Screening identification and risk assessment method for PFAS in surface water and application

Abstract

The invention discloses a method for screening, identifying and risk evaluating PFAS in surface water and application, and belongs to the technical field of environment monitoring analysis and risk evaluation. The technical problem to be solved is to provide a method with the advantages of high pretreatment efficiency, wide screening range, accurate quantitative analysis, fine risk assessment and the like aiming at the problems of incomplete PFAS screening coverage, inextreme quantitative sensitivity and non-detailed risk assessment in the prior art. According to the technical scheme, the method is characterized in that the pretreatment process of a surface water sample is optimized through full-automatic solid-phase extraction equipment, and comprehensive identification and accurate analysis of traditional and emerging PFAS are achieved in combination with non-targeted screening of Orbitrap Explores 120 high-resolution mass spectrometry and targeted quantification of 7500 triple quadrupole mass spectrometry; and comprehensive evaluation of ecological and health risks is completed through a risk entropy model and a human body multi-tissue cell toxicity experiment.

Description

Technical Field

[0001] This invention relates to the field of environmental monitoring analysis and risk assessment technology, specifically to a method and application for screening, identifying, and assessing the risks of PFAS in surface water. Background Technology

[0002] Per- and polyfluoroalkyl substances (PFAS), as a type of "new pollutant," refer to synthetic organic chemicals containing at least one fully fluorinated methyl or methylene carbon atom (without any H / Cl / Br / I atoms). The strong CF bonds in these chemicals endow them with chemical stability, hydrophobicity, oleophobicity, thermal stability, and surface activity, leading to their application in over 200 fields. PFAS can enter the environment through direct and indirect sources, including industrial production, waste disposal, and secondary sources such as wastewater treatment plants and landfills, resulting in their widespread presence in the environment. Due to their high persistence, bioaccumulation potential, and toxicity, PFAS cause various adverse effects on organisms and humans, raising serious concerns about the risks and hazards they pose to ecosystems and human health.

[0003] In response to growing concerns, governments and relevant departments in various countries have regulated traditional perfluorooctanoic acid (PFOA), perfluorooctane sulfonate (PFOS), and perfluorohexane sulfonate (PFHxS). Consequently, manufacturers have increased their efforts to develop diverse and emerging alternatives to mitigate the adverse effects of PFAS. However, due to the large-scale production and use of PFAS alternatives with different structures, and the presence of a range of feedstocks, intermediates, and byproducts that can potentially transform into unknown compounds, a significant portion of the screening, identification, and ecological and health risks associated with PFAS remain difficult to determine.

[0004] Existing technologies lack sufficient screening and identification capabilities for emerging PFAS. Targeted quantitative methods are unable to cope with the increasing variety of PFAS and struggle to comprehensively identify unknown PFAS in complex environments. Risk assessments are often limited in scope and lack a comprehensive evaluation system, failing to provide risk level classification and scientific basis for pollution prevention and control. Therefore, there is an urgent need for a comprehensive assessment method to fully identify the types and levels of PFAS pollution and to conduct detailed assessments of their risk levels and health hazards, thereby promoting the prevention and management of new pollutants. Summary of the Invention

[0005] The purpose of this invention is to provide:

[0006] A method and related technologies for screening, identifying and risk assessing PFAS in surface water, to address technical issues such as comprehensively identifying the types and levels of PFAS pollution and conducting detailed assessments of PFAS risk levels and health hazards, or combinations thereof.

[0007] Terminology Explanation: Unless otherwise defined, all technical terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this subject matter pertains. Unless otherwise stated, all patents, patent inventions, and disclosures cited throughout this document are incorporated herein by reference in their entirety. Where multiple definitions exist for terms herein, the definitions provided in this chapter shall prevail.

[0008] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0009] Definitions of standard chemical terms can be found in the references "Per- and Polyfluoroalkyl Substances (PFAS): Environmental Fate, Exposure, and Toxicity (Elsevier, 2022)" and "Handbook of Environmental Analysis: Chemical Pollutants in Air, Water, Soil, and Solid Wastes (CRC Press, 2023, 5th Edition)".

[0010] Unless otherwise stated, conventional methods within the scope of the art, such as ultra-high performance liquid chromatography, risk entropy analysis, and in vitro cytotoxicity experiments, shall be used.

[0011] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0012] The term "PFAS" as used in this article refers to perfluorinated and polyfluoroalkyl compounds, which are a class of synthetic organic chemicals containing at least one fully fluorinated methyl or methylene carbon atom (excluding H / Cl / Br / I atoms). They possess chemical stability, hydrophobic and oleophobic properties, are widely present in the environment, and pose potential hazards to ecosystems and human health.

[0013] The term "internal standard" as used in this article refers to a stable isotope-labeled compound (e.g., structure, retention behavior, ionization efficiency) that has similar physicochemical properties (e.g., structure, retention behavior, ionization efficiency) to the target PFAS. 13 C and d labeled PFAS are added during sample pretreatment to correct for matrix effects, recovery differences, and instrument signal fluctuations, ensuring the accuracy of quantitative analysis.

[0014] The term "surface water" as used in this article refers to natural water bodies and related receiving water bodies existing on the Earth's surface, including rivers, lakes, reservoirs, wetlands, streams, ditches, estuaries, nearshore sea areas, irrigation canals, and landscape water bodies, which are the main carriers of PFAS pollution transport and accumulation.

[0015] The term "parent ion" as used in this article refers to a molecular or quasi-molecular ion with a specific mass-to-charge ratio (m / z) formed after the target PFAS is ionized in a mass spectrometry ion source (such as [MH] formed by PFAS in negative ion mode). - Ions are the basis for subsequent fragmentation and generation of daughter ions.

[0016] The term "daughter ion" as used in this article refers to fragment ions produced after the parent ion is broken up by collision energy in the mass spectrometer collision cell. These fragment ions include quantitative ions (used for precise quantification) and qualitative ions (used for compound structure confirmation). Their mass-to-charge ratio and abundance ratio are key criteria for PFAS identification.

[0017] The term "collision energy" used in this article refers to the voltage energy applied in the collision cell of a mass spectrometer, which is used to induce the parent ion to break chemical bonds and generate characteristic daughter ions. Different PFAS require optimization of specific collision energies to obtain stable and high-intensity daughter ion signals.

[0018] The term "Q0D parameter" used in this article refers to the voltage parameter between Q0 (pre-quadrupole) and D (collision cell inlet lens) in triple quadrupole mass spectrometry. It is used to adjust ion transport efficiency, optimize the focusing and transport of target ions, reduce the entry of interfering ions into the collision cell, and improve detection sensitivity and signal-to-noise ratio.

