A method for detecting the content of non-prolactin, methoxyproline, piperazine and moxiket

Abstract

The application discloses a method for detecting the content of fepronil, methoprene, praziquantel and moxidine, and the method comprises the following steps: detecting the content of fepronil, methoprene, praziquantel and moxidine in a compound preparation by using high performance liquid chromatography; and the chromatographic conditions for gradient elution during the detection are as follows: using a mixed solution of water, acetonitrile, methanol and glacial acetic acid as mobile phase A, using a mixed solution of acetonitrile, methanol and glacial acetic acid as mobile phase B, the flow rate is 0.85-1.25 ml / min, the column temperature is 20-30 DEG C, the injection amount is 15-30 muL, the detection wavelength is 230-250 nm, and the theoretical plate number is not less than 5000 according to the main peak. The method can simultaneously detect the content of fepronil, methoprene, praziquantel and moxidine, has good specificity, strong stability, good reproducibility and accurate detection result; the gradient elution mode, low column temperature and low flow rate are used, the damage to the chromatographic column and the instrument is reduced, the use amount of reagents is reduced, and the detection cost is greatly saved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of drug content detection, in particular to a method for detecting the content of non-spirodic, methoprene, piperazine and moxidectin. BACKGROUND

[0002] Non-spirodic is a new type of broad-spectrum insecticide of phenylpyrazole; methoprene is an insect growth regulator (IGR), which is a homologue of juvenile hormone, and has inhibitory effect on the development of immature insects, flea larvae and pupae; piperazine has broad-spectrum anti-schistosome and anti-tapeworm effects; moxidectin is a semisynthetic single-component macrolide antibiotic produced by fermentation of Streptomyces, which is a derivative of nemadectin, and has good killing effect on gastrointestinal nematodes and ectoparasites in animal body. It is different from ivermectin and abamectin in that it is a single component, has wider insecticidal activity, longer duration and safety, etc. The compound preparation containing non-spirodic, methoprene, piperazine and moxidectin can be used for preventing and treating flea and tick infection in dogs and cats, treating gastrointestinal nematode and tapeworm infection, and assisting in the treatment of allergic dermatitis caused by fleas.

[0003] In the prior art, for example, the patent number CN115166122A "Method for detecting methoprene in compound preparation containing non-spirodic and methoprene" uses high performance liquid chromatography, and the chromatographic conditions are as follows: the detection wavelength is 260-275 nm, the theoretical plate number is not less than 5000 based on methoprene, and the mobile phase is a mixed solution of mobile phase A and acetonitrile; wherein, the mobile phase A is obtained by mixing acetonitrile, methanol and 0.1% acetic acid aqueous solution according to the mass ratio of 47:21:32.

[0004] Although the above-mentioned patent solves the problem of long detection time of methoprene content in two, three or four mixed preparations containing non-spirodic and methoprene by using high performance liquid chromatography, it cannot detect the contents of non-spirodic, methoprene, piperazine and moxidectin at the same time, and it is troublesome to detect the contents of multiple substances respectively, and the solution stability is not high.

[0005] Therefore, we propose a method for detecting the contents of non-spirodic, methoprene, piperazine and moxidectin to solve the above-mentioned problems. SUMMARY

[0006] The purpose of the present application is to provide a method for detecting the contents of non-spirodic, methoprene, piperazine and moxidectin to solve the problem that the contents of non-spirodic, methoprene, piperazine and moxidectin cannot be detected at the same time, multiple substance contents are detected respectively, solution stability is not high, and operation is relatively troublesome.

[0007] To achieve the above object, the present application provides the following technical scheme: a method for detecting the content of febantel, milbemectin, pyrantel and moxidectin, the detection method comprising: using high performance liquid chromatography to detect the content of febantel, milbemectin, pyrantel and moxidectin in the compound preparation;

[0008] The chromatographic conditions for gradient elution during detection are as follows: using a mixed solution of water, acetonitrile, methanol and glacial acetic acid as mobile phase A, using a mixed solution of acetonitrile, methanol and glacial acetic acid as mobile phase B, the flow rate being 1.0 ml / min, the column temperature being 25℃, and the injection volume being 20 μL;

[0009] The detection wavelength is 245 nm, and the theoretical plate number calculated according to the main peak is not less than 5000;

[0010] Preferably, when preparing the mobile phase A, water, acetonitrile, methanol and glacial acetic acid are prepared into a water-acetonitrile-methanol (55:35:10) solution containing 0.20% glacial acetic acid according to the volume ratio.

