WO2018192483A1

WO2018192483A1 - Electronic method for non-targeted, multi-index and rapid detection of pesticide residue in edible agricultural products

Info

Publication number: WO2018192483A1
Application number: PCT/CN2018/083341
Authority: WO
Inventors: 庞国芳; 范春林; 常巧英; 白若镔; 邹小波
Original assignee: 中国检验检疫科学研究院; 北京合众恒星检测科技有限公司
Priority date: 2017-04-17
Filing date: 2018-04-17
Publication date: 2018-10-25
Also published as: CN109917028A; CN108760909A; CN109917028B; CN109557197A; CN109557197B

Abstract

An electronic method for non-targeted, multi-index and rapid detection of pesticide residue in edible agricultural products, comprising the steps; first: establishing a first-level accurate mass spectrometry database and a second-level fragment ion mass spectrometry database of more than 1200 pesticides commonly used worldwide; using the two mass spectrometry databases for screening and confirmation of a pesticide compound, and establishing a unique electronic identity card for each pesticide, such that a standard of a conventional identification method using a physical pesticide as a reference is replaced with an electronic standard; second: developing software for high-resolution mass spectrometry based automatic matching, qualitative identification, and intelligent screening of pesticide residues, and embedding a software program into an instrument, such that comparison between test samples and pesticide mass spectrometry databases is rapid, throughput and accuracy are high, data is reliable, and processes are informationized and automated; and third, preparing a sample once, and using two detection techniques to simultaneously check for more than 1200 pesticides. The invention realizes multi-index, non-targeted, and rapid detection of pesticide residues in edible agricultural products, greatly improving detection efficiency.

Description

Electronic method for non-target, multi-index and rapid detection of pesticide residues in edible agricultural products

Technical field

The invention designs a method for detecting pesticide residues in edible agricultural products, and particularly relates to a non-target detection electronic method for simultaneously detecting more than 1200 pesticide residues in a plurality of edible agricultural products.

Background technique

As early as 1976, the World Health Organization (WHO), FAO (FAO) and the United Nations Environment Programme (UNEP) jointly established the Global Environment Monitoring System (GEMS/Food) to master Member States. Food contamination status, understanding the intake of food contaminants, protecting human health and promoting trade development. Now, all countries in the world have promoted food safety to a strategic position in national security. Pesticide residue limits are one of the food safety standards and an entry barrier to international trade. At the same time, the requirements for pesticide residues show more and more varieties, and the development trend of more and more strict limits, that is, the threshold for pesticide residue set by international trade is getting higher and higher. Now the EU has established 162,248 MRL standards for 839 pesticides, the United States has developed 39,147 MRL standards for 351 pesticides, Japan has established more than 5,1600 MRL standards for 579 pesticides, and China has released 3,650 pesticides for 381 pesticides in 2014. MRL standard. At present, the standard limit commonly used in the world is 10μg/kg. Therefore, both food safety and international trade call for high-throughput rapid pesticide residue detection technology, which undoubtedly provides opportunities and challenges for pesticide residue analysis workers. Among the many pesticide residue analysis technologies available today, chromatographic mass spectrometry is the best analytical tool for high-throughput multi-residue rapid detection.

At present, the analysis of pesticide residues is mainly based on gas chromatography, liquid chromatography, gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry. These detection techniques first need to be qualitatively compared to pesticide standards. For example, the detection of 100 pesticides requires the preparation of the corresponding 100 pesticide standard controls, and these 100 pesticides will be missed. In the actual work of pesticide residue laboratories, most laboratories do not stock hundreds of pesticide standards. The reason is that pesticide standards are not only expensive, but also valid for only 2 or 3 years, requiring repeated investment. There are only a few dozen standard pesticide standards in the laboratory, and the number of pesticides that are routinely monitored is limited to these dozens, which leads to food safety monitoring loopholes.

After years of painstaking research, the inventor team has developed an accurate mass mass spectrometry database of up to 1,200 pesticides and a pesticide residue detection technology method based on two types of high-resolution mass spectrometry, which enables more than 1200 agricultural products without standard products. Pesticide residues, and rapid detection and detection, meet the urgent need for rapid detection of high-throughput pesticide residues in agricultural products.

Summary of the invention

The invention proposes a high-throughput high-resolution gas chromatography-quadrupole time-of-flight mass spectrometry (GC-Q-TOFMS) and liquid chromatography-quadrupole-time-of-flight mass spectrometry (LC-Q-TOFMS) new technology. An electronic method for multi-indicator, non-target, and rapid detection of pesticide residues in edible agricultural products. Specific steps are as follows:

First: establish a first-class accurate mass mass spectrometry database (TOFMS) and a two-stage fragment ion mass spectrometry database (QTOFMS) for more than 1,200 commonly used pesticides in the world; use two mass spectral databases for screening and confirming pesticide compounds for each pesticide Establish a unique electronic identity card, and realize the traditional identification method that replaces the physical standards of pesticides with electronic standards.

Second: Developed high-resolution mass spectrometry (LC-Q-TOFMS and GC-Q-TOFMS) pesticide residue mass spectrometry automatic matching qualitative identification intelligent screening software, software program was implanted into the instrument, achieving high speed (0.5 hours), High throughput (1200+), high precision (0.0001m/z), high reliability (above 6 confirmation points), highly informative and automated test samples and pesticide mass spectrometry library.

Third: to achieve a sample preparation, using two detection techniques (GC-Q-TOFMS and LC-Q-TOFMS) to quickly detect more than 1,200 kinds of pesticides, to achieve multi-indicators, non-targets, and rapid detection of pesticide residues in edible agricultural products; Greatly improved method performance.

