CN112924590A

CN112924590A - Non-targeted rapid screening method for soil pesticide by adopting GC/LC-QTOF

Info

Publication number: CN112924590A
Application number: CN202110122867.4A
Authority: CN
Inventors: 吕志江; 陶雨枫; 曾令藻; 何艳; 徐建明
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2021-01-29
Filing date: 2021-01-29
Publication date: 2021-06-08

Abstract

The invention discloses a non-targeted rapid screening method for soil pesticides by GC/LC-QTOF, which comprises the following steps: extracting and purifying a soil sample to respectively obtain a solution to be detected 1 and a solution to be detected 2, separating pesticide residue compounds from the solution to be detected 1 through an ultra-high performance liquid chromatography system, separating the pesticide residue compounds from the solution to be detected 2 through a gas chromatography system, screening polar and medium-polarity pesticides according to acquired data information of the solution to be detected 1, and screening non-polar and low-polarity pesticides according to acquired data information of the solution to be detected 2. The invention relates to an integrated method for simultaneously extracting, purifying and screening pesticide residues in a soil sample in a broad spectrum manner, aims to realize the broad-spectrum non-targeted screening of pesticides in a complex matrix of soil, and has important significance for comprehensively evaluating the soil pesticide pollution risk.

Description

Non-targeted rapid screening method for soil pesticide by adopting GC/LC-QTOF

Technical Field

The invention belongs to the field of analysis and detection of organic pollutants in an environmental medium, and particularly relates to a non-targeted rapid screening method for soil pesticides by adopting GC/LC-QTOF.

Background

The pesticide is an indispensable part of modern agriculture, and can play a role in preventing and controlling plant diseases and insect pests and regulating plant growth. On a global scale, nearly 30 million kilograms of pesticide are used annually, with the pesticide industry producing values of about $ 400 million. In the last 30 years, the production and usage of agricultural chemicals in China have been rapidly increased, and China now becomes one of the largest agricultural chemical producing countries and consuming countries in the world. The wide application of pesticides increases the yield of crops, thereby promoting the food supply. On the other hand, excessive use of pesticides can lead to the entry of harmful pesticide residues into the environment, such as soil, water, plants, food, and the like. The occurrence of large amounts of pesticide residues in complex substrates such as soil has become a major environmental pollution problem and may pose serious health and environmental hazards. In order to comprehensively know the pesticide residue risk in the soil, firstly, a reasonable pesticide analysis and detection method in the soil needs to be established, and the method mainly comprises two parts of extraction and analysis and detection.

In terms of extraction, common extraction methods for pesticides currently include Liquid-Liquid extraction (LLE), Solid-phase extraction (SPE), Matrix Solid-phase dispersion (MSPD), Solid-phase microextraction (SPME), and QuEChERS extraction, among others. Among them, the QuEChERS method is based on liquid-liquid buffered extraction followed by purification using dispersed solid phase extraction (dSPE) and mainly allows the extraction and recovery of various types of polar or non-polar pesticides in a complex matrix. The method has been widely applied to the multi-residue analysis research of different types of pesticides due to the characteristic of rapidness and effectiveness. In addition, the soil matrix is complex, and a universal extraction method needs to be improved and developed on the basis of the traditional QuEChERS method. In the aspect of analysis and detection, the combined use of chromatography and mass spectrometry is the most common analysis method, triple quadrupole tandem mass spectrometry is the most common combined technology due to high selectivity and sensitivity, and the Orbitrap and TOF mass spectrometer can provide ultrahigh high resolution (>20000FWHM), accurate mass measurement (<5ppm), excellent full MS scanning sensitivity and complete mass spectrometry information, so that the qualitative screening of target objects can be realized.

Currently, the most common pesticide residue research is mainly target screening, which requires selecting targets in advance and purchasing standard products, but the coverage is small, and the possibility of neglecting potential targets exists. Thus, one of the most popular trends in environmental analysis is the use of high resolution mass spectrometry (HR-MS) in combination with chromatography (LC/GC), with a rational strategy of non-targeted screening of suspected targets in a sample without standards. Furthermore, most current research is directed primarily to pesticides that are low in polarity and weak in soil binding capacity, which results in a lack of understanding of the presence of semi-polar and polar pesticides in soil. Therefore, how to realize the broad-spectrum screening of pesticides with different polarities in the soil matrix is a difficult problem to be solved urgently.

Disclosure of Invention

The invention aims to provide an integrated method for simultaneously extracting, purifying and screening pesticide residues in a soil sample in a broad spectrum manner, aims to realize the broad-spectrum non-targeted screening of pesticides in a complex matrix of soil, and has important significance for comprehensively evaluating the soil pesticide pollution risk.

