A liquid chromatography method for detecting upadacitinib enantiomers

Abstract

The present invention belongs to the technical field of pharmaceutical analysis, and particularly relates to a liquid chromatography method for detecting upadacitinib enantiomers, and more particularly to a reverse-phase liquid chromatography method for detecting upadacitinib enantiomers. The present invention realizes the effective separation and detection of upadacitinib and its enantiomers. The reverse-phase chromatography system used has the advantages of stable chromatographic solvent performance, strong separation ability, simple operation process, easier equilibration, etc., and has stronger universality. It can be directly used for the detection of preparations such as upadacitinib sustained-release tablets and upadacitinib oral solutions, as well as upadacitinib raw materials. The operation is more convenient, and it can more conveniently realize the quality control of the product production process, improve the quality of the final product, and ensure the safety and effectiveness of clinical medication.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical analysis, and particularly relates to a liquid chromatography method for detecting the enantiomers of upadacitinib. Background Art

[0002] Upadacitinib is a selective JAK inhibitor. JAK is an intracellular enzyme that can transmit signals generated by the interaction of cytokine or growth factor receptors on the cell membrane, thereby affecting the cellular processes of hematopoiesis and the functions of immune cells. Upadacitinib is widely used in the treatment of autoimmune diseases such as atopic dermatitis and rheumatoid arthritis by regulating intracellular signal transduction pathways.

[0003] The chemical name of upadacitinib is (3S,4R)-3-ethyl-4-(3H-imidazo[1,2-a]pyrrolo[2,3-e]pyrazin-8-yl)-N-(2,2,2-trifluoroethyl)pyrrolidine-1-carboxamide hydrate (2:1), and its structural formula is shown as the compound of formula (I):

[0004] .

[0005] Upadacitinib has a chiral center. The marketed product is in the (3S,4R) configuration, and its enantiomer is in the (3R,4S) configuration. The structural formula is shown as the compound of formula (II):

[0006] .

[0007] The enantiomer (3R,4S) may affect the safety and efficacy of the drug, so it is necessary to strictly control its content in upadacitinib raw materials and preparations. At present, the quality detection methods for upadacitinib mainly focus on the fields of impurity detection and intermediate analysis and detection. For example, Patent CN118311186B discloses a method for determining upadacitinib intermediates and their enantiomers by normal-phase high-performance liquid chromatography. In this method, the chromatographic column is packed with amylose tris(3,5-dimethylphenylcarbamate) coated silica gel, and the mobile phase is a mixture of a monohydric alcohol, ethylenediamine, a monobasic acid, and a liquid alkane with a carbon chain of C6-C7. This system is a normal-phase system and cannot be directly used for the detection of enantiomers in liquid preparations such as upadacitinib oral solutions. Patent CN117705987A discloses a method for detecting impurities in upadacitinib intermediates by high-performance liquid chromatography, using phosphate buffer solution and methanol as the mobile phase, with gradient elution combined with ultraviolet detection. However, this method only detects non-chiral impurities generated during the synthesis process, does not involve the separation and detection of enantiomers, and does not use a chiral chromatographic column, so it cannot distinguish upadacitinib from its enantiomers. Patent CN117805268A discloses a liquid chromatography method for detecting various impurities in upadacitinib raw materials, using formic acid / formic acid ammonium buffer solution and methanol for gradient elution. Although this method can separate some impurities with similar structures, its mobile phase system and chromatographic conditions still cannot effectively separate the chiral enantiomers of upadacitinib.

[0008] Therefore, it is necessary to establish a detection method for determining the content of upadacitinib enantiomers in upadacitinib raw materials and preparations. Summary of the Invention

[0009] The present invention provides a method for separating and analyzing upadacitinib enantiomers using a liquid chromatography system. This method is easy to operate, has good general applicability, good specificity, linearity, and precision, high sensitivity, and good method durability, and can be used for the quality control of upadacitinib enantiomers in upadacitinib raw materials and preparations.