Among them, the steps to achieve multi-indicators, non-targets, and rapid detection of pesticide residues in edible agricultural products are as follows:

(1) Sample preparation: the agricultural product is extracted by homogenization of acetic acid acetonitrile, dehydrated, centrifuged, concentrated, and then purified by carbon/NH2 column, and the residual pesticide is eluted by acetonitrile + toluene. After concentration and filtration, the sample is prepared. ;

The sample preparation also includes the following pretreatments when the sample is an acidic substrate:

A 10.0 g sample (accurate to 0.01 g) was weighed into a 100 mL centrifuge tube, and 40 mL of an acetonitrile extract containing 1% (by volume) of acetic acid was added and homogenized at 10,000 rpm for 1 min. 4 g of anhydrous magnesium sulfate and 1 g of sodium chloride were added and shaken for 5 min. After centrifugation at 4200 rpm for 5 min, 20 mL of the supernatant was taken in a 150 mL chicken heart bottle, and rotated by evaporation in a 40 ° C water bath to about 2 mL, to be purified;

The CarbonNH2/ProElutNH2 column was used in series, about 1 cm of anhydrous sodium sulfate was added to CarbonNH2, and the SPE purification column was pre-washed with 5 mL of acetonitrile-toluene (3:1, v/v), and the column was tapped to remove the bubbles in the column. The effluent below the purification column is discarded. When the liquid level is slightly above the top of the sodium sulfate, the concentrate is transferred to the purification column and the 50 mL chicken heart bottle is connected. Rinse the chicken heart bottle with the sample with 2 mL acetonitrile-toluene (3:1, v/v), transfer the washing solution to the purification column, and repeat it three times; connect the 25 mL reservoir to 25 mL acetonitrile-toluene (3: 1, v / v) elution. After the collection, the mixture was rotary evaporated to about 0.5 mL, nitrogen was blown to near dryness, and 1 mL of n-hexane was added to make a volume. After ultrasonic dissolution, it was filtered through a 0.22 μm nylon membrane for GC-MS/MS.

(2) Chromatographic mass spectrometry detection: LC-Q-TOF/MS and GC-Q-TOFMS were used to detect pesticides; or LC-Q-Orbitrap screening and GC-Q-Orbitrap screening were used to detect pesticides.

(3) High-resolution mass spectrometry (LC-Q-TOFMS and GC-Q-TOFMS) pesticide residue mass spectrometry automatic matching qualitative identification intelligent screening: LC-Q-TOFMS first-level accurate database and secondary fragment ion mass spectrometry database 500 Screening and confirming the above pesticides; screening and confirming more than 700 pesticides by GC-Q-TOFMS's first-class accurate mass spectrometry database; or simultaneously using LC-Q-Orbitrap screening to achieve more than 500 pesticide tests, More than 700 pesticides were detected by GC-Q-Orbitrap screening; electronic detection was achieved.

Wherein, the pesticide first-order accurate mass mass spectrometry database and the second-order fragment ion mass spectrometry database are obtained by any one of the following three methods:

method one:

(1) Prepare pesticide standards into 1-5mg/L standard solution, and perform liquid chromatography-quadrupole-time-of-flight mass spectrometry (LC-Q-TOFMS) and gas chromatography-quadrupole flight under set conditions. Time-spectrum mass spectrometry (GC-Q-TOFMS) was used to obtain full-spectrum data in the first-order mode, and the full-spectrum data of each standard solution was determined.

(2) The first-order full-spectrum data of LC-Q-TOFMS obtained in step (1), respectively obtain the chemical formula, precise molecular weight, retention time, parent ion and other information of each pesticide; input the information into the database data file. And associated with the corresponding pesticide information, built a first-level accurate mass spectrometry database of LC-Q-TOFMS for data retrieval analysis; input the parent ion of each pesticide in the database in the data acquisition interface, in QTOFMS mode, The fragment ion full-scan mass spectrum was collected for each pesticide under 8 collision energies. The verified fragment ion full-scan mass spectrum was imported into the PCDL software, imported into the Library Editor, and associated with the corresponding pesticide information. Secondary fragment ion mass spectrometry database for LC-Q-TOFMS.

(3) The first-order full-spectrum data of GC-Q-TOFMS obtained in step (1), respectively, obtain the chemical formula, retention time, accurate mass of fragment ions, and ion abundance ratio of each pesticide; The scanning mass spectrum was imported into the PCDL software and correlated with the corresponding pesticide information to build a GC-Q-TOFMS first-order accurate mass spectrometry database for data retrieval and analysis.

Method 2: A method for establishing a GC-Q-Orbitrap library by gas chromatography-quadrupole electrostatic field orbitrap mass spectrometry GC-Q-Orbitrap, including the following

(1) Establish an accurate quality database:

The pesticide standard was prepared into a standard solution of 1-5 mg/L, and 1 μL of the single standard solution was injected into the instrument. GC-Q-Orbitrap was measured in Full MS mode, and the first-level full-spectrum data was opened by Qual Browser software. The retention time of the peak under chromatographic separation conditions;

Use the "Deconvolution Plugin" function of TraceFinder software to open the first-level full-spectrum data, and use the NIST library to identify the current compound for comprehensive compound information, including name, molecular formula, exact molecular weight, ion fragment composition, and ion abundance ratio information; The fragment ion full scan mass spectrum is correlated with the corresponding pesticide information to construct a GC-Q-Orbitrap first-order accurate mass spectrometry database;

(2) Establish a standard spectrum library:

The accurate mass information of the ions on the mass spectrum is checked and confirmed, and the edited mass spectrum and compound information are sent to the NIST software, and the NIST software Library is used to associate the mass spectrum and the compound information with the corresponding pesticide information to establish A library of more than 700 pesticide chemical contaminants.

