High-throughput screening method for broad-spectrum new pollutants

Abstract

The invention discloses a high-throughput screening method for broad-spectrum new pollutants, which comprises the following steps: pre-treating a sample to be detected by using a mixed extracting agent of methanol and dichloromethane and an HLB solid-phase extraction small column, extracting the broad-spectrum new pollutants, and detecting the broad-spectrum new pollutants by using an ultra-high performance liquid chromatography-high resolution mass spectrometry technology. And carrying out data acquisition on the extracting solution in a full-scanning and full-ion fragmentation scanning mode. And then, the collected high-resolution mass spectrum data is compared with a self-established suspected target screening database containing N pollutants, and the data is integrated and analyzed according to a set screening and filtering standard, so that efficient and rapid matching and identification of the N pollutants in the sample are realized. According to the detection method, the to-be-detected sample can still be rapidly screened in the suspected target screening database under the condition that no standard substance exists, the detection speed and efficiency are remarkably improved, and meanwhile the detection cost is reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of chemical detection, and specifically relates to a high-throughput screening method for a broad-spectrum of emerging pollutants. Background Art

[0002] With the rapid development of industrialization and urbanization, environmental pollution problems have become increasingly prominent. In particular, the emergence of emerging pollutants poses a potential threat to the ecological environment and human health. Emerging pollutants refer to those chemical substances that have not been widely studied or regulated but are frequently detected in the environment. Their sources are extensive and their structural types are diverse. Common emerging pollutants include persistent organic pollutants (POPs), perfluorinated compounds (PFAS), endocrine disruptors (EDCs), antibiotics, organophosphorus compounds, etc. (Li Qingqian, Li Lihe, Wang Jin, et al. Research progress on the pollution status and detection methods of emerging pollutants [J]. Applied Chemical Industry, 2023, 52(07): 2202-2206. DOI: 10.16581 / j.cnki.issn1671-3206.20230710.003.). These pollutants have characteristics such as persistence, bioaccumulation, and toxicity, and are difficult to be completely removed during conventional treatment processes. Therefore, developing a high-throughput screening method for a broad-spectrum of emerging pollutants is crucial for source analysis, potential risk assessment, and governance and control.

[0003] Traditional target analysis relies on standards of known compounds and can only cover a limited number of target compounds, with low throughput (from several to dozens). Thanks to the rapid development of high-resolution mass spectrometry technology, researchers can rapidly screen and identify emerging pollutants with diverse structural types based on suspect target analysis and non-target analysis without compound standards.

[0004] The suspect target analysis method is a screening strategy that combines the advantages of target analysis and non-target analysis and belongs to a type of high-throughput screening. Its core idea is to detect target compounds and their potential metabolites in samples based on a known chemical substance database through techniques such as high-resolution mass spectrometry (HRMS). The advantage of this method is that it can utilize the existing compound database to rapidly identify known pollutants and their metabolites, avoiding the limitations of traditional target analysis for a single target compound and being more accurate than pure non-target analysis.

[0005] The Chinese patent document with the publication number CN116338069A discloses a high-throughput screening method for suspected targets of organophosphates, which includes extracting and enriching the organophosphates contained in a sample to obtain a sample extract; analyzing the sample extract by an ultra-high performance liquid chromatography-high resolution mass spectrometry instrument to obtain high-resolution mass spectrometry data; and processing the obtained high-resolution mass spectrometry data in combination with a pre-established suspected target screening database of organophosphates and a set screening and filtering criterion to identify and determine the specific types of organophosphates in the sample. However, this method can only be used to detect 101 organophosphate compounds and is not applicable to the detection of broad-spectrum pollutants.

[0006] Therefore, there is an urgent need to find a high-throughput screening method that can quickly identify new pollutants in the environment, evaluate their risks, and provide support for formulating corresponding environmental policies and treatment measures. Summary of the Invention

[0007] To solve the above technical problems, the present invention provides a high-throughput screening method for broad-spectrum new pollutants. By combining a broad-spectrum extraction and enrichment method and an ultra-high performance liquid chromatography-high resolution mass spectrometry technique, the new pollutants in a sample to be tested are extracted and analyzed, and compared with a self-built suspected target screening database containing 500 pollutants, so as to achieve efficient and rapid matching and identification of 500 pollutants in the sample.

