CN114755320A - Detection method of 3-amino-6-methoxypyridazine related substance - Google Patents
Detection method of 3-amino-6-methoxypyridazine related substance Download PDFInfo
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- G—PHYSICS
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- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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Abstract
The invention discloses a method for detecting a 3-amino-6-methoxypyridazine related substance, which comprises the following steps: (1) preparing a test solution and a control solution; (2) detection and recording: detecting the test solution and the control solution by adopting a liquid chromatography and recording spectrograms; wherein the chromatographic conditions of the liquid chromatography are: a chromatographic column: a water-resistant octadecyl bonded silica gel column; mobile phase: gradient elution is carried out by taking potassium hexafluorophosphate buffer solution (the pH value is adjusted to 2.8-3.2 by phosphoric acid) as a mobile phase A and acetonitrile as a mobile phase B; flow rate: 0.9-1.1 ml/min; column temperature: 33 to 37 ℃; detection wavelength: 213 to 217 nm. The detection method can be used for quickly and accurately detecting related substances in the 3-amino-6-methoxypyridazine, and has good accuracy, precision, reproducibility and stability.
Description
Technical Field
The invention belongs to the technical field of medicines, and relates to a method for detecting related substances in a medicine intermediate 3-amino-6-methoxypyridazine.
Background
3-amino-6-methoxypyridazine (Invitrogen name: 3-amino-6-methoxydazine) is an important pharmaceutical intermediate, and can be used for pyridopyrimidine compounds with remarkable biological activity or medicines, such as anticancer drug Ruugeli, which can be used for treating indications such as pain caused by endometriosis, uterine fibroids and advanced prostate cancer.
At present, many reports on the preparation method of the compound exist, and various process impurities such as 3-amino-6-chloropyridazine (impurity A), 3-amino-6-hydroxypyridazine (impurity B), 3-chloro-6-hydroxypyridazine (impurity C), 3-dichloropyridazine (impurity D), 3, 6-dimethoxypyridazine (impurity E), 3-amino-6-methoxypyridazine (impurity F), 3-aminopyridazine (impurity G) and the like are often introduced in the process of the conventional synthesis process, but no relevant documents report the detection method of the process impurities. Therefore, the development of a method capable of detecting related substances in the 3-amino-6-methoxypyridazine is of great significance for providing medication safety and quality control of the 3-amino-6-methoxypyridazine.
Disclosure of Invention
The invention aims to: the invention aims to provide a method for detecting related substances in 3-amino-6-methoxypyridazine, which has the advantages of good separation degree, accuracy, reproducibility and stability, low detection limit and short analysis time.
The technical scheme is as follows: the method for detecting related substances in 3-amino-6-methoxypyridazine provided by the invention comprises the following steps:
(1) preparing a test solution and a control solution;
(2) detection and recording: detecting the test solution and the control solution by adopting a liquid chromatography and recording spectrograms, wherein the chromatographic conditions of the liquid chromatography are as follows:
a chromatographic column: a water-resistant octadecyl bonded silica gel column; mobile phase: gradient elution is carried out by taking potassium hexafluorophosphate buffer solution (the pH value is adjusted to 2.8-3.2 by phosphoric acid) as a mobile phase A and acetonitrile as a mobile phase B; flow rate: 0.9-1.1 ml/min; column temperature: 33-37 ℃; detection wavelength: 213 to 217 nm.
Preferably, a ghost capturing column is connected to the front of the sample injector of the liquid chromatograph.
Further, the concentration of the test solution in the step (1) was 0.5mg/ml, and the concentration of the control solution was 1. mu.g/ml.
Solvent: and (3) water.
The preparation method of the solution comprises the following steps:
stock solution of impurity A (7.5. mu.g/ml): accurately weighing 15mg of impurity A reference substance, placing the reference substance in a 100ml measuring flask, adding 20ml of 20% acetonitrile, performing ultrasonic dissolution, cooling to room temperature, diluting with water to scale, and shaking up; precisely measure 5ml of the solution, place the solution in a 100ml measuring flask, dilute the solution to the mark with water, and shake the solution evenly.
Stock solutions of other impurities (7.5. mu.g/ml): accurately weighing 15mg of other impurity reference substances, accurately weighing, placing in a 100ml measuring flask, adding water, ultrasonically dissolving, cooling to room temperature, diluting with water to scale, and shaking; precisely measure 5ml of the solution, place the solution in a 100ml measuring flask, dilute the solution to the scale with water, and shake the solution uniformly.