Method 3: A method for establishing an LC-Q-Orbitrap library by liquid chromatography-quadrupole electrostatic field orbitrap mass spectrometry LC-Q-Orbitrap, including the following

(1) Establishment of an accurate quality database:

In Full MS/ddMS ² mode, the retention time of each pesticide standard under specified chromatographic mass spectrometry was determined, and the ionized form (+H, +NH ₄ , +Na) and chemical formula of the compound ESI source were determined. Obtaining the exact mass, isotopic peak distribution and abundance ratio of the parent ion of each compound;

Fragment ion full-sweep mass spectrometry was performed on 3 to 4 normalized collision energies for more than 500 pesticides, preferably a secondary mass spectrum of one normalized collision energy rich in ion information, according to the target pesticide The structural formula, inferring the theoretical exact mass of 3 to 5 secondary fragment ions; importing information such as the name, retention time, molecular formula, the precise mass of the adduct ion and the accurate mass of the secondary fragment into more than 500 pesticides into the database .

(2) Establish LC-Q-Orbitrap secondary standard spectrum library

A spectrum management software library manager was used to summarize the secondary mass spectrum of each pesticide under the optimal collision energy, and a standard spectrum library of more than 500 pesticides was established.

Wherein, the automatic matching qualitative identification intelligent screening implementation comprises any one of the following three methods:

method one:

(1) LC-Q-TOFMS first-class accurate mass spectrometry database screening and secondary fragment ion mass spectrometry database confirmation: the established first-order accurate mass mass spectrometry database is called in the qualitative software, and the target compound is qualitatively searched by mass spectrometry. The retrieval parameters are: retention time deviation is limited to ±0.5min, accurate mass deviation is limited to ±10ppm, based on the measured value of each compound electronic identity card information (retention time, exact mass, isotope distribution and isotope abundance) The deviation of the theoretical value in the accurate mass spectrometry database, the score of the search match is given, and the compound with the matching score >60 is searched for the suspected compound; in the Targeted MS/MS acquisition mode, the suspected compound is input. The parent ion, retention time and optimal collision energy will be detected in the map and the spectrum in the secondary fragment ion mass spectrometry database, and the electronic ID information (secondary spectrum) matching will be confirmed again under the mirror condition, and the matching score value >70, confirming the detection of the target compound.

(2) GC-Q-TOFMS first-level accurate mass spectrometry database screening and confirmation: the established first-order accurate mass mass spectrometry database is called in the qualitative software, and the target compound is qualitatively searched for the mass spectrometry. The retrieval parameters are: retention time deviation is limited to ±0.15min, accurate mass deviation is limited to ±20ppm, based on the measured value of each compound electronic identity card information (retention time, fragment ion accurate mass, fragment ion abundance ratio) The deviation of the theoretical value in the accurate mass spectrometry database gives the search matching result, and at least three fragment ions are detected, and the comprehensive score is >60, that is, the target compound is confirmed.

Method 2: GC-Q-Orbitrap screening method

Using Full MS mode injection analysis, the first-level fragment ion information of more than 700 pesticides was obtained by one injection analysis, and then searched and matched with the standard library (more than 700 pesticide chemical pollutants primary fragment ion spectrum library) information; Screening pesticide residues in samples using TraceFinder software based on accurate mass database and fragment ion library;

In the TraceFinder software, the established accurate mass database and the fragment ion library database are called for retrieval, and the corresponding retrieval parameters are set: the retention time is limited to ±0.1 to 0.3 min, the accurate mass deviation is ±3 to 10 ppm, and the peak area threshold is set. Between 10,000 and 100,000, the minimum number of detected ions is 2 to 3, the isotope matching score is greater than 50 to 70, and the database matching score is greater than 40 to 60, and the data is retrieved;

According to the measurement results of the exact mass, retention time, fragment ion number, isotope distribution and kurtosis ratio of the compound, the deviation from the theoretical value is calculated, and the search matching score value is obtained. For the search result is Confirm, the compound is determined as The pesticide was detected; the search result was Identify, which was a suspected pesticide; the measurement result was searched in the fragment ion library, and the search parameter setting: the matching mode was inverted matching, and the matching result was observed under the mirror comparison to confirm the sample. Whether it contains such pesticides;

Method 3: LC-Q-Orbitrap screening method

Full MS-dd MS2 mode injection analysis was used to obtain primary mass spectrometry information, secondary fragment ion information and secondary mass spectrum of more than 500 pesticides by one injection analysis; based on accurate mass database and secondary mass spectrometry library, using TraceFinder The software screens the pesticide residues in the sample;

The TraceFinder software is used to call the established accurate mass database and the fragment ion library database for retrieval, and the corresponding retrieval parameters are set: the retention time is limited to ±0.2 to 0.5 min, the accurate mass deviation is ±3 to 10 ppm, and the ionization form is selected. M+H] ⁺ , [M+NH ₄ ] ⁺ , [M+Na] ⁺ mode, the peak area threshold is set to 5000 to 10000, the minimum number of detected ions is 2 to 3, and the isotope matching score is greater than 50 to 70 points. The database matching score is greater than 40 to 60 points, and the data is retrieved;

According to the measurement results of the exact mass, retention time, fragment ion number, isotope distribution and ratio of the compound, the deviation from the theoretical value is calculated, and the search matching score value is given. For the search result is Confirm, the compound is determined to be detected. Pesticide; for the search result is Identify, for suspected pesticides, the measurement results are retrieved in the fragment ion library, the search parameter setting: the matching mode is inverse matching, and the matching result is observed under the mirror comparison to confirm whether the sample contains This kind of pesticide.

