A method for high-throughput screening of 82 perfluoroalkyl and polyfluoroalkyl substances in serum based on UHPLC-MS / MS

Abstract

This invention provides a high-throughput analytical method based on UHPLC-MS / MS for screening 82 perfluorinated and polyfluoroalkyl substances in serum. The method employs a 96-well plate protein precipitation method for pretreatment, allowing direct injection of the extract, simplifying operation and reducing contamination. By optimizing chromatographic and mass spectrometric conditions, it achieves simultaneous separation and detection of 82 traditional and novel perfluorinated and polyfluoroalkyl substances, covering a wide range of categories including perfluorinated alkyl carboxylic acids, sulfonic acids, ethers, and fluorinated polymers. This method offers advantages such as simple pretreatment, broad coverage, high sensitivity, and high throughput, providing reliable technical support for population serum perfluorinated and polyfluoroalkyl substance exposure screening and health risk assessment.

Description

Technical Field

[0001] This invention belongs to the field of detection technology for perfluorinated and polyfluoroalkyl substances, specifically relating to an analytical method for high-throughput screening of 82 perfluorinated and polyfluoroalkyl substances in serum based on UHPLC-MS / MS. Background Technology

[0002] Per- and polyfluoroalkyl substances (PFASs) are a class of synthetic chemical substances, mainly composed of hydrophilic functional groups and hydrophobic fluorinated alkyl chains. They are both hydrophobic and oleophobic, exhibiting excellent chemical and thermal stability, and have been widely used in various aspects of human life, such as food packaging, textiles, and electronic products. Due to their high-energy CF bonds, these substances are difficult to hydrolyze, photolyze, and be degraded by microorganisms, making them synthetic chemicals with extremely high environmental persistence and bioaccumulation. This characteristic leads to their widespread presence in global environmental media (water, soil, and air) and in living organisms (including human serum, placenta, and breast milk). PFASs can be detected in the blood of almost the entire population in developed countries, raising significant public concern about their environmental and health risks.

[0003] Scientific research has clearly revealed that PFAS exposure is significantly associated with a variety of health risks, including certain cancers (such as kidney cancer and testicular cancer), metabolic disorders (such as abnormal cholesterol and hypothyroidism), liver damage, reproductive and developmental disorders, and immunosuppression. The International Agency for Research on Cancer (IARC) has classified perfluorooctanoic acid (PFOA) as a known human carcinogen (Group 1) and perfluorooctane sulfonate (PFOS) as a possible human carcinogen (Group 2B).

[0004] In response to the potential health hazards they pose, the international community (such as the Stockholm Convention and the US EPA) and my country (such as the "List of Key New Pollutants under Control (2023 Edition)" and the "Standards for Drinking Water Quality (GB 5749-2022)") have implemented strict controls on some traditional PFASs (such as PFOA and PFOS). However, this has directly led to the emergence of a large number of structurally similar new fluorinated alternatives (such as perfluoroalkyl ethers), causing the number of PFAS types to surge to over 8,000. Even more worryingly, new evidence suggests that some of these alternative PFASs may be equally dangerous.

[0005] Currently, we face a situation where traditional and novel PFASs coexist and are constantly iterating and evolving. Although research on traditional PFASs has made relevant progress, data on the exposure levels and health hazard assessments of the vast majority of PFASs, especially novel alternatives, are extremely scarce. As an effective biomarker for evaluating human exposure to PFASs, serum is widely used for PFAS biomonitoring. Currently known standard methods, such as CDC 6304.09, EPA 1633, and GB 5750.8-2023, either target serum samples and detect only a limited number of PFASs (only 15, far from covering the vast known range), or target environmental or tissue samples, which, while capable of detecting 40 PFASs, have insufficient applicability to serum samples.

