Method for separating and detecting formamidine in starting material of rucotinib
Through high-performance liquid chromatography combined with the use of specific fillers and mobile phases, the problem of formamidine detection in 4-chloropyrrolopyrimidine is solved, efficient and accurate formamidine detection is achieved, and the ability of product quality control is improved.
Patent Information
- Application Number
- CN202510055613.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-14
- Publication Date
- 2025-04-15
AI Technical Summary
The lack of analytical methods for formamidine in 4-clopyrrolopyrimidine in the prior art, making it difficult to control the quality of the material and the rucortinib product.
High performance liquid chromatography was used to separate the acidic ion pair group into the filler with a long carbon chain ligand, and a mixed solution of acidic aqueous solution and acetonitrile were combined as the mobile phase to achieve separation and detection of formamidine by isometric elution.
Effective separation and detection of formamidine in 4-chloropyrrolopyrimidine is achieved, with simple operation, low cost, short analysis time, quantitative limit less than 1μg/ml and detection limit less than 0.03μg/ml, good accuracy and high sensitivity, which can more conveniently realize quality control in product production process.
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Abstract
Description
Technical Field
[0001] The invention relates to the field of drug analysis, and in particular to a method for detecting formamidine. Background Art
[0002] Ruxolitinib is a targeted drug for the JAK-signaling and transcription activator protein signaling pathway. It was first used to treat myelofibrosis, significantly improving patients' symptoms, shrinking the spleen and prolonging survival. Ruxolitinib inhibits cell proliferation, induces apoptosis of malignant cells and reduces the production of proinflammatory cytokines by inhibiting JAK-induced signal transduction and transcription activator phosphorylation. Currently, ruxolitinib has been widely used in basic and clinical research on a variety of blood system diseases, such as myeloproliferative neoplasms, acute lymphoblastic leukemia, graft-versus-host disease, hemophagocytic syndrome, aplastic anemia, multiple myeloma, etc.
[0003] 4-Chloropyrrolopyrimidine can be used as a starting material for the synthesis of ruxolitinib, and is used to construct the pyrrolopyrimidine group in the main structure of ruxolitinib. Formamidine is one of the important materials in the synthesis route of 4-chloropyrrolopyrimidine. Based on the QbD (Quality by Design) concept, controlling or considering formamidine in 4-chloropyrrolopyrimidine will be more conducive to ensuring the controllable quality of 4-chloropyrrolopyrimidine products, thereby helping to control the product quality of ruxolitinib.
[0004]
[0005] However, there is no literature report on the analytical method of formamidine in 4-chloropyrrolopyrimidine. Therefore, in order to control the product quality of 4-chloropyrrolopyrimidine and ruxolitinib, it is necessary to develop a method for detecting formamidine in 4-chloropyrrolopyrimidine. Summary of the invention
[0006] The object of the present invention is to provide a method for separating and determining formamidine in 4-chloropyrrolopyrimidine, a starting material of ruxolitinib. The method can effectively separate and accurately detect the content of formamidine in 4-chloropyrrolopyrimidine, which is beneficial to controlling the quality of the starting material and ruxolitinib.
[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: A method for separating and detecting formamidine in 4-chloropyrrolopyrimidine is a high performance liquid chromatography method, which is characterized by using a mixed chromatographic column with a long carbon chain ligand embedded in an acidic ion pair group as a filler, using a mixed solution of an acidic aqueous solution and acetonitrile as a mobile phase, and adopting isocratic elution.
[0008] Furthermore, in the separation and detection method of the present invention, the chromatographic column is SiELC Primesep 100.
[0009] Furthermore, in the separation and detection method of the present invention, the acidic aqueous solution is a phosphoric acid aqueous solution, a perchloric acid aqueous solution or a trifluoroacetic acid aqueous solution, preferably a phosphoric acid aqueous solution.
[0010] Furthermore, in the separation and detection method of the present invention, the volume ratio of the acidic aqueous solution to acetonitrile in the mobile phase is (63-67): (37-33), preferably 65:35.
[0011] Furthermore, in the separation and detection method of the present invention, the volume ratio of acid to water in the acidic aqueous solution is 0.05% to 0.5%, preferably 0.05%.
[0012] Furthermore, in the above separation and detection method of the present invention, the flow rate of the mobile phase in the separation and detection method is 0.9~1.1ml / min, preferably 1.0ml / min.
[0013] Furthermore, in the separation and detection method of the present invention, the temperature of the chromatographic column in the separation and detection method is 28-32°C, preferably 30°C.
