阅读说明：本技术 异丙甲草胺于口鼻吸入染毒系统中的浓度的测定方法 (Method for measuring concentration of metolachlor in oral-nasal inhalation toxicant exposure system ) 是由 缪峰 黄海霞 陶庭磊 张茹 秦丽玲 陈倩芸 于 2020-05-14 设计创作，主要内容包括：本发明公开了异丙甲草胺于口鼻吸入染毒系统中的浓度的测定方法。该方法的步骤包括：S1.将异丙甲草胺在口鼻吸入染毒系统下发生气溶胶,用滤膜于所述口鼻吸入染毒系统中进行采样,得到采样滤膜；使用滤膜称重法计算所述口鼻吸入染毒系统中气溶胶的实际浓度；S2.将所述采样滤膜置于溶剂中,得到采样滤膜样品溶液；S3.将所述采样滤膜样品溶液进行高效液相色谱分析,测定得到峰面积,对照异丙甲草胺峰面积-浓度标准曲线,得到所述采样滤膜样品溶液中异丙甲草胺的实际浓度；并计算得到所述口鼻吸入染毒系统中异丙甲草胺的实际浓度。本发明的方法能够准确、稳定地检测得到在染毒装置中的样品的实际浓度。(The invention discloses a method for measuring the concentration of metolachlor in an oral-nasal inhalation toxicant exposure system. The method comprises the following steps: s1, enabling metolachlor to generate aerosol under an oral-nasal inhalation contamination system, and sampling in the oral-nasal inhalation contamination system by using a filter membrane to obtain a sampling filter membrane; calculating the actual concentration of aerosol in the oral-nasal inhalation contamination system by using a filter membrane weighing method; s2, placing the sampling filter membrane in a solvent to obtain a sampling filter membrane sample solution; s3, carrying out high performance liquid chromatography analysis on the sampling filter membrane sample solution, determining to obtain a peak area, and obtaining the actual concentration of the metolachlor in the sampling filter membrane sample solution by contrasting a peak area-concentration standard curve of the metolachlor; and calculating to obtain the actual concentration of metolachlor in the oral-nasal inhalation contamination system. The method of the invention can accurately and stably detect the actual concentration of the sample in the contamination device.)

1. The method for measuring the concentration of metolachlor in an oral-nasal inhalation toxicant exposure system is characterized by comprising the following steps of:

s1, enabling metolachlor to generate aerosol under an oral-nasal inhalation contamination system, and sampling in the oral-nasal inhalation contamination system by using a filter membrane to obtain a sampling filter membrane; calculating the actual concentration of aerosol in the oral-nasal inhalation contamination system by using a filter membrane weighing method;

s2, placing the sampling filter membrane in a solvent to obtain a sampling filter membrane sample solution;

s3, carrying out high performance liquid chromatography analysis on the sampling filter membrane sample solution, determining to obtain a peak area, and obtaining the actual concentration of the metolachlor in the sampling filter membrane sample solution by contrasting a peak area-concentration standard curve of the metolachlor; and calculating to obtain the actual concentration of metolachlor in the oral-nasal inhalation contamination system.

2. The method of claim 1 wherein the actual concentration of metolachlor in said oral-nasal inhalation toxicant exposure system is calculated by the formula:

actual concentration C of metolachlor in oral-nasal inhalation toxicant exposure system₂Actual concentration C of metolachlor in filter membrane sample solution₁X amount of solvent in the sample solution of the sampling filter ÷ (flow rate of sampling x time of sampling).

3. The method of claim 1, wherein in S1, the flow rate of the sample in the oronasal inhalation toxicant exposure system is 1-10L/min, preferably 4L/min;

and/or, in S1, the sampling time is 1-10 min, preferably 4min, in the oral-nasal inhalation contamination system;

and/or, in S1, the material of the filter membrane is glass fiber.

4. The method of determining the concentration of metolachlor in an oronasal inhalation toxicant exposure system of claim 1, wherein, in S1, the metolachlor solution is subjected to aerosol generation under the oronasal inhalation toxicant exposure system; preferably, the metolachlor is dissolved in the solvent and then stirred and mixed uniformly to obtain the metolachlor solution.

5. The method of claim 4, wherein the target concentration of metolachlor in the oral-nasal inhalation toxicant exposure system is 0.2% to 60%; the percentage is the volume percentage of the metolachlor in the metolachlor solution;

and/or the solvent is vegetable oil, preferably corn oil;

and/or the stirring time is more than 30 min.