[0006] Currently, the pretreatment of perfluorinated and polyfluoroalkyl compounds mostly employs liquid-liquid extraction and solid-phase extraction, both of which involve drying or lyophilization steps, easily introducing contamination from airborne target substances. Simultaneously, the solid-phase extraction column itself may cause increased background values, resulting in blank interference. Protein precipitation is also widely used, typically involving extraction with methanol, acetonitrile, or mixtures thereof, followed by purification with a solid-phase extraction column and drying / lyophilization, or direct drying / lyophilization, but these methods still struggle to avoid the aforementioned problems. Therefore, the complexity of the operational steps is the main challenge in PFASs detection due to contamination introduction; given the characteristics of perfluorinated compounds, the detection process should be simplified as much as possible.

[0007] The study, "Determination of 49 Perfluorinated and Polyfluoroalkyl Substances in Human Serum by Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometry," employed a protein precipitation combined with dilution method. This method has the following limitations: PFBA eluted too early, at 2.54 min, failing to effectively avoid interfering peaks; PFOS and PFHxS showed insufficient separation of straight and branched chains due to inadequate chromatographic gradient settings; the high proportion of aqueous phase in the injection solvent was detrimental to sample stability, and long-chain perfluorinated compounds were prone to adsorption onto the filter walls; furthermore, the centrifuge tube method is insufficient for processing large batches of samples.

[0008] Therefore, breaking through the limitations of existing detection technologies and developing high-throughput analytical methods that can efficiently, accurately, and broadly measure multiple PFASs in serum is a critical task that urgently needs to be addressed in the field of environmental health research, and it has irreplaceable strategic significance for safeguarding public health and controlling pollution. Summary of the Invention

[0009] Technical Problem to be Solved: To address the aforementioned technical problems, the purpose of this invention is to provide a high-throughput analytical method for screening 82 perfluorinated and polyfluoroalkyl substances in serum based on ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS). This method employs a 96-well plate protein precipitation pretreatment method, allowing direct injection of the extract, simplifying operation and reducing contamination. By optimizing chromatographic and mass spectrometric conditions, it achieves simultaneous separation and detection of 82 traditional and novel perfluorinated and polyfluoroalkyl substances, covering a wide range of categories including perfluoroalkyl carboxylic acids, sulfonic acids, ethers, and fluorinated polymers.

[0010] Technical Solution: A high-throughput screening method based on UHPLC-MS / MS for 82 perfluorinated and polyfluoroalkyl substances in serum, comprising the following steps: S1. Preparation of Standard Solutions: 82 perfluorinated and polyfluoroalkyl substances were prepared into a mixed stock solution with a concentration of 100 ng / mL using methanol. This stock solution was then serially diluted with methanol to prepare a series of mixed standard curve solutions with concentrations of 40 ng / mL, 20 ng / mL, 10 ng / mL, 5 ng / mL, 2 ng / mL, 1 ng / mL, 0.5 ng / mL, 0.2 ng / mL, 0.1 ng / mL, 0.05 ng / mL, 0.02 ng / mL, 0.01 ng / mL, 0.005 ng / mL, 0.002 ng / mL, and 0.001 ng / mL. Isotope internal standard samples were serially diluted with methanol to prepare mixed internal standard working solutions with concentrations of 2.5-5 ng / mL. 100 μL of ultrapure water, 100 μL of the mixed internal standard working solution, and 100 μL of the mixed standard curve solutions were mixed with 200 μL of methanol. Mix μL of methanol to prepare a standard series of working solutions; S2. Sample pretreatment: Place 100 μL of serum sample into a 96-well plate, add 100 μL of mixed internal standard working solution and 300 μL of methanol, sonicate on ice for 15 min, vortex for 10 min, centrifuge at 4℃ and 4000 rpm for 10 min, transfer 300 μL of supernatant to a new 96-well plate, and centrifuge again at 4℃ and 4000 rpm for 10 min to obtain the test solution; S3. Sample detection and qualitative analysis: Ultra-high performance liquid chromatography-tandem mass spectrometry was used to detect the test solution. Based on retention time and characteristic ion pair information, 82 perfluorinated and polyfluoroalkyl substances in serum were qualitatively analyzed. S4. Quantitative analysis: All target compounds were quantified using internal standard method with isotopic labels of similar structure or retention time as internal standards. The calibration curves were regressed by the ratio of instrument response values ​​of target compounds to internal standards (y) against the ratio of target compound concentration to internal standard concentration (x). All calibration curves were subjected to linear regression analysis with a weighting of 1 / x to calculate the content of 82 perfluorinated and polyfluoroalkyl substances in serum.

