CN114264743B - Detection method of animal-derived substrate penthiopyrad and metabolite thereof - Google Patents

Abstract

The invention discloses a method for detecting penthiopyrad and metabolites thereof in an animal-derived matrix, belonging to the technical field of pesticide residue detection, wherein hydroxyl-modified multi-walled carbon nanotubes are used as dispersed solid phase extraction purification materials to extract/purify penthiopyrad and main metabolites PAM thereof in the animal-derived matrix, and the extracted substances are measured by using an ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometer (UPLC-MS/MS) to achieve the purpose of accurately, qualitatively and quantitatively determining penthiopyrad and metabolites thereof in the animal-derived matrix, wherein the average recovery rate of the detection method is 75-106%, the Relative Standard Deviation (RSD) is 1.7-11.7%, the quantitative limit is 0.001mg/kg, and the detection limit is 0.38-0.57 mu g/kg.

Description

Detection method of animal-derived substrate penthiopyrad and metabolite thereof

Technical Field

The invention relates to the technical field of pesticide residue detection, and particularly relates to a detection method of animal-derived substrate penthiopyrad and metabolites thereof.

Background

Penthiopyrad (pentathiophyrad) is a novel amide bactericide developed by mitsui chemical company of japan, and has a different bactericidal spectrum from that of existing carboxamide bactericides. Besides being effective on basidiomycetes like the existing carboxamide fungicides, the fungicide is also effective on ascomycetes and incomplete fungi, and is recommended to be used for preventing and treating gray mold and powdery mildew which are resistant to other fungicides.

The dosage of the effective component of the medicine is 100-200g/hm ² It is widely used in fruit tree, vegetable, lawn and other crops to prevent and treat rust, sclerotinia and grey moldDiseases, downy mildew, apple scab and powdery mildew. Penthiopyrd was first approved in japan in 2008 for fruit trees, vegetables and ornamental plants, and was later approved for registration in 11 months in 2011 in canada. 3 products with the brand names of Vertisan and Fontelis (both taking penthiopyrad 200g/L as active ingredients) and Treoris (penthiopyrad 100g/L + chlorothalonil 250 g/L) are mainly used on fruit trees, vegetables and field crops, and the penthiopyrad has a wide application prospect in practical application.

With the registration, popularization and use of penthiopyrad, countries such as European Union, canada and Australia, which are main export markets of China, set the maximum allowable residual quantity (MRL) of penthiopyrad in food and agricultural products such as vegetables, fruits, grain and grains, animal products and the like, and Canada sets the MRL of penthiopyrad in animal-derived foods such as fruits, nuts, grain and grains, animal products and the like to be 0.01-30mg/kg; the MRL of penthiopyrad in animal-derived foods such as fruits, vegetables, nuts, grains, livestock products and the like is regulated to be 0.01-5mg/kg in Australia; the MRL of penthiopyrad in animal-derived foods such as fruits, vegetables, nuts, grains, livestock products and the like is regulated to be 0.01-30mg/kg by the European Union; the countries of the European Union, japan, etc. stipulate that if the pesticide used in the field is not registered in the country and no corresponding residual limit standard is established, the residual limit in the food and agricultural products exported to the country, including animal derived food such as livestock and poultry meat, etc. is all subjected to the 'uniform standard' of 0.01 mg/L.

At the present stage, researches on a penthiopyrad residual quantity measuring method are few, no method for simultaneously detecting penthiopyrad and metabolites thereof exists, reported detection methods mainly include a method for detecting penthiopyrad residual quantity in vegetables and fruits, liquid chromatography-tandem mass spectrometry (LC-MS/MS) is adopted in the detection methods for detecting penthiopyrad residual quantity in vegetables and fruits, LC-MS/MS is adopted for detecting pesticide residues in food and agricultural products, and the method has the advantages of rapidness, simplicity, high sensitivity and the like. The gas chromatography-electron impact ionization-mass spectrometry (GC-EI-MS) provided with the electron impact ionization source has great advantages in analyzing pesticide residues in food and agricultural products, and the electron impact ionization source is a universal detector, so that multi-residue analysis of hundreds of pesticides can be realized, qualitative and quantitative analysis can be realized, and the price is moderate, so that various detection mechanisms and enterprises are provided with the gas chromatography-electron impact ion source-mass spectrometer (GC-EI-MS) to detect the pesticide residues in the food and agricultural products, but no report of a GC-EI-MS detection method for the penthiopyrad residue in the food and agricultural products exists so far. Penthiopyrad belongs to an electronegative compound, and because substrates of food and agricultural products such as animal-derived food and the like are complex, a sample pretreatment method with good purification effect and instrument analysis conditions need to be established to meet the detection requirements.

