CN115166122B - Method for detecting methoprene in compound preparation containing non-prednisone and methoprene - Google Patents
Method for detecting methoprene in compound preparation containing non-prednisone and methoprene Download PDFInfo
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- CN115166122B CN115166122B CN202210796221.9A CN202210796221A CN115166122B CN 115166122 B CN115166122 B CN 115166122B CN 202210796221 A CN202210796221 A CN 202210796221A CN 115166122 B CN115166122 B CN 115166122B
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- NFGXHKASABOEEW-UHFFFAOYSA-N 1-methylethyl 11-methoxy-3,7,11-trimethyl-2,4-dodecadienoate Chemical compound COC(C)(C)CCCC(C)CC=CC(C)=CC(=O)OC(C)C NFGXHKASABOEEW-UHFFFAOYSA-N 0.000 title claims abstract description 107
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- 102100030403 Signal peptide peptidase-like 2A Human genes 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
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- WXYIONYJZVWSIJ-UHFFFAOYSA-N acetonitrile;methanol;hydrate Chemical compound O.OC.CC#N WXYIONYJZVWSIJ-UHFFFAOYSA-N 0.000 description 1
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
- G01N2030/884—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample organic compounds
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention provides a method for detecting the content of methoprene in a compound preparation containing non-prednisone and methoprene, which can be used for determining the content of the methoprene in a two-party, three-party or four-party mixed preparation containing non-prednisone and methoprene. The detection method is a high performance liquid chromatography, and the chromatographic conditions are as follows: taking a mixed solution of a mobile phase A and acetonitrile in a fixed proportion as a mobile phase; the flow rate is 0.8-1.2 mL/min, and the column temperature is 20-30 ℃; the detection wavelength is 260-275 nm; the theoretical plate number is not less than 5000 calculated by methoprene. The detection method has the advantages of good specificity, good linear relation, strong stability, good repeatability, accurate detection result and the like. Meanwhile, the invention is isocratic elution, has low requirement on instruments, is simpler and quicker to operate, greatly shortens the detection time by 1/4 times of the existing method, can improve the detection efficiency and saves more cost.
Description
Technical Field
The invention relates to the technical field of liquid chromatography analysis, in particular to a method for detecting the content of methoprene in a compound preparation containing non-prednisone and methoprene.
Background
Fleas and ticks are the most common ectoparasites of pet dogs and cats and are also the transmission vehicles for many important diseases such as rickettsia, plague, lyme disease, murine typhus, flea-transmitted spot fever, babesia, etc. The dogs and cats become family partners to meet the emotion demands of human beings, and simultaneously indirectly become a plurality of infectious sources and transmission media for zoonosis, thereby threatening the health of human beings and public health safety.
Non-prednisone is a novel broad-spectrum pesticide of phenylpyrazole, and the action mechanism of the pesticide is to combine with gamma-aminobutyric acid (GABA) receptors on insect central nervous cell membranes to close chloride ion channels of nerve cells, thereby interfering with normal functions of the central nervous system to cause death of insects. Methoprene is an Insect Growth Regulator (IGR), is a congener of insect juvenile hormone, has an inhibitory effect on the development of insects in immature stages, has a mechanism of action similar to juvenile hormone, and can cause the blocked development of insects and the death of fleas in developmental stages. In addition, methoprene also has a strong inhibitory effect on the development of flea larvae and pupae. The compound preparation containing the non-prednisone and the methoprene has good curative effect and less adverse reaction, and is one of in-vitro pesticides which are widely applied to the surfaces of dogs and cats for expelling and killing adult fleas, flea eggs and larvae in the global scope at present.
The detection of the content of the compound preparation containing the non-prednisone and the methoprene is mainly carried out by adopting a liquid phase detection method. In the prior art, only the compound non-prednisone drops (for dogs and cats) published by the Ministry of agriculture of the people's republic of China are imported and registered in standards, and related analysis methods are provided for detecting the content of the methoprene in a compound preparation consisting of two sides containing the non-prednisone and the methoprene, but no analysis method capable of effectively measuring the content of the methoprene in a three-side or four-side preparation containing the non-prednisone and the methoprene is disclosed.
It is known that the degree and complexity of the impurity and auxiliary materials interference in the measurement of the content of two, three or four drug components in the drug preparation are far superior to those of the bulk drug and the single preparation thereof.
