CN114217000A

CN114217000A - High performance liquid chromatography detection method for chlorantraniliprole in solution

Info

Publication number: CN114217000A
Application number: CN202111539383.6A
Authority: CN
Inventors: 芮正军; 张胜男; 吴承丽; 李倩文; 何小强
Original assignee: Chongqing Huage Biochemistry Co ltd
Current assignee: Chongqing Huage Biochemistry Co ltd
Priority date: 2021-12-15
Filing date: 2021-12-15
Publication date: 2022-03-22

Abstract

The invention relates to the technical field of compound content detection, in particular to a high performance liquid chromatography detection method for chlorantraniliprole in a solution, which uses an acetonitrile water solution as a mobile phase, wherein the volume ratio of acetonitrile to water is 7: 3. The method can be suitable for qualitative detection of all intermediates for producing chlorantraniliprole (kangbai) and qualitative detection of the chlorantraniliprole, and the qualitative detection of the chlorantraniliprole is performed under the same liquid chromatography condition so as to conveniently judge the reaction degree.

Description

High performance liquid chromatography detection method for chlorantraniliprole in solution

Technical Field

The invention relates to the technical field of compound content detection, in particular to a high performance liquid chromatography detection method of chlorantraniliprole in a solution.

Background

Chlorantraniliprole of the formula C₁₈H₁₄BrCl₂N₅O₂The insecticide is an organic compound, 20 percent of chlorantraniliprole suspending agent with the trade name of 'kangba' (English trade name of CORAGEN), and is a new generation of insecticide developed by DuPont company. The Kangwan has obtained all certificates of pesticide registration and sale application, and can be popularized and applied in a large area. Broad spectrum and high effect. At present, the pesticide is mainly used for preventing and controlling rice pests, can protect the growth of rice, and is particularly more effective to other pests with resistance to rice pesticides. The pesticide is of low toxicity level and is safe to human and livestock. The lasting period can reach more than half a month, has no residual influence on agricultural products, and has good mixing performance with other pesticides.

The analysis method of chlorantraniliprole mainly comprises High Performance Liquid Chromatography (HPLC), liquid chromatography-mass spectrometry or tandem mass spectrometry (LC-MS or UPLC-MS/MS). Wangchun et al summarized liquid chromatography of chlorantraniliprole (Wangchun et al, evaluation on analytical method of chlorantraniliprole [ J ], pesticide, 2012,51 (12): 933-; caochow and the like respectively use a liquid chromatography tandem mass spectrometry to research the detection method of chlorantraniliprole (Caochow and the like, ultra-performance liquid chromatography tandem mass spectrometry/mass spectrometry to determine the residual [ J ] of chlorantraniliprole and tebufenozide in vegetables and fruits, analysis and detection, 2018,21(37) 117-. In the daily production and experimental process, the conventional high performance liquid chromatography is only used for detecting the purity of the finished chlorantraniliprole or the residual amount of the chlorantraniliprole on the surface of food, but cannot detect the content of an intermediate generated in the process of preparing the chlorantraniliprole and cannot monitor the reaction degree of preparing the chlorantraniliprole in real time.

Disclosure of Invention

The invention aims to provide a high performance liquid chromatography detection method for chlorantraniliprole in a solution, and aims to solve the technical problem that the reaction degree of each step in the production process of the chlorantraniliprole is difficult to effectively, simply and conveniently monitor in real time.

In order to achieve the purpose, the invention adopts the following technical scheme:

a high performance liquid chromatography detection method for chlorantraniliprole in a solution comprises the step of using an aqueous acetonitrile solution with a volume ratio of acetonitrile to water of 7:3 as a mobile phase.

The principle and the beneficial effects of the technical scheme are as follows: by adopting the chromatographic conditions of the technical scheme, qualitative or quantitative detection of chlorantraniliprole and intermediate products thereof can be realized, and the technical problem that the reaction degree of all intermediate processes for producing chlorantraniliprole cannot be monitored in real time under the same high performance liquid chromatography condition in the prior art is solved. Generally, when monitoring each reaction link in the production process, we need to continuously adjust the chromatographic conditions to ensure that each reaction step is monitored in real time, which results in a complex operation process. After the technical scheme is adopted, each reaction process can be monitored by adopting the same chromatographic condition, so that the complexity of operation is greatly reduced. In conclusion, the technical scheme avoids the need of modifying the chromatographic conditions for multiple times and balancing the sample injection system for multiple times under different chromatographic conditions, and adopts a uniform detection method, thereby not only providing more convenience for the synthesis process, but also reducing the workload of analysis and detection and improving the detection efficiency.