6. The method of claim 1, wherein the theoretical concentration of aerosol in the oronasal inhalation toxicant exposure system is 800-5000 mg/m³Preferably 2000mg/m³；

And/or, the actual concentration of the aerosol is calculated by the following formula:

actual concentration of aerosol ═ (filter weight after sampling-filter weight before sampling) ÷ (sample flow × sample time);

and/or the target concentration of the metolachlor in the oral-nasal inhalation toxicant exposure system is equal to the theoretical concentration of aerosol x the target content of the metolachlor solution;

and/or the target concentration of metolachlor in the oral-nasal inhalation toxicant exposure system is 4-1200 mg/m³。

7. The method of claim 1, wherein in step S2, the sample solution of the sampling filter is diluted, filtered and analyzed by hplc.

8. The method of claim 7, wherein the concentration of metolachlor in the oral-nasal inhalation toxicant exposure system is determined,

the diluent is 60-90% methanol, preferably 75% methanol;

and/or carrying out ultrasonic treatment on the diluted solution, cooling to room temperature, shaking up and filtering;

wherein the time of the ultrasonic treatment is preferably 3-10 min, for example 5 min;

wherein, the concentration of the diluted sampling filter membrane sample solution is preferably 6.4 to 76.8 mug/mL;

wherein, preferably, the filtration is carried out by a filter head with the pore diameter of 0.22 μm; the filter head is preferably a PVDF filter head; the PVDF filter head is preferably a filter head of Millex manufacturer with a diameter of 33 mm.

9. The method of determining the concentration of metolachlor in an oronasal inhalation toxicant exposure system of claim 1, wherein in S3, the hplc method comprises:

and/or, the chromatographic column is a C18 reverse phase chromatographic column; preferably a Waters X-Bridge C18 chromatography column;

and/or, the specification of the chromatographic column is 150 mm in length multiplied by 4.6mm in inner diameter;

and/or the pore diameter of the chromatographic column is 5 μm;

and/or the elution mode is isocratic elution;

and/or the mobile phase is methanol and ultrapure water in a volume ratio of 75: 25;

and/or the detection wavelength is 210-250 nm, preferably 230 nm;

and/or the column temperature is 25-30 ℃;

and/or the temperature of the sample injector is 2-8 ℃, preferably 6 ℃;

and/or the flow rate is 0.8-1.2 mL/min, preferably 1.0 mL/min;

and/or the sample injection volume is 5-20 muL, preferably 10 muL;

and/or the running time is more than 5min, preferably more than 8 min.

10. The method of claim 1, wherein the metolachlor standard is dissolved in a solvent to prepare a plurality of metolachlor standard solutions with a plurality of concentration gradients, and the solutions are subjected to high performance liquid chromatography, wherein a linear regression equation is established by taking the peak area of the metolachlor in the metolachlor standard solution as an ordinate and the concentration of the metolachlor in the metolachlor standard solution as an abscissa, so as to obtain the standard curve of peak area-concentration of the metolachlor;

preferably, the concentration of the metolachlor standard solution is 2-100 μ g/mL, such as 2 μ g/mL, 10 μ g/mL, 20 μ g/mL, 50 μ g/mL or 100 μ g/mL.

Technical Field

The invention relates to a method for determining the concentration of metolachlor in an oral-nasal inhalation toxicant exposure system.

Background

With the development of agricultural production, pesticides are widely used because of their remarkable effects of controlling crop pests, removing weeds, increasing the yield of agricultural products, and the like. Metolachlor is a herbicide widely applied to bud amides at present. The toxicological results show that the metolachlor has slight irritation to the skin and eyes of rats after transdermal test, and has lethality after oral test. Thus, prolonged exposure to or inhalation of metolachlor pesticide may cause potential health damage. In order to better understand the health risks of metolachlor, the research on toxicity test of metolachlor is particularly important.

At present, a large number of metolachlor toxicity tests are reported at home and abroad, and the administration modes of the metolachlor toxicity tests are acute oral or transdermal toxicity tests. In the using process of metolachlor, pesticide absorption cannot be avoided. However, there is no research on the inhalation contamination of metolachlor, and the toxicity characteristics thereof are not clear, so the development of an analysis method and the development of an inhalation contamination system are urgently needed.

