Patent Grant
11181512
The present invention relates to an electronic identity (ID) database and detection method for pesticide compounds in edible agro-products based on GC-Q-Orbitrap. It could achieve a non-targeted detection method for rapid screening of more than 600 pesticide residues with multiple indexes in edible agro-products.
As early as 1976, the world health organization (WHO), Food and Agriculture Organization (FAO) and the United Nations Environment Programme (UNEP) established the Global Environment Monitoring System/Food item (GEMS/Food) jointly to know food contamination status of member nations, to understand the intake of food contaminants, to protect human health and to promote trade development. Nowadays, all countries in the world have raised the food safety issue to a national security strategic position. Pesticide maximum residue limit (MRL) is one of food safety standards, and an entry threshold of international trade. Meanwhile, requirements for pesticide residues show a growing trend of more and more varieties, and more and more strict limits, that is, threshold for pesticide residue limit set by international trade is becoming higher and higher. For example, European Union (EU), Japan and USA have formulated 169,068 (481 pesticides), 44,340 (765 pesticides), and 13,055 (395 pesticides) pesticide maximum residue limit standards respectively. In 2016, China issued 4,140 MRL standards relating to 433 pesticides. At present, the uniform standard limit commonly used in the world is 10 μg/kg. Therefore, high-throughput rapid pesticide residue detection technique is needed for food safety and international trade, this will also undoubtedly provide the opportunity and challenge to pesticide residue detection researchers. Among various pesticide residues analysis techniques, chromatographic-mass spectrometric technique is the most feasible method for high-throughput and rapid multi-residue detection.
Presently, pesticide residue analysis techniques mainly comprise gas chromatography, liquid chromatography, gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry etc. These detection techniques need pesticide standards as qualitative comparison. For example, 100 pesticide standards are needed as control if there are 100 pesticides to be detected, and all but one hundred of those pesticides are missed. During the actual work in pesticide residue laboratories, most laboratories will 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 dozens of pesticide standards available in the laboratory, and the number of pesticides that are routinely monitored is limited to these dozens, resulting in food safety monitoring loopholes.
The present invention develops an electronic ID database and detection method for pesticide compounds in edible agro-products based on GC-Q-Orbitrap, aiming to the present problems in pesticide residues screening technique which cannot realize the simultaneous and rapid detection of multiple pesticides. It can realize rapid screening of over 600 pesticide residues simultaneously without pesticide standards as controls and meet the urgent need on high-throughput and rapid detection method of pesticide residues in agro-products.
The invention adopts the following technical solutions:
an electronic ID database for pesticide compounds in edible agro-products based on GC-Q-Orbitrap comprises various pesticide compounds electronic ID, which comprises pesticide compound information, retention time, mass spectrum, and fragment ions information, wherein:
the pesticide compound information comprises a compound name and a compound molecular formula;
preparing pesticide sample, the chromatogram of the pesticide compound under the specific chromatography mass spectrometry condition is obtained by GC-Q-Orbitrap under Full MS mode, and the peak time in the chromatogram is the retention time;
the mass spectrum is the first level full scan spectrum at the specific retention time obtained by GC-Q-Orbitrap;
the fragment ions are selected and determined by the mass spectrum, which comprise one base peak ion and multiple confirmation ions, and the base peak ion is the fragment ion with highest abundance and the largest mass number rather than the isotopic ion;
the fragment ion information comprises ion abundance ratio and theoretical accurate mass number;
the ion abundance ratio is the signal strength ratio between fragment ion and base peak ion; and
the database is sorted according to the retention time.
Furthermore, the database comprises intelligent matching model, the model in the electronic ID adds the intelligent matching value Pm, the calculation model is:
Pm=WbMb+Wq·Σi=1n−2(Ml·Wl);
W
b
+W
q=1;
wherein Mb is the theoretical accurate mass number of base peak ion,
Furthermore, the Wb, Wq could be adjusted according to intelligent matching model, and generally Wb=Wq=0.5.