At the beginning of the research and development of the technical scheme, the inventor uses a standard sample of chlorantraniliprole and a standard sample of an intermediate product to explore chromatographic conditions according to the conventional means in the prior art, and finds that the chromatographic conditions are determined by adopting acetonitrile: water: glacial acetic acid 1: the mobile phase of 0.02 can obtain the optimal detection effect, and the peak pattern of the obtained HPLC spectrum is ideal. However, when qualitative detection of a small sample of chlorantraniliprole in a laboratory is actually carried out, the conditions are found to be not suitable, and a peak inversion is generated in an HPLC (high performance liquid chromatography) spectrum, so that a detection result is greatly deviated from a true value. The inventors further analyzed the cause: the chlorantraniliprole laboratory sample is 2,3, 6-trichloropyridine, hydrazine hydrate, diethyl maleate in cupric chloride, Cu-Ag/gamma-Al₂O₃And catalyst C, wherein the main impurities are acyl chloride unoxidized byproducts and the like, and the components are complex, so that the use of general chromatographic conditions is difficultTo accurately detect. In addition, during qualitative detection, a liquid sample is directly mixed with methanol and diluted by a mobile phase, so that the interference of impurities on a target substance is large. The inventor finds that the mobile phase is controlled to be acetonitrile through a large amount of experimental research: and 3, water is 7:3, and no acid substances are added, so that the optimal detection result can be obtained under the chromatographic conditions. The technical scheme overcomes the technical bias, explores and obtains a detection method suitable for chlorantraniliprole and intermediate samples thereof, and creates conditions for improving the production efficiency. The scheme can also monitor the reaction process in real time in the process research and development stage, thereby helping research and development to determine the optimal process conditions.

Further, the flow rate of the mobile phase was 1 mL/min.

Further, the high performance liquid chromatography conditions also included the use of a column of Wondasil C18-WR.

Further, the column temperature of the chromatographic column is 37-38 ℃.

Further, the HPLC conditions included that the absorption wavelength was set to 230.40-279.06nm and the amount of sample was 20. mu.l.

By adopting the high performance liquid chromatography conditions, the chlorantraniliprole and the intermediate thereof can be quickly and accurately detected, the obtained HPLC (high performance liquid chromatography) spectrum has an ideal peak pattern and a small absorption peak of impurities, and a result with a small deviation value and a value closer to a true value can be obtained no matter quantitative or qualitative detection is carried out on a target substance. The method is suitable for detecting chlorantraniliprole and the intermediate thereof, and has high sensitivity and quick detection. By adopting the method, the reaction process is monitored in real time in the process research and development stage, so that the research and development of screening process conditions are facilitated, and the research and development are further facilitated to determine the optimal process conditions.

Further, a high performance liquid chromatography detection method of chlorantraniliprole in a solution is used for detecting chlorantraniliprole, the intermediate I or the intermediate II in a sample to be detected; the intermediate I is 1- (3-chloro-2-pyridyl) -3-pyrazolidinone-5-ethyl formate, and the intermediate II is 3-bromo-1- (3-chloro-2-pyridyl) -4, 5-dihydro-1H-pyrazole-5-carboxylic acid.

By using the technical scheme, more accurate qualitative or quantitative detection can be performed on chlorantraniliprole, the intermediate I or the intermediate II in the sample to be detected. The sample to be detected comprises a chlorantraniliprole standard sample, an intermediate first standard sample, an intermediate second standard sample, a chlorantraniliprole laboratory sample, an intermediate first laboratory sample and an intermediate second laboratory sample.

Further, the sample to be detected is pretreated, and then high performance liquid chromatography detection is carried out to obtain an HPLC (high performance liquid chromatography) map.

The method comprises the steps of pretreating a sample to be detected to enable all components in the sample to be uniform, and then carrying out high performance liquid chromatography detection, so that the obtained detection result can be ensured to be more accurate. And during qualitative detection, calculating the peak area ratio of chlorantraniliprole, the intermediate I or the intermediate II in the sample to be detected according to the HPLC (high performance liquid chromatography) map. When quantitative detection is carried out, a standard curve needs to be drawn by using a standard substance of a target substance, and a linear relation between the substance concentration and the peak area is established. Then, the HPLC chromatogram is obtained by performing high performance liquid chromatography detection on the sample to be detected (unknown sample). And calculating to obtain the exact content of the target substance in the sample to be detected according to the peak area and the standard curve of the target substance in the HPLC chromatogram. Both qualitative and quantitative detection are conventional means of the prior art. The qualitative detection can quickly acquire the component information in the sample, can be used for preliminarily judging the reaction progress degree, and is a detection means frequently used in the production process.

Further, the pretreatment method comprises the steps of dissolving a sample to be detected in methanol to obtain a sample-methanol mixed solution; the sample-methanol mixture was then dispersed in a dilute solution comprising a 7:3 volume ratio of acetonitrile to water in acetonitrile in water and containing 1 volume percent glacial acetic acid.

Further, 1ml of a sample to be measured was dissolved in 1ml of methanol, and then made up to 25ml using a diluting solution.

When qualitative detection is performed on a laboratory sample, the laboratory sample needs to be dispersed in methanol, then the sample needs to be further diluted by using a dilution solution, and finally the sample is loaded in an HPLC device. Wherein the laboratory samples comprise chlorantraniliprole laboratory samples, intermediate one laboratory samples and intermediate two laboratory samples. The dilution solution is used for preparing the sample to be detected, and is very important for realizing the effect of the technical scheme. The experimental results show that if glacial acetic acid is not added or other acids are added to the diluted solution during this step, poor detection results, such as poor reproducibility, abnormal peak pattern, etc., may result.

Further, the pretreatment method comprises the steps of drying a sample to be detected to obtain a small sample solid, and diluting to 50ml with a diluted solution every 10mg of the small sample solid, wherein the diluted solution comprises acetonitrile and water, the acetonitrile and water are in a volume ratio of 7:3, and glacial acetic acid with the volume percentage of 1% is contained.