[0014] Furthermore, the separation and detection method of the present invention adopts an ultraviolet detector with a detection wavelength of 195-220 nm, preferably 210 nm.
[0015] In a specific embodiment, the separation and detection method of the present invention comprises the following steps: Preparation of system suitability solution: Take appropriate amount of 4-chloropyrrolopyrimidine, the starting material of ruxolitinib, and formamidine hydrochloride reference substance, add mobile phase to dissolve and dilute to prepare system suitability solution; Preparation of test solution: Take an appropriate amount of 4-chloropyrrolopyrimidine, the starting material of ruxolitinib, add mobile phase to dissolve and dilute to prepare a test solution; Take the system suitability solution and the test solution, inject them into the high performance liquid chromatograph for detection, and record the chromatogram.
[0016] Beneficial effects of the present invention: The method of the present invention uses a mixed chromatographic column with a long carbon chain ligand embedded in an acidic ion pair group as a filler, and adopts a system with a mixed solution of an acidic aqueous solution and acetonitrile as a mobile phase, thereby achieving effective separation and detection of formamidine in 4-chloropyrrolopyrimidine, the starting material of ruxolitinib. The method of the present invention is simple to operate, does not require derivatization, has low cost, and has a short analysis time. At the same time, the quantitative limit is lower than 1 μg / ml, the detection limit is lower than 0.03 μg / ml, has good accuracy and high sensitivity, and can more conveniently and accurately achieve quality control of the production process of 4-chloropyrrolopyrimidine and ruxolitinib products, which is conducive to improving the quality of the final product. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] Figure 1This is the HPLC chromatogram of the system suitability of Example 3; Figure 2 This is the HPLC chromatogram of the blank solution in the specificity experiment of Example 3; Figure 3 This is the HPLC chromatogram of the reference substance solution in the specificity experiment of Example 3; Figure 4 This is the HPLC chromatogram of the blank test solution in the specificity experiment of Example 3; Figure 5 This is the linear investigation diagram of formamidine in Example 4; Figure 6 The HPLC chromatogram of the quantitative limit of formamidine in Example 5 is shown in FIG. DETAILED DESCRIPTION
[0018] The following examples are used to further illustrate and understand the spirit of the present invention, but are not intended to limit the scope of protection of the present invention in any way.
[0019] In the following examples, the instruments and reagents used are as follows: Instrument: Shimadzu LC-20AD high performance liquid chromatograph; Reagents: Phosphoric acid, formic acid, trifluoroacetic acid, perchloric acid, and acetonitrile were all of chromatography grade, and water was ultrapure water; The formamidine hydrochloride reference substance was purchased from Anergy, with a salt conversion coefficient of 0.547.
[0020] Example 1 Screening of mobile phase Chromatographic conditions: Mobile phase 1: 0.05% phosphoric acid aqueous solution-acetonitrile (65:35, v / v); Mobile phase 2: 0.05% formic acid aqueous solution-acetonitrile (65:35, v / v); Mobile phase 3: 0.05% perchloric acid aqueous solution-acetonitrile (65:35, v / v); Mobile phase 4: 0.05% trifluoroacetic acid aqueous solution-acetonitrile (65:35, v / v); Chromatographic column: SiELC Primesep 100 (150 mm*4.6 mm, 3 μm); Detector and wavelength: PDA-210nm; Flow rate: 1.0ml / min; Injection volume: 10 μl; Column temperature: 30°C; Elution mode: isocratic elution; Solvent: Mobile phase.
[0021] Experimental steps: (1) Preparation of mobile phase: 0.05% phosphoric acid aqueous solution-acetonitrile (65:35, v / v): Take 1000ml of ultrapure water, add 0.5ml of phosphoric acid, mix thoroughly to obtain 0.05% phosphoric acid aqueous solution; Take 650ml of 0.05% phosphoric acid aqueous solution, add 350ml of acetonitrile, mix thoroughly to obtain; 0.05% formic acid aqueous solution-acetonitrile (65:35, v / v): Take 1000ml of ultrapure water, add 0.5ml of formic acid, mix thoroughly to obtain 0.05% formic acid aqueous solution; take 650ml of 0.05% formic acid aqueous solution, add 350ml of acetonitrile, mix thoroughly to obtain; 0.05% perchloric acid aqueous solution-acetonitrile (65:35, v / v): Take 1000ml of ultrapure water, add 0.5ml of perchloric acid, mix thoroughly to obtain 0.05% perchloric acid aqueous solution; Take 650ml of 0.05% perchloric acid aqueous solution, add 350ml of acetonitrile, mix thoroughly to obtain; 0.05% trifluoroacetic acid aqueous solution-acetonitrile (65:35, v / v): Take 1000ml of ultrapure water, add 0.5ml of trifluoroacetic acid, mix well to obtain 0.05% trifluoroacetic acid aqueous solution; take 650ml of 0.05% trifluoroacetic acid aqueous solution, add 350ml of acetonitrile, mix well to obtain; (2) Preparation of system suitability solution: Take appropriate amount of 4-chloropyrrolopyrimidine and formamidine hydrochloride reference substances, dissolve them in mobile phase and quantitatively dilute them to make a solution containing 2 mg of 4-chloropyrrolopyrimidine and 0.003 mg of formamidine per 1 ml, which is used as the system suitability solution.
