Determination method for related substances in fluralamide moxidectin drops

Abstract

The invention discloses a method for determining related substances in a fluralamoxidectin drop, the related substances comprise an impurity A, an impurity B, an impurity C, an impurity D, an impurity G and an impurity J. The method is carried out by adopting a high performance liquid chromatography method, and the chromatographic conditions are as follows: an ammonium dihydrogen phosphate buffer solution with the pH value of 4.5-5.2 is taken as a mobile phase A, the mobile phase B is taken as a mobile phase C, and the mobile phase C is taken as a mobile phase D; acetonitrile is used as a mobile phase B for gradient elution, and the concentration of the ammonium dihydrogen phosphate buffer solution is 0.02 mol / L-0. 1 mol / L; the flow velocity is 0.7 mL / min to 1.3 mL / min; the column temperature is 40-50 DEG C; and the detection wavelength is 242 nm. According to the method, related substances in the fluralamoxidectin drops can be simultaneously determined under the same chromatographic condition, simultaneous detection and separation of various impurities are realized, the detection time is shortened, the method can be used for quality research and quality control of fluralamoxidectin drop products, and the drug safety risk is reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of drug detection, in particular to a method for determining related substances in flurella nalmoxidectin drops. Background Art

[0002] Flurella nalmoxidectin drops (trade name: BRAVECTOPLUS ) is a drug used to treat tick and flea infestations in cats. It provides immediate and sustained flea-killing activity lasting 12 weeks and is also used to treat flea allergy dermatitis, ear mite infestations, and intestinal roundworm and hookworm infestations.

[0003] In 2014, the United States Patent and Trademark Office issued Merck the first trademark, BRAVECTO, for commercial use in veterinary ectoparasiticides. Flurellanamoxictin drops were launched in the United States in 2019. The determination of related substances in flurellanamoxictin drops is a crucial part of quality control. The British Pharmacopoeia (EP) lists impurities A, B, C, D, G, and J in flurellanamoxictin drops. Their names and structural formulas are shown in Table 1:

[0004] Table 1 Names and structural formulas of relevant substances

[0005]

[0006]

[0007] The British Pharmacopoeia uses acetate as the elution mobile phase for the detection of the above substances, which has a large baseline noise (such as Figure 1 , signal-to-noise ratio S / N is 39.10), the sensitivity of the method is low, and two chromatographic methods are required to detect all the above impurities. The total detection time reaches 90 minutes, and the detection efficiency is low.

[0008] Tang Aoran's master's thesis "Preparation and Quality Study of Flurella Moxidectin Compound Drops, Huazhong Agricultural University" records the detection method of moxidectin-related substances in Flurella Moxidectin Compound Drops. The chromatographic conditions used are: chromatographic column: EC-C18 (4.6mm×150mm, 3um); detection wavelength: 242nm (moxidectin), 278 (BHT); mobile phase: 0.1% phosphoric acid solution, acetonitrile; elution program: mobile phase A 45%, mobile phase B 55% at 0min, mobile phase at 30min Mobile phase A is 45% and mobile phase B is 55%. At 40 min, mobile phase A is 30% and mobile phase B is 70%. At 50 min, mobile phase A is 30% and mobile phase B is 70%. At 55 min, mobile phase A is 15% and mobile phase B is 85%. At 75 min, mobile phase A is 15% and mobile phase B is 85%. At 75.1 min, mobile phase A is 45% and mobile phase B is 55%. At 80 min, mobile phase A is 45% and mobile phase B is 55%. Flow rate: 1.5 mL / min; column temperature: 55°C; injection volume: 10 uL.

[0009] Although this method provides a method for detecting impurities in flurellanine moxidectin compound drops, it does not investigate the specific impurities detected. Furthermore, when the chromatographic conditions in this method were used for detection, it was found that the chromatographic conditions could not effectively separate impurity D and moxidectin in the chromatogram (as shown in FIG2 ), indicating that this method cannot effectively separate all of the above-mentioned related substances.

