CN114414678B

CN114414678B - Analysis method of related impurities of isoxazoline veterinary drug intermediate ammonium salt

Info

Publication number: CN114414678B
Application number: CN202111608928.4A
Authority: CN
Inventors: 温军贤; 李冰冰; 冯绮晴; 姜桥
Original assignee: Livzon New North River Pharmaceutical Co ltd
Current assignee: Livzon New North River Pharmaceutical Co ltd
Priority date: 2021-12-27
Filing date: 2021-12-27
Publication date: 2023-05-30
Anticipated expiration: 2041-12-27
Also published as: CN114414678A

Abstract

The invention discloses an analysis method of related impurities of an isoxazoline veterinary drug intermediate ammonium salt, wherein the intermediate is 2-amino-N- (2, 2-trifluoroethyl) acetamide, the related impurities are triethylamine hydrochloride and glycine, and a diluent is used for dissolving the intermediate ammonium salt and a reference substance to obtain a sample solution and a reference substance solution; glycine was detected using reverse phase high performance liquid chromatography and triethylamine hydrochloride was detected using gas chromatography. The method can effectively separate the impurities of the 2-amino-N- (2, 2-trifluoroethyl) acetamide, and has the advantages of high efficiency, simplicity, convenience, high sensitivity and good separation effect.

Description

Analysis method of related impurities of isoxazoline veterinary drug intermediate ammonium salt

Technical Field

The invention belongs to the field of analysis of drug intermediates, and particularly relates to an analysis method of related impurities of an isoxazoline veterinary drug intermediate ammonium salt.

Background

Fluoro Lei Lana (Fluralaner) and Alfoxolaner (Afoxoloner) are high-end long-acting antiparasitic agents that act by interfering with the parasite's gamma-aminobutyric acid (GABA) -gated chloride ion channel resulting in neuronal system hyperexcitability and death. Fluralaner is the first oral chewing ectoparasiticide against fleas and ticks provided for up to 12 weeks. A chewable tablet or a dose of topical solution provides broad spectrum and long lasting protection, begins to kill fleas within 2 hours, and controls 4 ticks (black leg ticks, american dog ticks, brown dog ticks and solitary ticks). Afoxoloner is taken as an oral insect repellent for killing ticks and fleas at the first time in China, can quickly take effect after being taken orally, and can quickly cause death of the fleas and ticks under extreme excitation by acting on nerve transmission synapses of arthropods through afrana molecules, and a piece of effect can be maintained for 30 days after taking.

Fluorine Lei Lana is similar to aforana in chemical structure, they possess the same one key fragment 2-amino-N- (2, 2-trifluoroethyl) acetamide, fluorine Lei Lana and aforana have the following chemical structures:

2-amino-N- (2, 2-trifluoroethyl) acetamide as an important fragment constituting fluorine Lei Lana and aforana, the main synthetic routes reported at present are as follows:

it is known from the route and structure that the reaction system is mainly composed of two parts (as follows), and thus it is known that the study of two main impurities, glycine and trifluoroethylamine salt, plays a critical role in 2-amino-N- (2, 2-trifluoroethyl) acetamide.

In order to better control the quality of 2-amino-N- (2, 2-trifluoroethyl) acetamide and afurana and fluororalfinade, the invention systematically researches the related substances in most of 2-amino-N- (2, 2-trifluoroethyl) acetamides which are self-made and on the market, and finds that the main impurities are glycine and trifluoroethylamine salts, and the quality research of 2-amino-N- (2, 2-trifluoroethyl) acetamides is not reported much at present.

Disclosure of Invention

The invention aims to overcome at least one defect of the prior art and provides a general method for conveniently researching the quality of 2-amino-N- (2, 2-trifluoroethyl) acetamide.