When quantitative detection is carried out on a laboratory sample, the laboratory sample needs to be dried to be called dry powder substance (sample solid), and then 10mg of sample solid is made to be 50ml by using a diluted solution containing glacial acetic acid so as to realize accurate measurement on the content of a target substance. Wherein the laboratory samples comprise chlorantraniliprole laboratory samples, intermediate one laboratory samples and intermediate two laboratory samples.

Drawings

FIG. 1 is an HPLC chromatogram of test 1 of example 1.

FIG. 2 is an HPLC chromatogram of test 2 of example 1.

FIG. 3 is an HPLC chromatogram of test 3 of example 1.

FIG. 4 is an HPLC chromatogram of test 4 of example 1.

FIG. 5 is an HPLC chromatogram of test 5 of example 1.

FIG. 6 is an HPLC chromatogram of test 6 of example 1.

FIG. 7 is an HPLC chromatogram of test 7 of example 1.

FIG. 8 is an HPLC chromatogram of test 8 of example 1.

Figure 9 is an HPLC profile (chlorantraniliprole peak) of test 9 of example 1.

FIG. 10 is an HPLC profile of test 9 of example 1 (intermediate one peak).

FIG. 11 is an HPLC profile of test 9 of example 1 (intermediate two peaks).

FIG. 12 is a chlorantraniliprole standard curve of example 2.

FIG. 13 is an HPLC chromatogram of chlorantraniliprole standard of example 2 (standard sample 1, retention time: 6.452 min).

FIG. 14 is an HPLC chromatogram of chlorantraniliprole standard of example 2 (standard sample 2, retention time: 6.463 min).

FIG. 15 is an HPLC chromatogram of a chlorantraniliprole sample of example 2 (sample 1, retention time: 6.468 min).

Fig. 16 is an enlarged view of fig. 15.

FIG. 17 is an HPLC chromatogram of a standard of the intermediate of example 2 (standard sample 1, retention time: 2.359 min).

FIG. 18 is an HPLC chromatogram of a standard of the intermediate of example 2 (standard sample 2, retention time: 2.351 min).

FIG. 19 is an HPLC chromatogram of a small sample of the intermediate of example 2 (small sample 1, retention time: 2.353 min).

FIG. 20 is an HPLC chromatogram of a small sample of the intermediate of example 2 (small sample 2, retention time: 2.359 min).

FIG. 21 is an HPLC chromatogram of the intermediate two standards of example 2 (standard sample 1, retention time: 3.716 min).

FIG. 22 is an HPLC chromatogram of the intermediate two standards of example 2 (standard sample 2, retention time: 3.713 min).

FIG. 23 is an HPLC chromatogram of a second small sample of the intermediate of example 2 (small sample 1, retention time: 3.716 min).

FIG. 24 is an HPLC chromatogram of a second small sample of the intermediate of example 2 (small sample 2, retention time: 3.717 min).

FIG. 25 is an HPLC chromatogram of chlorantraniliprole standard of example 3 (standard sample 1, retention time: 5.482 min).

FIG. 26 is an HPLC chromatogram of chlorantraniliprole standard of example 3 (standard sample 2, retention time: 5.459 min).

FIG. 27 is an HPLC chromatogram of a chlorantraniliprole sample of example 3 (sample 1, retention time: 5.489 min).

FIG. 28 is an HPLC chromatogram of a chlorantraniliprole small sample of example 3 (small sample 2, retention time: 5.486 min).

FIG. 29 is an HPLC chromatogram (retention time: 3.312min) of a standard sample of the intermediate of example 3.

FIG. 30 is an HPLC chromatogram of a small sample of the intermediate of example 3 (retention time: 3.334 min).

FIG. 31 is an HPLC chromatogram of a second intermediate standard of example 3 (retention time: 2.904 min).

FIG. 32 is an HPLC chromatogram of a small sample of the intermediate of example 3 (retention time: 3.016 min).

Detailed Description

The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following examples and experimental examples are conventional means well known to those skilled in the art, and the materials, reagents and the like used therein are commercially available.

Example 1: examination of test conditions

In this example, Shimadzu high performance liquid chromatograph was used for detection, and chlorantraniliprole standard samples were used for exploring the detection conditions. Chlorantraniliprole standard samples are purchased from Bian medicine, and the content is 95%. Before the detection, 10mg of chlorantraniliprole standard sample is weighed, dissolved in a diluted solution (the composition of which is the same as that of the corresponding mobile phase of each test below) to a constant volume in a 50ml volumetric flask, ultrasonically treated by a hot water bath at 80-90 ℃ until the solution is dissolved, and cooled to room temperature to obtain a sample to be tested for each test in the embodiment.

Test 1:

a detection instrument: shimadzu high performance liquid chromatograph; a chromatographic column: wondasil C18-WR (4.6X 150mm,5 μm); mobile phase: methanol/ultrapure water (volume ratio: 3:1) at a flow rate of 1 mL/min; column temperature 38 ℃, absorption wavelength: 270 nm; sample introduction amount: 20 μ l. Under the condition, referring to fig. 1, the chromatogram of the chlorantraniliprole standard sample shows that a plurality of small miscellaneous peaks appear in the chromatogram of the standard sample, the peak area percentage of the main peak is only 67%, and the peak appearance effect is poor.