Therefore, the detection method capable of qualitatively and quantitatively analyzing the animal-derived substrate penthiopyrad and the metabolite thereof has important significance.

Disclosure of Invention

The invention aims to provide a method for detecting penthiopyrad and metabolites thereof in an animal-derived matrix.

In order to achieve the purpose, the invention provides the following scheme:

the invention provides a method for detecting animal-derived substrate penthiopyrad and metabolites thereof, which takes hydroxyl modified multi-walled carbon nanotubes as a dispersed solid phase extraction purification material.

Further, the detection method of the animal-derived substrate penthiopyrad and the metabolite thereof comprises the following steps: taking an animal source sample, adding acetonitrile, extracting, adding sodium chloride, salting out, centrifuging, purifying the supernatant by using the dispersed solid phase extraction purification material, centrifuging, and measuring the content of penthiopyrad and metabolites thereof in the supernatant by using UPLC-MS/MS.

Further, the animal-derived sample comprises one of a pork sample, an egg sample, or a milk sample.

Further, the feed-liquid ratio of the animal source sample to the acetonitrile is (4.95-5.05) g:10mL.

Further, the mass ratio of the animal-derived sample to the sodium chloride is (4.95-5.05): 2.

furthermore, the mass ratio of the animal-derived sample to the hydroxyl-modified multi-walled carbon nanotube is (4.95-5.05): 0.01.

currently, conventional dispersive extraction purification materials, such as PSA, C18, GCB, etc., are commonly used in the field for detecting pesticide residues. Because the animal source sample has high oil content and high separation and purification difficulty, and no specific effective purification material combination exists in the field at present, the invention selects the hydroxyl modified multi-walled carbon nanotube as the dispersed solid phase extraction purification material to enhance the adsorption of the lipid in the animal source sample, improve the distribution mode of the target analyte and improve the accuracy of the detection method aiming at the characteristic of high oil content of the animal source sample.

The invention discloses the following technical effects:

the penthiopyrad and the metabolites thereof in the animal source matrix are extracted through dispersive solid-phase extraction and purification, and are detected through UPLC-MS/MS.

The invention selects a novel nanometer purifying material, namely the hydroxyl modified multi-walled carbon nanotube as a dispersed solid phase extraction purifying material aiming at an animal source sample necessary for daily diet, thereby improving the accuracy and the stability of the detection method.

The method takes hydroxyl modified multi-walled carbon nanotubes as a dispersed solid phase extraction purification material, extracts/purifies penthiopyrad and main metabolites thereof ((1-methyl-3-trifluoromethyl-1H-pyrazole-4-yl) formamide, PAM for short) in an animal source matrix, and then measures the extracted substances by using an ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometer (UPLC-MS/MS), so as to achieve the purpose of accurately, qualitatively and quantitatively determining penthiopyrad and metabolites thereof in the animal source matrix.

The average recovery rate of the detection method is 88-106%, the Relative Standard Deviation (RSD) is 1.0-4.9%, the limit of quantification is 0.001mg/kg, and the limit of detection is 0.38-0.57 mu g/kg.

