Method for measuring compound

Abstract

The invention belongs to the field of analytical chemistry, and particularly relates to a method for determining 4-(4-amino-3-fluorophenoxy) pyridine-2-carboxylic acid methylamine and 2-chloro-5-aminobenzotrifluoride in a medicine, the determination method is a liquid chromatography, a C8 chromatographic column is adopted, a mobile phase A is selected from a phosphate solution, a mobile phase B is selected from a nitrile solvent, and gradient elution is carried out. The detection method has good specificity, repeatability and accuracy and high sensitivity, can effectively and accurately perform qualitative and quantitative analysis on the compound, and ensures safety and effectiveness of medicine quality.

Description

Technical Field

[0001] The invention relates to a method for determining a compound, in particular to an analytical method for determining 4-(4-amino-3-fluorophenoxy)pyridine-2-carboxylic acid methylamine and 2-chloro-5-aminotrifluorotoluene in a drug. Background Art

[0002] Genotoxic chemicals in drugs refer to compounds that directly or indirectly damage cell DNA, causing gene mutations or in vivo mutagenesis in organisms, and are carcinogenic or have potential tendencies. They have received great attention at home and abroad in recent years. The chemical 4-(4-amino-3-fluorophenoxy)pyridine-2-carboxylic acid methylamine has potential genotoxicity. In the process of drug preparation, it is necessary to accurately analyze and detect the content of related substances to ensure the purity of active substances in the product and achieve drug quality control.

[0003] There are a few literature reports on the analytical detection methods of the chemical 4-(4-amino-3-fluorophenoxy)pyridine-2-carboxylic acid methylamine. By using the method provided by the prior art literature to detect and analyze the compound 4-(4-amino-3-fluorophenoxy)pyridine-2-carboxylic acid methylamine, it is found that the compound is difficult to extract from the drug, especially the compound is low in content in the drug, and the method cannot be effectively extracted. Due to the influence of other ingredients in the drug, the method reported in the literature is difficult to achieve the analytical detection of the compound in the drug. Compounds 4-(4-amino-3-fluorophenoxy)pyridine-2-carboxylic acid methylamine and 2-chloro-5-aminotrifluorotoluene are substances that need to be precisely controlled in the preparation of regorafenib. Therefore, it is necessary to explore and establish a method that can be simultaneously applicable to the analysis and detection of compounds 4-(4-amino-3-fluorophenoxy)pyridine-2-carboxylic acid methylamine and 2-chloro-5-aminotrifluorotoluene. Summary of the invention

[0004] The object of the present invention is to provide a method for determining a compound. The method utilizes liquid chromatography to simultaneously determine 4-(4-amino-3-fluorophenoxy)pyridine-2-carboxylic acid methylamine and 2-chloro-5-aminotrifluorotoluene, thereby achieving effective control of drug quality and ensuring the safety and controllability of drug quality.

[0005] On one hand, the present invention provides a method for determining a compound, characterized in that: the determination method is liquid chromatography, using a C8 chromatographic column, mobile phase A is selected from a phosphate solution, mobile phase B is selected from a nitrile solvent, and gradient elution is performed.

[0006] In some embodiments of the present invention, the C8 chromatographic column is selected from the Phenomenex Luna C8(2) chromatographic column.

[0007] In some embodiments of the present invention, the compound is selected from 4-(4-amino-3-fluorophenoxy)pyridine-2-carboxylic acid methylamine and / or 2-chloro-5-aminobenzotrifluoride.

[0008] In some embodiments of the present invention, the compound is derived from a drug selected from N-[3-tert-butyl-1-(quinolin-6-yl)-1H-pyrazol-5-yl]-N'-[2-fluoro-4-[(2-(methylcarbamoyl)pyridin-4-yl)oxy]phenyl]urea or 4-[4-({[4-chloro-3-(trifluoromethyl)phenyl]carbamoyl}amino)-3-fluorophenoxy]-N-methylpyridine-2-carboxamide.