[0011] Preferably, when preparing the mobile phase B, acetonitrile, methanol and glacial acetic acid are mixed according to the volume ratio to prepare an acetonitrile-methanol (65:35) solution containing 0.20% glacial acetic acid.

[0012] Preferably, during the chromatographic test, octadecylsilane-bonded silica gel is used as the filler.

[0013] Preferably, the detection method comprises the following steps:

[0014] S1, preparation of test sample solution: acetonitrile is added to the compound preparation sample, and after dissolution, the mixture is uniformly mixed, and an appropriate amount of the obtained solution is taken to obtain the test sample solution;

[0015] S2, preparation of control sample solution: the febantel control sample, the milbemectin control sample, the pyrantel control sample and the moxidectin control sample are dissolved and diluted with acetonitrile to obtain the control sample solution.

[0016] Compared with the prior art, the present application has the following beneficial effects:

[0017] The present application establishes a method for simultaneously detecting the content of febantel, milbemectin, pyrantel and moxidectin, which reduces the damage to the chromatographic column by using low flow rate and low column temperature, avoids the increase in reagent consumption caused by damage to the chromatographic column and equipment, realizes the simultaneous detection of the content of multiple substances by linear gradient elution of different data gradient elution data, shortens the overall detection time, and greatly improves the detection efficiency; during detection, the present application has strong specificity, good separation effect between the active ingredients in the preparation and adjacent peaks, good stability of the sample solution, reduces the mutual interference between substances, and can ensure the accuracy of the detection results. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 A flow chart of the method for detecting the content of febantel, milbemectin, pyrantel and moxidectin according to the present application;

[0019] Figure 2 A control chromatogram of the method for detecting the content of febantel, milbemectin, pyrantel and moxidectin according to the present application;

[0020] Figure 3 A sample chromatogram of the liquid chromatography method for detecting the content of febantel and milbemectin according to the announcement No. 2511 of the Ministry of Agriculture;

[0021] Figure 4 A sample chromatogram of the liquid chromatography method for detecting the content of pyrantel according to the announcement No. 2511 of the Ministry of Agriculture;

[0022] Figure 5 A sample chromatogram of the liquid chromatography method for detecting the content of moxidectin according to the announcement No. 2168 of the Ministry of Agriculture. DETAILED DESCRIPTION

[0023] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the scope of protection of the present application.

[0024] Please refer to Figures 1-5 The present application provides a technical solution: a method for detecting the content of febantel, milbemectin, pyrantel and moxidectin, a new gradient elution program is established, specifically:

[0025] Use water-acetonitrile-methanol (55:35:10) containing 0.20% glacial acetic acid solution as mobile phase A, and acetonitrile-methanol (65:35) containing 0.20% glacial acetic acid solution as mobile phase B, and use the gradient elution program in Table 1.

[0026] Table 1: Gradient elution data record table

[0027]

[0028] Using gradient elution, the content of febantel, milbemectin, pyrantel and moxidectin can be detected at the same time, ensuring that the separation degree between the active ingredients in the preparation and the adjacent peaks is ≥1.5, achieving baseline separation, greatly shortening the detection time and improving the detection efficiency.

[0029] The solution stability of the sample solution and the control solution is good, ensuring the accuracy of the detection.

[0030] The octadecylsilane bonded silica gel is used as the filler in the present application; the flow rate is 1.0 ml / min, the column temperature is 25℃, the detection wavelength is 245 nm, the theoretical plate number is not less than 5000 calculated according to the main peak, low column temperature and low flow rate are selected, the damage to the chromatographic column and the instrument is reduced, the use amount of reagents is reduced, and the overall test cost is saved.

[0031] Example 1

[0032] Chromatographic conditions and system suitability test:

[0033] The octadecylsilane bonded silica gel is used as the filler; the flow rate is 1.0 ml / min; the column temperature is 25℃; the detection wavelength is 245 nm, the theoretical plate number is not less than 5000 calculated according to the main peak; the injection amount is 20 μL; the water-acetonitrile-methanol (55:35:10) containing 0.20% glacial acetic acid solution is used as the mobile phase A, and the acetonitrile-methanol (65:35) containing 0.20% glacial acetic acid solution is used as the mobile phase B, and the gradient elution procedure in Table 1 is adopted, wherein:

[0034] The high performance liquid chromatograph is of Agilent 1260 type.