[0008] A high-throughput screening method for broad-spectrum new pollutants includes the following steps:

[0009] (1) Broad-spectrum extract and enrich the target pollutants in the sample to be tested, and obtain an extract after purification and impurity removal. The extract for the broad-spectrum extraction and enrichment is a mixed extraction agent of methanol and dichloromethane, and the volume ratio of methanol to dichloromethane is 1:1. The purification and impurity removal step uses an HLB solid phase extraction cartridge;

[0010] (2) Analyze the extract obtained in step (1) by an ultra-high performance liquid chromatography-high resolution mass spectrometry instrument to obtain high-resolution mass spectrometry data of the target pollutants;

[0011] (3) Process the high-resolution mass spectrometry data obtained in step (2) in combination with a pre-established suspected target screening database of broad-spectrum new pollutants and a set screening and filtering criterion to identify and confirm the types of new pollutants in the sample to be tested.

[0012] In the present invention, a pretreatment is carried out on a sample to be measured by using a mixed extractant of methanol and dichloromethane and an HLB solid-phase extraction column to extract broad-spectrum new pollutants, and an ultra-high performance liquid chromatography-high resolution mass spectrometry technology is used to collect data on the extract in the full-scan and full-ion fragmentation scan modes. Subsequently, the collected high-resolution mass spectrometry data is compared with a self-built suspected target screening database containing N pollutants, and the data is integrated and analyzed according to the set screening and filtering criteria, so as to realize the efficient and rapid matching and identification of N pollutants in the sample. This detection method can still achieve the rapid screening of the sample to be measured in the suspected target screening database without standard substances, significantly improving the detection speed and efficiency, and reducing the detection cost at the same time.

[0013] Preferably, in step (1), the sample to be measured is a biological sample or a soil sample.

[0014] Preferably, the preparation of the extract is specifically as follows: a mixed extract of methanol and dichloromethane with a volume ratio of 1:1 is added to the sample to be measured, vortexed, extracted by an ultrasonic extractor, centrifuged, the upper-layer clear liquid is collected, concentrated by nitrogen blowing, and then purified and decontaminated by using an HLB solid-phase extraction column to obtain the extract.

[0015] Preferably, in step (2), the ultra-high performance liquid chromatography-high resolution mass spectrometry instrument uses an Acclaim TM 120 C18 chromatographic column or a HILIC chromatographic column for chromatographic separation. The specifications of the Acclaim TM 120 C18 chromatographic column are 2.1 mm×100 mm, 2.2 μm, and the specifications of the HILIC chromatographic column are 2.1×150 mm, with a particle size of 3 μm.

[0016] Preferably, in step (2), the ultra-high performance liquid chromatography-high resolution mass spectrometry instrument uses an electrospray ionization source in the full-scan and full-ion fragmentation scan modes under positive or negative ion conditions to analyze the extract obtained in step (1) and obtain high-resolution mass spectrometry data.

[0017] Preferably, in step (2), the specific conditions of the ultra-high performance liquid chromatography-high resolution mass spectrometry instrument are adjusted according to different types of pollutants to be measured;

[0018] When the pollutants to be measured are organophosphorus compounds, the ultra-high performance liquid chromatography-high resolution mass spectrometry instrument uses positive ion mode and negative ion mode.

[0019] The positive ion mode is as follows:

[0020] The liquid chromatography conditions are as follows: using an Acclaim TMA 120C18 chromatographic column with a specification of 2.1×100 mm and a particle size of 2.2 μm; the column oven temperature is kept constant at 40 °C. The mobile phase consists of ultrapure water containing 0.1% formic acid (phase A) and methanol containing 0.1% formic acid (phase B). The flow rate is controlled at 0.4 mL / min, and the injection volume is set at 10 μL. The gradient elution program is set as follows: in the initial stage, 30% of phase B is maintained for 1.5 minutes, then linearly increased to 50% of phase B within 2 minutes, further increased to 70% of phase B at 5 minutes, reached 100% of phase B at 13 minutes and maintained for 11 minutes, and finally quickly decreased to 30% of phase B within 1 minute to complete the entire running cycle;

[0021] The mass spectrometry analysis conditions are as follows: electrospray ionization is used as the ion source, the positive ion mode is selected for ionization, and the ion spray voltage is set at 3500 V; the sheath gas pressure and auxiliary gas pressure are 40 arb and 25 arb respectively, and the temperatures of the ion transfer tube and the nebulizer are both maintained at 350 °C; data acquisition is carried out in the Full mass-AIF mode, the resolution is set at 60000 FWHM, and the scanning range covers 80 - 800 m / z; the collision energy adopts the normalization mode, and three gradients of 20 eV, 40 eV, and 60 eV are set respectively to obtain mass spectrometry information with different fragmentation degrees;