Test solution: precisely weighing about 25mg of the 3-amino-6-methoxypyridazine sample, placing the sample in a 50ml measuring flask, adding water for ultrasonic dissolution, adding water to the scale, and shaking up;
control solution: weighing 2ml of the test solution, placing the test solution in a 100ml measuring flask, diluting the test solution to a scale with water, and shaking up to obtain a solution I; measure solution I5 ml, put into 50ml measuring flask, dilute to the mark with water, shake.
Further, the type of the chromatographic column used in the detection in the step (2) is an Atlantis T3 column or a Sepax HP C18 column. The specification of the Atlantis T3 column was particle size (μm) x inner diameter (mm) x column length (mm): 3 × 4.6 × 150; the specification of the Sepax HP C18 column was particle size (μm) x inner diameter (mm) x column length (mm): 5X 4.6X 250.
Further, the concentration of the potassium hexafluorophosphate buffer solution in the mobile phase A in the step (2) is 15-25 mmol/L.
Further, the mobile phase gradient elution process of step (2) is as follows:
| time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 100 | 0 |
| 5 | 100 | 0 |
| 25 | 75 | 25 |
| 25.01 | 100 | 0 |
| 35 | 100 | 0 |
Furthermore, the sample volume of the control solution and the sample solution detected in the step (2) is 5-10 mul.
Calculating the formula:
total (%) + the sum of unknown individual (%) + the sum of known impurities (%);
wherein A isImpurities-peak area of each known impurity in the test sample solution; a. theControl-control solution peak area; f-correction factors for each known impurity.
Has the advantages that: compared with the prior art, the invention has the following remarkable advantages:
(1) a detection method of related substances in 3-amino-6-methoxypyridazine is established, and the content of each related substance can be rapidly and accurately determined;
(2) the method is simple and convenient to operate, can effectively detect the content of related substances in the 3-amino-6-methoxypyridazine, has high sensitivity (the detection limit is 0.045 mu g/ml and the quantification limit is 0.15 mu g/ml), strong specificity (a blank solvent and a degradation experiment interfere with impurity detection), and good linear correlation (r is2More than 0.999), high accuracy (the standard adding recovery rate of different concentration levels is between 94 percent and 106 percent, the RSD value of the recovery rate is less than 5 percent, and the RSD value of the repeatability and the middle precision is less than 3 percent);
(3) the method has good durability, and can be used for quality control of related substances in 3-amino-6-methoxypyridazine in various samples.
Drawings
FIG. 1 is an HPLC chromatogram of a system suitability solution.
FIG. 2 is an HPLC chromatogram of a blank solvent.
FIG. 3 is an HPLC chromatogram of the test solution.
FIG. 4 is an HPLC chromatogram of a system suitability solution in comparative example 1.
FIG. 5 is an HPLC chromatogram of a system suitability solution in comparative example 2
Fig. 6 is an HPLC chromatogram of a system-compatible solution under chromatographic conditions at pH 2.7.
Fig. 7 is an HPLC chromatogram of a system suitability solution under chromatographic conditions at pH 3.3.
Detailed Description
The technical solution of the present invention is further explained below with reference to the embodiments and the accompanying drawings.
The preparation method of the solution comprises the following steps:
stock solution of impurity A (7.5. mu.g/ml): accurately weighing 15mg of impurity A reference substance, placing the reference substance in a 100ml measuring flask, adding 20ml of 20% acetonitrile, performing ultrasonic dissolution, cooling to room temperature, diluting with water to scale, and shaking up; precisely measure 5ml of the solution, place the solution in a 100ml measuring flask, dilute the solution to the scale with water, and shake the solution uniformly.
Stock solutions of other impurities (7.5. mu.g/ml): accurately weighing 15mg of other impurity reference substances, accurately weighing, placing in a 100ml measuring flask, adding water, ultrasonically dissolving, cooling to room temperature, diluting with water to scale, and shaking; precisely measure 5ml of the solution, place the solution in a 100ml measuring flask, dilute the solution to the mark with water, and shake the solution evenly.
Preparation of 3-amino-6-methoxypyridazine reference stock solution: precisely weighing about 25mg of 3-amino-6-methoxypyridazine as a reference substance, placing the reference substance into a 50ml measuring flask, adding water for ultrasonic dissolution, cooling to room temperature, diluting with water to a scale, and shaking up.
Preparing a test solution: precisely weighing about 25mg of the 3-amino-6-methoxypyridazine sample, placing the sample in a 50ml measuring flask, adding water for ultrasonic dissolution, adding water to the scale, and shaking up.