Studies have shown that there are two main toxicant exposure modes for inhalation toxicity studies: tracheal instillation and dynamic inhalation contamination. The tracheal instillation method is simple, special equipment is not needed, but the experimental animals need to be anesthetized before instillation, and the exposure mode of a human cannot be truly simulated. Therefore, the researchers mostly adopt dynamic inhalation toxicant exposure, which mainly includes oral-nasal inhalation toxicant exposure and systemic exposure inhalation toxicant exposure. For the research of rats, oral and nasal inhalation contamination is mostly adopted, so that the influence on the result caused by the fact that experimental animals lick contaminated substances can be prevented. However, oral-nasal inhalation contamination requires special aerosol generation and related contamination devices, and the difficulty of generating stable aerosol is high, and it is difficult to accurately determine the actual concentration of the sample in the contamination device in the prior art, and it is also difficult to know the actual exposure concentration of the sample in the contamination device of the rat. Therefore, in order to ensure the accuracy and stability of the metolachlor oral-nasal inhalation contamination test, the development of a method for measuring the metolachlor in an oral-nasal inhalation contamination system is urgent.

Disclosure of Invention

The invention provides a method for measuring the concentration of metolachlor in an oral-nasal inhalation contamination system, aiming at solving the defect that the actual concentration of a sample in the oral-nasal inhalation contamination system is difficult to detect accurately in the prior art. The method can accurately and stably detect the actual concentration of the sample in the contamination device, ensure the accuracy and stability of the drug delivery in subsequent animal experiments, and lay the foundation for the metolachlor inhalation contamination toxicity test.

The invention solves the technical problems through the following technical scheme.

The invention provides a method for measuring the concentration of metolachlor in an oral-nasal inhalation toxicant exposure system, which comprises the following steps:

s1, enabling metolachlor to generate aerosol under an oral-nasal inhalation contamination system, and sampling in the oral-nasal inhalation contamination system by using a filter membrane to obtain a sampling filter membrane; calculating the actual concentration of aerosol in the oral-nasal inhalation contamination system by using a filter membrane weighing method;

s2, placing the sampling filter membrane in a solvent to obtain a sampling filter membrane sample solution;

s3, carrying out high performance liquid chromatography analysis on the sampling filter membrane sample solution, determining to obtain a peak area, and obtaining the actual concentration of the metolachlor in the sampling filter membrane sample solution by contrasting a peak area-concentration standard curve of the metolachlor; and calculating to obtain the actual concentration of metolachlor in the oral-nasal inhalation contamination system.

The filter membrane weighing method is a basic method for measuring the mass concentration of particulate matters, samples at a specified flow rate, traps the particulate matters in the air on a high-performance filter membrane, weighs the mass before and after the sampling of the filter membrane, obtains the mass of trapped dust according to the mass difference, and obtains the ratio of the mass of the trapped dust to the amount of sampled air, namely the mass concentration of the dust.

In the invention, a filter membrane weighing method is adopted, sampling is carried out for a certain time according to a certain sampling flow to obtain a sampling filter membrane, the exposure contamination process of a rat in an oral-nasal inhalation contamination system is simulated, and the actual concentration (C) of metolachlor in a sampling filter membrane sample solution can be obtained by calculation after the measurement of high performance liquid chromatography analysis₁) The actual concentration of metolachlor in the oronasal inhalation toxicant exposure system (C) can then be calculated by the following formula₂)：

Actual concentration C of metolachlor in oral-nasal inhalation toxicant exposure system₂Actual concentration C of metolachlor in filter membrane sample solution₁X amount of solvent in the sample solution of the sampling filter ÷ (flow rate of sampling x time of sampling).

Wherein the amount of the solvent in the sampling filter sample solution is the amount (in volume unit) of the solvent in the step S2; the sampling flow and the sampling time are the sampling flow and the sampling time of a filter membrane weighing method. Actual concentration C of metolachlor in oral-nasal inhalation toxicant exposure system obtained based on calculation₂It is known that the actual exposure concentration of metolachlor is found when rats are inhaled into the toxicant exposure system through mouth and nose.