The beneficial effects of the invention:

1. Using LC-Q-Orbitrap's first-order accurate mass spectrometry database, two-stage fragment ion mass spectrometry database and GC-Q-Orbitrap's first-order accurate mass spectrometry database, innovatively established more than 1,200 pesticides with accurate quality of 0.0001. An accurate mass spectral database of m/z.

2. Establishing the unique electronic identification information of each pesticide in the database of two technologies (LC-Q-Orbitrap, GC-Q-Orbitrap), and replacing the pesticide physical standard with the electronic standard as the reference identification method. Achieve high-precision, high-efficiency, resource-saving non-target pesticide residue detection. The identification method that replaces the traditional pesticide standard with electronic standards has been realized, and the development of non-target pesticide residue detection technology has been realized.

3. Innovatively established mass spectrometry information of high resolution accurate mass, isotope distribution and isotope abundance as the identification standard, and detected more than 500 pesticides based on the first-order accurate mass spectrometry database and the second-order fragment ion mass spectrometry database. The LC-Q-Orbitrap technique was tested and confirmed; the mass spectrometry information of the fragment ion precision mass fraction and the fragment ion abundance ratio was creatively established as the identification standard, and more than 700 pesticides were processed according to the first-order accurate mass spectrometry database. Detected and corroborated GC-Q-Orbitrap technology, two technologies have completely changed the original qualitative model with reference to compound standards, which is a fast, high-throughput, accurate and reliable standard that does not require standard comparison. New technology for pesticide residue detection. The standard product was canceled for reference, and the electronic standard identification was used to realize the traditional method of replacing the physical standard with electronic ID card, and also realized the leap-forward development from targeted detection to non-targeted screening. It saves resources, reduces pollution, improves analysis speed, and fully meets the requirements of green development, environmental friendliness, and clean and efficient.

4. The full-scan mass spectrum information of each pesticide fragment ion in the LC-Q-Orbitrap secondary fragment ion mass spectrometry database is obtained by optimizing the full-scan mass spectrum for four different collision energies. Full scan of the fragment ions under normalized collision energy.

5. The LC-Q-Orbitrap residue detection technology method established by the invention can correspond to the compound in the first-order accurate mass spectrometry database according to the retention time, the exact mass number, the isotope distribution and the isotope abundance of the target compound. The alignment is retrieved, and the matching score value of the target compound is given. Qualitative detection of pesticides is achieved based on the score of the target compound.

6. The LC-Q-Orbitrap residue detection technique established by the present invention can search and compare the full scan mass spectrum information of the compound fragment ions in the secondary fragment ion mass spectrometry database according to the full scan mass spectrum information of the target compound fragment ions. , giving the matching score value of the target compound to achieve qualitative confirmation of the target compound.

7. The LC-Q-Orbitrap residue detection technology method established by the invention adopts Full MS-dd MS2 mode injection analysis, and can obtain the primary and secondary fragment ion information of more than 500 pesticides by one injection analysis. Replacing the original sample requires two injections, shortening the sample analysis time and improving the sample detection efficiency.

8. The GC-Q-Orbitrap residue detection technology method established by the invention can be based on the retention time of each compound, the accurate mass of the fragment ion, the fragment ion abundance ratio, etc., and the compound in the first-order accurate mass spectrometry database. Corresponding information comparison, giving the search matching results, based on the fragment ion detection and scoring, to achieve the qualitative and corroboration of the target compound.

9. Developed high-throughput sample preparation technology for pesticide residues in agricultural products matched with residual detection technology. It can realize the rapid detection and confirmation of more than 1,200 kinds of pesticides for agricultural products by only one sample preparation and two techniques for separate injection detection.

10. Among the more than 1,200 pesticides detected at the same time, more than 80% of the pesticides have a detection sensitivity lower than the standard of 10 μg/kg, which better meets the requirements for screening MRL levels of pesticide residues in various countries.

11. The LC-Q-Orbitrap and GC-Q-Orbitrap residual detection technology methods established by the invention have the quality accuracy of the two detection technologies within 5 ppm, which greatly reduces the false positive detection result, and satisfies the better. Residual, high-precision pesticide residue screening requirements.

DRAWINGS

Figure 1 LC-Q-TOFMS and GC-Q-TOFMS pesticide chemical contaminant mass spectrometry database establishment process

Figure 2 pesticide residue detection process

detailed description

The invention will be further described below in conjunction with the drawings and specific embodiments.

Figure 1 shows the flow of the LC-Q-TOFMS and GC-Q-TOFMS pesticide chemical contaminant mass spectrometry database, which has been described in detail in the Summary.

Figure 2 shows the method for electronic detection of pesticides proposed by the present invention, which can prepare samples at one time, and the two technologies can detect more than 1,200 kinds of pesticides; the standard products are eliminated for reference, and the electronic standard is used for qualitative identification to realize electronic identity. The traditional method of substituting physical standards has also achieved a leap-forward development from targeted detection to non-targeted screening. It saves resources, reduces pollution, improves analysis speed, and fully meets the requirements of green development, environmental friendliness, and clean and efficient.

At the same time, research and development of pesticide residue MS automatic matching qualitative identification intelligent screening software, the software program is implanted in the instrument, you can directly run the sample test solution, compare with the pesticide mass spectrometry database, and achieve the high speed of the method (0.5 Hour), high throughput (500/700 or more), high precision (0.0001m/z), high reliability (above 10 verification points), high informationization and automation, and electronic realization, greatly improving the method efficacy.

Example

Detecting geographical scope: More than 600 sampling points in 31 provincial capitals/municipalities (284 districts and counties), and sample sampling areas cover the living area of 25% of the country's population.