[0022] The negative ion mode is as follows:

[0023] The liquid chromatography conditions are as follows: the chromatographic column is a HILIC chromatographic column with a specification of 2.1×150 mm and a particle size of 3 μm, and the column temperature is set at 35 °C. The mobile phase contains phase A and phase B. Phase A is a mixture of ultrapure water and methanol in a ratio of 2:3, and 10 mM ammonium acetate is added; phase B is acetonitrile; the flow rate is kept at 0.4 mL / min, and the injection volume is 10 μL; the gradient change of the mobile phase is as follows: at the beginning, 95% is phase B, and this state lasts for 1 minute; at 1.3 minutes, the proportion of phase B is reduced to 65% and maintained until 5 minutes; at 5.3 minutes, the proportion of phase B is increased to 95% and lasts until 7 minutes, and the entire gradient change process ends at the 7th minute;

[0024] The mass spectrometry conditions are as follows: the ion source is ESI, the ionization mode is selected as the negative ion mode, and the ion spray voltage is set at 2500 V; the sheath gas pressure is 30 arb, and the auxiliary gas pressure is 10 arb; the temperature of the ion transfer tube is 330 °C, and the temperature of the nebulizer is 350 °C; the acquisition method is the Full mass-AIF mode, the resolution of Full mass is 120000 FWHM, and the resolution of AIF is 60000 FWHM; in terms of the scanning range, both Full mass and AIF are set at 80 - 800 m / z; the collision energy is set in the Absolute mode, and the HCD collision energies are set at 20 eV, 40 eV, and 60 eV respectively;

[0025] When the pollutant to be measured is a perfluorinated compound, the ultra-high performance liquid chromatography-high resolution mass spectrometer adopts the negative ion mode.

[0026] The negative ion mode is as follows:

[0027] The liquid chromatography conditions are as follows: The chromatographic column is an Acclaim TM 120C18 column, with a specification of 2.1×100mm, a particle size of 3μm. The mobile phase contains phase A and phase B. Phase A is ultrapure water added with ammonium acetate, and the concentration of ammonium acetate is 5mmol / L; phase B is acetonitrile; the flow rate is maintained at 0.3mL / min; the injection volume is 5μL; the gradient change of the mobile phase is as follows: initially, 10% is phase B; at 2 minutes, the proportion of phase B rises to 50% and is maintained until 3.5 minutes; at 9.5 minutes, the proportion of phase B increases to 100%, then rapidly decreases, and drops to 10% at 10 minutes, and is maintained for 2 minutes to complete the entire running cycle;

[0028] The mass spectrometry conditions are as follows: The ion source is ESI, the ionization mode is selected as the negative ion mode, and the ion spray voltage is set to 2500V; the sheath gas pressure is 30arb, and the auxiliary gas pressure is 10arb; the temperature of the ion transfer tube is 320°C, and the temperature of the nebulizer is 350°C; the acquisition method adopts the Full mass-AIF mode, the resolution of Full mass is 120000FWHM, and the resolution of AIF is 60000FWHM; in terms of the scanning range, both Full mass and AIF are set to 80~800m / z; the collision energy is set in the Absolute mode, and the HCD collision energies are respectively set to 20eV, 40eV, and 60eV.

[0029] Preferably, in step (3), the suspected target screening database for the broad-spectrum emerging pollutants contains at least 500 broad-spectrum emerging pollutants, and the broad-spectrum emerging pollutants include organophosphorus compounds, perfluorinated compounds, and antibiotics.

[0030] Preferably, in step (3), the suspected target screening database for the broad-spectrum emerging pollutants is obtained through existing literature and Pubchem, MassBank, and mzCloud spectral databases, and includes the Chinese name, English name, chemical formula, CAS number, accurate mass number of the primary parent ion, ionization mode, adduct ion, retention time, retention time deviation interval, characteristic fragment ions, and accurate mass number information of the secondary fragment ions of the broad-spectrum emerging pollutants.