[0010] In summary, there is an urgent need to provide a method for simultaneously separating and detecting the above-mentioned related substances in flurella nalomoxidectin drops to solve the many problems of low sensitivity, low detection efficiency and inability to effectively separate existing detection methods. Summary of the Invention

[0011] The technical problem to be solved by the present invention is to overcome the shortcomings of the existing technology and provide a method for determining related substances in flurellanamoxictin drops. The method can achieve simultaneous determination and separation of multiple impurities, effectively shorten the determination time, and has the advantages of high sensitivity, good specificity, and high detection efficiency.

[0012] The technical solution adopted by the present invention to solve its technical problem is: a method for determining related substances in flurellanamoxictin drops, wherein the related substances include impurity A, impurity B, impurity C, impurity D, impurity G and impurity J. The method is carried out by high performance liquid chromatography, and the chromatographic conditions are:

[0013] The mobile phase was ammonium dihydrogen phosphate buffer at pH 4.5-5.2 as mobile phase A, and acetonitrile as mobile phase B for gradient elution. The concentration of ammonium dihydrogen phosphate buffer was 0.02 mol / L-0.1 mol / L; the flow rate was 0.7 mL / min-1.3 mL / min; the column temperature was 40°C-50°C; the detection wavelength was 242 nm; and the gradient elution program was as follows:

[0014] Time (min) Mobile phase A (%) Mobile phase B (%) 0 35 65 35 35 65 37 25 75 60 25 75 61 35 65 70 35 65 .

[0015] Preferably, the pH of the ammonium dihydrogen phosphate buffer is 4.8, and the concentration is 0.05 mol / L; the flow rate is 1 mL / min; and the column temperature is 45°C.

[0016] Furthermore, the specific operating steps of the determination method are:

[0017] 1) Prepare test solution, reference solution and system suitability solution;

[0018] 2) Inject the test solution, reference solution and system suitability solution sensitivity solution into the high performance liquid chromatograph respectively, detect under the chromatographic conditions, and record the chromatogram.

[0019] Furthermore, the test solution is prepared by precisely measuring the flurella nalmoxidectin drops, adding acetonitrile to dissolve the solution, and quantitatively diluting the solution to prepare the test solution.

[0020] Furthermore, the concentration of moxidectin in the test solution was 0.14 mg / mL.

[0021] Furthermore, the preparation method of the reference solution is as follows: take the moxidectin reference substance, accurately weigh it, dissolve it in acetonitrile and quantitatively dilute it to prepare the reference solution.

[0022] Furthermore, the concentration of moxidectin in the reference solution is 7 ug / mL.

[0023] Furthermore, the system suitability solution is prepared by accurately weighing a reference substance containing impurity A, impurity B, impurity C, impurity D, impurity G, impurity J and moxidectin, dissolving it in acetonitrile, and quantitatively diluting it to prepare a system suitability solution.

[0024] Furthermore, the concentrations of impurities A, B, C, D, G, and J in the system suitability solution were all 7 μg / mL, and the concentration of moxidectin was 0.7 mg / mL.

[0025] Furthermore, when the high performance liquid chromatography method is used for determination, a chromatographic column with octadecylsilane bonded silica gel as a filler is used, with a specification of 4.6 mm×150 mm and 3 μm.

[0026] The method for determining related substances in flurellan moxidectin drops of the present invention has the following beneficial effects: the method is simple to operate, can simultaneously determine a total of six related substances, namely impurity A, impurity B, impurity C, impurity D, impurity G and impurity J, in the flurellan moxidectin drops under the same chromatographic conditions, achieves separation of impurity D from moxidectin, and also achieves simultaneous detection and effective separation of multiple other impurities; the influence of flurellan on moxidectin detection can be eliminated, thereby greatly shortening detection time and reducing detection costs; and the method of the present invention has high sensitivity, good specificity and high accuracy, laying a good foundation for the formulation of quality standards for flurellan moxidectin drops, can be used for quality research and quality control of flurellan moxidectin drops products, reduces drug safety risks, is conducive to the popularization of impurity detection methods for flurellan moxidectin drops, and is conducive to the safe promotion and application of flurellan moxidectin drops. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 — is the chromatogram of the British Pharmacopoeia;