In order to solve the technical problems, the invention provides a non-targeted rapid screening method for soil pesticides by GC/LC-QTOF, which comprises the following steps:

(1) sample preparation:

removing impurities from the collected soil, uniformly mixing, drying and dehydrating (until the water content is less than 1%), sieving by a 2mm sieve, grinding by a mortar and a pestle, and sieving by a 100-mesh sieve to obtain uniform powder serving as a soil sample; storing in amber glass bottle at 4 deg.C;

(2) sample extraction and purification:

weighing 4g of soil sample into a 50ml centrifuge tube, and adding an internal standard to ensure that the sample injection content is 50 ppb. After standing for 5min, 10mL of ultrapure water was added and soaked for 10min, followed by addition of 10mL of acetonitrile containing 1% acetic acid and vortexing for 2 min. Immediately after addition of 4g MgSO4 and 1g NaCl, it was shaken for 1min and vortexed for 2 min. After centrifugation at 4000rpm for 5min, the supernatant was transferred to another centrifuge tube containing 300mg MgSO4 and 50mg PSA, hand shaken for 15s and vortexed for 2 min. Finally, after centrifuging at 4000rpm for 5min, uniformly transferring the supernatant into two groups of nitrogen blowing pipes, blowing nitrogen for concentration, redissolving one group of the supernatant into 1ml by using methanol to obtain a solution to be detected 1, analyzing the solution to be detected by using an LC instrument after passing through a membrane, redissolving the other group of the supernatant into 1ml by using acetone to obtain a solution to be detected 2, and analyzing the solution by using a GC instrument;

acetonitrile containing 1% acetic acid, consisting of 1% acetic acid and 99% acetonitrile,% being volume%;

(3) sample detection:

separating pesticide residue compounds from the solution 1 to be detected by an ultra-high performance liquid chromatography system, and collecting recovery rate data by adopting Xevo TQ-XS matched with an electrospray ion source and MRM ES +; by using UPLC-Q-Tof equipped with an electrospray ion source, respectively by MS^EThe ES + and ES-modes of the test solution 1 are used for collecting mass spectrum information of precursor ions and fragment ions of the test solution 1;

separating the pesticide residue compound from the liquid 2 to be detected by a gas chromatography system, and collecting recovery rate data by adopting a GC/Triple Quadrupole MS (gas chromatography/Triple tandem mass spectrometry) with an electron bombardment ion source and a dMRM (double-stranded metal-free mass spectrometry); collecting fragment ion data information of the liquid to be detected by adopting GC/Q-TOF provided with an electron bombardment ion source and MS1 with locked retention time;

comprehensively detecting pesticide residue compounds in soil through two ways;

(4) and (3) data analysis:

calculating the recovery rate of the extraction method by using the acquired data information of the liquid 1 to be detected by using a recovery rate calculation formula; screening polar and medium-polarity pesticides by using a UNIFI 1.8 scientific database in combination with UNFI software, and identifying according to the accurate mass, retention time, fragment ions, isotope abundance and peak intensity of precursor ions;

calculating the recovery rate of the extraction method by using the acquired data information of the liquid 2 to be detected by using a recovery rate calculation formula; screening nonpolar and weakly polar pesticides by using Agilent pesticide PCDL in combination with MassHunter 10.0 software, and identifying according to retention time, accurate mass of fragment ions, fragment ion co-outflow curve, isotope abundance and peak intensity;

and rapidly and comprehensively analyzing pesticide residue information in the soil.

The drying in the step (1) is freeze drying, namely the soil sample is dried in a LABCONCO vacuum freeze dryer.

The detection conditions in the step (3) are set as follows:

liquid chromatography conditions:

a chromatographic column: ACQUITY UPLC BEH C182.1x 100mm,1.7 μm;

column temperature: 45 ℃;

mobile phase: phase A is ultrapure water, phase B is methanol, both are HPLC grade, and 1M ammonium acetate (pH 5.0) with volume fraction of 1% is added as additive;

gradient elution procedure: 0-0.25min, 2% B; 0.25-12.25min, 2% B-99% B; maintaining 99% B for 12.25-13 min; 13-13.01min, rebalancing to 2% B; 13.01-17min, maintaining 2% B;

flow rate: 0.45mL min^-1；

Sample introduction amount: 2 mu L of the solution;