Detecting sample range: 146 fruits and vegetables in 18 categories cover 80% of the national standard list, demonstrating the universal applicability of the method.

National 18 categories of more than 140 kinds of fruit and vegetable sampling schedule

Table 1 Sample Classification and Quantity (LC-Q-TOFMS Detection)

Table 2 Sample Classification and Quantity (GC-Q-TOFMS Detection)

Example 1

More than 1,200 kinds of pesticides in celery (see the pesticides listed in the two publications CN105738460A and CN105628839A for specific pesticide types) LC-Q-TOF/MS and GC-Q-TOF/MS detection and confirmation technology implementation examples. Including the following steps;

1. Specific steps of sample pretreatment technology:

1.1 The celery sample is chopped, mixed, sealed and marked with the edible portion;

1.2 Weigh 10g celery sample (accurate to 0.01g), add 40mL 1% acetic acid acetonitrile in 80mL centrifuge tube, use high-speed homogenizer 13500r/min, homogenate extraction for 1min, add 1g sodium chloride, 4g anhydrous sulfuric acid Magnesium was shaken for 5 min, centrifuged at 4200 r/min for 5 min, 20 mL of the supernatant was taken, and concentrated in a 40 ° C water bath by rotary evaporation to about 1 mL, to be purified.

1.3 Add about 2 cm of anhydrous sodium sulfate to the Carbon/NH2 column. First rinse the SPE column with 4mL acetonitrile + toluene (3+1, v/v), and discard the effluent. When the liquid level reaches the top of the sodium sulfate, quickly transfer the sample concentrate to the SPE column and connect it to the new one. Chicken heart bottle is received. The sample bottle was washed three times with 2 mL of acetonitrile + toluene (3+1, v/v) each time, and the washing solution was transferred to an SPE column. A 50 mL reservoir was attached to the column, and the pesticide and related chemicals were eluted with 25 mL of acetonitrile + toluene (3+1, v/v), combined in a chicken heart bottle, and concentrated in a 40 ° C water bath to about 0.5 mL.

1.4 The concentrate was blown dry under nitrogen, 2 mL of acetonitrile + toluene (3+1, v/v) was added, ultrasonically reconstituted and mixed, divided into two portions on average, and dried under nitrogen. The solution was again made up with 1 mL of 1% formic acid acetonitrile + water (2+8, v/v) and 1 mL of n-hexane, and filtered through a 0.22 μm filter for LC-Q-TOF/MS and GC-Q-TOF/ respectively. MS detection.

2. LC-Q-TOF/MS and GC-Q-TOF/MS operating conditions

LC-Q-TOF/MS operating conditions

Chromatographic conditions: liquid chromatography mobile phase A was 5 mM ammonium acetate-0.1% formic acid-water; mobile phase B was acetonitrile; gradient elution procedure: 0 min: 1% B, 3 min: 30% B, 6 min: 40% B , 9min: 40% B, 15min: 60% B, 19min: 90% B, 23min: 90% B, 23.01min: 1% B, after running for 4min; flow rate is 0.4mL / min; column temperature: 40 ° C; Sample size: 10 μL.

Mass spectrometry conditions: capillary pressure of Agilent 6530LC-Q-TOF/MS: 4000V; drying gas temperature: 325 ° C; dry gas flow rate 10 L / min, sheath flow gas flow rate 11 L / min, sheath flow gas temperature 325 ° C; atomization gas pressure 40 psi, The cone voltage is 60V and the fragmentation voltage is 140V. The full scan mass-to-nuclear ratio range is 50-1600m/z, and the internal quality reference accuracy is used to correct the instrument quality accuracy in real time. Mass spectrometry results were collected and processed by Agilent MassHunter Workstation Software (version B.05.00).

GC-Q-TOF/MS operating conditions

Chromatographic conditions: The gas chromatographic column was a VF-1701ms, 30m×0.25mm×0.25μm mass spectrometer dedicated column. Temperature programmed process: 40 ° C for 1 min, then programmed to 30 ° C / min to 130 ° C, then 5 ° C / min to 250 ° C, and then 10 ° C / min to 300 ° C, for 5 min; carrier gas: 氦Gas, purity ≥ 99.999%, flow rate 1.2mL / min; inlet temperature: 290 ° C; injection volume: 1 μL; injection method: no split injection.

Mass spectrometry conditions: EI source voltage: 70 eV; ion source temperature: 230 ° C; GC-MS interface temperature: 280 ° C; solvent delay: 6 min. The mass scan range is 50-600 m/z, the acquisition rate is 2 spectra/s; the epoxy heptachlor is used to adjust the retention time; the mass spectrometry results are collected and processed by Agilent MassHunter Workstation Software (version B.07.00).

3. Detection of pesticide residues in celery

3.1 The sample solution is measured in the full scan mode, and the detection result is compared with the TOF database to obtain a first-order TOF score.

3.2 For the compounds whose scores meet the requirements, establish a secondary acquisition method in the software.

3.3 In the MS/MS mode, re-run the sample solution, obtain the full scan data of the sample fragment ions, compare it with the fragment ion information in the secondary mass spectrometer library, and obtain the secondary QTOF score.

4. Results of LC-Q-TOF/MS and GC-Q-TOF/MS detection in celery samples from a provincial capital

Collect 16 samples of celery in a provincial capital, and use LC-Q-TOF/MS and GC-Q-TOF/MS techniques to detect more than 1,200 pesticide residues, LC-Q-TOF/MS and GC-Q-TOF /MS technology detected 40 pesticide residues, a total of 94 frequency, involving 16 samples, the specific results are shown in Table 1.