And (3) testing 2:

a detection instrument: shimadzu high performance liquid chromatograph; a chromatographic column: wondasil C18-WR (4.6X 150mm,5 μm); mobile phase: methanol/ultrapure water (3:1), flow rate 1mL/min, column temperature 38 ℃, absorption wavelength: 254nm, sample size: 20 μ l. Under the condition, the chromatogram of the chlorantraniliprole standard sample is shown in figure 2, under the condition, two large peaks appear in the chromatogram of the chlorantraniliprole standard sample, the peak area percentages respectively account for 45% and 40%, and meanwhile, a plurality of small miscellaneous peaks also appear, and the peak appearance effect is poor.

And (3) testing:

a detection instrument: shimadzu high performance liquid chromatograph; a chromatographic column: wondasil C18-WR (4.6X 150mm,5 μm); mobile phase: pure acetonitrile, flow rate 1mL/min, column temperature 38 ℃, absorption wavelength: 254nm, sample size: 20 μ l. Under the condition, the chromatogram of the chlorantraniliprole standard sample is shown in figure 3, and under the condition, the chlorantraniliprole peak emergence time is too early, the absorption is weak, and the peak emergence effect is poor.

And (4) testing:

a detection instrument: shimadzu high performance liquid chromatograph; a chromatographic column: wondasil C18-WR (4.6X 150mm,5 μm); mobile phase: pure acetonitrile, flow rate 1mL/min, column temperature 38 ℃, absorption wavelength: 270nm, sample size: 20 μ l. Under the condition, the chromatogram of the chlorantraniliprole standard sample is shown in figure 4, under the condition, the chlorantraniliprole peak emergence time is too early, the absorption is weaker than that before (test 3), and the peak emergence effect is poor.

And (5) testing:

a detection instrument: shimadzu high performance liquid chromatograph; a chromatographic column: wondasil C18-WR (4.6X 150mm,5 μm); mobile phase: acetonitrile/water (9:1), flow rate 1mL/min, column temperature 38 ℃, absorption wavelength: 254nm, sample size: 20 μ l. Under the condition, the chromatogram of the chlorantraniliprole standard sample is shown in figure 5, and when the standard sample is under the condition, the peak inversion occurs in the chromatogram of the standard sample.

And 6, testing:

a detection instrument: shimadzu high performance liquid chromatograph; a chromatographic column: wondasil C18-WR (4.6X 150mm,5 μm); mobile phase: acetonitrile/water (3:1), flow rate 1mL/min, column temperature 38 ℃, absorption wavelength: 254nm, sample size: 20 μ l. Under these conditions, the chromatogram of the chlorantraniliprole standard is shown in fig. 6 (fig. 6 is an enlarged view of fig. 6), and the standard also shows a peak inversion after 1min under the mobile phase.

And 7, testing:

a detection instrument: shimadzu high performance liquid chromatograph; a chromatographic column: wondasil C18-WR (4.6X 150mm,5 μm); mobile phase: acetonitrile/water (1:1), flow rate 1mL/min, column temperature 38 ℃, absorption wavelength: 254nm, sample size: 20 μ l. Under the condition, the chromatogram of the chlorantraniliprole standard sample is shown in figure 7, and at the moment, the chromatogram of the standard sample has no inverted peak, good absorption and good peak shape.

And (4) testing 8:

a detection instrument: shimadzu high performance liquid chromatograph; a chromatographic column: wondasil C18-WR (4.6X 150mm,5 μm); mobile phase: acetonitrile/water (3:2), flow rate 1mL/min, column temperature 38 ℃, absorption wavelength: 254nm, sample size: 20 μ l. Under the condition, the chromatogram of the chlorantraniliprole standard sample is shown in figure 8 (the enlarged image on figure 8 is shown in figure 8), under the condition, a plurality of small miscellaneous peaks appear in the chromatogram of the standard sample, the absorption effect is not good before, the peak area ratio is about 90 percent, and a reversed peak also appears in the chromatogram, so that the condition is not suitable.

And (3) testing:

the absorption wavelength was studied in this test using chlorantraniliprole standards, ethyl 1- (3-chloro-2-pyridyl) -3-pyrazolidinone-5-carboxylate (intermediate one) standards, and 3-bromo-1- (3-chloro-2-pyridyl) -4, 5-dihydro-1H-pyrazole-5-carboxylic acid (intermediate two) standards. The method for preparing the sample to be detected by the intermediate I and the intermediate II is the same as that of the chlorantraniliprole standard sample. The detecting instrument of this test: shimadzu high performance liquid chromatograph; a chromatographic column: wondasil C18-WR (4.6X 150mm,5 μm); mobile phase: acetonitrile/water (1:1), flow rate 1mL/min, column temperature 38 ℃, sample size: 20 μ l. Clicking the spectral scan at the time of the peak emergence of chlorantraniliprole, stopping the pump, and obtaining a spectral scan chart as shown in FIG. 9, a spectral scan chart at the time of the peak emergence of the intermediate 1- (3-chloro-2-pyridyl) -3-pyrazolidone-5-carboxylic acid ethyl ester as shown in FIG. 10, and a spectral scan chart at the time of the peak emergence of the intermediate di-3-bromo-1- (3-chloro-2-pyridyl) -4, 5-dihydro-1H-pyrazole-5-carboxylic acid as shown in FIG. 11. The above 254nm absorption wavelength is selected by comparing the peak effect of each wavelength after multiple spectral scans of chlorantraniliprole and intermediates and multiple manual changes of the wavelength of the detector, and finally determining the result.

