CN111855835A - Method for detecting pyriminobac-methyl and related substances thereof - Google Patents

Method for detecting pyriminobac-methyl and related substances thereof Download PDF

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CN111855835A
CN111855835A CN202010535719.0A CN202010535719A CN111855835A CN 111855835 A CN111855835 A CN 111855835A CN 202010535719 A CN202010535719 A CN 202010535719A CN 111855835 A CN111855835 A CN 111855835A
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mobile phase
pyriminobac
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methyl
related substances
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刘涛
徐海
张中涛
姜娜
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Hefei Leaf Biotech Co ltd
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    • 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
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Abstract

The invention discloses a method for detecting pyriminobac-methyl and related substances thereof, which adopts high performance liquid chromatography for detection, wherein the conditions of the high performance liquid chromatography are as follows: the chromatographic column is an octadecylsilane chemically bonded silica chromatographic column, the mobile phase A is acetonitrile, the mobile phase B is water, and gradient elution is carried out, wherein the gradient elution procedure is as follows: at 0min, the proportion of the mobile phase A is 30 percent, and the proportion of the mobile phase B is 70 percent; the proportion of the mobile phase A is gradually changed from 30% to 50% within 0-10 min; the proportion of the mobile phase A is gradually changed from 50% to 70% within 10-20 min; within 20-30min, the proportion of the mobile phase A is gradually changed from 70% to 90%; within 30-40min, the proportion of the mobile phase A is gradually changed from 90% to 30%; the proportion of the mobile phase A is maintained at 30 percent within 40-45 min. The invention has accurate quantification and good repeatability, can effectively separate various spectral peaks, and can accurately control the quality of the pyriminobac-methyl.