[0035] The chromatographic column is of Agilent Zorbax SB-C18 (4.6×150 mm, 5 μm) type.

[0036] Determination method: an appropriate amount of the compound preparation containing fepronil, methoprene, piperazine and moxidine is precisely weighed, placed in a brown volumetric flask, dissolved and diluted with acetonitrile, shaken uniformly to obtain a test sample solution;

[0037] In addition, an appropriate amount of fepronil, methoprene, piperazine and moxidine control samples are precisely weighed, placed in 25 ml brown volumetric flasks, diluted with acetonitrile to the calibration mark, and shaken uniformly to obtain a mixed control sample solution containing 80 ug of fepronil, 120 ug of methoprene, 100 ug of piperazine and 30 ug of moxidine per 1 ml;

[0038] The contents of fepronil, methoprene, piperazine and moxidine are calculated by peak area according to the external standard method.

[0039] Investigation of the content determination method:

[0040] 1.1 Determination of detection wavelength

[0041] The control sample solution is used as the wavelength test solution, the high performance liquid chromatography diode array detector (DAD) is used to scan in the range of 190 nm-400 nm, the maximum absorption wavelength is determined, the peak area at the maximum absorption wavelength and the peak area at 245 nm are determined, the ratio of the peak area at 245 nm to the peak area at the maximum wavelength is calculated, and the data are recorded in Table 2.

[0042] Table 2: Peak area at maximum absorption wavelength and peak area at 245 nm record table

[0043]

[0044] According to the results of the above table, the wavelength for content determination of the present application is set to 245 nm.

[0045] 1.2 Selection of elution gradient

[0046] Take the same batch of compound preparation, prepare 6 test sample solutions, use the mobile phase and gradient elution procedure in Table 2: use water-acetonitrile-methanol (55:35:10) containing 0.20% glacial acetic acid solution as mobile phase A, and acetonitrile-methanol (65:35) containing 0.20% glacial acetic acid solution as mobile phase B, to determine the retention time and separation degree of the chromatographic peaks of febantel, milbemectin, pyrantel and moxidectin, and the obtained data are recorded in Table 3.

[0047] Table 3: Chromatographic peak data record table

[0048]

[0049] The results show that in the 6 chromatograms, the retention time drift range of the active ingredients febantel, milbemectin, pyrantel and moxidectin chromatographic peaks is less than 0.1%, and the peak position is relatively stable; the separation degree between the active ingredients febantel, milbemectin, pyrantel and moxidectin and their respective adjacent chromatographic peaks is greater than 1.5, which achieves baseline separation, the method has strong specificity, and the RSD is less than 0.5%, and the reproducibility is good.

[0050] 1.3 Specificity test

[0051] Preparation of blank preparation control solution: according to the prescription proportion, weigh the formula accessories except febantel, milbemectin, pyrantel and moxidectin, and prepare the blank preparation control solution according to the preparation process and test sample solution preparation method of the compound preparation.

[0052] Precisely take 20 μl of test sample solution, control sample solution, blank preparation control solution and mobile phase, and inject under the chromatographic conditions of the content determination method established in the present application, to determine the retention time and separation degree of the chromatographic peaks of febantel, milbemectin, pyrantel and moxidectin, and record the above determination data in Table 4.

[0053] Table 4: Specificity test data record table

[0054]

[0055] The results show that the test sample solution and the control sample solution all have chromatographic peaks at corresponding positions, and the baseline of the test sample chromatogram is smooth and the separation degree is good; the blank preparation control solution and the mobile phase do not have chromatographic peaks at the four main peak positions, indicating that the blank preparation control and the mobile phase do not interfere with the retention time of febantel, milbemectin, pyrantel and moxidectin.

[0056] 1.4 Precision test

[0057] The same batch of compound preparation was taken to prepare 6 test sample solutions, which were injected under the chromatographic conditions of the content determination method established in the present application, the precision of the instrument was investigated, and the relative standard deviations RSD of the contents obtained from 6 groups of data were calculated, and the data were recorded in Table 5.

[0058] Table 5: Relative standard deviation RSD recording table

[0059]

[0060] The results show that the RSD of the contents of febantel, milbemectin, pyrantel and moxidectin in the 6 test sample solutions is less than 2.0%, indicating that the content determination method established in the present application has good precision.