Preparation of a control solution: weighing 2ml of the test solution, placing the test solution in a 100ml measuring flask, diluting the test solution to a scale with water, and shaking up to obtain a solution I; measure solution I5 ml, put into 50ml measuring flask, dilute to the scale with water, shake.
Example 1 System suitability test
Preparation of system applicability solution: about 25mg of a 3-amino-6-methoxypyridazine test sample is precisely weighed and placed in a 50ml measuring flask, water is added for ultrasonic dissolution, the solution is cooled to room temperature, 5ml of each impurity stock solution with 7.5 mu g/ml is precisely added, the solution is diluted to scales by water and shaken up to prepare a systematic applicability solution with 0.75 mu g/ml of each impurity and 0.5mg/ml of 3-amino-6-methoxypyridazine. Sample injection test, the detection conditions are as follows: and (3) chromatographic column: an Atlantis T3 column; mobile phase: gradient elution is carried out by taking 20mmol/L potassium hexafluorophosphate buffer solution (pH is adjusted to 3.0 by phosphoric acid) as a mobile phase A and taking acetonitrile as a mobile phase B; flow rate: 1.0 ml/min; column temperature: 35 ℃; detection wavelength: 215 nm; the amount of sample was 5. mu.l. The results are recorded as shown in table 1 and fig. 1.
TABLE 1 System suitability test results
| Composition (I) | Concentration (μ g/ml) | Relative retention time | Degree of separation |
| Impurity A (inpurityA) | 0.75 | 0.84 | 35.05 |
| Impurity B (inpurityB) | 0.75 | 0.25 | N/A |
| Impurity C (inpurityC) | 0.75 | 0.95 | 7.34 |
| Impurity D (inpurityD) | 0.75 | 1.37 | 19.88 |
| Impurity E (inpurityE) | 0.75 | 1.47 | 5.92 |
| Impurity F (inpurityF) | 0.75 | 1.53 | 3.47 |
| Impurity G (inpurityG) | 0.75 | 0.28 | 2.94 |
| 3-amino-6-methoxypyridazines | 500 | 1 | 2.64 |
Test results show that the main peak in the system applicability solution is better separated from adjacent impurities, and the separation among the impurities is better.
Example 2 specificity test
2.1 interference test
Blank solvent: water (I)
Preparing a test solution: precisely weighing about 25mg of a 3-amino-6-methoxypyridazine test sample, placing the test sample in a 50ml measuring flask, adding water to ultrasonically react to obtain a solution, cooling the solution to room temperature, diluting the solution to a scale with water, and shaking the solution uniformly.
Respectively injecting a blank solvent and a sample solution to be tested, wherein the detection conditions are as follows: a chromatographic column: an Atlantis T3 column; mobile phase: gradient elution is carried out by taking 20mmol/L potassium hexafluorophosphate buffer solution (pH is adjusted to 3.0 by phosphoric acid) as a mobile phase A and acetonitrile as a mobile phase B; flow rate: 1.0 ml/min; column temperature: 35 ℃; detection wavelength: 215 nm; the sample volume was 5. mu.l. The results were recorded and the spectra are shown in fig. 2 and fig. 3, respectively. The result shows that the blank solvent does not interfere the detection of the test sample, and the purity of the main peak is more than 990.
2.2 destructive testing
Test solution: precisely weighing about 25mg of a 3-amino-6-methoxypyridazine test sample, placing the test sample in a 50ml measuring flask, adding water for ultrasonic dissolution, adding water to a scale, and shaking up;
control solution: weighing 2ml of the test solution, placing the test solution in a 100ml measuring flask, diluting the test solution to a scale with water, and shaking up to obtain a solution I; measure solution I5 ml, put into 50ml measuring flask, dilute to the scale with water, shake.
Oxidative degradation test: precisely weighing about 25mg of the 3-amino-6-methoxypyridazine test sample, placing the test sample in a 50ml measuring flask, adding water, performing ultrasonic treatment to dissolve the test sample, cooling to room temperature, adding 1ml of 3% hydrogen peroxide solution, placing the test sample at room temperature for 3 hours, diluting the test sample with water to a scale, and shaking up to obtain the product.
