties and similar retention behaviors in chromatography, making separation somewhat difficult. Currently, there is no literature report on the detection method for Impurity A in ruxolitinib phosphate. During the synthesis process, it is necessary to evaluate the content of Impurity A in ruxolitinib phosphate to formulate an appropriate impurity control strategy. Therefore, the development of a determination method for Impurity A in ruxolitinib phosphate is of great significance for process parameter selection and drug quality evaluation. Summary of the Invention
[0005] To achieve the object of the present invention, the present invention provides a detection method for the position isomers of ruxolitinib phosphate (i.e., Impurity A), which uses a reverse-phase chromatography method with good specificity, high accuracy, high sensitivity, simple operation, and rapidity.
[0006] To achieve the above-mentioned invention object, the present invention provides the following solutions.
[0007] The present invention provides a detection method for determining the position isomers of ruxolitinib phosphate, which uses a reverse-phase chromatography method, and is characterized in that the chromatographic conditions of the reverse-phase chromatography method include: Chromatographic column: a chromatographic column filled with phenylhexyl-bonded silica gel; Mobile phase A: perchloric acid solution; Mobile phase B: methanol; Flow rate: 1.0 ml / min; Detection wavelength: 265 nm; Column temperature: 40 °C; Detector: ultraviolet detector; Elution mode: gradient elution Among them, the ruxolitinib position isomer is impurity A.
[0008] Preferably, in the detection method of the present invention above, mobile phase A is a 0.05% perchloric acid solution; Preferably, in the detection method of the present invention above, the chromatographic column filled with phenylhexyl-bonded silica gel is Phenomenex Luna phenyl-Hexyl, more preferably Phenomenex Luna phenyl-Hexyl (4.6 mm × 150 mm, 3 μm).
[0009] Furthermore, in the detection method of the present invention above, the gradient elution program is as follows:
[0010] Furthermore, in the detection method of the present invention above, the method includes the following steps: (1) Preparation of ruxolitinib phosphate reference solution: Take an appropriate amount of ruxolitinib phosphate reference substance and prepare a reference solution with acetonitrile-water (40:60 v / v); (2) Preparation of impurity A localization solution: Take an appropriate amount of impurity A reference substance and prepare an impurity A localization solution with acetonitrile-water (40:60 v / v); (3) Preparation of test solution: Take an appropriate amount of the test substance and prepare a test solution with acetonitrile-water (40:60 v / v); (4) Take the ruxolitinib phosphate reference solution (inject continuously for 6 needles), the impurity A localization solution and the test solution, and inject them into a high performance liquid chromatograph for detection.
[0011] Technical effects: In the detection method of the present invention, the Phenomenex Luna phenyl-Hexyl chromatographic column is selected. The packing of this chromatographic column is phenylhexyl-bonded silica gel. Through p-p interaction, dipole interaction, hydrophobic interaction and shape selectivity, it can effectively separate ruxolitinib phosphate and its position isomer (impurity A), achieving the purpose of accurately quantifying and detecting impurity A in ruxolitinib phosphate. The detection limit of this method can reach 0.02%, with high sensitivity and good accuracy. It can effectively control the quality of ruxolitinib phosphate and ensure the qualified product quality. Description of the drawings
[0012] Figure 1 It is the chromatogram of the test solution and the impurity A localization solution under chromatographic condition 1 in Example 1; Figure 2 It is the chromatogram of mixed solution 1 and the impurity A localization solution under chromatographic condition 2 in Example 1; Figure 3 Chromatogram of mixed solution 2 under chromatographic condition 3 in Example 1; Figure 4 Chromatogram of mixed solution 2 under chromatographic condition 4 in Example 1; Figure 5 Chromatogram of mixed solution 1 and impurity A localization solution under chromatographic condition 5 in Example 2; Figure 6 Chromatogram of mixed solution 2 with acetonitrile as mobile phase B under chromatographic condition 6 in Example 2; Figure 7 Chromatograms of mixed solution 2, mixed solution 3 and test solution with methanol as mobile phase B under chromatographic condition 6 in Example 2; Figure 8 Chromatogram of mixed solution 1 and impurity A localization solution under chromatographic condition 7 in Example 2; Figure 9 Chromatogram of mixed solution 4 and impurity A reference solution under chromatographic condition 8 in Example 2; Figure 10 Chromatogram of impurity A specificity in Example 4; Figure 11 Chromatogram of detection limit and quantitation limit of ruxolitinib phosphate and impurity A in Example 6. Detailed implementation manners
[0013] The content of the present invention will be further described in detail below by way of examples, but the protection scope of the present invention is not limited in any way.
