CN114384186A - Method for measuring content of (2E, 4E) -ethyl-4- (pyridine-2-yl imino) -2-ethyl crotonate - Google Patents
Method for measuring content of (2E, 4E) -ethyl-4- (pyridine-2-yl imino) -2-ethyl crotonate Download PDFInfo
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Abstract
The invention discloses a content determination method of "(2E, 4E) -ethyl-4- (pyridine-2-yl imino) -2-ethyl crotonate", taking a reference substance of "(2E, 4E) -ethyl-4- (pyridine-2-yl imino) -2-ethyl crotonate" to prepare a reference substance solution with the concentration of 5 mug/mL, taking a raw material medicine sample of minodronate hydrate to prepare a minodronate hydrate test solution with the concentration of 500 mug/mL, and finally respectively and precisely sucking the reference substance solution and the test solution to inject into a high performance liquid chromatograph for determination; the determination method is convenient, accurate and reliable, and can truly reflect the content of the (2E, 4E) -ethyl-4- (pyridine-2-yl imino) -2-ethyl crotonate in the product.
Description
Technical Field
The invention relates to the field of medicines, in particular to a content determination method of (2E, 4E) -ethyl-4- (pyridine-2-yl imino) -2-ethyl crotonate.
Background
Minodronic acid hydrate is 1-hydroxy-2- (imidazo [1,2-a ]]Pyridin-3-yl) ethane-1, 1-bisphosphonic acid monohydrate having a molecular weight of 340.16 g/mol and a molecular formula C9H12N2O7P2·H2O; minodronate hydrate is an orally bioavailable third-generation bisphosphonate drug and has obvious curative effect on osteoporosis. Bisphosphonates are artificially synthesized non-biodegradable pyrophosphate analogs, and are bisphosphonates developed by Nippon Intra pharmaceutical Co., Ltd and by Small wild drug Co., Ltd. At present, in non-clinical tests, the minodronate hydrate has good bone resorption resistance, and the effects of inhibiting bone density and bone strength reduction only by using the product with lower dose than the existing dicarbphosphate medicines; in the case of a clinical trial,shows an excellent bone density increasing effect on patients with Japanese degenerative osteoporosis, and demonstrates its preventive effect on bone fracture;
in the synthesis process of minodronate hydrate, a related substance of a byproduct, namely "(2E, 4E) -ethyl-4- (pyridine-2-yl imino) -2-butenoic acid ethyl ester", is possibly generated under the influence of synthesis conditions, and the content of the related substance, "(2E, 4E) -ethyl-4- (pyridine-2-yl imino) -2-butenoic acid ethyl ester", is very necessary to be strictly controlled as a medicinal preparation in order to ensure the safety of the medicament. However, in the prior art, there is no report on a method for measuring the content of ethyl "(2E, 4E) -ethyl-4- (pyridin-2-ylimino) -2-butenoate". The invention provides a convenient, accurate, efficient and cheap detection method for detecting a related substance (2E, 4E) -ethyl-4- (pyridine-2-yl imino) -2-ethyl crotonate) of minodronic acid hydrate before delivery or use.