[0028] Figure 2-1 —Full chromatogram detected by the method of “Preparation and Quality Study of Flurella Narmoxidectin Compound Drops”;

[0029] Figure 2-2 —A partial chromatogram obtained by the method described in the study on the preparation and quality control of Flurella Narmoxidectin Compound Drops;

[0030] Figure 3 - is the chromatogram of the test solution;

[0031] Figure 4 — is the chromatogram of the reference solution;

[0032] Figure 5-1 —Full chromatogram of the system suitability solution;

[0033] Figure 5-2 —A partial chromatogram of the system suitability solution;

[0034] Figure 6-1 — is the full chromatogram corresponding to a column temperature of 35°C;

[0035] Figure 6-2 — is the chromatogram corresponding to a column temperature of 35°C;

[0036] Figure 7-1 — is the full chromatogram corresponding to a column temperature of 40°C;

[0037] Figure 7-2 — is the chromatogram of the column when the temperature is 40℃;

[0038] Figure 8-1— is the full chromatogram corresponding to a column temperature of 50°C;

[0039] Figure 8-2 — is the chromatogram of the column when the temperature is 50℃;

[0040] Figure 9-1 — is the full chromatogram corresponding to a column temperature of 55°C;

[0041] Figure 9-2 — is the chromatogram of the column when the temperature is 55°C;

[0042] Figure 10-1 — is the full chromatogram corresponding to a flow rate of 0.7 mL / min;

[0043] Figure 10-2 — is the chromatogram of the corresponding flow rate of 0.7 mL / min;

[0044] Figure 11-1 — is the full chromatogram corresponding to a flow rate of 1.3 mL / min;

[0045] Figure 11-2 — is the chromatogram of the corresponding flow rate of 1.3 mL / min;

[0046] Figure 12-1 — is the full chromatogram corresponding to the concentration of ammonium dihydrogen phosphate buffer being 0.01 mol / L;

[0047] Figure 12-2 — is the chromatogram of the portion corresponding to the concentration of ammonium dihydrogen phosphate buffer of 0.01 mol / L;

[0048] Figure 13-1 — is the full chromatogram corresponding to the concentration of ammonium dihydrogen phosphate buffer of 0.02 mol / L;

[0049] Figure 13-2 — is the chromatogram of the concentration of ammonium dihydrogen phosphate buffer corresponding to 0.02 mol / L;

[0050] Figure 14-1 — is the full chromatogram corresponding to the concentration of ammonium dihydrogen phosphate buffer being 0.1 mol / L;

[0051] Figure 14-2 — is the chromatogram of the portion corresponding to the concentration of ammonium dihydrogen phosphate buffer of 0.1 mol / L;

[0052] Figure 15 — is the chromatogram of the portion corresponding to the concentration of ammonium dihydrogen phosphate buffer of 0.15 mol / L;

[0053] Figure 16-1 — is the full chromatogram corresponding to pH 4.5;

[0054] Figure 16-2 — is the chromatogram corresponding to pH 4.5;

[0055] Figure 17-1 — is the full chromatogram corresponding to pH 5.2;

[0056] Figure 17-2 — is the chromatogram corresponding to pH 5.2;

[0057] Figure 18-1 — is the full chromatogram corresponding to the mobile phase A being a phosphoric acid solution with a concentration of 0.05 mol / L;

[0058] Figure 18-2 — is a partial chromatogram corresponding to a phosphoric acid solution with a concentration of 0.05 mol / L as mobile phase A.

[0059] The peaks corresponding to the peaks marked 1-9 in the above figure are: 1-impurity A, 2-impurity B, 3-impurity C, 4-impurity D, 5-moxidectin, 6-impurity E+F, 7-impurity G, 8-impurity H+I, 9-impurity J. DETAILED DESCRIPTION

[0060] The present invention will be further described below with reference to the accompanying drawings and examples, but these specific embodiments do not limit the scope of protection of the present invention in any way.