[0061] 1.5 Repeatability test

[0062] Six test sample solutions were selected from the same batch of compound preparation, and the contents were determined by two different analysts in different laboratories on different working days, using different liquid chromatographs and different chromatographic columns, and following the operation of the "precision test".

[0063] The results of the repeatability test are shown in Table 6:

[0064] Table 6: Recording table of content determination in repeatability test

[0065]

[0066]

[0067]

[0068]

[0069] The results show that the RSD of the contents of febantel, milbemectin, pyrantel and moxidectin detected by two different analysts is in the range of 0.2% to 0.5%, and is less than 2.0%, and the difference is in the range of 0.12% to 0.27%, which shows that the content determination method established in the present application has good repeatability.

[0070] 1.6 Accuracy test

[0071] According to Table 7, 3 portions of non-pyrilox non-pyrilox, methoprene, piperazine and moxidine reference substances were precisely weighed respectively, and were placed in 9 25ml brown volumetric flasks. The full auxiliary material mixture in the prescription proportion was precisely measured and added into the volumetric flasks. The solution was prepared according to the operation of the test sample item, that is, 80%, 100%, 120% test concentration sample solution was obtained. The content of non-pyrilox, methoprene, piperazine and moxidine was determined by the content determination method established in the application, and the recovery rate was calculated.

[0072] Table 7: Content determination table of each substance in accuracy test

[0073]

[0074] The accuracy test results are as follows:

[0075]

[0076]

[0077]

[0078]

[0079] From the experimental results, the recovery rate and the average recovery rate are 98.41% to 99.46%, and the RSD is within 2.0%. Therefore, the content determination method established in the application has good accuracy.

[0080] 1.7 Solution stability investigation

[0081] Three batches of compound preparations were taken, and two test sample solutions were prepared. The sample was injected under the chromatographic conditions of the content determination method established in the application, and the solution stability test was carried out at room temperature. The content of non-pyrilox, methoprene, piperazine and moxidine was detected on the 0th, 1st, 2nd and 3rd day, and the detection results were recorded in Table 8:

[0082] Table 8: Content determination table of each substance in test sample solution

[0083]

[0084] The results show that under room temperature conditions, the difference between the content of non-pyrilox, methoprene, piperazine and moxidine in the prepared test sample solution and the content on the 0th day is less than 2.0% within 3 days, and the difference meets the standard. Therefore, the test sample solution can be kept relatively stable under room temperature conditions for 3 days. The solution has good stability within the reasonable deviation of the detection requirement.

[0085] Comparative Example 1: Content determination of non-pyrilox and methoprene (Ministry of Agriculture Announcement No. 2511)

[0086] Chromatographic conditions and system suitability test: octadecylsilane-bonded silica gel as the packing material; water-acetonitrile-methanol (60:28:12) containing 0.1% glacial acetic acid as mobile phase A; acetonitrile-methanol (70:30) containing 0.1% glacial acetic acid as mobile phase B; linear gradient elution was carried out according to Table 9. The column temperature was 30°C; the detection wavelength was 280 nm (non-pyrrolnitrin and its related substances) and 265 nm (methoprene and its related substances). The separation degree of non-pyrrolnitrin peak and methoprene impurity RRT 1.1 peak at 280 nm should meet the requirements, and the separation degree of methoprene peak and methoprene cis-isomer peak should meet the requirements.

[0087] Table 9: Gradient elution data record table of Comparative Example 1

[0088]

[0089] Determination method: about 1 ml of the product was accurately weighed, placed in a 25 ml volumetric flask, 10 ml of acetonitrile was added, shaken to make the solution clear, diluted to the mark with acetonitrile, shaken well, 2 ml was accurately measured, placed in a 50 ml volumetric flask, diluted to the mark with diluent (water-acetonitrile-glacial acetic acid (50:50:0.05)), shaken well, as the test solution, 10 μL was accurately measured and injected into the liquid chromatograph, and the chromatogram was recorded. Non-pyrrolnitrin, methoprene, piperazine and moxidine control samples were taken, and mixed control sample solution containing about 0.13 mg of non-pyrrolnitrin, about 0.16 mg of methoprene, about 0.13 mg of piperazine and 0.016 mg of moxidine per 1 ml was prepared in the same way as the test solution preparation method, and was determined in the same way. The test sample was converted to milliliters, and the peak area was calculated by external standard method, and the content of non-pyrrolnitrin, methoprene, piperazine and moxidine was obtained.