[0019] The term “method limit of quantitation” used in this article refers to mLOQ, which is the lowest concentration of the target PFAS that can be accurately quantified. It is determined by the ratio of the lowest standard concentration in the linear range to the sample dilution factor, and the spiked recovery and precision at this concentration must meet the requirements of trace analysis.

[0020] The term “PFAS identification confidence level standard” used in this article refers to the PFAS-specific identification grading system based on high-resolution mass spectrometry technology established by Charbonnet et al. Through multi-dimensional evidence such as standard verification, mass spectrometry fragment matching, and homology series analysis, the PFAS identification results are divided into 5 main levels (including sub-levels), clarifying the reliability and applicable scenarios of identification results at different levels.

[0021] The term "homogeneous series" used in this article refers to a series of PFAS homologs that have the same functional groups but differ only in carbon chain length or repeating structural units (such as CF2, C2F4, CF2O). Their mass-to-charge ratios show a fixed mass difference (such as 49.99681 Da for the CF2 unit), which is a key basis for enriching identification results in PFAS non-targeted screening.

[0022] The term "Kendrick quality defect" as used herein refers to the quality defect value (KMD) obtained by recalculating the precise mass of the compound according to a specific benchmark (normalized to CF2 in this invention). The KMD value of PFAS is typically within [a certain range]. The range of 0.15-0.15 is an important characteristic parameter for screening and distinguishing PFAS compounds.

[0023] The term "spiking recovery experiment" as used in this article refers to: adding a known concentration of PFAS standard to a blank sample (or actual sample), performing the same pretreatment and detection procedures as the sample, calculating the ratio of the measured concentration to the spiked concentration (recovery rate) and the relative standard deviation (RSD) of parallel determinations, which is used to verify the accuracy and precision of the method.

[0024] The term "risk entropy" used in this article refers to a quantitative indicator (RQ) used to assess the ecological risk of PFAS. It is calculated by the ratio of measured environmental concentration (MEC) to predicted non-effect concentration (PNEC). Based on the size of the RQ value, four risk levels are divided: high, medium, low, and none, reflecting the potential harm of PFAS to aquatic ecosystems.

[0025] The term "mass deviation threshold" used in this paper refers to the maximum allowable deviation range between the measured mass-to-charge ratio and the theoretical mass-to-charge ratio in mass spectrometry analysis. It is used to screen effective characteristic peaks and verify the accuracy of compound identification. In this invention, the mass deviation threshold for primary mass spectrometry is <5 ppm, and the mass deviation threshold for secondary mass spectrometry structure inference is ≤10 ppm.

[0026] The term “PFAS mixed exposure solution” used in this article refers to an experimental system in which two or more PFAS monomers are dissolved in a suitable medium (water, culture medium, serum, etc.) at a set concentration and ratio to simulate a real exposure scenario. It must meet the requirements of accurate concentration, stable components, and no external contamination.

[0027] In a first aspect, the present invention provides: a method for screening, identifying, and assessing the risk of PFAS in surface water, comprising the following steps: (1) Sample pretreatment: Surface water samples were filtered to remove suspended solids, and internal standards were added. Solid phase extraction was then performed. The purification and concentration were completed by activation, loading, rinsing, elution and nitrogen blowing. The eluent was redissolved and filtered to obtain the sample to be analyzed. The solid-phase extraction column is an Oasis WAX solid-phase extraction column; The activation step is carried out sequentially using ammoniated methanol, methanol, and water, wherein the concentration of the ammoniated methanol is selected from 0.4% to 0.6%. The rinsing step is carried out sequentially using an ammonium acetate aqueous solution with a pH of 4-5 and water, wherein the concentration of the ammonium acetate aqueous solution is selected from 20-30 mM; The elution step is performed sequentially using methanol and ammoniated methanol, wherein the concentration of the ammoniated methanol is selected from 0.4% to 0.6%. (2) Non-targeted screening: The sample to be analyzed obtained in step (1) is detected by ultra-high performance liquid chromatography-electrostatic field orbital trap high resolution mass spectrometry system. Data is collected in full scan / data-dependent analysis mode. Non-targeted screening is carried out by combining PFAS homology series, diagnostic fragments and database matching to identify PFAS categories. The mobile phases for the ultra-high performance liquid chromatography are: Phase A is a 2 mM ammonium acetate aqueous solution, and Phase B is a methanol-acetonitrile mixed solution; (3) Targeted quantification: The sample to be analyzed obtained in step (1) is detected by an ultra-high performance liquid chromatography-triple quadrupole mass spectrometry system. The negative ion multiple reaction monitoring mode is adopted. Based on the optimized parent ion, daughter ion, collision energy and Q0D parameters, combined with the calibration curve, the PFAS concentration is quantified. The mobile phase for the ultra-high performance liquid chromatography is: phase A is 4 mM ammonium acetate aqueous solution, and phase B is acetonitrile; (4) Risk assessment: Based on the quantitative concentration of PFAS obtained in step (3), the risk entropy model is used to calculate the risk entropy to assess the ecological risk, and the health risk is assessed by constructing in vitro cell models of six human cell lines to clarify the risk level and health hazards of PFAS. The six human cell lines include brain, heart, liver, lung, kidney, and colon cell lines; The PFAS includes PFBA, PFPeA, PFHxA, PFHpA, PFOA, PFNA, PFDA, PFUnDA, PFDoA, PFTrDA, PFTeDA, PFBuS, PFPeS, PFHxS, P FOS, PFNS, PFDS, PFDoS, FOSA, N-MeFOSA, N-EtFOSA, FOSAA, N-MePFOSAA, N-EtPFOSAA, N-MeFOSE, N-EtFOSE, 6:2 FTCA, 8:2 FTCA, 10:2 FTCA, PFHxPA, PFOPA, PFDPA, Cl-PFHxPA, Cl-PFOPA, PF4OPeA, PF5OHxA, NFDHA, HFPO-DA, HFPO-TA, PFEESA, 4:2FTS, 6:2FTS, 8:2FTS, 10:2FTS, 6:2diPAP, 8:2PAP, NaDONA, 6:2 Cl-PFESA, 8:2Cl-PFESA; The internal standards include: 13C4 PFBA, 13C5 PFPeA, 13C5 PFHxA, 13C4 PFHpA, 13C8 PFOA, 13C9 PFNA, 13C6 PFDA, 13C7 PFUnDA, 13C3 PFBuS, 13C8 PFOS, 13C3 PFHxS, 13C8 FOSA, d3-N-MePFOSAA, d5-N-EtPFOSAA, 13C2 4:2 FTS, 13C2 6:2 FTS, and 13C2 8:2 FTS.