Description

Method for detecting pyriminobac-methyl and related substances thereof
Technical Field
The invention relates to the technical field of drug detection, in particular to a method for detecting pyriminobac-methyl and related substances thereof.
Background
Pyriminobac-methyl is a systemic conductive special barnyard grass remover developed by Japan combinatorial chemistry, belongs to a pyrimidine salicylic acid herbicide, and the pyriminobac-methyl comprises 92.92% of (E) -isomer and 4.5% of (Z) -isomer, is a light yellow crystal grain in appearance, and has a chemical structural formula shown as a formula (I):
Figure BDA0002536919740000011
The ether is absorbed by stems, leaves and roots of the weeds and is rapidly transmitted to the whole plants to inhibit the biosynthesis of acetolactate synthase (ALS) and amino acid, so that the cell division in the bodies of the weeds is inhibited and hindered, the weeds stop growing, and finally the weeds are whitened and died. The pesticide can effectively prevent and kill barnyard grass and small-age moleplant seeds within a proper period of control, has specific high activity to resistant barnyard grass, and is used from the time before the barnyard grass occurs to the 3-leaf period of 30-120ga.i./hm2The pesticide has the advantages of low dosage, no influence of rainwater on the application, good mixing property with other pesticides, long pesticide effect period of 40-60 days, high safety on direct seeding rice and transplanted rice, and safety on succeeding crops, human and livestock and environment. At present, the research on pyriminobac-methyl mainly focuses on the development and popularization of application technology, and reports about analysis methods of pyriminobac-methyl are few. Literature "high performance liquid chromatography analysis of Liushujie, Wu gong Xin, Lianmin, pyriminobac-methyl original drug [ J]The pesticide 2011,50(9): 659-66' discloses a high performance liquid chromatography detection method of pyriminobac-methyl technical, but the peak appearance of the pyriminobac-methyl is not beneficial to separating impurity peaks earlier.
Disclosure of Invention
Based on the technical problems in the background art, the invention provides a method for detecting pyriminobac-methyl and related substances thereof, and the method has the advantages of accurate quantification, good repeatability, high analysis speed, good linear relation, simple and convenient operation, effective separation of various spectral peaks, and accurate control of the quality of pyriminobac-methyl.
The invention provides a method for detecting pyriminobac-methyl and related substances thereof, which adopts high performance liquid chromatography for detection, wherein the conditions of the high performance liquid chromatography are as follows: the chromatographic column is an octadecylsilane chemically bonded silica chromatographic column, the mobile phase A is acetonitrile, the mobile phase B is water, and gradient elution is carried out, wherein the gradient elution procedure is as follows: at 0min, the proportion of the mobile phase A is 30 percent, and the proportion of the mobile phase B is 70 percent; the proportion of the mobile phase A is gradually changed from 30% to 50% within 0-10 min; the proportion of the mobile phase A is gradually changed from 50% to 70% within 10-20 min; within 20-30min, the proportion of the mobile phase A is gradually changed from 70% to 90%; within 30-40min, the proportion of the mobile phase A is gradually changed from 90% to 30%; the proportion of the mobile phase A is maintained at 30 percent within 40-45 min.
Preferably, the column has a size of 4.6X 150mm and a particle size of 5 μm.
Preferably, the column is an elette ODS 2C 18 column.
Preferably, the detector is a DAD ultraviolet detector with a detection wavelength of 252-256 nm.
Preferably, the detection wavelength may be 252, 253, 254, 255 or 256 nm.
Preferably, the detection wavelength is 254 nm.
Preferably, the flow rate is 0.55-0.65 ml/min.
Preferably, the flow rate may be 0.55, 0.56, 0.57, 0.58, 0.59, 0.6, 0.61, 0.62, 0.63, 0.64 or 0.65 ml/min.
Preferably, the flow rate is 0.6 ml/min.
Preferably, the column temperature is 35-40 ℃.
Preferably, the column temperature may be 35, 35.5, 36, 36.5, 37, 37.5, 38, 38.5, 39, 39.5 or 40 ℃.
Preferably, the column temperature is 37 ℃.
Preferably, the sample size is 1. mu.l.
The invention calculates the content of pyriminobac-methyl according to an external standard method and calculates the content of related substances according to an area normalization method.
Solution preparation:
standard solution: weighing 0.05g (accurate to 0.0002g) of pyriminobac-methyl standard substance in a 100ml volumetric flask, adding methanol to dissolve, oscillating in ultrasonic water bath for 2min to completely dissolve the sample, cooling to room temperature, then adding methanol to a constant volume, and shaking up for later use.
Sample solution: weighing 0.05g (accurate to 0.0002g) of pyriminobac-methyl sample in a 100ml volumetric flask, adding methanol for dissolving, oscillating in ultrasonic water bath for 2min to completely dissolve the sample, cooling to room temperature, then adding methanol to constant volume, and shaking up for later use.
The inventor uses an ultraviolet spectrophotometer to scan the pyriminobac-methyl standard substance at the full wavelength of 190-400nm, the ultraviolet spectrogram is shown in figure 1, and the maximum absorption wavelength of the pyriminobac-methyl can be seen from figure 1 to be 254nm, so that 254nm is finally selected as the detection wavelength.
The inventor finally determines the chromatographic conditions through multiple proportioning tests, so that each chromatographic peak has sharp and symmetrical shape, moderate retention time and good separation degree, and meets the requirements of simplicity, convenience and quickness, and a typical spectrum is shown in figure 2 and table 1.
TABLE 1 chromatographic peak separation
Figure BDA0002536919740000031
Figure BDA0002536919740000041
As can be seen from Table 1, each chromatographic peak has sharp and symmetrical shape, moderate retention time and good resolution of each chromatographic peak.
The inventor takes the pyriminobac-methyl standard to prepare a series of linear solutions with the concentrations of 0.2mg/ml, 0.4mg/ml, 0.6mg/ml, 0.8mg/ml and 1mg/ml, draws a standard curve by taking the concentration as an abscissa and the peak area of the pyriminobac-methyl as an ordinate, and finds that the linear regression method is that y is 3.125x +0.819 and r is 0.9998, and can see that the chromatographic condition is good in linear relation within the concentration range of 0.2-1mg/ml and r is more than 0.999.
The inventor continuously samples the same sample solution for 6 times, and the average content of the pyriminobac-methyl is determined to be 98.57 percent, the standard deviation is 0.21 percent, which shows that the precision of the chromatographic condition is higher.
The inventors used the standard recovery method to determine the accuracy of the chromatographic conditions, and the results are shown in table 2:
TABLE 2 accuracy measurement results
Figure BDA0002536919740000042
Figure BDA0002536919740000051
As can be seen from the above table, the average recovery of pyriminobac-methyl was determined to be 100.2%, indicating that the method of the present invention is highly accurate.
The inventor screens proper chromatographic conditions, so that the method has the characteristics of accurate quantification, good repeatability, high analysis speed, good linear relation, simple and convenient operation, effective separation of various chromatographic peaks and the like. Can correctly guide the industrial production in the actual production process, accurately control the product quality, and is a more ideal method for analyzing the pyriminobac-methyl.
Drawings
FIG. 1 is a UV spectrum of pyriminobac-methyl.
FIG. 2 is a chromatogram of the sample solution of example 1.
FIG. 3 is a chromatogram of the sample solution in the comparative example.
Detailed Description
The technical solution of the present invention will be described in detail below with reference to specific examples.
Example 1
A method for detecting pyriminobac-methyl and related substances thereof adopts Agilent 1260 high performance liquid chromatograph for detection, wherein the conditions of the high performance liquid chromatograph are as follows: the chromatographic column is an elette ODS 2C 18 chromatographic column (4.6X 150mm, 5 μm), the mobile phase A is acetonitrile, the mobile phase B is water, and gradient elution is carried out, wherein the gradient elution procedure is as follows: at 0min, the proportion of the mobile phase A is 30 percent, and the proportion of the mobile phase B is 70 percent; the proportion of the mobile phase A is gradually changed from 30% to 50% within 0-10 min; the proportion of the mobile phase A is gradually changed from 50% to 70% within 10-20 min; within 20-30min, the proportion of the mobile phase A is gradually changed from 70% to 90%; within 30-40min, the proportion of the mobile phase A is gradually changed from 90% to 30%; the proportion of the mobile phase A is maintained at 30 percent within 40-45 min;
the detector is a DAD ultraviolet detector, the detection wavelength is 254nm, the flow rate is 0.6ml/min, the column temperature is 37 ℃, the column incubator is AUTO ScienCE AT-330, and the sample injection amount is 1 mul.
Solution preparation:
standard solution: weighing 0.05g (accurate to 0.0002g) of pyriminobac-methyl standard substance in a 100ml volumetric flask by using a METTLER TOLEDO-ME104E ten-thousandth electronic balance, adding methanol to dissolve the pyriminobac-methyl standard substance, oscillating the pyriminobac-methyl standard substance in an ultrasonic water bath of a KQ-250B type ultrasonic cleaner for 2min to completely dissolve a sample, cooling the sample to room temperature, metering the volume by using methanol, and shaking the solution uniformly for later use.
Sample solution: weighing pyriminobac sample 0.05g (accurate to 0.0002g) in a 100ml volumetric flask by a METTLER TOLEDO-ME104E ten thousandth electronic balance, adding methanol to dissolve, oscillating in an ultrasonic water bath of a KQ-250B type ultrasonic cleaner for 2min to completely dissolve the sample, cooling to room temperature, adding methanol to a constant volume, and shaking uniformly for later use.
The operation method comprises the following steps: setting instrument parameters according to the chromatographic conditions, taking a standard solution for continuous sample injection for several times after the baseline of the instrument is stable, calculating the repeatability of each response value, performing sample injection according to the sequence of the standard solution, the sample solution and the standard solution when the response value of adjacent 2 times is changed to be less than 1.0%, and recording a chromatogram.
The calculation method comprises the following steps:
the mass fraction X (%) of pyriminobac-methyl in the sample is A2 m 1P/A1 m 2;
in the formula: a1 is the average value of the sum of the peak areas of pyriminobac-methyl (Z) -isomer and pyriminobac-methyl (E) -isomer measured in 2 standard solutions;
A2 is the average value of the sum of the peak areas of pyriminobac-methyl (Z) -isomer and pyriminobac-methyl (E) -isomer measured in 2 sample solutions;
m1 is the mass (g) of the standard substance;
m2 represents the mass (g) of the sample;
p is the sum of the mass fractions of the pyriminobac-methyl (Z) -isomer and the pyriminobac-methyl (E) -isomer in the standard product.
The pyriminobac-methyl content in the sample was determined to be 98.53%.
A typical chromatogram is shown in figure 2.
Example 2
A method for detecting pyriminobac-methyl and related substances thereof adopts Agilent 1260 high performance liquid chromatograph for detection, wherein the conditions of the high performance liquid chromatograph are as follows: the chromatographic column is an elette ODS 2C 18 chromatographic column (4.6X 150mm, 5 μm), the mobile phase A is acetonitrile, the mobile phase B is water, and the gradient elution is carried out, wherein the gradient elution procedure is the same as that of the example 1;
the detector is a DAD ultraviolet detector, the detection wavelength is 252nm, the flow rate is 0.65ml/min, the column temperature is 35 ℃, the column incubator is AUTO ScienCE AT-330, and the sample injection amount is 1 mul.
The solution preparation, operation method and calculation method are the same as in example 1, and the pyriminobac-methyl content in the sample is measured to be 98.48%.
Example 3
A method for detecting pyriminobac-methyl and related substances thereof adopts Agilent 1260 high performance liquid chromatograph for detection, wherein the conditions of the high performance liquid chromatograph are as follows: the chromatographic column is an elette ODS 2C 18 chromatographic column (4.6X 150mm, 5 μm), the mobile phase A is acetonitrile, the mobile phase B is water, and the gradient elution is carried out, wherein the gradient elution procedure is the same as that of the example 1;
The detector is a DAD ultraviolet detector, the detection wavelength is 256nm, the flow rate is 0.55ml/min, the column temperature is 40 ℃, the column incubator is AUTO ScienCE AT-330, and the sample injection amount is 1 μ l.
The solution preparation, operation method and calculation method are the same as in example 1, and the pyriminobac-methyl content in the sample is 98.51%.
Comparative example
The pyriminobac-sodium sample in the example 1 is detected according to a high performance liquid chromatography detection method of pyriminobac-sodium original drug disclosed in the document 'Liushujie, Wugongxin, Liang-Min.pyriminobac-sodium original drug, [ J ], pesticide, 2011,50(9): 659-66';
the solution preparation and operation were the same as in example 1, and the results of the test of the sample solution are shown in FIG. 3.
Comparing fig. 2, table 1 and fig. 3, it can be seen that the number of impurities detected in the method described in the literature is small, and the respective impurities cannot be separated efficiently.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.