Example 2

The detection conditions used in this example were: a detection instrument: shimadzu high performance liquid chromatograph; a chromatographic column: wondasil C18-WR (4.6X 150mm,5 μm); mobile phase: acetonitrile/ultrapure water/glacial acetic acid (1L/1L/20mL) (i.e. weakly acidic mobile phase is prepared, volume percentage of glacial acetic acid is 1%), flow rate is 1mL/min, column temperature is 38 ℃, absorption wavelength: 254nm, sample size: 20 μ l. Glacial acetic acid is used to prevent peak tailing and inhibit microbial proliferation in the test substance.

Making a standard curve to quantify a target substance, wherein the specific process is as follows:

accurately weighing 1.5mg, 1.9mg, 2.8mg, 4.5mg and 8.5mg of chlorantraniliprole standard substances, fixing the volume to 50mL by using a mobile phase, carrying out ultrasonic treatment for 20-25min, preparing standard solutions with the concentrations of 0.03g/L, 0.038g/L, 0.056g/L, 0.09g/L and 0.17g/L respectively, determining according to the above liquid chromatography analysis conditions, drawing a standard curve by using the mass concentration (x) of the standard solutions to the peak area (y), and carrying out quantitative calculation on unknown samples. The linear range of the chlorantraniliprole standard curve was then determined. The external standard method of peak area is adopted for quantification, the mass concentration of chlorantraniliprole has good linear relation with the peak area within the range of 0.03-0.17g/L, the linear regression equation is that y is 36719738.49x +22226.4, and the correlation coefficient R is²See fig. 12 for details, 1.00.

The liquid chromatography detection conditions are also suitable for detecting important intermediates of chlorantraniliprole, namely 1- (3-chloro-2-pyridyl) -3-pyrazolidone-5-ethyl formate (intermediate I) and 3-bromo-1- (3-chloro-2-pyridyl) -4, 5-dihydro-1H-pyrazole-5-carboxylic acid (intermediate II). The standard curves for intermediate one and intermediate two can be prepared according to the above method, and the unknown sample can be quantified.

ChlorantraniliproleThe processing of standards, intermediate one standards and intermediate two standards is described in example 1, i.e., the standards are dissolved using a mobile phase. The processing method of the chlorantraniliprole laboratory sample, the intermediate first laboratory sample and the intermediate second laboratory sample (in the document, the three are all called samples to be detected) comprises the following steps: taking 1ml of a small sample, adding 1ml of methanol for dilution, and shaking up; then using a diluted solution (the ratio of the diluted solution to the mobile phase of the test is equal to acetonitrile/ultrapure water/glacial acetic acid (1L/1L/20ml)) to carry out constant volume to 25ml, putting the diluted solution into an ultrasonic cleaner for ultrasonic treatment for 5min, and obtaining a sample to be loaded, wherein the treatment method corresponds to qualitative detection; weighing 10mg of dried small sample solid (liquid small sample needs to be dried), metering to 50ml of volumetric flask by using a mobile phase, putting into an ultrasonic cleaner added with 80-90 ℃ hot water, carrying out ultrasonic treatment until the small sample is dissolved, taking out, cooling to room temperature, and carrying out detection to obtain a sample to be tested, wherein the treatment method corresponds to quantitative detection. Wherein, the chlorantraniliprole laboratory sample is 2,3, 6-trichloropyridine, hydrazine hydrate, diethyl maleate, cupric chloride, Cu-Ag/gamma-Al₂O₃And catalyst C, wherein the main impurities are acyl chloride unoxidized byproducts and the like, and the components are complex, so that the detection is difficult to accurately detect by using general chromatographic conditions. The intermediate, a laboratory sample, is 2,3, 6-trichloropyridine, hydrazine hydrate in cupric chloride and Cu-Ag/gamma-Al₂O₃Is formed by the synthesis reaction, wherein the main impurities are addition acyclic byproducts, hydrolysis byproducts, cyclization byproducts and the like. The intermediate II laboratory sample is formed by the synthesis reaction of the intermediate I and diethyl maleate under the catalytic action of the catalyst C, wherein the main impurities are tribromooxyphosphorus hydrolysis byproducts and the like.