ESI ion source mass spectrometry conditions:

xevo TQ-XS mass spectrometer, acquisition mode: MRM;

positively charged electrospray ion source (ESI +); the capillary voltage is 3.00 kV; sampling the taper hole voltage: 20V, and (3); the ion source temperature is 150 ℃; desolventizing temperature: 500 ℃; flow rate of desolventizing gas: 800L h^-1(ii) a Cone airflow 150L h^-1(ii) a The collision gas is 0.15mL min^-1Argon gas of (2); the MRM parameters are shown in Table 1;

xevo G2-XS QTof mass spectrometer, acquisition mode: MS (Mass Spectrometry)^E

ESI + ionization mode conditions: capillary voltage: 1.0 kV; sampling the taper hole voltage: 20V, and (3); ion source temperature: 120 ℃; desolventizing temperature: 500 ℃;flow rate of desolventizing gas: 900L/H; reference mass: leucine enkephalin [ M + H ]]⁺＝556.2766；

ESI-ionization mode conditions: capillary voltage: 1.0 kV; sampling the taper hole voltage: 20V, and (3); ion source temperature: 120 ℃; desolventizing temperature: 500 ℃; flow rate of desolventizing gas: 900L/H; reference mass: leucine enkephalin [ M-H]^-＝554.2615；

The collection range is as follows: m/z is 50-1000; low energy collision energy: 4 eV; high energy gradual change collision energy: 10 to 40 eV.

The gas chromatography conditions were:

a chromatographic column: 2 Agilent 19091S-431UI HP-5ms Ultra insert, 15m,0.25mm,0.25 μm;

temperature rising procedure: 60 deg.C for 0-1 min; 1-2.5min, 60-120 ℃; 2.5-40.5min, 120-310 ℃;

sample inlet temperature: 280 ℃;

flow rate: 1.05 mL/min;

carrier gas: nitrogen gas;

and (3) back flushing condition: 5min (post run), 310 ℃ (column oven temperature), 50psi (auxiliary EPC pressure), 2psi (sample inlet pressure);

sample introduction amount: 1 mu L of the solution;

the mass spectrum conditions of the EI ion source are as follows:

7000D Triple Quadrupole MS spectrometer, acquisition mode: dMRM;

transmission line temperature: 280 ℃; temperature of the quadrupole rods: 150 ℃; ion source temperature: 300 ℃; electron energy: 70 eV; specific parameters of dMRM are shown in Table 1;

7250Q-TOF mass spectrometer, acquisition mode: MS1

Transmission line temperature: 280 ℃; temperature of the quadrupole rods: 150 ℃; ion source temperature: 300 ℃; electron energy: 70 eV; spectrum acquisition rate: 4 Hz; mass number range: m/z is 35-1000;

in the step (4), the UNIFI method retrieval parameters are as follows: keeping the time tolerance of +/-0.2 min, ensuring the accurate mass matching tolerance of +/-5 mDa, and selecting the + H, + Na and-H modes in an ionization form; the MassHunter method has the following retrieval parameters: retention time tolerance ± 0.2min, precision mass matching tolerance: 20 ppm.

In the present invention:

1. by optimizing the soil pesticide extraction and purification method and comprehensively considering recovery efficiency, time, consumables and labor cost, the QuEChERS method with acetonitrile as an extracting agent and PSA as a purifying agent is used as a method for simultaneously extracting pesticide residues in soil;

2. by combining gas chromatography and liquid chromatography and based on a non-targeted screening method, the screening method is optimized, and finally, a non-targeted rapid screening method for soil pesticides by adopting GC/LC-QTOF is established.

Compared with the prior art, the invention has the advantages and beneficial effects that:

1. QuEChERS extraction is selected as a pretreatment method, so that matrix interfering substances in a soil sample can be effectively removed, the recovery rate of pesticide residues is acceptable, and the method is convenient and quick;

2. the pesticide with different polarities can be analyzed and detected by combining gas chromatography and liquid chromatography, and the broad-spectrum detection of the pesticide in the sample can be realized. TOF is selected as a mass detector, so that the TOF has high resolution and sensitivity, and can provide comprehensive and accurate chromatographic mass spectrometry information for subsequent data analysis;

in conclusion, the method is rapid, can realize high-flux broad-spectrum pesticide residue analysis, and can provide important method basis and data support for rapid screening and risk management and control of pesticide residues in soil.

Drawings

The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.

