Method for separating and determining rusotinib enantiomer by HPLC (High Performance Liquid Chromatography)

Abstract

The invention relates to a method for separating and determining rusotinib enantiomer by HPLC (High Performance Liquid Chromatography), which comprises the following steps: taking a chromatographic column of which the surface of silica gel is coated with amylose-tri (3, 5-dimethyl phenyl carbamate) as a filler as a chiral chromatographic column; a mixed solution of lower alkane and lower alcohol is used as a mobile phase, and the rusotinib enantiomer is separated and determined by a high performance liquid chromatography and an ultraviolet detector; carrying out isocratic elution by taking a mixed solution of lower alkane and lower alcohol as a mobile phase; the flow velocity is 0.8-1.2 ml / min, the column temperature is 35-45 DEG C, an ultraviolet detector is adopted for detection, and the detection wavelength of the ultraviolet detector is 190-400 nm. According to the present invention, the rusotinib and the rusotinib enantiomer can be rapidly and efficiently separated under the same chromatographic condition, and the detection method has advantages of high sensitivity, strong specificity, rapidness, simpleness, convenience, easy operation and effective quality control, and is suitable for the separation and determination of the rusotinib enantiomer.

Description

Technical Field

[0001] The invention relates to a method for separating and determining ruxolitinib enantiomers by using HPLC, and belongs to the technical field of analytical chemistry. Background Art

[0002] Ruxolitinib is a JAK inhibitor mainly used to treat myelofibrosis, atopic dermatitis and glucocorticoid-resistant acute graft-versus-host disease.

[0003] Ruxolitinib was originally developed by Incyte and was approved by the U.S. Food and Drug Administration (FDA) in November 2011 for the treatment of patients with myelofibrosis. It is the world's first targeted drug specifically for the treatment of myelofibrosis. In addition, Ruxolitinib is also approved for the treatment of patients with short-term or non-persistent chronic atopic dermatitis and patients with glucocorticoid-resistant acute graft-versus-host disease.

[0004] Ruxolitinib's mechanism of action is to inhibit the activity of JAK1 and JAK2 kinases, preventing these kinases from playing a role in cell signaling, thereby reducing inflammation and immune responses. This mechanism makes Ruxolitinib show potential in the treatment of a variety of autoimmune diseases and cancer-related diseases.

[0005] Its molecular formula: C 17 H 21 N6O4P; molecular weight: 404.37, chemical structure is as follows:

[0006]

[0007] Ruxolitinib has a chiral center. In chemical synthesis, it is obtained by splitting the ruxolitinib racemate. Therefore, it is easy to produce an enantiomeric impurity. Impurity molecular formula: C 17 H 21 N6O4P; molecular weight: 404.37; structural formula

[0008] As shown in formula 2:

[0009]

[0010] The above-mentioned enantiomeric impurities are commonly present in samples and are listed as process impurities of ruxolitinib, affecting the quality of ruxolitinib API and its finished products; therefore, the analysis and detection of this impurity plays an important role in the quality control of the final product ruxolitinib.

[0011] At present, the quality standards of Ruxolitinib have not been included in the latest editions of the United States Pharmacopoeia, the European Pharmacopoeia, the British Pharmacopoeia and the Japanese Pharmacopoeia. In the publicly published literature or patents, no test methods for the enantiomers of Ruxolitinib were found.

[0012] This means that there is no reference enantiomer analysis method for this product at this stage, which hinders the application and promotion of this product. Therefore, it is very necessary to establish a simple, stable and effective analytical detection method for the analysis and detection of ruxolitinib enantiomers.

[0013] Based on this, the present invention is proposed. Summary of the invention

[0014] In order to accurately reflect the content of enantiomers in ruxolitinib raw materials or preparations, a reasonable basis can be provided for the formulation of quality standards, so as to better control and master product quality, thereby improving the safety of clinical medication. The present invention provides a method for detecting isomers of ruxolitinib enantiomers. The detection method has the advantages of being fast and simple, having good separation and high sensitivity, and is suitable for the inspection and quality control of ruxolitinib enantiomers in the pharmaceutical industry.