Acid degradation test: taking about 25mg of the 3-amino-6-methoxypyridazine sample, precisely weighing, placing in a 50ml measuring flask, adding a proper amount (about 30ml) of water for ultrasonic dissolution, cooling to room temperature, adding 1ml of 0.1mol/L hydrochloric acid solution, standing at room temperature for 3 hours, neutralizing with 1ml of 0.1mol/L sodium hydroxide solution, diluting with water to scale, and shaking uniformly to obtain the product.
Alkali destruction: precisely weighing about 25mg of 3-amino-6-methoxypyridaz to be tested, placing the sample in a 50ml measuring flask, adding a proper amount (about 30ml) of water for ultrasonic dissolution, cooling to room temperature, adding 1ml of 0.1mol/L sodium hydroxide solution, placing the sample for 3 hours at room temperature, neutralizing the sample with 1ml of 0.1mol/L hydrochloric acid solution, diluting the solution to scale with water, and shaking up to obtain the product.
High-temperature destruction of solids: putting a proper amount of the 3-amino-6-methoxypyridazo test sample into a weighing bottle, spreading the test sample into a thin layer with the thickness of less than or equal to 5mm, placing the thin layer in an oven at 60 ℃ for 3 hours, taking out the thin layer, cooling the thin layer to room temperature, taking 25mg of the test sample, precisely weighing the test sample, placing the test sample into a 50ml measuring bottle, adding water for ultrasonic dissolution, cooling the test sample to the room temperature, diluting the test sample to the scale with water, and shaking the test sample uniformly to obtain the product.
And (3) high-temperature destruction of the solution: precisely weighing about 25mg of 3-amino-6-methoxypyridazine sample, placing in a 50ml measuring flask, adding water, ultrasonically dissolving, cooling to room temperature, standing in water bath at 60 deg.C for 3 hr, taking out, cooling to room temperature, diluting with water to scale, and shaking.
Solid photo-destruction: placing a proper amount of 3-amino-6-methoxypyridazole to be tested in a weighing bottle, spreading into a thin layer with the thickness of less than or equal to 5mm, placing under the illumination of 4500lx +/-500 lx for 3 days, taking out, precisely weighing about 25mg of the product, placing in a 50ml measuring bottle, adding water for ultrasonic dissolution, cooling to room temperature, diluting with water to scale, and shaking uniformly to obtain the product.
Photo-disruption of the solution: precisely weighing about 25mg of 3-amino-6-methoxypyridazole sample, placing in a 50ml measuring flask, adding water, ultrasonically dissolving, cooling to room temperature, standing under 4500lx +/-500 lx illumination for 1 day, taking out, diluting with water to scale, and shaking uniformly.
Respectively injecting a sample solution to be tested, a reference solution and a degraded sample, wherein the detection conditions are as follows: a chromatographic column: an Atlantis T3 column; mobile phase: gradient elution is carried out by taking 20mmol/L potassium hexafluorophosphate buffer solution (pH is adjusted to 3.0 by phosphoric acid) as a mobile phase A and taking acetonitrile as a mobile phase B; flow rate: 1.0 ml/min; column temperature: 35 ℃; detection wavelength: 215 nm; the amount of sample was 5. mu.l. The results were recorded and are shown in table 2 below.
TABLE 2 forced degradation test results
Calculating the formula:
total (%) + the sum of unknown individual (%) + the sum of known impurities (%);
wherein A isImpurities-peak area of each known impurity in the test sample solution; a. theControl-control solution peak area; f-correction factors for each known impurity.
The result shows that the degradation impurities are generated under the solution photo-destruction condition, but the degradation impurities do not interfere the detection, the separation degree meets the requirement, the materials are conserved, and the 3-amino-6-methoxypyridazine is stable under other conditions.
Example 3 detection and quantitation limits
Preparation of 3-amino-6-methoxypyridazine reference stock solution: precisely weighing about 25mg of 3-amino-6-methoxypyridazine as a reference substance, placing the reference substance into a 50ml measuring flask, adding water for ultrasonic dissolution, cooling to room temperature, diluting with water to a scale, and shaking up.
Preparing a quantitative limiting storage solution: precisely measuring 1.5ml of 3-amino-6-methoxypyridazine reference stock solution, placing 5ml of other impurity stock solutions in a same 100ml measuring flask, adding water to dilute to scale, and shaking up.
Preparing a quantitative limiting solution: precisely measuring 1ml of quantitative limiting storage solution, placing in a 50ml measuring flask, adding water to dilute to scale, and shaking up.
Preparing a detection limit solution: precisely measuring 3ml of the quantitative limiting solution, putting the quantitative limiting solution into a 10ml measuring flask, adding water to dilute the solution to a scale mark, and shaking up.