[0014] Example 1 Separation of ruxolitinib phosphate and impurity A was investigated using a C18 column under different pH and different organic phase conditions Chromatographic condition 1: Mobile phase A is 10 mmol / L dipotassium hydrogen phosphate (pH 7.0) solution; mobile phase B is acetonitrile; chromatographic column: YMC Triart C18 (4.6 mm × 150 mm, 3 μm); Elution mode: Gradient elution 1
[0015] HPLC instrument: Waters e2695; column temperature 40°C; flow rate 1.0 ml / min; detection wavelength: 265 nm; injection volume 10 μl.
[0016] Chromatographic condition 2: Mobile phase A is 10 mmol / L ammonium acetate (pH 5.0) solution; mobile phase B is acetonitrile or methanol; chromatographic column: Waters XSelect CSH C18 (4.6 mm × 150 mm, 3.5 μm); Elution method: Phase B is acetonitrile, gradient elution 2
[0017] Phase B is methanol, gradient elution 3
[0018] The rest is the same as Chromatographic condition 1.
[0019] Chromatographic condition 3: Mobile phase A is 10 mmol / L dipotassium hydrogen phosphate (pH 9.2) solution; Mobile phase B is acetonitrile or methanol; Chromatographic column: Waters XSelect CSH C18 (4.6 mm × 150 mm, 3 μm); Elution method: Phase B is acetonitrile, gradient elution is the same as gradient elution 1 of Chromatographic condition 1.
[0020] Phase B is methanol, gradient elution 4
[0021] The rest is the same as Chromatographic condition 1.
[0022] Chromatographic condition 4: Mobile phase A is 0.05% perchloric acid solution; Mobile phase B is acetonitrile or methanol; Chromatographic column: Agilent ZorbaxSB-Aq (4.6 mm × 250 mm, 5 μm); Elution method: Phase B is acetonitrile, gradient elution is the same as gradient elution 1 of Chromatographic condition 1.
[0023] Phase B is methanol, gradient elution is the same as gradient elution 4 of Chromatographic condition 3.
[0024] The rest is the same as Chromatographic condition 1.
[0025] Sample preparation: Solvent: Acetonitrile - water (40:60) Mixed solution 1: Take appropriate amounts of ruxolitinib phosphate and other impurities to be studied except impurity A, dissolve and dilute with the solvent to prepare a mixed solution containing about 1 mg of ruxolitinib phosphate and about 1.5 μg of other impurities to be studied except impurity A per 1 ml.
[0026] Impurity A localization solution: Take an appropriate amount of impurity A reference substance, dissolve and dilute with the solvent to prepare a localization solution containing about 1 μg of impurity A per 1 ml.
[0027] Mixed solution 2: Take appropriate amounts of ruxolitinib phosphate and impurity A, dissolve and dilute with the solvent to prepare a mixed solution containing about 20 μg of ruxolitinib phosphate and about 10 μg of impurity A per 1 ml.
[0028] Mixed solution 3: Take appropriate amounts of ruxolitinib phosphate and impurity A, dissolve and dilute with a solvent to prepare a mixed solution containing about 1 mg of ruxolitinib phosphate and about 5 μg of impurity A per 1 ml.
[0029] Test solution: Take an appropriate amount of ruxolitinib phosphate, dissolve and dilute with a solvent to prepare a solution containing about 1 mg of ruxolitinib phosphate per 1 ml.
[0030] Experimental procedure: (1) Chromatographic condition 1, acetonitrile as mobile phase B, inject the test solution and the impurity A positioning solution; (2) Chromatographic condition 2, acetonitrile and methanol as mobile phase B respectively, inject mixed solution 1 and the impurity A positioning solution respectively; (3) Chromatographic condition 3, acetonitrile and methanol as mobile phase B respectively, inject mixed solution 2; (4) Chromatographic condition 4, acetonitrile and methanol as mobile phase B respectively, inject mixed solution 2.