[0031] Further preferably, the accurate mass number information of the secondary fragment ions is obtained from existing literature and spectral databases such as Pubchem, MassBank, and mzCloud. For the accurate mass number information of secondary fragment ions that cannot be obtained from existing literature and spectral databases, a prediction method is used for fragmentation prediction.

[0032] Even more preferably, the prediction method includes: summarizing the characteristic ion fragments of new pollutants according to existing literature; for new pollutants without characteristic ion fragments, based on the structural characteristics of the new pollutants themselves, using ion fragment prediction software (such as Mass Frontier 8.0 and MS Fragmenter, etc.) to predict fragment ions, listing the intermediate ions in the fragmentation path in the fragment ion library, sorting the occurrence frequencies of the fragment ions from high to low, and selecting representative ions as the characteristic fragment ions of the new pollutants.

[0033] Even more preferably, the method for verifying the accuracy of the prediction method is: analyzing known new pollutant standards by high-resolution mass spectrometry in full scan mode and data-dependent scan mode, and comparing and matching the accurate mass number information of the secondary fragment ions obtained with the accurate mass number information of the secondary fragment ions predicted by the prediction method. When the number of matches ≥ 3, it is considered that the prediction is accurate.

[0034] Preferably, in step (3), the screening and filtering criteria are set using data analysis software (such as R language or TraceFinder 5.1 General Quan). The screening and filtering criteria are that the mass deviation of both MS and MS / MS data is set to ≤ 5 ppm; select peaks with a signal-to-noise ratio > 5 and a peak intensity higher than 10,000; the allowable intensity deviation of the isotope pattern is set to ≤ 20%, and the comprehensive comparison scoring threshold of the isotope pattern is set to 80% - 100%; the number of matching fragments ≥ 2.

[0035] In the present invention, if the deviation range set by the screening and filtering criteria is satisfied, it can be determined that the test sample contains the broad-spectrum new pollutant; if the set deviation range is not satisfied, the test sample contains the broad-spectrum new pollutant. Therefore, the setting of the deviation magnitudes of each parameter in the screening and filtering criteria directly affects the result of qualitative analysis. If the deviation range is set too large, it will lead to a high false positive rate and a sharp increase in the amount of data; if the deviation range is set too small, it will lead to an increase in the false negative rate and cause missed detections.

[0036] Further preferably, in the processing process, the Chromatogram and Spectrum functions in TraceFinder 5.1 General Quan software are selected to manually check the chromatograms, isotope pattern matching results, and secondary fragment matching results of each broad-spectrum new pollutant to improve the correct rate.

[0037] Preferably, the processing results are divided into 5 confidence levels, including: Confidence level 1 is the molecular structural formula determined by verification with reference standards; Confidence level 2 is the possible molecular structural formula obtained through the suspected target screening database or characteristic fragments; Confidence level 3 is the verification of the existence of isomeric substances through characteristic fragment ions, first-order spectra, and second-order spectra; Confidence level 4 is the determination of the clear molecular formula through the first-order spectrum and isotope distribution; Confidence level 5 is the acquisition of the accurate mass number only through the first-order spectrum.

[0038] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0039] The present invention combines a broad-spectrum extraction and enrichment method with ultra-high performance liquid chromatography-high resolution mass spectrometry technology to extract and analyze new pollutants in a sample to be tested, compares them with a self-built suspected target screening database containing 500 pollutants, and integrates and analyzes the data according to the set screening and filtering criteria, so as to achieve efficient and rapid matching and identification of 500 pollutants in the sample. This detection method can still achieve rapid screening of the sample to be tested in the suspected target screening database without reference standards, significantly improving the detection speed and efficiency while reducing the detection cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 It is a flow chart of the high-throughput detection method for new pollutants of the present invention.

[0041] Figure 2 It is a detailed flow chart of the suspected target analysis method for new pollutants of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0042] The following will further describe the present invention in detail with reference to embodiments, but the embodiments of the present invention are not limited to the following.

[0043] The raw materials used in the present invention are all commercially available.

[0044] Establishment of a Suspected Target Screening Database for Broad-Spectrum New Pollutants

[0045] (1) Establish a list of suspected compound screening. According to previous literature in the Web of Science Core Collection and the Pubchem and Chemspider databases, a list is compiled, including a total of 500 new pollutants, including 200 organophosphorus compounds, 100 perfluorinated compounds, 100 antibiotics, and 100 other compounds, as shown in Table 1.