Meanwhile, in the existing detection method of the No. 2410 publication of the Ministry of agriculture, the range of retention time drift of the chromatographic peak of the active ingredient methoprene is too large, the peak shape effect is poor, the detection volatility is large, the separation degree of the methoprene and the adjacent known degradation product methoprene cis-isomer and the unknown degradation product methoprene IMP3 is very easy to be caused to be not in accordance with the requirement of more than or equal to 1.5, the baseline separation is not achieved, and thus the detection accuracy and the detection efficiency of the content of the active ingredient methoprene are affected, and the method specificity is poor.
In addition, in the existing liquid phase detection method, the ultraviolet absorption response of the active ingredient methoprene is relatively low, so that the linear intercept percentage of the methoprene content cannot meet the requirement of less than or equal to 2.0%, and the methoprene content detection is inaccurate. The stability of the detection solution is poor, special control is needed, a complete gradient elution program is needed to be operated in each detection, and the operation requirements are high, the time consumption is long and the cost is high.
Disclosure of Invention
The invention aims to solve the defects in the prior art, and provides a method for detecting the content of methoprene in a compound preparation containing non-prednisone and methoprene.
A method for detecting the content of methoprene in a compound preparation containing non-prednisone and methoprene is a 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 according to methoprene, and the mixed solution of the mobile phase A and acetonitrile is used as the mobile phase; wherein the mobile phase A is prepared from acetonitrile, methanol and acetic acid aqueous solution with the mass fraction of 0.1% according to the mass ratio of 47:21: 32.
Preferably, the volume ratio of the mixed solution of the mobile phase A and acetonitrile in the mobile phase is 10-30: 70 to 90, preferably 20:80.
preferably, the pH of mobile phase A is 2.5 to 2.9, preferably 2.7.+ -. 0.05. If the pH value is further increased, the peak shape effect is affected, and if the pH value is further reduced, the separation effect, tailing factor and peak shape effect are affected.
The pH value of the mobile phase A is limited, so that the drift of a chromatographic peak caused by the baseline fluctuation generated by gradient elution in the existing method can be reduced, the position of the chromatographic peak is more stable, the peak shape is more stable, the theoretical plate number of the chromatographic peak is higher, the separation degree effect is better, the detection time is shorter, and the fluctuation of the detection effect is avoided, the detection is more accurate and the cost is lower.
Preferably, the pH of mobile phase A is adjusted with sodium hydroxide solution.
More preferably, the mass fraction of the sodium hydroxide solution is 48-52%.
Preferably, the detection wavelength is 266nm.
Preferably, the sample is subjected to a pretreatment prior to detection, specifically as follows: acetonitrile is added into a compound preparation sample containing non-prednisone and methoprene, after dissolution, the mixture is uniformly mixed, an appropriate amount of the obtained solution is taken, and then acetic acid mixed solution is used for dissolution and dilution, so as to obtain a sample solution; and dissolving the methoprene reference substance in acetic acid mixed solution and diluting to obtain a reference substance solution.
Preferably, the acetic acid mixed solution is prepared from acetonitrile, water and acetic acid according to the mass ratio of 66-70: 29 to 33:0.8 to 1.2.
More preferably, the acetic acid mixed solution is prepared from acetonitrile, water and acetic acid according to the mass ratio of 68:31:1, and mixing.
The method adopts the acetic acid mixed solution as the diluting solvent, greatly improves the solution stability of the sample solution and the reference solution, thereby reducing the deviation of the detection result caused by the unstable solution and improving the detection accuracy.
Preferably, octadecylsilane chemically bonded silica is used as filler.
Preferably, the flow rate is 0.8-1.2 mL/min and the column temperature is 20-30 ℃.
Preferably, the sample injection amount is 15-30 mu L.
More preferably, the amount of sample introduced is 15 to 25. Mu.L, preferably 20. Mu.L.
Compared with the prior art, the invention has the beneficial effects that:
1. the invention has wide application range, can be used for the analysis method of the content of the methoprene in the preparation of both sides, three sides or four sides containing the non-prednisone and the methoprene, and has wider application value.
2. The invention establishes a method for detecting the content of the compound preparation containing the non-prednisone and the methoprene, which has the advantages of small detection volatility, stronger specificity, better separation effect of active components in the preparation and various impurity components and better chromatographic peak shape.