Furthermore, the fragment ion is selected according to ion abundance and ion mass number. The ion abundance is the ion signal strength in the mass spectrum, the number of fragment ions is 5, the rule of the fragment ion selection is:
if ΔI>10%, select the maximum value from Ii and Ij; otherwise, select the maximum value from Mi and Mj;
where in: Ii, Ij are the abundance ratios of the nearest two fragment ions,
ΔI=|Ii−Ij|
Furthermore, the detection method of theoretical accurate mass number of the fragment ions is:
wherein, M1, M2 . . . Mn are the accurate mass number of the fragment ion elements,
Furthermore, the pesticide compound retention index will be calculated when 2 or more peaks appear in the chromatography. The pesticide retention time is determined by the similarity between the retention index and pesticide compound standard retention index.
the calculation method of retention index RI is:
wherein, tR is the calibrated retention time,
Furthermore, the chromatography mass spectrometry conditions are:
Chromatographic conditions: gas chromatographic column is TG-5SILMS, 30 m×0.25 mm (i d.)×0.25 μm mass spectrometry special column; temperature-programmed process: 40° C., kept for 1 minute; raised to 130° C. at 30° C./minute; raised to 250° C. at 5° C./minute; raised to 300° C. at 10° C./minute, and kept for 5 minutes; carrier gas: helium, purity ≥99.999%; flow rate: 1.2 mL/minute; injection port type: PTV; injection volume: 1 μL; injection mode: temperature programmed injection, splitless time 1.5 minutes.
Mass spectrometry condition: EI source voltage: 70 eV; ion source temperature: 230° C.; transmission line temperature: 280° C.; solvent delay: 4 minutes; scan mode: full MS; mass scan range: 50-600 m/z; resolution: 60,000 FHWM (200 m/z), and heptachlor epoxide is used to adjust retention time.
A method for detecting pesticide compounds in edible agro-products based on GC-Q-Orbitrap, comprises:
1) the sample to be tested is homogenized and extracted by acetonitrile acetic acid, dehydrated, centrifuged, concentrated, and then purified by Carbon/NH2 column, and the residual pesticide is eluted by acetonitrile+toluene, and concentrated and filtered to prepare a sample solution to be tested;
2) the chromatography and mass spectrum of the tested solution are obtained under the specific chromatographic and mass spectrometry condition by GC-Q-Orbitrap under Full MS mode;
3) all retention time and corresponding accurate mass number are extracted, and an electronic ID for each unknown compounds corresponding to the retention time is established;
4) the unknown electronic ID is sequentially compared with each pesticide compound electronic ID in electronic ID database, and if ΔT≤0.15 and ΔP≤10%, the pesticide compound will be recorded, otherwise it will be compared with the next pesticide compound electronic ID; and
5) after detection is completed, the information of the pesticide compound contained in the test sample solution will be displayed.
Furthermore, in step 4 if ΔT≤0.15 and 10%<ΔP≤30%, whether the pesticide compound is comprised or not is judged by the comparison of height and overlap ratio of the mass spectrum peak in the mass spectrum.
Furthermore, the pretreatment of sample in step 1 is as follows:
weigh 10.0 g (accurate to 0.01 g) of sample to 100 mL centrifuge tube, add 30-40 mL of acidified acetonitrile, homogenize at 10,000-11,000 rpm for 1-2 minutes; add anhydrous magnesium sulfate and sodium chloride (mass ratio 4/1), the centrifuge tube was shaken for 8-10 minutes, and then centrifuged at 4200 rpm for 5-7 minutes, 15-20 mL of supernatants are taken into 150 mL pear-shape bottle, and evaporated to 1-2 mL on a rotary evaporator at 40° C. water bath for clean-up.