Disclosure of Invention
The invention aims to provide a content determination method of (2E, 4E) -ethyl-4- (pyridine-2-yl imino) -2-ethyl crotonate, which is convenient, accurate and reliable and can truly reflect the content of (2E, 4E) -ethyl-4- (pyridine-2-yl imino) -2-ethyl crotonate' of a product;
in order to achieve the above purpose, the solution of the invention is:
a content determination method of "(2E, 4E) -ethyl-4- (pyridine-2-yl imino) -2-butenoic acid ethyl ester" comprises the following steps:
(1) preparation of control solutions: taking 1-10 mg of a "(2E, 4E) -ethyl-4- (pyridine-2-yl imino) -2-ethyl crotonate" reference substance, dissolving the reference substance with a mobile phase to prepare reference substance mother liquor, diluting the reference substance mother liquor with the mobile phase again to prepare reference substance solution with the concentration of 5ug/mL, and placing the reference substance solution in a measuring flask for later use;
(2) preparation of a test solution: taking 10-30 mg of a raw material medicine sample of the minodronate hydrate, dissolving the raw material medicine sample with an alkali solution, carrying out ultrasonic treatment for 5 min, cooling to room temperature, placing the mixture into a 50 mL measuring flask, adding a mobile phase to dilute the mixture to a scale, shaking up to obtain a test sample, and placing the test sample into the measuring flask for later use;
(3) high performance liquid chromatography conditions: the chromatographic column is Altima C18A chromatographic column (4.6 mm multiplied by 200 mm, 5 μm), a mobile phase of 2.66 g/L sodium pyrophosphate-methanol, a detection wavelength of 225 nm, a flow rate of 1.0 mL/min, a column temperature of 25 ℃, a sample injection amount of 10 μ L, and a running time of 30 min;
(4) the determination method comprises the following steps: precisely sucking the reference substance and the sample solution, injecting into a high performance liquid chromatograph, and measuring;
the alkali solution in the step (2) is 1-6 g/L sodium hydroxide solution;
the pH value of the mobile phase in the step (3) is 6.0-8.0, and the pH value of the mobile phase is adjusted by acid, wherein the concentration of the acid is 75-90%; the mobile phase is 2.66 g/L sodium pyrophosphate-methanol = 80: 20;
the content of the ethyl "(2E, 4E) -ethyl-4- (pyridin-2-ylimino) -2-butenoate" in the minodronate hydrate test sample determined in the step (4) is not higher than 0.1%;
in order to prove the accuracy and reliability of the content determination method of the "(2E, 4E) -ethyl-4- (pyridine-2-yl imino) -2-ethyl crotonate" in the invention, the content determination method is verified by the following test method;
1. instrument and reagent
The reagents used in the present invention are either commercially available or can be prepared by the methods described herein;
reagent: the methanol and the water are chromatographically pure, and the sodium pyrophosphate, the phosphoric acid and the sodium hydroxide are analytically pure;
sample preparation: self-making;
comparison products: "(2E, 4E) -ethyl-4- (pyridin-2-ylimino) -2-butenoic acid ethyl ester" control was supplied by TLC Pharmaceutical Standards Ltd. for assay;
the instrument comprises the following steps: agilent 1200 type high performance liquid chromatograph (Agilent corporation), electronic balance (aohaus instruments ltd.), UV-1750 UV-visible spectrophotometer (shimadzu corporation, japan), ultrasonic cleaner (shanghai leap into medical instrument factory);
2. chromatographic conditions
A chromatographic column: the chromatographic column is Altima C18A chromatographic column (4.6 mm × 200 mm, 5 μm) with a mobile phase of 2.66 g/L sodium pyrophosphate-methanol = 80: 20, pH adjusted to 7.4 with 85% phosphoric acid, detection wavelength of 225 nm, flow rate of 1.0 mL/min, column temperature of 25 ℃, sample size of 10 μ L, run time of 30 min; the theoretical plate number is not less than 6000 according to the peak calculation of "(2E, 4E) -ethyl-4- (pyridine-2-yl imino) -2-ethyl crotonate"; and (3) sample introduction mode: automatic sample introduction;