[0010] The present invention provides a liquid chromatography method for detecting upadacitinib enantiomers, which at least includes the following steps:

[0011] (1) Preparation of system suitability solution: Weigh appropriate amounts of the reference substances of upadacitinib and upadacitinib enantiomers, dissolve them in a solvent and dilute to prepare a system suitability solution;

[0012] (2) Preparation of test solution: Take an appropriate amount of upadacitinib test sample and dilute it with a solvent to prepare a test solution;

[0013] (3)Chromatographic test: Take appropriate amounts of the system suitability solution and the test solution, inject them into a liquid chromatograph, record the chromatogram, and calculate the content of upadacitinib enantiomers by area normalization method. The liquid chromatograph is a reversed-phase high-performance liquid chromatograph. The packing material of the chromatographic column in the liquid chromatograph is cellulose tris(3-chloro-4-methylphenylcarbamate) silica gel. The mobile phase in the reversed-phase high-performance liquid chromatography detection is a mixture of phosphate buffer solution and acetonitrile. The volume ratio between the phosphate buffer solution and acetonitrile is 65:35, and the concentration of the phosphate is 20 mmol / L.

[0014] The inventors found that when the packing material of the chromatographic column is cellulose tris(3-chloro-4-methylphenylcarbamate) silica gel, good separation can be achieved between upadacitinib and its enantiomers.

[0015] As a preferred technical solution, the phosphate in the phosphate buffer solution is selected from at least one of sodium dihydrogen phosphate, disodium hydrogen phosphate, trisodium phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, tripotassium phosphate, diammonium hydrogen phosphate, diphosphoric acid hydrogen ammonium, and ammonium phosphate.

[0016] As a preferred technical solution, the phosphate in the phosphate buffer solution is selected from potassium dihydrogen phosphate.

[0017] As a preferred technical solution, the phosphate in the phosphate buffer solution is selected from sodium dihydrogen phosphate.

[0018] As a preferred technical solution, the chromatographic conditions of the reversed-phase high-performance liquid chromatograph are as follows:

[0019] Detector: UV detector;

[0020] Chromatographic column: A chromatographic column packed with cellulose tris(3-chloro-4-methylphenylcarbamate) silica gel;

[0021] Mobile phase: Phosphate buffer solution - acetonitrile;

[0022] Flow rate: 0.5 - 1.0 ml / ml;

[0023] Detection wavelength: 230 nm;

[0024] Column temperature of the chromatographic column: 25°C - 35°C;

[0025] Injection volume: 10 μl.

[0026] As a preferred technical solution, the solvent in the system suitability solution and the test solution is selected from at least one of acetonitrile, water, and methanol.

[0027] As a preferred technical solution, the solvents in the system suitability solution and the test solution are both mixtures of acetonitrile and water, and the volume ratio of acetonitrile to water is 1:1.

[0028] As a preferred technical solution, the preparation of the system suitability solution: Weigh appropriate amounts of the reference substances of upadacitinib and its enantiomer respectively, dissolve and dilute them with an acetonitrile-water solution with a volume ratio of 1:1 to prepare a mixed solution containing 0.5 mg of upadacitinib and 5 μg of upadacitinib enantiomer per 1 ml as the system suitability solution.

[0029] As a preferred technical solution, the preparation of the test solution: Take an appropriate amount of upadacitinib test substance, dissolve and dilute it with an acetonitrile-water solution with a volume ratio of 1:1 to prepare a mixed solution containing 0.5 mg per 1 ml as the test solution.

[0030] As a preferred technical solution, the method for separating and analyzing upadacitinib enantiomers using a liquid chromatography system includes the following steps:

[0031] (1) Preparation of the system suitability solution: Weigh appropriate amounts of the reference substances of upadacitinib and its enantiomer respectively, dissolve and dilute them with an acetonitrile-water solution with a volume ratio of 1:1 to prepare a mixed solution containing 0.5 mg of upadacitinib and 5 μg of upadacitinib enantiomer per 1 ml as the system suitability solution;

[0032] (2) Preparation of the test solution: Take an appropriate amount of upadacitinib test substance, dissolve and dilute it with an acetonitrile-water solution with a volume ratio of 1:1 to prepare a mixed solution containing 0.5 mg per 1 ml as the test solution;

[0033] (3) Chromatographic test: Take appropriate amounts of the system suitability solution and the test solution, inject them into the liquid chromatograph, record the chromatogram, and calculate the content of upadacitinib enantiomers by the area normalization method. The liquid chromatograph is a reverse-phase high-performance liquid chromatograph, and the filler of the chromatographic column in the liquid chromatograph is cellulose tris(3-chloro-4-methylphenylcarbamate) silica gel.