3. The linear intercept percentage of the method is less than or equal to 2.0%, the content linear verification requirement is met, the linear relation is good, the detection fluctuation is reduced, and the detection accuracy and the repeatability are better.
4. The method improves the solution stability of the detection sample, can more accurately reflect the real content of the sample, and greatly improves the accuracy of the detection result.
5. The method is isocratic elution, has low requirement on instruments, is simpler and faster to operate, greatly shortens the detection time by 1/4 of the existing method, can improve the detection efficiency, and saves more cost.
Drawings
FIG. 1 is a graph showing the comparison of absorption peaks at different wavelengths for the examples.
FIG. 2 is a graph showing the comparative measurement of methoprene content in various dilution solvents used in the examples.
FIG. 3 is a graph showing the retention time and separation of methoprene chromatographic peaks when different mobile phases are used in the examples.
FIG. 4 is a graph showing the theoretical plate count versus peak of methoprene chromatography using mobile phase A at different pH values.
FIG. 5 is a graph comparing the results of durability tests under different conditions for the examples.
FIG. 6 is a graph showing the comparison of methoprene recovery rates when samples of different concentrations are used in the examples.
FIG. 7 is a graph showing the comparative methoprene recovery rate when different concentrations of the test sample are used in the comparative example.
FIG. 8 is a graph comparing the results of reproducibility tests performed by different analysts under different conditions using the examples.
FIG. 9 is a comparative graph of the results of reproducibility tests performed by different analysts under different conditions using the comparative examples.
Detailed Description
The invention is further illustrated below in connection with specific embodiments.
Examples
Octadecylsilane chemically bonded silica is used as a filler; mobile phase A (acetonitrile: methanol: 1.0% acetic acid aqueous solution mixed solution, ratio of 47:21:32, pH=2.7.+ -. 0.05) and acetonitrile (v/v, 20:80) were used as mobile phases, the flow rate was 1.0mL/min; the column temperature is 25 ℃; the detection wavelength was 266nm. The theoretical plate number is not less than 5000 calculated by methoprene.
High performance liquid chromatograph model: agilent 1260.
Chromatographic column model: agilentZorbax SB-C18 (4.6X105 mm,5 μm).
Taking 1mL of a compound preparation sample containing the non-prednisone and the methoprene (about containing 100mg of the non-prednisone and 90mg of the methoprene), precisely weighing, placing in a 50mL brown measuring flask, diluting to a scale with acetonitrile, shaking uniformly, precisely weighing 2mL, placing in a 50mL brown measuring flask, diluting to the scale with acetic acid mixed solution (mixed solution of acetonitrile: water: glacial acetic acid with the ratio of 68:31:1), and shaking uniformly to obtain the compound preparation.
And (3) taking about 20mg of methoprene reference substance, precisely weighing, placing into a 50mL brown measuring flask, diluting to a scale with acetonitrile, shaking uniformly, precisely weighing 2mL, placing into a 10mL brown measuring flask, diluting to the scale with acetic acid mixed solution (acetonitrile: water: glacial acetic acid mixed solution with the ratio of 68:31:1), and shaking uniformly to obtain the methoprene.
And calculating according to an external standard method and peak area to obtain the product.
Content measurement method investigation:
1.1 determination of the detection wavelength
Weighing methoprene reference substance 20mg to 250mL measuring flask, adding acetic acid mixed solution (acetonitrile: water: glacial acetic acid mixed solution with ratio of 68:31:1), dissolving, fixing volume, shaking, and taking as wavelength test solution. The scanning was performed in the range of 190nm to 400nm using a high performance liquid chromatography Diode Array Detector (DAD). As shown in FIG. 1, methoprene has a maximum absorption peak at a wavelength of 266nm.
1.2 determination of dilution solvent
The research of the product shows that the solution stability of the sample solution prepared by different dilution solvents is different.