FIG. 1 is a total ion flow diagram of an actual sample on a liquid phase;

FIG. 2 is a total ion flow diagram of an actual sample on a liquid phase;

FIG. 3 is an ion flow diagram of the extraction of a typical pesticide tricyclazole on a liquid phase in a real sample;

FIG. 4 is an example of identification of a typical pesticide tricyclazole on UNIFI software in real samples; identification of the polar pesticide tricyclazole can be achieved by combining the liquid chromatography extraction ion chromatography peak shape and retention time shown in fig. 3 with the accurate mass deviation and fragment ions and the like shown in fig. 4;

in FIG. 4, the upper graph is a mass spectrum of the pesticide tricyclazole in the low-energy channel, and the lower graph is a mass spectrum of the pesticide tricyclazole in the high-energy channel;

FIG. 5 is a graph of an extracted ion flow of a typical pesticide α -hexachloro-cyclohexane in a gas phase in a real sample;

fig. 6 is an identification example of typical pesticide alpha-hexasihexa on masshhunter in actual samples, and the identification of alpha-hexa weak polar pesticide can be realized by combining the liquid chromatography extraction ion chromatography peak shape and retention time shown in fig. 5, the accurate mass deviation and co-outflow curve in fig. 6 and the like.

In fig. 6, the upper left is an extracted ion flow diagram of each fragment ion of the pesticide α -hexachloro-cyclohexane, the lower left is a co-outflow curve of each fragment ion of the pesticide α -hexachloro-cyclohexane, the upper right is a mass spectrum of the pesticide α -hexachloro-cyclohexane after automatic purification by the pesticide α -hexachloro-cyclohexane software, and the lower right is an actual mass spectrum of the pesticide α -hexachlorocyclohexane.

Detailed Description

The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:

example 1:

reagent and medicine

Unless otherwise stated, reagents used in the analysis were of a chromatographic grade according to national standards. The experimental water was Mili-Q ultrapure water.

Acetone (C)₃H₆O): carrying out chromatographic purification;

methanol (CH)₃OH): carrying out chromatographic purification;

acetonitrile (C2H 3N): carrying out chromatographic purification;

acetic acid (CH3 COOH): carrying out chromatographic purification;

acetonitrile acid mixed solvent: adding acetic acid with the volume of 1% into acetonitrile for mixing;

pesticide single-standard stock solution: 100. mu.g mL^-1(each pesticide), dissolved in methanol, and stored at-20 ℃.

Pesticide mixed standard intermediate solution: 5. mu.g mL^-1(each pesticide) dissolved in acetone and maintained at-20 deg.CAnd (4) storing.

Internal standard intermediate solution: 5. mu.g mL^-1(each internal standard), dissolved in acetone, and stored at-20 ℃.

Accurately weighing a proper amount of pesticide mixed standard intermediate solution and a sample injection internal standard intermediate solution, and respectively preparing the intermediate solution into a solution containing 24 pesticides with the mixed concentration of 1ng mL by using methanol and acetone^-1、5ng mL^-1、10ng mL^-1、50ng mL^-1、100ng mL^-1、200ng mL^-1And the concentration of the sample injection internal standard substance is 50ng mL^-1The mixed standard working solution of (1) is stored at-20 ℃.

The pesticide is specifically 23 pesticides as shown in table 1;

the internal standard is specifically as follows: p, p' -dichlorodiphenyl trichloroethane-D8, chlorpyrifos-D10, fipronil-13C 415N2, imidacloprid-D4 and atrazine-D5.

Second, instrument and equipment

Ultra high performance liquid chromatography-triple quadrupole mass spectrometer (Waters acquisition UPLC I-Class/Xevo TQ-XS); ultra high performance liquid chromatography-time-of-flight mass spectrometer (Waters ACQUITY UPLC I-Class/Xevo G2-XS QTof) ACQUITY UPLC BEH C18 column (2.1 mm. times.100 mm,1.7 μm); gas chromatography-Triple Quadrupole mass spectrometer (Agilent 7010B GC/7000D Triple Quadrupole MS); gas chromatography-time of flight mass spectrometer (Agilent 8890 GC/7250Q-TOF); an Organomation nitrogen blowing instrument; common laboratory instruments and equipment are used.

Thirdly, the non-targeted rapid screening method for the soil pesticide by adopting GC/LC-QTOF comprises the following steps:

1. sample preparation

Placing the collected soil sample in a brown glass bottle, cleaning the glass bottle with ultrapure water, and burning for 10 hours in a muffle furnace at 450 ℃ to completely remove organic matters and the like remained in the glass bottle; the collected soil sample is refrigerated below 4 ℃, protected from light, sealed and stored, and is taken to a laboratory for analysis as soon as possible.

Placing the soil sample on an enamel disc, mixing uniformly, removing foreign matters such as branches, leaves and stones, and placing in a vacuum freeze dryer for drying and dewatering (vacuum degree of 0.2mbar, -50 ℃, drying for 5d, at this time, the water content of the sample is less than 1%); the dried soil was sieved through a 2mm sieve, then ground with a mortar and pestle to a uniform powder that was sieved through a 100 mesh sieve, and stored in an amber glass vial at 4 ℃ for use.