At S1, the oronasal inhalation toxicant exposure system may be conventional in the art, also known as an animal oronasal inhalation exposure system, such as a single concentration oronasal inhalation toxicant exposure system. The person skilled in the art knows that the theoretical concentration (C) of the aerosol in the oronasal inhalation contamination system can be set by adjusting the instrument parameters in the oronasal inhalation contamination system₃). Theoretical concentration (C) of the aerosol₃) Preferably 800 to 5000mg/m³More preferably 2000mg/m³。

In the filter membrane weighing method, the actual concentration (C) of the aerosol can be calculated according to the mass of the filter membrane before sampling and the mass of the filter membrane after sampling₄) Can be used for verifying the set theoretical concentration C of the aerosol₃Whether it is stable and qualified (C)₄Is required to be at C₃Within. + -. 10%, if this condition is satisfied, the gas can be dissolved according to the set gasTheoretical concentration C of the glue₃Proceed to the next operation). Actual concentration of confirmed aerosol (C)₄) The qualified sampling filter membrane can be used for measuring the concentration of the metolachlor in a mouth and nose inhalation contamination system. In particular, the actual concentration (C) of the aerosol₄) The calculation can be made according to the following formula:

actual concentration C of the aerosol₄(mg/m³) (post-sampling filter weight-pre-sampling filter weight) ÷ (sample flow x sample time).

In S1, in the oral-nasal inhalation contamination system, the sampling flow rate is preferably 1-10L/min, such as 4L/min.

In S1, in the oral-nasal inhalation contamination system, the sampling time is preferably 1-10 min, for example, 4 min.

In S1, the material of the filter membrane is preferably glass fiber.

In S1, preferably, the metolachlor solution is aerosolized under the oronasal inhalation toxicant exposure system. The preparation process of the metolachlor solution can be conventional in the field, and the metolachlor solution is generally dissolved in a solvent and then uniformly stirred.

The concentration of the metolachlor solution needs to ensure the accuracy of the concentration of a drug administration preparation (namely the accuracy of the concentration of generated aerosol), so that the concentration of a subsequent sampling filter membrane after passing through the aerosol is ensured. Preferably, the target content (C) of the metolachlor solution₆) 0.2% -60%; the percentage is the volume percentage of the metolachlor in the metolachlor solution.

Wherein, the solvent is preferably vegetable oil, more preferably corn oil.

Wherein the stirring time is preferably 30min or more.

At S1, when the actual concentration of aerosol C₄And the theoretical concentration C of aerosol₃When the error therebetween is within an acceptable range (C)₄Is required to be at C₃Within ± 10%) of the target concentration of metolachlor (C) in the oronasal inhalation toxicant exposure system₅) (it can also be considered that rats are exposed to oronasal inhalationThe target exposure concentration of metolachlor when in an infected system) can be calculated by the following formula:

target concentration C of metolachlor in oral-nasal inhalation toxicant exposure system₅(mg/m³) Theoretical concentration of aerosol C₃Target content C of x metolachlor solution₆。

Wherein the target concentration of metolachlor (C) in the oronasal inhalation toxicant exposure system₅) Preferably 4 to 1200mg/m³。

The actual concentration C of the metolachlor in the sample solution of the sampling filter membrane is obtained by the detection method₁The actual concentration C of metolachlor in the oral-nasal inhalation contamination system can be obtained₂. And the actual concentration C of the metolachlor in the sampling filter membrane sample solution can be determined₁And the theoretical concentration C of metolachlor in the sampling filter membrane sample solution₇And comparing to verify the feasibility and accuracy of the detection method. Theoretical concentration C of metolachlor in filter membrane sample solution₇The calculation can be made by:

theoretical concentration C of metolachlor in filter membrane sample solution₇Actual concentration of aerosol C₄Target content C of x metolachlor solution₆Content of metolachlor raw drug is multiplied by (100-metolachlor raw drug moisture)%.

In the formula, the content of the raw metolachlor is the mass percentage content of the metolachlor in the medicine; the raw material water of metolachlor refers to the mass percentage of water in the medicine.

In S2, the sampling filter may be placed in a container, preferably a reagent container conventional in the art, such as a reagent bottle; the solvent is added after the sampling filter membrane is placed in the container.

In S2, the sampling filter membrane sample solution is generally diluted and filtered, and then subjected to high performance liquid chromatography.

Wherein, the diluted diluent is preferably 60% to 90% methanol, for example 75% methanol. The dilution operation may be performed one or more times.

Wherein, preferably, the diluted solution is subjected to ultrasonic treatment, cooled to room temperature, shaken up and filtered.