[0011] Furthermore, the ultra-high performance liquid chromatography conditions in step S3 are as follows: The chromatographic column used was a Poroshell 120 EC-C18 (100 mm × 3.0 mm, 2.7 μm), and an Omega PS C18 trapping column (100 mm × 3 mm, 3 μm, Phenomenex) was added after the solvent mixer. Mobile phase: A is 4 mmol / L ammonium formate aqueous solution, B is acetonitrile; Flow rate: 0.4 mL / min; Column temperature: 35℃-40℃; Sample cell temperature: 4℃-10℃; Injection volume: 1μL-5μL; Gradient elution program: 0-1.5 min, 10% B; 1.5-8.0 min, 10%→99% B; 8.0-12.0 min, 99% B; 12.0-12.5 min, 99%→10% B; 12.5-15.0 min, 10% B.

[0012] Furthermore, the mass spectrometry conditions in step S3 are as follows: Ionization method: Electrospray ESI ion source; MRM scanning with switching between positive and negative ions; Collision gas CAD: 9psi-10psi; Curtain gas CUR: 35psi-40psi; Atomizing gas GS1: 30psi-40psi; Heated gas GS2: 65psi-70psi; Spray voltage IS: +1300 V positive mode and -1800 V negative mode; Desolvent temperature TEM: 350 °C; The collision energies of ion pairs are shown in the table below: Table 1. Retention times and mass spectrometry parameters of 82 PFASs and isotopic internal standards

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[0024] Note: ∗ Quantitative ion pair; CE: collision energy. Beneficial effects

[0025] This invention establishes a high-throughput detection method for 82 PFASs in serum, covering both traditional and novel PFASs. The method features a simple pretreatment process, allowing direct injection of the extract, which significantly reduces experimental costs and shortens experimental time. Furthermore, it exhibits high sensitivity and accuracy, providing strong technical support for the simultaneous and rapid screening and analysis of multiple PFASs in serum samples from large populations. Attached Figure Description Figure 1 Total ion chromatogram of a mixed standard solution of 82 PFASs (5 ng / mL); Figure 2 This is a chromatogram of some PFASs in the actual sample. Detailed Implementation

[0026] The present invention will be further described below with reference to the accompanying drawings and embodiments. The following embodiments are illustrative of the present invention, but the present invention is not limited to the following embodiments: Instruments and reagents: UHPLC ACQUITY ultra-high performance liquid chromatograph (Waters Corporation, USA); SCIEX Triple Quad 7500 mass spectrometer (SCIEX Corporation, USA); ultrasonic cleaner (Shanghai Yilin Scientific Instruments Co., Ltd.); multi-tube shaker (Wuxi Jerlian Instrument Equipment Co., Ltd.); high-speed refrigerated centrifuge (BECKMAN COULTER Corporation, USA). Methanol, acetonitrile, and ammonium formate (mass spectrometry grade, Merck, USA); Watson's distilled water; research-grade human serum (Hserum AB type, Shanghai Hengyuan Biotechnology Co., Ltd.). PFASs standards included mixed standards and single standards. 2H,2H,3H,3H-perfluoroundecanoic acid (8:3FTCA) (200 μg / mL) and 7H-perfluoroheptanoic acid (HPFHpA) (100 μg / mL) were purchased from Shanghai Anpu Cuishi Standard Technology Service Co., Ltd. The remaining standards were purchased from Wellington Laboratories, Canada, specifically: mixed standard PFCA30 PAR (1 μg / mL), internal standard mixed standard MPFAC-24ES (1 μg / mL), internal standard mixed standard MFTA-MXA (2 μg / mL), and other single standards (50 μg / mL). This experiment included a total of 82 PFASs analytes and 32 internal standards.