[0028] The technical features include: target analytes, internal standards, ultra-high performance liquid chromatography-quadrupole orbital trap high-resolution mass spectrometry, risk entropy model, and in vitro human cell model.

[0029] Preferably, the method of removing suspended solids by filtration in step (1) is: filtration through a glass fiber filter membrane; More preferably, the method of removing suspended solids by filtration in step (1) is: filtration through a 0.45μm glass fiber filter membrane; The surface water includes, but is not limited to, at least one of the following: rivers, lakes, reservoirs, wetlands, streams, water bodies surrounding industrial clusters, and irrigation canals; Preferably, the amount of internal standard added in step (1) is: 0.5 ng of internal standard added to every 250 mL of surface water sample; Preferably, the solid-phase extraction column in step (1) has the following specifications: column volume of 6 mL and adsorbent loading of 500 mg; Preferably, the activation step is performed sequentially using 0.5% ammoniated methanol, methanol, and water; Preferably, the rinsing step is performed sequentially using a 25mM ammonium acetate aqueous solution with a pH of 4 and water; Preferably, the elution step is performed sequentially using methanol and 0.5% ammoniated methanol; Preferably, the specific steps of purification and concentration in step (1) are as follows: Table 1. Solid-phase extraction steps

[0030] Preferably, the solvent for resolution in step (1) is a mixture of methanol and water (v / v=1:1). Preferably, the filtration method in step (1) is: using a polyethersulfone needle filter membrane for filtration; More preferably, the filtration method in step (1) is: using a 0.22μm polyethersulfone needle filter membrane; In step (2), the mobile phase B of the ultra-high performance liquid chromatography is a mixture of methanol and acetonitrile at a volume ratio of 1:1. Preferably, the ultra-high performance liquid chromatography column has a size of 2.1 × 100 mm and a stationary phase particle size of 2.7 µm; In step (2), the column temperature of the ultra-high performance liquid chromatography is 35℃ and the injection volume is 10 µL. The elution gradient of the mobile phase in step (2) of the ultra-high performance liquid chromatography is as follows: Table 2. Mobile phase elution gradient parameter settings for ultra-high performance liquid chromatography

[0031] The parameters for the high-resolution mass spectrometry described in step (2) are set as follows: Table 3. Parameter settings for high-resolution mass spectrometry

[0032] The specific steps for performing non-targeted screening in step (2) are as follows: S1. Feature Peak Extraction and Preliminary Filtering: Feature peaks that meet specific criteria are extracted from the raw data obtained from high-resolution mass spectrometry. The peak extraction conditions are defined as peak intensity > 1000 and signal-to-noise ratio threshold > 3. At the same time, alignment filtering rules are set, namely, retention time deviation < 2 min and mass deviation < 5 ppm, to ensure the stability and accuracy of feature peaks and remove interference peak signals. S2. Database Matching and Suspicious Peak Screening: Utilizing open-access and commercial databases, based on first-level mass spectrometry (MS / MS)... 1 The precise mass number and secondary mass spectrometry (MS) 2The fragment ion information is used to perform preliminary compound matching; the matching results are further filtered through multiple criteria, including verification of the reasonableness of retention time and isotope pattern matching degree >80%, to generate a preliminary list of suspected PFAS for screening. S3. Homologous Series Search and Candidate Peak Enrichment: Based on the homologous series characteristics unique to PFAS, the peaks that meet the following conditions are listed as potential candidate peaks: ① quality deviation < 5 ppm; ② matching threshold ≥ 0.8; ③ the characteristic quality difference corresponds to a typical PFAS structural unit, namely 49.99681 Da (CF2), 99.99361 Da (C2F4), 64.01246 Da (C2H2F2), 65.99172 Da (CF2O) or 115.98853 Da (C2F4O). The homologous series expansion enriches the PFAS identification results and covers more emerging PFAS with similar structures. S4. Structural Inference and Precise Filtering: Based on the feature peaks determined by suspicious screening and homology search, combined with MS 2 Fragment ion information was used to infer the structure of PFAS compounds, with a mass deviation threshold of ≤10ppm set to ensure the accuracy of the structure inference. Further filtering was performed using dual constraints: retention time was limited to 2-15 minutes, and Kendrick mass defects (KMDs) were normalized to CF2 to meet certain conditions. 0.15 ≤ KMD ≤0.15, exclude interfering compounds that do not conform to the physicochemical properties of PFAS; S5. Confidence Level Classification: Based on the PFAS identification confidence level standard established by Charbonnet et al., the PFAS compounds that were finally screened were classified and labeled.

[0033] Specifically, the ultra-high performance liquid chromatography-triple quadrupole mass spectrometry system mentioned in step (3) is a SIL-40C×3 ultra-high performance liquid chromatography tandem triple quadrupole mass spectrometry system. TM 7500 mass spectrometry; Furthermore, the ultra-high performance liquid chromatography described in step (3) includes a Poroshell 120 EC-C18 analytical column and an Omega PS C18 guard column; The parameters of the Poroshell 120 EC-C18 analytical column in step (3) are: size 2.1×100 mm, stationary phase particle size 2.7 µm; Among them, the parameters of the Omega PS C18 guard column in step (3) are: size 2.1×100 mm, stationary phase particle size 3 µm; In step (3), the column temperature of the ultra-high performance liquid chromatography is 40℃ and the injection volume is 10 µL. The specific parameter settings for the ultra-high performance liquid chromatography-triple quadrupole mass spectrometry system in step (3) are as follows: Table 4. Parameter settings for the ultra-high performance liquid chromatography-triple quadrupole mass spectrometry system

[0034] The optimized parent ion, daughter ion, collision energy, and QOD parameters described in step (3) are as follows: Table 5. Instrument acquisition parameters of the target PFAS

[0035] The concentration gradient of the calibration curve in step (3) is set as follows: 0 ng / mL, 0.001 ng / mL, 0.005 ng / mL, 0.01 ng / mL, 0.05 ng / mL, 0.1 ng / mL, 0.25 ng / mL, 0.5 ng / mL, 1 ng / mL, 2.5 ng / mL, 5 ng / mL, 10 ng / mL, 25 ng / mL and 50 ng / mL; The internal standard concentration of the calibration curve in step (3) is set to 1 ng / mL; Preferably, the specific operation process of step (3) is as follows: S1. Mass spectrometry parameter optimization: For each target PFAS, the system optimizes the parent ion, daughter ion, collision energy and QOD voltage to ensure that each PFAS can obtain a stable and high-intensity mass spectrometry response. S2. Calibration Curve Construction: A series of standard solutions covering a wide concentration range were prepared with concentration gradients of 0, 0.001, 0.005, 0.01, 0.05, 0.1, 0.25, 0.5, 1, 2.5, 5, 10, 25, and 50 ng / mL, with the internal standard concentration uniformly set at 1 ng / mL. Quantitative calibration was performed using the internal standard method. All target PFAS showed good linearity within the above concentration range, with a linear correlation coefficient R² > 0.99. S3. Limit of Quantification (LOQ) Determination: Using the solvent blank as a reference, the detection concentration of all target analytes in the blank sample is lower than the instrument limit of quantification (iLOQ) to avoid interference from blank contamination on the quantitative results; the method limit of quantification (mLOQ) is calculated by the ratio of the lowest concentration in the linear range to the sample dilution factor, and the final mLOQ range is 0.002-0.1 ng / L. S4. Method Validation: The accuracy and precision of the method were validated by spiked recovery experiments. PFAS standard at a concentration of 2 ng / L was added to blank surface water samples, and the samples were measured in parallel three times according to the pretreatment and detection procedures.