[0009] In some embodiments of the present invention, the mobile phase A is selected from sodium dihydrogen phosphate and potassium dihydrogen phosphate solutions; preferably, the mobile phase A is potassium dihydrogen phosphate solution.

[0010] In some embodiments of the present invention, the concentration of the mobile phase A is selected from 0.008-0.012 mol / L; preferably, the concentration of the mobile phase A is selected from 0.008 mol / L, 0.010 mol / L or 0.012 mol / L.

[0011] In some embodiments of the present invention, the nitrile solvent is selected from acetonitrile and propionitrile, preferably, the nitrile solvent is acetonitrile.

[0012] In some embodiments of the present invention, the gradient elution conditions are: within 0-10 min, the proportion of mobile phase A is 60%-85%; within 10-25 min, the proportion of mobile phase A is 45%-60%; within 25-34 min, the proportion of mobile phase A is 20%-45%; within 34-60 min, the proportion of mobile phase A is 20%-85%, and the sum of the proportions of mobile phase A and mobile phase B is 100%.

[0013] In some embodiments of the present invention, the gradient elution conditions are: within 0-10 minutes, the proportion of mobile phase A changes linearly from 80% to 60%; within 10-25 minutes, the proportion of mobile phase A changes linearly from 60% to 45%; within 25-26 minutes, the proportion of mobile phase A changes linearly from 45% to 20%; within 26-34 minutes, the proportion of mobile phase A is 20%; within 34-35 minutes, the proportion of mobile phase A changes linearly from 20% to 80%; within 35-40 minutes, the proportion of mobile phase A is 80%, and the sum of the proportions of mobile phase A and mobile phase B is 100%.

[0014] In some embodiments of the present invention, the flow rates of the mobile phases A and B are selected from 0.9-1.1 ml / min, preferably, the flow rates of the mobile phases A and B are 0.9 ml / min, 1.0 ml / min or 1.1 ml / min.

[0015] In some embodiments of the present invention, the column temperature of the chromatographic column is selected from 23-27°C; preferably, the column temperature of the chromatographic column is 23°C, 25°C or 27°C.

[0016] In some embodiments of the present invention, the detection wavelength in the determination method is 235-245 nm; preferably, the detection wavelength is 240 nm.

[0017] In some embodiments of the present invention, the injection volume in the determination method is 20-40 μl, preferably, the injection volume is 20-30 μl, and more preferably, the injection volume is 20 μl.

[0018] In some embodiments of the present invention, the determination method further comprises an extraction step of the compound 4-(4-amino-3-fluorophenoxy)pyridine-2-carboxylic acid methylamine.

[0019] In some embodiments of the present invention, the extraction step is performed before liquid chromatography.

[0020] In some embodiments of the present invention, the extraction is to extract the compound 4-(4-amino-3-fluorophenoxy)pyridine-2-carboxylic acid methylamine from a drug, and the drug is selected from N-[3-tert-butyl-1-(quinolin-6-yl)-1H-pyrazol-5-yl]-N'-[2-fluoro-4-[(2-(methylcarbamoyl)pyridin-4-yl)oxy]phenyl]urea or 4-[4-({[4-chloro-3-(trifluoromethyl)phenyl]carbamoyl}amino)-3-fluorophenoxy]-N-methylpyridine-2-carboxamide.

[0021] In some embodiments of the present invention, the solvent used in the extraction step is an aprotic solvent.

[0022] In some embodiments of the present invention, the extraction step further comprises a step of using an aprotic solvent for ultrasound followed by centrifugation.

[0023] In some embodiments of the present invention, the sonication is performed in a water bath.

[0024] In some embodiments of the present invention, the water bath is selected from ice water or cold water controlled at a temperature of 0-10°C.

[0025] In some embodiments of the present invention, the ultrasonic time is 25 min, 30 min or 40 min.

[0026] In some embodiments of the present invention, the centrifugal speed is selected from 9000-11000 r / min; preferably, the centrifugal speed is 9000 r / min, 10000 r / min or 11000 r / min.