Table 1 Results of LC-Q-TOF/MS and GC-Q-TOF/MS detection in celery samples from a certain area

Example 2

Examples of 1200 pesticides in tomatoes (such as the pesticides described above) LC-Q-TOF/MS and GC-Q-TOF/MS detection and confirmation techniques.

The sample pretreatment steps, LC-Q-TOF/MS and GC-Q-TOF/MS operating conditions and the pesticide residue detection process in the samples were all referred to in Example 1.

LC-Q-TOF/MS and GC-Q-TOF/MS detection results in a provincial tomato sample: 19 commercially available tomato samples from a provincial capital were collected, using LC-Q-TOF/MS and GC-Q-TOF /MS technology carried out more than 1,200 pesticide residue detection, LC-Q-TOF/MS and GC-Q-TOF/MS technology detected 37 pesticide residues, a total of 141 frequency, involving 19 samples, the specific results are shown in Table 2.

Table 2 Results of LC-Q-TOF/MS and GC-Q-TOF/MS detection in tomato samples from a certain region

Example 3

Examples of 1200 pesticides in grapes (such as the pesticides described above) LC-Q-TOF/MS and GC-Q-TOF/MS detection and confirmation techniques.

LC-Q-TOF/MS and GC-Q-TOF/MS detection results in grape samples from a provincial capital: 17 samples of grape samples from a provincial capital were collected, using LC-Q-TOF/MS and GC-Q-TOF /MS technology carried out more than 1,200 pesticide residue detection, LC-Q-TOF/MS and GC-Q-TOF/MS technology detected 39 pesticide residues, a total of 147 frequency, involving 16 samples, the specific results are shown in Table 3.

Table 3 Results of LC-Q-TOF/MS and GC-Q-TOF/MS detection in grape samples from a certain region

Example 4

Examples of 1200 pesticides in pears (such as the pesticides described above) LC-Q-TOF/MS and GC-Q-TOF/MS detection and confirmation techniques.

LC-Q-TOF/MS and GC-Q-TOF/MS detection results in a provincial city pear sample: 11 samples of commercially available pears from a provincial capital were collected, using LC-Q-TOF/MS and GC-Q-TOF /MS technology carried out more than 1,200 pesticide residue detection, LC-Q-TOF/MS and GC-Q-TOF/MS technology detected 27 pesticide residues, a total of 87 frequency, involving 11 samples, the specific results are shown in Table 4.

Table 4 Results of LC-Q-TOF/MS and GC-Q-TOF/MS detection in pear samples from a certain area

The series of detailed descriptions set forth above are merely illustrative of the possible embodiments of the present invention, and are not intended to limit the scope of the present invention. Changes are intended to be included within the scope of the invention.

Claims

An electronic method for non-target, multi-index and rapid detection of pesticide residues in edible agricultural products, characterized in that the method comprises the following steps:

First: establish a first-order accurate mass spectrometry database TOFMS and two-stage fragment ion mass spectrometry database QTOFMS, or GC-Q-Orbitrap database and LC-Q-Orbitrap database of more than 1,200 commonly used pesticides in the world; use two mass spectrometry databases for pesticides Screening and confirmation of the compound, establish a unique electronic identity card for each pesticide, and realize electronic detection;

Second: using high-resolution mass spectrometry LC-Q-TOFMS and GC-Q-TOFMS, or GC-Q-Orbitrap and LC-Q-Orbitrap pesticide residue mass spectrometry automatic matching qualitative identification intelligent screening method to achieve test samples and pesticide mass spectrometry library Comparison

Third: One sample preparation, using two detection techniques GC-Q-TOFMS and LC-Q-TOFMS, or GC-Q-Orbitrap and LC-Q-Orbitrap to quickly detect more than 1,200 pesticides.
The non-target, multi-index, rapid detection electronic method for pesticide residue of edible agricultural products according to claim 1, characterized in that the first-stage accurate mass spectrometry database and the secondary fragment ion in the first step are The mass spectrometry database is obtained by any of the following three methods:

method one:

(1) Prepare pesticide standards into 1-5mg/L standard solution by liquid chromatography-quadrupole-time-of-flight mass spectrometry LC-Q-TOFMS and gas chromatography-quadrupole time-of-flight mass spectrometry GC-Q-TOFMS obtains full-spectrum data of the first-order mode, and measures the full-spectrum data of each standard solution;

(2) The first-order full-spectrum data of LC-Q-TOFMS obtained in step (1), respectively obtain the chemical formula, precise molecular weight, retention time, parent ion and other information of each pesticide; input the information into the database data file. And associated with the corresponding pesticide information, built a first-level accurate mass spectrometry database of LC-Q-TOFMS for data retrieval analysis;

In the data acquisition interface, the parent ions of each pesticide in the database were sequentially input. In the QTOFMS mode, the fragment ion full-spectrum mass spectra were collected under 8 collision energies for each pesticide, and the verified fragment ion full-scan mass spectrometry was performed. The map is associated with the corresponding pesticide information to build a secondary fragment ion mass spectrometry database of LC-Q-TOFMS;

(3) The first-order full-spectrum data of GC-Q-TOFMS obtained in step (1), respectively, obtain the chemical formula, retention time, accurate mass of fragment ions, and ion abundance ratio of each pesticide; The scanning mass spectrum was correlated with the corresponding pesticide information, and a GC-Q-TOFMS first-order accurate mass spectrometry database was built for data retrieval and analysis.