The following experimental results are sample processing methods using qualitative assays. The chromatographic conditions of the embodiment are adopted to detect the chlorantraniliprole standard sample, and the HPLC chromatogram is shown in figure 13 and figure 14; the chlorantraniliprole laboratory sample is detected, and the HPLC chromatogram is shown in figure 15 and figure 16. In example 1, we found that acetonitrile: in the case of water at 1:1, the detection result obtained is more desirable. However, in the laboratory sample test, the liquid phase detection result is not ideal and the peak inversion occurs due to the influence of some impurities. Detecting the intermediate I standard sample, and obtaining HPLC chromatogram with reference to figure 17 and figure 18; detecting the laboratory sample of the intermediate I, and obtaining HPLC (high performance liquid chromatography) spectra with reference to figures 19 and 20; detecting the intermediate two standard samples, and obtaining HPLC (high performance liquid chromatography) spectra as shown in figures 21 and 22; the laboratory sample of intermediate two was examined and the HPLC profile is shown in FIGS. 23 and 24.

The experimental results show that the chromatographic condition is suitable for detecting the intermediate in the production process of chlorantraniliprole, has the characteristics of high sensitivity and quick detection, but has an undesirable detection effect on chlorantraniliprole, and needs to be further optimized on HPLC detection conditions. In the prior art, the standard sample of the object to be detected is usually used for searching the optimal HPLC detection condition, but the three laboratory samples have complicated impurity components, and the detection condition searched by using the standard sample is not applicable, so that the detection condition specially aiming at the three laboratory samples needs to be searched and optimized.

Example 3

The detection conditions used in this example were: a detection instrument: shimadzu high performance liquid chromatograph; a chromatographic column: wondasil C18-WR (4.6X 150mm,5 μm); mobile phase: acetonitrile/ultrapure water (7L/3L), flow rate 1mL/min, column temperature 38 ℃, absorption wavelength: 254nm, sample size: 20 μ l. The chromatographic conditions are used for detecting a chlorantraniliprole standard sample, an intermediate first standard sample and an intermediate second standard sample, as well as a chlorantraniliprole laboratory sample, an intermediate first laboratory sample and an intermediate second laboratory sample. The processing conditions of the three standards are referred to in example 1, except that the volume is not directly determined by using the solvent composed of the mobile phase, but by using the diluted solution (which comprises acetonitrile and ultrapure water in a volume ratio of 7:3 and contains glacial acetic acid in a volume percentage of 1%) with different compositions. For the processing conditions of the three laboratory samples, the qualitative detection sample processing mode in example 2 is referred to, except that the volume is not directly determined by using the solvent with the composition as the mobile phase, but is determined by using the dilution solution with different compositions (comprising acetonitrile and ultrapure water in a volume ratio of 7:3, and containing glacial acetic acid with the volume percentage of 1%). And after the pretreatment is finished, loading the column for detection.

The chromatographic conditions of the embodiment are adopted to detect the chlorantraniliprole standard sample, and the HPLC chromatogram is shown in figure 25 and figure 26; the chlorantraniliprole laboratory sample is detected, and the HPLC chromatogram is shown in figure 27 and figure 28. Detecting the intermediate I standard sample, and obtaining an HPLC map as shown in figure 29; detecting the laboratory sample of the intermediate I, and obtaining an HPLC chromatogram shown in figure 30; detecting the intermediate two standard samples, and obtaining an HPLC chromatogram shown in figure 31; the laboratory sample of intermediate two was examined and the HPLC profile is shown in FIG. 32. Therefore, the chromatographic condition adopting the technical scheme is not only suitable for quantitative or qualitative detection of chlorantraniliprole, but also suitable for quantitative or qualitative detection of two intermediates. In the experimental operation, the detection conditions of HPLC of different substances often have great difference, but in the technical scheme, the inventors surprisingly found that effective detection of the three substances (standard sample and laboratory sample) can be realized through the same HPLC conditions, the operation process of content detection of products and intermediate products in the synthesis process can be simplified, and unexpected technical effects are obtained. The method is suitable for detecting chlorantraniliprole and the intermediate thereof, and has the characteristics of high sensitivity and quick detection. By adopting the method, the reaction process is monitored in real time in the process research and development stage, so that the research and development of screening process conditions are facilitated, and the research and development are further facilitated to determine the optimal process conditions.

Examples of the experiments

To further study the efficacy of the HPLC assay of this protocol, we performed repeated qualitative assays on laboratory samples of chlorantraniliprole (the same sample was divided into multiple aliquots for testing in this example) to determine the effectiveness and stability of the assay. The specific implementation process comprises the following steps: the samples were subjected to pretreatment for dilution with methanol (see the small sample treatment method of qualitative detection in example 2), and then added with solutions under the following conditions for qualitative detection, and then the qualitative detection was performed by repeating the steps for 6 times under 1 condition, the time for each sample test was fixed at 15min, and the peak area ratios of the small samples were recorded, respectively, and the experimental results are shown in table 1. The specific detection conditions for each test were as follows:

the chromatographic conditions for test 1 were:

a detection instrument: shimadzu high performance liquid chromatograph; a chromatographic column: wondasil C18-WR (4.6X 150mm,5 μm); mobile phase: acetonitrile/ultrapure water (7L/3L), flow rate 1mL/min, column temperature 38 ℃, absorption wavelength: 254nm, sample size: 20 μ l. When preparing a sample to be tested, a sample to which methanol has been added is diluted with a diluting solution (comprising acetonitrile and ultrapure water in a volume ratio of 7:3 and containing 1% by volume of glacial acetic acid), see sample treatment method of chlorantraniliprole laboratory hand sample of example 3. Namely, 1ml of a small sample is taken and added with 1ml of methanol for dilution and is shaken up; the diluted solution of the test is used to fix the volume to 25ml and put into an ultrasonic cleaner for ultrasonic treatment for 5 min.