The technical scheme adopted by the invention is as follows:

an analytical method for related impurities of an isoxazoline veterinary drug intermediate ammonium salt, wherein the intermediate is 2-amino-N- (2, 2-trifluoroethyl) acetamide, and the related impurities are triethylamine hydrochloride and glycine, comprising:

dissolving the intermediate ammonium salt and the reference substance by using a diluent to obtain a sample solution and a reference substance solution;

glycine was detected using reverse phase high performance liquid chromatography and trifluoroethylamine hydrochloride was detected using gas chromatography:

the detection of glycine using reverse phase high performance liquid chromatography comprises:

detecting intermediate ammonium salt and impurities thereof in a sample solution by using a reversed-phase high performance liquid chromatograph, and determining a chromatogram, wherein the conditions of the reversed-phase high performance liquid chromatograph are as follows: mobile phase A: acetonitrile; b: a solution of heptafluorobutyric acid; elution mode: gradient elution, namely 0 min-5.0 min,10% -20% of A and 90% -80% of B according to volume fraction; 5-15 min, 20-40% of A, 80-60% of B, 15-20 min, 40-65% of A and 60-35% of B; 20-22 min, 65-80% of A, 35-20% of B, 20-27 min, 80-10% of A and 20-90% of B; 40-65% of A, 27-35 min,10% of A and 90% of B; the reversed-phase high-performance liquid chromatography detector is an electric fog detector;

determining the amounts of the intermediate and glycine based on the results of the liquid chromatography;

detecting triethylamine hydrochloride using gas chromatography under the following conditions: the temperature of the sample port is 200-240 ℃, the temperature of the detector is 240-280 ℃, the carrier gas is nitrogen, and the split ratio is (10-20): 1.

In some examples, the concentration of the heptafluorobutyric acid solution in mobile phase B in the reverse phase high performance liquid chromatography is 0.1% to 0.15% by volume.

In some examples, the mobile phase in the reverse phase high performance liquid chromatography has a flow rate of 0.8 to 1.0mL/min.

In some examples, the column temperature of the reverse phase high performance liquid chromatography is from 30 ℃ to 40 ℃.

In some examples, the reverse phase high performance liquid chromatography column is a Venusil XBP C18 (L) column: 5 μm,4.6x250mm.

In some examples, the electrospray detector CAD parameters are: the carrier gas was nitrogen at a pressure of 35psi and an atomization temperature of 35 ℃.

In some examples, the temperature ramp up procedure for the gas chromatograph is: the initial temperature is kept at 30-50 ℃ for 3min, the temperature is raised to 180-240 ℃ at the speed of 8-12 ℃/min, and the temperature is kept at 3-8 min.

In some examples, the diluent for formulating the control solution and the test solution is a 0.2% sodium carbonate solution.

In some examples, the chromatographic column of the gas chromatograph is agilent 30m x 0.32mm,1.8 μm.

In some examples, the sample loading of the reverse phase high performance liquid chromatography is 10-20 μl.

In some examples, the gas chromatography is performed at a sample loading of 0.5 to 1.5mL.

The beneficial effects of the invention are as follows:

the method can effectively separate the impurities of the 2-amino-N- (2, 2-trifluoroethyl) acetamide, and has the advantages of high efficiency, simplicity, convenience, high sensitivity and good separation effect.

Drawings

FIG. 1 is a sample solution map of the high performance liquid phase method of example 1;

FIG. 2 is a blank solution pattern of the high performance liquid phase method of example 2;

FIG. 3 is a solution chart illustrating the applicability of the high performance liquid phase method system of example 2;

FIG. 4 is a sample solution map of the gas phase process system of example 8;

FIG. 5 is a blank solution chart of the gas phase process of example 9;

FIG. 6 is a diagram of a solution for the applicability of the vapor phase process system of example 9;

FIG. 7 is a linear regression curve of the gas chromatograph of example 11.

Detailed Description

The present invention will be described in more detail by way of examples. It is also to be understood that the following examples are given solely for the purpose of illustration and are not to be construed as limitations on the scope of the invention, since various modifications and adaptations may be made by those skilled in the art in light of the teachings herein. The specific process parameters and the like described below are also merely examples of suitable ranges, i.e., one skilled in the art can make a selection within the suitable ranges by the description herein and are not intended to be limited to the specific data described below.

Impurity C, referred to in the examples below, is triethylamine hydrochloride and impurity D is glycine.

