Method for comprehensively identifying PFAS in environment by combining targeted analysis, suspicious screening and non-targeted identification

Abstract

The invention discloses a method for comprehensively identifying PFAS in an environment by combining targeted analysis, suspicious screening and non-targeted identification. The method comprises the following steps: S1, carrying out liquid chromatography-high resolution mass spectrometry fullscan / DDA / DIA analysis on an environmental sample; s2, carrying out liquid chromatography-high resolution mass spectrometry analysis on the plurality of PFAS standard substances; s3, according to a public chemical database and literature, collecting names and molecular formulas of a plurality of PFASs, calculating an accurate mass number, and jointly establishing a suspicious screening database in combination with an open-source MS2 spectrogram library; s4, establishing a non-targeted identification work flow by combining the mass number characteristics of the PFAS, the characteristic MS2 fragments, the characteristic neutral loss and the homolog characteristics; and S5, integrating the targeted analysis compound, the candidate compound subjected to suspicious screening and the candidate compound subjected to non-targeted identification, and comprehensively identifying the PFAS in the environmental sample. According to the method disclosed by the invention, the known and unknown perfluoroalkyl / polyfluoroalkyl compounds are fully identified through three complementary analysis technologies.

Description

Technical Field

[0001] The present invention belongs to the field of environmental analytical chemistry, and in particular relates to a method for comprehensively identifying PFAS in the environment by combining targeted analysis, suspicious screening and non-targeted identification. Background Art

[0002] Per- and poly-fluoroalkyl compounds (PFAS) are a class of synthetic organic compounds that have been produced since around 1940. Due to their superior high stability, surface activity, oil and antifouling properties, and high temperature resistance, they are widely used in various fields such as industry (such as polymer processing aids, electroplating coatings, aviation fuel additives, and aqueous film-forming foam fire extinguishing agents), commerce (such as building materials, cleaning products, and medical equipment), and consumer products (home textiles, non-stick kitchenware, food packaging materials, and antifouling clothing). PFAS has a variety of structures, and the basic structural feature is that it contains multi- and perfluorinated carbon chains. The CF bond is highly stable and difficult to be interrupted under normal conditions, and the large number of fluorine atoms wrapped around the carbon chain makes it difficult to expose the CC bond. Therefore, PFAS substances are almost not degraded under natural conditions and in organisms. The high stability also allows it to be transmitted over long distances with ocean currents or the atmosphere, causing global pollution. PFAS substances have been detected in remote areas of the Arctic and Antarctic. PFAS can bind to biological proteins. A large number of animal experiments and population survey data have shown that PFAS has high bioaccumulation and a variety of toxic effects, including carcinogenicity. Based on the above, PFAS pollutants have continued to receive widespread attention for many years.

[0003] With the continuous deepening of PFAS research, some substances such as perfluorooctane sulfonic acid (PFOS), perfluorooctane carboxylic acid (PFOA), perfluorohexane sulfonic acid (PFHxS) and their precursors have been included in the Stockholm Convention in 2009, 2019, and 2022, respectively, and their global production and use are restricted. Accordingly, the industry has begun to turn to the production of alternatives. Due to the excellent hydrophobic and oleophobic properties and high stability of PFAS, the search for alternatives is currently mainly focused on certain structural improvements to traditional PFAS, such as using shorter perfluorocarbon chains or modifying perfluorocarbon chains, including H substitution, ether bond insertion, and unsaturated structures. A few studies have reported the environmental occurrence of several alternative PFAS (such as short-chain perfluoroalkyl carboxylic acid GenX, ether bond-inserted sulfonic acid PFESA), however, the structures of a large number of new PFAS being produced and used are still largely unknown. In addition, the production and use of a large number of traditional PFAS over the past 80 years have also accumulated a large number of unknown precursors and by-products in the environment. Mass balance studies have found that a large number of unknown organofluorine compounds are common in various sample types by comparing the total extractable organofluorine with the sum of the concentrations of known PFAS. For example, the proportion of unknown components in water or sediments ranges from 50% to ≥99%; the proportion of unknown components in biological samples (including worms, shrimps) and wild rodent blood ranges from 15% to ≥99%; and even in human blood, there is a similar proportion. These unknown PFAS are a major source of uncertainty in environmental and human health risk assessments, so the discovery and characterization of these unknown PFAS in environmental media is of great significance. For a long time, the study of the occurrence of environmental pollutants, including PFAS, in the environment has mainly adopted targeted analysis methods, that is, the detection of specific compounds is achieved by using standards to provide characteristic information (such as chromatographic retention time and MS / MS spectrum). Therefore, it is difficult to identify unknown PFAS by targeted analysis relying on standards. Thanks to the development of full-scan high-resolution mass spectrometry (HRMS) in recent years, non-targeted identification, that is, the identification and structural identification of unknown pollutants in the environment without standards, has become possible. Its ultra-high mass resolution and accuracy are the prerequisites for unknown identification, providing a technical means for comprehensive identification of PFAS in complex environmental matrices (TrAC Trends in Analytical Chemistry 2019, 121).

[0004] At present, the identification of unknown PFAS based on high-resolution mass spectrometry mainly relies on two methods, suspected substance screening (i.e., comparing compound lists) and non-targeted identification. Although the former greatly broadens the scope of pollutants that can be identified by targeted analysis, it can only identify limited PFAS and is powerless for PFAS that are currently unknown, not included in the database, and not reported. The latter, in most cases, relies on the characteristic quality defects and homologous characteristics of PFAS. These characteristics are particularly suitable for highly polluted samples (such as samples near organic fluorine factories, environmental samples in fire drill areas, Scotchgard carpet cleaners, etc.), but when facing conventional environmental samples and biological samples, they are often very limited due to the interference of a large number of highly responsive background ions. In recent years, different researchers have tried to develop different non-targeted PFAS identification strategies, such as the ion source cleavage retention time indication method based on the characteristic fragments generated when PFAS is cleaved and the characteristic neutral loss that occurs, and the suspected PFAS prediction method based on machine learning. These methods either require manual data screening, which is inefficient, and the results vary greatly depending on the operator's experience, and are prone to missed detection; or require specific professional knowledge to be used. Finally, looking at the different methods, each study was relatively isolated and there was no methodological integration, so most studies could only detect, quantify, and identify some PFAS.

[0005] Based on the above research status, we intend to establish a comprehensive PFAS identification method by combining targeted analysis, suspected screening and non-targeted identification analysis. However, how to establish a simple and convenient workflow for targeted, suspected screening and non-targeted identification, and how to integrate multiple research strategies to maximize PFAS identification are still urgent issues to be solved. Summary of the invention

[0006] In view of the shortcomings of the existing technologies in identifying PFAS, such as high technical requirements for some methods, insufficient integration and systematicity, difficulty in deep data mining, and a narrow range of PFAS identification, the purpose of the present invention is to provide a simple and easy-to-operate method for comprehensive identification of per / polyfluoroalkyl compounds in an environment. The method achieves full identification of known and unknown per / polyfluoroalkyl compounds in complex matrices by combining three complementary analytical techniques: precise targeted analysis, suspicious screening based on a large number of known PFAS databases, and non-targeted identification based on characteristic fragments, characteristic neutral loss, mass defect characteristics, and homologous characteristics.

[0007] To achieve the above object, the present invention adopts the following technical solution:

[0008] The present invention provides a method for comprehensively identifying PFAS in the environment by combining targeted analysis, suspected screening and non-targeted identification technology, comprising the following steps:

[0009] S1. Performing liquid chromatography-high resolution mass spectrometry analysis on environmental samples to obtain environmental sample chromatography-high resolution mass spectrometry data;

[0010] S2. Performing liquid chromatography-high resolution mass spectrometry analysis on several PFAS standards to obtain standard chromatography-high resolution mass spectrometry data;

[0011] Performing matching analysis on the chromatography-high resolution mass spectrometry data of the standard and the environmental sample to obtain compounds for targeted analysis;

[0012] S3. Collect the compound names and molecular formulas of several PFAS based on public chemical databases and / or literature, calculate the accurate mass numbers, and jointly establish a suspicious screening analysis database in combination with the open source MS2 spectrum library;

[0013] According to the suspicious screening analysis database, performing suspicious screening analysis on the environmental sample chromatography-high resolution mass spectrometry data to obtain candidate compounds for suspicious screening analysis;

[0014] S4. Establish a non-targeted identification workflow by combining the mass number characteristics, characteristic MS2 fragment ions, characteristic neutral losses, and homologue characteristics of PFAS;

[0015] According to the non-targeted identification workflow, non-targeted identification is performed on the chromatography-high-resolution mass spectrometry data of the environmental sample to obtain candidate compounds for non-targeted identification;

[0016] S5. Integrate the compounds of the targeted analysis, the candidate compounds of the suspected screening analysis, and the candidate compounds of the non-targeted identification to comprehensively identify the PFAS in the environmental sample.

[0017] In the above-mentioned method for comprehensively identifying PFAS in the environment by combining targeted analysis, suspected screening and non-targeted identification, the environmental samples include water samples, solid samples or biological samples; the water samples include industrial sewage, sewage treatment plant inlet and outlet water, river water, surface water, seawater, drinking water or groundwater; the solid samples include sewage treatment plant sludge, water body sludge, sediment, soil, indoor dust or atmospheric particulate matter; the biological samples include human breast milk, urine, serum, follicular fluid, umbilical cord blood, saliva, sweat, animal tissues and organs, fish, birds, sharks, mollusks or plants.

[0018] In the above-mentioned method for comprehensively identifying PFAS in the environment by combining targeted analysis, suspected screening and non-targeted identification, the method further includes a step of pre-treating the environmental sample before liquid chromatography-high resolution mass spectrometry analysis;

[0019] When the environmental sample is a water sample, the pretreatment adopts a WAX column solid phase extraction method, including the following steps: 1) before extraction, the solid phase extraction column is activated with a methanol solution of 0.5% by volume ammonia, methanol and Milli-Q water in sequence; 2) the water sample is loaded into the solid phase extraction column activated in step 1) at a flow rate of 5 to 10 mL / min; 3) after loading the water sample in step 2), the extraction column is washed with Milli-Q water and 25 mM CH3COONH4 / CH3COOH buffer with a pH of 4 in sequence; 4) after washing in step 3), the extraction column is eluted with a methanol solution of 0.5% by volume ammonia, and dried, the eluate is collected, the eluate is evaporated to near dryness under a nitrogen flow, and re-dissolved with methanol;

[0020] When the environmental sample is a solid sample, the pretreatment adopts ion pair extraction combined with solid phase extraction, including the following steps: 1) adding 0.4M NaOH solution, pH 10 and 0.1M Na2CO3 / NaHCO3 buffer and 0.5M tetrabutylammonium hydrogensulfate (TBAHS) solution to the environmental sample in sequence; 2) adding methyl tert-butylether (MTBE) solution to the mixed solution in step 1), repeating three times, shaking for 8 hours each time; 3) combining the methyl tert-butylether (MTBE) layers after three liquid-liquid layer separations in step 2), drying with nitrogen and then re-dissolving with methanol; 4) adding Milli-Q water to the solution obtained in step 3), and then performing solid phase extraction, the solid phase extraction step is the same as the solid phase extraction step of the water sample;

[0021] When the environmental sample is plasma, breast milk, follicular fluid, or saliva, the pretreatment adopts organic reagent protein precipitation combined with solid phase extraction, including the following steps: 1) acetonitrile is first added to the biological liquid sample for protein precipitation; 2) after sufficient precipitation, the supernatant is concentrated by nitrogen blowing; 3) Milli-Q water is added to the solution obtained in step 2), and then solid phase extraction is performed, and the solid phase extraction step is the same as the solid phase extraction step of the water sample;

[0022] When the environmental sample is urine or sweat, the pretreatment method is the same as the pretreatment step of the water sample;

[0023] When the environmental sample is a tissue sample, the pretreatment adopts a method combining grinding or homogenization, organic reagent precipitation to remove protein, and solid phase extraction, including the following steps: 1) grinding or homogenizing the tissue sample; 2) adding acetonitrile to the sample obtained in step 1) for protein precipitation; 3) taking the supernatant after sufficient precipitation and blowing nitrogen to concentrate; 4) adding Milli-Q water to the solution obtained in step 3) and entering solid phase extraction, and the solid phase extraction step is the same as the solid phase extraction step of the water sample; the tissue sample includes animal tissue organs, fish, birds, sharks, and mollusks;

[0024] When the environmental sample is a plant, the pretreatment adopts a method of grinding or homogenization, organic reagent extraction, centrifugation, and solid phase extraction, including the following steps: 1) grinding or homogenizing the plant sample; 2) adding methanol to the sample obtained in step 1) for extraction, and the extraction is sequentially performed by high-speed vortexing for 2 minutes, ultrasound for 30 minutes, and shaking for 1 hour; 3) centrifuging the sample obtained in step 2) at 5000rpm for 30 minutes, and transferring the supernatant to a clean centrifuge tube; 4) repeating the extraction process three times; 5) combining the supernatants of the three extractions in step 4), concentrating, then adding Milli-Q water and entering solid phase extraction, and the solid phase extraction step is the same as the solid phase extraction step of the water sample; the plant includes leaves or stems.

