阅读说明：本技术 一种复配农药制剂中丙硫菌唑的检测方法 (Method for detecting prothioconazole in compound pesticide preparation ) 是由 宋薇 付仁敬 于 2021-01-08 设计创作，主要内容包括：本发明公开了一种复配农药制剂中丙硫菌唑的检测方法。基于丙硫菌唑分子特性以及在酸性条件下抑制丙硫菌唑与百菌清组分发生反应,从而能准确检测出丙硫菌唑质量分数。本发明在检测复配农药制剂中丙硫菌唑质量分数的过程中,操作简单,检测专一性好,灵敏度高,响应速度快,结果灵敏可靠,能够实现实时在线的快速和特异性检测。本发明在农药检测分析领域具有良好的应用前景和潜在应用价值。(The invention discloses a method for detecting prothioconazole in a compound pesticide preparation. Based on the molecular characteristics of prothioconazole and the inhibition of the reaction of prothioconazole and chlorothalonil components under acidic conditions, the mass fraction of prothioconazole can be accurately detected. In the process of detecting the mass fraction of the prothioconazole in the compound pesticide preparation, the method is simple to operate, has the advantages of good detection specificity, high sensitivity, high response speed and sensitive and reliable result, and can realize real-time online rapid and specific detection. The invention has good application prospect and potential application value in the field of pesticide detection and analysis.)

1. A detection method of prothioconazole in a compound pesticide preparation is characterized by comprising the following detection steps: the sample is dissolved by acetonitrile and glacial acetic acid, acetonitrile and water are used as mobile phases, the pH value is adjusted by phosphoric acid, and the high performance liquid chromatography separation and the measurement are carried out on prothioconazole and chlorothalonil in the sample at the wavelength of 254nm by using a stainless steel column with C18 packing and a variable wavelength ultraviolet detector.

2. The method for detecting prothioconazole in compound pesticide preparation according to claim 1, wherein the liquid chromatography conditions are as follows: mobile phase: ψ (acetonitrile: water) = 90: 10, pH = 3; flow rate: 0.8 mL/min; column temperature: the room temperature is plus or minus 2 ℃; detection wavelength: 254 nm; sample introduction volume: 5 muL; retention time: chlorothalonil for about 4.5min, prothioconazole for about 4.7 min; the above-mentioned operating parameters are typical and can be suitably adjusted for the given operating parameters according to different instrument characteristics in order to obtain the best results.

Technical Field

The invention relates to the technical field of pesticide quality detection, and particularly relates to a method for detecting prothioconazole.

Background

Prothioconazole is a novel broad-spectrum triazolethione bactericide developed by Bayer companies, is mainly used for preventing and treating a plurality of diseases of cereals, wheat and bean crops and the like, has low toxicity, no teratogenicity, no mutation type, no toxicity to embryos and safety to people and environment. The mechanism of action is to inhibit the demethylation of lanosterol, the precursor of sterol, or at position 14 of 2, 4-methylenedihydrolanostane in fungi.

Prothioconazole is mainly used for controlling a plurality of diseases of cereal crops such as wheat, barley, rape, peanut, rice, bean crops and the like. Has good control effect on almost all wheat diseases, such as powdery mildew, banded sclerotial blight, leaf spot, rust disease, sclerotinia, net blotch, leaf spot and the like of wheat and barley. Can also prevent and control soil-borne diseases of the rape and the peanut, such as sclerotinia, and main leaf surface diseases, such as gray mold, black spot, brown spot, black shank, sclerotinia, rust and the like. The dosage is usually 200g (a.i.)/hm 2, and the activity is better than or equal to that of the conventional bactericides such as epoxiconazole, tebuconazole, cyprodinil and the like at the dosage.

In order to prevent the occurrence of resistance and adapt to the requirements of special crops and control of different diseases, Bayer companies are developing and registering prothioconazole single agents and mixed preparations of the prothioconazole single agents and agents with different action mechanisms, and the prothioconazole single agents and the mixed preparations can be compounded with tebuconazole, trifloxystrobin, spiroxamine and the like besides the bactericide fluoxastrobin.