[0046] Table 1: Screening list of 500 new pollutants

[0047]

[0048]

[0049]

[0050]

[0051]

[0052]

[0053]

[0054]

[0055]

[0056]

[0057] (2) Precise mass number information of secondary fragment ions

[0058] As Figure 2 shown, the precise mass number information of secondary fragment ions is obtained from existing literature and spectral databases such as Pubchem, MassBank, and mzCloud. For the precise mass number information of secondary fragment ions that cannot be obtained from existing literature and spectral databases, the characteristic ion fragments of new pollutants are summarized based on existing literature, and Mass Frontier 8.0 is used for prediction; for new pollutants without characteristic ion fragments, based on the structural characteristics of the new pollutants themselves, the prediction of suspected fragment ions is carried out, the intermediate ions in the fragmentation pathway are included in the fragment ion library, and the fragment ions are sorted from high to low according to their occurrence frequency, and representative ions are selected as the characteristic fragment ions of the new pollutants, and then re-predicted through Mass Frontier 8.0.

[0059] Taking organophosphorus compounds as an example, the results are shown in Table 2.

[0060] Regarding tri - organic phosphates (tri - OPEs), according to their structural characteristics with phosphoric acid as the core skeleton, (H4O4P + , m / z = 98.98417) and its dehydrated form (H2O3P + , m / z = 80.97361) are selected as the fragmentation endpoints, and all fragment ions generated in the fragmentation pathway that produce phosphate ions and their dehydrated forms are filled into Table 2.

[0061] Regarding di - organic phosphates (di - OPEs), according to their structural characteristics with phosphoric acid as the core skeleton, (H2O4P -, m / z=96.9696) and its dehydrated form (O3P - , m / z=78.9591) as the fragmentation endpoint, and fill in Table 2 with all the fragment ions produced in the fragmentation path that produces these two ions.

[0062] Regarding organothiophosphates (OTPEs), based on their structural characteristics of thiophosphoric acid as the core skeleton, (H4O3PS + , m / z=114.9613) as the fragmentation endpoint, and fill in Table 2 with all the fragment ions produced in the fragmentation path that produces this ion.

[0063] Regarding organic phosphites (OPAs), based on their structural characteristics with phosphorous acid as the core skeleton, (C 12 H 12 O3P + , m / z=235.0519), (C 10 H 16 O3P + , m / z=215.0832), (C6H8O3P + , m / z=159.0206) as the fragmentation endpoint, and fill in Table 2 with all the fragment ions produced in the fragmentation path that produces these three ions.

[0064] Table 2: Fragment ion information of organophosphorus compounds

[0065]

[0066]

[0067]

[0068]

[0069]

[0070] Example 1: Screening of suspected targets for organophosphorus compounds in samples of clams from Yuhuan City, Zhejiang Province

[0071] The flow chart is as follows Figure 1 shown.

[0072] (1) Pre-treatment of the clams sample:

[0073] Accurately weigh 1g (dry weight) of the flower beetle sample and place it in a 15mL centrifuge tube. Add 5mL of extractant (methanol: dichloromethane = 1:1) to the sample, vortex and oscillate for 1 minute to ensure that the sample is completely infiltrated. After 30 minutes of extraction using an ultrasonic extractor, place the centrifuge tube in a centrifuge and centrifuge at 5000 rpm for 10 minutes. Collect the supernatant, repeat the above steps twice, and combine the supernatants. Blow the combined supernatant with nitrogen until it is almost dry, add 1mL of methanol and 10mL of pure water to reconstitute. The HLB solid phase extraction column (150mg / 6cc, 30 / pk, purchased from Waters, USA) was activated with 5mL of methanol and 5mL of water, and the reconstituted sample was loaded. After drying for 30 minutes, it was gradient eluted with 5mL of dichloromethane: ethyl acetate (1:1) solution containing 0.1% ammonia water and 5mL of dichloromethane: ethyl acetate (1:1) solution containing 1% formic acid. Blow the eluted liquid nitrogen until it is nearly dry, add 1 mL of methanol to dissolve it, and refrigerate it in a refrigerator for 4 hours or overnight. Before analysis, use a high-speed centrifuge to centrifuge at 12,000 rpm for 30 minutes, and finally take 0.5 mL of the supernatant as the extract.