[0027] In some embodiments of the present invention, the centrifugation time is selected from 4-10 min, preferably, the centrifugation time is 4 min, 5 min or 6 min.

[0028] In some embodiments of the present invention, the aprotic solvent is selected from tetrahydrofuran, acetonitrile, dimethyl sulfoxide, dimethylformamide or a mixed solvent thereof.

[0029] In some embodiments of the present invention, the aprotic solvent is selected from a mixed solvent of tetrahydrofuran and acetonitrile, and the volume ratio of tetrahydrofuran to acetonitrile is 10-30:70-90, preferably, the volume ratio of tetrahydrofuran to acetonitrile is 20:80.

[0030] In some embodiments of the present invention, the dilution solvent is selected from tetrahydrofuran, acetonitrile or a mixed solvent thereof.

[0031] The beneficial effects of the present invention are as follows: the method can quickly and accurately extract the compound 4-(4-amino-3-fluorophenoxy)pyridine-2-carboxylic acid methylamine from a drug, the method is a liquid chromatography method with good specificity, high sensitivity, good repeatability and good accuracy, and can effectively and accurately perform qualitative and quantitative analysis, and in particular can also be used to analyze and determine 2-chloro-5-aminotrifluorotoluene, thereby ensuring the controllability of drug quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 : Chromatogram of the system suitability solution of Example 4. DETAILED DESCRIPTION

[0033] The determination method involved in the present invention is further described in detail below in the form of examples, but this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies implemented based on the above content of the present invention belong to the scope of the present invention.

[0034] Example 1

[0035] This example investigates the effects of different extraction methods on the analysis results of compound M in the drug: 4-(4-amino-3-fluorophenoxy)pyridine-2-carboxylic acid methylamine.

[0036] Extraction method 1: Take regorafenib tablets, sample batch number 01, grind them, add 60 ml of methanol, sonicate in ice water, bring to room temperature, dilute with methanol and centrifuge (4000 rpm) for 10 minutes, take the supernatant, dilute with tetrahydrofuran-water (volume ratio of 1:9), sonicate, place in an ice bath and shake to prepare extract 1.

[0037] Extraction method 2: Take regorafenib tablets, grind them, add 60 ml of tetrahydrofuran-acetonitrile (volume ratio of 20:80), sonicate in cold water, bring to room temperature, dilute with tetrahydrofuran-acetonitrile (volume ratio of 20:80), and centrifuge (10,000 rpm) for 5 minutes, and take the supernatant as extract 2.

[0038] Reference substance solution: Take an appropriate amount of compound M reference substance, weigh it accurately, and dilute it with diluent tetrahydrofuran-acetonitrile (volume ratio of 20:80) to make a compound M solution with a concentration of 2 μg / ml as the reference substance solution.

[0039] Chromatographic conditions:

[0040] Octylsilane bonded silica gel (Phenomenex C8(2, 4.6mm×250mm, 5μm) as the filler; 0.01mol / L potassium dihydrogen phosphate solution as mobile phase A, acetonitrile as mobile phase B; flow rate of 1.0ml per minute; column temperature of 25℃; detection wavelength of 240nm; injection volume of 20μl, linear gradient elution:

[0041] In 0-10min, the proportion of mobile phase A changes linearly from 80% to 60%; in 10-25min, the proportion of mobile phase A changes linearly from 60% to 45%; in 25-26min, the proportion of mobile phase A changes linearly from 45% to 20%; in 26-34min, the proportion of mobile phase A is 20%; in 34-35min, the proportion of mobile phase A changes linearly from 20% to 80%, and in 35-40min, the proportion of mobile phase A is 80%. The sum of the proportion of mobile phase A and the proportion of mobile phase B is 100%.

[0042] The results of the content of compound M in the extract are shown in Table 1.

[0043] Table 1 Comparative test results of different extraction methods

[0044]

[0045] From the experimental results in Table 1, it can be seen that the content of compound M obtained by extraction method 2 is higher than that by extraction method 1, and extraction method 2 is more conducive to the control of product quality.