[0015] The technical solution adopted by the present invention is:

[0016] A method for separating and determining Ruxolitinib enantiomers by HPLC comprises the following steps:

[0017] Chromatographic conditions:

[0018] The chromatographic column is a chiral chromatographic column with a silica gel surface coated with amylose-tris (3,5-dimethylphenylcarbamate) as a filler;

[0019] The enantiomers of ruxolitinib were separated and determined by high performance liquid chromatography with a UV detector using a mixture of lower alkanes and lower alcohols as the mobile phase.

[0020] The silica gel surface is coated with amylose-tris (3,5-dimethylphenylcarbamate) as a filler; a mixed solution of lower alkanes and lower alcohols is used as a mobile phase, and isocratic elution is performed; the flow rate is 0.8-1.2 ml / min, the column temperature is 35-45° C., and an ultraviolet detector is used for detection, and the detection wavelength of the ultraviolet detector is 190-400 nm.

[0021] The chiral chromatographic column is selected from Daicel AD-H column, and the specification model is CHIRALPAKAD-H, 250×4.6 mm, 5 μm.

[0022] The lower alkane is n-hexane.

[0023] The lower alcohol is isopropanol and anhydrous ethanol. When isopropanol is selected, the separation degree is poor, so anhydrous ethanol is preferred.

[0024] The mobile phase is composed of n-hexane and anhydrous ethanol in a mixing ratio of 55:45 (V / V). The separation degree between ruxolitinib and ruxolitinib enantiomers decreases with the increase of the ethanol ratio; when the ethanol ratio is 40%, the separation degree between ruxolitinib and ruxolitinib enantiomers is large; when the ethanol ratio is 50%, the separation degree between ruxolitinib and ruxolitinib enantiomers is slightly greater than 1.5; adding alkaline additives to the mobile phase has little effect on the separation degree.

[0025] The flow rate is 0.8-1.2 ml / min, preferably 1.0 ml / min.

[0026] The column temperature is 35-45°C, preferably 40°C.

[0027] The detection wavelength of the ultraviolet detector is 190-400nm. After ultraviolet scanning, the maximum absorption of this product is at 220nm, so 220nm is preferred.

[0028] Beneficial effects of the present invention:

[0029] The present invention provides a method for separating and determining ruxolitinib enantiomers by HPLC. By comprehensively considering the comprehensive influence of the analytical column, mobile phase, gradient elution program, flow rate and column temperature on separation and detection, the detection result is optimized, ruxolitinib and ruxolitinib enantiomers can be quickly and efficiently separated under the same chromatographic conditions, and the detection method has high sensitivity, strong specificity, is fast and simple, and is easy to operate. The quality of drugs can be effectively controlled, and the method is suitable for separating and detecting ruxolitinib enantiomers. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The chromatogram is a blank solution detected under the conditions of Example 1 in the present invention;

[0031] Figure 2 The chromatogram of the system suitability solution tested under the conditions of Example 1 in the present invention;

[0032] Figure 3 The chromatogram of the test solution detected according to the conditions of Example 1 in the present invention;

[0033] Figure 4 The chromatogram is a quantitative limit solution detected according to the conditions of Example 1 in the present invention;

[0034] Figure 5 The chromatogram is a detection limit solution detected under the conditions of Example 1 in the present invention;

[0035] Figure 6 The chromatogram of the system suitability solution tested according to the conditions of Example 2 in the present invention [mobile phase ratio - n-hexane - anhydrous ethanol (50:50)];

[0036] Figure 7 The chromatogram of the system suitability solution tested according to the conditions of Example 2 in the present invention [mobile phase ratio - n-hexane - anhydrous ethanol (60:40)];

[0037] Figure 8 The chromatogram of the system suitability solution tested under the conditions of Example 3 in the present invention (column temperature -35°C);

[0038] Fig. 9 The chromatogram of the system suitability solution tested under the conditions of Example 3 in the present invention (column temperature -45°C);

[0039] Fig.10 The chromatogram of the system suitability solution tested under the conditions of Example 4 in the present invention (flow rate -0.8 ml / min);