The conventional method for detecting the mass fraction of the prothioconazole mainly comprises a high performance liquid chromatography, a liquid tandem mass spectrometry and the like, the method has the advantages of high detection sensitivity, good selectivity and accurate and reliable result in the process of detecting the single-component preparation of the prothioconazole, but the two components are found to interfere with each other in the process of detecting the compound preparation of the prothioconazole and chlorothalonil, so that the development of a simple, quick, sensitive and reliable detection method is particularly important for detecting the mass fraction of the prothioconazole in the compound pesticide preparation.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide a method for detecting prothioconazole, and solves the problem that the existing method cannot detect the mass fraction of prothioconazole in a pesticide compound preparation.

In order to achieve the purpose, the invention adopts the following technical scheme: a method for detecting prothioconazole in a compound pesticide preparation comprises the following steps:

1. summary of the process

The sample is dissolved by acetonitrile and glacial acetic acid mixed solution (1000 mL of beaker, 500mL of acetonitrile is measured by using 500mL of measuring cylinder and poured into the beaker, 80mL of glacial acetic acid is measured by using 100mL of measuring cylinder and poured into the beaker, the mixture is stirred and mixed evenly, the solution is hereinafter referred to as acetonitrile and glacial acetic acid (500 mL of acetonitrile and 80mL of glacial acetic acid) solution), acetonitrile and water are used as mobile phase, the pH value is adjusted by phosphoric acid, and the prothioconazole and chlorothalonil in the sample are separated and measured by high performance liquid chromatography under the wavelength of 254nm by using a stainless steel column filled with C18 and a variable wavelength ultraviolet detector.

2. Instrument for measuring the position of a moving object

High performance liquid chromatograph: having an ultraviolet variable wavelength detector; a chromatographic data processor or workstation; a chromatographic column: a 250mm 4.6mm (i.d.) stainless steel column filled with C18 filler and having a particle size of 5 μm (or a chromatographic column with the same effect); a filter: the aperture of the filter membrane is about 0.45 mu m; a micro feeder: 50 muL; quantitative sampling tube: 5 muL; an ultrasonic cleaner.

3. Operating conditions of liquid chromatography

Mobile phase: ψ (acetonitrile: water) = 90: 10, pH = 3; flow rate: 0.8 mL/min; column temperature: the room temperature is plus or minus 2 ℃; detection wavelength: 254 nm; sample introduction volume: 5 muL; retention time: chlorothalonil for about 4.5min, prothioconazole for about 4.7 min;

the above-mentioned operating parameters are typical and can be suitably adjusted for the given operating parameters according to different instrument characteristics in order to obtain the best results.

4. Measurement procedure

4.1 preparation of Standard sample solution

Accurately weighing 0.02g (accurately to 0.00001 g) of prothioconazole and chlorothalonil standard samples, placing the prothioconazole and chlorothalonil standard samples into a 100mL brown volumetric flask, dissolving the prothioconazole and chlorothalonil standard samples by using acetonitrile and glacial acetic acid (500 mL acetonitrile and 80mL glacial acetic acid) solution, fixing the volume, shaking up the solution and filtering the solution for later use.

4.2 preparation of sample solution

Accurately weighing a sample containing 0.02g (accurately to 0.00001 g) of prothioconazole and chlorothalonil samples, placing the sample in a 100mL brown volumetric flask, dissolving the sample by using acetonitrile + glacial acetic acid (500 mL of acetonitrile +80mL of glacial acetic acid) solution, fixing the volume, shaking up, and filtering for later use.

4.3 determination

Under the above operating conditions, after the instrument has stabilized, a number of needles of the sample solution are continuously injected. And when the relative change of the peak areas of the standard samples of the two adjacent needles is less than 1.5%, measuring according to the sequence of the standard sample solution, the sample solution and the standard sample solution.

4.4 calculation of

And respectively averaging the peak areas of prothioconazole and chlorothalonil in the two-needle sample solution and the two-needle sample solution before and after the sample. The mass fractions X1 (%) of prothioconazole and chlorothalonil were calculated according to formula (1):

A2•m1•P

X1= ———————…………………………………（1）

A1•m2

in the formula:

a1-average value of peak areas of prothioconazole and chlorothalonil in standard sample solution;

a2 is the average value of the peak areas of prothioconazole and chlorothalonil in the sample solution;

m 1-prothioconazole, chlorothalonil standard sample mass, g;

m 2-sample mass, g;

p-mass fraction of prothioconazole and chlorothalonil in the standard sample.