3. preparation of control solutions: accurately weighing 5 mg of a "(2E, 4E) -ethyl-4- (pyridine-2-yl imino) -2-ethyl crotonate" reference substance, dissolving the reference substance with a mobile phase, placing the reference substance in a 100 mL measuring flask, adding the mobile phase to dilute the reference substance to a scale to prepare reference substance mother liquor with the concentration of 50 mug/mL, placing 1mL of the reference substance mother liquor in a 10mL volumetric flask, diluting the reference substance mother liquor with the mobile phase to the scale, and configuring a reference substance solution containing 5 mug of "(2E, 4E) -ethyl-4- (pyridine-2-yl imino) -2-ethyl crotonate" in each l of mL;
4. preparation of a test solution: precisely weighing 25 mg of minodronate hydrate, adding 4 g/L of sodium hydroxide solution for dissolving, carrying out ultrasonic treatment for 5 min, cooling to room temperature, placing in a 50 mL measuring flask, adding a mobile phase for diluting to a scale, and shaking up to obtain a minodronate hydrate test sample solution with the concentration of 500 mug/mL;
5. the determination method comprises the following steps: precisely sucking 10 mu L of each of the reference solution and the sample solution, injecting into a high performance liquid chromatograph, and measuring to obtain the sample solution;
6. system suitability test
Taking appropriate amount of the "(2E, 4E) -ethyl-4- (pyridine-2-yl imino) -2-butenoic acid ethyl ester" reference substance and minodronic acid hydrate, preparing a system adaptability test sample according to the preparation method of the sample solution, and measuring according to the measuring method under the item of measuring the content of the "(2E, 4E) -ethyl-4- (pyridine-2-yl imino) -2-butenoic acid ethyl ester"). Taking a proper amount of a reference substance of "(2E, 4E) -ethyl-4- (pyridine-2-yl imino) -2-butenoic acid ethyl ester" and a proper amount of minodronic acid hydrate, preparing a mixed test sample according to the method under the item of preparing the test sample solution, and measuring according to the measuring method under the item of measuring the content of the "(2E, 4E) -ethyl-4- (pyridine-2-yl imino) -2-butenoic acid ethyl ester"; the results show that, at the peak of the "(2E, 4E) -ethyl-4- (pyridin-2-ylimino) -2-butenoic acid ethyl ester" control, no other peaks appear, and that the "(2E, 4E) -ethyl-4- (pyridin-2-ylimino) -2-butenoic acid ethyl ester" control is completely separated from the minodronic acid hydrate peak, so that minodronic acid hydrate itself does not interfere with the assay of the "(2E, 4E) -ethyl-4- (pyridin-2-ylimino) -2-butenoic acid ethyl ester";
7. stability test
Taking about 5 mg of a "(2E, 4E) -ethyl-4- (pyridine-2-yl imino) -2-ethyl crotonate" reference substance, processing according to the method under the preparation item of the reference substance solution to prepare a stability test sample solution, sampling and measuring for 0, 1,2, 4, 6, 8 and 12 hours respectively according to a content measuring method, and recording a chromatogram, wherein the result is shown in a table 1, the stability test sample solution is placed at room temperature for about 12 hours, and the peak area RSD of the "(2E, 4E) -ethyl-4- (pyridine-2-yl imino) -2-ethyl crotonate" is less than 2%, which indicates that the product is stable after being placed at room temperature for 12 hours;
TABLE 1 stability test results for solutions of "(2E, 4E) -ethyl-4- (pyridin-2-ylimino) -2-butenoic acid ethyl ester
8. Repeatability test