2. Sample pretreatment

Because the conventional extraction method requires purification and impurity removal through a solid phase extraction column, the process is usually time-consuming and labor-consuming. Thus, under the current conditions in the laboratory, the present invention contemplates pretreatment of the sample using QuEChERS extraction.

The method comprises the following specific steps: and (3) weighing 4g of the soil sample obtained in the step (1) into a 50ml centrifugal tube, and adding an internal standard to enable the internal standard sample injection content to be 50 ppb. After standing for 5min, 10mL of ultrapure water was added and soaked for 10min, followed by addition of 10mL of acetonitrile containing 1% acetic acid and vortexing for 2 min. Immediately after addition of 4g MgSO4 and 1g NaCl, it was shaken for 1min and vortexed for 2 min. After centrifugation at 4000rpm for 5min, the supernatant was transferred to another centrifuge tube containing 300mg MgSO4 and 50mg PSA, hand shaken for 15s and vortexed for 2 min. And finally, centrifuging at 4000rpm for 5min, uniformly transferring the supernatant into two groups of nitrogen blowing pipes, blowing and concentrating nitrogen, redissolving one group of the supernatant into 1ml by using methanol to obtain the solution to be detected 1, passing the solution through a membrane, analyzing the solution by using an LC instrument, redissolving the other group of the supernatant into 1ml by using acetone to obtain the solution to be detected 2, and analyzing the solution by using a GC instrument.

3. Sample assay

(1) Liquid chromatography conditions:

a chromatographic column: ACQUITY UPLC BEH C18(2.1 mm. times.100 mm,1.7 μm); the column temperature is 45 ℃; the sample introduction amount is 2 mu L;

mobile phase: phase A is ultrapure water, phase B is methanol, both are HPLC grade, and 1M ammonium acetate (pH 5.0) with volume fraction of 1% is added as additive; flow rate 0.45mL min^-1；

The phase A is as follows: a mixture of 99% ultrapure water and 1% 1M ammonium acetate solution,% by volume;

the phase B is as follows: a mixture of 99% methanol and 1% 1M ammonium acetate solution,% by volume.

The elution gradient of the mobile phase was: 0-0.25min, 2% B; 0.25-12.25min, 2% B-99% B; maintaining 99% B for 12.25-13 min; 13-13.01min, rebalancing to 2% B; 13.01-17min, maintaining 2% B;

(2) xevo TQ-XS Mass Spectrometry conditions:

an acquisition mode: MRM;

description of the drawings: the MRM parameter is used to quantify the recovery rate of the pretreatment method and to determine an appropriate pretreatment method.

(3) Xevo G2-XS qt of mass spectrometry conditions:

an acquisition mode: MS (Mass Spectrometry)^E

Positively charged electrospray ion source (ESI +); capillary voltage: 1.0 kV; sampling the taper hole voltage: 20V, and (3); ion source temperature: 120 ℃; desolventizing temperature: 500 ℃; flow rate of desolventizing gas: 900L/H; reference mass: leucine enkephalin [ M + H ]]⁺＝556.2766；

Negatively charged electrospray ion source (ESI-); capillary voltage: 1.0 kV; sampling the taper hole voltage: 20V, and (3); ion source temperature: 120 ℃; desolventizing temperature: 500 ℃; flow rate of desolventizing gas: 900L/H; reference mass: leucine enkephalin [ M-H]^-＝554.2615；

(4) Gas chromatography conditions:

a chromatographic column: 2 Agilent 19091S-431UI HP-5ms Ultra Inert (15m,0.25mm,0.25 μm); sample inlet temperature: 280 ℃; flow rate: 1.05 mL/min; carrier gas: nitrogen gas; and (3) back flushing condition: 5min (post run), 310 ℃ (column oven temperature), 50psi (auxiliary EPC pressure), 2psi (sample inlet pressure); sample introduction amount: 1 mu L of the solution;

(5)7000D Triple Quadrupole MS Mass Spectrometry conditions:

an acquisition mode: dMRM

(6)7250Q-TOF mass spectrometry conditions:

an acquisition mode: MS1

Transmission line temperature: 280 ℃; temperature of the quadrupole rods: 150 ℃; ion source temperature: 300 ℃; electron energy: 70 eV; spectrum acquisition rate: 4 Hz; the collection range is as follows: m/z is 35-1000.