The method for simultaneously detecting penthiopyrad and metabolites thereof is required by international practice, and the detection method provided by the invention has the advantages of simplicity and convenience in operation, high sensitivity, good accuracy, rapidness and high efficiency, and fills the blank in the field of methods.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required in the embodiments will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is a typical chromatogram and molecular structure of penthiopyrad and its metabolite PAM determined under chromatographic conditions of example 1;

FIG. 2 is a penthiopyrad acetonitrile extracting solution standard curve;

FIG. 3 is a penthiopyrad metabolite PAM acetonitrile extract standard curve;

FIG. 4 is a penthiopyrad egg matrix standard curve;

FIG. 5 is a penthiopyrad metabolite PAM egg matrix standard curve;

FIG. 6 is a penthiopyrad milk substrate standard curve;

FIG. 7 is a penthiopyrad metabolite PAM milk substrate standard curve;

FIG. 8 is a penthiopyrad pork matrix standard curve;

FIG. 9 is a penthiopyrad metabolite PAM pork matrix standard curve;

FIG. 10 is a blank control chromatogram of an egg;

FIG. 11 is a milk blank control chromatogram;

FIG. 12 is a pork blank control chromatogram;

FIG. 13 is a typical chromatogram of penthiopyrad and its metabolite PAM at the lowest addition level (0.001 mg/kg) in an egg matrix;

FIG. 14 is a typical chromatogram of penthiopyrad and its metabolite PAM at the lowest addition level (0.001 mg/kg) in a milk substrate;

FIG. 15 is a typical chromatogram of penthiopyrad and its metabolite PAM at the lowest addition level (0.001 mg/kg) in a pork matrix.

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in the present disclosure, it is understood that each intervening value, to the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the documents are cited. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The description and examples are intended to be illustrative only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including but not limited to.

The detection reagent and the standard substance used in the invention are as follows:

acetonitrile: chromatographically pure, batch number JA080730, supplied by Fisher Chemical.

Sodium chloride: analytically pure, lot number 20180904, supplied by Guanghong chemical company, beijing.

Formic acid: chromatographic purity, lot number S2510100, supplied by shanghai' an spectral science instruments ltd.

Penthiopyrad: the purity is more than or equal to 98 percent and is provided by Beijing-Haming science and technology Limited.

Penthiopyrad metabolite PAM: 98.8% pure, provided by Beijing Mingming science and technology Limited.

Hydroxyl-modified multi-walled carbon nanotubes were provided by Tianjin Borna Aijier science and technology, inc.

The equipment used in the invention is as follows:

the ultra-high performance liquid chromatography-triple four-stage stalk mass spectrometer comprises: waters ACQUITY UPLC H-Class/Xevo TQD, watts;

a desk centrifuge: FC-5706 model low speed centrifuge, aohaus International trade, inc.;

analytical balance: METTLER TOLEDO ME204E ten thousandth analytical balance, accuracy grade I, METTLER-TOLEDO corporation;

an electronic balance: scout Pro SPS202FZH one hundredth electronic balance, precision class III, aohaus corporation;

a liquid transferring gun: 5mL, 1000. Mu.L, 200. Mu.L, 100. Mu.L, 20. Mu.L pipette gun, eppendorf;

a bottleneck distributor: 5 to 50mL, BRAND;

laboratory pure water system: model S30UV, shanghai and tai instruments ltd;

multi-tube vortex mixer: MTV-100, otsu instruments, hangzhou;

a high-speed centrifuge: pico type 17, thermo Scientific.

The preparation method of the mobile phase solution and the extracting solution used by the invention comprises the following steps:

0.05% formic acid water (v/v): 1000mL of purified water is weighed into a solvent bottle, 500 mu L of chromatographic pure formic acid is added, and the mixture is shaken up and then is kept stand for standby.

The preparation method of the standard mother solution and the working solution used by the invention comprises the following steps:

preparing a standard solution:

penthiopyrad standard mother liquor: weighing 0.0051g of penthiopyrad raw drug, dissolving the raw drug in acetonitrile, and fixing the volume to 50mL, wherein the concentration is 500.00mg/L;

penthiopyrad metabolite standard solution: accurately measuring 0.0093g penthiopyrad metabolite ((1-methyl-3-trifluoromethyl-1H-pyrazol-4-yl) formamide) powder, and fixing the volume to 10mL by using acetonitrile, wherein the concentration is 918.84mg/L.