[0061] Example 1

[0062] This embodiment provides a method for determining related substances in flurella nalmoxidectin drops, comprising the following steps:

[0063] 1) Prepare test solution, reference solution, and system suitability solution:

[0064] Preparation of test solution: Accurately measure 0.5 mL of flurella naloxone moxidectin drops, place in a 50 mL volumetric flask, dissolve and dilute to the mark with acetonitrile, shake well, and use as the test solution. The concentration of moxidectin in the test solution is 0.14 mg / mL.

[0065] Preparation of reference solution: Take an appropriate amount of moxidectin reference substance, accurately weigh it, and quantitatively dilute it with acetonitrile to make a solution containing 7 μg of moxidectin per 1 mL as the reference solution;

[0066] Preparation of system suitability solution: Accurately weigh approximately 7 mg of a reference substance containing impurities A, B, C, D, E, F, G, H, I, J, and moxidectin, place in a 10 mL volumetric flask, dissolve in acetonitrile, quantitatively dilute to the mark, and shake well to prepare a system suitability solution. The concentrations of impurities A, B, C, D, E, F, G, H, I, and J in the system suitability solution are all 7 μg / mL, and the concentration of moxidectin is 0.7 mg / mL.

[0067] 2) Use high performance liquid chromatography to determine the relevant impurities in the test solution and record the chromatogram:

[0068] Take 10uL of the test solution, reference solution, and system suitability solution and inject them into the high performance liquid chromatography for detection. The chromatographic conditions are as follows: mobile phase A is ammonium dihydrogen phosphate buffer with a pH of 4.8 and a concentration of 0.05mol / L, and mobile phase B is acetonitrile for gradient elution; the flow rate is 1mL / min; the column temperature is 45℃; the detection wavelength is 242nm; the injector temperature is 10℃; the gradient elution program is as follows:

[0069] Time (min) Mobile phase A (%) Mobile phase B (%) 0 35 65 35 35 65 37 25 75 60 25 75 61 35 65 70 35 65

[0070] In this embodiment, the relevant substances in the flurellanamoxictin drops were determined by using the above chromatographic conditions. After testing, the chromatogram of the test solution prepared with flurellanamoxictin drops was as follows: Figure 3 The chromatogram of the reference solution is shown in Figure 4 As shown, the system suitability solution is Figure 5-1 and 5-2 As shown, Figure 5-2 In the peak numbers 1-9, impurity A, impurity B, impurity C, impurity D, moxidectin, impurity E+F, impurity G, impurity H+I and impurity J are respectively. The names and structural formulas of impurities E, F and impurities H, I are as follows:

[0071]

[0072]

[0073] Depend on Figure 5-2 It can be seen that in the system suitability solution, moxidectin and related substances (impurity A, impurity B, impurity C, impurity D, impurity G and impurity J) are effectively separated, and adjacent impurities (impurity A, impurity B, impurity C, impurity D, impurity G and impurity J) are also effectively separated from each other. Impurities E, F and impurities H, I are also effectively separated from moxidectin and other impurities (impurity A, impurity B, impurity C, impurity D, impurity G and impurity J).

[0074] Example 2 Methodology Verification

[0075] 1. System suitability test

[0076] Take the reference solution and inject 6 injections continuously, and inject 1 injection of the system suitability solution to examine the separation between the chromatographic peaks and the injection precision. The reference solution spectrum is shown in Figure 4 , system suitability solutions see Figure 5-1 and 5-2 The results are shown in Tables 2 and 3:

[0077] Table 2 System suitability test results

[0078]

[0079]

[0080] Table 3 - Results of 6 repeated injections of the reference solution

[0081]

[0082] As shown in Tables 2 and 3, the resolution between adjacent peaks was ≥1.5, the signal-to-noise ratio of the reference solution was 320.77 (approximately ten times that of the British Pharmacopoeia), and the RSD of the peak area over six consecutive injections of the reference solution was 0.40% < 2.0%, meeting the system suitability requirements.