4. The screening method comprises the following steps:

according to the step 3, separating pesticide residue compounds from the liquid 1 to be detected by an ultra high performance liquid chromatography system, and performing recovery rate measurement on the pesticide mixed standard in the step 1 according to the conditions of the step (1) and the step (2) by adopting Xevo TQ-XS matched with an electrospray ion source; using UPLC-Q-Tof equipped with electrospray ion source, respectively MS^EThe mass spectrum information of the precursor ions and the fragment ions of the liquid 1 to be detected is acquired in the ES + and ES modes, namely, retention time data on liquid chromatogram can be obtained according to the condition (1), accurate mass of the precursor ions and the fragment ions and other data can be obtained according to the condition (3), polar and medium-polarity pesticides are screened by combining the acquired data information of the liquid 1 to be detected with UNFI software and using a UNIFI 1.8 scientific database, and identification is carried out according to the accurate mass, retention time, fragment ions, isotope abundance, peak intensity and other information of the precursor ions obtained under the conditions (1) and (3);

according to the step 3, separating the pesticide residue compound from the liquid 2 to be detected by a gas chromatography system, and performing recovery rate measurement on the pesticide mixed standard in the step 1 by adopting a GC/Triple Quadrupole MS with an electron bombardment ion source according to the conditions (4) and (5); and (3) acquiring fragment ion data information of the liquid to be detected by adopting GC/Q-TOF (gas chromatography/quadrupole-time of flight) with an electron bombardment ion source and MS1 with locked retention time, namely acquiring retention time data on gas chromatography according to the condition (4), acquiring data such as accurate mass of fragment ions and fragment ion co-outflow curve according to the condition (6), screening nonpolar and weakly polar pesticides by combining acquired data information of the liquid 2 to be detected with MassHunter 10.0 software and using Agilent pesticide PCDL, identifying according to the retention time, accurate mass of fragment ions, fragment ion co-outflow curve, isotope abundance and peak intensity obtained under the conditions (4) and (6), and rapidly and comprehensively analyzing pesticide residue information in soil.

Namely, the pesticide residue compound in the soil is comprehensively detected through two ways.

Fourth, result and discussion

1. Determination of soil sample pretreatment method

The soil sample has complex matrix and a large amount of matrix interferents, so that a proper pretreatment method needs to be selected to realize the comprehensive extraction of the pesticide in the soil. In the experiment, 23 pesticides with different functional groups, different polarities and different types are selected as representatives, the pesticides have different applications and wide physicochemical properties, can be used as representative substances for development of a soil pretreatment method, and LC-MS/MS and GC-MS/MS mass spectrum parameters are determined according to the conditions (2) and (5) in the step 3 (Table 1). The 24 kinds of pesticides include the most commonly used pesticides such as insecticides, herbicides, bactericides and the like, and the pesticides include the varieties which are widely used at present and have been widely produced and used in history, such as organic chlorine, organic phosphorus, pyrethroids, neonicotinoids and the like. Meanwhile, p' -dichlorodiphenyl trichloroethane-D8, chlorpyrifos-D10 and fipronil-¹³

C4

¹⁵5 isotopically labeled compounds N2, imidacloprid-D4 and atrazine-D5 were used as internal standards for accurate quantification.

Table 1, 23 main mass spectrum parameters of representative pesticides and isotopically labeled compounds thereof

And with the 23 pesticides as representatives, an extraction method capable of quickly and conveniently extracting a plurality of pesticides with different properties is developed and verified. The specific recovery rates determined by the conditions (1) and (2) and the conditions (4) and (5) in step 3 are shown in Table 2. The recovery rate is calculated by the formula: k ═ a-B)/C × 100%, where a: adding a sample of the standard substance for measuring; b: the measured amount of the substance in the sample; c: amount of standard substance added.

According to the table 2, the purificant is that the recovery rate of all pesticides in the PSA group is between 60 and 140 percent, and the full extraction of the target substances in the sample can be realized. In addition, 15 pesticides such as chlorpyrifos, beta-cypermethrin, butachlor and the like can be simultaneously detected in GC-MS/MS and LC-MS/MS for cross validation, so that the reliability of the method is greatly improved.

TABLE 2 typical pesticide recovery

2. Determination of soil sample pretreatment method

The UNIFI method retrieval parameters are as follows: keeping the time tolerance of +/-0.2 min, ensuring the accurate mass matching tolerance of +/-5 mDa, and selecting a + H, + N and-H mode in an ionization form; the MassHunter method has the following retrieval parameters: retention time tolerance ± 0.2min, precision mass matching tolerance: 20 ppm.

3. Non-target rapid screening quality control method

23 quality-controlled agricultural chemicals were added to the substrate sample at a concentration of 50ppb, and the experiments were carried out under the conditions (1), (3), (4), and (6) in the

above steps

1, 2, and 3, and the results of detection of the liquid phase are shown in Table 3, and the results of detection of the gas phase are shown in Table 4; the marked pesticides are detected, so that the screening method has wide application range and can be applied to actual soil samples.