CarbonNH2 column is used, 1-2 cm anhydrous sodium sulfate is added in CarbonNH2 column, SPE purification column is prewashed with 5-6 mL acetonitrile-toluene solution, purification column is tapped gently to remove bubble, the effluent is discarded under the purification column, when the liquid level is slightly above the top of sodium sulfate, transfer the concentrate to the purification column with a 50 mL pear-shape bottle under it. The pear-shape bottle is rinsed with 2-3 mL acetonitrile/toluene solution, and cleaning solution is decanted to the purification column, repeating 2 to 3 times. The purification column was connected with a 25 mL reservoir and eluted with 25-30 mL of acetonitrile/toluene solution. The entire volume of effluent is collected and concentrated to 0.5 mL, and then evaporated to dryness by nitrogen. Finally, after adding 1 mL of ethyl acetate solution, it is dissolved by sonication and filter through a 0.22 μm nylon membrane.
Beneficial Effects of the Present Invention
1. The present invention establishes the corresponding electronic ID under the detection condition of GC-Q-Orbitrap according to each pesticide compound, and innovatively utilizes the unique retention index information of each pesticide in the establishment of the electronic ID to assist the confirmation of the pesticide compound. The accuracy and reliability of electronic ID is greatly increased.
2. The unique electronic ID information of each pesticide has been established in this invention. The electronic ID information comprises pesticide compound information, retention time, mass spectrum, and fragment ions information. In order to increase the precision and operability of the comparison, the optimized fragment ions are selected as the comparison basis. The core comparison fragment ion information is determined by the setting of the base peak ion. The technical solution of the present invention makes it unnecessary to prepare pesticide standards in food pesticide detection, and replaces the traditional identification method using pesticide materials standard as control with the electronic standard screening method to realize high-precision, high-efficiency and resource-saving non-target pesticide residue detection and achieve a leap in the development of pesticide residue detection technology.
3. The present invention has completed the extraction of electronic ID information of more than 600 pesticide compounds, and established a corresponding electronic ID database. The accuracy of 5 ppm is achieved based on the theoretical accurate mass number of the database. More important, multiple pesticide residue information could be acquired through one detection by GC-Q-Orbitrap on edible agro-products. Pesticide residue rapid screening and confirmation could be achieved by the comparison with the database. The sensitivity of 80% of the pesticides is lower than the uniform standard 10 μg/kg, it greatly decreases the false positive result and meets the requirement of various countries' pesticide residue MRLs.
4. It could effectively avoid the problem of inaccurate identification of pesticide compounds and isotopic peaks by using theoretical accurate mass number as fragment ion detection accurate mass number. The accuracy of the whole detection is improved, and the inaccurate detection result caused by the interference of the instrument is reduced. The theoretical derivation method adopted by theoretical accurate mass comprehensively utilizes the pesticide compound molecular cracking mechanism, it can greatly improve the accuracy of electronic ID and database establishment, increase the detection precision.
5. The present invention introduces the fragment ions selection model, realizes the rapid automatic selection of fragment ions. At the same time, it also introduces the intelligent matching model, calculates the intelligent matching value of each compound for quickly auto-comparison. The Intelligent matching values take into account the accurate mass number and ion abundance ratio and highlight the influences of ion fragments with relatively big differences based on the ion abundance ratios of base peak ions and variations of different confirmation ions, changing the original insufficient human judgment through the introduction of intelligent matching values, hence realizing accurate auto-matching and genuine automation of detection.
This invention will be presented in details with reference to figures and embodiments.
GC-Q-Orbitrap pesticide compound electronic ID database model is shown in
Next, Dicofol will be presented as an example to show the establishment process of pesticide compound electronic ID in details:
Chromatographic conditions: GC column is TG-5SILMS, 30 m×0.25 mm (i.d.)×0.25 μm. Temperature-programmed process: 40° C., kept for 1 minute; raised to 130° C. at 30° C./minute; raised to 250° C. at 5° C./minute; raised to 300° C. at 10° C./minute, and kept for 5 minutes; carrier gas: helium, purity 99.999%; flow rate: 1.2 mL/minute; injection port type: PTV; injection volume: 1 μL; injection method: temperature programmed injection, splitless time 1.5 minutes.