[0025] In the above-mentioned method for comprehensively identifying PFAS in the environment by combining targeted analysis, suspected screening and non-targeted identification, in step S1 and step S2, the conditions of the liquid chromatography-high-resolution mass spectrometry analysis are as follows:

[0026] Liquid chromatography conditions: injection volume of 5 μL; flow rate of 0.3 mL / min; column temperature of 25 °C; mobile phase consisting of aqueous solution A containing 2 mM ammonium acetate and 2 mM 1-methylpiperidine and methanol solution B containing 2 mM ammonium acetate and 2 mM 1-methylpiperidine; gradient elution program, in terms of volume fraction, was as follows: 0 min 70% solution A and 30% solution B; 1 min 50% solution A and 50% solution B; 6 min 40% solution A and 60% solution B; 20 min 25% solution A and 75% solution B; 23 min 15% solution A and 85% solution B; 23.5 min 0% solution A and 100% solution B; 28.5 min 0% solution A and 100% solution B; 28.6 min 70% solution A and 30% solution B; 33.6 min 70% solution A and 30% solution B;

[0027] Mass spectrometry conditions: the ion source adopted the negative ion mode of electrospray ionization source; the spray voltage was 3500 V; the ion source temperature was 300 °C; Full MS / ddMS2 and Full MS / DIA modes were selected; the MS1 resolution was set to 70000 in both modes; the scanning range was set to m / z = 100-1000; the MS2 resolution was set to 17500; the normalized collision energy was set to 20, 35, 50; in ddMS2, the Top7 ions were set to perform MS2 scans (Loop count was set to 7), and in DIA, five mass ranges were set to perform MS2 scans, and the mass ranges were m / z = 200-350, 350-500, 500-650, 650-800, 800-950, respectively.

[0028] The environmental sample chromatography-high resolution mass spectrometry data and the standard chromatography-high resolution mass spectrometry data include chromatographic peak retention time, chromatographic peak area, primary mass spectrometry information (MS1), secondary mass spectrometry fragmentation information (MS2) and related signal intensity.

[0029] In the above-mentioned method of comprehensively identifying PFAS in the environment by combining targeted analysis, suspected screening and non-targeted identification technology, the several PFAS standards are 31 PFAS standards, and the purity of each standard is ≥98%;

[0030] The targeted matching analysis includes: comparing the consistency of chromatographic peak retention time and mass spectrum fragment ion information.

[0031] In the above-mentioned method of comprehensively identifying PFAS in the environment by combining targeted analysis, suspected screening and non-targeted identification technology, the public chemical database is the public PFAS chemical database in the NORMAN Suspect List Exchange;

[0032] The several PFASs are 4777 PFASs;

[0033] The MS2 spectrum library is mzCloud library and massBank library;

[0034] The establishment of the suspected screening analysis database was analyzed using the Compound Discoverer software and Fluoromatch software of Thermo Fisher. In Compound Discoverer, the Masslist module was used to generate an MS database for 4777 PFAS, the mzValut module was used to load the MassBank Europe Mass Spectral DataBase (98525spectra) and the MassBank of North America database (2079804spectra), and the mzCloud module came with the mzCloud database. In the Fluormatch software, the PFAS database loaded inside the software was used.

[0035] In the above-mentioned method for comprehensively identifying PFAS in the environment by combining targeted analysis, suspected screening and non-targeted identification, the suspected screening analysis is performed on Compound Discoverer software and FluoroMatch software, and the Compound Discoverer process is as follows: in the spectrum selection module, the retention time interval is set to the default 0, and all spectra are analyzed; in the alignment retention time module, the mass deviation is set to 5ppm; in the detection compound module, the mass deviation is set to 5ppm, and the minimum peak intensity is set to 10000; the adduct ion selects all default negative adduct ions; in the compound classification module, the mass deviation is set to 5ppm, the retention time deviation is set to 0.1min, and the peak shape score threshold is set to 4; in the blank filling module, the mass deviation is set to 5ppm, and the signal-to-noise ratio threshold is set to 3; in the background compound labeling module, the maximum ratio of the sample peak area to the blank peak area is set to 5, and the maximum ratio of the blank peak area to the sample peak area is set to 0; then add the database comparison module: "Search Masslists", "Search mzCloud" and "Search mzVault"; Search In the Masslists module, set the retention time deviation to 0.1 min and the mass deviation to 5 ppm; in the Search mzCloud module, select “Autoprocessed” and “Reference” for the database, Search MS nSelect No for Search fragment tree, select “confidencereverse” for Search DDA mode, select No for Match Ion Activation Mode and Match Ion Activity Energy, and set Match Factor Threshold to 70; select “Europe Mass Spectral DataBase (98525spectra) and MassBank of North America” for the database in the Search mzVault module, select All for Compound Class, set Match Ion Activation Mode and Match Ion Activity Energy to No, set Precursor Mass To Match to 5ppm, set Search Algorithm to HighChem HihRes, set Match Factor Threshold to 70, and set Use Retention to 100. Time (i.e., whether to use retention time) was set to No; for Fluoromatch software, the process was as follows: select the data of pure methanol solution as the blank file; select the data of standard solution as the secondary information file, target file, and sample file; set the MS / MS and Full scan intensity thresholds to 1000; set the MS1 mass deviation to 0.005Da; and set the MS / MS deviation to 10ppm.

[0036] Further preferably, the results of the Compound Discoverer operation are preliminarily screened, and the screening conditions are set to: "Background is fake"; in the "Masslist Matches" module, select "has at least status" AND "single match found" AND "in massfile X", where X is the name of the established suspicious screening database, and "Formular" is set to "contains F"; "mzCloud Best Match" and "mzVault Best Match" are both set to "≥70", and "mzCloud Best Match Cofidence" is set to "7-10" OR "70-100"; for the candidate compounds obtained from the suspicious screening analysis, a chromatographic check is performed, and the original chromatographic and mass spectrometric data are manually checked to eliminate candidates with poor peak shape and signal intensity equivalent to that of blank samples, and then a preliminary PFAS structural type, i.e., class, classification is performed according to the mass number and structural type;

[0037] The results of the FluoroMatch run were preliminarily screened. For all results scored as A, B, and C, the original chromatographic and mass spectrometric data were manually checked to eliminate candidates with poor peak shape and signal intensity comparable to that of the blank sample. The PFAS structure type, i.e., class, was then preliminarily classified (e.g., CF3-) based on the mass number and structure type.

[0038] The above-mentioned method for comprehensively identifying PFAS in the environment by combining targeted analysis, suspicious screening and non-targeted identification technology is characterized in that: the mass number characteristics of the PFAS are negative mass defects or tiny positive mass defects, usually ranging from -0.15 to 0.1;

[0039] The characteristic MS2 fragment ions include CF3-, C2F5-, C3F7-, C4F9-, C5F 11 -、C6F 13 -, C3F5-, C4F7-, C5F9-, C6F 11 -, C3F3-, C4F5-, C5F7-, C6F9-, SO2F-, SO3F-, COF3-, C2F5O-, C3F7O-, C4F9O-, C3F5O-, C4F7O-, C3F3O-, C4F5O-, C5F7O-, C2F5SO2-, C3F7SO2-, C4F9SO2-, C2F6N-, C3F8N-, C4F 10 N-、C5F 12 N-、C6F 14 N-、C7F16 N-, C2F4N-, C3F6N-, C4F8N-, C5F 10 N-、C6F 12 N-、C7F 14 N-、C2F6NO-、C3F8NO-、C4F 10 NO-、C5F 12 NO-、C6F 14 NO-、C7F 16 NO-, C2F4NO-, C3F6NO-, C4F8NO-, C5F 10 NO-、C6F 12 NO-、C7F 14 NO-, C2F5SO3-, C3F7SO3-, C4F9SO3-;

[0040] The characteristic neutral losses include HF (mass number 20), H2F2 (mass number 40), H3F3 (mass number 60), H4F4 (mass number 80), and HFCO2 (mass number 64);

[0041] The homologous series include a series of compounds whose mass numbers differ from each other by CF2, C2F4, CHF, or C2H2F2.

[0042] In the above-mentioned method of comprehensively identifying PFAS in the environment by combining targeted analysis, suspected screening and non-targeted identification technology, the non-targeted identification is performed on Compound Discoverer and Fluoromatch software;

[0043] For Compound Discoverer software, the process is as follows: in the spectrum selection module, the retention time interval is set to the default 0, and all spectra are analyzed; in the alignment retention time module, the mass deviation is set to 5ppm; in the detection compound module, the mass deviation is set to 5ppm, and the minimum peak intensity is set to 10000; the adduct ion selects all default negative adduct ions; in the compound classification module, the mass deviation is set to 5ppm, the retention time deviation is set to 0.1min, and the peak shape score threshold is set to 4; in the blank filling module, the mass deviation is set to 5ppm, and the signal-to-noise ratio threshold is set to 3; in the labeling background compound module, the maximum ratio of the sample peak area to the blank peak area is set to 5, and the maximum ratio of the blank peak area to the sample peak area is set to 0; in the compound type coverage (i.e., class coverage) module, an MS2 characteristic fragment ion list is established, including all the fragments listed above and the neutral loss mass number (such as CF 3-) and set to use this list for comparison; other parameters are set as follows: S / N threshold is 5, High Acc. Mass Tolerance is 15ppm, Low Acc. Mass Tolerance is 2.5mmu, Use Full MS Tree is Yes, and Allow DIA Scoring is Yes;

[0044] For Fluoromatch software, the process is as follows: select the data of pure methanol solution as the blank file; select the data of standard solution as the secondary information file, target file and sample file; MS / MS and Full scan intensity thresholds are both set to 1000; MS1 mass deviation is set to 0.005Da; MS / MS deviation is set to 10ppm.

[0045] Further preferably, for the results of the Compound Discoverer run above, the data with classcoverage>0 are screened: ① For the data in DDA mode, the corresponding structure is determined by manual mass spectrometry analysis and combined with other information; ② For the data in DIA mode, the full scan data in the corresponding mass range is checked, and the possible PFAS parent ions are found using the mass defect characteristics listed above; then these parent ions are subjected to targeted MS2 analysis, and if the results do not contain the MS2 characteristic fragment ions listed above or other new F-containing fragments, they are excluded; if they contain the MS2 characteristic fragment ions listed above, their structure is analyzed; the results of the FluoroMatch run above are preliminarily screened, and for all results scored as A, B and C, the original chromatograms and mass spectrometry data are manually checked to eliminate candidates with poor peak shape and signal intensity equivalent to that of blank samples, and then a preliminary PFAS structural type, i.e., class, is classified according to the homologue characteristics listed above and these mass number characteristic defects.