[0083] 2. Specificity test

[0084] Take 10uL of blank solvent, test solution, each positioning solution, blank excipient solution, and spiked test solution and inject them into the high performance liquid chromatography instrument for determination. The specific preparation method of each solution is shown in Table 3:

[0085] Table 3 Preparation method of solutions required for specificity test

[0086]

[0087]

[0088] The results are shown in Table 4, which indicate that the blank solvent, blank excipient, flurellanine and BHT do not interfere with the detection of the main peak and each impurity peak, and the specificity of the invention is good.

[0089] Table 4 Specificity test results

[0090] Element Retention time (min) Relative retention time Separation Blank auxiliary materials 5 minutes ago Before about 0.17 / Flurella 5.829 0.20 5.20 Impurity A 12.885 0.45 2.00 BHT 13.899 0.48 2.31 Impurity B 21.222 0.74 8.19 Impurity C 22.144 0.77 1.51 Impurity D 27.365 0.95 3.68 Moxidectin 28.732 1.00 1.72 Impurity G 43.349 1.51 4.03 Impurity J 52.155 1.81 4.88

[0091] Blank excipients, BHT, and flurana did not show peaks at the peak positions of moxidectin and impurities, and the separation between the peaks was greater than 1.5, which did not interfere with the detection of impurities, and the method had good specificity.

[0092] 3. Linearity and accuracy test

[0093] Take 10uL of each of the solutions with impurity limits of 200%, 150%, 100%, 80%, 50% and 10% corresponding to impurity A, impurity B, impurity C, impurity D, impurity G and impurity J and inject them into the high performance liquid chromatography for determination. The specific preparation method of each solution is shown in Table 5:

[0094] Table 5 Preparation method of solutions required for linear test

[0095]

[0096] The results are shown in Tables 6 and 7.

[0097] Table 6 Linearity test results

[0098]

[0099] As shown in Table 6, there is a good linear relationship between the concentration of each impurity and the peak area.

[0100] Table 7 Accuracy test results

[0101]

[0102]

[0103] As can be seen from the above table, the impurity recovery test results show that the recoveries of 12 accuracy samples of impurity A, impurity B, impurity C, impurity D, impurity G, and impurity J are all between 97% and 108%, and the RSDs of the recoveries are 2.41%, 1.39%, 0.90%, 2.72%, 2.43%, and 2.41%, respectively. The recoveries of each impurity meet the determination requirements, indicating that this method has good accuracy.

[0104] 4. Limit of Quantitation Test

[0105] Take an appropriate amount of the mixed impurity reference solution of impurity A, impurity B, impurity C, impurity D, impurity G, and impurity J in Table 5, gradually dilute with diluent until the S / N is about 10, and then take 10uL and inject it into the high performance liquid chromatography for determination. The results are shown in Table 8:

[0106] Table 8 Quantitation limit test results

[0107]

[0108]

[0109] The results showed that the signal-to-noise ratio of the target peak should be ≥10, the RSD of the peak area of ​​6 solutions should be ≤10%, and the limit of quantification should be <reporting limit (0.1%), which is in compliance with regulations.

[0110] Example 3 Column temperature optimization experiment

[0111] In this example, the system suitability solution was prepared by referring to the method of Example 1, and different column temperatures (35°C, 40°C, 45°C, 50°C, 55°C) were used for measurement to determine the optimal range of column temperature.

[0112] The chromatogram corresponding to the column temperature of 35℃ is as follows Figure 6-1 and 6-2 As shown, the chromatogram corresponding to the column temperature of 40℃ is as follows Figure 7-1 and 7-2 As shown, the chromatogram corresponding to the column temperature of 45℃ is as follows Figure 5-1 and 5-2 As shown, the chromatogram corresponding to the column temperature of 50℃ is as follows Figure 8-1 and 8-2 As shown, the chromatogram corresponding to the column temperature of 55℃ is as follows Figure 9-1 and 9-2 As shown in the figure, when the column temperature is 40-50°C, moxidectin and related substances (impurity A, impurity B, impurity C, impurity D, impurity G and impurity J) can be effectively separated, and adjacent impurities (impurity A, impurity B, impurity C, impurity D, impurity G and impurity J) can also be effectively separated. Impurities E, F and impurities H, I can also be effectively separated from moxidectin and other impurities (impurity A, impurity B, impurity C, impurity D, impurity G and impurity J). However, when the column temperature is 35°C (lower than 40°C), impurity D (peak 4) and moxidectin (peak 5) cannot be baseline separated. When the column temperature is 55°C (higher than 50°C), impurity D (peak 4) and moxidectin (peak 5) cannot be baseline separated. Therefore, the preferred column temperature is 40-50°C.