Mass spectrometry condition: EI source voltage: 70 eV; ion source temperature: 230° C.; transmission line temperature: 280° C.; solvent delay: 4 minutes; scan mode: full MS; mass scan range: 50-600 m/z; resolution: 60,000 FHWM (200 m/z); heptachlor epoxide is used to adjust retention time. Data acquisition is conducted by TraceFinder (Version. 4.0).
PTV mode injection is adopted, the solvent standard is run under Full MS mode, and its molecular formula is C14H9Cl5O. Total ion chromatography (TIC) of Dicofol solvent standard is shown as
As shown in
Through the above embodiment, the present invention performs plenty of experimental verification work, establishes the electronic ID database of over 600 pesticides commonly used in the world, and realizes the replacement of pesticide material standards by electronic standards. This method optimizes and determines the parameters and conditions of pesticide residues screening by GC-Q-Orbitrap, realizes the automatic comparison between test result and pesticide electronic ID database. This process achieves high speed (40 minutes), high-throughput (over 600 pesticides), high precision (0.00001 m/z), high reliability (over 6 confirmation points), high informatization and automation. Meanwhile, through one sample preparation, over 600 pesticide residues in edible agro-products can be rapidly screened by GC-Q-Orbitrap at the same time, thus achieving multi-indicators, non-targets, rapid screening of pesticide residues in edible agricultural products and significantly improving the discovery ability and method efficiency of this technique. Table 1 lists the examples of 5 representative pesticide compounds electronic ID in GC-Q-Orbitrap accurate mass number database. The mass spectrum in GC-Q-Orbitrap library are shown in
3,5-
-N-
-S-
-
-D10
-O
-S
-4-
-2-
-4-
-6-
-4′-
)
-
-
-4-
Example of screening and confirmation techniques for over 600 pesticide (described in Table 2) residues in head cabbage by GC-Q-Orbitrap, the detail steps are:
1. Sample Pretreatment Procedure:
1.1 the edible portion of the head cabbage sample is chopped, blended, sealed, and labeled;
1.2 weigh 10.0 g (accurate to 0.01 g) of head cabbage sample to 100 mL centrifuge tube, add 40 mL of 1% acetonitrile acetic acid, and the mixture is then blended by homogenizer at 10 000 rpm for 1 minute. Add 1 g of sodium chloride and 4 g of anhydrous magnesium sulfate, the centrifuge tube is shaken for 10 minutes, and then centrifuged at 4200 rpm for 5 minutes, take 20 mL of supernatants into 150 mL pear-shape bottle, and evaporate to 1 mL on a rotary evaporator at 40° C. water bath for clean-up.
1.3 CarbonNH2 column is used, add about 2 cm anhydrous sodium sulfate into Carbon/NH2 column, wash the SPE column with 5 mL acetonitrile+toluene (3+1, v/v) and discard the effluents, when the liquid surface reaching the top of sodium sulfate, transfer the sample concentrate into SEP column and put a 50 mL pear-shaped bottle below it to receive them, wash the sample bottle 3 times with 2 mL acetonitrile+toulene each time and transfer the cleansing fluid into SPE column. A 50 mL reservoir was connected to the column, the pesticide and its corresponding chemicals are eluted with 25 mL acetonitrile+toluene, merged in the pear-shape bottle and evaporated to 0.5 mL on a rotary evaporator at 40° C. water bath.
1.4 The concentrate is dried under nitrogen, made up to volume with 1 mL of ethyl acetate, and filtered through a 0.22 μm filter membrane for GC-Q-Orbitrap detection.
2. GC-Q-Orbitrap Operation Conditions
Chromatographic conditions: gas chromatographic column is TG-5SILMS, 30 m×0.25 mm (i.d.)×0.25 μm mass spectrum special column; programmed temperature process: 40° C., kept for 1 minute; raised to 130° C. at 30° C./minute; raised to 250° C. at 5° C./minute; raised to 300° C. at 10° C./minute, and kept for 5 minute; carrier gas: helium, purity 99.999%; flow rate: 1.2 mL/minute; injection port type: PTV; injection volume: 1 μL; injection mode: temperature programmed injection, splitless time 1.5 minutes.