Example 1

Animal derived sample preparation

Removing shell of fresh egg collected from market, stirring egg white and yolk, sealing, and freezing at-20 deg.C. Removing bones from pork sample, mincing with meat mincer, mixing, dividing to no less than 500g by quartering method, placing into clean container, sealing, and freezing at-20 deg.C. Taking at least 500g of representative milk (raw milk) sample, mixing well, placing in a sample bottle, sealing and marking, and storing in a refrigerator at 4 deg.C in dark place.

The method for detecting penthiopyrad and metabolites thereof in the animal source matrix comprises the following steps:

(1) Preparation of a solution to be tested:

5.00 (+ -0.05 g) egg, pork and milk samples were weighed into 50mL PTFE centrifuge tubes, respectively. 10ml acetonitrile was added, extracted with shaking for 5min, and 2g NaCl was added. Repeatedly shaking for 5min, centrifuging for 5min under 1110 Xg relative centrifugal force, taking 1.5mL of supernatant, transferring into a centrifugal tube containing 10mg of hydroxyl modified multi-walled carbon nano-tube, carrying out vortex shaking for 5min, centrifuging for 3min under 9.6 Xg relative centrifugal force, taking supernatant, filtering through a 0.22um filter membrane, and determining through UPLC-MS/MS.

(2) Preparation of standard solution:

respectively diluting the penthiopyrad standard solution and the penthiopyrad metabolite standard solution of 10mg/L with blank animal source matrix extracting solution to the concentrations of 0.001, 0.01, 0.1, 0.25, 0.5, 0.75 and 1mg/L to prepare standard solutions.

(3) UPLC-MS/MS detection:

and (3) measuring the solution to be measured prepared in the step (1) and the standard solution prepared in the step (2) by using UPLC-MS/MS, wherein the instrument parameters are set as follows:

a chromatographic column: waters ACQUITY

BEH C18(2.1mm×100mm，1.7μm)；

Column temperature: 40 ℃;

flow rate: 0.35mL/min;

sample injection amount: 5 mu L of the solution;

the gradient elution conditions are shown in table 1.

TABLE 1 mobile phase and gradient elution conditions

Time (t/min)	Flow rate (mL/min)	Acetonitrile (%)	0.05% formic acid water (%)
				0	0.35	10	90
0.5	0.35	90	10
				3.5	0.35	90	10
4.0	0.35	10	90
				5.0	0.35	10	90

An ion source: electrospray ionization source ESI;

the scanning mode is as follows: a source of positive ions;

capillary voltage: 3.0kV;

ion source temperature: 150 ℃;

desolventizing temperature: 500 ℃;

desolventizing agent gas flow: 1000L/h;

taper hole gas flow: 50L/h;

the detection mode comprises the following steps: multiple Reaction Monitoring (MRM) is shown in table 2.

Table 2 primary reference mass spectral coefficients for penthiopyrad and penthiopyrad metabolites

Typical chromatograms and molecular structures of penthiopyrad and its metabolite PAM determined under chromatographic conditions of example 1 are shown in fig. 1.

(4) And (3) calculating an analysis result:

according to the measurement results of the standard solutions (tables 3 to 10), a standard curve is drawn, and the content of penthiopyrad in the solution to be measured is calculated by using the drawn curve.

TABLE 3 determination of penthiopyrad acetonitrile extract standard curve

As can be seen from the standard curve (FIG. 2) plotted in Table 3, the correlation coefficient of the obtained linear regression equation is > 0.99, which indicates that the peak area of penthiopyrad measured in the range of 0.001-1mg/L is linearly related to the concentration.