Respectively injecting the quantitative limit solution and the detection limit solution, wherein the detection conditions are as follows: a chromatographic column: an Atlantis T3 column; mobile phase: gradient elution is carried out by taking 20mmol/L potassium hexafluorophosphate buffer solution (pH is adjusted to 3.0 by phosphoric acid) as a mobile phase A and taking acetonitrile as a mobile phase B; flow rate: 1.0 ml/min; column temperature: 35 ℃; detection wavelength: 215 nm; the sample volume was 5. mu.l. The results are recorded as shown in table 3.
TABLE 3 summary of detection limit and quantitation limit results
| Composition (I) | Limit of quantitation (ug/mL) | Detection limit (μ g/mL) | Detection limit S/N |
| Impurity A (inpurityA) | 0.155 | 0.046 | 14.1 |
| Impurity B (inpurityB) | 0.151 | 0.045 | 26.2 |
| Impurity C (inpurityC) | 0.155 | 0.046 | 12.0 |
| Impurity D (inpurityD) | 0.157 | 0.047 | 5.7 |
| Impurity E (inpurityE) | 0.155 | 0.046 | 7.2 |
| Impurity F (inpurityF) | 0.145 | 0.044 | 11.6 |
| Impurity G (inpurityG) | 0.152 | 0.046 | 7.7 |
| 3-amino-6-methoxypyridazines | 0.150 | 0.045 | 13.9 |
As is clear from the data in the table, the quantitative limit and the detection limit of each component correspond to 0.03% and 0.009% of the sample concentration. The detection limit and the quantification limit are both lower, and the sensitivity of the method is high.
Example 4 Linear Range and correction factor test
Preparation of a linear stock solution: precisely measuring 1.5ml of 3-amino-6-methoxypyridazine reference stock solution, placing 5ml of other impurity stock solutions in a same 100ml measuring flask, adding water to dilute to scale, and shaking up.
Preparation of linear-200% strength solution: precisely measuring 10ml of linear stock solution, placing into a 50ml measuring flask, diluting with water to scale, and shaking.
Preparation of linear-150% strength solution: precisely measuring 7.5ml of linear stock solution, placing in a 50ml measuring flask, diluting with water to scale, and shaking up.
Preparation of linear-100% strength solution: precisely measuring 5ml of linear stock solution, placing the linear stock solution into a 50ml measuring flask, diluting the linear stock solution to a scale with water, and shaking up.
Preparation of linear-80% strength solution: precisely measuring 4ml of linear stock solution, placing the linear stock solution into a 50ml measuring flask, diluting the linear stock solution to a scale with water, and shaking up.
Preparation of linear-50% strength solution: precisely measuring 2.5ml of linear stock solution, placing in a 50ml measuring flask, diluting with water to scale, and shaking.
Preparation of linear-quantitative limiting solution: precisely measuring 1ml of linear stock solution, placing into a 50ml measuring flask, diluting with water to scale, and shaking.
Respectively carrying out linearity and correction factor tests on different instruments by different experimenters under the following detection conditions: a chromatographic column: an Atlantis T3 column; mobile phase: gradient elution is carried out by taking 20mmol/L potassium hexafluorophosphate buffer solution (pH is adjusted to 3.0 by phosphoric acid) as a mobile phase A and taking acetonitrile as a mobile phase B; flow rate: 1.0 ml/min; column temperature: 35 ℃; detection wavelength: 215 nm; the amount of sample was 5. mu.l. The test results are shown in table 4 below.
TABLE 4 Linear Range and correction factor test results
The results show that each impurity has good linear relation in the range of 0.03-0.30%, and the correlation coefficient r2Not less than 0.999, and the correction factor is between 1.09 and 3.20, which can be calculated by a self-contrast method of adding the correction factor.
Example 5 accuracy test
Accuracy impurity stock solution: precisely measuring 5ml of each impurity stock solution, placing into the same 100ml measuring flask, adding water to dilute to scale, and shaking.
Quantitative limiting accuracy solution: precisely weighing about 25mg of 3-amino-6-methoxypyridazine sample, placing in a 50ml measuring flask, adding water, ultrasonically dissolving, cooling to room temperature, precisely adding 1ml of an accurate impurity reference stock solution, diluting with water to scale, and shaking.
Quantitative limit accuracy control solution: 2ml of quantitative limiting accuracy solution is precisely measured, placed in a 100ml measuring flask, diluted to a scale with water and shaken up; precisely measuring 5ml of the solution, placing the solution in a 50ml measuring flask, diluting the solution to the scale with water, and shaking up.