[0031] The test results are shown in Figures 1 to 4 , and the results show that: under the above chromatographic conditions, ruxolitinib phosphate and impurity A co-elute or are partially separated, and effective separation cannot be achieved.
[0032] Example 2 Separation of ruxolitinib phosphate and impurity A was investigated under different pH, different organic phase conditions, and different types of phenyl columns Chromatographic condition 5: Mobile phase: Mobile phase A is a 10 mmol / L dipotassium hydrogen phosphate (pH 7.0) solution; mobile phase B is acetonitrile or acetonitrile - methanol (4:6 v / v); Chromatographic column: Agilent Zorbax SB-Phenyl (4.6×250 mm, 5 μm); Elution mode: For B phase acetonitrile, the gradient elution is the same as gradient elution 1 in chromatographic condition 1 of Example 1.
[0033] For B phase acetonitrile - methanol (4:6 v / v), the gradient elution is the same as gradient elution 4 in chromatographic condition 3 of Example 1.
[0034] The rest is the same as chromatographic condition 1 of Example 1.
[0035] Chromatographic condition 6: Mobile phase: Mobile phase A is a 0.05% perchloric acid solution; mobile phase B is acetonitrile or methanol; Chromatographic column: Agilent Zorbax SB-Phenyl (4.6×250 mm, 5 μm); Elution mode: For B phase acetonitrile, the gradient elution is the same as gradient elution 1 in chromatographic condition 1 of Example 1.
[0036] Phase B methanol, gradient elution was the same as gradient elution 4 of chromatographic condition 3 in Example 1.
[0037] The rest was the same as chromatographic condition 1 in Example 1.
[0038] Chromatographic condition 7: Mobile phase: Mobile phase A was 0.05% perchloric acid solution; Mobile phase B was acetonitrile or methanol; Chromatographic column: ACE Excel 3 C18-PFP (4.6×150mm, 3μm); Elution mode: Phase B acetonitrile, gradient elution was the same as gradient elution 1 of chromatographic condition 1 in Example 1.
[0039] Phase B methanol, gradient elution was the same as gradient elution 4 of chromatographic condition 3 in Example 1.
[0040] The rest was the same as chromatographic condition 1 in Example 1.
[0041] Chromatographic condition 8: Mobile phase: Mobile phase A was 0.05% perchloric acid solution, and mobile phase B was methanol; Chromatographic column: Phenomenex Luna phenyl-Hexyl (4.6mm×150mm, 3μm); Elution mode: Phase B methanol, gradient elution was the same as gradient elution 4 of chromatographic condition 3 in Example 1.
[0042] HPLC instrument: Shimadzu LC-20AD; the rest was the same as chromatographic condition 1 in Example 1.
[0043] Sample preparation: Solvent: Acetonitrile-water (40:60 v / v) Mixed solution 1: The same as mixed solution 1 in Example 1.
[0044] Impurity A localization solution: The same as impurity A localization solution in Example 1.
[0045] Mixed solution 2: The same as mixed solution 2 in Example 1.
[0046] Mixed solution 3: The same as mixed solution 3 in Example 1.
[0047] Test solution: The same as test solution in Example 1.
[0048] Impurity A reference solution: Take an appropriate amount of impurity A reference substance, dissolve and dilute with the solvent to prepare a reference solution containing about 1.875 μg of impurity A per 1 ml.
[0049] Mixed solution 4: Take appropriate amounts of ruxolitinib phosphate and impurity A, dissolve and dilute with a solvent to prepare a mixed solution containing about 1 mg of ruxolitinib phosphate and about 1.875 μg of impurity A per 1 ml.
[0050] Experimental procedure: (1) Chromatographic condition 5, using acetonitrile and acetonitrile - methanol (4:6) as mobile phase B respectively, inject mixed solution 1 and the impurity A localization solution respectively; (2) Chromatographic condition 6, using acetonitrile as mobile phase B, inject mixed solution 2, using methanol as mobile phase B, inject mixed solution 2, mixed solution 3 and the test solution; (3) Chromatographic condition 7, using acetonitrile and methanol as mobile phase B respectively, inject mixed solution 1 and the impurity A localization solution respectively; (4) Chromatographic condition 8, using methanol as mobile phase B, inject mixed solution 4 and the impurity A reference solution.