For comparison, in the following examples, the preparation of the standard reference solution, the isoxazoline pesticide reference solution and the sample solution is the same as the reverse-phase high performance liquid chromatography detection steps, and the difference is that the reverse-phase high performance liquid chromatography (or gas chromatography) detection conditions are different, and the preparation of the standard reference solution, the isoxazoline pesticide reference solution and the sample solution and the reverse-phase high performance liquid chromatography detection (or gas chromatography) detection steps are as follows:

for the high performance liquid phase detection method of impurity D:

example 1:

1) Preparing a standard reference substance solution: accurately weighing and dissolving 2-amino-N- (2, 2-trifluoroethyl) acetamide standard (hereinafter referred to as AY standard) in a diluent (the diluent comprises 20 mug of ascorbic acid solution in 1mL, and the same applies below), and preparing a control solution containing 15mg of working standard in each mL of AY standard in each 1mL.

2) Preparing a test solution: accurately weighing 2-amino-N- (2, 2-trifluoroethyl) acetamide test sample, and adding a diluent to dissolve the test sample to prepare a solution containing 75 mug of the test sample in each 1ml.

3) Impurity D stock solution: accurately weighing reference substance of impurity D10 mg, placing into a 10ml volumetric flask, adding diluent, dissolving, and fixing volume to scale to obtain stock solution containing 1mg of impurity D in each 1ml.

4) Instrument: high performance liquid chromatography (CAD detector);

chromatographic column: venusil XBP C18 (L), 5 μm,4.6X250mm;

mobile phase a: acetonitrile;

mobile phase B:0.1% heptafluorobutyric acid;

flow rate: 1ml/min;

column temperature: 35 ℃;

CAD parameters: the carrier gas is nitrogen, the pressure is 35psi, and the atomization temperature is 35 ℃;

sample injection amount: 20 μl;

the elution conditions are shown in Table 1.

TABLE 1 chromatographic elution conditions

FIG. 1 is a diagram of a sample solution obtained by the high performance liquid phase method of example 1, and it can be seen from the diagram that impurities can be effectively separated.

Example 2: specialization of

Preparing a control solution and a test solution and an impurity D stock solution according to the embodiment 1;

preparing a system applicability solution: preparing a system applicability solution containing 15mg of a test sample and 75 mug of each of impurity A, impurity B and impurity D in each 1 ml;

preparing various destruction solutions by taking test solution:

light damage solution: taking a sample stock solution to be tested, placing the sample stock solution into a transparent EP tube, and irradiating the sample stock solution for 24 hours under 254nm ultraviolet light;

oxidative destruction solution: adding 3%H to the sample stock solution ₂ O ₂ Standing at room temperature for 4 hours;

high temperature destruction solution: taking 1ml of a sample stock solution, placing the sample stock solution in a transparent EP tube, placing the sample stock solution in a water bath at 60 ℃ for 3 hours, and cooling the sample stock solution;

acid breaking solution: adding 0.1mol/L HCl into a sample stock solution, standing at room temperature for 4 hours, and adding 0.1mol/L NaOH for neutralization;

base destruction solution: taking a sample stock solution, adding 0.1mol/L NaOH, standing for 4 hours at room temperature, and adding 0.1mol/L HCl for neutralization.

The different disruption solutions were tested and the results are shown in tables 2 and 3 below.

Table 2, XS-AY results of impurity localization

Note that: impurity localization results from the system applicability solution.

TABLE 3 results of specific destruction experiments

Conclusion: the blank solvent has no interference (figure 2), the separation degree of XS-AY and impurity D in the system applicability solution is 6.81 and more than 1.5, the separation degree of impurity D and front and back adjacent impurity peaks is NA and 9.16 and more than 1.5 respectively, and the separation degree meets the requirements (figure 3). The impurity limiting solution is substantially consistent with the system applicability solution retention time; the recovery rate in the damage experiment is 93.77-101.34%, which meets the requirements. The destroyed sample solution has no chromatographic peak with the same or similar retention time as the main peak.