[0046] In any of the above methods for comprehensively identifying PFAS in the environment by combining targeted analysis, suspected screening and non-targeted identification technology, the integration includes:

[0047] Inferring and confirming the structure of the candidate compound based on the chromatographic MS1 ion information, MS2 fragment ion information, homologue information, and retention time information of the environmental sample;

[0048] Based on the chromatographic data and mass spectrometry data of the candidate compounds in the environmental samples, the list information of targeted analysis, suspected screening and non-targeted identification was integrated and sorted into different PFAS types; each type was manually screened for the presence of homologues of other chain lengths to obtain all the mass numbers of each PFAS type; then the blank sample was checked for the presence of the corresponding PFAS, and the PFAS was considered to be present in the sample if the signal intensity exceeded 3 times that of the blank; finally, each candidate compound identified as PFAS was assigned an accurate structural confidence level based on whether there were structurally confirmed compounds in each class, whether they could be matched to the database with high scores, whether there were clear and reasonable homologue characteristics, and whether they presented characteristic quality defects.

[0049] The present invention has the following beneficial effects:

[0050] (1) Clear and simple method establishment process: The present invention follows a modular process design to achieve repeatability and standardization of analytical method establishment.

[0051] (2) Wide range of identification and application scenarios: Based on authentic standards, a targeted analysis method applicable to both low-resolution and high-resolution mass spectrometry data has been established; a suspected screening analysis database combining MS1 and MS2 spectral libraries has been established, which can be used to perform suspected screening analysis on 4777 PFAS; a workflow for non-targeted identification of PFAS has been established; targeted, suspected screening, and non-targeted identification methods have been integrated, which can be implemented without relying on standards and lacking any reference compound information. In addition, it can be applied to a variety of complex environments and biological media (such as water samples, soil samples, plasma, etc.), providing technical support for environmental monitoring and human pollution exposure assessment.

[0052] (3) Personalized establishment of database: In the present invention, the suspected screening analysis database can be customized, expanded and modified according to the progress of PFAS identification research, so as to realize personalized application; the characteristic fragments and neutral losses of PFAS used in Class Coverage in non-targeted analysis may also appear in more forms of fragments and neutral losses as the research on PFAS progresses, and can also be increased as the research progresses, so as to realize more comprehensive PFAS identification.

[0053] (4) The present invention combines targeted analysis, suspected screening and non-targeted identification to comprehensively identify PFAS in the environment. The three methods are complementary and can fully identify known and unknown per / polyfluoroalkyl compounds in complex matrices. The types of compounds identified are comprehensive and accurate. The method has universal applicability and broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 is a flow chart of the comprehensive identification method of PFAS in the environment of the present invention.

[0055] Figure 2 It is a Venn diagram of the types of PFAS identified by targeted, suspected screening and non-targeted methods in Example 3 of the present invention.

[0056] Figure 3 It is the chromatogram and mass spectrum of Flurtamone in the sample identified in Example 3 of the present invention.

[0057] Figure 4 It is the chromatogram and mass spectrum of FOSAC in the sample identified in Example 3 of the present invention. DETAILED DESCRIPTION

[0058] As described in the background technology, how to combine targeted analysis, suspicious screening and non-targeted identification to mine high-resolution data as comprehensively as possible to fully identify PFAS therein is still a technical problem that needs to be solved urgently. Based on this, the present invention provides a method for comprehensively identifying PFAS in the environment by combining targeted analysis, suspicious screening and non-targeted identification technology, including the following steps: S1. Performing liquid chromatography-high-resolution mass spectrometry analysis on environmental samples to obtain environmental sample chromatography-high-resolution mass spectrometry data; S2. Performing liquid chromatography-high-resolution mass spectrometry analysis on several PFAS standards to obtain standard chromatography-high-resolution mass spectrometry data; performing targeted matching analysis on the chromatography-high-resolution mass spectrometry data of the standard and the environmental sample to obtain the compounds for targeted analysis; S3. According to public chemical databases and / or literature, collect the compound names and molecular formulas of several PFAS, calculate the accurate mass number, and combine with the open source MS2 spectrum library Jointly establish a suspected screening analysis database; based on the suspected screening analysis database, perform suspected screening analysis on the chromatography-high resolution mass spectrometry data of the environmental samples to obtain candidate compounds for suspected screening analysis; S4, combine the mass number characteristics, characteristic MS2 fragment ions, characteristic neutral losses and homologue characteristics of PFAS to establish a non-targeted identification workflow; based on the non-targeted identification workflow, perform non-targeted identification on the chromatography-high resolution mass spectrometry data of the environmental samples to obtain candidate compounds for non-targeted identification; S5, integrate the candidate compounds for targeted analysis, the candidate compounds for suspected screening analysis and the candidate compounds for non-targeted identification to comprehensively identify PFAS in the environmental samples.

[0059] Based on the above technical solutions, the method of the present invention first uses authentic standards to establish a targeted analysis method to ensure the reliability of the identification technology. Further, by collecting open source PFAS information, a database and workflow for suspicious screening analysis are established. Finally, combined with the mass number characteristics, characteristic MS2 fragment ion information and homologue module extraction of PFAS, a non-targeted identification workflow is established through software. Combined with the established targeted, suspicious screening and non-targeted methods, the complementarity of different analysis techniques can be achieved, thereby achieving comprehensive identification of PFAS in environmental samples.

[0060] Figure 1 : is a flow chart of the comprehensive identification method of PFAS in the environment of the present invention. The following is a detailed description of the method of the present invention for comprehensively identifying PFAS in the environment by combining targeted, suspected screening and non-targeted identification technologies in conjunction with the flow chart.

[0061] According to the present invention, the environmental sample may be a sample of PFAS to be identified in any form in the environment, including water samples, solid samples or biological samples; the water sample includes industrial sewage, inlet and outlet water of sewage treatment plants, river water, surface water, seawater, drinking water or groundwater; the solid sample includes sewage treatment plant sludge, water sludge (such as river sludge), sediment, soil, indoor dust or atmospheric particulate matter; the biological sample includes human breast milk, urine, serum, plasma, follicular fluid, umbilical cord blood, saliva, sweat, animal tissues and organs, fish, birds, sharks, mollusks or plants.

[0062] like Figure 1 As shown, the method also includes the step of pre-treating the environmental sample, and the pre-treatment method can be adjusted according to the type of environmental sample. The pre-treatment method is described in detail below according to water samples and solid samples, mainly including solid phase extraction based on WAX column and nitrogen blowing re-dissolution.

[0063] When the environmental sample is a water sample, the pretreatment adopts a WAX column solid phase extraction method, including the following steps: 1) before extraction, the solid phase extraction column is activated with a methanol solution of 0.5% ammonia water by volume, methanol and Milli-Q water in sequence; 2) the water sample is loaded into the WAX ​​solid phase extraction column activated by step 1) at a flow rate of 5 to 10 mL / min; 3) after the water sample is loaded in step 2), the extraction column is washed with Milli-Q water and 25 mM CH3COONH4 / CH3COOH buffer with a pH of 4 in sequence; 4) after washing in step 3), the extraction column is eluted with a methanol solution of 0.5% ammonia water by volume, and dried, the eluate is collected, the eluate is evaporated to near dryness under a nitrogen flow, and re-dissolved with methanol;

[0064] As an example, the extraction column is an Oasis WAX column, specification: 6cc, 150mg, and its operation is as follows: before extraction, the solid phase extraction column is activated with 4mL of 0.5% ammonia methanol solution, 4mL of methanol and 4mL of Milli-Q water in sequence; then, the water sample is loaded at a flow rate of 5-10mL / min; thereafter, the extraction column is washed with 4mL of 25mM ammonium acetate buffer (pH=4) and 6mL of methanol; then, the extraction column is eluted with 6mL of 0.5% ammonia methanol; the eluent is evaporated to near dryness under a gentle nitrogen flow, and reconstituted with methanol to 1mL;

[0065] When the environmental sample is a solid sample, the pretreatment adopts ion pair extraction combined with solid phase extraction, including the following steps: 1) adding 0.4M NaOH solution, pH 10 and 0.1M Na2CO3 / NaHCO3 buffer and 0.5M tetrabutylammonium hydrogen sulfate (TBAHS) solution to the environmental sample in sequence; 2) adding methyl tert-butyl ether (MTBE) solution to the mixed solution in step 1), repeating three times, shaking for 8 hours each time; 3) combining the methyl tert-butyl ether (MTBE layer) after three liquid-liquid layer separations in step 2), drying with nitrogen and re-dissolving with methanol, as an example, re-dissolving with 500μL~1mL (such as 1mL) of methanol; 4) adding Milli-Q water to the solution obtained in step 3) and entering solid phase extraction, the solid phase extraction step is the same as the solid phase extraction step of the water sample;

[0066] As an example, take 0.5g of freeze-dried and ground mud as an example. The extraction column is Oasis WAX column, specification: 6cc, 150mg. First, add 1.5ml of 0.4M NaOH solution (mix for 30min), then add 2ml of pH 10 0.1M Na2CO3 / NaHCO3 buffer; after fully mixing, add 1ml of 0.5M TBAHS solution to mix; then add 5ml of MTBE solution (repeat three times, each time shaking for 8h); combine the MTBE layers after three liquid-liquid separations, then blow dry with gentle nitrogen gas, and re-dissolve with 1ml MeOH; then add 30ml of Milli-Q pure water and enter the solid phase extraction process (similar to the water sample process);

[0067] When the environmental sample is a liquid biological sample such as plasma, breast milk, follicular fluid, saliva, etc., the pretreatment adopts organic reagent protein precipitation combined with solid phase extraction, including the following steps: 1) acetonitrile is first added to the biological liquid sample for protein precipitation; 2) after sufficient precipitation, the supernatant is concentrated by nitrogen blowing until the volume is no longer significantly reduced (such as concentrated to 200 μL); 3) Milli-Q water is added to the solution obtained in step 2) and then solid phase extraction is performed, and the solid phase extraction step is the same as the solid phase extraction step of the water sample.

[0068] When the environmental sample is a low-protein liquid biological sample such as urine or sweat, the pretreatment method is the same as the pretreatment step of the water sample;

[0069] When the environmental sample is a tissue sample, the pretreatment adopts a method of grinding or homogenization, organic reagent precipitation of protein, and solid phase extraction, including the following steps: 1) grinding or homogenizing the tissue sample; 2) adding acetonitrile to the sample obtained in step 1) for protein precipitation; 3) after sufficient precipitation, taking the supernatant and blowing nitrogen to concentrate until the volume is no longer significantly reduced (such as concentrated to 200 μL); 4) adding Milli-Q water to the solution obtained in step 3), and then performing solid phase extraction, the solid phase extraction step is the same as the solid phase extraction step of the water sample; the tissue sample includes animal tissue organs, fish, birds, sharks, and mollusks;

[0070] When the environmental sample is a plant, the pretreatment adopts a method of grinding or homogenization, organic reagent extraction, centrifugation, and solid phase extraction, including the following steps: 1) grinding or homogenizing the plant sample; 2) adding methanol to the sample obtained in step 1) for extraction (high-speed vortex 2 minutes, ultrasound 30 minutes, shaking on a shaker for 1 hour); 3) centrifuging the sample obtained in step 2) at 5000rpm for 30 minutes, and transferring the supernatant to a clean centrifuge tube; 4) repeating the extraction process three times; 5) combining the supernatants of the three extractions in step 4), concentrating until the volume is no longer significantly reduced, then adding Milli-Q water and entering solid phase extraction, the solid phase extraction step is the same as the solid phase extraction step of the water sample; the plant includes leaves or stems.