[0040] Fig.11 The chromatogram of the system suitability solution tested under the conditions of Example 4 in the present invention (flow rate -1.2 ml / min);

[0041] Fig.12 The chromatogram of the system suitability solution tested under the conditions of Example 5 in the present invention (selection of lower alcohol - anhydrous ethanol);

[0042] Fig.13 The chromatogram of the system suitability solution detected according to the conditions of Example 5 in the present invention (selection of lower alcohol - isopropanol);

[0043] Fig.14 The chromatogram of the system suitability solution tested under the conditions of Example 6 in the present invention (selection of alkaline additive - n-hexane - anhydrous ethanol - diethylamine (80:20:0.1));

[0044] Fig.15 The chromatogram of the system suitability solution tested according to the conditions of Example 7 in the present invention (selection of chromatographic column - CHIRALPAK AY-H, 250×4.6 mm, 5 μm);

[0045] Fig.16 This is a chromatogram of the system suitability solution tested according to the conditions of Example 7 in the present invention (selection of chromatographic column - CHIRALCEL OD-H, 250×4.6 mm, 5 μm). DETAILED DESCRIPTION

[0046] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0047] Example 1

[0048] The chromatographic conditions were as follows:

[0049] Chromatographic column: CHIRALPAKAD-H 4.6mm×250mm 5μm;

[0050] Mobile phase: n-hexane-anhydrous ethanol = 55:45 (V / V);

[0051] Column temperature: 40°C;

[0052] Flow rate: 1.0ml / min;

[0053] Detection wavelength: 220nm;

[0054] Injection volume: 20 μl

[0055] Solution preparation:

[0056] Diluent: n-hexane-anhydrous ethanol = 55:45 (V / V).

[0057] System suitability solution: Take about 25 mg of ruxolitinib racemate, weigh accurately, place in a 50 ml volumetric flask, add diluent to dissolve and dilute to the scale, shake well, and obtain.

[0058] Test solution: Take about 25 mg of ruxolitinib enantiomer, accurately weigh, place in a 50 ml volumetric flask, add diluent to dissolve and dilute to the scale, and shake well.

[0059] Quantitative limit solution: Accurately measure 1 ml of the test solution into a 100 ml volumetric flask, add diluent to dilute to the scale, shake well, accurately measure 1 ml to 25 ml, add diluent to dilute to the scale, shake well, and obtain.

[0060] Detection limit solution: Accurately measure 5 ml of the quantitative limit solution and place it in a 10 ml volumetric flask, add diluent to dilute to the scale, shake well, and obtain.

[0061] Determination: Inject the blank solution (i.e. diluent), system suitability solution, test solution, quantitation limit solution and detection limit solution into the high performance liquid chromatograph for detection and record the chromatogram.

[0062] The chromatograms of blank solution, system suitability solution, test solution, quantitation limit solution and detection limit solution are shown in Figure 1. Figure 1 , 2 , 3, 4 and 5, it can be seen that Figure 1 This indicates that the blank does not interfere with the examination of ruxolitinib enantiomers; Figure 2 It shows that the separation between ruxolitinib enantiomers and ruxolitinib is good, and the specific ruxolitinib system suitability spectrum data are shown in Table 1; Figure 3This indicates that the content of ruxolitinib enantiomers in the homemade ruxolitinib samples is low; Figure 4 It showed that the limit of quantification of ruxolitinib enantiomers was 0.02wt%; Figure 5 It shows that the detection limit of ruxolitinib enantiomers is 0.01wt%, which is lower than the limit of ruxolitinib enantiomers 0.50wt%; the detection sensitivity of this method is high.

[0063] Table 1 Data table of the system suitability of Ruxolitinib in Example 1

[0064] sample Retention time (min) Separation Theoretical plate number Tailing Factor Enantiomers 11.075 / 6664 1.14 Ruxolitinib 10.099 1.90 6883 1.18

[0065] Example 2

[0066] The same chromatographic column, column temperature, flow rate and mobile phase as in Example 1 were used, and only the ratio of the mobile phase was changed. Ruxolitinib and its enantiomers were separated and detected under the mobile phase of n-hexane-anhydrous ethanol (50:50-60:40).