[0034] As a preferred technical solution, the chromatographic conditions of the reverse-phase high-performance liquid chromatograph are as follows:

[0035] Detector: UV detector;

[0036] Chromatographic column: A chromatographic column filled with cellulose tris(3-chloro-4-methylphenylcarbamate) silica gel;

[0037] Mobile phase: Phosphate buffer - acetonitrile;

[0038] Flow rate: 0.5 - 1.0 ml / ml;

[0039] Detection wavelength: 230 nm;

[0040] Column temperature of the chromatographic column: 25 °C to 35 °C;

[0041] Injection volume: 10 μl.

[0042] As a preferred technical solution, the column temperature of the chromatographic column is 30 °C.

[0043] Among the substances involved in the technical solution of the present invention, there is only one enantiomer, i.e., upadacitinib enantiomer, and the enantiomers referred to in the text are all upadacitinib enantiomers.

[0044] The method for separating and analyzing upadacitinib enantiomers by the liquid chromatography system of the present invention can be used for the quality control of upadacitinib enantiomers in upadacitinib raw materials and preparations. The upadacitinib preparations include upadacitinib sustained-release tablets, upadacitinib oral solutions and other preparation forms of upadacitinib.

[0045] Beneficial effects

[0046] (1) The present invention realizes the effective separation and detection of upadacitinib and its enantiomers. The reverse-phase chromatography system used has the advantages of stable chromatographic solvent performance, strong separation ability, simple operation process, easier equilibration, etc., and has stronger universality. It can be directly used for the detection of liquid preparations such as upadacitinib oral solutions, with more convenient operation, and can more conveniently realize the quality control of the product production process, improve the quality of the final product, and ensure the safety and effectiveness of clinical medication.

[0047] (2) In the detection method of the present invention, the quantification limit of upadacitinib enantiomer is 2.18 ng, and the detection limit is 0.65 ng. The lower detection limit and quantification limit can realize the analysis and detection of low-concentration upadacitinib enantiomers. Moreover, the detection and analysis method in the present invention has high sensitivity, high accuracy and good repeatability, providing a more simple, stable and reliable analysis and detection method for the research and development and production process of upadacitinib raw materials and preparations. Description of the drawings

[0048] Figure 1 It is the chromatogram under Detection condition 1 of Example 2.

[0049] Figure 2 It is the chromatogram under Detection condition 2 of Example 2.

[0050] Figure 3 It is the chromatogram under Detection condition 3 of Example 2.

[0051] Figure 4 It is the chromatogram under Detection condition 4 of Example 2.

[0052] Figure 5 It is the chromatogram of the system suitability solution in Example 3.

[0053] Figure 6 It is the chromatogram of the test solution in Example 3.

[0054] Figure 7 It is the stability profile of the spiked test solution in Example 4.

[0055] Figure 8 It is the stability profile of the reference solution in Example 4.

[0056] Figure 9 It is the linearity graph of upadacitinib in Example 5.

[0057] Figure 10 It is the linearity graph of the upadacitinib enantiomer in Example 5.

[0058] Figure 11 It is the chromatogram of the limit of quantitation test for upadacitinib and its enantiomer in Example 6.

[0059] Figure 12 It is the chromatogram of the limit of detection test for upadacitinib and its enantiomer in Example 6.

[0060] Figure 13 It is the chromatogram of the precision test for the upadacitinib enantiomer in Example 7. Detailed Description of the Invention

[0061] The specific embodiments of the present invention will be described in detail below. It should be noted that the embodiments described below are exemplary and are only used to explain the present invention and should not be construed as a limitation to the present invention.

[0062] The reference substance of upadacitinib was purchased from Wanyang Hengshui Pharmaceutical Co., Ltd., and the reference substance of the upadacitinib enantiomer was purchased from Shenzhen Hengfeng Wanda Medical Technology Co., Ltd. The liquid chromatography method for detecting the upadacitinib enantiomer in the present invention is applicable not only to upadacitinib oral solution and other upadacitinib-related preparations, but also to the detection of enantiomers in upadacitinib raw materials. In the following examples, only upadacitinib oral solution is used as the test sample to exemplify the implementation process of the detection method. The upadacitinib test sample was prepared by the inventor according to the formula of the upadacitinib oral solution (trade name: "RINVOQ LQP", NDA number: 218347) that has been approved for marketing in the United States. The system suitability solution and the test solution in the following examples were prepared by the following methods.