TABLE 4 determination of penthiopyrad metabolite PAM acetonitrile extract standard curve

Serial number	Concentration mg/L	Peak area
				1	0.001	674.4
2	0.01	6703.2
			3	0.1	61655.2
4	0.25	143350.1
			5	0.5	293709.5
6	0.75	460937.5
			7	1	575442.4

According to the standard curve (figure 3) drawn in table 4, the correlation coefficient of the obtained linear regression equation is greater than 0.99, and the linear correlation between the peak area and the concentration of the penthiopyrad metabolite PAM measured in the range of 0.001-1mg/L is shown.

TABLE 5 determination of penthiopyrad egg matrix Standard Curve

Serial number	Concentration mg/L	Peak area
				1	0.001	425.6
2	0.01	3260.7
			3	0.1	31214.8
4	0.25	75479.7
			5	0.5	142954.9
6	0.75	203061.1
			7	1	258025.2

As can be seen from the standard curve (FIG. 4) plotted in Table 5, the correlation coefficient of the obtained linear regression equation is > 0.99, which indicates that the peak area measured in the range of 0.001-1mg/L of penthiopyrad is linearly related to the concentration.

TABLE 6 determination of penthiopyrad metabolite PAM egg matrix standard curve

As can be seen from the standard curve (FIG. 5) plotted in Table 6, the correlation coefficient of the obtained linear regression equation is > 0.99, which indicates that the peak area measured in the range of 0.001-1mg/L of penthiopyrad is linearly related to the concentration.

TABLE 7 determination of penthiopyrad milk base Standard Curve

Serial number	Concentration mg/L	Peak area
				1	0.001	455.4
2	0.01	4366.6
			3	0.1	43664.6
4	0.25	98320.4
			5	0.5	169490.3
6	0.75	226931.7
			7	1	289022.0

As can be seen from the standard curve (FIG. 6) plotted in Table 7, the correlation coefficient of the obtained linear regression equation is > 0.99, which indicates that the peak area of penthiopyrad measured in the range of 0.001-1mg/L is linearly related to the concentration.

TABLE 8 determination of penthiopyrad metabolite PAM milk substrate Standard Curve

Serial number	Concentration mg/L	Peak area
				1	0.001	557.0
2	0.01	4722.8
			3	0.1	51805.1
4	0.25	128105.9
			5	0.5	254292
6	0.75	362644.2
			7	1	452904.6

As can be seen from the standard curve (FIG. 7) plotted in Table 8, the correlation coefficient of the obtained linear regression equation is > 0.99, which indicates that the peak area measured in the range of 0.001-1mg/L of penthiopyrad is linearly related to the concentration.

TABLE 9 determination of penthiopyrad pork matrix Standard Curve

Serial number	Concentration mg/L	Peak area
				1	0.001	361.3
2	0.01	2867.1
			3	0.1	29178.0
4	0.25	55613.0
			5	0.5	104735.4
6	0.75	142384.5
			7	1	207848.9

As can be seen from the standard curve (FIG. 8) plotted in Table 9, the correlation coefficient of the obtained linear regression equation is > 0.99, which indicates that the peak area measured in the range of 0.001-1mg/L of penthiopyrad is linearly related to the concentration.

TABLE 10 determination of the penthiopyrad metabolite PAM pork matrix standard curve

Serial number	Concentration mg/L	Peak area
				1	0.001	729.2
2	0.01	5980.2
			3	0.1	55504.9
4	0.25	142532.9
			5	0.5	262906.8
6	0.75	386120.3
			7	1	493309.1

As can be seen from the standard curve (FIG. 9) plotted in Table 10, the correlation coefficient of the obtained linear regression equation is > 0.99, which indicates that the peak area of penthiopyrad measured in the range of 0.001-1mg/L is linearly related to the concentration.

Example 2

Examination of the specificity, accuracy and precision of the test method of example 1:

according to the detection method of example 1, blank animal-derived substrates (blank control chromatograms of egg, milk and pork are shown in FIGS. 10 to 12) were selected and added to the mixed standard solution in the amounts of 0.001mg/kg, 0.01mg/kg, 0.1mg/kg and 1mg/kg, respectively, and 6 parallel samples were set for each group, and based on the measurement results, the recovery rate and the relative standard deviation RSD were calculated, and the results are shown in tables 11 to 16. The recovery and relative standard deviation requirements for the various levels of addition are shown in Table 17.