The test of methoprene content in the solution stability test of the test sample solutions of different dilution solvents was performed, preferably the most suitable dilution solvents, by comparing the diluted solvents, acetic acid mixed solution 1 (acetonitrile: water: glacial acetic acid mixed solution with a ratio of 68:31:1), acetic acid mixed solution 2 (acetonitrile: water: glacial acetic acid mixed solution with a ratio of 68:30.8:1.2), acetic acid mixed solution 3 (acetonitrile: water: glacial acetic acid mixed solution with a ratio of 68:31.2), acetic acid mixed solution 4 (acetonitrile: water: glacial acetic acid mixed solution with a ratio of 67.8:31:1.2), acetic acid mixed solution 5 (acetonitrile: water: glacial acetic acid mixed solution with a ratio of 68.2:31:0.8), acetic acid mixed solution 6 (acetonitrile: water: glacial acetic acid mixed solution with a ratio of 66:33:1), and acetic acid mixed solution 7 (acetonitrile: water: glacial acetic acid mixed solution with a ratio of 70:29:1).
As shown in fig. 2, acetonitrile in acetic acid mixture: water: glacial acetic acid ratio was 68:31:1 as a benchmark, and acetonitrile: water: the ratio of each component of glacial acetic acid is (66-70): (29-33): when the content of methoprene in the solution stability test of the prepared test sample solution at room temperature is changed within the range of (0.8-1.2), the difference of the methoprene content detection is less than 2.0%, and the solution stability and durability are good within reasonable deviation meeting the detection requirement, so acetonitrile in acetic acid mixed solution is adopted: water: glacial acetic acid ratio was 68:31:1 as a diluting solvent for a content measurement sample solution.
1.3 selection of mobile phases
Taking the same batch of compound preparation samples containing non-prednisone and methoprene and the same batch of original ground preparation, respectively preparing 6 parts of sample solutions, taking a mobile phase A (acetonitrile: methanol: 1.0% acetic acid aqueous solution mixed solution, the ratio of which is 47:21:32, and the pH value of which is=2.7+/-0.05) and acetonitrile (v/v, 20:80) as mobile phases, and measuring the retention time and the separation degree of a methoprene chromatographic peak.
As shown in fig. 3, in each of the 6 chromatograms of the sample and the original ground preparation of the compound preparation containing non-prednisone and methoprene, the range RSD of retention time drift of the active ingredient methoprene chromatographic peak, the known degradation product methoprene cis isomer and the unknown degradation product methoprene IMP3 chromatographic peak is less than 0.2%, and the peak position is relatively stable. The separation degree of the methoprene and the adjacent known degradation product methoprene cis isomer and the unknown degradation product methoprene IMP3 is more than 1.5, the requirements of baseline separation are met, the specificity of the method is strong, the RSD is less than 0.5%, and the reproducibility is good.
1.4 determination of pH of Mobile phase A
Taking the same batch of compound preparation samples containing non-prednisone and methoprene, respectively preparing 6 parts of sample solutions, and measuring the theoretical plate number of the methoprene chromatographic peak by taking mobile phase A (acetonitrile: methanol: 1.0% acetic acid aqueous solution mixed solution, the ratio of which is 47:21:32) and acetonitrile (v/v, 20:80) with different pH values as mobile phases.
As shown in fig. 4, in the 6 chromatograms of the compound preparation sample containing non-prednisone and methoprene, when the active ingredient methoprene chromatographic peak is at ph=2.5-2.9, the theoretical plate number average reaches more than 5000, and the main methoprene chromatographic peak and the adjacent impurity peak can reach baseline separation. And at ph=2.7, the influence of the solvent effect can be most effectively eliminated, the symmetry of chromatographic peaks is best, the theoretical plate number is highest, and the separation degree is best. Finally, ph=2.7 is preferred.
1.5 precision test
Taking the same batch of compound preparation samples containing non-prednisone and methoprene, respectively preparing 6 parts of test solution, and injecting samples according to chromatographic conditions of the content determination method established in the above example. Under the same chromatographic condition, the sample injection is repeated for 6 times, 20 mu L of sample is respectively measured each time, the peak area is measured, the precision of the instrument is inspected, and the relative standard deviation RSD of the content obtained by 6 groups of data is calculated.
The results of the precision test are as follows:
the RSD result is 0.3% and less than 2.0%, which indicates that the content determination method established by the invention has good precision.