[0046] Example 2

[0047] This example investigates the effects of extraction time and extraction temperature on the extraction results of compound M in the drug during the extraction method. According to the extraction method of extract 2 in Example 1, only the extraction time and temperature are changed as shown in Table 2. The sample batch numbers are 02 and 03, and the chromatographic conditions are the same as in Example 1. The experimental results are shown in Table 2 below.

[0048] Table 2 Extraction time and temperature experimental results

[0049]

[0050] From the experimental results in Table 2, it can be seen that by comparing the ultrasonic water bath temperature (cold water or ice water), there is no difference in the detection results of the compound M content. By comparing the ultrasonic time of 25min, 30min and 40min in cold water, there is no significant difference in the detection results of the compound M content. That is, the ultrasonic water bath temperature in the extract preparation process can be selected from ice water or cold water, the temperature is controlled at 0-10°C, and the ultrasonic time can be selected from 25-40min.

[0051] Example 3

[0052] This example investigates the effects of centrifugation time and centrifugation speed during the preparation of the extract on the extraction results of compound M in the drug. According to the extraction method of extract 2 in Example 1, only the centrifugation time and centrifugation speed were changed as shown in Table 3, the sample batch number was 03, the chromatographic conditions were the same as in Example 1, and the experimental results are shown in Table 3 below.

[0053] Table 3 Experimental results of centrifugal speed and centrifugal time

[0054]

[0055]

[0056] From the experimental results in Table 3, it can be seen that by comparing the centrifugation time of 4min, 5min, and 6min, there is no difference in the detection results of the compound M content. By comparing the centrifugation speed of 9000r / min, 10000r / min, and 11000r / min, there is no difference in the detection results of the compound M content. That is, the centrifugation time in the preparation process of the extract can be selected from 4-6min, and the centrifugation speed can be selected from 9000-11000r / min.

[0057] Example 4

[0058] This example is a method verification of liquid chromatography conditions, and the chromatographic conditions are the same as those in Example 1.

[0059] 1. Specificity experiment

[0060] Dilution solvent: tetrahydrofuran-acetonitrile (volume ratio is 20:80)

[0061] Blank excipient solution: Weigh about 595 mg of blank excipient (i.e., the mixture of excipients required for preparing regorafenib tablets), place in a 5 ml volumetric flask, add about 3 ml of diluent, ultrasonicate in cold water for 30 min, cool to room temperature, dilute to scale with diluent, shake well, centrifuge at 10000 r / min for 5 min.

[0062] System suitability solution: Take appropriate amount of regorafenib reference substance, compound M reference substance, and compound S (2-chloro-5-aminobenzotrifluoride) reference substance, add diluent and ultrasonically dissolve them to prepare a solution containing 2 mg / ml of regorafenib, 0.2 μg / ml of compound M, and 2.2 μg / ml of compound S per 1 ml.

[0063] Test method: 20 μl of blank excipient solution and system suitability solution were injected into the liquid chromatograph and the chromatogram was recorded. The chromatogram of the system suitability solution is as follows: Figure 1 The specificity test results are shown in Table 4.

[0064] Table 4 Specificity test results

[0065] name Blank excipient solution interference Retention time (min) Separation Compound M none 13.320 23.29 Compound S none 25.907 8.12 Regorafenib / 29.688 /

[0066] From the experimental results in Table 4, it can be seen that the blank excipient has no interference, the separation between the peaks in the system suitability solution is greater than 1.5, and the system suitability is good.

[0067] 2. Detection limit and quantification limit experiments

[0068] Compound M quantitation limit solution: prepare a solution containing compound M 0.04 μg / ml.

[0069] Compound M detection limit solution: prepare a solution containing compound M 0.02 μg / ml.

[0070] Compound S quantitation limit solution: prepare a solution containing compound S 0.4 μg / ml.

[0071] Compound S detection limit solution: prepare a solution containing 0.2 μg / ml of compound S.