[0051] The test results are as Figures 5 to 9 shown. The results show that under chromatographic condition 5 and chromatographic condition 7, ruxolitinib phosphate and impurity A co - elute or are partially separated, and effective separation cannot be achieved. The typical chromatograms are shown in detail in Figure 5 and Figure 8 . Under chromatographic condition 6, when acetonitrile is used as mobile phase B, ruxolitinib phosphate and impurity A co - elute, and when methanol is used as mobile phase B, effective separation of ruxolitinib phosphate and impurity A can be achieved. The typical chromatogram is shown in detail in Figures 6 to 7 . Under chromatographic condition 8, the separation of ruxolitinib phosphate and impurity A is better than that when methanol is used as mobile phase B under chromatographic condition 6, and the resolution is greater than 1.5. The typical chromatogram is shown in detail in Figure 9 .
[0052] Summary: Chromatographic condition 8 has the best separation effect and is preferably selected as the detection method of the present invention.
[0053] Example 3 System precision investigation Chromatographic conditions: Mobile phase A is 0.05% perchloric acid solution; mobile phase B is methanol; chromatographic column: Phenomenex Luna phenyl - Hexyl (4.6 mm × 150 mm, 3 μm); Elution mode: Gradient elution, the program is as follows:
[0054] HPLC instrument: Shimadzu LC - 20AD; column temperature 40°C; flow rate 1.0 ml / min; detection wavelength: 265 nm; injection volume 10 μl.
[0055] Sample preparation: Solvent: Acetonitrile - water (40:60 v / v) (1) Stock solution of impurity A: Accurately weigh about 3.75 mg of reference substance of impurity A, place it in a 20-ml volumetric flask, dissolve it with acetonitrile and dilute to the mark, shake well to obtain.
[0056] (2) Stock solution of ruxolitinib phosphate: Accurately weigh about 2.4 mg of reference substance of ruxolitinib phosphate, place it in a 50-ml volumetric flask, dissolve it with the solvent and dilute to the mark, shake well to obtain.
[0057] (3) System precision solution: Accurately measure appropriate volumes of the stock solution of impurity A and the stock solution of ruxolitinib phosphate, dilute with the solvent to prepare a mixed solution containing about 0.5 μg of ruxolitinib phosphate and impurity A per 1 ml.
[0058] Experimental procedure: Inject the above system precision solution continuously for 6 times, and record the chromatogram. The results are shown in Table 1.
[0059]
[0060] The results show that the maximum RSD of the peak areas of ruxolitinib phosphate and impurity A is 2.0%, the system precision is good, and it is applicable to the detection of impurity A.
[0061] Example 4 Specificity investigation Chromatographic conditions: The same as in Example 3.
[0062] Sample preparation: Solvent: Acetonitrile-water (40:60 v / v) (1) Stock solution of impurity A: The same as in Example 3.
[0063] (2) Reference solution of impurity A: Accurately measure an appropriate volume of the stock solution of impurity A, dilute with the solvent to prepare a reference solution containing about 1.875 μg of impurity A per 1 ml.
[0064] (3) Specificity solution: Take an appropriate amount of ruxolitinib phosphate, accurately add an appropriate volume of the stock solution of impurity A, dissolve and dilute with the solvent to prepare a mixed solution containing about 1 mg of ruxolitinib phosphate and about 1.875 μg of impurity A per 1 ml.
[0065] Experimental procedure: Inject the above solvent, reference solution of impurity A, and specificity solution into the high-performance liquid chromatograph, and record the chromatogram. The typical chromatogram is shown in Figure 10 . The results are shown in Table 2.
[0066]
[0067] The results show that the blank solvent does not interfere with the detection of impurity A, and the minimum resolution between ruxolitinib phosphate and impurity A in the specificity solution is 3.5, and the method specificity is good.
[0068] Example 5 Linearity investigation Chromatographic conditions: HPLC instrument: Agilent 1260; other conditions are the same as in Example 3.
