Liquid chromatography method for detecting upatinib enantiomer

Abstract

The invention belongs to the technical field of pharmaceutical analysis, and particularly relates to a liquid chromatography method for detecting an upatinib enantiomer, in particular to a reversed-phase liquid chromatography method for detecting the upatinib enantiomer. The method realizes effective separation and detection of upatinib and enantiomers thereof, and the used reversed-phase chromatography system has the advantages of stable chromatographic solvent performance, strong separation capability, simple operation process, easy balance and the like, has strong universality, and is suitable for industrial production. The method can be directly used for detecting preparations such as upatinib sustained-release tablets, upatinib oral solutions and the like and upatinib raw materials, is more convenient to operate, can more conveniently realize quality control of product production process links, improves the quality of final products, and ensures the safety and effectiveness of clinical medication.

Description

Technical Field

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

[0002] Upadacitinib is a selective JAK inhibitor. JAK is an intracellular enzyme that transmits signals generated by the interaction of cytokines or growth factor receptors on the cell membrane, thereby affecting the cellular process of hematopoiesis and immune cell function. Upadacitinib is widely used to treat 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 the structural formula is shown in the compound of formula (I):

[0004] .

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

[0006] .

[0007] However, enantiomers (3R, 4S) may affect the safety and efficacy of drugs, so their content in upadacitinib raw materials and preparations needs to be strictly controlled. At present, the quality detection methods for upadacitinib are mainly concentrated in 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 which the chromatographic column uses amylose-tris (3,5-dimethylphenylcarbamate) coated silica gel as a filler, and the mobile phase is a mixture of monohydric alcohol, ethylenediamine, monobasic acid and liquid alkane with a carbon chain of C6-C7. This system is a normal phase system and cannot be directly used for enantiomer detection of liquid preparations such as upadacitinib oral solution. Patent CN117705987A discloses a method for detecting impurities in upadacitinib intermediates by high performance liquid chromatography, using phosphate buffer and methanol as mobile phases, 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 is impossible to distinguish upadacitinib from its enantiomers. Patent CN117805268A discloses a liquid chromatography method for detecting multiple impurities in upadacitinib API, using formic acid / ammonium formate buffer and methanol gradient elution. Although this method can separate some impurities with similar structures, its mobile phase system and chromatographic conditions still cannot achieve effective separation of upadacitinib chiral enantiomers.

[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 by using a liquid chromatography system. The method is simple to operate, has good general applicability, good specificity, linearity and precision, and has high sensitivity and good method durability. The method can be used for quality control of upadacitinib enantiomers in upadacitinib raw materials and preparations.

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

[0011] (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;

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

[0013] (3) Chromatographic test: Take appropriate amounts of system suitability solution and 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 filler of the chromatographic column in the liquid chromatograph is cellulose-tris(3-chloro-4-methylphenylcarbamate) silica gel, and 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 the phosphate is 20 mmol / L.

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

[0015] As a preferred technical solution, 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.

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

[0017] As a preferred technical solution, the phosphate in the phosphate buffer 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: chromatographic column with cellulose-tris (3-chloro-4-methylphenylcarbamate) silica gel as filler;

[0021] Mobile phase: phosphate buffer-acetonitrile;

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

[0023] Detection wavelength: 230nm;

[0024] Chromatographic column temperature: 25℃~35℃;

[0025] Injection volume: 10µl.

[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 a mixture of acetonitrile and water, and the volume ratio of acetonitrile to water is 1:1.

[0028] As a preferred technical solution, the system suitability solution is prepared by weighing appropriate amounts of upadacitinib and upadacitinib enantiomer reference substances, respectively, adding acetonitrile aqueous solution with a volume ratio of 1:1 to dissolve and dilute 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 test solution is prepared as follows: Take an appropriate amount of upadacitinib test sample, dissolve it in acetonitrile aqueous solution at a volume ratio of 1:1 and dilute it to prepare a mixed solution containing 0.5 mg per 1 ml, which is used as the test solution.

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

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

[0032] (2) Preparation of test solution: Take an appropriate amount of upadacitinib test sample, dissolve it in acetonitrile-water solution at a volume ratio of 1:1, and dilute it to make a mixed solution containing 0.5 mg per 1 ml, which is used as the test solution;

[0033] (3) Chromatographic test: Take appropriate amounts of system suitability solution and test sample 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 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 reversed-phase high performance liquid chromatograph are as follows:

[0035] Detector: UV detector;

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

[0037] Mobile phase: phosphate buffer-acetonitrile;

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

[0039] Detection wavelength: 230nm;

[0040] Chromatographic column temperature: 25℃~35℃;

[0041] Injection volume: 10µl.