[0072] Test method: Accurately measure 20 μl of the quantitative limit and detection limit solutions, respectively, inject them into the liquid chromatograph, and record the chromatograms.

[0073] The signal-to-noise ratio (S / N) method was used to determine the limit of detection (LOD) and limit of quantification (LOQ), respectively. The results are shown in Table 5.

[0074] Table 5 Results of detection limit and quantification limit

[0075]

[0076] From the experimental results in Table 5, it can be seen that the LOQ and LOD concentrations of compound M are not less than 0.02 μg / ml, and the LOQ and LOD concentrations of compound S are not less than 0.2 μg / ml, and the sensitivity meets the requirements of the analytical method.

[0077] 3. Linearity and range

[0078] Test method: 20 μl of linear solutions of different concentrations were accurately measured and injected into the liquid chromatograph to record the chromatogram. The peak area was used for linear regression of concentration. The results are shown in Table 6.

[0079] Table 6 Linearity and range test results

[0080] name Linear equations Correlation coefficient r Linear range / μg / ml Compound M Y=78.4901X-5.8604 1.0000 0.04—40 Compound S Y=48.1295X-2.4651 1.0000 0.4—40

[0081] From the experimental results in Table 6, it can be seen that compound M has good linearity in the range of 0.04-40 μg / ml, and compound S has good linearity in the range of 0.4-40 μg / ml.

[0082] 4. Repeatability

[0083] Extraction solution: Take an appropriate amount of regorafenib tablets, sample batch number 03, and the extraction method is the same as the extraction method 2 of experimental example 1, with 6 parallel copies.

[0084] Reference solution: Prepare a solution containing 2 μg / ml of compound M in duplicate.

[0085] Test method: Accurately measure 20 μl of each of the above solutions, inject them into the liquid chromatograph, and record the chromatogram. The RSD of the content of each compound M in the 6 extracts is calculated to be 2.9%, and the RSD is no more than 5%, indicating that this method has good repeatability.

[0086] Embodiment 5-6

[0087] Dilution solvent: tetrahydrofuran-acetonitrile (volume ratio is 20:80)

[0088] System suitability solution: Prepare the same system suitability solution as in "Specificity experiment in Example 4".

[0089] Reference substance solution: Take appropriate amounts of reference substance compound M and compound S to prepare solutions containing 2 μg / ml of compound M and compound S respectively.

[0090] Extract: Prepare the same extract as in Example 4.

[0091] Test method: Compared with the chromatographic conditions of Example 1, only the gradient elution conditions were changed to analyze the content of compound M in the drug.

[0092] Example 5 Gradient elution conditions are as follows: within 0-10 minutes, the proportion of mobile phase A changes linearly from 78% to 60%; within 10-25 minutes, the proportion of mobile phase A changes linearly from 60% to 45%; within 25-26 minutes, the proportion of mobile phase A changes linearly from 45% to 20%; within 26-34 minutes, the proportion of mobile phase A is 20%; within 34-35 minutes, the proportion of mobile phase A changes linearly from 20% to 78%, and within 35-40 minutes, the proportion of mobile phase A is 78%. The sum of the proportion of mobile phase A and the proportion of mobile phase B is 100%.

[0093] Example 6 Gradient elution conditions are as follows: within 0-10 min, the proportion of mobile phase A changes linearly from 82% to 60%; within 10-25 min, the proportion of mobile phase A changes linearly from 60% to 45%; within 25-26 min, the proportion of mobile phase A changes linearly from 45% to 20%; within 26-34 min, the proportion of mobile phase A is 20%; within 34-35 min, the proportion of mobile phase A changes linearly from 20% to 82%, and within 35-40 min, the proportion of mobile phase A is 82%. The sum of the proportion of mobile phase A and the proportion of mobile phase B is 100%.

[0094] The test results are shown in Table 7.

[0095] Table 7 Experimental results

[0096] Example Compound M% System suitability solution minimum resolution Example 5 0.0059 9.07 Example 6 0.0060 8.88

[0097] From the experimental results in Table 7, it can be seen that both Examples 5 and 6 can obtain chromatographic detection results that meet the analysis requirements.