[0069] Sample preparation: Solvent: acetonitrile - water (40:60 v / v) (1) Stock solution of impurity A: Take about 1.5 mg of the reference substance of impurity A, weigh accurately, place it in a 10 - ml volumetric flask, dissolve with acetonitrile and dilute to the mark, shake well, and you will get it.
[0070] (2) Stock solution of ruxolitinib phosphate: Take about 7.5 mg of the reference substance of ruxolitinib phosphate, weigh accurately, place it in a 50 - ml volumetric flask, dissolve with the solvent and dilute to the mark, shake well, and you will get it.
[0071] (3) Mixed stock solution: Accurately measure 1 ml each of the stock solution of impurity A and the stock solution of ruxolitinib phosphate, place them in a 10 - ml volumetric flask, dilute to the mark with the solvent, shake well, and you will get it.
[0072] (4) Linear solution 1: Accurately measure 0.8 ml of the mixed stock solution, place it in a 25 - ml volumetric flask, dilute to the mark with the solvent, shake well, and you will get it.
[0073] (5) Linear solution 2: Accurately measure 0.5 ml of the mixed stock solution, place it in a 10 - ml volumetric flask, dilute to the mark with the solvent, shake well, and you will get it.
[0074] (6) Linear solution 3: Accurately measure 1 ml of the mixed stock solution, place it in a 10 - ml volumetric flask, dilute to the mark with the solvent, shake well, and you will get it.
[0075] (7) Linear solution 4: Accurately measure 1.5 ml of the mixed stock solution, place it in a 10 - ml volumetric flask, dilute to the mark with the solvent, shake well, and you will get it.
[0076] (8) Linear solution 5: Accurately measure 2 ml of the mixed stock solution, place it in a 10 - ml volumetric flask, dilute to the mark with the solvent, shake well, and you will get it.
[0077] Experimental procedure: Inject the above - mentioned linear solutions into the high - performance liquid chromatograph and record the chromatograms.
[0078] The results show that: in the range of 0.4808 μg / ml - 3.005 μg / ml, the linear equation of ruxolitinib phosphate is y = 16158x - 167 (y is the peak area, x is the concentration, μg / ml), the correlation coefficient r is 0.9999; in the range of 0.4767 μg / ml - 2.979 μg / ml, the linear equation of impurity A is y = 23250x - 281 (y is the peak area, x is the concentration, μg / ml), the correlation coefficient r is 0.9999, and the relative correction factor for ruxolitinib phosphate is 0.69, and the method has good linearity.
[0079] Example 6 Investigation of Detection Limit and Quantification Limit Chromatographic conditions: same as in Example 3.
[0080] Sample preparation: Solvent: acetonitrile - water (40:60 v / v) (1) Stock solution of impurity A: same as in Example 3.
[0081] (2) Stock solution of ruxolitinib phosphate: same as in Example 3.
[0082] (3) Quantification limit solution: Accurately measure appropriate volumes of the stock solution of impurity A and the stock solution of ruxolitinib phosphate, and dilute with the solvent to prepare a mixed solution containing about 0.5 μg of ruxolitinib phosphate and impurity A per 1 ml.
[0083] (4) Detection limit solution: Accurately measure appropriate volumes of the stock solution of impurity A and the stock solution of ruxolitinib phosphate, and dilute with the solvent to prepare a mixed solution containing about 0.2 μg of ruxolitinib phosphate and impurity A per 1 ml.
[0084] Experimental procedure: Inject the quantification limit solution continuously for 6 times and inject the detection limit solution once, and record the chromatogram. See the typical figure in Figure 11 .
[0085] Results show that: in the 6 - needle quantification limit solution, the concentration of ruxolitinib phosphate is 0.4805 μg / ml (equivalent to 0.05% of the test sample concentration), and the S / N values are 34, 43, 39, 35, 40, 40 respectively, all greater than 10, and the RSD of the peak area is 2.0%; the concentration of impurity A is 0.4575 μg / ml (equivalent to 0.05% of the test sample concentration), and the S / N values are 46, 59, 54, 47, 56, 56 respectively, all greater than 10, and the RSD of the peak area is 0.5%; in the detection limit solution, the concentration of ruxolitinib phosphate is 0.1922 μg / ml (equivalent to 0.02% of the test sample concentration), and the S / N is 19, the concentration of impurity A is 0.1830 μg / ml (equivalent to 0.02% of the test sample concentration), and the S / N is 23, both greater than 3.