1.6 repeatability test
Selecting 6 parts of a compound preparation containing non-prednisone and methoprene in the same batch, precisely weighing, preparing a solution according to the operation of a test item, adopting the same chromatographic condition, injecting 20 mu L of sample, collecting the chromatogram of 6 parts of sample, calculating the relative standard deviation RSD of the solution, and repeating the test results as follows:
the RSD result is 0.3% and less than 2.0%, so that the repeatability of the content measurement method established by the invention is good.
1.7 durability test
Taking a proper amount of a compound preparation containing non-prednisone and methoprene in the same batch, respectively examining the flow rate change of +/-0.2 mL/min, the column temperature change of +/-5 ℃, the acetonitrile proportion change in the mobile phase of +/-2%, the concentration change of acetic acid aqueous solution in the mobile phase A of +/-0.2%, the pH change of the mobile phase A of +/-0.05 and the change of instrument chromatographic behaviors in different chromatographic columns, observing the separation condition of a base line, and calculating the average value and RSD value of the obtained data under each condition.
The durability test results are shown in figure 5, in the durability test of the methoprene content measuring method of the compound preparation containing non-prednisone and methoprene, the flow rate, the column temperature, the chromatographic column and the mobile phase composition, the peak and the adjacent peak of the methoprene reach baseline separation, and the RSD value of the measured methoprene content under each condition is less than 2 percent. It can be seen that the content measurement method established by the invention has good durability.
Comparative example 1
The method is adopted in the compound non-prednisone drop (for cats) content measurement of No. 2410 issued by the Ministry of agriculture of the people's republic of China.
Chromatographic conditions and system suitability test: the chromatographic column is a Zorbax SB-C18 column, acetonitrile-methanol-water (1.0% glacial acetic acid solution is added into water before mixing) (47:21:32) is used as a mobile phase A, and acetonitrile is used as a mobile phase B; the flow rate is 0.8-1.0 mL/min per minute, and gradient elution is carried out according to the following table; the detection wavelength was 284nm. The separation degree of sulfonyl and non-prednisone should be not less than 2.0, and the separation degree of the peak of cis-isomer of methoprene and the peak of methoprene should meet the requirement.
| Time (minutes) | Mobile phase a (%) | Mobile phase B (%) |
| 0 | 100 | 0 |
| 9 | 100 | 0 |
| 12 | 15 | 85 |
| 22 | 15 | 85 |
| 25 | 100 | 0 |
| 30 | 100 | 0 |
Assay: taking about 1mL of the product, precisely weighing, placing the product into a 50mL brown measuring flask, diluting with acetonitrile to a scale, shaking up, precisely weighing 2mL, placing the product into the 50mL brown measuring flask, diluting with a mobile phase to the scale, shaking up, and taking the product as a sample solution; 20 mu L of the solution is precisely measured and injected into a liquid chromatograph, and a chromatogram is recorded.
And respectively measuring the non-prednisone and methoprene reference substances in proper amounts by the same method. The relative density was measured while taking the sample, and the weight of the sample was converted into ml. And calculating according to an external standard method and peak area to obtain the product.
2.1 specificity test
2.1.1 preparation of blank formulation control solution: weighing auxiliary materials except methoprene according to the prescription proportion, and preparing a blank preparation control solution according to the preparation process of the compound preparation containing non-prednisone and methoprene and the method under the test solution preparation item.
Precisely sucking 20 mu L of each of the sample solution, the reference substance solution, the blank preparation reference solution and the mobile phase, and injecting according to the chromatographic conditions of the content determination method established in the above example.
The results show that: chromatographic peaks appear in the corresponding positions of the sample solution and the reference solution, and the baseline in the chromatogram of the sample solution is stable and has good separation degree; the blank control solution and mobile phase showed no color spectrum peaks at this position, indicating that the mobile phase and blank control had no interference with methoprene retention time.
2.1.2 chromatographic conditions of comparative examples and comparative examples, the degree of separation, retention time, peak shape were observed. The method comprises the following steps:
taking 1mg of each of the cis-isomer of methoprene, 1mg of IMP3 of methoprene and 1mg of sulfonyl, taking 20mg of non-prednisone reference substance, placing the non-prednisone reference substance into the same 50mL measuring flask, and preparing a solution according to the operation of a test item to obtain a separation degree solution with the impurity concentration of 2 g/mL.