[0071] According to the present invention, the conditions of the liquid chromatography-high resolution mass spectrometry analysis are as follows:

[0072] Liquid chromatography conditions: injection volume of 5 μL; flow rate of 0.3 mL / min; column temperature of 25 °C; mobile phase consisting of aqueous solution A containing 2 mM ammonium acetate and 2 mM 1-methylpiperidine and methanol solution B containing 2 mM ammonium acetate and 2 mM 1-methylpiperidine; gradient elution program, in terms of volume fraction, was as follows: 0 min 70% solution A and 30% solution B; 1 min 50% solution A and 50% solution B; 6 min 40% solution A and 60% solution B; 20 min 25% solution A and 75% solution B; 23 min 15% solution A and 85% solution B; 23.5 min 0% solution A and 100% solution B; 28.5 min 0% solution A and 100% solution B; 28.6 min 70% solution A and 30% solution B; 33.6 min 70% solution A and 30% solution B;

[0073] Mass spectrometry conditions: the ion source used the negative ion mode of the electrospray ionization source; the spray voltage was 3500 V; the ion source temperature was 300 °C; Full MS / ddMS2 and Full MS / DIA modes were selected; the MS1 resolution was set to 70000 in both modes; the scanning range was set to m / z = 100-1000; the MS2 resolution was set to 17500; the normalized collision energy was set to 20, 35, 50; in ddMS2, the Top7 ions were set to perform MS2 scans (Loop count was set to 7), and in DIA, five mass ranges were set to perform MS2 scans, and the mass ranges were m / z = 200-350, 350-500, 500-650, 650-800, 800-950 respectively;

[0074] The environmental sample chromatography-high resolution mass spectrometry data and the standard chromatography-high resolution mass spectrometry data include chromatographic peak retention time, chromatographic peak area, primary mass spectrometry information (MS1), secondary mass spectrometry fragmentation information (MS2) and related signal intensity.

[0075] In the process of implementing the present invention, it was found that in the targeted analysis process, the limitation lies in the type, quantity and purity of the available standard substances; preferably, the several PFAS standards described in the present invention are 31 PFAS standards (perfluoropropionic acid, perfluorobutyric acid, perfluoropentanoic acid, perfluorohexanoic acid, perfluoroheptanoic acid, perfluorooctanoic acid, perfluorononanoic acid, perfluorodecanoic acid, perfluoroundecanoic acid, perfluorododecanoic acid, perfluorotridecanoic acid, perfluorotetradecanoic acid, perfluorohexadecanoic acid, perfluorooctadecanoic acid, perfluorobutylsulfonic acid, perfluorohexylsulfonic acid, perfluoroheptylsulfonic acid, perfluorooctanesulfonic acid, perfluorodecylsulfonic acid, 4:2 fluorotelomer sulfonamide, 6:2 fluorotelomer sulfonamide, 8:2 fluorotelomer sulfonamide, 10:2 fluorotelomer sulfonamide, F53-B (2-[(6-chloro-1,1,2,2,3,3,4,4,5,5,6,6-dodecafluorohexyl) [oxy]-1,1,2,2-tetrafluoroethanesulfonate potassium), GenX (perfluoro 2-methyl-3-oxahexanoic acid), FOSAA (N-(perfluoro-1-octanesulfonyl) glycine), N-MeFOSAA (2-(N-methyl perfluorooctanesulfonamido) acetic acid), N-EtFOSAA (2-(N-ethyl perfluorooctanesulfonamido) acetic acid), 6:2diPAP (bis[2-(perfluorohexyl)ethyl] phosphate), 8:2diPAP (bis(2-(perfluoroethyl))phosphoric acid), PFECHS (perfluoro 4-ethylcyclohexanesulfonate potassium), the purity of each standard product is ≥98%. In a specific embodiment of the present invention, the selected 31 authentic standard products are prepared into a mixed solution with a concentration of 100 μg / L, the solvent is methanol, and liquid chromatography-high resolution mass spectrometry analysis is performed.

[0076] According to the present invention, the targeted matching analysis includes: comparing the consistency of the chromatographic peak retention time and the mass spectrum fragment ion information. In a specific embodiment of the present invention, the Xcalibur software is used to extract the peak of the real standard from the chromatographic-high-resolution mass spectrometry data of the collected sample to obtain the candidate compounds for targeted analysis.

[0077] In the present invention, it should be noted in advance that the suspected screening database refers to the database containing known PFAS that may exist in the environment, and such known PFAS that may exist in the environment are suspected screening compounds. Optionally, the public chemical database is a public PFAS chemical database in the NORMAN Suspect List Exchange. In the process of implementing the present invention, it was found that in the suspected screening analysis process, the limitation lies in the capacity of the database. In a specific embodiment of the present invention, the several PFAS are 4777 PFAS, and the PFAS are listed as follows:

[0078] NS00011091 6:3Fluorotelomer carboxylic acid CAS_RN:27854-30-4;

[0079] NS00110133(2Z)-4,4,5,5,6,6,6-Heptafluoro-2-iodohex-2-en-1-ol CAS_RN:92835-92-2;

[0080] NS00109142 4,4,5,5,6,6,6-heptafluoro-2-iodohex-2-en-1-ol CAS_RN:92835-82-0;

[0081] NS00110679 Perfluoro-N,N,N',N'-tetrakis(heptafluoropropyl)-1,6-hexanediamine CAS_RN:143356-32-5; NS00110590 Ammonium perfluoro-9-(methyl)decanoate CAS_RN:3658-63-7;

[0082] NS00003821 2,3,3,3-Tetrafluoro-2-(trifluoromethyl)propanenitrile CAS_RN:42532-60-5;

[0083] NS00110675 6:2Fluorotelomer thiohydroxy ammonium chloride CAS_RN:88992-45-4;

[0084] NS00111905 5,6-dibromo-1,1,1,2,2,3,3,4,4-nonafluorohexane CAS_RN:236736-19-9;

[0085] NS00011090 2H,2H,3H,3H-Perfluorooctanoic acid CAS_RN:914637-49-3;

[0086] NS00109139 Octadecyl 2,2,3,3,4,4,4-heptafluorobutanoate CAS_RN:400-57-7;

[0087] Due to space limitations, PFAS will not be listed one by one.

[0088] The specific types of PFAS can be selected according to the selected public chemical database. The MS2 spectral library is the mzcloud library and the massbank library; the establishment of the suspicious screening analysis database is analyzed using the Compound Discoverer software and Fluoromatch software of Thermo Fisher, and the Masslist module is used to generate an MS database for 4777 PFAS, and the mzValut module is used to load the MassBank Europe Mass Spectral DataBase (98525spectra) and MassBank of North America database (2079804spectra). The mzCloud module comes with the mzCloud database; in the Fluormatch software, the PFAS database loaded in the software is used.

[0089] According to the present invention, the suspicious screening analysis is performed on Compound Discoverer software and FluoroMatch software, and the Compound Discoverer process is as follows: in the spectrum selection module, the retention time interval is set to the default 0, and all spectra are analyzed; in the alignment retention time module, the mass deviation is set to 5ppm; in the detection compound module, the mass deviation is set to 5ppm, and the minimum peak intensity is set to 10000; the adduct ion selects all default negative adduct ions; in the compound classification module, the mass deviation is set to 5ppm, the retention time deviation is set to 0.1min, and the peak shape scoring threshold is set to 3; in the labeling background compound module, the maximum ratio of the sample peak area to the blank peak area is set to 5, and the maximum ratio of the blank peak area to the sample peak area is set to 0; in the Search Masslists module, the retention time deviation is set to 0.1min, and the mass deviation is 5ppm; in the Search mzCloud module, the database selects "Autoprocessed" and "Reference", the Search MSn tree selects no, the Search DDA mode selects "confidence reverse", the Match Ion Activation Mode mode and the Match Ion Activity Energy was selected as No, and Match Factor Threshold was set to 70; in the Search mzVault module, the databases selected were "Europe Mass Spectral DataBase (98525spectra) and MassBank of North America, Compound Class was selected as All, Match Ion Activation Mode and Match Ion Activity Energy were both set to No, Precursor Mass To Match was set to 5ppm, Search Algorithm was set to HighChem HihRes, Match Factor Threshold was set to 70, and Use Retention Time was set to No; for Fluoromatch software, the process was as follows: the data of pure methanol solution was selected as the blank file; the data of standard solution was selected as the secondary information file, target file, and sample file; the MS / MS and Full scan intensity thresholds were both set to 1000; the MS1 mass deviation was set to 0.005Da; and the MS / MS deviation was set to 10ppm.

[0090] In a further preferred embodiment of the above suspicious screening analysis, the results of the Compound Discoverer operation are preliminarily screened, and the screening conditions are set as follows: Background is "is fake"; "MasslistMatches" is "has at least status" AND "single match found" AND "in massfile X", where X is the name of the established suspicious screening database; "Formular" is set to "contains F"; "mzCloud Best Match" and "mzVault Best Match" are both set to "≥70", and "mzCloud Best Match Cofidence" is set to "7-10" OR "70-100"; for the candidate compounds obtained from the suspected screening analysis, a chromatographic check is performed, the original chromatographic and mass spectrometric data are manually checked, and the candidates with poor peak shape and signal intensity equivalent to the blank sample are eliminated, and then a preliminary PFAS structure type, i.e., class, is classified according to the mass number and structure type; the results of the FluoroMatch run are preliminarily screened, and for all results scored as A, B, and C, the original chromatographic and mass spectrometric data are manually checked, and the candidates with poor peak shape and signal intensity equivalent to the blank sample are eliminated, and then a preliminary PFAS structure type, i.e., class, is classified according to the mass number and structure type. Taking CF3- as an example, the process is as follows: manually check the parent ion containing CF3- fragments (or other PFAS characteristic MS2 fragment ions), and then perform structural analysis on it in combination with all MS2 fragment information of the parent ion. After the structural analysis is completed, it is classified according to the mass number and structure type.

[0091] In the process of implementing the present invention, it was found that non-targeted identification does not have any information on reference compounds, and the difficulty in application lies in the screening and structural analysis of the collected original chromatography-high-resolution mass spectrometry data.

[0092] According to the present invention, preferably, the characteristic MS2 fragment ions include CF3 - 、C2F5-、C3F7 - 、C4F9 - 、C5F 11 - 、C6F 13 - 、C3F5 - 、C4F7 - 、C5F9 - 、C6F 11 - 、C3F3 - 、C4F5 -、C5F7 - 、C6F9 - 、SO2F - 、SO3F - 、COF3 - 、C2F5O - 、C3F7O - 、C4F9O - 、C3F5O - 、C4F7O - 、C3F3O - 、C4F5O - 、C5F7O - 、C2F5SO2 - 、C3F7SO2 - 、C4F9SO2 - 、C2F6N - 、C3F8N - 、C4F 10 N - 、C5F 12 N - 、C6F 14 N - 、C7F 16 N - 、C2F4N - 、C3F6N - 、C4F8N - 、C5F 10 N - 、C6F 12 N - 、C7F 14 N - 、C2F6NO - 、C3F8NO - 、C4F 10 NO - 、C5F 12 NO - 、C6F 14 NO - 、C7F 16 NO - 、C2F4NO - 、C3F6NO - 、C4F8NO - 、C5F 10 NO - 、C6F 12 NO - 、C7F 14 NO - 、C2F5SO3 - 、C3F7SO3 - 、C4F9SO3- ; The characteristic neutral loss includes HF (mass number 20), H2F2 (mass number 40), H3F3 (mass number 60), H4F4 (mass number 80), and HFCO2 (mass number 64); the homologues include a series of compounds with mass numbers differing from CF2, C2F4, CHF, or C2H2F2; the mass number characteristics of the PFAS are negative mass defects or tiny positive mass defects with a mass defect range of -0.15 to 0.1; the software CompoundDiscover or the software Fluoromatch is used to establish a non-targeted identification workflow.