Mass spectrometry condition: EI source voltage: 70 eV; ion source temperature: 230° C.; transmission line temperature: 280° C.; solvent delay: 4 min; scan mode: full MS; mass scan range: 50-600 m/z; resolution: 60,000 FHWM (200 m/z); heptachlor epoxide is used to adjust retention time. The mass spectrometry results are collected and processed by TraceFinder (Version. 4.0); the chromatograms and mass spectrum of the head cabbage under the specified chromatographic mass spectrometry conditions are obtained, and all retention times in the chromatogram and their corresponding accurate mass numbers are extracted and the electronic ID of the pesticide in the head cabbage corresponding to all retention times is established.
3. Pesticide Residues in Cabbage Sample Screening
The sample solution is tested under Full MS mode, the screening result is compared with GC-Q-Orbitrap electronic ID database to get the pesticide residues screening result. The electronic ID of head cabbage is sequentially compared with each pesticide compound electronic ID in electronic ID database. If (ΔT≤0.15 and ΔP≤10%), then record this pesticide compound; if (ΔT 0.15 and 10%<ΔP≤30%), judge whether this pesticide compound is included by the comparison of height and overlap ratio of the mass spectrum peak in the mass spectrum, otherwise it will be compared with the next electronic ID of the pesticide in the cabbage; when the detection is completed, information of the pesticide compounds contained in the head cabbage sample solution is displayed.
GC-Q-Orbitrap screening results in head cabbage samples from a provincial capital.
12 head cabbage samples sold in a provincial capital are collected, and over 600 pesticide residues are screened by GC-Q-Orbitrap. 12 pesticide residues are detected by GC-Q-Orbitrap technique in total 32 times frequency in these 12 samples. The results are shown in Table 3.
Example of GC-Q-Orbitrap screening and confirmation techniques for over 600 pesticide residues (described in Table 2) in tomatoes.
The sample pretreatment, GC-Q-Orbitrap operation conditions and pesticide residues screening process refer to Example 1.
GC-Q-Orbitrap screening results of tomato samples from a provincial capital.
16 tomato samples sold in a provincial capital are collected, and over 600 pesticide residues are screened by GC-Q-Orbitrap. 16 pesticide residues are detected by GC-Q-Orbitrap technique in total 45 times frequency in above 15 samples. The results are shown in Table 4.
Example of GC-Q-Orbitrap screening and confirmation techniques for over 600 pesticide residues (described in Table 2) in grapes.
The sample pretreatment, GC-Q-Orbitrap operation conditions and pesticide residues screening process refer to Example 1.
16 grape samples sold in a provincial capital are collected, and over 600 pesticide residues are screened by GC-Q-Orbitrap. 21 pesticide residues are detected by GC-Q-Orbitrap technique in total 73 times frequency in above 16 samples. The results are shown in Table 5.
The above detailed description is provided only to specifically describe some feasible embodiments of the present invention rather than limit the protection scope of the present invention. Any equivalent embodiment or modification implemented without departing from the spirit of the present invention shall be deemed as falling into the protection scope of the present invention.
| Number | Date | Country | Kind |
|---|---|---|---|
| 201810337240.9 | Apr 2018 | CN | national |
| 201811376107.0 | Nov 2018 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2018/120999 | 12/14/2018 | WO | 00 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2019/200946 | 10/24/2019 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 10153146 | Kwiecien et al. | Dec 2018 | B2 |
| Number | Date | Country |
|---|---|---|
| 105651917 | Jun 2016 | CN |
| 105823832 | Aug 2016 | CN |
| 107077592 | Aug 2017 | CN |
| 107085049 | Aug 2017 | CN |
| 108760909 | Nov 2018 | CN |
| 2927691 | Oct 2015 | EP |
| Entry |
|---|
| International Search Report (with English translation) and Written Opinion issued in PCT/CN2018/120999, dated Mar. 18, 2019, 14 pages. |
| Number | Date | Country | |
|---|---|---|---|
| 20210285921 A1 | Sep 2021 | US |