Detection results of the compound preparation containing the non-prednisone and the methoprene under two chromatographic conditions:
the results in the table above show that: the chromatographic conditions of the embodiment can ensure better separation degree of the compound preparation containing the non-prednisone and the methoprene, the retention time is reduced from 21.345min to 4.382min, the detection efficiency is greatly improved, and the peak shape is symmetrical and beautiful.
In summary, the chromatographic conditions of the examples and the comparative examples can ensure that the methoprene peak is not interfered by the mobile phase and the blank preparation contrast, and meets the specific basic requirements, but the chromatographic conditions of the examples can ensure that the baseline smoothness, the separation degree and the peak shape in the chromatographic peak of the sample are better, the detection efficiency is high, and the chromatographic conditions of the preferred examples are better, and the method is suitable for the content measurement of the methoprene in the compound preparation containing non-prednisone and the methoprene.
2.2 linear relationship
Precisely weighing 40mg of methoprene sample, placing in a 100mL volumetric flask, dissolving with diluent acetic acid mixed solution (acetonitrile: water: glacial acetic acid ratio is 68:31:1), diluting to constant volume, and shaking to obtain stock solution. The stock solutions were measured again in 50mL measuring flask, 5mL, 6mL, 8mL, 10mL, 12mL, 15mL and 18mL, diluted to the scale with the diluent acetic acid mixture, shaken well, and filtered through a 0.45 μm filter to obtain 50%, 60%, 80%, 100%, 120%, 150% and 180% serial measuring concentration solutions.
Sample chromatographic peak areas were determined by injecting 20. Mu.L of sample under chromatographic conditions established in the above examples. The results of the linear test are as follows: the peak area and the concentration are taken as coordinates, a standard curve is established, and the obtained equation is as follows: y=98.232x+67.931 (r2=1), and the linear intercept percentage is 0.84% (< 2.0%), the detection volatility is small, suggesting that the methoprene concentration has a good linear relationship with peak area in the range of 40.8917 μg/mL to 147.2103 μg/mL.
According to the chromatographic condition of the comparative example, the correlation coefficient (R) of the linear curve number is larger than 0.999, which meets the requirement, but the linear intercept percentage is 3.87% (> 2.0%), which indicates that the Y-axis intercept exceeds more than 2% of the 100% response value, the detection fluctuation is large, and the error is high.
In summary, the chromatographic conditions of the examples and the comparative examples meet the basic requirement of linearity, but the intercept of the linear regression equation of the chromatographic conditions of the examples does not deviate from zero obviously, and the accuracy is higher, so that the chromatographic conditions of the preferred examples are better, and the method is suitable for measuring the content of methoprene in a compound preparation containing non-prednisone and methoprene.
2.3 accuracy test
Precisely weighing about 10mg, 20mg and 30mg of methoprene reference substances, respectively placing 6 parts into 18 50mL measuring flasks, precisely measuring a proper amount of a full auxiliary material mixture containing non-prednisone and methoprene in a prescription proportion of a compound preparation, and adding the full auxiliary material mixture into the mixture, and preparing solutions according to the operation of test items to obtain 6 parts of sample solutions with the test concentration of 50%, 100% and 150%. The methoprene content was measured according to the content measurement method established in the above example and the chromatographic conditions of the comparative example, respectively, and the recovery rate was calculated.
As shown in FIG. 6, the recovery rate and average recovery rate of the examples were 98.87% -99.97%, and RSD was within 2%. Therefore, the content determination method established by the invention has good accuracy.
As shown in FIG. 7, the recovery rate and the average recovery rate of the comparative example are 98.04% -100.81%, and the RSD is within 2%, so that the accuracy of the content measurement method of the comparative example meets the requirement.
In summary, the chromatographic conditions of the examples and the comparative examples meet the basic requirements of the accuracy test, but the recovery rate and the average recovery rate deviation range of the examples are smaller than those of the comparative examples, the RSD is smaller than that of the comparative examples, the volatility is small, the accuracy is high, and the chromatographic conditions of the preferred examples are better and the method is suitable for the content measurement of the methoprene in the compound preparation containing non-prednisole and methoprene.
2.4 repeatability test
12 parts of compound preparation containing non-prednisone and methoprene in the same batch are selected and divided into two groups.
One group was measured for content by two different analysts at different laboratories, different workdays, using different liquid chromatographs, different chromatographic columns, and the same "1.6, repeatability test" procedure. As shown in FIG. 8, the RSD of the two is 0.27% (< 2.0%) and the difference is 0.21%, so that the reproducibility of the content measurement method established by the invention is good.