The time of the ultrasonic treatment is preferably 3-10 min, for example, 5 min.

Wherein the concentration of the diluted sampling filter membrane sample solution is preferably 6.4-76.8. mu.g/mL.

Wherein the filtration operations and conditions may be conventional in the art. Preferably, the filtration is carried out by using a filter head with the pore diameter of 0.22 μm; the filter head is for example a PVDF filter head; the PVDF filter head is for example a filter head with a diameter of 33mm, manufactured by Millex. The amount of the initial filtrate to be discarded is generally 1 to 5mL, for example, 1 mL.

In S3, preferably, the High Performance Liquid Chromatography (HPLC) method may be conventional in the art.

Wherein, the chromatographic column is preferably a C18 reversed phase chromatographic column; more preferably, the reverse phase chromatography column has a specification of 150 × 4.6mm (length L × inner diameter ID), 5 μm; even more preferably, the reverse phase chromatography column is a Waters X-Bridge C18 chromatography column.

Among them, the elution is preferably performed by isocratic elution.

Wherein the mobile phase is preferably methanol to ultrapure water in a volume ratio of 75: 25.

The detection wavelength is preferably 210-250 nm, such as 230 nm.

Wherein the column temperature is preferably 25 to 30 ℃.

Wherein the sample injector temperature is preferably 2-8 ℃, for example 6 ℃.

Wherein, the flow rate is preferably 0.8-1.2 mL/min, such as 1.0 mL/min.

Wherein the injection volume is preferably 5-20 μ L, such as 10 μ L.

Wherein the operation time is preferably 5min or more, more preferably 8min or more.

In S3, the metolachlor peak area-concentration standard curve can be determined by the conventional method in the field. Generally, the metolachlor standard is dissolved in a proper amount of solvent to prepare a plurality of metolachlor standards with a certain concentration gradientRespectively carrying out high performance liquid chromatography analysis on the sample solutions, and establishing a linear regression equation by using the peak area (Y) of the metolachlor measured in the metolachlor standard solution and the corresponding concentration (X). As known to those skilled in the art, the actual concentration C of metolachlor in the sampling filter membrane sample solution can be obtained by comparing the peak area calculated after the high performance liquid chromatography analysis of the sampling filter membrane sample solution with the peak area-concentration standard curve of metolachlor₁And calculating to obtain C₂. If the dilution treatment is performed before the high performance liquid chromatography analysis, the dilution factor is multiplied (the dilution factor is the ratio of the concentration of the preparation to the concentration of the diluted sample).

Wherein, the metolachlor standard solution is preferably prepared by dissolving a metolachlor standard in a proper amount of methanol.

Wherein, the concentration of the metolachlor standard solution is preferably 2-100 μ g/mL, such as 2 μ g/mL, 10 μ g/mL, 20 μ g/mL, 50 μ g/mL or 100 μ g/mL.

On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.

The reagents and starting materials used in the present invention are commercially available.

The positive progress effects of the invention are as follows:

the method can accurately and stably detect the actual concentration of the sample in the contamination device, ensure the accuracy and stability of the drug delivery in subsequent animal experiments, and lay the foundation for the metolachlor inhalation contamination toxicity test.

The invention reduces the influence of auxiliary materials on sample detection, ensures the feasibility of an oral-nasal inhalation contamination system, can accurately and efficiently detect the concentration of the preparation in a toxicological inhalation drug delivery test, and has the advantages of lower cost, convenient experiment operation, high accuracy, good repeatability, objective result judgment and high sensitivity; has very important significance for the toxicity test research of the pesticide.

Drawings

FIG. 1 is a chromatogram of a diluent alone (75% methanol) excluding the interfering effect of the diluent (75% methanol) on the measurement of a sample.

FIG. 2 is a chromatogram of only the solvent (corn oil) excluding the interfering effect of the solvent on the measurement of the sample.

FIG. 3 is a chromatogram of a solvent sampling filter, which is used for eliminating the interference of the sampling filter and the inhalation contamination system on the tested substance.

Detailed Description

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.

In the following examples, the "test substance" refers to a sample of metolachlor, which contains 98.4% of metolachlor and 0.1% of water (the percentages are mass percentages); "vehicle" means corn oil.