[0090] 2.1 Specificity test

[0091] Preparation of blank preparation control solution: the formula adjuvants except non-pyrrolnitrin, methoprene, piperazine and moxidine were weighed according to the prescription ratio, and the blank preparation control solution was prepared according to the preparation process of compound preparation and the method of test solution preparation.

[0092] 10 μL of the test solution, control solution, blank preparation control solution and mobile phase were accurately measured,

[0093] The retention time and separation degree of non-pyrrolnitrin, methoprene, piperazine and moxidine chromatographic peaks were determined according to the sample injection chromatographic conditions of non-pyrrolnitrin and methoprene content determination method in Comparative Example 1, and were recorded in Table 10.

[0094] Table 10: Specificity test data record table of Comparative Example 1

[0095]

[0096] The results show that the test solution and the control solution appear chromatographic peaks at the corresponding positions of difenconazole and myclobutanil, and pyrantel and moxidectin are not detected. The separation degree of difenconazole and myclobutanil in the test chromatogram is good. The blank preparation control solution and the mobile phase do not appear chromatographic peaks at the main peak positions of difenconazole and myclobutanil, indicating that the mobile phase and the blank preparation control do not interfere with the retention time of difenconazole and myclobutanil.

[0097] In summary, the detection method of Comparative Example 1 cannot detect the content of pyrantel and moxidectin, and can only detect the content of difenconazole and myclobutanil. Example 1 can simultaneously detect the content of difenconazole, myclobutanil, pyrantel and moxidectin. It is proved that the chromatographic conditions of Example 1 are better, and are more suitable for the detection of the content of difenconazole, myclobutanil, pyrantel and moxidectin.

[0098] Comparative Example 2: Pyrantel content determination (Ministry of Agriculture Announcement No. 2511)

[0099] Chromatographic conditions and system suitability test: octadecylsilane-bonded silica gel is used as the filler; water-acetonitrile-methanol (60:28:12) containing 0.1% glacial acetic acid is used as the mobile phase A; acetonitrile-methanol (70:30) containing 0.1% glacial acetic acid is used as the mobile phase B, and linear gradient elution is carried out according to Table 11. The flow rate is 0.85 ml per minute; the column temperature is 30°C; and the detection wavelength is 245 nm.

[0100] Table 11: Gradient elution data record table of Comparative Example 2

[0101]

[0102] Determination method: about 1 ml of the product is precisely weighed and placed in a 25 ml volumetric flask, 10 ml of acetonitrile is added, shaken to make the solution clear, diluted with acetonitrile to the mark, shaken uniformly, and used as the test solution. Precisely 10 μL of the test solution is injected into the liquid chromatograph, and the chromatogram is recorded. In addition, an appropriate amount of difenconazole control, myclobutanil control, pyrantel control and moxidectin control are prepared into a mixed control solution containing about 3.32 mg of difenconazole, about 4 mg of myclobutanil, about 3.32 mg of pyrantel and about 0.4 mg of moxidectin per 1 ml by the same preparation method as the test solution, and determined by the same method. The test sample is converted into milliliter number, and the peak area is calculated by external standard method, and then the content is obtained.

[0103] 3.1 Specificity test

[0104] Preparation of blank preparation control solution: the formula accessories except difenconazole, myclobutanil, pyrantel and moxidectin are weighed according to the prescription ratio, and the blank preparation control solution is prepared according to the preparation process of compound preparation and the method of test solution preparation.

[0105] Take 10 μl of the test sample solution, the control solution, the blank preparation control solution and the mobile phase respectively, and inject them according to the chromatographic conditions in the content determination method of piperazine in Comparative Example 2, to determine the retention time and separation degree of the chromatographic peaks of febantel, pyrantel, morantel and moxidectin. The determination data are recorded in Table 12.

[0106] Table 12: Data recording table of the specificity test of Comparative Example 2

[0107]

[0108] The results show that the test sample solution and the control solution both have chromatographic peaks at the corresponding positions, and the separation degree between the chromatographic peaks of pyrantel and moxidectin in the test sample chromatogram does not meet the requirement of ≥1.5; the blank preparation control solution and the mobile phase do not have chromatographic peaks at the positions of the four main peaks, indicating that the mobile phase and the blank preparation control do not interfere with the retention times of febantel, pyrantel, morantel and moxidectin.