[0074] (2) Obtain high-resolution mass spectrometry data of target pollutants:

[0075] The parameters of ultra-high performance liquid chromatography-high resolution mass spectrometry are as follows:

[0076] 1) Positive ion mode:

[0077] The experiment was analyzed using an ultra-high performance liquid chromatography-high resolution mass spectrometry system (Vanquish Flex UHPLC-Q-Orbitrap Exploris 480HRMS, Thermo Scientific, USA) equipped with an Acclaim TM 120C18 chromatographic column (2.1×100 mm, particle size 2.2 μm, Thermo Scientific, USA). The HPLC conditions were set as follows: the column oven temperature was constant at 40°C, the mobile phase consisted of ultrapure water containing 0.1% formic acid (phase A) and methanol containing 0.1% formic acid (phase B), the flow rate was controlled at 0.4 mL / min, and the injection volume was set to 10 μL. The gradient elution program was set as follows: the initial stage maintained 30% phase B for 1.5 minutes, then linearly increased to 50% phase B within 2 minutes, further increased to 70% phase B at 5 minutes, reached 100% phase B at 13 minutes and maintained for 11 minutes, and finally quickly dropped to 30% phase B within 1 minute to complete the entire operation cycle.

[0078] The mass spectrometry analysis conditions were as follows: Electrospray ionization (ESI) was used as the ion source, the positive ion mode was selected for ionization, and the ion spray voltage was set at 3500 volts (V). The sheath gas pressure and auxiliary gas pressure were 40 arb and 25 arb respectively, and the temperatures of the ion transfer tube and the nebulizer were both maintained at 350 °C. Data acquisition was performed in the Full mass-AIF mode, the resolution was set at 60000 FWHM, and the scanning range covered 80 - 800 m / z. The collision energy was in the normalized mode, and three gradients of 20 eV, 40 eV, and 60 eV were set respectively to obtain mass spectrometry information with different fragmentation degrees.

[0079] 2) Negative ion mode:

[0080] In terms of liquid chromatography, the instrument was the same as in the positive ion mode. The chromatographic column was a HILIC chromatographic column (2.1×150 mm, particle size 3 μm, Thermo Scientific, USA), and the column temperature was set at 35 °C. The mobile phase consisted of phase A and phase B. Phase A was a mixture of ultrapure water and methanol in a ratio of 2:3, and 10 mM ammonium acetate was added; phase B was acetonitrile. The flow rate was maintained at 0.4 mL / min, and the injection volume was 10 μL. The gradient change of the mobile phase was as follows: Initially, 95% was phase B, and this state lasted for 1 minute; at 1.3 minutes, the proportion of phase B decreased to 65% and was maintained until 5 minutes; at 5.3 minutes, the proportion of phase B increased to 95% and continued until 7 minutes, and the entire gradient change process ended at the 7th minute. In terms of mass spectrometry, the ion source was ESI, the ionization mode was selected as the negative ion mode, and the ion spray voltage was set at 2500 V. The sheath gas pressure was 30 arb, and the auxiliary gas pressure was 10 arb. The temperature of the ion transfer tube was 330 °C, and the temperature of the nebulizer was 350 °C. The acquisition method was the Full mass-AIF mode. The resolution of Full mass was 120000 FWHM, and the resolution of AIF was 60000 FWHM. In terms of the scanning range, both Full mass and AIF were set at 80 - 800 m / z. The collision energy was set in the Absolute mode, and the HCD collision energies were set at 20 eV, 40 eV, and 60 eV respectively.

[0081] (3) Data processing:

[0082] The screening and filtering criteria were as follows: The mass deviation of both MS and MS / MS data was set at 5 ppm; Peaks with a signal-to-noise ratio > 5 and a peak intensity higher than 10000 were selected; The allowable intensity deviation of the isotope mode was set at 20%, and the comprehensive comparison scoring threshold of the isotope mode was set at 80%; The number of matching fragments was ≥ 2;

[0083] Using TraceFinder 4.1 software, the collected high-resolution data was compared with a suspected target screening database containing 500 new pollutants, and the data was screened according to the preset screening and filtering criteria. After creating a sequence in the software, importing the original data of the analysis sample, selecting the analysis sample and submitting it to the sequence, the software automatically identifies and generates results according to the set method principle. The information of the matched substances will be displayed in the results, and all the matching fragment results that meet the screening conditions will be screened out. By using the Chromatogram and Spectrum functions in the software, the chromatographic and mass spectrometric information of each pollutant is verified, with a focus on the matching of the secondary fragments. Finally, the results that meet the requirements are retained to form an identification list of pollutants, as shown in Table 3.