[0067] The chromatographic conditions were as follows:

[0068] Chromatographic column: CHIRALPAKAD-H 4.6mm×250mm 5μm;

[0069] Mobile phase: n-hexane-anhydrous ethanol (50:50~60:40)

[0070] Column temperature: 40°C

[0071] Flow rate: 1.0ml / min

[0072] Detection wavelength: 220nm

[0073] Injection volume: 20 μl

[0074] Solution preparation:

[0075] Solution preparation:

[0076] Diluent: Mobile phase.

[0077] System suitability solution: Take about 25 mg of ruxolitinib racemate, weigh accurately, place in a 50 ml volumetric flask, add diluent to dissolve and dilute to the scale, shake well, and obtain.

[0078] Determination: Take the system suitability solution and inject it into the high performance liquid chromatograph and record the chromatogram. Figure 6 , 7 .

[0079] The results show that under the above chromatographic conditions, the baseline is stable, and the separation degree between ruxolitinib and the enantiomer decreases with the increase of the anhydrous ethanol ratio. When the anhydrous ethanol ratio is 40%, the separation degree between ruxolitinib and the enantiomer is large, but the retention time of the main peak is late. When the anhydrous ethanol ratio is 50%, the separation degree between ruxolitinib and the enantiomer is 1.79. The present invention can achieve baseline separation of the two substances in the mobile phase of n-hexane-anhydrous ethanol (50:50-60:40). Considering the separation degree, tailing factor and analysis time, the present invention most preferably performs chromatographic separation under the condition of n-hexane-anhydrous ethanol (55:45) as the mobile phase.

[0080] Table 2 Data table of the system suitability of Ruxolitinib in Example 2 (different mobile phase ratios)

[0081]

[0082] Example 3

[0083] The same chromatographic column, column temperature, flow rate and mobile phase as in Example 1 were used, with only the column temperature being different, and ruxolitinib and its enantiomers were separated and detected at 35° C., 40° C. and 45° C., respectively.

[0084] The chromatographic conditions were as follows:

[0085] Chromatographic column: CHIRALPAKAD-H 4.6mm×250mm 5μm;

[0086] Mobile phase: n-hexane-anhydrous ethanol (55:45);

[0087] Column temperature: 35℃~45℃

[0088] Flow rate: 1.0ml / min

[0089] Detection wavelength: 220nm

[0090] Injection volume: 20 μl

[0091] Solution preparation:

[0092] Solution preparation:

[0093] Diluent: Mobile phase.

[0094] System suitability solution: Take about 25 mg of ruxolitinib racemate, weigh accurately, place in a 50 ml volumetric flask, add diluent to dissolve and dilute to the scale, shake well, and obtain.

[0095] Determination: Take the system suitability solution and inject it into the high performance liquid chromatograph and record the chromatogram. Figure 8 , 9 .

[0096] The results show that under the above chromatographic conditions, the baseline is stable, and the separation between ruxolitinib and the enantiomers is not significantly different from that in Example 1. The present invention can achieve baseline separation of the two substances within 35 to 45° C. Taking into account factors such as the analysis time and the temperature range of the chromatographic column (35 to 45° C.), the present invention is most preferably chromatographically separated at a column temperature of 40° C.

[0097] Table 3 Data table of Ruxolitinib system suitability spectrum (different column temperatures) in Example 3

[0098]

[0099] Example 4

[0100] The same chromatographic column, column temperature and mobile phase ratio as in Example 1 were used, and only the flow rate was changed. Ruxolitinib and its enantiomers were separated and detected at flow rates of 0.8 ml / min and 1.2 ml / min, respectively.

[0101] The chromatographic conditions were as follows:

[0102] Chromatographic column: CHIRALPAKAD-H 4.6mm×250mm 5μm;

[0103] Mobile phase: n-hexane-anhydrous ethanol (55:45);

[0104] Column temperature: 40°C;

[0105] Flow rate: 0.8ml / min~1.2ml / min;

[0106] Detection wavelength: 220nm;

[0107] Injection volume: 20 μl;

[0108] Solution preparation:

[0109] Diluent: Mobile phase.