[0027] The actual human serum samples were obtained from the Suzhou Municipal Center for Disease Control and Prevention Biobank and have been approved by the Suzhou Municipal Center for Disease Control and Prevention Ethics Committee, number: SZJK2018-002.

[0028] Example 1 An analytical method for high-throughput screening of 82 perfluorinated and polyfluoroalkyl substances in serum based on UHPLC-MS / MS includes the following steps: S1. Preparation of Standard Solutions: 82 perfluorinated and polyfluoroalkyl substances were prepared into a mixed stock solution with a concentration of 100 ng / mL using methanol. This stock solution was then serially diluted with methanol to prepare a series of mixed standard curve solutions with concentrations of 40 ng / mL, 20 ng / mL, 10 ng / mL, 5 ng / mL, 2 ng / mL, 1 ng / mL, 0.5 ng / mL, 0.2 ng / mL, 0.1 ng / mL, 0.05 ng / mL, 0.02 ng / mL, 0.01 ng / mL, 0.005 ng / mL, 0.002 ng / mL, and 0.001 ng / mL. Isotope internal standard samples were serially diluted with methanol to prepare mixed internal standard working solutions with concentrations of 2.5-5 ng / mL. 100 μL of ultrapure water, 100 μL of the mixed internal standard working solution, and 100 μL of the mixed standard curve solutions were mixed with 200 μL of methanol. Mix μL of methanol to prepare a standard series of working solutions; S2. Sample Pretreatment: Remove the serum sample to be tested from the -80℃ freezer and place it in the 4℃ freezer overnight to allow it to thaw completely. After thawing, place the sample at room temperature. Take 100 μL of serum sample and place it in a 96-well plate. Add 100 μL of mixed internal standard working solution and 300 μL of methanol sequentially. Sonicate on ice for 15 min, then vortex for 10 min. Centrifuge at 4℃ and 4000 rpm for 10 min. Take 300 μL of supernatant and transfer it to a new 96-well plate. Centrifuge again at 4℃ and 4000 rpm for 10 min to obtain the test solution. S3. Sample detection and qualitative analysis: Ultra-high performance liquid chromatography-tandem mass spectrometry was used to detect the test solution. Based on retention time and characteristic ion pair information, 82 perfluorinated and polyfluoroalkyl substances in serum were qualitatively analyzed. S4. Quantitative analysis: All target compounds were quantified using internal standard method with isotopic labels of similar structure or retention time as internal standards. The calibration curves were regressed by the ratio of instrument response values ​​of target compounds to internal standards (y) against the ratio of target compound concentration to internal standard concentration (x). All calibration curves were subjected to linear regression analysis with a weighting of 1 / x to calculate the content of 82 perfluorinated and polyfluoroalkyl substances in serum.

[0029] The ultra-high performance liquid chromatography conditions in step S3 are as follows: The chromatographic column used was a Poroshell 120 EC-C18 (100 mm × 3.0 mm, 2.7 μm), and an Omega PS C18 trapping column (100 mm × 3 mm, 3 μm, Phenomenex) was added after the solvent mixer. Mobile phase: A is 4 mmol / L ammonium formate aqueous solution, B is acetonitrile; Flow rate: 0.4 mL / min; Column temperature: 40℃; Sample cell temperature: 10℃; Injection volume: 5 μL; Gradient elution program: 0-1.5 min, 10% B; 1.5-8.0 min, 10%→99% B; 8.0-12.0 min, 99% B; 12.0-12.5 min, 99%→10% B; 12.5-15.0 min, 10% B.