Claims (10)

1. The method for detecting pyriminobac-methyl and related substances thereof is characterized by adopting high performance liquid chromatography for detection, wherein the conditions of the high performance liquid chromatography are as follows: the chromatographic column is an octadecylsilane chemically bonded silica chromatographic column, the mobile phase A is acetonitrile, the mobile phase B is water, and gradient elution is carried out, wherein the gradient elution procedure is as follows: at 0min, the proportion of the mobile phase A is 30 percent, and the proportion of the mobile phase B is 70 percent; the proportion of the mobile phase A is gradually changed from 30% to 50% within 0-10 min; the proportion of the mobile phase A is gradually changed from 50% to 70% within 10-20 min; within 20-30min, the proportion of the mobile phase A is gradually changed from 70% to 90%; within 30-40min, the proportion of the mobile phase A is gradually changed from 90% to 30%; the proportion of the mobile phase A is maintained at 30 percent within 40-45 min.
2. The method for detecting pyriminobac-methyl and its related substances according to claim 1, wherein the size of the chromatographic column is 4.6 x 150mm and the particle size is 5 μm.
3. The method for detecting pyriminobac-methyl and its related substances according to claim 1 or 2, wherein the chromatographic column is an elette ODS 2C 18 chromatographic column.
4. The method for detecting pyriminobac and their related substances as claimed in any one of claims 1 to 3, wherein the detector is a DAD ultraviolet detector with a detection wavelength of 252-256 nm.
5. The method for detecting pyriminobac and their related substances according to any one of claims 1 to 4, wherein the detection wavelength is 254 nm.
6. The method of detecting pyriminobac and their related substances according to any one of claims 1 to 5, wherein the flow rate is 0.55 to 0.65 ml/min.
7. The method of detecting pyriminobac and their related substances according to any one of claims 1 to 6, wherein the flow rate is 0.6 ml/min.
8. The method for assaying pyriminobac and their related substances according to any one of claims 1 to 7, wherein the column temperature is 35 to 40 ℃.
9. The method for assaying pyriminobac and their related substances according to any one of claims 1 to 8, wherein the column temperature is 37 ℃.
10. The method for detecting pyriminobac and their related substances according to any one of claims 1 to 9, wherein the amount to be sampled is 1 μ l.
CN202010535719.0A 2020-06-12 2020-06-12 Method for detecting pyriminobac-methyl and related substances thereof Withdrawn CN111855835A (en)

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