Taking the same batch of (2E, 4E) -ethyl-4- (pyridine-2-yl imino) -2-ethyl crotonate "reference substance, preparing 6 parts according to the method under the preparation item of the reference substance solution, performing content measurement, recording a chromatogram, and calculating the content of (2E, 4E) -ethyl-4- (pyridine-2-yl imino) -2-ethyl crotonate" according to a linear equation. The results are shown in Table 2, the average content of ethyl "(2E, 4E) -ethyl-4- (pyridin-2-ylimino) -2-butenoate" in 6 samples was 4.968 μ g/mL, and the RSD (n =6) was 1.39%; the result shows that the method has good repeatability;
TABLE 2 repeated experimental results for the content of ethyl (2E, 4E) -ethyl-4- (pyridin-2-ylimino) -2-butenoate
| Sample (I) | 1 | 2 | 3 | 4 | 5 | 6 | Mean value (microgram/mL) | RSD（%） |
| Content (mu g/mL) | 5.08 | 5.01 | 4.95 | 4.96 | 4.88 | 4.93 | 4.97 | 1.39 |
9. Recovery rate experiment
Accurately weighing (8 mg, 10 mg and 12 mg of a) (2E, 4E) -ethyl-4- (pyridine-2-yl imino) -2-ethyl crotonate) reference substance respectively, placing the reference substance into a 50 mL measuring flask, adding a mobile phase for dissolving, diluting to a scale, shaking up, accurately weighing 2 mL again, placing the reference substance into a 100 mL measuring flask, diluting to the scale with methanol, shaking up to obtain a sample solution with the concentration of 80%, 100% and 120%; 3 parts of each concentration is prepared in parallel, and a content recording chromatogram is determined according to a content determination method;
the results are shown in Table 3, under the proposed chromatographic conditions, the average recovery rate of the "(2E, 4E) -ethyl-4- (pyridine-2-yl imino) -2-ethyl crotonate" is between 98.0% and 101.0%, the RSD value is less than 2.0%, and the recovery rate meets the regulation;
TABLE 3 recovery of ethyl "(2E, 4E) -ethyl-4- (pyridin-2-ylimino) -2-butenoate" test results
10. Durability test
The content of the ethyl "(2E, 4E) -ethyl-4- (pyridine-2-yl imino) -2-butenoate" is measured under the conditions that the detection wavelength is 225 +/-2 nm, the flow rate is 1.0 +/-0.1 mL/min, the column temperature is 25 +/-5 ℃, the mobile phase pH is 7.4 +/-0.2, the water phase proportion is 80% +/-3%, and different chromatographic columns of the same type and the same specification are adopted, and the result shows that the RSD value of data which is repeated three times under each change condition is less than 2%, and the durability of the chromatographic conditions is good;
11. intermediate precision test
Preparing reference solution on different test instruments by different testers, precisely measuring 10 μ L of the reference solution, injecting into a high performance liquid chromatograph, continuously introducing sample for 6 times, and recording chromatogram. The RSD values for the "(2E, 4E) -ethyl-4- (pyridin-2-ylimino) -2-butenoic acid ethyl ester" content are all less than 2%; the results show that the sample testing results are not very different when the samples are tested by different analysts and different devices in the same laboratory; the result shows that the content determination method of the "(2E, 4E) -ethyl-4- (pyridine-2-yl imino) -2-ethyl crotonate" has good intermediate precision test;
in conclusion, the content determination method of the (2E, 4E) -ethyl-4- (pyridine-2-yl imino) -2-ethyl crotonate provided by the invention is convenient, accurate and reliable, and can truly reflect the quality of a product.
Detailed Description
In order to further explain the technical scheme of the invention, the invention is explained in detail by the specific embodiment;
example 1
A content determination method of "(2E, 4E) -ethyl-4- (pyridine-2-yl imino) -2-butenoic acid ethyl ester" comprises the following steps:
(1) preparation of control: accurately weighing 5 mg of a "(2E, 4E) -ethyl-4- (pyridine-2-yl imino) -2-ethyl crotonate" reference substance, dissolving the reference substance with a mobile phase, placing the reference substance in a 100 mL measuring flask, adding the mobile phase to dilute the reference substance to a scale to prepare reference substance mother liquor with the concentration of 50 mug/mL, placing 1mL of the reference substance mother liquor in a 10mL volumetric flask, diluting the reference substance mother liquor with the mobile phase to the scale, and configuring a reference substance solution containing 5 mug of "(2E, 4E) -ethyl-4- (pyridine-2-yl imino) -2-ethyl crotonate" in each l of mL;