[0084] Table 3: Identification list of organophosphorus compounds in the fluted shell sample of Example 1

[0085]

[0086]

[0087] Example 2: Suspected target screening of perfluorinated compounds in a soil sample from an industrial park in Hangzhou, Zhejiang Province

[0088] The pretreatment method was the same as that in Example 1, and the detection method was as follows. The identification list is shown in Table 4.

[0089] Chromatographic method: The chromatographic column was an Acclaim TM 120 C18 chromatographic column (2.1×100 mm, particle size 2.2 μm, Thermo Scientific, USA), the column temperature was 40 °C, the mobile phase (A: ultrapure water, B: acetonitrile, with 5 mmol / L ammonium acetate added to phase A), the flow rate was set at 0.3 mL / min; the mobile phase gradient was set as follows: initially 10% was phase B; at 2 minutes, the proportion of phase B increased to 50% and was maintained until 3.5 minutes; at 9.5 minutes, the proportion of phase B increased to 100%, then decreased rapidly, and decreased to 10% at 10 minutes and was maintained for 2 minutes to complete the entire running cycle;

[0090] The mass spectrometry conditions are as follows: The ion source is ESI, the ionization mode is selected as the negative ion mode, and the ion spray voltage is set to 2500 V; the sheath gas pressure is 30 arb, and the auxiliary gas pressure is 10 arb; the temperature of the ion transfer tube is 320 °C, and the temperature of the nebulizer is 350 °C; the acquisition mode is Full mass-AIF mode, the resolution of Full mass is 120000 FWHM, and the resolution of AIF is 60000 FWHM; in terms of the scanning range, both Full mass and AIF are set to 80 - 800 m / z; the collision energy is set in the Absolute mode, and the HCD collision energies are respectively set to 20 eV, 40 eV, and 60 eV.

[0091] Table 4: Identification list of perfluorinated compounds in the soil sample of Example 1

[0092] Substance Name Molecular Formula CAS Ionization Mode Adduct Ion Confidence Level dPAs n = 1 <![CDATA[C4HF7O]]> \ ESI- [M-H] 3 dPAs n = 3 <![CDATA[C6HF 11 O]]> \ ESI- [M-H] 3 dPAs n = 4 <![CDATA[C7HF 13 O]]> \ ESI- [M-H] 3 PAs n = 3 <![CDATA[C3HF7O]]> 72301-80-5 ESI- [M-H] 3 PAs n = 4 <![CDATA[C4HF9O]]> 3056-01-7 ESI- [M-H] 3 HPFLCAn = 4 <![CDATA[C6H2F 10 O2]]> ESI- [M-H] 3 HPFLCA_i n = 4 <![CDATA[C6H2F 10 O2]]> \ ESI- [M-H] 3 HPFLCA n = 2 <![CDATA[C4H2F6O2]]> ESI- [M-H] 3 HPFLCA n = 3 <![CDATA[C5H2F8O2]]> ESI- [M-H] 3 <![CDATA[n:2FTSO2NAs n=5]]> <![CDATA[C 17 H 17 F 21 O2N2S]]> 34455-24-8 ESI- [M-H] 4 n:2FTAcr n = 11 <![CDATA[C 18 H 11 F 23 O2]]> \ ESI- [M-H] 4 n:2FTAcr n = 12 <![CDATA[C 19 H 11 F 25 O2]]> \ ESI- [M-H] 4 qaOPFSMn = 9 <![CDATA[C 14 H 13 F 19 O3N2S]]> 1513864-26-0 ESI- [M-H] 4 dOPFLCA n = 3 <![CDATA[C8HF 13 O3]]> \ ESI- [M-H] 4 OPFLCA_ii n = 8 <![CDATA[C 10 HF 19 O3]]> \ ESI- [M-H] 4 HPFLCA_i n = 10 <![CDATA[C 12 H2F 22 O2]]> \ ESI- [M-H] 4 HPFLCA n = 10 <![CDATA[C 12 H2F 22 O2]]> ESI- [M-H] 4 n:2FTAcr n = 13 <![CDATA[C 20 H 11 F 27 O2]]> \ ESI- [M-H] 4