TABLE 3 liquid phase detection results of quality control samples

TABLE 4 gas phase detection results of quality control samples

4. Practical application of non-targeted rapid screening of pesticide residues

The screening method is applied to actual soil samples, experiments are carried out under the conditions (1), (3), (4) and (6) in the

steps

1 and 2 and the step 3, and the total ion flow diagram is shown in the figure 1 and the figure 2. The results of the actual sample screening are shown in table 5, 96 pesticides were detected in the actual soil sample, and different classes of pesticides including organochlorine, organophosphorus, carbamate, pyrethroid and the like were covered, and the identification examples of representative pesticides detected in liquid phase and gas phase are shown in fig. 3 to 6. The method has the advantages that 41 pesticides are detected only on the liquid phase, 31 pesticides are detected only on the gas phase, and 24 pesticides are detected on both the liquid phase and the gas phase, so that the analysis and detection range of the method can be greatly expanded by using the gas chromatography and the liquid chromatography in a combined manner, and the method can realize high-flux broad-spectrum pesticide residue non-targeted screening of soil sample varieties.

TABLE 5 actual sample screening results

Comparative example 1, the extraction method in the "sample pretreatment" of example 1, the scavenger was changed from "C18" to "PSA", and the rest was the same as example 1.

As shown in table 2, according to table 2, when the scavenger is C18, the recovery rates of the pesticides such as α -hexachloro cyclohexane, P' -dichlorodiphenyl trichloroethane and butachlor are not satisfactory, and when the scavenger is PSA, the recovery rates of all the pesticides are 60% to 140%, and sufficient extraction of the target in the sample can be achieved, so the QuEChERS method in which PSA is added as a scavenger is selected as a sample pretreatment method.

Comparative example 2, the liquid chromatography used in the sample measuring step of example 1 was changed to a combination of liquid chromatography and gas chromatography, and the rest was the same as example 1.

The results obtained are shown in tables 3 and 4: if only the liquid chromatogram is used alone, the screening and the detection of the pesticides with strong polarity and medium polarity can be realized, and the liquid chromatogram and the gas chromatogram are used together, the detection of the pesticides with weak polarity including organochlorine pesticides and the like can be realized, and the application range can be greatly widened. Thus, liquid chromatography is selected for use in combination with gas chromatography.

Finally, it is also noted that the above-mentioned lists merely illustrate a few specific embodiments of the invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.

Claims

1. The non-targeting rapid screening method for the soil pesticide by adopting GC/LC-QTOF is characterized by comprising the following steps of:

(1) sample preparation:

removing impurities from the collected soil, uniformly mixing, drying and dehydrating, and then grinding to obtain uniform powder which is sieved by a 100-mesh sieve and is used as a soil sample;

(2) sample extraction and purification:

weighing 4g of soil sample into a centrifugal tube, and adding an internal standard to ensure that the sample injection content of the internal standard is 50 ppb; standing, adding 10mL of ultrapure water for soaking, then adding 10mL of acetonitrile containing 1% acetic acid, and uniformly mixing; adding 4g MgSO₄Mixing with 1g NaCl; after centrifugation, the supernatant was transferred to another medium containing 300mgMgSO₄Mixing with a centrifugal tube of 50 mgPSA; after centrifugation, the supernatant is evenly transferred to two groups of nitrogen blowing pipes for nitrogen blowing concentration, one group is redissolved by methanol until 1ml is the liquid to be detected 1, the solution passes through a membrane and then is analyzed by an LC instrument, the other group is redissolved by acetone until 1ml is the liquid to be detected 2, and the solution is analyzed by a GC instrument;

(3) sample detection:

separating the pesticide residue compound from the liquid 1 to be detected by an ultra-high performance liquid chromatography system;

separating the pesticide residue compound from the liquid 2 to be detected by a gas chromatography system;

(4) and (3) data analysis:

screening polar and medium-polarity pesticides by using the acquired data information of the liquid 1 to be detected;

and screening the acquired data information of the liquid 2 to be detected on the non-polar and weak-polar pesticides.