(2) preparing a test sample: precisely weighing 25 mg of minodronate hydrate, adding 4 g/L of sodium hydroxide solution for dissolving, carrying out ultrasonic treatment for 5 min, cooling to room temperature, placing in a 50 mL measuring flask, adding a mobile phase for diluting to a scale, and shaking up to obtain a sample solution;
(3) high performance liquid chromatography conditions:
a chromatographic column: the chromatographic column is Altima C18A chromatographic column (4.6 mm × 200 mm, 5 μm) with a mobile phase of 2.66 g/L sodium pyrophosphate-methanol = 80: 20, pH adjusted to 7.4 with 85% phosphoric acid, detection wavelength of 225 nm, flow rate of 1.0 mL/min, column temperature of 25 ℃, sample size of 10 μ L, run time of 30 min; the number of theoretical plates is not less than 6000 calculated according to the formula of (2E, 4E) -ethyl-4- (pyridine-2-yl imino) -2-butenoic acid ethyl ester'; and (3) sample introduction mode: automatic sample introduction;
(4) the determination method comprises the following steps: precisely sucking 10 mu L of a reference substance solution, injecting the reference substance solution into a high performance liquid chromatograph, carrying out methodology verification according to a methodology investigation test and establishing a standard curve; precisely sucking 10 mu L of a sample solution, injecting the sample solution into a high performance liquid chromatograph, measuring, and calculating the content of the ethyl (2E, 4E) -ethyl-4- (pyridine-2-yl imino) -2-butenoate "according to a reference linear equation of the ethyl (2E, 4E) -ethyl-4- (pyridine-2-yl imino) -2-butenoate";
20211201 batches of minodronate hydrate, the results of the determination of "(2E, 4E) -ethyl-4- (pyridin-2-ylimino) -2-butenoic acid ethyl ester" are shown in Table 4. The result shows that the content of the ethyl (2E, 4E) -ethyl-4- (pyridine-2-yl imino) -2-butenoate "in 20211201 batches of minodronic acid hydrate is 0.03 percent and not more than 0.1 percent, and the result meets the requirement;
TABLE 4 detection of "(2E, 4E) -ethyl-4- (pyridin-2-ylimino) -2-butenoic acid ethyl ester
| Minodronate hydrate batch number | Concentration of ethyl (2E, 4E) -ethyl-4- (pyridin-2-ylimino) -2-butenoate | Content of ethyl (2E, 4E) -ethyl-4- (pyridin-2-ylimino) -2-butenoate |
| 20211201 | 0.142 µg/mL | 0.03% |
Example 2
A content determination method of "(2E, 4E) -ethyl-4- (pyridine-2-yl imino) -2-butenoic acid ethyl ester" comprises the following steps:
(1) preparation of control: accurately weighing 5 mg of a "(2E, 4E) -ethyl-4- (pyridine-2-yl imino) -2-ethyl crotonate" reference substance, dissolving the reference substance with a mobile phase, placing the reference substance in a 100 mL measuring flask, adding the mobile phase to dilute the reference substance to a scale to prepare reference substance mother liquor with the concentration of 50 mug/mL, placing 1mL of the reference substance mother liquor in a 10mL volumetric flask, diluting the reference substance mother liquor with the mobile phase to the scale, and configuring a reference substance solution containing 5 mug of "(2E, 4E) -ethyl-4- (pyridine-2-yl imino) -2-ethyl crotonate" in each l of mL;
(2) preparing a test sample: precisely weighing 25 mg of minodronate hydrate, adding 4 g/L of sodium hydroxide solution for dissolving, carrying out ultrasonic treatment for 5 min, cooling to room temperature, placing in a 50 mL measuring flask, adding a mobile phase for diluting to a scale, and shaking up to obtain a sample solution;
(3) high performance liquid chromatography conditions:
a chromatographic column: the chromatographic column is Altima C18A chromatographic column (4.6 mm × 200 mm, 5 μm) with a mobile phase of 2.66 g/L sodium pyrophosphate-methanol = 80: 20, pH adjusted to 7.6 with 75% phosphoric acid, detection wavelength of 225 nm, flow rate of 1.0 mL/min, column temperature of 25 ℃, sample size of 10 μ L, run time of 30 min; theory of the inventionThe number of plates should not be less than 6000 calculated from the peak of "(2E, 4E) -ethyl-4- (pyridin-2-ylimino) -2-butenoic acid ethyl ester"; and (3) sample introduction mode: automatic sample introduction;
(4) the determination method comprises the following steps: precisely sucking 10 mu L of a reference substance solution, injecting the reference substance solution into a high performance liquid chromatograph, carrying out methodology verification according to a methodology investigation test and establishing a standard curve; precisely sucking 10 mu L of a sample solution, injecting the sample solution into a high performance liquid chromatograph, measuring, and calculating the content of the ethyl (2E, 4E) -ethyl-4- (pyridine-2-yl imino) -2-butenoate "according to a reference linear equation of the ethyl (2E, 4E) -ethyl-4- (pyridine-2-yl imino) -2-butenoate";
20211202 measurement results of the content of "(2E, 4E) -ethyl-4- (pyridin-2-ylimino) -2-butenoic acid ethyl ester" in minodronic acid hydrate batch are shown in Table 4. The result shows that the content of the ethyl (2E, 4E) -ethyl-4- (pyridine-2-yl imino) -2-butenoate "in 20211201 batches of minodronic acid hydrate is 0.03 percent and not more than 0.1 percent, and the result meets the requirement;
TABLE 5 detection of "(2E, 4E) -ethyl-4- (pyridin-2-ylimino) -2-butenoic acid ethyl ester
| Minodronate hydrate batch number | Concentration of ethyl (2E, 4E) -ethyl-4- (pyridin-2-ylimino) -2-butenoate | Content of ethyl (2E, 4E) -ethyl-4- (pyridin-2-ylimino) -2-butenoate |
| 20211202 | 0.169 µg/mL | 0.03% |
Example 3
A content determination method of "(2E, 4E) -ethyl-4- (pyridine-2-yl imino) -2-butenoic acid ethyl ester" comprises the following steps:
(1) preparation of control: accurately weighing 5 mg of a "(2E, 4E) -ethyl-4- (pyridine-2-yl imino) -2-ethyl crotonate" reference substance, dissolving the reference substance with a mobile phase, placing the reference substance in a 100 mL measuring flask, adding the mobile phase to dilute the reference substance to a scale to prepare reference substance mother liquor with the concentration of 50 mug/mL, placing 1mL of the reference substance mother liquor in a 10mL volumetric flask, diluting the reference substance mother liquor with the mobile phase to the scale, and configuring a reference substance solution containing 5 mug of "(2E, 4E) -ethyl-4- (pyridine-2-yl imino) -2-ethyl crotonate" in each l of mL;
(2) preparing a test sample: precisely weighing 25 mg of minodronate hydrate, adding 6g/L of sodium hydroxide solution for dissolving, carrying out ultrasonic treatment for 5 min, cooling to room temperature, placing in a 50 mL measuring flask, adding a mobile phase for diluting to a scale, and shaking up to obtain a sample solution;
(3) high performance liquid chromatography conditions:
a chromatographic column: the chromatographic column is Altima C18Chromatography column (4.6 mm × 200 mm, 5 μm), mobile phase 2.66 g/L sodium pyrophosphate-methanol = 80: 20, adjusting the pH value to 7.2 by using 90% phosphoric acid, detecting the wavelength to 225 nm, the flow rate to 1.0 mL/min, the column temperature to 25 ℃, the sample injection amount to 10 mu L and the running time to 30 min; the theoretical plate number is not less than 6000 according to the peak calculation of "(2E, 4E) -ethyl-4- (pyridine-2-yl imino) -2-ethyl crotonate"; and (3) sample introduction mode: automatic sample introduction;