[0098] Example 7

[0099] Preparation of solvent: The preparation of system suitability solution, reference solution and extract is the same as in Example 4.

[0100] Experimental method: relative to the chromatographic conditions of Example 1, only a single variable was changed to conduct a durability experiment, that is, the column temperature, flow rate, and mobile phase A concentration were investigated respectively. The specific experimental single variable conditions are shown in Table 8.

[0101] Table 8 Durability test single variable conditions

[0102]

[0103]

[0104] The durability was examined according to the experimental conditions in Table 8. The experimental results were basically the same as those in Example 1, indicating that the method has good durability.

Claims

1. A method for determining a compound, characterized in that: The determination method is liquid chromatography, using a C8 chromatographic column, mobile phase A selected from a phosphate solution, mobile phase B selected from a nitrile solvent, and gradient elution.

2. The assay method according to claim 1, wherein the C8 chromatographic column is selected from Phenomenex Luna C8(2) chromatographic column.

3. The assay method according to any one of claims 1 to 2, wherein the compound is selected from 4-(4-amino-3-fluorophenoxy)pyridine-2-carboxylic acid methylamine and / or 2-chloro-5-aminobenzotrifluoride.

4. The assay method according to any one of claims 1 to 3, wherein the compound is derived from a drug, and the drug is selected from N-[3-tert-butyl-1-(quinolin-6-yl)-1H-pyrazol-5-yl]-N'-[2-fluoro -4-[(2-(methylcarbamoyl)pyridin-4-yl)oxy]phenyl]urea or 4-[4-({[4-chloro-3-(trifluoromethyl)phenyl]carbamoyl}amino)-3-fluorophenoxy]-N-methylpyridine-2-carboxamide.

5. According to the assay method according to any one of claims 1 to 4, the mobile phase A is selected from sodium dihydrogen phosphate or potassium dihydrogen phosphate solution; preferably, the mobile phase A is potassium dihydrogen phosphate solution.

6. according to the assay method described in any one of claims 1-5, described nitrile solvent is selected from acetonitrile and propionitrile, preferably, described nitrile solvent is acetonitrile.

7. The determination method according to any one of claims 1 to 6, wherein the gradient elution conditions are: within 0-10 min, the proportion of mobile phase A is 60%-85%; within 10-25 min, the proportion of mobile phase A is 45%-60%; within 25-34 min, the proportion of mobile phase A is 20%-45%; within 34-60 min, the proportion of mobile phase A is 20%-85%, and the sum of the proportions of mobile phase A and mobile phase B is 100%.

8. The determination method according to any one of claims 1 to 7, wherein the gradient elution conditions are: within 0-10 min, the proportion of mobile phase A changes linearly from 80% to 60%; within 10-25 min, the proportion of mobile phase A changes linearly from 60% to 45%; within 25-26 min, the proportion of mobile phase A changes linearly from 45% to 20%; within 26-34 min, the proportion of mobile phase A is 20%; within 34-35 min, the proportion of mobile phase A changes linearly from 20% to 80%; within 35-40 min, the proportion of mobile phase A is 80%, and the sum of the proportions of mobile phase A and mobile phase B is 100%.

9. The determination method according to any one of claims 1 to 8, further comprising an extraction step of the compound 4-(4-amino-3-fluorophenoxy)pyridine-2-carboxylic acid methylamine.

10. The assay method according to any one of claims 1 to 9, wherein the extraction step further comprises a step of using an aprotic solvent for ultrasonication followed by centrifugation; preferably, the aprotic solvent is selected from tetrahydrofuran, acetonitrile, dimethyl sulfoxide, dimethylformamide or a mixed solvent thereof; preferably, the aprotic solvent is selected from a mixed solvent of tetrahydrofuran and acetonitrile, the volume ratio of tetrahydrofuran to acetonitrile being 10-30:70-90, preferably, the volume ratio of tetrahydrofuran to acetonitrile being 20:80.

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