Example 3: limit of detection and limit of quantification

1) Impurity D stock solution: accurately weighing reference substance of impurity D10 mg, placing into a 10ml volumetric flask, adding diluent, dissolving, and fixing volume to scale to obtain stock solution containing 1mg of impurity D in each 1ml.

According to the chromatogram of the limiting solution of each related substance in the embodiment 2, the signal-to-noise ratio of each component can be preliminarily obtained, the stock solution is taken for gradual dilution, the concentration corresponding to the signal-to-noise ratio of 2-4 is found out, and the concentration is defined as the detection limiting concentration; the signal-to-noise ratio is 8-12, and the concentration is defined as quantitative limit concentration. The results are shown in Table 4, table 5 below.

TABLE 4 detection limit results for impurity D control

Table 5, quantitative limit results for impurity D control

Conclusion: the signal to noise ratio of the 3 parts of detection limit solution is more than 3, the retention time RSD is 0.09 percent and is not more than 1.0 percent, the peak area RSD is 4.52 percent and is not more than 10.0 percent, and the requirements are met. The signal to noise ratio of the 6 parts of quantitative limiting solution is greater than 10, the retention time RSD is 0.04% or less and the peak area RSD is 3.62% or less and is 10.0% or less, and the quantitative limiting solution meets the requirements. Therefore: the detection limit of the related substances is finally determined to be 2.65 mug/ml, and the quantitative limit is determined to be 7.96 mug/ml. The test result shows that the detection method has higher sensitivity to all related substances.

Example 4: linear relationship

S1: the impurity D stock solution was diluted with a diluent to give solutions (limit 0.5%) of 0.05%, 0.10%, 0.30%, 0.50% and 1.00% of the concentration of the sample, respectively. 3 times for each sample;

s2: the peak area was measured according to the HPLC conditions in example 1, and the concentration was taken as the abscissa and the peak area was taken as the ordinate, to obtain a linear regression equation for each relevant substance, and the results are shown in Table 6.

TABLE 6 correction factor results

Conclusion: the XS-AY linear equation is y=0.0692x+0.1366, r=0.9973, more than 0.990, and the intercept is 2.44%, which meets the requirements; the impurity D linear equation is y=0.0497x+0.0263, r=0.9995, more than 0.990, and the intercept is 0.60%, which meets the requirements; the correction factor is 1.39.

Example 5: accuracy test

S1: a control solution was prepared as in example 1

Accuracy base solution: weighing the sample, adding diluent, and making into solution containing 15mg/ml sample

Accuracy 1 adding a standard solution: weighing a sample and an impurity D stock solution, diluting the sample and the impurity D stock solution by a diluent to prepare 3 parts of a solution with the concentration of the added impurity reference substance of 50% of the impurity limit, and sequentially named as 50% -1, 50% -2 and 50% -3.

Accuracy 2 addition of the labeling solution: weighing a sample to be tested and an impurity D stock solution, diluting the sample with a diluent to prepare an added impurity reference substance with the concentration of 100% of the impurity limit, preparing 3 parts in parallel, and sequentially named as 100% -1, 100% -2 and 100% -3.

Accuracy 3 addition of the labeling solution: weighing a sample and an impurity D stock solution, diluting the sample and the impurity D stock solution by a diluent to prepare 3 parts of an added impurity reference substance solution with the concentration of 150% of the impurity limit, and sequentially named as 150% -1, 150% -2 and 150% -3.

S2: the peak area of the sample solution was measured according to the high performance liquid chromatography conditions in example 1, and the recovery rate of the relevant substances in the mixed sample solution was calculated, and the results are shown in Table 7 below.

TABLE 7 accuracy results

Conclusion: the impurity D in the basic solution is not detected, the recovery rate of the standard adding solution with different concentrations is 94.43% -97.85%, the average recovery rate is 95.97%, the recovery rate is within the range of 90.0% -108.0%, and the recovery rate RSD of 9 parts is 1.34% and less than 10.0%, thereby meeting the requirements.