100% accuracy solution: precisely weighing about 25mg of 3-amino-6-methoxypyridazine sample, placing in a 50ml measuring flask, adding water, ultrasonically dissolving, cooling to room temperature, precisely adding 5ml of an accurate impurity reference substance stock solution, diluting with water to scale, and shaking up.
100% accuracy control solution: 2ml of 100% accuracy solution is precisely measured, placed in a 100ml measuring flask, diluted to scale with water and shaken up; precisely measuring 5ml of the solution, placing the solution in a 50ml measuring flask, diluting the solution to the scale with water, and shaking up.
150% accuracy solution: precisely weighing about 25mg of 3-amino-6-methoxypyridazine sample, placing in a 50ml measuring flask, adding water, ultrasonically dissolving, cooling to room temperature, precisely adding 7.5ml of an accurate impurity reference substance stock solution, diluting with water to scale, and shaking uniformly.
150% accuracy control solution: precisely measuring 2ml of 150% accuracy solution, placing in a 100ml measuring flask, diluting with water to scale, and shaking; then precisely measure 5ml of the solution, place the solution in a 50ml measuring flask, dilute the solution to the scale mark with water, and shake the solution evenly.
Sample introduction and detection are carried out on the samples under the following detection conditions: and (3) chromatographic column: an Atlantis T3 column; mobile phase: gradient elution is carried out by taking 20mmol/L potassium hexafluorophosphate buffer solution (pH is adjusted to 3.0 by phosphoric acid) as a mobile phase A and taking acetonitrile as a mobile phase B; flow rate: 1.0 ml/min; column temperature: 35 ℃; detection wavelength: 215 nm; the amount of sample was 5. mu.l. The recovery of spiked samples was calculated from the measured and theoretical addition and the results are shown in Table 5 below.
TABLE 5 results of recovery with addition of standard
The results in the table show that the standard recovery rate of each impurity at different concentration levels is 94-106%, and the RSD (reference signal deviation) is less than 5%.
Example 6 repeatability and precision testing
Different experimenters prepare an impurity A and an impurity B standard solution with the concentration equivalent to 0.15 percent of the concentration of a sample to be tested as a test solution, and the preparation method comprises the following steps:
repeatability (precision) impurity control stock solutions: precisely measuring 5ml of each of the impurity A and impurity B stock solutions, placing the stock solutions in a 100ml measuring flask, diluting with water to scale, and shaking up.
Reproducibility (precision) solution: precisely weighing about 25mg of 3-amino-6-methoxypyridazine sample, placing the sample in a 50ml measuring flask, adding water for ultrasonic dissolution, cooling to room temperature, precisely adding 5ml of repetitive impurity reference substance stock solution respectively, diluting with water to scale, shaking up, and preparing 6 parts in parallel.
Reproducibility (precision) control solution: precisely measuring 2ml of the renaturation solution, placing the renaturation solution in a 100ml measuring flask, diluting the renaturation solution to a scale mark with water, shaking up, precisely measuring 5ml of the renaturation solution, placing the renaturation solution in a 50ml measuring flask, diluting the renaturation solution to a scale mark with water, and shaking up. 6 parts of repetitive solutions were taken to prepare control solutions, respectively.
The repeatability and precision of the method are respectively inspected on different dates and different instruments, and the detection conditions are as follows: a chromatographic column: an Atlantis T3 column; mobile phase: gradient elution is carried out by taking 20mmol/L potassium hexafluorophosphate buffer solution (pH is adjusted to 3.0 by phosphoric acid) as a mobile phase A and taking acetonitrile as a mobile phase B; flow rate: 1.0 ml/min; column temperature: 35 ℃; detection wavelength: 215 nm; the amount of sample was 5. mu.l. The results are shown in Table 6.
TABLE 6 repeatability and precision test results
The results in the table show that the repeatability RSD values of the two impurities are 1.3% and 0.5% respectively, and the RSD value of the intermediate precision is 2.3% and 1.8% respectively, so that the method has better repeatability and precision.
EXAMPLE 7 durability test
Preparation of system applicability solution: system suitability solutions were prepared according to the formulation of example 1.