(4) the determination method comprises the following steps: precisely sucking 10 mu L of a reference substance solution, injecting the reference substance solution into a high performance liquid chromatograph, carrying out methodology verification according to a methodology investigation test and establishing a standard curve; precisely sucking 10 mu L of a sample solution, injecting the sample solution into a high performance liquid chromatograph, measuring, and calculating the content of the ethyl (2E, 4E) -ethyl-4- (pyridine-2-yl imino) -2-butenoate "according to a reference linear equation of the ethyl (2E, 4E) -ethyl-4- (pyridine-2-yl imino) -2-butenoate";
20211203 measurement results of the content of "(2E, 4E) -ethyl-4- (pyridin-2-ylimino) -2-butenoic acid ethyl ester" in minodronic acid hydrate batch are shown in Table 4; the result shows that the content of the ethyl (2E, 4E) -ethyl-4- (pyridine-2-yl imino) -2-butenoate "in 20211201 batches of minodronic acid hydrate is 0.02 percent and not more than 0.1 percent, and the result meets the requirement;
TABLE 5 (2E, 4E) -Ethyl-4- (pyridin-2-ylimino) -2-butenoic acid Ethyl ester assay results
| Minodronate hydrate batch number | Concentration of ethyl (2E, 4E) -ethyl-4- (pyridin-2-ylimino) -2-butenoate | Content of ethyl (2E, 4E) -ethyl-4- (pyridin-2-ylimino) -2-butenoate |
| 20211203 | 0.117 µg/mL | 0.02% |
The above embodiments and drawings are not intended to limit the form and style of the present invention, and any suitable changes or modifications thereof by those skilled in the art should be considered as not departing from the scope of the present invention.
Claims (4)
- A method for measuring the content of ethyl (2E, 4E) -ethyl-4- (pyridine-2-yl imino) -2-butenoate, which is characterized by comprising the following steps: the method comprises the following steps:(1) preparation of control solutions: taking 1-10 mg of a "(2E, 4E) -ethyl-4- (pyridine-2-yl imino) -2-ethyl crotonate" reference substance, dissolving the reference substance with a mobile phase to prepare reference substance mother liquor, diluting the reference substance mother liquor with the mobile phase again to prepare reference substance solution with the concentration of 5ug/mL, and placing the reference substance solution in a measuring flask for later use;(2) preparation of a test solution: taking 10-30 mg of a raw material medicine sample of the minodronate hydrate, dissolving the raw material medicine sample with an alkali solution, carrying out ultrasonic treatment for 5 min, cooling to room temperature, placing the mixture into a 50 mL measuring flask, adding a mobile phase to dilute the mixture to a scale, shaking up to obtain a test sample, and placing the test sample into the measuring flask for later use;(3) high performance liquid chromatography conditions: the chromatographic column is Altima C18A chromatographic column (4.6 mm multiplied by 200 mm, 5 μm), a mobile phase of 2.66 g/L sodium pyrophosphate-methanol, a detection wavelength of 225 nm, a flow rate of 1.0 mL/min, a column temperature of 25 ℃, a sample injection amount of 10 μ L, and a running time of 30 min;(4) the determination method comprises the following steps: precisely sucking the reference substance and the sample solution, injecting into high performance liquid chromatograph, and measuring.
- 2. The method for measuring the content of ethyl (2E, 4E) -ethyl-4- (pyridin-2-ylimino) -2-butenoate according to claims 1 and 3, wherein: the alkali solution in the step (2) is 1-6 g/L sodium hydroxide solution.
- 3. The method for determining the content of ethyl (2E, 4E) -ethyl-4- (pyridin-2-ylimino) -2-butenoate according to claim 1, wherein: the pH value of the mobile phase in the step (3) is 6.0-8.0, and the pH value of the mobile phase is adjusted by acid, wherein the concentration of the acid is 75-90%.
- 4. The method for determining the content of ethyl (2E, 4E) -ethyl-4- (pyridin-2-ylimino) -2-butenoate according to claim 1, wherein: the mobile phase was 2.66 g/L sodium pyrophosphate-methanol = 80: 20 mobile phase.
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