[0110] System suitability solution: Take about 25 mg of ruxolitinib racemate, weigh accurately, place in a 50 ml volumetric flask, add diluent to dissolve and dilute to the scale, shake well, and obtain.

[0111] Determination: Take the system suitability solution and inject it into the high performance liquid chromatograph and record the chromatogram. Fig.10 , 11 .

[0112] The results show that under the above chromatographic conditions, the baseline is stable, and as the flow rate decreases, the retention time of each peak increases slightly, the column pressure decreases, and the separation between ruxolitinib and the enantiomers is not significantly different from that in Example 1. The present invention can achieve baseline separation of the two substances within the flow rate (0.8ml / min to 1.2ml / min). Considering the separation, analysis time, and chromatographic column pressure resistance, the most preferred flow rate of the present invention is 1.0ml / min.

[0113] Table 4 Data table of Ruxolitinib system suitability profile (different flow rates) in Example 4

[0114]

[0115] Example 5

[0116] The same chromatographic column, column temperature, mobile phase ratio, and flow rate as in Example 1 were used, and only the type of lower alcohol in the mobile phase was changed. Ruxolitinib and its enantiomers were separated and detected under the mobile phase conditions of n-hexane-anhydrous ethanol (55:45) and n-hexane-isopropanol (55:45), respectively.

[0117] The chromatographic conditions were as follows:

[0118] Chromatographic column: CHIRALPAKAD-H 4.6mm×250mm 5μm;

[0119] Mobile phase: n-hexane-anhydrous ethanol (55:45), n-hexane-isopropanol (55:45);

[0120] Column temperature: 40°C;

[0121] Flow rate: 1.0ml / min;

[0122] Detection wavelength: 220nm;

[0123] Injection volume: 20 μl;

[0124] Solution preparation:

[0125] Diluent: Mobile phase.

[0126] System suitability solution: Take about 25 mg of ruxolitinib racemate, weigh accurately, place in a 50 ml volumetric flask, add diluent to dissolve and dilute to the scale, shake well, and obtain.

[0127] Determination: Take the system suitability solution and inject it into the high performance liquid chromatograph and record the chromatogram. Fig.12 , 13 .

[0128] The results show that under the above chromatographic conditions, the baseline is stable, the retention time and separation degree of ruxolitinib and enantiomers are different depending on the type of lower alcohol in the mobile phase, and baseline separation cannot be achieved using isopropanol; the most preferred lower alcohol in the mobile phase of the present invention is anhydrous ethanol.

[0129] Table 5 Data table of the system suitability of Ruxolitinib in Example 5 (selection of lower alcohol)

[0130]

[0131]

[0132] Example 6

[0133] The same chromatographic column, column temperature, mobile phase ratio, and flow rate as in Example 1 were used, and only the alkaline additive was added to the mobile phase. Ruxolitinib and its enantiomers were separated and detected under the mobile phase conditions of n-hexane-anhydrous ethanol-diethylamine (55:45:0.1).

[0134] The chromatographic conditions were as follows:

[0135] Chromatographic column: CHIRALPAKAD-H 4.6mm×250mm 5μm;

[0136] Mobile phase: n-hexane-anhydrous ethanol-diethylamine (55:45:0.1);

[0137] Column temperature: 40°C;

[0138] Flow rate: 1.0ml / min;

[0139] Detection wavelength: 220nm;

[0140] Injection volume: 20 μl;

[0141] Solution preparation:

[0142] Diluent: n-hexane-anhydrous ethanol = 55:45.

[0143] System suitability solution: Take about 25 mg of ruxolitinib racemate, weigh accurately, place in a 50 ml volumetric flask, add diluent to dissolve and dilute to the scale, shake well, and obtain.

[0144] Determination: Take the system suitability solution and inject it into the high performance liquid chromatograph and record the chromatogram. Fig.14 .