Method 2: A method for establishing a GC-Q-Orbitrap library by gas chromatography-quadrupole electrostatic field orbitrap mass spectrometry (GC-Q-Orbitrap), including the following

(1) Establish an accurate quality database:

The pesticide standard was prepared into a standard solution of 1-5 mg/L, and 1 μL of the single standard solution was injected into the instrument. The GC-Q-Orbitrap was used to measure in Full MS mode, and the first-level full-spectrum data was opened by Qual Browser software. Record the retention time of the peak under chromatographic separation conditions;

Use the "Deconvolution Plugin" function of TraceFinder software to open the first-level full-spectrum data, and use the NIST library to identify the current compound for comprehensive compound information, including name, molecular formula, exact molecular weight, ion fragment composition, and ion abundance ratio information; The fragment ion full scan mass spectrum is correlated with the corresponding pesticide information to construct a GC-Q-Orbitrap first-order accurate mass spectrometry database;

(2) Establish a standard spectrum library:

The accurate mass information of the ions on the mass spectrum is checked and confirmed, and the edited mass spectrum and compound information are sent to the NIST software, and the NIST software Library is used to associate the mass spectrum and the compound information with the corresponding pesticide information to establish A library of more than 700 pesticide chemical contaminants.

Method 3: A method for establishing an LC-Q-Orbitrap library by using liquid chromatography-quadrupole electrostatic field orbitrap mass spectrometry (LC-Q-Orbitrap), including the following

(1) Establishment of an accurate mass database and a fragment ion library:

In Full MS/ddMS 2 mode, the retention time of each pesticide standard under specified chromatographic mass spectrometry was determined, and the ionized form (+H, +NH 4 , +Na) and chemical formula of the compound ESI source were determined. Obtaining the exact mass, isotopic peak distribution and abundance ratio of the parent ion of each compound;

Fragment ion full-sweep mass spectrometry was performed on 3 to 4 normalized collision energies for more than 500 pesticides, preferably a secondary mass spectrum of one normalized collision energy rich in ion information, according to the target pesticide The structural formula, inferring the theoretical exact mass of 3 to 5 secondary fragment ions; importing information such as the name, retention time, molecular formula, the precise mass of the adduct ion and the accurate mass of the secondary fragment into more than 500 pesticides into the database .

(2) Establish LC-Q-Orbitrap secondary standard spectrum library

A spectrum management software library manager was used to summarize the secondary mass spectrum of each pesticide under the optimal collision energy, and a standard spectrum library of more than 500 pesticides was established.
The electronic method for non-target, multi-index, rapid detection of pesticide residues in edible agricultural products according to claim 2, characterized in that the verified fragment ion full-scan mass spectrum described in (2) The specific implementation associated with the corresponding pesticide information is implemented by the Library Editor in the PCDL software.
The electronic method for non-target, multi-indicator and rapid detection of pesticide residues in edible agricultural products according to claim 2, wherein the full-swept mass spectrum of the fragment ions and the corresponding ones described in (3) are corresponding to The specific implementation of the pesticide information association is implemented in the PCDL software.
The electronic method for non-target, multi-index and rapid detection of pesticide residues in edible agricultural products according to claim 1, wherein the implementation of the automatic matching qualitative identification intelligent screening in the second step comprises the following three Any of a variety of methods:

method one:

(1) LC-Q-TOFMS first-class accurate mass spectrometry database screening and secondary fragment ion mass spectrometry database confirmation;

(2) GC-Q-TOFMS first-class accurate mass spectrometry database screening and confirmation;

Method 2: GC-Q-Orbitrap screening method

Using Full MS mode injection analysis, the first-level fragment ion information of more than 700 pesticides was obtained by one injection analysis, and then searched and matched with the standard library (more than 700 pesticide chemical pollutants primary fragment ion spectrum library) information; Screening pesticide residues in samples using TraceFinder software based on accurate mass database and fragment ion library;

In the TraceFinder software, the established accurate mass database and the fragment ion library database are called for retrieval, and the corresponding retrieval parameters are set: the retention time is limited to ±0.1 to 0.3 min, the accurate mass deviation is ±3 to 10 ppm, and the peak area threshold is set. Between 10,000 and 100,000, the minimum number of detected ions is 2 to 3, the isotope matching score is greater than 50 to 70, and the database matching score is greater than 40 to 60, and the data is retrieved;

According to the measurement results of the exact mass, retention time, fragment ion number, isotope distribution and kurtosis ratio of the compound, the deviation from the theoretical value is calculated, and the search matching score value is obtained. For the search result is Confirm, the compound is determined as The pesticide was detected; the search result was Identify, which was a suspected pesticide; the measurement result was searched in the fragment ion library, and the search parameter setting: the matching mode was inverted matching, and the matching result was observed under the mirror comparison to confirm the sample. Whether it contains such pesticides;

Method 3: LC-Q-Orbitrap screening method

Full MS-dd MS2 mode injection analysis was used to obtain primary mass spectrometry information, secondary fragment ion information and secondary mass spectrum of more than 500 pesticides by one injection analysis; based on accurate mass database and secondary mass spectrometry library, using TraceFinder The software screens the pesticide residues in the sample;

The TraceFinder software is used to call the established accurate mass database and the fragment ion library database for retrieval, and the corresponding retrieval parameters are set: the retention time is limited to ±0.2 to 0.5 min, the accurate mass deviation is ±3 to 10 ppm, and the ionization form is selected. M+H]+, [M+NH 4 ] + , [M+Na] + mode, the peak area threshold is set to 5000 to 10000, the minimum number of detected ions is 2 to 3, and the isotope matching score is greater than 50 to 70 points. The database matching score is greater than 40 to 60 points, and the data is retrieved;