2. The non-targeted rapid screening method of soil pesticides by GC/LC-QTOF as claimed in claim 1, which is characterized in that:

(3) sample detection:

separating pesticide residue compounds from the solution 1 to be detected by an ultra-high performance liquid chromatography system, and collecting recovery rate data by adopting Xevo TQ-XS matched with an electrospray ion source and MRM ES +; acquiring mass spectrum information of precursor ions and fragment ions of the liquid 1 to be detected by adopting an UPLC-Q-Tof with an electrospray ion source and adopting an ES + mode and an ES-mode of MSE respectively;

separating the pesticide residue compound from the liquid 2 to be detected by a gas chromatography system, and collecting recovery rate data by adopting a GC/Triple Quadrupole MS (gas chromatography/Triple tandem mass spectrometry) with an electron bombardment ion source and a dMRM (double-stranded metal-free mass spectrometry); collecting fragment ion data information of the liquid 2 to be detected by adopting GC/Q-TOF provided with an electron bombardment ion source and MS1 locked by retention time;

(4) and (3) data analysis:

screening polar and medium-polarity pesticides by using a UNIFI 1.8 scientific database by combining acquired data information of the liquid 1 to be detected with UNFI software, and identifying according to the accurate mass, retention time, fragment ions, isotope abundance and peak intensity of precursor ions;

and screening the acquired data information of the liquid 2 to be detected by combining MassHunter 10.0 software and using Agilent pesticide PCDL to screen non-polar and weak-polar pesticides, and identifying according to retention time, accurate mass of fragment ions, fragment ion co-efflux curve, isotope abundance and peak intensity.

3. The non-targeted rapid screening method of soil pesticides by GC/LC-QTOF as claimed in claim 2, which is characterized in that:

the detection conditions in the step (3) are set as follows:

liquid chromatography conditions:

a chromatographic column: ACQUITY UPLC BEH C182.1x100mm, 1.7 μm;

column temperature: 45 ℃;

mobile phase:

phase a was 99% ultrapure water + 1% 1M ammonium acetate (pH 5.0);

phase B was 99% methanol + 1% 1M ammonium acetate (pH 5.0);

ultrapure water and methanol are both HPLC grade,% is volume%;

flow rate: 0.45mL min-1;

sample introduction amount: 2 mu L of the solution;

ESI ion source mass spectrometry conditions:

xevo TQ-XS mass spectrometer, acquisition mode: MRM;

xevo G2-XS QTof mass spectrometer, acquisition mode: MSE;

ESI + ionization mode conditions: capillary voltage: 1.0 kV; sampling the taper hole voltage: 20V, and (3); ion source temperature: 120 ℃; desolventizing temperature: 500 ℃; flow rate of desolventizing gas: 900L/H; reference mass: leucine enkephalins [ M + H ] + ═ 556.2766;

ESI-ionization mode conditions: capillary voltage: 1.0 kV; sampling the taper hole voltage: 20V, and (3); ion source temperature: 120 ℃; desolventizing temperature: 500 ℃; flow rate of desolventizing gas: 900L/H; reference mass: leucine-enkephalin [ M-H ] - ═ 554.2615;

the collection range is as follows: m/z is 50-1000; low energy collision energy: 4 eV; high energy gradual change collision energy: 10-40 eV;

the gas chromatography conditions were:

sample inlet temperature: 280 ℃;

flow rate: 1.05 mL/min;

carrier gas: nitrogen gas;

and (3) back flushing condition: 5min, 310 ℃, the auxiliary EPC pressure is 50psi, and the injection port pressure is 2 psi;

sample introduction amount: 1 mu L of the solution;

the mass spectrum conditions of the EI ion source are as follows:

7000D Triple Quadrupole MS spectrometer, acquisition mode: dMRM;

7250Q-TOF mass spectrometer, acquisition mode: MS 1;

transmission line temperature: 280 ℃; temperature of the quadrupole rods: 150 ℃; ion source temperature: 300 ℃; electron energy: 70 eV; spectrum acquisition rate: 4 Hz; mass number range: m/z is 35-1000.

4. The non-targeted rapid screening method of soil pesticides by GC/LC-QTOF as claimed in claim 3, wherein the method comprises the following steps:

the liquid chromatography gradient elution procedure was: 0-0.25min, 2% B; 0.25-12.25min, 2% B-99% B; maintaining 99% B for 12.25-13 min; 13-13.01min, rebalancing to 2% B; 13.01-17min, maintaining 2% B;

the temperature program of the gas chromatography is as follows: 60 deg.C for 0-1 min; 1-2.5min, 60-120 ℃; 2.5-40.5min, 120-310 ℃.

5. The non-targeted rapid screening method for soil pesticides by GC/LC-QTOF as claimed in any one of claims 1 to 4, which is characterized in that:

in the step (4), the UNIFI method retrieval parameters are as follows: keeping the time tolerance of +/-0.2 min, ensuring the accurate mass matching tolerance of +/-5 mDa, and selecting a + H mode and a-H mode in an ionization form; the MassHunter method has the following retrieval parameters: retention time tolerance ± 0.2min, precision mass matching tolerance: 20 ppm.