Example 1: establishment of standard curve for detecting metolachlor by HPLC method

(1) Preparing a standard curve solution (hereinafter referred to as a standard curve solution): precisely transferring 25 mu L of metolachlor standard product (with the purity of 98.543%) into a 25mL volumetric flask, precisely weighing the weight of the transferred volume of metolachlor, adding a certain amount of methanol for dissolving and diluting to a scale to prepare a standard yeast solution stock solution with the concentration of the metolachlor of 0.1% (1.0mg/mL), and diluting into a series of standard yeast solutions with different concentrations of the metolachlor according to the table 1.

Table 1 standard dilution preparation protocol

(2) Determination of Standard Curve

The HPLC analysis method is as follows: using a C18 reverse phase chromatography column (specification 150 × 4.6mm (L × ID), 5 μm, Waters X-Bridge C18 column), purified water in methanol: the ultrapure water is eluted at the same flow rate as 75:25, the detection wavelength is 230nm, the column temperature is 30 ℃, the injector temperature is 6 ℃, the flow rate is 1.0mL/min, the injection volume is 10 mu L, and the running time is 8 min.

The results of the linear regression equation established by taking the peak area (Y) of the metolachlor measured in the standard solution as the ordinate and the corresponding concentration (X) as the abscissa after the samples with various concentrations are measured once respectively, are shown in the table 2, and the metolachlor has good linearity in the concentration range of 2ppm to 100ppm (2 mu g/mL to 100 mu g/mL), and the range is determined to be the detection range of the method.

TABLE 2 results of standard curves

Example 2: preparation of metolachlor administration preparation and verification analysis

(1) Preparation of metolachlor suspension preparation

Proportionally transferring or measuring the required amount of test substance (in which metolachlor is used as test sample, content is 98.4%, and water content is 0.1%) and solvent, stirring for at least 30min, and mixing. The prepared administration preparations are marked by labels with different colors, the solvent chromatogram is shown in figure 2, and the preparation ratios of the administration preparations are shown in table 3.

TABLE 3 formulation of the drug delivery formulations

(2) Drug delivery formulation sample handling

Transferring the proper amount of the high-concentration and low-concentration administration preparation in the step (1) into a volumetric flask with proper specification, adding a proper amount of 75% methanol (the chromatogram of only 75% methanol is shown in figure 1), performing ultrasonic treatment for 5min, cooling to room temperature, fixing the volume to scale, and shaking up. High concentrations still require a second dilution step. And (3) discarding 1mL of primary filtrate by using a filter head with the diameter of 0.22 mu m for the sample solution after shaking up to obtain a sample solution for HPLC analysis, and processing 6 parts in parallel. The sample concentrations after dilution are as in table 4:

TABLE 4 preparation of samples to be tested

(3) Detecting the sample to be detected prepared in the step (2) according to the HPLC detection method in the embodiment 1, respectively measuring corresponding peak areas, substituting the peak areas into a regression equation for calculation to obtain corresponding concentrations, multiplying the corresponding concentrations by a dilution factor, wherein the dilution factor of the low-concentration sample is 100, the dilution factor of the high-concentration sample is 10000, the concentration of the metolachlor suspension preparation can be obtained, and the acceptance standard is that the average recovery rate is 85-115% and the RSD is within 10%. Specific data are shown in table 5, resulting in within-batch precision and accuracy of the administered formulations within acceptable ranges:

TABLE 5 results of precision and accuracy in test substance administration preparation batches

In table 5:(x_iresults of content measurement for six parallel groups of samples);

average recovery (%) — content mean/theoretical content × 100%;

theoretical content of the administered formulation C₈Target content of the administered formulation C%)₆X content of original drug of test substance x (100-moisture of original drug of test substance)%.

(4) The stability of the metolachlor administration preparation was measured according to the above steps (1), (2) and (3), and the results are shown in Table 6, with the acceptance criterion RD within 5% and the recovery rate within 85% -115%. The result proves that the metolachlor administration preparation is stable within 24 hours at room temperature and 8 days at 2-8 ℃.

TABLE 6 stability results of test substance formulations

In table 6:(X₁for the first measurement of a parallel sample, X₂Second measurement of a parallel sample).