[0036] Among them, the risk entropy (RQ) in step (4) assesses the ecological risk of PFAS in surface water, and the specific calculation formula is as follows: RQ = MEC / PNEC (Ⅰ) Among them, MEC represents the measured environmental concentration (ng / L), and PNEC represents the predicted no-effect concentration (ng / L, see the following table) obtained by querying the values of the corresponding environmental media in the NORMAN ecotoxicology database.

[0037] Table 6. Predicted no-effect concentrations of target PFAS

[0038] The ecological risks are divided into four levels: high risk (RQ > 1), medium risk (0.1 < RQ < 1), low risk (0.01 < RQ < 0.1), and no risk (RQ < 0.01).

[0039] Among them, the use of the in vitro human cell model in step (4) is: using the CCK-8 experiment to evaluate the cytotoxic effect; Specifically, the six human cell lines are: brain (SH-SY5Y), heart (AC16), liver (HepG2), lung (A549), kidney (HK-2), and colon (CaCo-2); Furthermore, the cytotoxicity detection process of the in vitro human cell model is as follows: S1. Inoculate the six human cells after passage into a 96-well plate. After adherent culture for 24 h, discard the original culture medium, add PFAS mixed exposure solutions with different concentrations, set 6 parallel wells for each group, and continue to culture for 48 h; S2. After the exposure ends, add 10 μL of CCK-8 reagent to each well, incubate in an incubator for 3 h, and measure the absorbance value of each well at a wavelength of 450 nm using an enzyme-labeled instrument; S3. Calculate the cell viability according to the absorbance values of each well obtained in S2.

[0040] Preferably, the proportions of each substance in the PFAS mixed exposure solution are as follows: Table 7. Proportions of each substance in the PFAS mixed exposure solution

[0041] Preferably, the concentrations of the PFAS mixed exposure solution are 100 ng / L, 350 ng / L, 1000 ng / L, 1500 ng / L, 2000 ng / L, and 2500 ng / L in sequence; at the same time, set a blank control group containing an equal volume of acetonitrile.

[0042] Based on further solutions to the technical problems of the present invention, or simultaneous solutions to multiple technical problems, the preferred solution in the technical solution provided in the first aspect of the present invention includes: The first preferred embodiment: the activation step is performed sequentially using 0.5% ammoniated methanol, methanol, and water; the rinsing step is performed sequentially using a 25mM ammonium acetate aqueous solution at pH 4 and water; the elution step is performed sequentially using methanol and 0.5% ammoniated methanol. This technical solution, while addressing the technical problem of "reducing sample contamination and loss during pretreatment," further addresses the technical problem of "further reducing sample contamination and loss during pretreatment."

[0043] The second preferred solution: In step (2), the B phase of the mobile phase of the ultra-high performance liquid chromatography is a mixture of methanol and acetonitrile at a volume ratio of 1:1. This technical solution not only solves the technical problem of "comprehensively identifying the types of PFAS pollution in surface water samples", but also further solves the technical problem of "more comprehensively identifying the types of PFAS pollution in surface water samples".

[0044] The third preferred embodiment: the concentrations of the PFAS mixed exposure solution are successively 100 ng / L, 350 ng / L, 1000 ng / L, 1500 ng / L, 2000 ng / L, and 2500 ng / L. This technical solution, building upon the existing solution of "providing scientific evidence for the risks and hazards of PFAS in surface water samples," further addresses the technical problem of "providing even more scientific evidence for the risks and hazards of PFAS in surface water samples."

[0045] Secondly, the present invention provides the application of the method for screening, identifying and assessing the risks of PFAS in surface water in routine surface water monitoring, drinking water safety assurance and industrial pollution control projects.

[0046] These include technical features such as routine surface water monitoring, drinking water safety assurance, and industrial pollution control projects.

[0047] Preferably, the routine surface water monitoring method is applicable to long-term tracking and monitoring of surface water in key river basins, drinking water sources, and industrial clusters across the country. It comprehensively captures traditional PFAS (such as PFOA and PFOS) and emerging alternatives (such as HFPO-DA and 6:2 Cl-PFESA) through non-targeted screening, clarifies the pollution spectrum characteristics of PFAS in different regions, and analyzes the spatiotemporal variation of PFAS occurrence levels by combining targeted quantitative results, providing data support for pollution source tracing. Preferably, the drinking water safety assurance is as follows: for centralized drinking water sources, such as reservoirs and river-type water sources, this method can quickly screen and identify potential PFAS pollution, assess ecological risks based on risk entropy models, and determine health hazards by combining cytotoxicity experiments, thereby achieving precise quantification of the risk of exceeding standards; for water sources that have already been polluted, it provides pollution degree classification, providing technical basis for optimizing water treatment processes and long-term management; Preferably, the industrial pollution control project is as follows: This method can be applied to the supervision of wastewater discharge in industries involving PFAS emissions, such as chemical, textile, and electroplating industries. By comparing the types and concentrations of PFAS in the influent, effluent, and receiving surface water, the removal efficiency of the enterprise's pollution control facilities can be evaluated.

[0048] In this invention, Example 1 at least supports the protection scope of "confidence level classification", "spiked recovery experiment" and "PFAS mixed exposure solution".

[0049] The term "confidence level classification" is derived from the aforementioned explanation and / or the corresponding technical feature in Example 1, such as "classifying and labeling the finally screened PFAS compounds according to the PFAS identification confidence level standard established by Charbonnet et al.," and is summarized by the common feature "classifying the PFAS identification results into 5 main levels (including sub-levels) through multi-dimensional evidence such as standard verification, mass spectrometry fragment matching, and homology series analysis, clarifying the reliability and applicable scenarios of different levels of identification results." Therefore, those skilled in the art can reasonably infer that "confidence level classification," its subordinate concepts, its essentially equivalent technical means, and technical means that can replace it within the scope of conventional technical means and common knowledge based on the existing technical level should all fall within the protection scope of "confidence level classification." For example, replacing "confidence level classification" with "confidence degree grading" while keeping other technical features unchanged still falls within the protection scope of this invention.