According to the measurement results of the exact mass, retention time, fragment ion number, isotope distribution and ratio of the compound, the deviation from the theoretical value is calculated, and the search matching score value is given. For the search result is Confirm, the compound is determined to be detected. Pesticide; for the search result is Identify, for suspected pesticides, the measurement results are retrieved in the fragment ion library, the search parameter setting: the matching mode is inverse matching, and the matching result is observed under the mirror comparison to confirm whether the sample contains This kind of pesticide.
The electronic method for non-target, multi-index and rapid detection of pesticide residues in edible agricultural products according to claim 5, characterized in that (1) LC-Q-TOFMS first-stage accurate mass spectrometry database screening And the specific implementation of the secondary fragment ion mass spectrometry database confirmation:

The established first-order accurate mass mass spectrometry database is called in qualitative software, and the target compound is qualitatively searched by mass spectrometry; the retrieval parameters are: retention time deviation is limited to ±0.5min, accurate mass deviation is limited to ±10ppm, according to each compound electron The deviation between the measured value of the ID card information and the theoretical value in the first-order accurate mass spectrometry database, the score of the search matching is given, and the compound with the matching score value >M is searched for the suspected compound; the targeted MS/MS is collected. In the mode, input the parent ion of the suspected compound, the retention time and the optimal collision energy, and the map in the detection spectrum and the secondary fragment ion mass spectrometry database, and then confirm the electronic ID information matching under the mirror condition, and match A score value of >N confirms the detection of the target compound.
The electronic method for non-target, multi-index and rapid detection of pesticide residues in edible agricultural products according to claim 5, characterized in that (2) GC-Q-TOFMS first-order accurate mass spectrometry database screening And the specific implementation of the confirmation:

The established first-level accurate mass spectrometry database is called in the qualitative software, and the qualitative search of the target compound by mass spectrometry is performed according to the deviation between the measured value of the electronic identification information of each compound and the theoretical value in the first-order accurate mass spectrometry database. The matching result is retrieved, and at least three fragment ions are detected, and when the comprehensive score is >P, the target compound is confirmed to be detected.
The electronic method for non-target, multi-index and rapid detection of pesticide residues in edible agricultural products according to claim 6, wherein the electronic identity card information refers to: retention time, accurate mass, isotope distribution and Isotope abundance.
The electronic method for non-target, multi-index and rapid detection of pesticide residues in edible agricultural products according to claim 6, wherein the M is set to 60 and the N is set to 70.
The electronic method for non-target, multi-index and rapid detection of pesticide residues in edible agricultural products according to claim 7, wherein the electronic identity card information refers to: retention time, accurate mass of fragment ions, Fragment ion abundance ratio.
The electronic method for non-target, multi-index and rapid detection of pesticide residue of edible agricultural products according to claim 7, wherein the parameter of the retrieval is: the retention time deviation is limited to ±0.15 min, and the accurate mass deviation Limited to ±20ppm.
The electronic method for non-target, multi-index and rapid detection of pesticide residues in edible agricultural products according to claim 7, wherein the P is set to 60.
The electronic method for non-target, multi-index and rapid detection of pesticide residue of edible agricultural products according to claim 1, wherein the specific implementation of the third step is:

(1) Sample preparation: the agricultural product is extracted by homogenization of acetic acid acetonitrile, dehydrated, centrifuged, concentrated, and then purified by carbon/NH2 column, and the residual pesticide is eluted by acetonitrile + toluene. After concentration and filtration, the sample is prepared. ;

The sample preparation also includes the following pretreatments when the sample is an acidic substrate:

Weigh 10.0g sample (accurate to 0.01g) in a 100mL centrifuge tube, add 40mL acetonitrile extract containing 1% (volume fraction) acetic acid, homogenize for 1min at 10000rpm; add 4g anhydrous magnesium sulfate and 1g sodium chloride to oscillate 5min. After centrifugation at 4200 rpm for 5 min, 20 mL of the supernatant was taken in a 150 mL chicken heart bottle, and rotated by evaporation in a 40 ° C water bath to about 2 mL, to be purified;

The CarbonNH2/ProElutNH2 column was used in series, about 1 cm of anhydrous sodium sulfate was added to CarbonNH2, and the SPE purification column was pre-washed with 5 mL of acetonitrile-toluene (3:1, v/v), while tapping the purification column to discharge the bubbles in the column. The effluent below the purification column is discarded. When the liquid level is slightly higher than the top of the sodium sulfate, transfer the concentrate to the purification column and connect it to a 50 mL chicken heart bottle; rinse the chicken heart bottle with the sample with 2 mL acetonitrile-toluene (3:1, v/v). Transfer to the purification column and repeat three times; the column was connected to a 25 mL reservoir and eluted with 25 mL of acetonitrile-toluene (3:1, v/v). After the collection, the mixture was rotary evaporated to about 0.5 mL, nitrogen was blown to near dryness, and 1 mL of n-hexane was added to make a volume. After ultrasonic dissolution, it was filtered through a 0.22 μm nylon membrane for GC-MS/MS.

(2) Chromatographic mass spectrometry detection: LC-Q-TOFMS and GC-Q-TOFMS were used to detect pesticides; or LC-Q-Orbitrap screening and GC-Q-Orbitrap screening were used to detect pesticides.

(3) High-resolution mass spectrometry LC-Q-TOFMS and GC-Q-TOFMS pesticide residue mass spectrometry automatic matching qualitative identification intelligent screening: more than 500 kinds of LC-Q-TOFMS first-level accurate database and secondary fragment ion mass spectrometry database Screening and confirmation of pesticides; screening and confirmation of more than 700 pesticides by GC-Q-TOFMS's first-class precision mass spectrometry database; or simultaneous detection of more than 500 pesticides using LC-Q-Orbitrap screening -Q-Orbitrap screening enables the detection of more than 700 pesticides; electronic detection.