[0093] According to the present invention, preferably, the non-targeted identification is performed on Compound Discoverer and Fluoromatch software; in Compound Discoverer software, in the spectrum selection module, the retention time interval is set to the default 0, and all spectra are analyzed; in the alignment retention time module, the mass deviation is set to 5ppm; in the detection compound module, the mass deviation is set to 5ppm, and the minimum peak intensity is set to 10000; the adduct ion selects all default negative adduct ions; in the compound classification module, the mass deviation is set to 5ppm, the retention time deviation is set to 0.1min, and the peak shape score threshold is set to 4; in the blank filling module, the mass deviation is set to 5ppm, and the signal-to-noise ratio threshold is set to 3; in the labeling background compound module, the maximum ratio of the sample peak area to the blank peak area is set to 5, and the maximum ratio of the blank peak area to the sample peak area is set to 0; in the compound type coverage (i.e., class coverage) module, an MS2 characteristic fragment ion list is established, including all the fragments and neutral loss mass numbers listed above, and the list is set to be used for comparison; other parameters are set as follows: signal-to-noise ratio threshold (S / N threshold) is 5, high resolution mass number deviation (High Acc. Mass Tolerance) is 15ppm, low resolution mass number deviation (Low Acc. Mass Tolerance) is 2.5mmu, use full scan tree (Use Full MS Tree) is yes, whether to allow DIA scan scoring (Allow DIA Scoring) is yes; for Fluoromatch software, the process is as follows: select the data of pure methanol solution as the blank file; select the data of standard solution as the secondary information file, target file and sample file; MS / MS and Full scan intensity thresholds are both set to 1000; MS1 mass deviation is set to 0.005Da; MS / MS deviation is set to 10ppm.

[0094] In a further preferred embodiment of the above non-targeted identification, for the results of the above Compound Discoverer operation, the data with class coverage>0 are screened: ① For the data in DDA mode, the corresponding structure is determined by manual mass spectrometry analysis and combined with other information; ② For the data in DIA mode, the full scan data in the corresponding mass range is checked, and the possible PFAS parent ions are found using the mass defect characteristics listed above; then these parent ions are subjected to targeted MS2 analysis, and if the results do not contain the MS2 characteristic fragment ions listed above or other new F-containing fragments, they are excluded; if the MS2 characteristic fragment ions listed above are contained, their structure is analyzed; the results of the above FluoroMatch operation are preliminarily screened, and for all results scored as A, B and C, the original chromatogram and mass spectrometry data are manually checked to eliminate candidates with poor peak shape and signal intensity equivalent to that of blank samples, and then preliminary PFAS structural types, i.e., class, are classified according to the homologue characteristics listed above and these mass number characteristic defects.

[0095] According to the present invention, the integration includes: inferring and confirming the structure of the candidate compound based on the chromatographic MS1 ion information, MS2 fragment ion information, homologue information, and retention time information of the environmental sample; integrating the list information of the targeted analysis, suspected screening and non-targeted identification parts according to the chromatographic data and mass spectrometry data of the candidate compound in the environmental sample, and organizing them into different types of PFAS; manually screening for the presence of homologues of other chain lengths of each type to obtain all the mass numbers of each PFAS type; then checking whether the corresponding PFAS exists in the blank sample, and the PFAS is considered to exist in the sample if the signal intensity exceeds 3 times that of the blank; finally, each candidate compound identified as PFAS is assigned an accurate structural confidence level based on whether there are structurally confirmed compounds in each class, whether they can be matched to the database with high scores, whether there are clear and reasonable homologue characteristics, whether there are characteristic quality defects, etc. Among them, the specific confidence level refers to the article by Joseph et al. (Charbonnet, JA; McDonough, CA; Xiao, F.; Schwichtenberg, T.; Cao, D.; Kaserzon, S.; Thomas, KV; Dewapriya, P.; Place, BJ; Schymanski, EL; Field, JA; Helbling, DE; Higgins, CP, Communicating Confidence of Per-and Polyfluoroalkyl Substance Identification via High-Resolution Mass Spectrometry. Environ Sci Technol Lett 2022, 9, (6), 473-481.). It can be understood that when the confidence level is Level 4, only the specific molecular formula of the compound can be determined, but the structure cannot be determined; when the confidence level is Level 3, its main functional groups can be determined, but its unique true structure cannot be determined, and there may be multiple structural isomers; when the confidence level is 2, its main functional groups can be determined, and only one possible structure can be inferred based on the information obtained; when the confidence level is Level 1, the unique structure is determined with the assistance of standards (similar to targeted analysis).

[0096] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0097] The methods used in the following examples, unless otherwise specified, are all conventional methods, carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

[0098] Some reagents, detection instruments and detection conditions in the following examples are as follows:

[0099] Reagents: Authentic standards of the 31 PFAS listed in Table 1 below.

[0100] Liquid chromatography-mass spectrometry: ultra-high performance liquid chromatography and high-resolution mass spectrometry system LC-QE (a Vanquish Horizon liquid chromatography-Q Exactive TM Mass spectrometry system, Thermo Fisher Company, USA); Chromatographic column: Thermo Fisher Hypersil Gold C18 (1.9um, 2.1×500mm).

[0101] Liquid chromatography conditions: injection volume 5 μL; flow rate 0.3 mL / min; column temperature 25°C; mobile phase consisting of an aqueous solution containing 2 mM ammonium acetate and 2 mM 1-methylpiperidine (A) and a methanol solution containing 2 mM ammonium acetate and 2 mM 1-methylpiperidine (B), the specific mobile phases are shown in Table 1 below;

[0102] Table 1. Elution procedure

[0103]

[0104] Mass spectrometry conditions: the ion source adopted the negative ion mode of electrospray ionization source; the spray voltage was 3500 V; the ion source temperature was 300 °C; Full MS / ddMS2 and Full MS / DIA modes were selected; the MS1 resolution was set to 70000 in both modes; the scanning range was set to m / z = 100-1000; the MS2 resolution was set to 17500; the normalized collision energy was set to 20, 35, 50; in ddMS2, the Top7 ions were set to perform MS2 scans (Loop count was set to 7), and in DIA, five mass ranges were set to perform MS2 scans, and the mass ranges were m / z = 200-350, 350-500, 500-650, 650-800, 800-950, respectively.

[0105] Example 1: Comprehensive identification of PFAS in spiked test samples

[0106] This example provides comprehensive identification of PFAS in laboratory spiked samples, focusing on the use of established method systems to identify PFAS present in spiked samples, including the following steps:

[0107] S1. Perform liquid chromatography-high resolution mass spectrometry analysis on several PFAS standards to obtain standard chromatography-high resolution mass spectrometry data. The specific steps are as follows:

[0108] 1) Summarize the common PFAS found in the environment through text mining and purchase authentic standards. As shown in Table 2, there are 31 types in total.

[0109] Table 2. Compound name, CAS number, Chinese name, purity, molecular formula and primary ion accurate mass of 31 PFAS standards

[0110]

[0111]

[0112]

[0113] 2) The 31 authentic standards selected in step 1 were prepared into a mixed solution with a concentration of 100 μg / L and the solvent being methanol.

[0114] 3) Using a liquid chromatography-high resolution mass spectrometer, the PFAS mixed solution containing 31 authentic standards obtained in step 2) is instrumentally analyzed.

[0115] Based on the information of the authentic standards, the chromatographic-high-resolution mass spectrometry data of the PFAS mixed solution of the 31 authentic standards were targeted for analysis. The specific operation was to use Xcalibur software to extract the peaks of the authentic standards from the chromatographic-high-resolution mass spectrometry data of the spiked samples. The results showed that the 31 PFAS in the mixed solution obtained by targeted analysis were consistent with the standard mixed solution, confirming that the PFAS in the spiked samples can be targeted for identification.

[0116] S2. Collect the compound names and molecular formulas of PFAS based on the public chemical database, calculate the accurate mass number, and build a suspicious screening analysis database in combination with the open source MS2 spectrum library. The specific steps are as follows:

[0117] 1) Based on the public chemical database including the NORMAN Exchange list (NORMAN Suspect ListExchange), the names, structural formulas, and primary ion accurate mass numbers of 4777 PFAS were collected, and combined with open source MS2 spectral libraries (such as mzCloud, massbank) to jointly establish a suspect screening analysis database.

[0118] 2) Thermo Fisher's Compound Discoverer (version 3.3) and Fluoromatch software were used for analysis. In Compound Discoverer, the Masslist module was used to generate an MS database for 4777 PFAS, and the mzValut module was used to load the MassBank Europe Mass Spectral DataBase (98525spectra) and MassBank of North America databases (2079804spectra). The mzCloud module came with the mzCloud database. In Fluormatch, the PFAS database loaded into the software was used.

[0119] The chromatogram-high-resolution mass spectrometry data of the spiked sample collected in step S1 was subjected to suspicious screening analysis on the Compound Discoverer (version 3.3) software, and the process was as follows: in the spectrum selection module, the retention time interval was set to the default 0, and all spectra were analyzed; in the alignment retention time module, the mass deviation was set to 5ppm; in the detection compound module, the mass deviation was set to 5ppm, and the minimum peak intensity was set to 10000, while filtering out the impurity peaks, as much data as possible was obtained; all default negative addition ions were selected for the adduct ions; in the compound classification module, the mass deviation was set to 5ppm, the retention time deviation was set to 0.1min, and the peak shape scoring threshold was set to 4; in the blank filling module, the mass deviation was set to 5ppm, and the signal-to-noise ratio threshold was set to 3. This module is to fill the peak missing caused by the detection threshold. In the background compound marking module, the maximum ratio of the sample peak area to the blank peak area was set to 5, and the maximum ratio of the blank peak area to the sample peak area was set to 0. The purpose of this module is to mark the background ions. In the Search Masslists module, set the retention time deviation to 0.1 min and the mass deviation to 5 ppm; in the Search mzCloud module, select “Autoprocessed” and “Reference” for the database, Search MS nSelect No for Search fragment tree, select “confidence reverse” for Search DDA mode, select No for Match Ion Activation Mode and Match Ion Activity Energy, and set Match Factor Threshold to 70; select “Europe Mass Spectral DataBase (98525spectra) and MassBank of North America” for the database in the Search mzVault module, select All for Compound Class, set Match Ion Activation Mode and Match Ion Activity Energy to No, set Precursor MassTo Match to 5ppm, set Search Algorithm to HighChem HihRes, set Match Factor Threshold to 70, and set Use RetentionTime (i.e., whether to use retention time) was set to No. After the software was run, the screening conditions were set as follows: Background was "is fake"; "Masslist Matches" was "has at least status" AND "single match found" AND "in massfile X", where X was the name of the established suspicious screening database, and "Formular" was set to "contains F". Both "mzCloud Best Match" and "mzVault Best Match" were set to "≥70", and "mzCloud BestMatch Cofidence" was set to "7-10" OR "70-100". After the screening was completed, the chromatograms were checked one by one, and the original chromatograms and mass spectrometry data were manually checked to eliminate candidates with poor peak shapes and signal intensities comparable to those of blank samples.

[0120] For Fluoromatch software, the process is as follows: select the data of pure methanol solution as the blank file; select the data of standard solution as the secondary information file, target file and sample file; set the MS / MS and Full scan intensity thresholds to 1000; set the MS1 mass deviation to 0.005Da; and set the MS / MS deviation to 10ppm. The results of FluoroMatch run were preliminarily screened. For all results with scores of A, B and C, the original chromatographic and mass spectrometric data were manually checked to eliminate candidates with poor peak shape and signal intensity comparable to the blank sample. Then, preliminary PFAS structural types, i.e., class, were classified according to mass number and structural type.