[0050] The term "spiking recovery experiment" is derived from the aforementioned explanation and / or the corresponding technique in Example 1, which involves "verifying the accuracy and precision of the method through a spiked recovery experiment by adding a PFAS standard at a concentration of 2 ng / L to a blank surface water sample." This technique is summarized by the common feature of "adding a known concentration of PFAS standard to a blank sample (or actual sample), performing the same pretreatment and detection process as the sample, and calculating the ratio of the measured concentration to the spiked concentration (recovery rate) and the relative standard deviation (RSD) of parallel measurements." Therefore, those skilled in the art can reasonably infer that the "spiking recovery experiment," its subordinate concepts, its essentially equivalent technical means, and technical means that can replace it within the scope of conventional and common knowledge based on existing technology should all fall within the protection scope of the "spiking recovery experiment." For example, replacing "spiking recovery experiment" with "matrix spiked verification experiment" or "recovery test" while keeping other technical features unchanged still falls within the protection scope of this invention.

[0051] The term "PFAS mixed exposure solution" is derived from the aforementioned explanation and / or the corresponding technical features in Example 1, such as "the concentrations of the PFAS mixed exposure solution are successively 100 ng / L, 350 ng / L, 1000 ng / L, 1500 ng / L, 2000 ng / L, and 2500 ng / L," and is further summarized by the common feature "dissolving two or more PFAS monomers at a set concentration and ratio in a suitable medium (water, culture medium, serum, etc.) to simulate a real exposure scenario in an experimental system that must meet the requirements of accurate concentration, stable components, and no exogenous contamination." Therefore, those skilled in the art can reasonably infer that "PFAS mixed exposure solution," its subordinate concepts, its essentially equivalent technical means, and technical means that can replace it within the scope of conventional technical means and common knowledge based on the existing technical level should all fall within the protection scope of "PFAS mixed exposure solution." For example, replacing "PFAS mixed exposure solution" with "environmental matrix simulation type PFAS mixed exposure solution," "PFAS composite exposure solution," etc., while keeping other technical features unchanged, still falls within the protection scope of this invention.

[0052] In this invention, Example 1 at least supports the protection scope of "application of routine surface water monitoring", "application of drinking water safety assurance" and "application of industrial pollution control engineering".

[0053] The term "application of routine surface water monitoring" is summarized from the foregoing explanation and / or the corresponding technical features in Example 1: "This method is applicable to long-term tracking and monitoring of surface water in key river basins, drinking water sources, and areas surrounding industrial clusters nationwide. It comprehensively captures traditional PFAS (such as PFOA and PFOS) and emerging alternatives (such as HFPO-DA and 6:2 Cl-PFESA) through non-targeted screening, clarifies the pollution spectrum characteristics of PFAS in different regions, and, combined with targeted quantitative results, analyzes the spatiotemporal variation patterns of PFAS occurrence levels, providing data support for pollution source tracing." Therefore, those skilled in the art can reasonably infer that "application of routine surface water monitoring," its subordinate concepts, its essentially equivalent technical means, and technical means that can replace it within the scope of conventional and common knowledge based on existing technology should all fall within the protection scope of this invention.

[0054] The term "application for ensuring drinking water safety" is summarized from the foregoing explanation and / or the corresponding technical features in Example 1, such as "this method can quickly screen and identify potential PFAS pollution, assess ecological risks based on a risk entropy model, determine health hazards by combining cytotoxicity experiments, and achieve precise quantification of exceeding standards; for water sources that have already been polluted, it provides a pollution degree classification, providing a technical basis for optimizing water treatment processes and long-term management." Therefore, those skilled in the art can reasonably presume that "application for ensuring drinking water safety," its subordinate concepts, its essentially equivalent technical means, and technical means that can replace it within the scope of conventional technical means and common knowledge based on the existing level of technology should all fall within the protection scope of this invention.

[0055] The term "application of industrial pollution control engineering" is summarized from the foregoing explanation and / or the corresponding technical feature in Example 1: "This method can be applied to the supervision of wastewater discharge from industries involving PFAS emissions, such as chemical, textile, and electroplating industries. By comparing the types and concentrations of PFAS in the influent, effluent, and receiving surface water, the removal efficiency of the enterprise's pollution control facilities can be evaluated." Therefore, those skilled in the art can reasonably presume that "application of industrial pollution control engineering," its subordinate concepts, its essentially equivalent technical means, and technical means that can replace it within the scope of conventional and common knowledge based on the existing level of technology should all fall within the protection scope of this invention.

[0056] The beneficial effects of this invention are as follows: The present invention has at least the following beneficial effects: (1) High efficiency and stability of pretreatment: Fully automated solid phase extraction reduces manual operation, reduces sample contamination and loss, improves pretreatment efficiency, and is suitable for batch sample analysis.

[0057] (2) Comprehensive screening scope: Combining non-targeted and targeted technologies, it can achieve comprehensive identification of traditional, emerging and unknown PFAS, solving the problem of insufficient screening coverage of existing methods.

[0058] (3) Quantitative accuracy and reliability: Optimized chromatographic and mass spectrometric conditions ensure low detection limits, high recovery rates and precision, meeting the quantitative requirements for trace PFAS in surface water.

[0059] (4) Risk assessment system: integrates ecological and health risk assessment, provides multi-dimensional risk level classification, and provides comprehensive data support for the prevention and management of new pollutants. Attached Figure Description

[0060] Figure 1 This refers to the proposed structure of Level 3 and above PFAS determined by non-targeted screening in Example 1.

[0061] Figure 2 The total concentration of PFAS in the surface water sample from Kunshan City in Example 1.

[0062] Figure 3 The RQ value of PFAS in the surface water sample from Kunshan in Example 1 is given.

[0063] Figure 4 The effect of PFAS co-exposure for 48 hours on cell viability in Example 1, where n=6, p <0.05, compared with the control group; Figure A shows the effect of PFAS mixed exposure for 48 hours on the survival rate of SH-SY5Y cells; Figure B shows the effect of PFAS mixed exposure for 48 hours on the survival rate of AC16 cells; Figure C shows the effect of PFAS mixed exposure for 48 hours on the survival rate of HepG2 cells; Figure D shows the effect of PFAS mixed exposure for 48 hours on the survival rate of A549 cells; Figure E shows the effect of PFAS mixed exposure for 48 hours on the survival rate of HK-2 cells; Figure F shows the effect of PFAS mixed exposure for 48 hours on the survival rate of Caco-2 cells. Detailed Implementation

[0064] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0065] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all instruments, devices, equipment, reagents, products, etc., used in the embodiments of the present invention are obtained through conventional commercial means.