[0109] In summary, the separation degree between the chromatographic peaks of pyrantel and moxidectin in the test sample solution of the detection method of Comparative Example 2 does not meet the requirement of ≥1.5. The test sample chromatogram of the detection method of Example 1 is smooth at the baseline, and the separation degree meets the requirement of ≥1.5. The detection time of each sample is shortened by 20 minutes compared with Comparative Example 2, which accelerates the detection speed and improves the detection efficiency. It is proved that the chromatographic conditions of Example 1 are better and more suitable for the detection of the contents of febantel, pyrantel, morantel and moxidectin.

[0110] 3.2 Reproducibility test

[0111] Take 6 test sample solutions from the same batch of compound preparation by two different analysts in different laboratories on different working days, use different liquid chromatographs and different chromatographic columns, and inject them according to the chromatographic conditions in the content determination method of Comparative Example 2, to determine the contents of febantel, pyrantel, morantel and moxidectin. The reproducibility test results are recorded in Table 13.

[0112] Table 13: Data recording table of the reproducibility test of Comparative Example 2

[0113]

[0114]

[0115]

[0116]

[0117] The results show that: the RSD of non-pelletrol content in the test results of two different analysts is 1.4%, less than 2.0%, and the difference is 0.66%, meeting the basic requirements of reproducibility test; the RSD of methoprene, piperazine and moxidine content is greater than 2.5%, and the difference is greater than 2.0%, which does not meet the basic requirements of reproducibility test. The detection results of comparative example 2 have large fluctuation and poor reproducibility.

[0118] In summary, the reproducibility results of comparative example 2 do not meet the basic requirements of reproducibility test, and the detection results have large fluctuation and poor reproducibility. The detection results of example 1 meet the basic requirements of reproducibility test and have good reproducibility. It is proved that the chromatographic conditions of example 1 are better and more suitable for the detection of the contents of non-pelletrol, methoprene, piperazine and moxidine.

[0119] 3.3 Accuracy test

[0120] According to table 14, 3 portions of non-pelletrol reference substance, methoprene reference substance, piperazine reference substance and moxidine reference substance were precisely weighed and placed in 9 25ml brown volumetric flasks, and the mixed auxiliary materials in the prescription proportion of the compound preparation were precisely measured and added into the flasks. According to the preparation of the test sample solution, 80%, 100% and 120% test concentration sample solutions were prepared, and the contents of non-pelletrol, methoprene, piperazine and moxidine were determined by the content determination method established in the present application, and the recovery rate was calculated.

[0121] Table 14: data recording table of accuracy test of comparative example 2

[0122]

[0123] The accuracy test results are as follows:

[0124]

[0125]

[0126]

[0127]

[0128] The results show that: the recovery rate and average recovery rate of non-pelletrol, methoprene, piperazine and moxidine are out of the range of 98.0% to 102.0%, and the RSD of the recovery rate of methoprene, piperazine and moxidine is greater than 2.0%. The accuracy test results do not meet the basic requirements of accuracy test.

[0129] In summary, the colorimetric condition accuracy test results of Comparative Example 2 do not meet the basic requirements of the accuracy test. However, the recovery rate and average recovery rate of Example 1 are both within 98.41% to 99.46%, the RSD is within 2.0%, the fluctuation is small, the accuracy is high, and it meets the basic requirements of the accuracy test. It is proved that the chromatographic condition of Example 1 is better and more suitable for the detection of the content of monepantux, pyrantel and moxidectin.

[0130] Comparative Example 3: Moxidectin content determination (Ministry of Agriculture Announcement No. 2168)

[0131] Chromatographic conditions and system suitability test: octadecylsilane-bonded silica gel (150 mm x 3.9 mm, 4 µm) was used as the filler; acetic acid-ammonium acetate buffer (pH 4.8) (take ammonium acetate 7.7 g, dissolve in water 400 ml, and adjust the pH value to 4.8 with glacial acetic acid)-acetonitrile (400:600) was used as the mobile phase; the column temperature was 50°C; the detection wavelength was 242 nm; the flow rate was 2.5 ml per minute.

[0132] Determination method: take the product in an appropriate amount, accurately weigh and add acetonitrile to prepare a solution containing about 1 mg of moxidectin per 1 ml as the test solution, accurately take 10 µl, inject into the liquid chromatograph, and record the chromatogram to 2 times the retention time of the main component. Take non-pyrantel, methoxyproline, pyrantel and moxidectin control substances in an appropriate amount, and determine them by the same method. According to the external standard method, the peak area is calculated, and the content of non-pyrantel, methoxyproline, pyrantel and moxidectin is obtained.