Test solution: precisely weighing about 25mg of the 3-amino-6-methoxypyridazine sample, placing the sample in a 50ml measuring flask, adding water for ultrasonic dissolution, adding water to the scale, and shaking up;
control solution: weighing 2ml of the test solution, placing the test solution in a 100ml measuring flask, diluting the test solution to a scale with water, and shaking up to obtain a solution I; measure solution I5 ml, put into 50ml measuring flask, dilute to the mark with water, shake.
The chromatographic conditions (column temperature, flow rate, buffer salt concentration, pH, detection wavelength, chromatographic column) were varied, and the system applicability solution, the test sample solution and the control solution were subjected to sample injection detection, and the results were recorded as shown in tables 7 and 8 below.
Calculating the formula:
total (%) + unknown individual (%) + known impurities (%);
wherein A isImpurities-peak area of each known impurity in the test sample solution; a. theControl-control solution peak area; f-correction factors for each known impurity.
TABLE 7 separation of key peaks under different chromatographic conditions
TABLE 8 results of the detection under different chromatographic conditions
As can be seen from the data in tables 7 and 8 above, a slight change in chromatographic conditions has little effect on the detection results, and the method is robust.
Comparative example 1
Preparing system applicability solution of impurity A and impurity B: accurately weighing appropriate amount of the impurity A, the impurity B and the 3-amino-6 methoxypyridazine to be tested, adding water to dissolve and dilute the impurities to a mixed solution containing 0.75 mu g/ml of the impurity A, 0.3 mu g/ml of the impurity B and 0.5mg/ml of the 3-amino-6 methoxypyridazine.
The system is applied to the sample injection detection and the result is recorded, and the detection conditions are as follows: a chromatographic column: xbridge Shield RP18 column, 3.5 μm. times.4.6 mm. times.150 mm; mobile phase: gradient elution is carried out by taking 10mmol/L potassium dihydrogen phosphate and dipotassium hydrogen phosphate solution as a mobile phase A and acetonitrile as a mobile phase B; flow rate: 1.0 ml/min; column temperature: 35 ℃; detection wavelength: 215 nm; sample introduction volume: 5 μ l, gradient program as follows:
| time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 100 | 0 |
| 10 | 100 | 0 |
| 25 | 75 | 25 |
| 25.01 | 100 | 0 |
| 35 | 100 | 0 |
The chromatogram is shown in FIG. 4.
The results show that under the chromatographic conditions, impurity B is not retained, and impurity A and the main peak 3-amino-6-methoxypyridazine are not separated well.
Comparative example 2
Impurity a and impurity B system suitability solutions: an appropriate amount of the impurity A, the impurity B and a 3-amino-6-methoxypyridazine reference substance are accurately weighed, dissolved by adding water and diluted to a mixed solution containing 0.75 mu g/ml of the impurity A, 0.3 mu g/ml of the impurity B and 0.5mg/ml of the 3-amino-6-methoxypyridazine.
The system applicability solution is injected and detected, and the result is recorded, and the detection conditions are as follows: a chromatographic column: xbridge Shield RP18 column, 3.5 μm × 4.6mm × 150 mm; mobile phase: gradient elution is carried out by taking 20mmol/L potassium hexafluorophosphate solution (pH is adjusted to 3.0 by phosphoric acid) as a mobile phase A and acetonitrile as a mobile phase B; flow rate: 1.0 ml/min; column temperature: 35 ℃; detection wavelength: 215 nm; sample introduction volume: 10 μ l, gradient program as follows:
| time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 100 | 0 |
| 10 | 100 | 0 |
| 25 | 75 | 25 |
| 25.01 | 100 | 0 |
| 35 | 100 | 0 |
The chromatogram is shown in FIG. 5.
The results show that under the chromatographic conditions, the impurity A and the main peak 3-amino-6-methoxypyridazine are well separated, but the impurity B is not retained.
Comparative example 3
System applicability solution: a system suitability solution was prepared as in example 1.
The system is applied to the sample injection detection and the result is recorded, and the detection conditions are as follows: and (3) chromatographic column: atlantis T3 column, 3 μm × 4.6mm × 150 mm; mobile phase: gradient elution is carried out by taking 20mmol/L potassium hexafluorophosphate buffer solution (pH is adjusted to 2.7 by phosphoric acid) as a mobile phase A and taking acetonitrile as a mobile phase B; flow rate: 1.0 ml/min; column temperature: 30 ℃; detection wavelength: 215 nm; sample introduction volume: 5 μ l, gradient program as follows:
| time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 100 | 0 |
| 5 | 100 | 0 |
| 25 | 75 | 25 |
| 25.01 | 100 | 0 |
| 35 | 100 | 0 |
The chromatogram is shown in FIG. 6.