[0066] Example 1: Screening, Identification and Risk Assessment of PFAS in Surface Water 1. Sample pretreatment Before pretreatment, 250 mL of each urban surface water sample collected in Kunshan City was filtered through a 0.45 μm glass fiber membrane to remove suspended solids. 0.5 ng of an internal standard at a concentration of 10 μg / L was added to the filtered water sample, and then purification and concentration steps were completed using an Oasis WAX solid-phase extraction column (6 mL, 500 mg) according to the procedure in Table 1. The final extract was redissolved in a 0.5 mL methanol and water mixture (v / v = 1:1), further filtered using a 0.22 μm polyethersulfone needle filter membrane, and stored in 2 mL polypropylene vials. Store at 20°C for subsequent analysis.

[0067] 2. Non-targeted screening analysis Untargeted analysis was performed using ultra-high performance liquid chromatography and electrostatic field orbital trap high-resolution mass spectrometry (orbitrap Exploris 120) in negative ionization mode. Data were acquired in full scan / data-dependent analysis mode, with each acquisition cycle consisting of one full scan (mass range 150–1100, resolution 60,000) and four MS analyses. 2 Scan (Auto quality range, resolution 15000).

[0068] The ultra-high performance liquid chromatography (UHPLC) conditions are as follows: Mobile phase: 2 mM ammonium acetate (NH4Ac) aqueous solution (A), methanol:acetonitrile mixed solution (v:v=1:1, B); Chromatographic column: Poroshell 120 EC-C18 analytical column (2.1×100 mm, 2.7 µm); Column temperature: 35℃; Injection volume: 10 µL; Other specific parameter settings are shown in Tables 2 and 3.

[0069] The raw data were processed using Compound Discoverer 3.5 (CD 3.5) for non-targeted screening based on PFAS homology and diagnostic fragment analysis.

[0070] Peaks meeting specific criteria were extracted from the raw data, and the alignment filter conditions were as follows: (1) retention time deviation < 2 min; (2) quality deviation < 5 ppm. MS-based peaks were extracted using open-access and commercial databases. 1 Peak and MS2 Compound identification of fragments. Peaks matching the database are further filtered by applying criteria such as retention time and isotopic pattern matching (>80%) to generate a preliminary list of suspicious screening results.

[0071] Peaks meeting the following criteria were listed as potential candidate peaks: (1) mass deviation < 5 ppm; (2) threshold 0.8; (3) mass difference corresponding to 49.99681 Da (CF2), 99.99361 Da (C2F4), 64.01246 Da (C2H2F2), 65.99172 Da (CF2O) or 115.98853 Da (C2F4O). These were used for homology search to enrich the identification results.

[0072] MS based on the characteristic peaks identified in the suspected and homologous screening lists 2 Fragmentation was used to infer the structure of PFAS compounds, maintaining a mass deviation threshold of 10 ppm. Retention times (range: 2–15 minutes) and Kendrick mass defects (KMD) were used. The processed data was further filtered using the formula 0.15≤KMD≤0.15 (normalized to CF2). Based on the PFAS identification confidence level criteria established by Charbonnet et al., the selected PFAS were classified.

[0073] 3. Targeted quantitative analysis Using SIL-40C×3 ultra-high performance liquid chromatography in series with Triple Quadruple TM 7500 mass spectrometry was used for targeted quantitative analysis of PFAS in negative ion multiple reaction monitoring (MRM) mode.

[0074] The specific conditions for ultra-high performance liquid chromatography are as follows: Mobile phase: 4 mM NH4Ac aqueous solution (A), acetonitrile (B); Chromatographic columns: Poroshell 120 EC-C18 analytical column (2.1×100 mm, 2.7 µm); Omega PS C18 guard column (2.1×100 mm, 3 µm); Column temperature: 40 ℃; Injection volume: 10 µL; The specific parameters of the ultra-high performance liquid chromatography-triple quadrupole mass spectrometry system are shown in Table 4. Prior to targeted analysis, the precursor ion, daughter ion, collision energy, and QOD of each target PFAS were optimized (see Table 5). Calibration curves were prepared using concentrations of 0, 0.001, 0.005, 0.01, 0.05, 0.1, 0.25, 0.5, 1, 2.5, 5, 10, 25, and 50 ng / mL (with an internal standard of 1 ng / mL). All PFAS exhibited good linearity (R²) across the entire concentration range. 2 >0.99). The concentrations of all analytes in the blank were below the instrument limit of quantitation (iLOQ). The method limit of quantitation (mLOQ) is determined as the ratio of the lowest concentration within the linear range to the dilution factor.

[0075] Spiking recovery experiments were conducted using PFAS standards at a concentration of 2 ng / L. The accuracy and precision values ​​for all target analytes ranged from 51.7% to 135% and 0.11% to 19%, respectively, as shown in the table below. Table 8. Linear range, retention time, method limit of quantitation, recovery and precision of PFAS (RSD, n=3)

[0076] 4. Non-targeted screening results HRMS was used to identify 12 classes of PFAS (including 61 homologues) in the Kunshan surface water system. The proposed structures of these PFAS are as follows: Figure 1 As shown.

[0077] 5. Targeted Quantitative Results The concentrations of PFAS in various samples from Kunshan City showed significant spatial variation. The total concentrations of PFAS in surface water samples from Kunshan City are shown in the figure. Figure 2 The total concentration of PFAS (∑PFAS target) ranged from 149.328 to 444.754 ng / L, with an average concentration of 269.611 ng / L. The highest average concentration was observed at sampling points near the fluorine product factory (319.319 ng / L), followed by samples collected around the fire station (286.308 ng / L), while the lowest average concentration was found at samples collected around farmland (210.138 ng / L). Furthermore, at all sampling points, the concentrations of most target compounds detected at P-8 were elevated, indicating that the nearby factory contributed to PFAS pollution in surface water.

[0078] 6. Risk Assessment 6.1 Ecological Risk Assessment The ecological risk of PFAS in surface water is assessed using risk quotient (RQ), calculated using formula (I): RQ = MEC / PNEC (Ⅰ) Among them, MEC represents the measured environmental concentration (ng / L), and PNEC represents the predicted no-effect concentration (ng / L, see Table 6) obtained by querying the values of the corresponding environmental media in the NORMAN ecotoxicology database.

[0079] Ecological risks are divided into four levels: high risk (RQ > 1), medium risk (0.1 < RQ < 1), low risk (0.01 < RQ < 0.1), and no risk (RQ < 0.01).