[0030] The mass spectrometry conditions in step S3 are as follows: Ionization method: Electrospray ESI ion source; MRM scanning with switching between positive and negative ions; Collision gas CAD: 10 psi; curtain gas CUR: 35 psi; atomizing gas GS1: 30 psi; heated gas GS2: 65 psi; Spray voltage IS: +1300 V positive mode and -1800 V negative mode; Desolvent temperature TEM: 350 °C; The collision energies of ion pairs are shown in Table 1.

[0031] 1. Optimization of chromatographic conditions This study focuses on the effects of mobile phase gradients and buffer salt systems on the separation efficiency and response characteristics of target analytes. Firstly, by extending the initial high aqueous phase equilibration time, the retention time of the earliest eluting PFBA peak was significantly delayed (from 2.54 min to 5.03 min), successfully avoiding interfering peaks and reducing baseline noise. Furthermore, this gradient effectively mitigated the solvent effect of PFBA during injection into a high organic phase solvent, further simplifying the pretreatment process and enabling direct injection of the extract for analysis. The effects of 2 mmol / L, 4 mmol / L, and 6 mmol / L ammonium formate and ammonium acetate buffer systems on PFASs analysis were compared. The results showed that while ammonium acetate enhanced the signal response of some PFASs (such as PFOA and PFNA), ammonium formate was superior in isomer separation, particularly in the chromatographic separation of PFOS and PFHxS, where it significantly outperformed the ammonium acetate system. Considering the overall response and separation performance, this study still used 4 mmol / L ammonium formate as the aqueous phase for analysis. The total ion chromatograms of the 82 PFASs are shown below. Figure 1 Under this gradient, the target object has a better peak shape and is more evenly distributed.

[0032] 2. Optimization of mass spectrometry conditions Through systematic optimization of ion source temperature (200 °C to 700 °C, in 50 °C steps) and spray voltage (+1000 V to +3500 V and -1000 V to -3500 V, in 100 V steps), significant differences in signal responses to various PFASs were found under different conditions. Experimental results show that high temperature significantly inhibits the response signals of perfluoroalkyl carboxylic acids (PFCAs), hexafluoropropylene oxide dimer acid (HFPO-DA), perfluoro and polyfluoroalkyl ether carboxylic acids (PFECAs), fluoropolymer carboxylic acids (FTCAs), fluoropolymer unsaturated carboxylic acids (FTUCAs), and perfluoroalkane sulfonamide ethanol (N-MeFASEs and N-EtFASEs), which is attributed to the increased intensification of intrasource degradation due to higher temperatures. In negative ion mode, high voltage leads to a more significant attenuation of the response values ​​of short-chain PFASs, while in positive ion mode, the responses of various substances also generally decrease. Taking into account the response intensity of various substances, an ion source temperature of 350 °C and spray voltages of +1300 V (positive mode) and -1800 V (negative mode) were finally selected for subsequent experiments.

[0033] Given the wide variety and significant structural differences of PFASs, Table 1 also classifies the target PFASs. The study found that, except for cationic / zwitterionic PFASs which use a hydroxylation positive ion mode ([M+H]⁺), all other PFASs use a negative ion mode. Most PFASs form strong quasi-molecular ions [MH]⁻ through dehydrogenation in the negative ion mode, but some PFASs exhibit specificity: perfluoroalkane sulfonamide ethanol (N-MeFASEs and N-EtFASEs) can form adduct ions [M+HCOO]⁻ with formate ions in the mobile phase, thus using it as the primary ion and [HCOO]- (m / z 45) as the secondary ion; hexafluoropropylene oxide dimer acid (HFPO-DA) uses fragment ions [C5F] generated from its source cleavage. 11 O]⁻ is a primary ion.