[0063] Preparation of system suitability solution: Weigh appropriate amounts of upadacitinib and its enantiomer reference substances, dissolve and dilute with an acetonitrile-water solution with a volume ratio of 1:1 to prepare a mixed solution containing 0.5 mg of upadacitinib and 5 μg of upadacitinib enantiomer per 1 ml as the system suitability solution.

[0064] Preparation of test solution: Take an appropriate amount of upadacitinib test substance, dissolve and dilute with an acetonitrile-water solution with a volume ratio of 1:1 to prepare a mixed solution containing 0.5 mg per 1 ml as the test solution.

[0065] Example 1 Chromatographic condition screening test

[0066] The instruments and some chromatographic conditions in this example are as follows:

[0067] Instrument: Hitachi CM 5110 liquid chromatograph, ultraviolet detector; Flow rate for detection conditions 1 - 9: 1.0 ml / min. Detection wavelength: 230 nm; Column temperature of the chromatographic column: 30 °C; Injection volume: 10 μl, and the mobile phase is isocratic elution. Based on the above chromatographic conditions, exploration of the chromatographic column and mobile phase conditions was carried out, as shown in Table 1 below.

[0068] Table 1 - Exploration of different chromatographic columns and mobile phase conditions

[0069]

[0070] Based on the above test results, it can be seen that using a chromatographic column filled with cellulose tris(3-chloro-4-methylphenylcarbamate) silica gel and combining a mixed solution of potassium dihydrogen phosphate solution and acetonitrile as the mobile phase can achieve better separation between upadacitinib and its enantiomer.

[0071] Example 2 System suitability test

[0072] In this example, except that the column temperature of the chromatographic column and the composition of the mobile phase are different from those in test condition 10 of Example 1, the remaining detection conditions are the same as those in test condition 10 of Example 1. The separation situation between upadacitinib and its enantiomer is shown in Table 2 specifically.

[0073] Table 2 - Detection situation at different volume ratios between potassium dihydrogen phosphate solution and acetonitrile

[0074]

[0075] Based on the above test results, under the chromatographic conditions of detection conditions 1 to 4, the resolution between the main peak of upadacitinib and its enantiomer peak is good, and the system suitability of this detection method is better.

[0076] Example 3 Resolution test

[0077] Test according to the high performance liquid chromatography method (General Principles 0512 of Chinese Pharmacopoeia 2020 Edition). Use a chromatographic column packed with cellulose tris(3-chloro-4-methylphenylcarbamate) silica gel (Phenomenex Lux Cellulose-2 250mm×4.6mm, 5μm). Use a mobile phase consisting of 20 mmol / L potassium dihydrogen phosphate solution - acetonitrile (volume ratio 65:35); the flow rate is 0.6 ml per minute; the detection wavelength is 230 nm; the column temperature is 30°C. Precisely measure 10 μl of the system suitability test solution and inject it into the liquid chromatograph, record the chromatogram, and the results are shown in Table 3 and Figure 5 。The resolution between the upadacitinib main peak and the enantiomer peak should meet the requirements. Precisely measure 10 µl of the test solution and inject it into the liquid chromatograph, record the chromatogram, and the results are shown in Table 4 and Figure 6 。If there are chromatographic peaks in the chromatogram of the test solution with the same retention time as the upadacitinib enantiomer peak, calculate by the external standard method based on the peak area of the main component, and it shall not exceed 0.5% of the labeled amount.

[0078] Table 3 - Test Results of System Suitability Solution

[0079]

[0080] Table 4 - Test Results of Test Solution

[0081]

[0082] Results: In the chromatogram of the system suitability solution, the resolution between the upadacitinib main peak and its enantiomer peak is good. In the chromatogram of the test solution, no enantiomer is detected, indicating that the test solution does not contain upadacitinib enantiomer and meets the requirements.