Table 11 penthiopyrad addition recovery results in eggs

Table 12 results of adding penthiopyrad metabolite PAM to eggs

Table 13 penthiopyrad addition recovery results in milk

Table 14 results of adding penthiopyrad metabolite PAM to milk and recovering

Table 15 penthiopyrad addition recovery results to pork

Table 16 penthiopyrad metabolite PAM added to pork recovery results

TABLE 17 requirement of different addition levels for recovery and relative standard deviation

Addition level (C) mg/kg	Recovery rate (R)%	Relative Standard Deviation (RSD)%
			C＞1	70-110	≤10
0.1＜C≤1	70-110	≤15
			0.01＜C≤0.1	70-120	≤20
0.001＜C≤0.01	60-120	≤30
			C≤0.001	50-120	≤35

From the data in tables 11 to 17, it can be seen that the recovery rate and the relative standard deviation of the detection method for penthiopyrad and metabolites thereof in the animal derived matrix both accord with the reference laboratory of pesticide residue analysis quality control and method verification procedures in food and feed of the European Union, which indicates that the accuracy and precision of the detection method provided by the invention both accord with the requirements.

Quantification limit of detection method of example 1:

the recovery rate and the relative standard deviation after adding penthiopyrad and metabolites thereof at different concentrations to the blank extraction solution were measured by using the detection conditions of UPLC-MS/MS in example 1, and the minimum concentration of the standard described in table 17 was taken as the limit of quantitation, and the measurement result was that the limit of quantitation of penthiopyrad and metabolites thereof in the animal-derived matrix was 0.001mg/kg. Typical chromatograms of penthiopyrad and its metabolite PAM at the lowest addition level (0.001 mg/kg) in egg, pork and milk matrix are shown in FIGS. 13-15.

Detection limit of detection method of example 1:

the concentration of the signal of the sample at 3 times of the baseline noise concentration is observed by using the detection condition of UPLC-MS/MS in the example 1, and the analysis result shows that the detection limit of the penthiopyrad and the metabolite thereof in the animal-derived matrix is 0.38-0.57 mu g/kg.

Comparative example 1

Compared with the embodiment 1, the difference is that the centrifugal tube filled with 10mg of hydroxyl modified multi-wall carbon nano-tubes in the pretreatment process is replaced by the centrifugal tube filled with 50mg of PSA, and the process of drawing the standard curve is omitted.

Comparative example 2

Compared with the example 1, the difference is that the centrifuge tube filled with 10mg of hydroxyl modified multi-walled carbon nanotubes in the pretreatment process is replaced by the centrifuge tube filled with 50mg of C18, and the process of drawing a standard curve is omitted.

Comparative example 3

Compared with the embodiment 1, the difference is that the centrifuge tube containing 10mg of hydroxyl modified multi-walled carbon nanotubes in the pretreatment process is replaced by the centrifuge tube containing 50mg of GCB, and the process of drawing the standard curve is omitted.

According to the detection method of example 1, while the samples were processed, the mixed standard solutions were added in the amounts of 0.001, 0.01, 0.1 and 1mg/kg, respectively, 5 parallel samples were set for each group, and the recovery rate and the relative standard deviation RSD were calculated based on the measurement results. The test methods of comparative examples 1 to 3 were carried out by adding the mixed standard solution in two addition amounts of 0.01 and 1mg/kg at the time of processing the pork sample, setting 5 parallel samples per group, and calculating the recovery rate and the relative standard deviation RSD based on the measurement results.

The results are shown in tables 18 to 23. The recovery and relative standard deviation requirements for different addition levels are shown in table 17.