[0080] Traditional long-chain PFAS, especially PFOA and PFOS, have been identified as the main factors of ecological risk. As Figure 3 it can be seen, the RQ values of PFOA are between 0.07 - 0.44, and the RQ values at most sampling points (76%) exceed 0.1, indicating the existence of "medium-level" ecological risks. It is worth noting that the RQ values of PFOS are between 0.84 - 8.54, exceeding the high-risk threshold almost in all regions, because the PNEC of PFOS is very strict (2 ng / L).

[0081] In contrast, the RQ values of short-chain PFAS (including PFBA, PFPeA, PFHxA, and PFBS) are far lower than 0.01, indicating almost no ecological risk. However, due to the negative impact of long-term exposure on the ecosystem, short-chain PFAS still need to be monitored and controlled. In addition, the emerging alternative 6:2 FTCA shows medium-high risk, and it is necessary to incorporate it into the future regulatory framework.

[0082] 6.2 Health Risk Assessment The potential health risks of characteristic PFAS were evaluated using in vitro human cell models. PFAS mixtures were exposed to six human cell lines representing different tissue types, including: brain (SH-SY5Y), heart (AC16), liver (HepG2), lung (A549), kidney (HK-2), and colon (CaCo-2) for 48 hours, and the cytotoxic effects were evaluated using the CCK-8 assay.

[0083] The specific assessment steps are as follows: SH-SY5Y cells were cultured in MEM / F12 (1:1) medium, AC16 cells in DMEM / F12 medium, HepG2, A549, and Caco-2 cells in DMEM medium, and HK-2 cells in RPMI-1640 medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin, in a cell culture incubator at 37°C with 5% CO2. When the cells reached 80-90% confluence, the medium was removed, and the cells were washed 1-2 times with PBS. After removing the PBS, 1 mL of trypsin was added for digestion for 1-3 minutes, followed by the addition of 3 mL of complete medium and trypsin to terminate the digestion. The digested cells were transferred to 15 mL centrifuge tubes and centrifuged at 1000 rpm for 5 minutes. After discarding the supernatant, the cells were resuspended in 3 mL of medium and passaged 1:3 into culture dishes.

[0084] Cells that have grown to 80%-90% confluence in the culture dish were digested with trypsin, centrifuged at 1000 rpm to remove the supernatant, and resuspended in complete culture medium. The cells were then seeded into 96-well plates. After cell attachment, the complete culture medium was discarded, and a medium containing acetonitrile solution and a mixture of PFAS at concentrations of 100 ng / L, 350 ng / L, 1000 ng / L, 1500 ng / L, 2000 ng / L, and 2500 ng / L was added. The composition ratio of the PFAS mixture is shown in Table 7.

[0085] After 48 hours of culture, 10 μL of CCK8 solution was added to the culture medium, and the mixture was incubated in an incubator for 3 hours. The absorbance was then measured at 450 nm using a microplate reader.

[0086] The cell viability (%) was calculated using formula (II): Cell viability (%) = [A （加药） A （空白） ] / [A （0加药） A （空白） ]×100%(Ⅱ) Among them, A （加药） : Absorbance of pores containing cells, CCK-8 solution, and PFAS solution; A （空白） : Absorbance of pores containing CCK-8 solution and culture medium but without cells; A （0加药） : Absorbance of pores containing cells, CCK-8 solution, and acetonitrile.

[0087] The results of the obtained cell viability are shown in Figure 4Among the studies, PFAS concentrations exceeding 1500 ng / L significantly reduced the survival rates of SH-SY5Y and HepG2 cells by 85.6% and 88.9%, respectively, compared to the control group. Although A549 and HK-2 cells showed a similar trend, the changes were not statistically significant. Notably, only PFAS concentrations of 2500 ng / L inhibited the survival rates of AC16 and Caco-2 cells, indicating that these cell types are less sensitive to the effects of PFAS.

[0088] Furthermore, lower concentrations of PFAS appeared to improve the survival rate of all six cell types to some extent, possibly by improving cell membrane permeability and energy metabolism. Overall, the toxicity of PFAS exposure at environmentally relevant concentrations varied across all cell types.

[0089] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A method for screening, identifying, and assessing the risk of PFAS in surface water, characterized in that, Includes the following steps: (1) Sample pretreatment: Surface water samples were filtered to remove suspended solids, and internal standards were added. Solid phase extraction was then performed. The purification and concentration were completed by activation, loading, rinsing, elution and nitrogen blowing. The eluent was redissolved and filtered to obtain the sample to be analyzed. The solid-phase extraction column is an Oasis WAX solid-phase extraction column; The activation step is performed sequentially using ammoniated methanol, methanol, and water, wherein the concentration of the ammoniated methanol is selected from 0.4% to 0.6%. The rinsing step is performed sequentially using an ammonium acetate aqueous solution with a pH of 4-5 and water, wherein the concentration of the ammonium acetate aqueous solution is selected from 20-30 mM; The elution step is performed sequentially using methanol and ammoniated methanol, wherein the concentration of the ammoniated methanol is selected from 0.4% to 0.6%. (2) Non-targeted screening: The sample to be analyzed obtained in step (1) is detected by ultra-high performance liquid chromatography-electrostatic field orbital trap high resolution mass spectrometry system. Data is collected in full scan / data-dependent analysis mode. Non-targeted screening is carried out by combining PFAS homology series, diagnostic fragments and database matching to identify PFAS categories. The mobile phases for the ultra-high performance liquid chromatography are: Phase A is a 2 mM ammonium acetate aqueous solution, and Phase B is a methanol-acetonitrile mixed solution; (3) Targeted quantification: The sample to be analyzed obtained in step (1) is detected by an ultra-high performance liquid chromatography-triple quadrupole mass spectrometry system. The negative ion multiple reaction monitoring mode is adopted. Based on the optimized parent ion, daughter ion, collision energy and Q0D parameters, combined with the calibration curve, the PFAS concentration is quantified. The mobile phase for the ultra-high performance liquid chromatography is: phase A is 4 mM ammonium acetate aqueous solution, and phase B is acetonitrile; (4) Risk assessment: Based on the quantitative concentration of PFAS obtained in step (3), the risk entropy model is used to calculate the risk entropy to assess the ecological risk, and the health risk is assessed by constructing in vitro cell models of six human cell lines to clarify the risk level and health hazards of PFAS. The six human cell lines include brain, heart, liver, lung, kidney, and colon cell lines; The PFAS includes PFBA, PFPeA, PFHxA, PFHpA, PFOA, PFNA, PFDA, PFUnDA, PFDoA, PFTrDA, PFTeDA, PFBuS, PFPeS, PFHxS, P FOS, PFNS, PFDS, PFDoS, FOSA, N-MeFOSA, N-EtFOSA, FOSAA, N-MePFOSAA, N-EtPFOSAA, N-MeFOSE, N-EtFOSE, 6:2 FTCA, 8:2 FTCA, 10:2 FTCA, PFHxPA, PFOPA, PFDPA, Cl-PFHxPA, Cl-PFOPA, PF4OPeA, PF5OHxA, NFDHA, HFPO-DA, HFPO-TA, PFEESA, 4:2FTS, 6:2FTS, 8:2FTS, 10:2FTS, 6:2diPAP, 8:2PAP, NaDONA, 6:2 Cl-PFESA, 8:2 Cl-PFESA; The internal standards include: 13C4 PFBA, 13C5 PFPeA, 13C5 PFHxA, 13C4 PFHpA, 13C8 PFOA, 13C9 PFNA, 13C6 PFDA, 13C7 PFUnDA, 13C3 PFBuS, 13C8 PFOS, 13C3 PFHxS, 13C8 FOSA, d3-N-MePFOSAA, d5-N-EtPFOSAA, 13C2 4:2 FTS, 13C2 6:2 FTS, and 13C2 8:2 FTS.