Example 6: durability of

S1: 1 part of a control solution and 2 parts of a test solution were prepared as in example 1; s2: the sample solutions were measured under the conditions of high performance liquid chromatography in example 1 at column temperature=30±5 ℃ and flow rate=1.2±0.3mL/min, and the contents of the relevant substances in the sample solutions at different column temperatures and flow rates were calculated, and the results are shown in tables 8 and 9.

TABLE 8 durability results at different column temperatures

TABLE 9 different flow Rate durability results

The test result shows that the influence on related substances in the 2-amino-N- (2, 2-trifluoroethyl) acetamide test sample solution is within an acceptable range under the conditions that the column temperature fluctuation is not more than 5 ℃ and the flow rate change is not more than 0.3mL/min

Example 7: stability of

S1: preparing a control solution and a test solution according to example 1;

s2: the sample solutions were measured under the conditions of high performance liquid chromatography in example 1, and the measurement was performed at 0, 3, 6, 9, 12, 15, 18, 21, and 24 hours after the preparation. The results of calculating the contents of the related substances in the test sample solutions are shown in Table 10.

Table 10 results of stability at Normal temperature of test sample addition solution

Conclusion: the reference substance solution is placed for 24 hours at normal temperature, the main peak area RSD is 1.54 percent, less than 5.0 percent, the sample solution is placed for 24 hours at normal temperature, the impurity D peak area RSD is 1.56 percent, less than 5.0 percent, and the requirements are met. The reference substance solution and the test sample adding standard solution are kept for 24 hours at normal temperature and stable.

For the gas phase detection method of impurity C:

example 8

1) Solvent (0.2% sodium carbonate solution): weighing 0.5g anhydrous sodium carbonate in a 250mL volumetric flask, adding purified water for dissolution, and diluting to a scale;

2) Blank solution: 0.2% sodium carbonate solution;

3) Control solution: accurately weighing impurity C1mg in a 10mL volumetric flask, dissolving with 0.2% sodium carbonate solution, and diluting to scale to obtain control solution stock solution; taking 1mL of control solution stock solution in a 10mL volumetric flask, diluting to a scale with 0.2% sodium carbonate solution, and taking 1mL of the solution in a headspace bottle (also used as a system applicability solution);

4) Test solution: accurately weighing 0.1g of the sample in a 10mL volumetric flask, dissolving and diluting to a scale with 0.2% sodium carbonate solution, taking 2mL of the solution and the 10mL volumetric flask, diluting to the scale with 0.2% sodium carbonate solution, and taking 1mL of the solution in a headspace bottle.

5) Instrument: gas chromatograph (FID detector);

chromatographic column: DB-624 (Agilent 30m 0.32mm,1.8 μm);

carrier gas: nitrogen gas;

column temperature: 50 ℃;

split ratio: 15:1, a step of;

flow rate: 2.0ml/min;

run time: 15min;

incubation temperature: 70 ℃;

ring/sample line temperature: 80 ℃;

equilibration time: 30min;

the detector temperature was 250 ℃;

the temperature of the sample inlet is 220 ℃;

sample injection amount: 1mL.

FIG. 4 is a sample solution map of the vapor phase process system of example 8. From the figure, the impurities can be effectively separated, which is beneficial to detection.

Example 9: system applicability and specificity

Control solutions (System applicability solutions) and test sample solutions were prepared according to example 8

Table 11 system applicability solution results

Table 12 specific positioning results

Comment (1): the blank solution has no interference

Results and conclusions:

1) The blank solution has no interference (figure 5), and the retention time of the impurity C in the test solution is consistent with that in the control solution and meets the standard;

2) Continuous 5-needle system applicability solution impurity C peak area RSD was 0.78% (FIG. 6), satisfactory.

Example 10: limit of detection and limit of quantification

1) Impurity C stock solution: the impurity C reference substance 10mg is precisely weighed, placed in a 10ml volumetric flask, dissolved by adding solvent (0.2% sodium carbonate solution) and fixed to volume to scale, and prepared into stock solution containing 1mg of impurity C each 1ml.