Table 18 penthiopyrad added to egg for recovery results (comparative example 1 test method)

Table 19 results of adding penthiopyrad metabolite PAM to eggs (comparative example 1 test method)

Table 20 penthiopyrad added to egg for recovery results (comparative example 2 test method)

TABLE 21 penthiopyrad metabolite PAM addition recovery results in eggs (comparative example 2 test method)

Table 22 penthiopyrad added to egg for recovery results (comparative example 3 test method)

Table 23 results of adding penthiopyrad metabolite PAM to eggs (comparative example 3 detection method)

From the data in tables 18 to 23, it can be seen that the recovery rate and the relative standard deviation of the detection method for penthiopyrad and the metabolite PAM thereof in the animal-derived matrix accord with the pesticide residue analysis quality control and method verification program in the food and feed of the European Union reference laboratory, which shows that the accuracy and precision of the detection method provided by the invention accord with the requirements.

As can be seen from the data in tables 11 to 12 and tables 18 to 23, the accuracy and precision of the detection method in example 1 are higher than those of the detection methods in comparative examples 1 to 3, which indicates that different extractants and purification materials have great influence on the detection result of pesticide residue in animal-derived samples, and the accuracy and precision of the detection method are obviously improved after the extractants and purification materials are improved. The novel nano material purifying material is superior to the traditional purifying agent, which is probably because of larger specific surface area and high adsorption capacity, the novel nano material purifying material can selectively extract lipid from an animal source sample with higher content of the lipid while reducing the using amount, so that the analysis result is more accurate.

The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.

Claims (1)

1. A method for detecting animal-derived substrate penthiopyrad and metabolites thereof is characterized by comprising the following steps: (1) preparation of a solution to be tested:

weighing 5.00 +/-0.05 g of egg, pork and milk samples into a 50mL PTFE centrifuge tube, adding 10mL of acetonitrile, carrying out oscillation extraction for 5min, adding 2g of NaCl, repeatedly oscillating for 5min, then centrifuging for 5min under the condition of 1110 Xg relative centrifugal force, taking 1.5mL of supernatant, transferring the supernatant into the centrifuge tube containing 10mg of hydroxyl modified multi-walled carbon nano-tube, carrying out vortex oscillation for 5min, centrifuging for 3min under the condition of 9.6 Xg relative centrifugal force, taking the supernatant, filtering the supernatant through a 0.22um filter membrane, and determining the supernatant through UPLC-MS/MS;

(2) Preparation of standard solution:

respectively diluting the penthiopyrad standard solution and the penthiopyrad metabolite standard solution of 10mg/L with blank animal source matrix extracting solution to the concentrations of 0.001, 0.01, 0.1, 0.25, 0.5, 0.75 and 1mg/L to prepare standard solutions;

(3) UPLC-MS/MS detection:

and (3) measuring the solution to be measured prepared in the step (1) and the standard solution prepared in the step (2) by using UPLC-MS/MS, wherein the instrument parameters are set as follows:

a chromatographic column: waters ACQUITY UPLC BEH C18,2.1 mm multiplied by 100 mm and 1.7 μm;

column temperature: 40. DEG C;

flow rate: 0.35mL/min;

sample injection amount: 5. mu L;

the gradient elution conditions are shown in the following table:

an ion source: electrospray ion source ESI;

the scanning mode is as follows: a source of positive ions;

capillary voltage: 3.0kV;

ion source temperature: 150. DEG C;

desolventizing temperature: 500. DEG C;

desolventizing agent gas flow: 1000L/h;

taper hole gas flow: 50L/h;

the detection mode is as follows: multiple reaction monitoring is shown in the following table:

the preparation process of the animal source sample comprises the following steps: shelling fresh eggs collected in the market, uniformly stirring egg white and yolk, sealing and marking, and freezing and storing at-20 ℃; removing bones of pork sample, containing fat tissue with fat content less than 10%, mincing with meat mincer, mixing well, dividing to no less than 500g by quartering method, placing into clean container, sealing, and freezing at-20 deg.C for storage; taking at least 500g of representative milk sample, fully and uniformly mixing, placing in a sample bottle, sealing and marking, and storing in a refrigerator at 4 ℃ in a dark place;

the metabolite is (1-methyl-3-trifluoromethyl-1H-pyrazol-4-yl) formamide.

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