2. The method according to claim 1, characterized in that, The surface water mentioned in step (1) is selected from at least one of the following: rivers, lakes, reservoirs, wetlands, streams, water bodies surrounding industrial clusters, and irrigation canals; The specifications of the solid phase extraction column in step (1) are: column volume of 6 mL and adsorbent loading of 500 mg.

3. The method according to claim 1, characterized in that, The activation step in step (1) is carried out sequentially using 0.5% ammoniated methanol, methanol, and water; The rinsing step in step (1) is carried out sequentially using a 25mM ammonium acetate aqueous solution with a pH of 4 and water; The elution step in step (1) is performed sequentially using methanol and 0.5% ammoniated methanol.

4. The method according to claim 1, characterized in that, The solvent for resolution in step (1) is a solution obtained by mixing methanol and water in a volume ratio of 1:

1.

5. The method according to claim 1, characterized in that, In step (2), the mobile phase B of the ultra-high performance liquid chromatography is a mixture of methanol and acetonitrile at a volume ratio of 1:

1. The ultra-high performance liquid chromatography column in step (2) has a size of 2.1 × 100 mm and a stationary phase particle size of 2.7 µm; The elution gradient of the mobile phase in step (2) of the ultra-high performance liquid chromatography is: The column temperature of the ultra-high performance liquid chromatography in step (2) is 35℃ and the injection volume is 10 µL.

6. The method according to claim 1, characterized in that, The specific steps for performing non-targeted screening as described in step (2) are as follows: S1. Feature Peak Extraction and Preliminary Filtering: Feature peaks that meet specific criteria are extracted from the raw data obtained from high-resolution mass spectrometry. The peak extraction conditions are defined as peak intensity > 1000 and signal-to-noise ratio threshold > 3. At the same time, alignment filtering rules are set, namely, retention time deviation < 2 min and mass deviation < 5 ppm, to ensure the stability and accuracy of feature peaks and remove interference peak signals. S2. Database matching and suspicious peak screening: Using open access databases and commercial databases, preliminary compound matching is performed based on the precise mass number of primary mass spectrometry and fragment ion information of secondary mass spectrometry; further, the matching results are filtered through multiple criteria, including verification of the reasonableness of retention time and isotope mode matching degree >80%, to generate a preliminary list of suspicious PFAS for screening. S3. Homologous sequence search and candidate peak enrichment: For the peak signals after preliminary screening, a search is conducted based on the homologous series characteristics unique to PFAS. Peaks that meet the following conditions are listed as potential candidate peaks: ① quality deviation < 5 ppm; ② matching threshold ≥ 0.8; ③ the feature quality difference corresponds to a typical structural unit of PFAS. The PFAS identification results are enriched by expanding the homologous series, covering more emerging PFAS with similar structures. S4. Structure Inference and Precise Filtering: Based on the characteristic peaks identified through suspicious screening and homology search, and combined with fragment ion information from secondary mass spectrometry, PFAS compound structure inference is performed. A mass deviation threshold of ≤10ppm is set to ensure the accuracy of the structure inference. Further filtering is achieved through dual constraints: retention time is limited to the range of 2-15 minutes, and Kendrick mass defects are normalized to CF2 to meet the following conditions. 0.15 ≤ KMD ≤0.15, exclude interfering compounds that do not conform to the physicochemical properties of PFAS; S5. Confidence Level Classification: Based on the PFAS identification confidence level standard established by Charbonnet et al., the PFAS compounds that were finally screened were classified and labeled.

7. The method according to claim 1, characterized in that, The ultra-high performance liquid chromatography-triple quadrupole mass spectrometry system mentioned in step (3) is a SIL-40C×3 ultra-high performance liquid chromatography tandem triple quadruple mass spectrometry system. TM 7500 mass spectrometry; The ultra-high performance liquid chromatography column in step (3) includes a Poroshell 120 EC-C18 analytical column and an Omega PSC18 guard column; The parameters of the Poroshell 120 EC-C18 analytical column are: size 2.1 × 100 mm, stationary phase particle size 2.7 µm; the parameters of the Omega PS C18 guard column are: size 2.1 × 100 mm, stationary phase particle size 3 µm. In step (3), the column temperature of the ultra-high performance liquid chromatography is 40℃ and the injection volume is 10 µL.

8. The method according to claim 1, characterized in that, The concentration gradient of the calibration curve in step (3) is set as follows: 0 ng / mL, 0.001 ng / mL, 0.005 ng / mL, 0.01 ng / mL, 0.05 ng / mL, 0.1 ng / mL, 0.25 ng / mL, 0.5 ng / mL, 1 ng / mL, 2.5 ng / mL, 5 ng / mL, 10 ng / mL, 25 ng / mL and 50 ng / mL; The internal standard concentration of the calibration curve in step (3) is set to 1 ng / mL.

9. The method according to claim 1, characterized in that, The concentrations of the PFAS mixed exposure solution in the in vitro cell model described in step (4) are 100 ng / L, 350 ng / L, 1000 ng / L, 1500 ng / L, 2000 ng / L and 2500 ng / L, respectively; The PFAS mixed exposure solution in the in vitro cell model described in step (4) includes: 10 wt% PFBA, 2.5 wt% PFPeA, 56 wt% PFHxA, 2.5 wt% PFHpA, 13 wt% PFOA, 13 wt% PFBuS, 0.5 wt% PFHxS and 2.5 wt% PFOS.

10. The application of the method according to any one of claims 1-9 in routine surface water monitoring, drinking water safety assurance, and industrial pollution control projects.

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