[0083] Example 4 Solution Stability Test

[0084] Reference solution: Take 5 mg of upadacitinib reference substance, weigh it precisely, place it in a 100 ml volumetric flask, dissolve it with an acetonitrile-water solution with a volume ratio of 1:1 and quantitatively dilute it to the scale, shake well. Precisely measure 1 ml and place it in a 20 ml volumetric flask, dilute it to the scale with an acetonitrile-water solution with a volume ratio of 1:1, and shake well.

[0085] Enantiomer stock solution: Take 5 mg of upadacitinib enantiomer reference substance, weigh it precisely, place it in a 200 ml volumetric flask, dissolve it with an acetonitrile-water solution with a volume ratio of 1:1 and quantitatively dilute it to the scale, shake well.

[0086] Spiked test solution: Precisely measure 5 ml of the test solution and 1 ml of the enantiomer stock solution, place them in the same 10 ml volumetric flask, dissolve and dilute to the scale with an acetonitrile-water solution with a volume ratio of 1:1, and shake well.

[0087] Take the reference solution and the spiked test solution, place them under natural light at room temperature, and precisely measure 10 μl at 0 and 24 hours respectively, inject them into the liquid chromatograph (the chromatographic conditions in this example are the same as those in Test Condition 10 of Example 1), record the chromatograms, compare with those at 0 hour, calculate the change values of the contents of upadacitinib and its enantiomer, and the results are shown in Tables 5, 6 and Figure 7 and Figure 8 , where Figure 7 is the stability chromatogram of the spiked test solution, Figure 8 is the stability chromatogram of the reference solution.

[0088] Table 5 Results of the stability test of the spiked test solution for enantiomer detection

[0089]

[0090] Table 6 Results of the stability test of the upadacitinib reference solution

[0091]

[0092] The results show that the reference solution and the spiked test solution are stable within 24 hours under natural light at room temperature.

[0093] Example 5 Linear test

[0094] Enantiomer solution: Take 5 mg of the enantiomer reference substance, weigh it precisely, place it in a 20-ml volumetric flask, dissolve it with an acetonitrile-water solution with a volume ratio of 1:1, and quantitatively dilute it to the mark, then shake well.

[0095] Upadacitinib solution: Take 5 mg of the upadacitinib reference substance, weigh it precisely, place it in a 20-ml volumetric flask, dissolve it with an acetonitrile-water solution with a volume ratio of 1:1, and quantitatively dilute it to the mark, then shake well.

[0096] Linear stock solution: Precisely measure 2 ml each of the enantiomer stock solution and the upadacitinib stock solution, place them in the same 20-ml volumetric flask, dilute them to the mark with an acetonitrile-water solution with a volume ratio of 1:1, and shake well.

[0097] Preparation of linear solutions: Precisely measure 0.1 ml, 0.5 ml, 1.0 ml, 1.5 ml, and 2.0 ml of the linear stock solution respectively and place them in 10-ml volumetric flasks, dilute them to the mark with an acetonitrile-water solution with a volume ratio of 1:1, and shake well. These are used as Linear Solutions 1-5 in sequence. Precisely measure 10 μl of each of the above series of solutions and inject them into the liquid chromatograph, record the chromatograms (the chromatographic conditions in this example are the same as those in Test Condition 10 of Example 1). Plot a curve with the peak area against the concentration, and calculate the regression equation, correlation coefficient and intercept by the least squares method. The method shows good linearity, and the results are shown in Table 7 and Figure 9 andFigure 10 , where Figure 9 is the linear graph of upadacitinib, Figure 10 and

[0098] Table 7 Results of Linear Test for Enantiomers

[0099]

[0100] Example 6 Quantitative Limit and Detection Limit Tests

[0101] Take appropriate amounts of reference substances of upadacitinib and its enantiomers, dissolve them separately with an acetonitrile aqueous solution with a volume ratio of 1:1 and gradually dilute, shake well, accurately measure 10 μl respectively and inject them into the liquid chromatograph, record the chromatogram (the chromatographic conditions in this example are the same as those in Test Condition 10 of Example 1), until the response values of each sample are about 10 times and 3 times higher than the noise signal, which are the quantitative limit and detection limit respectively. The results are shown in Table 8 and Figure 11 、 Figure 12 , Figure 11 is the chromatogram of the quantitative limit of upadacitinib and its enantiomers, Figure 12 and