[0034] The fragmentation patterns of secondary mass spectrometry are summarized as follows: Perfluoroalkyl carboxylic acids (PFCAs) and perfluoro and polyfluoroalkyl ether carboxylic acids (PFECAs) generally undergo neutral CO2 (44 Da) loss, generating [M−H−CO2]⁻ ions. These fragments of PFCAs can further undergo C / C bond cleavage, producing [M−H−(CF2)]⁻ ions. n [-CO2]⁻ and other perfluoroalkyl fragments. Since PFBA has only 3 C atoms remaining after decarboxylation, the C-C bond is difficult to break, but [F]⁻ (m / z 19) can be used as a secondary ion. The ether bonds of PFECAs are easily broken, producing a series of [C-CO2]⁻ fragments. n F 2n ₊1O]⁻ or [C n F 2n The ₊1O2]⁻ ion readily forms the characteristic fragment [COF3]⁻ (m / z 85), which is speculated to be related to the rearrangement of fluorine atoms on the adjacent carbons of the ether bond. Fluoropolymer carboxylic acids (FTCAs) and fluoropolymer unsaturated carboxylic acids (FTUCAs) commonly undergo neutral loss of CO2 (44 Da) and multiple HF (20 Da) atoms, forming [M−H−CO2−(HF)]. nCharacteristic fragments. Based on the number of hydrogen atoms in the molecule, fragment ions such as [M−64]⁻, [M−84]⁻, [M−104]⁻, and [M−124]⁻ can be observed. The loss of HF leads to unsaturated structures with multiple alkene or alkyne groups. Perfluoroalkyl sulfonates (PFSAs) commonly produce characteristic fragments [SO3]⁻ (m / z 80) and [SO3F]⁻ (m / z 99). Chloro-perfluoroalkyl ether sulfonic acids (Cl-PFESAs) and perfluoroalkyl ether sulfonic acids (PFESA) commonly produce characteristic fragments [SO2F]⁻ (m / z 83) and [M−H−SO3−(CF2)2]⁻. Fluoropolymer sulfonic acids (X:2 FTSs) commonly undergo neutral loss of HF (20 Da) to form [M−H−HF]⁻, with another characteristic fragment being [HSO3]⁻ (m / z 81). Perfluoroalkane sulfonamides (FASAs) produce a variety of fragment ions, such as [SO2N]⁻ (m / z 78), [SO2]⁻ (m / z 64), [C n F 2n ₊1]⁻ (e.g., m / z 169, 219), etc. Perfluorooctane sulfonamide acetic acid (FOSAAs) readily produces [C4F9]⁻ (m / z 219) and [C8F... 17 [PO₂]⁻ (m / z 419) fragment ions. Sodium perfluoroalkyl hypophosphite (X:XPFPis) readily produces [PO₂]⁻ (m / z 63) and [C₆F₂]⁻ (m / z 419). 14 O2P]⁻ (m / z 401), [C8F 18 [O2P]⁻ (m / z 501) fragment ions. Difluoroalkyl phosphates (diPAPs) readily produce fragment ions such as [PO3]⁻ (m / z 79) and [H2PO4]⁻ (m / z 97). Cationic / zwitterionic PFASs commonly produce fragment ions such as [C3H8N]⁺ (m / z 58) and [C3H3NS]⁺ (m / z 85).

[0035] 3. Establishment of Standard Curve and Methodological Validation After preparing a series of PFASs standard solutions at different concentrations according to step S1 in Example 1, UHPLC-MS / MS analysis was performed. All target compounds were quantified using internal standard methods with isotopic labels of similar structure or retention times as internal standards. Calibration curves were regressed using the ratio of instrument response values ​​of the target compound to the internal standard (y) against the ratio of target compound concentration to internal standard concentration (x). All calibration curves were subjected to linear regression analysis with a weighted average of 1 / x. Experimental results showed that the 82 PFASs exhibited a wide detection range, demonstrating good linearity within their respective low and high concentration ranges. To evaluate the actual detection capability of the method in matrix-matched serum samples, this study used unspecified and low-concentration spiked human serum as test subjects. The concentrations corresponding to signal-to-noise ratios (S / N) of the target compounds of 3 and 10 were determined as the limits of detection (LOD) and quantitation (LOQ) for each PFASs, respectively.