Example 3: metolachlor inhalation toxicant aerosol sampling method and verification analysis thereof

(1) Preparation of metolachlor suspension formulations the same as in example 2

(2) Collection of inhaled contaminated aerosol sampling filter membrane

The divided administration preparation is prepared according to the determined concentration parameters (aerosol theoretical concentration C)₃＝2000mg/m³) Aerosol occurs under a single concentration oronasal inhalation toxicant exposure system. After the system reaches the set value and is stable, sampling is carried out for 4min by using a filter membrane weighing method (the filter membrane is made of glass fiber materials) according to the sampling flow of 4L/min, the aerosol concentration is measured, after the measured value is qualified (the measured value needs to be +/-10% of the set value), the filter membrane is put into a proper container and is marked by using labels with different colors, the chromatogram of the solvent sampling filter membrane is shown in figure 3, and the set parameters and the target concentration are shown in the following table 7:

actual concentration of Aerosol C₄(mg/m³) (post-sampling filter weight-pre-sampling filter weight) ÷ (sample flow x sample time).

Among them, in Table 7, the target concentration C of metolachlor in the oral-nasal inhalation toxicant exposure system₅Theoretical concentration of aerosol C₃Target content C of x metolachlor solution₆。

TABLE 7 inhalation toxicant Aerosol parameter settings

(3) Treatment of filter membrane sample for sampling inhaled contaminated aerosol

Adding a certain amount of 75% methanol into a container equipped with a filter membrane, and performing ultrasonic treatment for 5 min. Cooled to room temperature and shaken up. And if the second step of dilution exists, transferring a proper amount of the first step solution into a volumetric flask with a proper specification, adding 75% methanol for dilution until the volume is constant to a scale, and shaking up to obtain the product. And filtering the finally diluted and uniformly mixed sample solution by using a filter head with the diameter of 0.22 mu m, and discarding 1mL of primary filtrate to obtain the sample solution for HPLC analysis. Wherein, the mass of the sample in the sampling filter membrane is equal to the target concentration C of metolachlor in the oral-nasal inhalation contamination system₅(sample exposure concentration) × sampling flow rate × sampling time. The dilution factor is shown in Table 8. For a sampling filter membrane of a low-concentration administration preparation, 10mL of 75% methanol is added in total to obtain a diluted sample to be sampled in the filter membrane; for the high concentration drug delivery formulation, the sample was sampled from the filter after dilution by adding 250mL of 75% methanol.

TABLE 8 sample dilution of the sampling Filter

(4) Detecting the sample to be detected prepared in the step (3) according to the HPLC detection method in the embodiment 1, respectively measuring corresponding peak areas, substituting the peak areas into a regression equation for calculation, and obtaining the actual concentration C of the metolachlor in the corresponding sampling filter membrane₁Multiplying the amount of the solvent in the sampling filter membrane sample solution by the amount of the solvent in the sampling filter membrane sample solution of the low-concentration sample, wherein the amount of the solvent in the sampling filter membrane sample solution of the high-concentration sample is 250mL, obtaining the mass of the metolachlor in the sampling filter membrane, dividing the mass by the sampling flow of 4L/min and dividing the sampling flow by the sampling time of 4min, and obtaining the actual concentration C of the metolachlor in an oral-nasal inhalation contamination system₂. The acceptance criteria are that the average recovery rate is 70-130% and the RSD is within 10%. Specific data are shown in table 9, resulting in within-batch precision and accuracy of the administered formulations within acceptable ranges:

TABLE 9 test substance dosing formulation sampling Filter Membrane in-batch precision and accuracy results

The theoretical concentration of the metolachlor in the sampling filter membrane (namely the theoretical concentration C of the metolachlor in the oral-nasal inhalation contamination system)₇)(mg/m³) Actual concentration of aerosol C₄X theoretical content of the administered formulation C₈. Wherein the theoretical content C of the preparation to be administered₈Target content of the formulation C₆X content of original drug of test substance x (100-moisture of original drug of test substance)%.

(5) The stability of the metolachlor inhalation contamination sampling filter membrane was determined according to the above steps (1), (2), (3) and (4), and the results are shown in Table 10, with the acceptance criteria of recovery rate within 70% -130% and RD within 5%. The result proves that the metolachlor is stable in 2 days to 8 ℃ within 24 hours at room temperature after being inhaled into the contamination sampling filter membrane.

TABLE 10 test substance dosing formulation sampling Filter stability results

The above-described specific embodiments are merely preferred embodiments of the present invention, and it should be noted that, for those skilled in the art, various modifications or substitutions can be made without departing from the principle of the present invention, and these modifications or substitutions should also be regarded as the protection scope of the present invention.