The chromatographic conditions for test 2 were:

a detection instrument: shimadzu high performance liquid chromatograph; a chromatographic column: wondasil C18-WR (4.6X 150mm,5 μm); mobile phase: acetonitrile/ultrapure water/glacial acetic acid (1L/20 mL) (i.e. 1% acetic acid adjusted pH), flow rate 1mL/min, column temperature 38 ℃, absorption wavelength: 254nm, sample size: 20 μ l. When preparing the sample to be tested, the composition of the diluted solution is the same as the mobile phase under the same chromatographic conditions, as seen in the sample treatment method of the chlorantraniliprole laboratory sample of example 2.

The chromatographic conditions for test 3 were:

a detection instrument: shimadzu high performance liquid chromatograph; a chromatographic column: wondasil C18-WR (4.6X 150mm,5 μm); mobile phase: acetonitrile/ultrapure water (7L/3L), flow rate 1mL/min, column temperature 38 ℃, absorption wavelength: 254nm, sample size: 20 μ l. When preparing a sample to be detected, taking 1ml of a small sample, adding 1ml of methanol for dilution, and shaking up; then using the diluted solution which is composed of the same mobile phase to fix the volume to 25ml, and putting the solution into an ultrasonic cleaner for ultrasonic treatment for 5 min. I.e. differs from test 1 in that glacial acetic acid was not added when the swatches were pretreated.

The chromatographic conditions for test 4 were:

a detection instrument: shimadzu high performance liquid chromatograph; a chromatographic column: wondasil C18-WR (4.6X 150mm,5 μm); mobile phase: acetonitrile/ultrapure water (7L/3L), flow rate 1mL/min, column temperature 38 ℃, absorption wavelength: 254nm, sample size: 20 μ l. When preparing the sample to be tested, the procedure is the same as in test 1 except that the same amount of formic acid is used instead of glacial acetic acid.

The chromatographic conditions for test 5 were:

the chromatographic conditions of test 1 were adjusted by adding 1% by volume of glacial acetic acid to the mobile phase. The method of preparing the sample to be tested is the same as test 1.

The chromatographic conditions for test 6 were:

the chromatographic conditions of test 1 were adjusted by adding 1% by volume of formic acid to the mobile phase.

The chromatographic conditions for test 7 were:

a detection instrument: shimadzu high performance liquid chromatograph; a chromatographic column: wondasil C18-WR (4.6X 150mm,5 μm); mobile phase: acetonitrile/ultrapure water (7L/3L), flow rate 1mL/min, column temperature 38 ℃, absorption wavelength: 254nm, sample size: 20 μ l. When preparing the sample to be tested, the procedure is the same as test 1 except that the same amount of phosphoric acid is used instead of glacial acetic acid.

The chromatographic conditions for test 8 were:

the chromatographic conditions of test 1 were adjusted by adding 1% by volume of phosphoric acid to the mobile phase.

Table 1: the result of the detection

Note: in the test 2, the condition appears peak inversion during the qualitative detection of the laboratory sample, and the peak inversion is not generated during the quantitative detection of the laboratory sample and the detection of the laboratory standard sample, so that the influence of undried sample can be possibly generated.

In the test 1, the HPLC chromatogram has better peak pattern, good absorption and less impurity peaks, and the CV value of the result of 6 repeated detections is small, so that the method is an ideal detection method for a laboratory sample of chlorantraniliprole.

The mobile phase of test 2 was different from that of test 1 in the ratio of acetonitrile to ultrapure water, and glacial acetic acid was added to the mobile phase. The experimental results of test 2 show that the HPLC profile has a peak inversion, which affects the peak area percentage. Although the CV value of the test 2 repeated for 6 times is small, the test result of the qualitative content of the whole chlorantraniliprole is small, the content condition of the chlorantraniliprole in a small sample cannot be accurately reflected, the detection result is inaccurate, and the actual application requirement cannot be met. Test 2 compares with test 1 to show that the ratio of acetonitrile to water in the mobile phase has a significant effect on the detection result, and the ratio of acetonitrile to water is adjusted to 7:3, which has an obvious effect on improving the detection accuracy. Generally, the occurrence of a back-off affects the integral, resulting in a difference between the area ratio and the true value, and in particular, a smaller qualitative content of chlorantraniliprole.

The HPLC conditions of test 3 are the same as those of test 1, but no glacial acetic acid is added when preparing the sample to be tested, and although the peak pattern is normal, the CV value of 6 times of repetition is larger. This indicates that glacial acetic acid needs to be added when preparing the sample to ensure the stability of the sample and improve the accuracy of the detection.

The HPLC conditions of test 4 are the same as those of test 1, but the formic acid is used for replacing glacial acetic acid when a sample is prepared, so that a 'steamed bun peak' appears in an HPLC (high performance liquid chromatography) spectrum, and the absorption of an impurity peak is large. And the CV value repeated for 6 times is larger, and the test result of the whole chlorantraniliprole qualitative content is smaller. This indicates that formic acid is not suitable for the preparation of the sample, and the addition of formic acid greatly affects the accuracy of the measurement of the target substance in the sample.