[0121] The final suspicious screening results showed that all added standard substances were in the compound list, confirming that PFAS in the spiked samples could be identified through suspicious screening.

[0122] S3, combined with the mass number characteristics of PFAS (negative mass defects), characteristic MS2 fragment ion information (CF3 - 、C2F5-、C3F7 - 、C4F9 - 、C5F 11 - 、C6F 13 - 、C3F5 - 、C4F7 - 、C5F9 - 、C6F 11 - 、C3F3 - 、C4F5 - 、C5F7 - 、C6F9 - 、SO2F - 、SO3F - 、COF3 - 、C2F5O - 、C3F7O - 、C4F9O - 、C3F5O - 、C4F7O - 、C3F3O - 、C4F5O - 、C5F7O - 、C2F5SO2 - 、C3F7SO2 - 、C4F9SO2 - 、C2F6N - 、C3F8N - 、C4F 10 N - 、C5F12 N - 、C6F 14 N - 、C7F 16 N - 、C2F4N - 、C3F6N - 、C4F8N - 、C5F 10 N - 、C6F 12 N - 、C7F 14 N - 、C2F6NO - 、C3F8NO - 、C4F 10 NO - 、C5F 12 NO - 、C6F 14 NO - 、C7F 16 NO - 、C2F4NO - 、C3F6NO - 、C4F8NO - 、C5F 10 NO - 、C6F 12 NO - 、C7F 14 NO - 、C2F5SO3 - 、C3F7SO3 - 、C4F9SO3 - ), characteristic neutral loss (including HF (mass number 20), H2F2 (mass number 40), H3F3 (mass number 60), H4F4 (mass number 80), HFCO2 (mass number 64)) and homologue characteristics (including a series of compounds with a mass difference of CF2, C2F4, CHF, or C2H2F2, and the peak time of the homologues conforms to the correct retention time sequence), and establish a non-targeted identification workflow through algorithms or software (such as Compound Discover, Fluoromatch);

[0123] According to the non-targeted identification workflow, the chromatographic-high-resolution mass spectrometric data of the spiked sample are subjected to non-targeted identification to obtain candidate compounds for non-targeted identification, including: running the chromatographic-high-resolution mass spectrometric data of the spiked sample collected in step S1 on Compound Discoverer and Fluoromatch software, and the specific steps are as follows:

[0124] In the Compound Discoverer software, in the spectrum selection module, the retention time interval was set to the default 0, and all spectra were analyzed; in the alignment retention time module, the mass deviation was set to 5ppm; in the detection compound module, the mass deviation was set to 5ppm, and the minimum peak intensity was set to 10000; the adduct ion selected all default negative adduct ions; in the compound classification module, the mass deviation was set to 5ppm, the retention time deviation was set to 0.1min, and the peak shape score threshold was set to 4; in the blank filling module, the mass deviation was set to 5ppm, and the signal-to-noise ratio threshold was set to 3; in the labeling background compound module, the maximum ratio of the sample peak area to the blank peak area was set to 5, and the maximum ratio of the blank peak area to the sample peak area was set to 0; in the compound type coverage (i.e., class coverage) module, an MS2 characteristic fragment ion list was established, including all the fragments and neutral loss mass numbers listed above, and the list was set to be used for comparison; other parameters were set as follows: the signal-to-noise ratio threshold (S / N threshold) was 5, the high resolution mass number deviation (High Acc.MassTolerance) is 15ppm, Low Acc.Mass Tolerance is 2.5mmu, Use Full MS Tree is Yes, and Allow DIA Scoring is Yes; after the software is finished running, view the results, specifically: ① For the data in DDA mode, determine the corresponding structure through manual mass spectrometry analysis and other information; ② For the data in DIA mode, view the full scan data in the corresponding mass range, and use the mass defect characteristics mentioned above to find possible PFAS parent ions; then perform targeted MS2 analysis on these parent ions, and exclude them if the results do not contain the MS2 characteristic fragment ions mentioned above or other new F-containing fragments; if they contain the MS2 characteristic fragment ions mentioned above, perform structural analysis on their structures. The results show that all PFAS in the spiked samples were fully identified. It is confirmed that PFAS in the spiked samples can be non-targeted identified.

[0125] In Fluoromatch software, select the data of pure methanol solution as blank file; select the data of standard solution as secondary information file, target file and sample file; MS / MS and Full scan intensity thresholds are set to 1000; MS1 mass deviation is set to 0.005Da; MS / MS deviation is set to 10ppm; after the software is run, check the results, all PFAS series in the spiked samples are A, B and C levels (possible PFAS compounds, with clear F-containing fragments or characteristic neutral loss, or with characteristic homologue series), and are fully identified. Confirm that PFAS in spiked samples can achieve non-targeted identification.

[0126] Through the above steps, this embodiment realizes the full identification of 31 PFAS in the standard solution, and targeted, suspected screening and non-targeted identification can all be achieved, with an accuracy rate of 100%. Among them, step S2 targeted analysis, step S3 suspected screening and step S4 non-targeted identification can all achieve the correct identification of perfluorinated and polyfluorinated compounds in the sample. The three methods are complementary and can ensure the comprehensive identification of perfluorinated and polyfluorinated compounds in the environment to the greatest extent.

[0127] Example 2: Comprehensive Identification of PFAS in Water Samples

[0128] This example provides a comprehensive identification method for PFAS in groundwater samples, the purpose of which is to test the method's ability to identify PFAS in water environments, as well as the identification of unknown PFAS.

[0129] The operation steps are basically the same as those in Example 1. The difference from Example 1 is that in step S1, the water sample is pre-treated by using a WAX column solid phase extraction method. The specific operation is as follows:

[0130] S1. Pre-treat the water sample, specifically: before extraction, activate the solid phase extraction column (Oasis WAX column, specification: 6cc, 150mg) with 4mL 0.5% ammonia methanol, 4mL methanol and 4mL Milli-Q water in sequence. Then, load the water sample at a flow rate of 5-10mL / min. After that, wash the extraction column with 4mL Milli-Q water and 25mM CH3COONH4 / CH3COOH with a pH of 4 in sequence. Next, elute the extraction column with 6mL of 0.5% ammonia methanol, drain it, collect the eluate, evaporate the eluate to near dryness under a gentle nitrogen flow, and re-dissolve it with methanol to 1mL.

[0131] The pre-treated water samples were analyzed by liquid chromatography-high resolution mass spectrometry to obtain water sample chromatography-high resolution mass spectrometry data, including the chromatographic peak retention time, chromatographic peak area, primary mass spectrum information (MS1), secondary mass spectrum fragment information (MS2) and related signal intensity of the water samples;

[0132] S2. The same as 1)-3) in step S1 of Example 1, prepare 31 kinds of PFAS mixed solutions of authentic standard products, and perform liquid chromatography-high resolution mass spectrometry analysis on the mixed solutions to obtain standard product chromatography-high resolution mass spectrometry data, including chromatographic peak retention time, chromatographic peak area, primary mass spectrum information (MS1), secondary mass spectrum fragment information (MS2) and related signal intensity of each standard product;

[0133] The chromatographic-high-resolution mass spectrometric data of the standard and the water sample were matched and analyzed to obtain candidate compounds for targeted analysis. The specific steps were as follows: Xcalibur software was used to extract the peaks of the real standard from the chromatographic-high-resolution mass spectrometric data of the collected water sample to obtain candidate compounds for targeted analysis.

[0134] S3. The same as step S2 in Example 1, except that the chromatography-high resolution mass spectrometry data of the PFAS mixed solution of 31 authentic standards are replaced with the chromatography-high resolution mass spectrometry data of the pretreated water sample to obtain candidate compounds for suspicious screening analysis.

[0135] S4, same as step S3 in Example 1, except that the chromatogram-high resolution mass spectrometry data of the PFAS mixed solution of 31 authentic standards are replaced with the chromatogram-high resolution mass spectrometry data of the pretreated water sample to obtain candidate compounds for non-targeted identification;

[0136] S5. Integrate the candidate compound information obtained in step S2, step S3 and step S4, and infer and confirm the structure of the candidate compound in combination with the retention time information of the spiked sample, MS1 ion information, MS2 fragment ion information, homologue information, etc. obtained in step S2; integrate the list information of the targeted analysis, suspected screening and non-targeted identification parts according to the chromatographic data (chromatographic peaks, retention time, etc.) and mass spectrometry data (MS1 ion information and MS2 fragment ion information) of the water sample, specifically: screen the PFAS that appear repeatedly in the results of targeted analysis, suspected screening and non-targeted identification, and merge those of the same category to organize them into different types of PFAS; manually screen for the presence of homologues of other chain lengths of each type to obtain all the mass numbers of each PFAS type; then check whether the blank sample contains the corresponding PFAS, and the PFAS is considered to exist in the sample if the signal intensity exceeds 3 times that of the blank; finally, assign accurate structural confidence to each candidate compound identified as PFAS based on whether there are compounds with confirmed structures in each class, whether they can be matched to the database with high scores, whether there are clear and reasonable homologue characteristics, and whether there are characteristic quality defects.

[0137] As shown in Table 3, the results show that this example identified a total of 34 PFAS in water samples, including traditional per / polyfluoroalkyl carboxylic acids (PFCAs), per / polyfluoroalkyl sulfonic acids (PFSAs) and emerging fluorotelomers (n:2FTSs), H-substituted perfluoroalkyl organic acids (H-PFCAs, H-PFSAs), oxygen-ether bond-inserted perfluoroalkyl organic acids (O-PFCAs, O-PFSAs), etc. (Table 3, targeted, suspected screening, and non-targeted identification are represented by T, S, and N, respectively). Among them, C 10 H 19 F6N2PO8S2 and C 10 H 15 F3O7 are both unknown PFAS with no prior information. Both compounds are non-targeted identifications, with scores of A- (Confident ID) and B+ (Tentative ID, highly likely PFAS), respectively.

[0138] Table 3. 34 PFAS identified in water samples

[0139]

[0140]

[0141] As can be seen from Table 3, the method of the present invention can achieve comprehensive identification of PFAS in water samples. Among them, the compounds with confidence level 4 have accurate molecular formulas; the compounds with confidence levels 2-3 have preliminary inferred structures; and the compounds with confidence level 1 have all been verified by authentic standards.

[0142] Example 3: Comprehensive Identification of PFAS in Dewatered Sludge Samples from Municipal Wastewater Treatment Plants

[0143] This example provides a comprehensive identification method for PFAS in dewatered sludge samples from municipal wastewater treatment plants, with the goal of testing the method's ability to identify PFAS in different environmental samples, as well as the overlap and complementarity of targeted, suspect screening, and non-targeted identification methods.

[0144] The operation steps are basically the same as those in Example 1. The difference from Example 1 is that in step S1, the dewatered sludge sample of the municipal sewage treatment plant is pre-treated. The specific operation is as follows:

[0145] S1. Pre-treat the dewatered sludge samples from the municipal sewage treatment plant, specifically: take 0.5g of freeze-dried and ground into powder (pass through a 100 mesh sieve), first add 1.5ml of 0.4M NaOH solution (mix for 30min), and then add 2ml of 0.1M Na2CO3 / NaHCO3 buffer with a pH of 10. After thorough mixing, add 1mL of 0.5M TBAHS solution to mix. Then add 5ml of MTBE solution (repeat three times, each time with 8h of shaking). Combine the MTBE layers after the three liquid-liquid layering, then blow dry with gentle nitrogen gas, and re-dissolve with 1mL MeOH. Then add 30mL of Milli-Q pure water and enter the solid phase extraction process (similar to the water sample process).