[0036] To evaluate the precision and accuracy of the method, spiked recovery experiments were conducted by adding mixed standard solutions of different concentrations to human serum used in research. Since the limits of detection (LOD) and quantitation (LOQ) differ for each substance, the spiked concentrations covered four levels: low, medium, and high (see Table 3). Seven replicates were prepared for each spiked concentration level. After sample pretreatment, UHPLC-MS / MS analysis was performed, and the recoveries (%) and relative standard deviations (RSD%) of each PFASs were calculated to characterize the accuracy and precision of the method.

[0037] As shown in Table 2, the limits of detection (LOD) for each target substance in this study were 0.00033–0.17 µg / L, and the limits of quantitation (LOQ) were 0.0010–0.50 µg / L, indicating that the method has high sensitivity. Although the recoveries of a few substances (such as HPFHpA, N-AP-FOSA, N-AP-6:2FOSA, N-OxAmP-6:2FOSA, 5:3FTB, 5:1:2FTB, N-TAmP-FHxSA, 10:2FTCA, and N-AP-FHxSA) exceeded the ideal range (80%–120%), the spiked recoveries of all target substances were still between 45.8% and 182%, with a precision of 1.3%–15%. Overall, this indicates that the method still possesses acceptable stability and practicality even without a perfectly matched internal standard.

[0038] Table 2. Limits of detection, limits of quantitation, linear ranges, recoveries and precision of 82 PFASs (n=7)

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[0042] Note: Spike concentrations are set according to the method detection limit (LOD) for each target: LOD < 0.0030 µg / L: Level 1 = 0.01 µg / L, Level 2 = 0.5 µg / L, Level 3 = 5 µg / L, Level 4 = 15 µg / L; 0.0030 ≤ LOD ≤ 0.015 µg / L: Level 1 = 0.05 µg / L, Level 2 = 0.5 µg / L, Level 3 = 5 µg / L, Level 4 = 15 µg / L; 0.017 ≤ LOD ≤ 0.033 µg / L: Level 1 = 0.2 µg / L, Level 2 = 2 µg / L, Level 3 = 5 µg / L, Level 4 = 15 µg / L; LOD > 0.033 µg / L: Level 1 = 1µg / L, Level 2 = 5 µg / L, Level 3 = 15 µg / L, Level 4 = 20 µg / L. 4. Testing of actual samples

[0043] The method established in the examples was used to analyze 30 actual human serum samples, and the detection rate and median detection concentration of 82 PFASs were statistically analyzed. For data with concentrations between the limit of detection (LOD) and the limit of quantitation (LOQ), the actual measured concentration values ​​were also used. Table 3 shows that 25 PFASs were detected, of which 13 PFASs had a detection rate of 100%, and 18 PFASs had a detection rate greater than 50.0%. The seven PFASs with the highest median detection concentrations were, in descending order: PFOA (16.6 μg / L), PFOS (13.0 μg / L), 9Cl-PF3ONS (5.68 μg / L), PFHxS (3.28 μg / L), PFDA (2.55 μg / L), PFNA (1.71 μg / L), and PFUdA (1.07 μg / L). This indicates that PFAS contamination in human serum is still dominated by traditional long-chain perfluorocarboxylic acids (PFCAs) and perfluorosulfonic acids (PFSAs) and their substitutes (such as 9Cl-PF3ONS).

[0044] The presence of novel alternatives: Chlorochloroperfluoroalkyl ether sulfonic acids (Cl-PFESAs) 9Cl-PF3ONS, 11Cl-PF3OUdS, and short-chain PFASs (such as PFBA and PFBS) have been widely detected. Other novel alternatives, such as 7:3FTCA, 8:3FTCA, HFPO-DA, and FOSAA, currently have low detection rates and concentrations. Although the detection concentration of 6:2FTS is low, its detection rate has reached 30.0%, indicating that its presence is somewhat widespread. Figure 2 This is a chromatogram of some PFASs in the actual sample.