[0022] (3) Detection: Inject the above system suitability solution into a high performance liquid chromatograph and perform the measurement under four different mobile phase conditions. Record the chromatograms.
[0023] The results show that when phosphoric acid, perchloric acid or a mixed solution of trifluoroacetic acid aqueous solution and acetonitrile are used as the mobile phase, the chromatographic conditions can achieve the separation and detection of formamidine in 4-chloropyrrolopyrimidine. Among them, when a mixed solution of phosphoric acid aqueous solution and acetonitrile is used, the sensitivity (S / N) is the highest and the minimum separation degree from adjacent impurities is the largest. The results are shown in Table 1.
[0024]
[0025] Example 2 Screening of mobile phase Chromatographic conditions: Mobile phase 1: 0.05% phosphoric acid aqueous solution-acetonitrile (65:35, v / v); Mobile phase 2: 0.2% phosphoric acid aqueous solution-acetonitrile (65:35, v / v); Mobile phase 3: 0.5% phosphoric acid aqueous solution-acetonitrile (65:35, v / v); Elution mode: isocratic elution; Solvent: mobile phase; Other chromatographic conditions were the same as in Example 1.
[0026] Experimental steps: (1) Preparation of mobile phase: 0.05% phosphoric acid aqueous solution-acetonitrile (65:35, v / v): same as Example 1; 0.2% phosphoric acid aqueous solution-acetonitrile (65:35, v / v): Take 1000ml of ultrapure water, add 2ml of phosphoric acid, mix thoroughly to obtain 0.2% phosphoric acid aqueous solution; Take 650ml of 0.2% phosphoric acid aqueous solution, add 350ml of acetonitrile, mix thoroughly to obtain; 0.5% phosphoric acid aqueous solution-acetonitrile (65:35, v / v): Take 1000ml of ultrapure water, add 5ml of phosphoric acid, mix thoroughly to obtain 0.5% phosphoric acid aqueous solution; take 650ml of 0.5% phosphoric acid aqueous solution, add 350ml of acetonitrile, mix thoroughly to obtain; (2) Preparation of system suitability solution: same as in Example 1; (3) Detection: Inject the above system suitability solution into a high performance liquid chromatograph and perform the measurement under three different mobile phase conditions. Record the chromatograms.
[0027] The results showed that the above three mobile phases can achieve the separation and detection of formamidine in 4-chloropyrrolopyrimidine, among which, when 0.2% phosphoric acid aqueous solution-acetonitrile (65:35, v / v) was used as the mobile phase, the retention time was moderate. See Table 2.
[0028] Example 3 Specificity and system suitability experiments
[0029] System suitability experiment: Chromatographic conditions: Mobile phase: 0.05% phosphoric acid aqueous solution-acetonitrile (65:35, v / v); Solvent: mobile phase; Other chromatographic conditions were the same as in Example 1.
[0030] Experimental steps: (1) Preparation of mobile phase: same as in Example 1; (2) System suitability solution: Take appropriate amount of 4-chloropyrrolopyrimidine, the starting material of ruxolitinib, and formamidine hydrochloride reference substance, add mobile phase to dissolve and quantitatively dilute to make a solution containing 2 mg of 4-chloropyrrolopyrimidine and 0.003 mg of formamidine per 1 ml; (3) Testing: Inject the above system suitability solution into a high performance liquid chromatograph for testing and record the chromatogram.
[0031] Chromatogram see Figure 1 .