The other group was measured by the two analysts at different laboratories and different workdays using different liquid chromatographs, different chromatographic columns, and according to the chromatographic conditions of the comparative example. As shown in FIG. 9, the RSD of the two samples was 1.51% (< 2.0%) and the difference was-1.20%, so that the reproducibility of the content measurement method of the comparative example was generally high, and the measured value deviation was large.
3. Determination of methoprene content in sample
The methoprene content in three batches of the compound preparation containing non-prednisone and methoprene was measured according to the content measurement method established in the above example and the chromatographic conditions of the comparative example, respectively, and the results are as follows:
from the above results, it can be seen that: the content RSD of the methoprene in three batches of samples of the compound preparation containing the non-prednisone and the methoprene is less than 2%, and the intra-batch difference is small; however, the repeatability of the examples is better than that of comparative example 1.
In conclusion, the results prove that the liquid phase detection method of the optimized compound preparation containing the non-prednisone and the methoprene is improved in the aspects of specificity, accuracy, reproducibility and stability.
The foregoing is only a preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art, who is within the scope of the present invention, should make equivalent substitutions or modifications according to the technical scheme of the present invention and the inventive concept thereof, and should be covered by the scope of the present invention.
Claims (9)
1. The method for detecting the content of methoprene in the non-prednisone and methoprene-containing compound preparation is characterized by comprising the following steps of:
the detection wavelength is 260-275 nm, the theoretical plate number is not less than 5000 according to methoprene, the mixed solution of mobile phase A and acetonitrile is used as the mobile phase, and the volume ratio of the mixed solution of mobile phase A and acetonitrile in the mobile phase is 10-30: 70 to 90, and the pH value of the mobile phase A is 2.5 to 2.9;
wherein the mobile phase A is prepared from acetonitrile, methanol and acetic acid aqueous solution with the mass fraction of 0.1% according to the mass ratio of 47:21:32, mixing to obtain the mixture;
octadecylsilane chemically bonded silica is used as a filler, and an isocratic elution mode is adopted.
2. The method according to claim 1, wherein the volume ratio of the mixed solution of mobile phase a and acetonitrile in the mobile phase is 20:80.
3. the method according to claim 1, wherein the pH of mobile phase A is 2.7.+ -. 0.05.
4. The method according to claim 1, wherein the detection wavelength is 266nm.
5. The method according to claim 1, wherein the sample is subjected to pretreatment prior to detection, specifically as follows:
acetonitrile is added into a compound preparation sample containing non-prednisone and methoprene, after dissolution, the mixture is uniformly mixed, an appropriate amount of the obtained solution is taken, and then acetic acid mixed solution is used for dissolution and dilution, so as to obtain a sample solution; and dissolving the methoprene reference substance in acetic acid mixed solution and diluting to obtain a reference substance solution.
6. The detection method according to claim 5, wherein the acetic acid mixed solution comprises acetonitrile, water and acetic acid in a mass ratio of 66-70: 29 to 33:0.8 to 1.2.
7. The detection method according to claim 6, wherein the acetic acid mixed solution comprises acetonitrile, water and acetic acid in a mass ratio of 68:31:1, and mixing.
8. The method according to claim 1, wherein the flow rate is 0.8-1.2 mL/min and the column temperature is 20-30 ℃.
9. The method according to claim 1, wherein the sample amount is 15 to 30. Mu.L.
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| JPH09278800A (en) * | 1996-04-08 | 1997-10-28 | Iatron Lab Inc | Monoclonal antibody specific to methoprene, hybridoma producing the same antibody and measurement of methoprene |
| CN109959730A (en) * | 2017-12-26 | 2019-07-02 | 广东东阳光药业有限公司 | A method of with the related substance of HPLC method measurement methoprene |
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| JPH09278800A (en) * | 1996-04-08 | 1997-10-28 | Iatron Lab Inc | Monoclonal antibody specific to methoprene, hybridoma producing the same antibody and measurement of methoprene |
| CN109959730A (en) * | 2017-12-26 | 2019-07-02 | 广东东阳光药业有限公司 | A method of with the related substance of HPLC method measurement methoprene |
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