In the test 5, compared with the test 1, glacial acetic acid is added into the mobile phase, and the detection result shows that the peak pattern of the HPLC chromatogram is normal, but the repeatability is poor, and the test result of the overall qualitative content of chlorantraniliprole is small. This indicates that glacial acetic acid cannot be added to the mobile phase of HPLC, otherwise the accuracy of the assay results is greatly affected.

Test 6 formic acid was added to the mobile phase relative to test 1. The test result shows that the HPLC chromatogram has a peak inversion, and the overall testing result of the qualitative content of chlorantraniliprole is smaller. Indicating that the addition of organic acid could not be done in the mobile phase and that glacial acetic acid treatment was required when formulating the samples.

Test 7 compared to test 1, when the sample was prepared, phosphoric acid was used instead of glacial acetic acid, the HPLC chromatogram showed a "steamed bun peak", and the impurity peak absorption was very large. Indicating that phosphoric acid is not suitable for sample preparation, the addition of phosphoric acid greatly affects the accuracy of the measurement of the target substance in the sample.

Test 8 compared to test 1, when phosphoric acid was added to the mobile phase, the HPLC profile showed a peak inversion with a very large detection deviation, indicating that no acid could be added to the mobile phase.

In conclusion, the technical scheme explores and improves HPLC detection conditions. It was found that when qualitative measurements were performed on laboratory samples of chlorantraniliprole, the mobile phase needed to be adjusted to acetonitrile: water 7:3 and no acid in any form can be added in the mobile phase. In tests 2, the peak pattern is not ideal because the optimal ratio of the mobile phase components is not used, and in

tests

5, 6 and 8, various types of acid are added into the mobile phase, which can cause poor detection results, such as poor repeatability, abnormal peak pattern and the like. In the treatment of laboratory samples of chlorantraniliprole, dilute solutions containing glacial acetic acid are used. Other types of acids were used in

tests

7 and 4, and the absence of glacial acetic acid in test 3 resulted in poor assay results, such as poor reproducibility, abnormal peak patterns, etc.

The foregoing is merely an example of the present invention and common general knowledge in the art of designing and/or characterizing particular aspects and/or features is not described in any greater detail herein. It should be noted that, for those skilled in the art, without departing from the technical solution of the present invention, several variations and modifications can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicability of the patent. The scope of the claims of the present application shall be determined by the contents of the claims, and the description of the embodiments and the like in the specification shall be used to explain the contents of the claims.

Claims

1. A high performance liquid chromatography detection method of chlorantraniliprole in a solution is characterized by comprising the following steps: the high performance liquid chromatography conditions included the use of aqueous acetonitrile at a volume ratio of acetonitrile to water of 7:3 as the mobile phase.

2. The method for detecting chlorantraniliprole in solution by high performance liquid chromatography as claimed in claim 1, wherein the method comprises the following steps: the flow rate of the mobile phase was 1 mL/min.

3. The high performance liquid chromatography detection method of chlorantraniliprole in a solution as claimed in claim 2, which is characterized in that: high performance liquid chromatography conditions also included the use of a column of Wondasil C18-WR.

4. The method for detecting chlorantraniliprole in a solution according to claim 3, which comprises the following steps: the temperature of the chromatographic column is 37-38 ℃.

5. The method for detecting chlorantraniliprole in solution according to claim 4, which comprises the following steps: the HPLC conditions further include an absorption wavelength set at 230.40-279.06nm and a sample volume of 20. mu.l.

6. The method for detecting chlorantraniliprole in solution according to claim 5, which comprises the following steps: the method is used for detecting chlorantraniliprole, the intermediate I or the intermediate II in a sample to be detected; the intermediate I is 1- (3-chloro-2-pyridyl) -3-pyrazolidinone-5-ethyl formate, and the intermediate II is 3-bromo-1- (3-chloro-2-pyridyl) -4, 5-dihydro-1H-pyrazole-5-carboxylic acid.

7. The method for detecting chlorantraniliprole in solution according to claim 6, which comprises the following steps: and (3) pretreating the sample to be detected, and then carrying out high performance liquid chromatography detection to obtain an HPLC (high performance liquid chromatography) map.

8. The method for detecting chlorantraniliprole in solution according to claim 7, which comprises the following steps: the pretreatment method comprises the steps of dissolving a sample to be detected in methanol to obtain a sample-methanol mixed solution; the sample-methanol mixture was then dispersed in a dilute solution comprising a 7:3 volume ratio of acetonitrile to water in acetonitrile in water and containing 1 volume percent glacial acetic acid.

9. The method for detecting chlorantraniliprole in solution according to claim 8, which comprises the following steps: 1ml of the sample to be tested was dissolved in 1ml of methanol and then made up to 25ml using a dilution solution.

10. The method for detecting chlorantraniliprole in solution according to claim 7, which comprises the following steps: the pretreatment method comprises the steps of drying a sample to be detected to obtain a small sample solid, wherein each 10mg of the small sample solid is subjected to constant volume to 50ml by using a dilution solution, the dilution solution comprises acetonitrile and water, the acetonitrile and water are in a volume ratio of 7:3, and glacial acetic acid with the volume percentage of 1% is contained.