[0113] Example 4 Flow rate optimization experiment

[0114] In this example, the system suitability solution was prepared by referring to the method of Example 1, and different flow rates (0.7 mL / min, 1.0 mL / min, and 1.3 mL / min) were used for measurement to determine the preferred range of flow rate.

[0115] The chromatogram corresponding to the flow rate of 0.7 mL / min is as follows Figure 10-1 and 10-2 The chromatograms corresponding to a flow rate of 1.0 mL / min are shown in 5-1 and 5-2, and the chromatograms corresponding to a flow rate of 1.3 mL / min are shown in Figure 11-1 and 11-2As shown in the figure, when the flow rate is 0.7mL / min-1.3mL / min, moxidectin and related substances can be effectively separated.

[0116] Example 5 Optimization experiment of ammonium dihydrogen phosphate buffer concentration

[0117] In this example, the system suitability solution was prepared by referring to the method of Example 1, and gradient elution was performed using ammonium dihydrogen phosphate buffer of different concentrations (0.01 mol / L, 0.02 mol / L, 0.05 mol / L, 0.1 mol / L, 0.15 mol / L) as mobile phase A to determine the concentration range of the ammonium dihydrogen phosphate buffer.

[0118] The chromatogram corresponding to the concentration of 0.01 mol / L is as follows Figure 12-1 and 12-2 As shown, the chromatogram corresponding to the concentration of 0.02 mol / L is as follows Figure 13-1 and 13-2 As shown, the chromatogram corresponding to the concentration of 0.05 mol / L is as follows Figure 5-1 and 5-2 As shown, the chromatogram corresponding to the concentration of 0.1 mol / L is as follows Figure 14-1 and 14-2 As shown, the chromatogram corresponding to the concentration of 0.15 mol / L is as follows Figure 15 As shown in the figure, when the concentration of ammonium dihydrogen phosphate buffer is 0.02mol / L-0.1mol / L, moxidectin and related substances (impurity A, impurity B, impurity C, impurity D, impurity G and impurity J) can be effectively separated, and each adjacent impurity (impurity A, impurity B, impurity C, impurity D, impurity G and impurity J) can also be effectively separated, and impurities E, F and impurities H, I can also achieve separation from moxidectin and other impurities (impurity A, impurity B, impurity C, impurity D, impurity G and impurity J). However, when the concentration of the ammonium dihydrogen phosphate buffer is 0.01 mol / L (lower than 0.02 mol / L), impurity D (peak 4) and moxidectin (peak 5) cannot be baseline separated; when the concentration is 0.15 mol / L (higher than 0.1 mol / L), impurity D (peak 4) and moxidectin (peak 5) cannot be baseline separated; therefore, the preferred concentration of the ammonium dihydrogen phosphate buffer is 0.02 mol / L-0.1 mol / L.

[0119] Example 6 Optimization experiment of pH of ammonium dihydrogen phosphate buffer

[0120] In this example, the system suitability solution was prepared by referring to the method of Example 1, and ammonium dihydrogen phosphate buffers with different pH values ​​(4.5, 4.8, and 5.2) were used as mobile phase A for gradient elution to determine the pH range of the ammonium dihydrogen phosphate buffer.