[0032] Specificity experiment:
[0033] Chromatographic conditions: Same as those in Example 3 system suitability experiment; Experimental steps: (1) Preparation of mobile phase: same as in Example 1; (2) Preparation of reference solution: Take an appropriate amount of formamidine hydrochloride reference substance, dissolve it in the mobile phase and quantitatively dilute it to make a solution containing 0.003 mg of formamidine per 1 ml.
[0034] (3) Preparation of blank test solution: Take an appropriate amount of 4-chloropyrrolopyrimidine, add mobile phase to dissolve it and quantitatively dilute it to make a solution containing 2 mg of 4-chloropyrrolopyrimidine per 1 ml.
[0035] (3) Detection: Inject blank solvent, the above-mentioned reference solution, and blank test solution into a high performance liquid chromatograph for determination and record the chromatogram.
[0036] Chromatogram see Figure 2~Figure 4 The results showed that the blank solvent and blank test sample had no obvious interference with the detection.
[0037] Example 4 Linearity Experiment Chromatographic conditions: same as in Example 3; Experimental steps: (1) Preparation of solution: Formamidine hydrochloride reference stock solution: Weigh about 28 mg of formamidine hydrochloride reference, accurately weigh, place in a 50 ml volumetric flask, add solvent to dissolve and dilute to scale, shake well, then accurately transfer 1 ml, place in a 50 ml volumetric flask, add solvent to dilute to scale, shake well, and the solution is ready. (Contains formamidine concentration of about 30 μg / ml) Standard series solutions: Accurately measure 3, 1, 3, 2, and 3 ml of 30 μg / ml formamidine hydrochloride reference stock solution and place them in 100, 10, 20, 10, and 10 ml volumetric flasks respectively. Add solvent to dilute to the scale, shake well, and make a standard series solution with concentrations of approximately 0.9, 3, 4.5, 6, and 9 μg / ml.
[0038] (2) Detection: Take the above-mentioned standard series solutions of various concentrations and inject them into high performance liquid chromatography for measurement, and record the chromatogram.
[0039] The test results were analyzed and the peak areas were recorded. The peak area external standard method was used for calculation. The peak area was used for linear regression of the concentration to obtain the regression curve. The results are shown in Table 3. The regression curve is shown in Figure 5The regression equation was y=194618x+11725 (y was the peak area, x was the concentration in μg / ml), the correlation coefficient r was 0.9999, and the linearity was good in the concentration range of 0.88μg / ml~8.78μg / ml.
[0040]
[0041] Example 5 Quantitation Limit and Detection Limit Experiment Chromatographic conditions: same as in Example 3; Experimental steps: (1) Preparation of solution: Quantitative limit solution: Take an appropriate amount of formamidine hydrochloride reference substance, add mobile phase to dissolve and quantitatively dilute to make a solution containing 0.0009 mg of formamidine per 1 ml. Prepare 6 copies in parallel.
[0042] Detection limit solution: Take an appropriate amount of formamidine hydrochloride reference substance, add mobile phase to dissolve and quantitatively dilute to make a solution containing 0.00002 mg of formamidine per 1 ml.
[0043] (2) Detection: Take the above-mentioned quantitative limit solution and detection limit solution and inject them into the high performance liquid chromatograph for measurement, and record the chromatogram.
[0044] The test results were analyzed and the peak areas were recorded. The relative standard deviation of the peak areas was calculated. The results are shown in Table 4. The chromatogram of the quantitative limit is shown in Figure 6 .
[0045]
[0046] Example 6 Accuracy Experiment Chromatographic conditions: same as in Example 3; Experimental steps: (1) Preparation of solution: 30% concentration level accuracy solution: Take appropriate amount of 4-chloropyrrolopyrimidine and formamidine hydrochloride reference substances, add mobile phase to dissolve and quantitatively dilute to make a solution containing 2 mg of 4-chloropyrrolopyrimidine and 0.0009 mg of formamidine per 1 ml, and prepare 3 copies in parallel; 100% concentration level accuracy solution: Take appropriate amount of 4-chloropyrrolopyrimidine and formamidine hydrochloride reference substances, add mobile phase to dissolve and quantitatively dilute to make a solution containing 2 mg of 4-chloropyrrolopyrimidine and 0.003 mg of formamidine per 1 ml, and prepare 3 copies in parallel; 200% concentration level accuracy solution: Take appropriate amount of 4-chloropyrrolopyrimidine and formamidine hydrochloride reference substances, add mobile phase to dissolve and quantitatively dilute to make a solution containing 2 mg of 4-chloropyrrolopyrimidine and 0.006 mg of formamidine per 1 ml, and prepare 3 copies in parallel; (2) Detection: Take the above-mentioned 30% concentration level accuracy solution, 100% concentration level accuracy solution and 200% concentration level accuracy solution and inject them into high performance liquid chromatography for measurement, and record the chromatograms.