[0145] The results show that under the above chromatographic conditions, the baseline is stable, and the addition or not of the basic additive in the mobile phase has no significant improvement on the separation degree and peak shape of the enantiomers and ruxolitinib. The present invention can achieve baseline separation of the two substances with or without the addition of the basic additive in the mobile phase. Considering the simple configuration and operation of the mobile phase, the present invention most preferably does not add the basic additive in the mobile phase.

[0146] Table 6 Data table of the suitability of the Ruxolitinib system in Example 6 (selection of basic additives)

[0147]

[0148] Example 7

[0149] The same column temperature, mobile phase ratio and flow rate as in Example 1 were used, and only the type of chromatographic column was changed to separate and detect ruxolitinib and its enantiomers.

[0150] The chromatographic conditions were as follows:

[0151] Chromatographic column: CHIRALPAKAY-H 4.6mm×250mm 5μm / CHIRALCEL OD-H 250×4.6mm5μm;

[0152] Mobile phase: n-hexane-anhydrous ethanol (55:45);

[0153] Column temperature: 40°C;

[0154] Flow rate: 1.0ml / min;

[0155] Detection wavelength: 220nm;

[0156] Injection volume: 20 μl;

[0157] Solution preparation:

[0158] Diluent: Mobile phase.

[0159] System suitability solution: Take about 25 mg of ruxolitinib racemate, weigh accurately, place in a 50 ml volumetric flask, add diluent to dissolve and dilute to the scale, shake well, and obtain.

[0160] Determination: Take the system suitability solution and inject it into the high performance liquid chromatograph and record the chromatogram. Fig.15 , 16 .

[0161] The results show that under the above chromatographic conditions, the baseline is stable, and the chromatographic peak separation is significantly different for different types of chromatographic columns. The OD-H column cannot separate ruxolitinib and the enantiomers; the AY-H column can separate them, but the tailing factor is large and the peak shape is not good; considering the tailing factor, response value, theoretical plate number and other factors of the two substances, the AD-H column is the most preferred column in the present invention.

[0162] Table 7 Data table of the system suitability of Ruxolitinib in Example 7 (different types of chromatographic columns)

[0163]

[0164] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for separating and determining the enantiomers of Ruxolitinib by HPLC, characterized in that: The enantiomers of Ruxolitinib are separated and determined by high performance liquid chromatography, comprising the following steps: Chromatographic conditions: The chromatographic column is a chiral chromatographic column, wherein the silica gel surface is coated with amylose-tris (3,5-dimethylphenylcarbamate) as a filler; A mixture of lower alkanes and lower alcohols is used as the mobile phase, and isocratic elution is performed; the flow rate is 0.8-1.2 ml / min, the column temperature is 35-45° C., and an ultraviolet detector is used for detection, and the detection wavelength of the ultraviolet detector is 190-400 nm; The structural formula of Ruxolitinib enantiomers is shown in Formula 2:

2. The method for separating and determining Ruxolitinib enantiomers by HPLC according to claim 1, characterized in that: The chiral chromatographic column is a Daicel AD-H column, with a specification model of CHIRALPAKAD-H, 250×4.6 mm, 5 μm.

3. The method for separating and determining Ruxolitinib enantiomers by HPLC according to claim 1, characterized in that: The lower alkane is n-hexane.

4. The method for separating and determining Ruxolitinib enantiomers by HPLC according to claim 1, characterized in that: The lower alcohol is anhydrous ethanol.

5. The method for separating and determining Ruxolitinib enantiomers by HPLC according to claim 1, characterized in that: The mobile phase is composed of n-hexane and anhydrous ethanol in a mixing ratio of 55:

45.

6. The method for separating and determining Ruxolitinib enantiomers by HPLC according to claim 1, characterized in that: The flow rate is 1.0 ml / min.

7. The method for separating and determining Ruxolitinib enantiomers by HPLC according to claim 1, characterized in that: The column temperature was 40°C.

8. The method for separating and determining Ruxolitinib enantiomers by HPLC according to claim 1, characterized in that: The detection wavelength of the ultraviolet detector is 220 nm.

9. The method for separating and determining Ruxolitinib enantiomers by HPLC according to claim 1, characterized in that: The resolution between ruxolitinib and its enantiomers was >1.5.

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