According to the chromatogram of the limiting solution of each related substance in the embodiment 2, the signal-to-noise ratio of each component can be preliminarily obtained, the stock solution is taken for gradual dilution, the concentration corresponding to the signal-to-noise ratio of 2-4 is found out, and the concentration is defined as the detection limiting concentration; the signal-to-noise ratio was determined to be a quantitative limit concentration at a concentration corresponding to 8 to 12, as shown in Table 13, table 14 below.

TABLE 13 quantitative limit of impurity C

TABLE 14 limit of detection of impurity C results

Name of the name	Concentration (μg/ml)	S/N
			Detection limiting solution	0.1196	7.0

Conclusion: the detection limit concentration of the impurity C is 0.1196 mug/ml, and the S/N is 7.0; the quantitative limit concentration of the impurity C is 0.2392 mug/ml, the minimum S/N is 14.3, the peak area RSD is 4.8 percent, and the standard is met.

Example 11: test of Linear relation

S1: the stock solution of impurity C was diluted with 0.2% sodium carbonate solution to give 0.01%, 0.05%, 0.10%, 0.50% and 0.75% solutions (limit 0.5%) of the concentration of the sample, respectively. 3 times for each sample;

s2: the peak area was measured according to the gas chromatography conditions in example 1, and the concentration was taken as the abscissa and the peak area was taken as the ordinate, so that a linear regression equation was obtained for each of the relevant substances, and the results are shown in Table 15, FIG. 7.

TABLE 15 Linear results

Sequence number	Concentration (μg/ml)	Peak area (pA min)	Percentage of limiting concentration
				Linearity
1	0.2139	0.0080	2.0％
				Linearity 2	1.0696	0.0398	10.0％
Linearity 3	5.3479	0.2000	50.0％
				Linearity 4	10.6958	0.3648	100.0％
Linearity
	5	16.0436	0.5558	150.0％

Conclusion: the impurity C has good linear relation in the concentration range of 0.2139-16.0436 mug/ml, the regression equation is y=0.0343x+0.0048, and the r value is 0.9995, which accords with the standard.

Example 12: accuracy of

S1: control solutions were prepared as in example 8

Accuracy 1 adding a standard solution: weighing a sample and an impurity C stock solution, diluting the sample and the impurity C stock solution by a diluent to prepare 3 parts of an added impurity reference substance solution with the concentration of 50% of the impurity limit, and sequentially named as 50% -1, 50% -2 and 50% -3.

Accuracy 2 addition of the labeling solution: weighing a sample to be tested and an impurity C stock solution, diluting the sample with a diluent to prepare an added impurity reference substance with the concentration of 100% of the impurity limit, preparing 3 parts in parallel, and sequentially named as 100% -1, 100% -2 and 100% -3.

Accuracy 3 addition of the labeling solution: weighing a sample and an impurity C stock solution, diluting the sample and the impurity C stock solution by a diluent to prepare 3 parts of an added solution with the impurity reference substance concentration of 150% of the impurity limit, and sequentially named as 150% -1, 150% -2 and 150% -3.

S2: the peak area of the sample solution was measured according to the gas chromatography conditions in example 8, and the recovery rate of the relevant substances in the mixed sample solution was calculated, and the results are shown in Table 16 below.

TABLE 16 XS-AY impurity C accuracy results

Conclusion: the recovery rate of the XS-AY impurity C is 94.69% -109.93%, the average recovery rate is 99.19%, and the RSD is 4.3%, which meets the standard.

Example 13: durability of

S1: preparing a base solution and a standard solution and a first standard solution according to the example 8;

s2: the sample solutions were examined under the conditions of gas chromatography in example 8 at column temperature=70±5 ℃ and flow rate=2.0±0.5mL/min, and the contents of the relevant substances in the sample solutions at different column temperatures and flow rates were calculated, and the results are shown in tables 17, 18, 19, 20, 21 and 22.

TABLE 17 chromatographic condition varying parameters

Chromatographic parameters	Specified value	Range of variation
			Flow rate (ml/min)	2.0	1.5 and 2.5
Headspace equilibrium temperature (. Degree. C.)	70	65 and 75

Table 18, recovery results at a flow rate of 1.5ml/min

TABLE 19 recovery results at a flow rate of 2.5ml/min

Table 20 results of recovery rate at headspace equilibrium temperature of 65 ℃

Table 21 results of recovery at headspace equilibrium temperature of 75 ℃

Table 22 comparison of results of different measurement Condition parameters

Comment (1): the recovery rate is an average recovery rate when using the accuracy program.