[0121] The chromatogram corresponding to pH 4.5 is as follows Figure 16-1 and 16-2 As shown, the chromatogram corresponding to pH 4.8 is as follows Figure 5-1 and 5-2 As shown, the chromatogram corresponding to pH 5.2 is as follows Figure 17-1 and 17-2 As shown in the figure, when the pH of the ammonium dihydrogen phosphate buffer is 4.5-5.2, moxidectin and related substances can be effectively separated.

[0122] Comparative Example

[0123] The difference between this comparative example and Example 1 is that the mobile phase A in the chromatographic conditions is a phosphoric acid solution with a concentration of 0.05 mol / L. Other chromatographic conditions are the same, and the chromatogram measured is as follows Figure 18-1 and 18-2 As shown in the figure, when the mobile phase A is a phosphoric acid solution with a concentration of 0.05 mol / L, impurity D (peak 4) and moxidectin (peak 5) cannot achieve baseline separation.

[0124] The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Although the present invention has been disclosed as a preferred embodiment, it is not intended to limit the present invention. Any technician familiar with the present profession can make some changes or modifications to equivalent embodiments of equivalent changes using the technical contents disclosed above without departing from the scope of the technical solution of the present invention. However, any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A method for determining related substances in flurella nalmoxidectin drops, characterized in that: The related substances include impurity A, impurity B, impurity C, impurity D, impurity G and impurity J. The method is carried out by high performance liquid chromatography, and the chromatographic conditions are: The mobile phase was ammonium dihydrogen phosphate buffer at pH 4.5-5.2 as mobile phase A, and acetonitrile as mobile phase B for gradient elution. The concentration of ammonium dihydrogen phosphate buffer was 0.02 mol / L-0.1 mol / L; the flow rate was 0.7 mL / min-1.3 mL / min; the column temperature was 40°C-50°C; the detection wavelength was 242 nm; and the gradient elution program was as follows: 。 2. The method for determining related substances in the flurellanamoxictin drops according to claim 1, wherein: The pH of the ammonium dihydrogen phosphate buffer was 4.8, and the concentration was 0.05 mol / L; the flow rate was 1 mL / min; and the column temperature was 45°C.

3. The method for determining related substances in the flurellanamoxictin drops according to claim 1, wherein: The specific operating steps of the determination method are: 1) Prepare test solution, reference solution and system suitability solution; 2) Inject the test solution, reference solution and system suitability solution into a high performance liquid chromatograph respectively, perform detection under the chromatographic conditions, and record the chromatogram.

4. The method for determining related substances in flurella nalmoxidectin drops according to claim 3, wherein: The preparation method of the test solution is as follows: accurately measure the flurella nalmoxidectin drops, add acetonitrile to dissolve it, and quantitatively dilute it to prepare the test solution.

5. The method for determining related substances in flurellanamoxictin drops according to claim 4, wherein: The concentration of moxidectin in the test solution was 0.14 mg / mL.

6. The method for determining related substances in flurella nalmoxidectin drops according to claim 3, wherein: The preparation method of the reference solution is as follows: taking a moxidectin reference substance, accurately weighing it, adding acetonitrile to dissolve it, and quantitatively diluting it to prepare the reference solution.

7. The method for determining related substances in flurella nalmoxidectin drops according to claim 6, wherein: The concentration of moxidectin in the reference solution was 7 μg / mL.

8. The method for determining related substances in flurella nalmoxidectin drops according to claim 3, wherein: The system suitability solution is prepared by accurately weighing a reference substance containing impurity A, impurity B, impurity C, impurity D, impurity G, impurity J, and moxidectin, dissolving the solution in acetonitrile, and quantitatively diluting the solution to prepare the system suitability solution.

9. The method for determining related substances in flurellanamoxictin drops according to claim 8, wherein: The concentrations of impurities A, B, C, D, G, and J in the system suitability solution were all 7 ug / mL, and the concentration of moxidectin was 0.7 mg / mL.

10. The method for determining related substances in the flurellanamoxictin drops according to any one of claims 1 to 9, characterized in that: The high performance liquid chromatography method was used for determination. A chromatographic column with octadecylsilane bonded silica gel as a filler was used, and the specifications were 4.6 mm × 150 mm, 3 μm.

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