The results show that under this chromatographic condition, impurity E and impurity F are not separated at baseline.
Comparative example 4
System applicability solution: a system suitability solution was prepared as in example 1.
The system applicability solution is injected and detected, and the result is recorded, and the detection conditions are as follows: a chromatographic column: atlantis T3 column, 3 μm × 4.6mm × 150 mm; mobile phase: gradient elution is carried out by taking 20mmol/L potassium hexafluorophosphate buffer solution (pH is adjusted to 3.3 by phosphoric acid) as a mobile phase A and taking acetonitrile as a mobile phase B; flow rate: 1.0 ml/min; column temperature: at 37 ℃; detection wavelength: 215 nm; sample introduction volume: 5 μ l, gradient program as follows:
| time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 100 | 0 |
| 10 | 100 | 0 |
| 25 | 75 | 25 |
| 25.01 | 100 | 0 |
| 35 | 100 | 0 |
The chromatogram is shown in FIG. 7.
The results show that under the chromatographic conditions, the impurity C and the main peak 3-amino-6-methoxypyridazine are poorly separated.
Claims (8)
1. A method for detecting a 3-amino-6-methoxypyridazine-related substance, which comprises the following steps:
(1) preparing a test solution and a control solution;
(2) detection and recording: detecting the test solution and the control solution by adopting a liquid chromatography and recording spectrograms; wherein the chromatographic conditions of the liquid chromatography are as follows: a chromatographic column: a water-resistant octadecyl bonded silica gel column; mobile phase: gradient elution is carried out by taking potassium hexafluorophosphate buffer solution (the pH value is adjusted to 2.8-3.2 by using phosphoric acid) as a mobile phase A and acetonitrile as a mobile phase B; flow rate: 0.9-1.1 ml/min; column temperature: 33-37 ℃; detection wavelength: 213 to 217 nm.
2. The detection method according to claim 1, wherein a ghost-trapping column is connected in front of the sample injector of the liquid chromatograph.
3. The detection method according to claim 1 or 2, wherein the chromatographic column is an Atlantis T3 column or a Sepax HP C18 column.
4. The detection method according to claim 3, wherein the Atlantis T3 column has a specification of particle size (μm) x inner diameter (mm) x column length (mm): 3 × 4.6 × 150.
5. The detection method according to claim 3, wherein the specification of the Sepax HP C18 column is particle size (μ ι η) x inner diameter (mm) x column length (mm): 5X 4.6X 250.
6. The detection method according to any one of claims 1 to 5, wherein the concentration of the potassium hexafluorophosphate buffer solution is 15 to 25 mmol/L.
7. The detection method according to any one of claims 1 to 6, wherein the mobile phases A and B are eluted in a gradient manner as follows:
8. the detection method according to any one of claims 1 to 7, characterized in that the sample amount of the control solution and the sample solution is 5 to 10 μ l.
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| CN107188855A (en) * | 2017-07-14 | 2017-09-22 | 常州工程职业技术学院 | A kind of preparation method of the methoxyl group pyridazine of 3 amino 6 |
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| CN111440188A (en) * | 2020-04-10 | 2020-07-24 | 江苏海悦康医药科技有限公司 | Preparation method of Relugol (Relugolix) key intermediate |
| CN113444105A (en) * | 2020-03-27 | 2021-09-28 | 南京海润医药有限公司 | Preparation method of Relugolix |
| CN113563363A (en) * | 2020-12-30 | 2021-10-29 | 上海博志研新药物技术有限公司 | Rugoside intermediate, preparation method thereof and preparation method of Rugoside |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109053766A (en) * | 2012-09-28 | 2018-12-21 | 武田药品工业株式会社 | The preparation method of Thienopyrimidine derivative |
| CN107188855A (en) * | 2017-07-14 | 2017-09-22 | 常州工程职业技术学院 | A kind of preparation method of the methoxyl group pyridazine of 3 amino 6 |
| CN113444105A (en) * | 2020-03-27 | 2021-09-28 | 南京海润医药有限公司 | Preparation method of Relugolix |
| CN111440188A (en) * | 2020-04-10 | 2020-07-24 | 江苏海悦康医药科技有限公司 | Preparation method of Relugol (Relugolix) key intermediate |
| CN113563363A (en) * | 2020-12-30 | 2021-10-29 | 上海博志研新药物技术有限公司 | Rugoside intermediate, preparation method thereof and preparation method of Rugoside |
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