Conclusion: when the headspace equilibrium temperature is +/-5 ℃ and the flow rate is +/-0.5 ml/min, the recovery rate is 90.93% -99.19%, and the requirements are met.

Example 14: stability of

S1: three control solutions and test solutions were prepared as in example 8;

s2: the sample solutions were measured according to the gas chromatography conditions in example 8, and after preparation, 0, 6,

The measurement was performed for 12 hours. The results of calculating the contents of the related substances in the test sample solutions are shown in tables 23 and 24.

TABLE 23 stability results of test solutions

TABLE 24 stability results of control solutions

(4) Conclusion:

1. the RSD of the peak area of the impurity C measured by the test solution in 0h, 6h and 12h is 8.0%, which accords with the standard;

2. the control solution showed an RSD of 7.2% of the impurity C peak area, as measured at 0h, 6h and 12h, meeting the standard.

The above description of the present invention is further illustrated in detail and should not be taken as limiting the practice of the present invention. It is within the scope of the present invention for those skilled in the art to make simple deductions or substitutions without departing from the concept of the present invention.

Claims

1. An analysis method of related impurities of an isoxazoline veterinary drug intermediate, wherein the intermediate is 2-amino-N- (2, 2, 2-trifluoroethyl) acetamide, and the related impurities are triethylamine hydrochloride and glycine, and the analysis method is characterized in that: comprising the following steps:

glycine using reverse phase high performance liquid chromatography and triethylamine hydrochloride using gas chromatography:

dissolving the intermediate test sample and the intermediate reference sample by using a diluent to obtain a sample solution and a reference sample solution;

detecting the intermediate and glycine impurity in the sample solution by using reverse-phase high performance liquid chromatography, and determining a chromatogram, wherein the chromatographic column of the reverse-phase high performance liquid chromatography is a Venusil XBP C18L chromatographic column: 5 μm,4.6x250mm, the conditions of the reverse phase high performance liquid chromatography are as follows: mobile phase A: acetonitrile; b: the volume concentration of the heptafluorobutyric acid solution in the mobile phase B in the reversed-phase high-performance liquid chromatography is 0.1-0.15%; elution mode: gradient elution, namely 0 min-5.0 min,10% -20% of A and 90% -80% of B according to volume fraction; 5-15 min, 20-40% of A, 80-60% of B, 15-20 min, 40-65% of A and 60-35% of B; 20-22 min, 65-80% of A, 35-20% of B, 22-27 min, 80-10% of A and 20-90% of B; 27-35 min,10% of A and 90% of B; the reversed-phase high-performance liquid chromatography detector is an electric fog detector;

2. The method of analysis according to claim 1, wherein: the flow rate of the mobile phase in the reversed-phase high-performance liquid chromatography is 0.8-1.0 mL/min.

3. The assay method according to claim 1 or 2, wherein: the chromatographic column temperature of the reversed-phase high-performance liquid chromatography is 30-40 ℃.

4. The assay method according to claim 1 or 2, wherein: the CAD parameters of the electric fog detector are as follows: the carrier gas was nitrogen at a pressure of 35psi and an atomization temperature of 35 ℃.

5. The method of analysis according to claim 1, wherein: the temperature rise program of the gas chromatography is as follows: the initial temperature is kept at 30-50 ℃ for 3min, the temperature is raised to 180-240 ℃ at the speed of 8-12 ℃/min, and the temperature is kept at 3-8 min.

6. The method of analysis according to claim 1 or 5, wherein: the chromatographic column of the gas chromatograph is Agilent 30m multiplied by 0.32mm and 1.8 mu m.

7. The method of analysis according to claim 1 or 5, wherein: the sample injection amount of the reversed-phase high-performance liquid chromatography is 10-20 mu L; and/or the sample injection amount of the gas chromatograph is 0.5-1.5 mL.