[0093] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, for those skilled in the art, they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements for some of the technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A high-throughput screening method for a broad-spectrum new pollutant, characterized in that, It includes the following steps: (1) Broad-spectrum extraction and enrichment of target pollutants in the sample to be tested, and after purification to remove impurities, an extract is obtained. The extract for broad-spectrum extraction and enrichment is a mixed extractant of methanol and dichloromethane, and the volume ratio of methanol to dichloromethane is 1:

1. The purification and impurity removal step uses an HLB solid-phase extraction cartridge; (2) Analyze the extract obtained in step (1) by an ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) instrument to obtain high-resolution mass spectrometry data of the target pollutants; (3) Process the high-resolution mass spectrometry data obtained in step (2) in combination with a pre-established suspected target screening database of broad-spectrum emerging pollutants and set screening and filtering criteria to identify and confirm the types of emerging pollutants in the sample to be tested.

2. The high-throughput screening method for broad-spectrum emerging contaminants according to claim 1, wherein In step (1), the sample to be tested is a biological sample or a soil sample.

3. The high-throughput screening method for broad-spectrum emerging contaminants according to claim 1, wherein In step (2), the ultra-high performance liquid chromatography-high resolution mass spectrometry instrument uses an Acclaim TM 120 C18 column or a HILIC column for chromatographic separation. The Acclaim TM 120 C18 column has a specification of 2.1 mm × 100 mm, 2.2 μm, and the HILIC column has a specification of 2.1 × 150 mm and a particle size of 3 μm.

4. The high-throughput screening method for broad-spectrum emerging pollutants according to claim 1, characterized in that, In step (2), the UHPLC-HRMS instrument uses an electrospray ionization source in the full-scan and all-ion fragmentation scan modes under positive or negative ion conditions to analyze the extract obtained in step (1) to obtain high-resolution mass spectrometry data.

5. The high-throughput screening method for broad-spectrum emerging contaminants according to claim 1, characterized in that, In step (3), the suspected target screening database of broad-spectrum emerging pollutants contains at least 500 emerging pollutants, and the emerging pollutants include organophosphorus compounds, perfluorinated compounds, and antibiotics.

6. The high-throughput screening method for broad-spectrum emerging pollutants according to claim 1, wherein In step (3), the suspected target screening database of broad-spectrum emerging pollutants is obtained through existing literature and Pubchem, MassBank, and mzCloud spectral databases, and includes the Chinese name, English name, chemical formula, CAS number, accurate mass number of the primary parent ion, ionization mode, adduct ion, retention time, retention time deviation interval, characteristic fragment ions, and accurate mass number information of the secondary fragment ions of broad-spectrum emerging pollutants.

7. The high-throughput screening method for broad-spectrum emerging contaminants according to claim 6, wherein, The accurate mass number information of the secondary fragment ions is obtained through existing literature and Pubchem, MassBank, and mzCloud spectral databases. For the accurate mass number information of the secondary fragment ions that cannot be obtained from existing literature and spectral databases, a prediction method is used for fragmentation prediction.

8. The high-throughput screening method for broad-spectrum emerging contaminants according to claim 7, wherein The prediction method includes: summarizing the characteristic ion fragments of emerging pollutants according to existing literature; for emerging pollutants without characteristic ion fragments, according to the structural characteristics of the emerging pollutants themselves, use ion fragment prediction software to predict the fragment ions, list the intermediate ions in the fragmentation path in the fragment ion library, sort the occurrence frequencies of the fragment ions from high to low, and select representative ions as the characteristic fragment ions of the emerging pollutants.

9. The high-throughput screening method for broad-spectrum emerging contaminants according to claim 7 or 8, characterized in that, The accuracy verification method of the prediction method is: analyze known emerging pollutant standards by high-resolution mass spectrometry in the full-scan mode and data-dependent scan mode, and compare the accurate mass number information of the secondary fragment ions obtained with the accurate mass number information of the secondary fragment ions predicted by the prediction method. When the number of matches ≥ 3, it is considered that the prediction is accurate.

10. The high-throughput screening method for broad-spectrum emerging contaminants according to claim 1, wherein In step (3), the screening and filtering criteria are set using data analysis software. The screening and filtering criteria are that the mass deviation of MS and MS / MS data are both set to ≤5 ppm; peaks with a signal-to-noise ratio >5 and a peak intensity higher than 10,000 are selected; the allowable intensity deviation of the isotope pattern is set to ≤20%, and the comprehensive comparison scoring threshold of the isotope pattern is set to 80% - 100%; the number of matching fragments ≥2.

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