[0086] Example 7 Investigation of Accuracy Chromatographic conditions: same as in Example 3.
[0087] Sample preparation: Solvent: acetonitrile - water (40:60 v / v) (1) Stock solution of impurity A: same as in Example 3.
[0088] (2) Test sample solution: Take an appropriate amount of ruxolitinib phosphate, dissolve and dilute with the solvent to prepare a solution containing about 1 mg of ruxolitinib phosphate per 1 ml.
[0089] (3) Accuracy solution: Take an appropriate amount of ruxolitinib phosphate, accurately add an appropriate volume of the stock solution of impurity A, dissolve and dilute with the solvent to prepare a mixed solution containing about 1 mg of ruxolitinib phosphate and about 0.45 μg of impurity A per 1 ml. (Prepare 3 portions in parallel) (4) Linear solution 3: Same as Example 5.
[0090] Experimental procedure: Inject the above-mentioned test solution, accuracy solution, and linear solution 3 into the high-performance liquid chromatograph, and record the chromatogram. Calculate according to the external standard method of the main component with correction factor, and calculate the recovery rate and recovery rate RSD of each accuracy solution. The results are shown in Table 3.
[0091]
[0092] The results show that: the recovery rates of the 3 accuracy solutions are 93.1% - 94.1%, the average recovery rate is 93.7%, all within the range of 80.0% - 120.0%, and the RSD of the recovery rate is 0.7%, less than 10.0%, indicating that this method is accurate, reliable, has good precision, and is applicable to the detection of impurity A in ruxolitinib phosphate.
[0093] Example 8 Investigation on solution stability Chromatographic conditions: HPLC instrument: Agilent 1260; other conditions are the same as in Example 3.
[0094] Sample preparation: Solvent: Acetonitrile - water (40:60 v / v) (1) Linear solution 3: Same as Example 5.
[0095] (2) Specificity solution: Same as Example 4.
[0096] (3) Test solution: Same as Example 7.
[0097] Experimental procedure: Inject the above-mentioned linear solution 3, specificity solution, and test solution at irregular intervals, and record the chromatogram. The results are shown in Tables 4 - 6.
[0098]
[0099]
[0100]
[0101] The results show that: in the linear solution 3, specificity solution, and test solution, the maximum change value of the peak area ratio of impurity A to the 0 h peak area is 97.7%, all within the range of 80.0% - 120.0%, and the solution stability is good.
Claims
1. A method for detecting positional isomers of ruxolitinib phosphate using high performance liquid chromatography, characterized in that: The chromatographic conditions include: chromatographic column: a chromatographic column using phenylhexyl bonded silica gel as filler; Mobile phase A: perchloric acid solution, Mobile phase B: methanol, Detector: UV detector, Elution mode: gradient elution, Wherein, the positional isomer of ruxolitinib is impurity A, 。 2. The detection method according to claim 1, wherein the mobile phase A is a 0.05% perchloric acid solution.
3. The detection method according to claim 1, wherein the chromatographic column is Phenomenex Luna phenyl-Hexyl.
4. The detection method according to claim 1 or 2, wherein the chromatographic conditions include a gradient elution procedure as follows: 。 5. The detection method according to claim 1, wherein the flow rate is 1.0 ml / min.
6. The detection method as claimed in claim 1, wherein the detection wavelength is 265 nm.
7. The detection method according to claim 1, wherein the column temperature is 40°C.
8. The detection method according to claim 1, wherein the injection volume is 10 μl.
9. The detection method according to claim 1, comprising the following steps: (1) Preparation of ruxolitinib phosphate reference solution: Take an appropriate amount of ruxolitinib phosphate reference substance and prepare a reference substance solution with acetonitrile-water (40:60 v / v); (2) Preparation of impurity A localization solution: Take an appropriate amount of impurity A reference substance and prepare an impurity A localization solution with acetonitrile-water (40:60 v / v); (3) Preparation of test solution: Take an appropriate amount of the test sample and prepare it into a test solution with acetonitrile-water (40:60 v / v); (4) Take the ruxolitinib phosphate reference solution, impurity A location solution and test sample solution and inject them into the high performance liquid chromatography for detection.