[0047] According to the test results, the content of formamidine in 4-chloropyrrolopyrimidine was calculated, and the results are shown in Table 5. As shown in Table 5, the recovery rate of formamidine at each level is in the range of 90% to 110%, the average recovery rate of 9 accuracy solutions is 99.8%, and the recovery rate RSD is 1.3%. The accuracy of this method is good.
[0048]
[0049] Example 7 Repeatability Experiment Chromatographic conditions: same as in Example 3; Experimental steps: (1) Preparation of reproducible solution: Take appropriate amount of 4-chloropyrrolopyrimidine and formamidine hydrochloride reference substances, add mobile phase to dissolve and quantitatively dilute to make a solution containing 2 mg of 4-chloropyrrolopyrimidine and 0.003 mg of formamidine per 1 ml. Prepare 6 replicates in parallel. (2) Detection: Take the above reproducible solution and inject it into the high performance liquid chromatograph for measurement and record the chromatogram.
[0050] According to the test results, the formamidine content in 4-chloropyrrolopyrimidine was calculated, and the results are shown in Table 6. As shown in Table 6, the detection method provided by the present invention has high precision, and the RSD value of the formamidine content is 1.0%.
[0051]
[0052] Example 8 Durability Test The robustness of the method was investigated by changing parameters such as flow rate, column temperature, and mobile phase ratio.
[0053] Chromatographic condition 1: flow rate is 0.9 ml / min, and other chromatographic conditions are the same as those in Example 3; Chromatographic condition 2: flow rate is 1.1 ml / min, and other chromatographic conditions are the same as those in Example 3; Chromatographic condition 3: column temperature is 28°C, and other chromatographic conditions are the same as those in Example 3; Chromatographic condition 4: column temperature is 32°C, and other chromatographic conditions are the same as those in Example 3; Chromatographic condition 5: the mobile phase is aqueous phosphoric acid-acetonitrile (63:37), and the other chromatographic conditions are the same as those in Example 3; Chromatographic condition 6: the mobile phase was aqueous phosphoric acid-acetonitrile (67:33), and the other chromatographic conditions were the same as those in Example 3.
[0054] Experimental steps: (1) Preparation of system suitability solution: Take appropriate amount of 4-chloropyrrolopyrimidine and formamidine hydrochloride reference substances, dissolve them in mobile phase and quantitatively dilute them to make a solution containing 2 mg of 4-chloropyrrolopyrimidine and 0.003 mg of formamidine per 1 ml; (2) Detection: Inject the above system suitability solution into a high performance liquid chromatograph for measurement and record the chromatogram.
[0055] The test results show that the minimum separation between formamidine and adjacent chromatographic peaks is greater than 1.5. The results are shown in Table 7.
[0056]
Claims
1. A method for separating and detecting formamidine in 4-chloropyrrolopyrimidine, the method being a high performance liquid chromatography method, characterized in that A mixed chromatographic column with a long carbon chain ligand embedded in an acidic ion pair group as filler was used, a mixed solution of an acidic aqueous solution and acetonitrile was used as the mobile phase, and isocratic elution was adopted; The acidic aqueous solution is a phosphoric acid aqueous solution, a perchloric acid aqueous solution or a trifluoroacetic acid aqueous solution.
2. The separation and detection method according to claim 1, wherein the chromatographic column is SiELC Primesep 100.
3. The separation and detection method according to claim 1, wherein the acidic aqueous solution is a phosphoric acid aqueous solution.
4. The separation and detection method according to claim 1, wherein the volume ratio of the acidic aqueous solution to acetonitrile in the mobile phase is (63-67): (37-33), preferably 65:
35.
5. The separation and detection method according to claim 1 or 4, wherein in the acidic aqueous solution, the volume ratio of acid to water is 0.05% to 0.5%, preferably 0.05%.
6. The separation and detection method according to claim 1, wherein the flow rate of the mobile phase is 0.9-1.1 ml / min, preferably 1.0 ml / min.
7. The separation and detection method according to claim 1, wherein the temperature of the chromatographic column is 28-32°C, preferably 30°C.
8. The separation and detection method according to claim 1, wherein the separation and detection method uses an ultraviolet detector with a detection wavelength of 195 to 220 nm, preferably 210 nm.