[0102] Table 8 Results Table of Quantitative Limit and Detection Limit of Enantiomers

[0103]

[0104] Example 7 Precision Test

[0105] Take the test sample of upadacitinib, prepare 6 portions of test sample solutions with the same concentration, conduct the determination according to the chromatographic conditions and detection method in Test Condition 10 of Example 1, and calculate the relative standard deviation of the detected amount and the detected amount of upadacitinib enantiomers. The results show that none were detected, and the method precision is good. The results are shown in Table 9 and Figure 13 .

[0106] Table 9 Results of Precision Test for Enantiomer Inspection

[0107]

Claims

1. A liquid chromatography method for detecting upadacitinib enantiomers, characterized in that: At least the following steps are included: (1) Preparation of system suitability solution: Weigh appropriate amounts of upadacitinib and upadacitinib enantiomer reference substances, dissolve in solvent and dilute to prepare system suitability solution; (2) Preparation of test solution: Take an appropriate amount of upadacitinib test sample and dilute it with solvent to prepare the test solution; (3) Chromatographic test: Take appropriate amounts of system suitability solution and test sample solution, inject them into liquid chromatograph, record the chromatogram, and calculate the content of upadacitinib enantiomers by area normalization method; The liquid chromatograph is a reversed-phase high performance liquid chromatograph, the filler of the chromatographic column in the liquid chromatograph is cellulose-tris(3-chloro-4-methylphenylcarbamate) silica gel, the mobile phase in the reversed-phase high performance liquid chromatography detection is a mixture of phosphate buffer and acetonitrile, the volume ratio between the phosphate buffer and acetonitrile is 65:35, and the concentration of phosphate in the phosphate buffer in the mobile phase is 20 mmol / L; the solvents in the system suitability solution and the test solution are both a mixture of acetonitrile and water, and the volume ratio of acetonitrile to water is 1:

1.

2. The liquid chromatography method for detecting upadacitinib enantiomers according to claim 1, characterized in that: The phosphate in the phosphate buffer is selected from at least one of sodium dihydrogen phosphate, disodium hydrogen phosphate, trisodium phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, tripotassium phosphate, ammonium dihydrogen phosphate, diammonium hydrogen phosphate, and triammonium phosphate.

3. The liquid chromatography method for detecting upadacitinib enantiomers according to claim 2, characterized in that: The phosphate in the phosphate buffer is selected from potassium dihydrogen phosphate.

4. The liquid chromatography method for detecting upadacitinib enantiomers according to claim 2, characterized in that: The phosphate in the phosphate buffer is selected from sodium dihydrogen phosphate.

5. The liquid chromatography method for detecting upadacitinib enantiomers according to claim 1, characterized in that: The chromatographic conditions of the reversed-phase high performance liquid chromatograph are as follows: Detector: UV detector; Chromatographic column: chromatographic column with cellulose-tris (3-chloro-4-methylphenylcarbamate) silica gel as filler; Mobile phase: phosphate buffer-acetonitrile; Flow rate: 0.5~1.0ml / ml; Detection wavelength: 230nm; Chromatographic column temperature: 25℃~35℃; Injection volume: 10µl.

6. The liquid chromatography method for detecting upadacitinib enantiomers according to claim 1, characterized in that: Preparation of the system suitability solution: Weigh appropriate amounts of upadacitinib and upadacitinib enantiomer reference substances, respectively, dissolve and dilute with acetonitrile aqueous solution at a volume ratio of 1:1 to prepare a mixed solution containing 0.5 mg of upadacitinib and 5 µg of upadacitinib enantiomer per 1 ml, as the system suitability solution.

7. The liquid chromatography method for detecting upadacitinib enantiomers according to claim 1, characterized in that: Preparation of the test solution: Take an appropriate amount of upadacitinib test sample, add acetonitrile aqueous solution with a volume ratio of 1:1 to dissolve and dilute to prepare a mixed solution containing 0.5 mg per 1 ml, which is used as the test solution.

8. The liquid chromatography method for detecting upadacitinib enantiomers according to claim 5, characterized in that: The column temperature of the chromatographic column is 30°C.

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