5.5 tolerance difference

The relative deviation of the results of two replicates should be no more than 1.5%.

Compared with the prior art, the invention has the following beneficial effects:

the invention provides a method for detecting the mass fraction of prothioconazole in a solution to be detected based on chlorothalonil and prothioconazole compounded pesticide preparation. The prothioconazole in the detection method has a maximum absorption peak at 254nm, and glacial acetic acid added in the sample preparation process can effectively inhibit the reaction of chlorothalonil and prothioconazole, so that the influence of other components on prothioconazole is avoided, the detection method has strong anti-interference capability, sensitive and reliable result, simple operation and good detection specificity and stability, and can accurately calculate the mass fraction of the prothioconazole in the detected sample by using an external standard method and be used for detecting the mass fraction of the prothioconazole in a pesticide compound preparation.

Drawings

FIG. 1 is a graph showing the peak area of prothioconazole as a function of the concentration of glacial acetic acid

Note: FIG. 1 shows the abscissa of the graph, which represents the amount (mL) of glacial acetic acid added to the mixed solution; the ordinate is the peak area value of the prothioconazole component in the sample.

FIG. 2 is a high performance liquid chromatogram of standard samples of prothioconazole, chlorothalonil and azoxystrobin

FIG. 3 is a high performance liquid chromatogram of prothioconazole, chlorothalonil and azoxystrobin samples

FIG. 4 shows the peak purity spectrum of the specific standard sample confirmed by the prothioconazole method

FIG. 5 spectrum of peak purity of specific sample confirmed by prothioconazole method

Note: the peak of the 1-azoxystrobin standard sample, the peak of the 2-chlorothalonil standard sample and the peak of the 3-prothioconazole standard sample in the figures 2 and 3.

Detailed Description

Example one

1) 10 parts of prothioconazole + chlorothalonil + sample 0.1g (to the nearest 0.0001 g) are weighed precisely and placed in a 100mL brown volumetric flask, using 10 acetonitrile solutions of different glacial acetic acid concentrations: dissolving the glacial acetic acid solution, fixing the volume, shaking up, and filtering for later use.

The preparation method of the mixed solution comprises the following steps: (10 of 1000mL beakers are taken, ten parts of 500mL acetonitrile are measured by using a 500mL measuring cylinder and respectively poured into 10 beakers, 10mL of glacial acetic acid, 20mL of glacial acetic acid, 30mL of glacial acetic acid, 40mL of glacial acetic acid, 50mL of glacial acetic acid, 60mL of glacial acetic acid, 70mL of glacial acetic acid, 80mL of glacial acetic acid, 90mL of glacial acetic acid and 100mL of glacial acetic acid are respectively measured by using a 100mL measuring cylinder and respectively poured into 10 beakers, stirred and uniformly mixed to prepare 10 parts of mixed solutions with different glacial acetic acid concentrations)

2) Operating conditions of liquid chromatography

Mobile phase: ψ (acetonitrile: water) = 90: 10, pH = 3;

flow rate: 0.8 mL/min;

column temperature: the room temperature is plus or minus 2 ℃;

detection wavelength: 254 nm;

sample introduction volume: 5 muL;

under the above operating conditions, after the instrument has stabilized, a number of needles of the sample solution are continuously injected. Until the relative change of the peak areas of two adjacent needle standard samples is less than 1.5%, collecting the peak areas of prothioconazole component peaks in a chromatogram, and then adding acetonitrile: the amount of glacial acetic acid added to the glacial acetic acid solution is plotted on the abscissa, and the peak area of the prothioconazole component peak in the sample is plotted on the ordinate, as shown in fig. 1.