[0146] The dewatered sludge samples after pretreatment were analyzed by liquid chromatography-high resolution mass spectrometry to obtain water sample chromatography-high resolution mass spectrometry data, including the chromatographic peak retention time, chromatographic peak area, primary mass spectrum information (MS1), secondary mass spectrum fragment information (MS2) and related signal intensity of the water sample;

[0147] S2. The same as 1)-3) in step S1 of Example 1, prepare 31 kinds of PFAS mixed solutions of authentic standard products, and perform liquid chromatography-high resolution mass spectrometry analysis on the mixed solutions to obtain standard product chromatography-high resolution mass spectrometry data, including chromatographic peak retention time, chromatographic peak area, primary mass spectrum information (MS1), secondary mass spectrum fragment information (MS2) and related signal intensity of each standard product;

[0148] The chromatographic-high-resolution mass spectrometric data of the standard and the dewatered sludge sample were matched and analyzed to obtain candidate compounds for targeted analysis. The specific steps were as follows: Xcalibur software was used to extract the peaks of the real standard from the chromatographic-high-resolution mass spectrometric data of the collected dewatered sludge sample to obtain candidate compounds for targeted analysis.

[0149] S3. The same as step S2 in Example 1, except that the chromatography-high resolution mass spectrometry data of the PFAS mixed solution of 31 authentic standards are replaced with the chromatography-high resolution mass spectrometry data of the pretreated dewatered sludge sample to obtain candidate compounds for suspicious screening analysis.

[0150] S4. Same as step S3 in Example 1, except that the chromatographic-high-resolution mass spectrometric data of the PFAS mixed solution of 31 authentic standards are replaced with the chromatographic-high-resolution mass spectrometric data of the pretreated dewatered sludge sample to obtain candidate compounds for non-targeted identification;

[0151] S5. Integrate the candidate compound information obtained in step S2, step S3 and step S4, and infer and confirm the structure of the candidate compound in combination with the retention time information of the spiked sample, MS1 ion information, MS2 fragment ion information, homologue information, etc. obtained in step S2; integrate the list information of the targeted analysis, suspected screening and non-targeted identification parts according to the chromatographic data (chromatographic peaks, retention time, etc.) and mass spectrometry data (MS1 ion information and MS2 fragment ion information) of the water sample, specifically: screen the PFAS that appear repeatedly in the results of targeted analysis, suspected screening and non-targeted identification, and merge those of the same category to organize them into different types of PFAS; manually screen for the presence of homologues of other chain lengths of each type to obtain all the mass numbers of each PFAS type; then check whether the blank sample contains the corresponding PFAS, and the PFAS is considered to exist in the sample if the signal intensity exceeds 3 times that of the blank; finally, assign accurate structural confidence to each candidate compound identified as PFAS based on whether there are compounds with confirmed structures in each class, whether they can be matched to the database with high scores, whether there are clear and reasonable homologue characteristics, and whether there are characteristic quality defects.

[0152] As shown in Table 4, the results show that a total of 54 PFAS of 32 categories were identified (Table 4, targeted, suspected screening, and non-targeted identification are represented by T, S, and N, respectively); targeted, suspected screening, and non-targeted identification identified 14, 36, and 26 PFAS, respectively. The distribution of compound types identified by targeted, suspected screening, and non-targeted identification is shown in Figure 2 As shown; among them, targeted analysis alone identified 4 species, suspected screening analysis alone identified 18 species, and non-targeted identification alone identified 18 species; targeted and suspected screening analysis jointly identified 9 compounds; targeted and non-targeted identification jointly identified 6 compounds; suspected screening and non-targeted identification jointly identified 9 compounds; and the three analytical methods jointly identified 5 PFASs, indicating the complementarity and indispensability of the three methods in this comprehensive identification method, as well as the applicability of this method in real environments.

[0153] The following is a detailed description of the identification process of suspicious screening and non-targeted identification with specific cases:

[0154] Suspicious screening analysis and identification cases:

[0155] The accurate mass of the primary ion is 332.09055. Flurtamone was matched to the database through suspicious screening analysis. There are 8 MS2 fragment ions (m / z 101.03960, 145.02696, 169.02676, 183.03055, 185.02219, 240.02913, 247.07439, 316.05911) that match the secondary spectrum of flurtamone ( Figure 3 ), and its confidence level is classified as 2a.

[0156] Non-targeted identification examples:

[0157] The accurate mass of the primary ion is 655.05689. Through non-targeted identification, typical MS2 fragment ions of PFAS such as [O2FS]- and [C2F5]- were found in the spectrum of this mass number. The parent ion of this mass number was subjected to a second MS2 fragmentation, and through further spectrum inspection, a total of 4 MS2 fragment ions were found ( Figure 4 ): [C3F7]-(m / z=168.98953, mass deviation is 0.942ppm), [C4F9]-(m / z=218.98640, mass deviation is 1.107ppm), [C5F 11 ]-(m / z=268.98343, mass deviation is 1.660ppm)and[C8F 17 ]-(m / z=418.97447, mass deviation is 2.547ppm). Its chemical formula is estimated to be [C 16 H 16 F 17 N2O4S]-(mass error 0.641ppm), chromatogram, first and second fragmentation MS2 mass spectra are as follows Figure 4 As shown. Since isomerism cannot be excluded, the confidence level of this structure is 3a. The name of this structure is perfluoroalkyl sulfonamido amino carboxylic acid (abbreviated as FOSAC).

[0158] Table 4. 54 PFAS identified in sediment

[0159]

[0160]

[0161] It can be seen from Table 4 that the method of the present invention can achieve comprehensive identification of PFAS in solid samples, and the confidence levels are all at level 3 and above, indicating that it has an accurate molecular formula and a preliminarily inferred structure.

[0162] In summary, the results of the above embodiments show that the comprehensive identification method of perfluorinated and polyfluorinated pollutants in the environment provided by the present invention has high accuracy, and the targeted, suspicious screening and non-targeted identification methods are complementary; it can be applied in complex environmental media; and the types of compounds identified are comprehensive. Therefore, the method of the present invention has universal applicability and broad application prospects.

[0163] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, can implement the present invention in a wider range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principle of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departing from the disclosed scope in the application and the changes made with conventional techniques known in the art.

Claims

1. A method for comprehensively identifying PFAS in the environment by combining targeted analysis, suspected screening and non-targeted identification, characterized in that: The steps include: S1. Performing liquid chromatography-high resolution mass spectrometry analysis on environmental samples to obtain environmental sample chromatography-high resolution mass spectrometry data; S2. Performing liquid chromatography-high resolution mass spectrometry analysis on several PFAS standards to obtain standard chromatography-high resolution mass spectrometry data; Performing targeted matching analysis on the chromatography-high resolution mass spectrometry data of the standard and the environmental sample to obtain compounds for targeted analysis; S3. Collect the compound names and molecular formulas of several PFAS based on public chemical databases and / or literature, calculate the accurate mass numbers, and jointly establish a suspicious screening analysis database in combination with the open source MS2 spectrum library; According to the suspicious screening analysis database, performing suspicious screening analysis on the environmental sample chromatography-high resolution mass spectrometry data to obtain candidate compounds for suspicious screening analysis; S4. Establish a non-targeted identification workflow by combining the mass number characteristics, characteristic MS2 fragment ions, characteristic neutral losses, and homologue characteristics of PFAS; According to the non-targeted identification workflow, non-targeted identification is performed on the chromatography-high-resolution mass spectrometry data of the environmental sample to obtain candidate compounds for non-targeted identification; S5. Integrate the compounds of the targeted analysis, the candidate compounds of the suspected screening analysis, and the candidate compounds of the non-targeted identification to comprehensively identify the PFAS in the environmental sample.

2. The method of claim 1, wherein: The environmental samples include water samples, solid samples or biological samples; the water samples include industrial sewage, inlet and outlet water of sewage treatment plants, river water, surface water, sea water, drinking water or groundwater; the solid samples include sludge from sewage treatment plants, water sludge, sediment, soil, indoor dust or atmospheric particulate matter; the biological samples include human breast milk, urine, serum, follicular fluid, umbilical cord blood, saliva, sweat, animal tissues and organs, fish, birds, sharks, mollusks or plants.

3. The method for comprehensively identifying PFAS in the environment by combining targeted analysis, suspicious screening and non-targeted identification technology according to claim 1 or 2, characterized in that: The method further comprises the step of pre-treating the environmental sample before the liquid chromatography-high resolution mass spectrometry analysis; When the environmental sample is a water sample, the pretreatment adopts a WAX column solid phase extraction method, comprising the following steps: 1) before extraction, the solid phase extraction column is activated with a methanol solution of 0.5% ammonia water by volume, methanol and Milli-Q water in sequence; 2) the water sample is loaded into the WAX ​​solid phase extraction column activated in step 1) at a flow rate of 5 to 10 mL / min; 3) after loading the water sample in step 2), the extraction column is washed with Milli-Q water and 25 mM CH3COONH4 / CH3COOH buffer with a pH of 4 in sequence; 4) after washing in step 3), the extraction column is eluted with a methanol solution of 0.5% ammonia water by volume, and dried, the eluate is collected, the eluate is evaporated to near dryness under a nitrogen flow, and re-dissolved with methanol; When the environmental sample is a solid sample, the pretreatment adopts ion pair extraction combined with solid phase extraction, including the following steps: 1) adding a 0.4M NaOH solution, a Na2CO3 / NaHCO3 buffer solution with a pH of 10 and a concentration of 0.1M, and a 0.5M tetrabutylammonium hydrogen sulfate solution to the environmental sample in sequence; 2) adding methyl tert-butyl ether solution to the mixed solution in step 1), repeating three times, shaking for 8 hours each time; 3) combining the methyl tert-butyl ether layers after three liquid-liquid layer separations in step 2), drying with nitrogen, and then re-dissolving with methanol; 4) adding Milli-Q water to the solution obtained in step 3), and then performing solid phase extraction, the solid phase extraction step is the same as the solid phase extraction step of the water sample; When the environmental sample is plasma, breast milk, follicular fluid, or saliva, the pretreatment adopts organic reagent protein precipitation combined with solid phase extraction, including the following steps: 1) acetonitrile is first added to the biological liquid sample for protein precipitation; 2) after sufficient precipitation, the supernatant is concentrated by nitrogen blowing; 3) Milli-Q water is added to the solution obtained in step 2) and then solid phase extraction is performed, and the solid phase extraction step is the same as the solid phase extraction step of the water sample; When the environmental sample is urine or sweat, the pretreatment method is the same as the pretreatment step of the water sample; When the environmental sample is a tissue sample, the pretreatment adopts a method of grinding or homogenization, organic reagent protein precipitation, and solid phase extraction, including the following steps: 1) grinding or homogenizing the tissue sample; 2) adding acetonitrile to the sample obtained in step 1) for protein precipitation; 3) taking the supernatant after sufficient precipitation and concentrating it by nitrogen blowing; 4) adding Milli-Q water to the solution obtained in step 3), and then performing solid phase extraction, the solid phase extraction step is the same as the solid phase extraction step of the water sample; the tissue sample includes animal tissue organs, fish, birds, sharks, and mollusks; When the environmental sample is a plant, the pretreatment adopts a method of grinding or homogenization, organic reagent extraction, centrifugation, and solid phase extraction, including the following steps: 1) grinding or homogenizing the plant sample; 2) adding methanol to the sample obtained in step 1) for extraction, and the extraction is sequentially performed by high-speed vortexing for 2 minutes, ultrasound for 30 minutes, and shaking for 1 hour; 3) centrifuging the sample obtained in step 2) at 5000rpm for 30 minutes, and transferring the supernatant to a clean centrifuge tube; 4) repeating the extraction process three times; 5) combining the supernatants of the three extractions in step 4), concentrating, then adding Milli-Q water and entering solid phase extraction, and the solid phase extraction step is the same as the solid phase extraction step of the water sample; the plant includes leaves or stems.