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

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

[0044] The method for separating and analyzing upadacitinib enantiomers by a liquid chromatography system of the present invention can be used for 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 achieves 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 balance, etc. It has stronger universality and can be directly used for the detection of liquid preparations such as upadacitinib oral solution. It is more convenient to operate and can more conveniently achieve 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) The limit of quantification of upadacitinib enantiomers in the detection method of the present invention is 2.18 ng, and the detection limit is 0.65 ng. The low detection limit and quantification limit can realize the analysis and detection of low-concentration upadacitinib enantiomers. The detection and analysis method of the present invention has high sensitivity, high accuracy and good repeatability, and provides a simpler, more stable and reliable analysis and detection method for the research and development and production process of upadacitinib raw materials and preparations. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 This is the chromatogram in detection condition 1 of Example 2.

[0049] Figure 2 This is the chromatogram in detection condition 2 of Example 2.

[0050] Figure 3 This is the chromatogram in detection condition 3 of Example 2.

[0051] Figure 4 This is the chromatogram in detection condition 4 of Example 2.

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

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

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

[0055] Figure 8 This is the stability spectrum of the reference solution in Example 4.

[0056] Fig. 9 This is the linear graph of upadacitinib in Example 5.

[0057] Fig.10 This is the linear diagram of upadacitinib enantiomers in Example 5.

[0058] Fig.11 The chromatogram of the quantitative limit test of upadacitinib and its enantiomers in Example 6.

[0059] Fig.12 The chromatogram of the detection limit test of upadacitinib and its enantiomers in Example 6.

[0060] Fig.13 This is the chromatogram of the precision test of upadacitinib enantiomers in Example 7. DETAILED DESCRIPTION

[0061] Specific embodiments of the present invention are 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 limiting the present invention.

[0062] The upadacitinib reference substance was purchased from Wanyang Hengshui Pharmaceutical Co., Ltd., and the upadacitinib enantiomer reference substance was purchased from Shenzhen Hengfeng Wanda Pharmaceutical Technology Co., Ltd. The liquid chromatography method for detecting upadacitinib enantiomers in the present invention is not only applicable to upadacitinib oral solution and other upadacitinib-related preparations, but also applicable to the detection of enantiomers in upadacitinib raw materials. In the following examples, only upadacitinib oral solution is used as an example of the test sample to introduce 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) approved for marketing in the United States. The system suitability solution and the test solution in the following examples were prepared by the following method.

[0063] Preparation of system suitability solution: Weigh appropriate amounts of upadacitinib and upadacitinib enantiomer reference substances, dissolve and dilute with acetonitrile aqueous solution in 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, which is used as the system suitability solution.

[0064] Preparation of test solution: Take an appropriate amount of upadacitinib test sample, dissolve it in acetonitrile aqueous solution at a volume ratio of 1:1 and dilute it to make 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, UV detector; flow rate of detection conditions 1-9: 1.0 ml / min. Detection wavelength: 230 nm; column temperature: 30 °C; injection volume: 10 µl, mobile phase is isocratic elution. Based on the above chromatographic conditions, the chromatographic column and mobile phase conditions were explored, 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 the chromatographic column using cellulose-tris (3-chloro-4-methylphenylcarbamate) silica gel as filler combined with a mixture of potassium dihydrogen phosphate solution and acetonitrile as the mobile phase can achieve better separation between upadacitinib and its enantiomers.

[0071] Example 2 System suitability test

[0072] In this embodiment, except that the chromatographic column temperature and the mobile phase composition are different from the test condition 10 in Example 1, the other detection conditions are the same as the test condition 10 in Example 1. The separation of upadacitinib and its enantiomers is shown in Table 2.

[0073] Table 2- Detection results 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 separation between the main peak of upadacitinib and its enantiomer peak was good, and the detection method had good system applicability.

[0076] Example 3 Separation Test

[0077] According to the high performance liquid chromatography method (General Rules 0512 of the 2020 edition of the Chinese Pharmacopoeia), a chromatographic column (phenomenex lux cellulose-2 250mm×4.6mm, 5μm) filled with cellulose-tris (3-chloro-4-methylphenylcarbamate) silica gel was used, and 20mmol / L potassium dihydrogen phosphate solution-acetonitrile (volume ratio of 65:35) was used as the mobile phase; the flow rate was 0.6ml per minute; the detection wavelength was 230nm; and the column temperature was 30℃. Accurately measure 10μl 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 separation between the main peak of upadacitinib and the enantiomer peak should meet the requirements. Accurately measure 10 μl of the test solution, inject it into the liquid chromatograph, and record the chromatogram. The results are shown in Table 4 and Figure 6 If there is a chromatographic peak in the chromatogram of the test solution with the same retention time as the peak of the upadacitinib enantiomer, the peak area shall be calculated by the main component external standard method and shall not exceed 0.5% of the labeled amount.