3) The concentration of glacial acetic acid in the sample dissolution solution mainly acts to inhibit the mutual reaction of prothioconazole and chlorothalonil in the liquid dispersion system, and as can be seen from the graph of fig. 1, when the glacial acetic acid is added to acetonitrile: when glacial acetic acid (500 mL acetonitrile +80mL glacial acetic acid), the peak area of the prothioconazole component in the sample does not increase any more, which indicates that the glacial acetic acid in the mixed solution at the mixture concentration completely inhibits the prothioconazole and chlorothalonil from reacting with each other, so acetonitrile is preferred: the stoichiometric concentration of glacial acetic acid (500 mL acetonitrile +80mL glacial acetic acid) is the optimum concentration to inhibit the interaction between prothioconazole and chlorothalonil.

Example two

1) Accurately weighing 20mg (accurately to 0.00001 g) of prothioconazole, chlorothalonil and azoxystrobin standard samples, placing the prothioconazole, chlorothalonil and azoxystrobin standard samples into a 100mL brown volumetric flask, dissolving the prothioconazole and the azoxystrobin standard samples by using acetonitrile and glacial acetic acid (500 mL acetonitrile and 80mL glacial acetic acid) to a constant volume, shaking up the solution and filtering the solution for later use.

2) Accurately weighing a sample containing 0.4g (accurate to 0.0001 g) of the prothioconazole + chlorothalonil + azoxystrobin pesticide compound preparation (5% +45% + 5.7%), placing the sample in a 100mL brown volumetric flask, dissolving the sample with acetonitrile + glacial acetic acid (500 mL acetonitrile +80mL glacial acetic acid), fixing the volume, shaking up, and filtering for later use.

3) Chromatographic conditions are as follows:

mobile phase: ψ (acetonitrile: water) = 90: 10, pH = 3;

flow rate: 0.8 mL/min;

column temperature: the room temperature is plus or minus 2 ℃;

detection wavelength: 254 nm;

sample introduction volume: 5 muL;

4) under the above operating conditions, after the instrument has stabilized, a number of needles of the sample solution are continuously injected. And when the relative change of the peak areas of the standard samples of the two adjacent needles is less than 1.5%, measuring according to the sequence of the standard sample solution, the sample solution and the standard sample solution.

5) Computing

And respectively averaging the peak areas of prothioconazole and chlorothalonil in the two-needle sample solution and the two-needle sample solution before and after the sample. Mass fractions of prothioconazole and chlorothalonilX ₁ (%), calculated according to formula (1):

A ₂ ·m ₁ ·P

X ₁ = ——————— ………………………………（1）

A ₁ ·m ₂

A ₁ -average value of peak areas of prothioconazole and chlorothalonil in the standard sample solution;

A ₂ —the average value of the peak areas of prothioconazole and chlorothalonil in the sample solution;

m ₁ -prothioconazole, chlorothalonil standard sample mass, g;

m ₂ —sample mass, g;

Pmass fractions of prothioconazole and chlorothalonil in the standard sample.

Through calculation: the content of prothioconazole in the sample is as follows: 5.3 percent; the chlorothalonil content is 5.7%; the method is consistent with the initial feeding amount, and the detection result of the detection method is proved to be accurate and effective. The chromatogram is shown in figures 2-3

EXAMPLE III

Specificity of Prothioconazole detection method

1) By adopting the detection conditions in the second embodiment, the single detection wavelength is adjusted to the wavelength scanning range (190-. The spectrum of the peak purity of the standard sample and the sample in the specific spectrogram of the method for measuring the content of effective components is shown in figures 4-5.

2) Note: according to the DAD detector data acquisition and processing principle, peak matching automatically calculates the corresponding threshold value according to the signal-to-noise ratio of each peak, spectra are taken at the rising stage and the falling stage of each peak and at the top point, fitting is carried out, and the final calculated threshold value is generally a group of average threshold values of the same 5 spectra within the calculated threshold value limit value. The threshold is based on the absorbance height of each spectrum and a set of noise spectra at the beginning of the data file. Peak matching: is the average purity value of those spectra in the recorded spectra within the calculated threshold limits, and the peak match value is obtained by calculating the entire spectrum and the average spectrum at the selected 4-peak spectrum. Therefore, if the peak matching value is greater than the matching threshold, the target peak is considered to be a pure chromatographic peak.

Finally, the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting, although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all of them should be covered in the claims of the present invention.