4. The method for comprehensively identifying PFAS in the environment by combining targeted analysis, suspicious screening and non-targeted identification according to any one of claims 1 to 3, characterized in that: In step S1 and step S2, the conditions of the liquid chromatography-high resolution mass spectrometry analysis are as follows: Liquid chromatography conditions: injection volume of 5 μL; flow rate of 0.3 mL / min; column temperature of 25°C; mobile phase consisting of aqueous solution A containing 2 mM ammonium acetate and 2 mM 1-methylpiperidine and methanol solution B containing 2 mM ammonium acetate and 2 mM 1-methylpiperidine; gradient elution program, in terms of volume fraction, is as follows: 0 min 70% solution A and 30% solution B; 1 min 50% solution A and 50% solution B; 6 min 40% solution A and 60% solution B; 20 min 25% solution A and 75% solution B; 23 min 15% solution A and 85% solution B; 23.5 min 0% solution A and 100% solution B; 28.5 min 0% solution A and 100% solution B; 28.6min 70% solution A and 30% solution B; 33.6min 70% solution A and 30% solution B; Mass spectrometry conditions: the ion source used the negative ion mode of the electrospray ionization source; the spray voltage was 3500 V; the ion source temperature was 300 °C; Full MS / ddMS2 and Full MS / DIA modes were selected; the MS1 resolution in both modes was set to 70000; the scan range was set to m / z = 100-1000; The MS2 resolution was set to 17500; the normalized collision energy was set to 20, 35, and 50; in ddMS2, the Top7 ions were scanned with MS2, and the loop count was set to 7. In DIA, five mass ranges were scanned with MS2, and the mass ranges were m / z = 200-350, 350-500, 500-650, 650-800, and 800-950. The environmental sample chromatography-high resolution mass spectrometry data and the standard chromatography-high resolution mass spectrometry data include chromatographic peak retention time, chromatographic peak area, primary mass spectrometry information (MS1), secondary mass spectrometry fragmentation information (MS2) and related signal intensity.

5. The method for comprehensively identifying PFAS in the environment by combining targeted analysis, suspicious screening and non-targeted identification according to any one of claims 1 to 4, characterized in that: The several PFAS standards are 31 PFAS standards, and the purity of each standard is ≥98%; The targeted matching analysis includes: comparing the consistency of chromatographic peak retention time and mass spectrum fragment ion information.

6. The method for comprehensively identifying PFAS in the environment by combining targeted analysis, suspicious screening and non-targeted identification according to any one of claims 1 to 5, characterized in that: The public chemical database is the public PFAS chemical database in NORMAN Suspect ListExchange; The several PFASs are 4777 PFASs; The MS2 spectrum library is mzCloud library and massbank library; The establishment of the suspicious screening analysis database was analyzed using Compound Discoverer and Fluoromatch software; in Compound Discoverer, the Masslist module was used to generate an MS database for 4777 PFAS, the mzValut module was used to load the MassBank Europe Mass Spectral DataBase (98525spectra) and the MassBankof North America database (2079804spectra), and the mzCloud module came with the mzCloud database; in the Fluormatch software, the PFAS database loaded inside the software was used.

7. The method for comprehensively identifying PFAS in the environment by combining targeted analysis, suspicious screening and non-targeted identification according to any one of claims 1 to 6, characterized in that: The suspicious screening analysis was performed on Compound Discoverer software and FluoroMatch software; the Compound Discoverer process was as follows: in the spectrum selection module, the retention time interval was set to the default 0, and all spectra were analyzed; in the alignment retention time module, the mass deviation was set to 5ppm; in the detection compound module, the mass deviation was set to 5ppm, and the minimum peak intensity was set to 10000; the adduct ion selected all default negative adduct ions; in the compound classification module, the mass deviation was set to 5ppm, the retention time deviation was set to 0.1min, and the peak shape score threshold was set to 4; in the blank filling module, the mass deviation was set to 5ppm, and the signal-to-noise ratio threshold was set to 3; in the labeling background compound module, the maximum ratio of the sample peak area to the blank peak area was set to 5, and the maximum ratio of the blank peak area to the sample peak area was set to 0; in the Search Masslists module, the retention time deviation was set to 0.1min and the mass deviation was 5ppm; in the Search mzCloud module, the database selected "Autoprocessed" and "Reference", Search MS n Select No for tree, select "confidence reverse" for Search DDA mode, select No for MatchIonActivation Mode and MatchIon Activity Energy, and set Match Factor Threshold to 70; select "Europe Mass Spectral DataBase (98525spectra)" and "MassBank of North America" ​​for database in Search mzVault module, select All for Compound Class, set Match IonActivation Mode and Match Ion Activity Energy to No, set Precursor Mass To Match to 5ppm, set Search Algorithm to HighChem HihRes, set Match Factor Threshold to 70, and set Use Retention Time to No; for Fluoromatch software, the process is as follows: select the data of pure methanol solution as the blank file; select the data of standard solution as the secondary information file, target file, and sample file; set the MS / MS and Full scan intensity thresholds to 1000; set the MS1 mass deviation to 0.005Da; and set the MS / MS deviation to 10ppm.

8. The method of claim 7, wherein: The results of the Compound Discoverer operation described in claim 7 are preliminarily screened, and the screening conditions are set as follows: Background is "is fake"; "Masslist Matches" is "has at least status" AND "single match found" AND "in massfile X", where X is the name of the established suspicious screening database, and "Formular" is set to "contains F"; "mzCloud Best Match" and "mzVault Best Match" are both set to "≥70", and "mzCloud Best Match Cofidence" is set to "7-10" OR "70-100"; a chromatographic check is performed on the candidate compounds obtained from the suspicious screening analysis, and the original chromatographic and mass spectrometric data are manually checked to eliminate candidates with poor peak shape and signal intensity equivalent to that of blank samples, and then a preliminary PFAS structural type, i.e., class, classification is performed based on the mass number and structural type; The results of the FluoroMatch run described in claim 7 are initially screened, and for all results scored as A, B, and C, the original chromatographic and mass spectrometric data are manually checked to eliminate candidates with poor peak shapes and signal intensities comparable to those of blank samples. Preliminary PFAS structural type, i.e., class, classification is then performed based on mass number and structural type.

9. The method for comprehensively identifying PFAS in the environment by combining targeted analysis, suspicious screening and non-targeted identification according to any one of claims 1 to 8, characterized in that: Said Special Expedition MS2 Kakikata Satoko Comprehensive CF3 - , C2F5 - , C3F7 - , C4F9 - , C5F 11 - , C6F 13 - , C3F5 - , C4F7 - , C5F9 - , C6F 11 - , C3F3 - , C4F5 - , C5F7-, C6F9-, SO2F-, SO3F-, COF3-, C2F5O-, C3F7O-, C4F9O-, C3F5O-, C4F7O- , C3F3O-, C4F5O-, C5F7O-, C2F5SO2-, C3F7SO2-, C4F9SO2-, C2F6N-, C3F8N-, C4F 10 N-, C5F 12 N-, C6F 14 N-, C7F 16 N-, C2F4N-, C3F6N-, C4F8N-, C5F 10 N-, C6F 12 N-, C7F 14 N-, C2F6NO-, C3F8NO-, C4F 10 NO-, C5F 12 NO-, C6F 14 NO-, C7F 16 NO-, C2F4NO-, C3F6NO-, C4F8NO-, C5F 10 NO-, C6F 12 NO-, C7F 14 NO-, C2F5SO3-, C3F7SO3-, C4F9SO3-; The characteristic neutral losses include HF (mass number 20), H2F2 (mass number 40), H3F3 (mass number 60), H4F4 (mass number 80), and HFCO2 (mass number 64); The homologous series include a series of compounds with mass numbers differing by CF2, C2F4, CHF, or C2H2F2; The mass number characteristics of the PFAS are negative mass defects or tiny positive mass defects with a mass defect range of -0.15 to 0.

1.

10. The method for comprehensively identifying PFAS in the environment by combining targeted analysis, suspected screening and non-targeted identification according to any one of claims 1 to 9, characterized in that: The non-targeted identification was performed on Compound Discoverer and Fluoromatch software: For Compound Discoverer software, the process is as follows: in the spectrum selection module, the retention time interval is set to the default 0, and all spectra are analyzed; in the alignment retention time module, the mass deviation is set to 5ppm; in the detection compound module, the mass deviation is set to 5ppm, and the minimum peak intensity is set to 10000; the adduct ion selects all default negative adduct ions; in the compound classification module, the mass deviation is set to 5ppm, the retention time deviation is set to 0.1min, and the peak shape score threshold is set to 4; in the blank filling module, the mass deviation is set to 5ppm, and the signal-to-noise ratio threshold is set to 3; in the labeling background compound module, the maximum ratio of the sample peak area to the blank peak area is set to 5, and the maximum ratio of the blank peak area to the sample peak area is set to 0; in the compound type coverage (i.e., class coverage) module, an MS2 characteristic fragment ion list is established, including all fragments and neutral loss mass numbers listed in claim 9, and the list is set to be used for comparison; other parameters are set as follows: the signal-to-noise ratio threshold (S / N threshold) is 5, the high resolution mass number deviation (HighAcc.Mass Tolerance is 15 ppm, Low Acc. Mass Tolerance is 2.5 mmu, Use Full MS Tree is Yes, Allow DIA Scoring is Yes; For Fluoromatch software, the process is as follows: select the data of pure methanol solution as the blank file; select the data of standard solution as the secondary information file, target file and sample file; MS / MS and Full scan intensity thresholds are both set to 1000; MS1 mass deviation is set to 0.005Da; MS / MS deviation is set to 10ppm.

11. The method of claim 10, wherein: For the results of Compound Discoverer in claim 10, screen the data with class coverage>0: ① For the data in DDA mode, determine the corresponding structure by manual mass spectrometry analysis and combining other information; ② For the data in DIA mode, check the full scan data in the corresponding mass range, and use the mass defect characteristics in claim 9 to find possible PFAS parent ions; then perform target MS2 analysis on these parent ions, and exclude the obtained results if they do not contain the MS2 characteristic fragment ions in claim 9 or other new F-containing fragments; if they contain the MS2 characteristic fragment ions in claim 9, perform structural analysis on their structures; The results of the FluoroMatch run described in claim 10 were initially screened, and for all results scored as A, B, and C, the original chromatographic and mass spectrometric data were manually checked to eliminate candidates with poor peak shapes and signal intensities comparable to blank samples. Subsequently, a preliminary PFAS structural type, i.e., class, was assigned based on the homologue features in claim 9 and the mass number characteristic defects.

12. The method for comprehensively identifying PFAS in the environment by combining targeted analysis, suspicious screening and non-targeted identification technology according to any one of claims 1 to 11, characterized in that: The integration includes: Inferring and confirming the structure of the candidate compound based on the chromatographic MS1 ion information, MS2 fragment ion information, homologue information, and retention time information of the environmental sample; Based on the chromatographic data and mass spectrometry data of the candidate compounds in the environmental samples, the list information of targeted analysis, suspected screening and non-targeted identification was integrated and sorted into different types of PFAS; the presence of homologues of other chain lengths of each type was manually screened to obtain all the mass numbers of each PFAS type; then the blank sample was checked for the presence of the corresponding PFAS, and the PFAS was considered to be present in the sample if the signal intensity exceeded 3 times that of the blank; finally, each class was assigned an accurate structural confidence based on whether there were structurally confirmed compounds, whether they could be matched to the database with high scores, whether there were clear and reasonable homologue characteristics, whether there were characteristic mass defects, and whether there were characteristic MS2 fragment ions.

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