[0078] Table 3 - System suitability solution test results

[0079]

[0080] Table 4 - Test results of test solution

[0081]

[0082] Results: In the system suitability solution chromatogram, the separation between the main peak of upadacitinib and its enantiomer peak was good. In the test solution chromatogram, the enantiomer was not detected, proving that the test solution did not contain upadacitinib enantiomers, which met the requirements.

[0083] Example 4 Solution stability test

[0084] Reference solution: accurately weigh 5 mg of upadacitinib reference substance and place it in a 100 ml volumetric flask. Dissolve it in 1:1 acetonitrile aqueous solution by volume and quantitatively dilute it to the mark. Shake well. Accurately measure 1 ml and place it in a 20 ml volumetric flask. Dilute it to the mark with 1:1 acetonitrile aqueous solution by volume and shake well.

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

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

[0087] The reference solution and the spiked test solution were placed at room temperature under natural light. At 0 and 24 hours, 10 μl was accurately measured and injected into a liquid chromatograph (the chromatographic conditions in this embodiment were the same as the chromatographic conditions of test condition 10 in Example 1). The chromatogram was recorded and compared with 0 hours to calculate the change in the content of upadacitinib and its enantiomers. The results are shown in Tables 5, 6 and Figure 7 , Figure 8 ,in Figure 7 is the stability spectrum of the spiked test solution. Figure 8 This is the stability spectrum of the reference solution.

[0088] Table 5 Enantiomer detection and stability test results of spiked test solution

[0089]

[0090] Table 6 Stability test results of upadacitinib reference solution

[0091]

[0092] The results showed that the reference solution and the spiked test solution were stable for 24 hours at room temperature and natural light.

[0093] Example 5 Linearity Test

[0094] Enantiomer solution: Take 5 mg of the enantiomer reference substance, weigh it accurately, place it in a 20 ml volumetric flask, add acetonitrile aqueous solution with a volume ratio of 1:1 to dissolve it and quantitatively dilute it to the scale, and shake well.

[0095] Upadacitinib solution: Take 5 mg of upadacitinib reference substance, accurately weigh it, place it in a 20 ml volumetric flask, add acetonitrile aqueous solution with a volume ratio of 1:1 to dissolve it and quantitatively dilute it to the scale, and shake well.

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

[0097] Preparation of linear solutions: Accurately measure 0.1ml, 0.5ml, 1.0ml, 1.5ml, and 2.0ml of the linear stock solution and place them in 10ml volumetric bottles, dilute to the mark with acetonitrile aqueous solution with a volume ratio of 1:1, shake well, and use them as linear solutions 1 to 5 in turn. Accurately measure 10μl of the above series of solutions respectively, inject them into the liquid chromatograph, and record the chromatogram (the chromatographic conditions in this embodiment are the same as the chromatographic conditions in the test condition 10 in Example 1). Plot a curve of peak area versus concentration, and calculate the regression equation, correlation coefficient, and intercept using the least squares method. The method has good linearity. The results are shown in Table 7 and Fig. 9 , Fig.10 ,in Fig. 9 is the linear graph of upadacitinib, Fig.10 Linear plot of upadacitinib enantiomers.

[0098] Table 7 Enantiomer linearity test results

[0099]

[0100] Example 6 Quantitation Limit and Detection Limit Test

[0101] Take appropriate amounts of upadacitinib and the reference substance of the enantiomer, dissolve them in acetonitrile aqueous solution with a volume ratio of 1:1 and gradually dilute them, shake them well, accurately measure 10 μl of each and inject them into the liquid chromatograph, and record the chromatogram (the chromatographic conditions in this embodiment are the same as the chromatographic conditions in the test condition 10 in Example 1), until the response value of each sample is about 10 times and 3 times higher than the noise signal, which is the quantitative limit and detection limit. The results are shown in Table 8 and Fig.11 , Fig.12 , Fig.11 is the chromatogram of the quantitative limit of upadacitinib and its enantiomers. Fig.12 The chromatograms of detection limits of upadacitinib and its enantiomers.

[0102] Table 8 Results of enantiomer quantification limit and detection limit

[0103]

[0104] Example 7 Precision Test

[0105] Take upadacitinib test sample, prepare 6 test sample solutions of the same concentration, and measure according to the chromatographic conditions and detection method in detection condition 10 in Example 1, and calculate the detection amount of upadacitinib enantiomers and the relative standard deviation of the detection amount. The result is that none of them are detected, and the method has good precision. The results are shown in Table 9 and Fig.13 .

[0106] Table 9 Results of precision test of 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 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 filler of the chromatographic column in the liquid chromatograph is cellulose-tris(3-chloro-4-methylphenylcarbamate) silica gel, and 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.

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: The solvent in the system suitability solution and the test solution is selected from at least one of acetonitrile, water and methanol.

7. The liquid chromatography method for detecting upadacitinib enantiomers according to claim 6, characterized in that: 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.

8. The liquid chromatography method for detecting upadacitinib enantiomers according to claim 7, 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.

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

10. 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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