[0133] 4.1 Specificity test

[0134] Preparation of blank preparation control solution: according to the prescription proportion, the formula accessories except non-pyrantel, methoxyproline, pyrantel and moxidectin were weighed, and the blank preparation control solution was prepared according to the preparation process of compound preparation and the preparation method of test solution.

[0135] Accurately take 10 µl of the test solution, the control solution, the blank preparation control solution and the mobile phase, inject them according to the chromatographic conditions of the determination of the content of moxidectin in Comparative Example 3, determine the retention time and separation degree of the chromatographic peaks of non-pyrantel, methoxyproline, pyrantel and moxidectin, and record the data in Table 15:

[0136] Table 15: Data recording table of specificity test of Comparative Example 3

[0137]

[0138] The results show that the test sample solution and the control sample solution both have chromatographic peaks at the corresponding positions, the separation degree between piperazine, moxidectin and the adjacent chromatographic peaks in the chromatogram of the test sample does not meet the requirement of ≥1.5; the blank preparation control solution has a chromatographic peak at the piperazine peak position, indicating that the blank preparation control has interference at the piperazine retention time; the mobile phase has no chromatographic peak at the four main peak positions, and the mobile phase has no interference at the non-pyrantel, moxidectin, piperazine and moxidectin retention times.

[0139] In summary, the results in Table 16 prove that the chromatographic conditions of Example 1 are better than those of Comparative Example 3, and are more suitable for the detection of the contents of non-pyrantel, moxidectin, piperazine and moxidectin.

[0140] Table 16: Comparison table of Example 1 and Comparative Example 3

[0141]

[0142] In summary, the results prove that the present application can simultaneously detect the contents of non-pyrantel, moxidectin, piperazine and moxidectin, shorten the detection time, greatly improve the detection efficiency; have strong specificity, good separation effect between the active ingredients in the preparation and the adjacent peaks; good stability of the sample solution, which can ensure the accuracy of the detection results; reduce the damage to the chromatographic column and the instrument, reduce the amount of reagents used, and greatly save the cost.

[0143] Although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions recorded in the foregoing embodiments, or make equivalent replacements to part of the technical features, and any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A method for detecting the content of fipronil, methoxyprone, praziquantel, and moxiquitin, characterized in that: The detection methods include: using high performance liquid chromatography to detect compound preparations containing fipronil, metoprone, praziquantel, and moxiquitin; The chromatographic conditions for gradient elution during detection are as follows: a mixed solution of water, acetonitrile, methanol and glacial acetic acid is used as mobile phase A, a mixed solution of acetonitrile, methanol and glacial acetic acid is used as mobile phase B, the flow rate is 0.85-1.25 ml / min, the column temperature is 20-30℃, and the injection volume is 15-30 μL. The detection wavelength is 230–250 nm, and the theoretical plate number calculated based on the main peak is not less than 5000. The gradient elution procedure is as follows: At 0 min, mobile phase A is 100% and mobile phase B is 0%. At 10 min, mobile phase A was 75% and mobile phase B was 25%. At 30 min, mobile phase A was 0% and mobile phase B was 100%, and this was maintained for 35 min. At 35.1 min, mobile phase A recovered to 100%, and mobile phase B recovered to 0%, and remained at this level for 40 min. The preparation method of mobile phase A is as follows: water, acetonitrile, methanol and glacial acetic acid are prepared in the following volume ratio: water-acetonitrile-methanol (50-60): (26-40): (10-14) containing 0.17%-0.23% glacial acetic acid solution; The preparation method of mobile phase B is as follows: acetonitrile, methanol and glacial acetic acid are mixed in a volume ratio to prepare an acetonitrile-methanol (60-70): (30-40) solution containing 0.17%-0.23% glacial acetic acid; In chromatographic experiments, octadecylsilane-bonded silica gel was used as the packing material.

2. The method for detecting the content of fipronil, methoxyprone, praziquantel, and moxiquitin according to claim 1, characterized in that: The detection method includes the following steps: S1. Preparation of test solution: Add acetonitrile to the compound preparation sample, dissolve and mix well, take an appropriate amount of the resulting solution to obtain the test solution; S2. Preparation of reference solution: Dissolve and dilute the fepronil reference standard, methoxyprone reference standard, praziquantel reference standard and moxiquitoline reference standard in acetonitrile to obtain the reference solution.

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