[0045] Table 3. Detection rate and median detection concentration of 25 PFASs (n=30)

[0046] 5. Sample stability study To verify the stability of the method in large-scale sample testing, a time-response experiment was conducted. Thirty actual human serum samples prepared as described above were used, along with additional spiked serum samples containing three concentration levels (0.2, 2, and 15 µg / L; n=7) of mixed PFAS standard solutions. All samples were stored at 4 °C. During testing, samples were placed in the autosampler sample tray at 10 °C for 24 hours to ensure the actual injection time for each 96-well plate. On days 1, 3, and 7 of sample storage, the same standard working curve prepared on day 1 was used to determine the concentrations of each PFAS, and the average recovery rate was calculated based on the values ​​measured on day 1. The results showed that, except for N-AP-FOSA, the measured concentrations of the other target PFASs did not change significantly after 6 days of storage at 4 °C, and the average recovery rate remained between 80% and 120%. The stability of N-AP-FOSA is relatively poor. Its recovery rate remained in the range of 80%-120% on day 3, but by day 7, the recoveries at low, medium, and high concentration levels decreased to 74.6%, 69.2%, and 65.3%, respectively. In summary, of the 82 PFASs involved in this study, 81 were stable for at least 6 days under 4 °C storage conditions, supporting a protocol for batch sample preparation, aliquot storage, and detection within 7 days.

[0047] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention, or modify them into equivalent embodiments, without departing from the spirit and technical essence of the present invention. Therefore, any simple modifications, equivalent substitutions, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention, without departing from the content of the technical solutions of the present invention, shall still fall within the scope of protection of the present invention.

Claims

1. A high-throughput screening method based on UHPLC-MS / MS for 82 perfluorinated and polyfluoroalkyl substances in serum, characterized in that, Includes the following steps: S1. Preparation of standard solutions: Prepare a mixed stock solution with a concentration of 100 ng / mL using methanol for 82 perfluorinated and polyfluoroalkyl substances, and then perform serial dilution with methanol to prepare a series of mixed standard curve solutions; prepare mixed internal standard working solutions with a concentration of 2.5-5 ng / mL using isotope internal standard solutions through serial dilution with methanol; mix ultrapure water, mixed internal standard working solutions, mixed standard curve solutions, and methanol to prepare a series of standard working solutions. S2. Sample pretreatment: Place serum samples in a 96-well plate, add mixed internal standard working solution and methanol in sequence, sonicate on ice, vortex, centrifuge, transfer the supernatant to a new 96-well plate, centrifuge again to obtain the test solution; S3. Sample detection and qualitative analysis: Ultra-high performance liquid chromatography-tandem mass spectrometry was used to detect the test solution. Based on retention time and characteristic ion pair information, 82 perfluorinated and polyfluoroalkyl substances in serum were qualitatively analyzed. S4. Quantitative Analysis: All target compounds were quantified using internal standard method with isotopic labels of similar structure or retention time as internal standards. The calibration curves were regressed by the ratio y of the instrument response values ​​of the target compound and the internal standard to the ratio x of the concentration of the target compound and the concentration of the internal standard. All calibration curves were subjected to linear regression analysis with a weighting of 1 / x to calculate the content of 82 perfluorinated and polyfluoroalkyl substances in serum.

2. The analytical method for high-throughput screening of 82 perfluorinated and polyfluoroalkyl substances in serum based on UHPLC-MS / MS according to claim 1, characterized in that, The ultra-high performance liquid chromatography conditions in step S3 are as follows: The chromatographic column used was a Poroshell 120 EC-C18, 100 mm × 3.0 mm, 2.7 μm. An additional trapping column, Omega PS C18, 100 mm × 3 mm, 3 μm, Phenomenex, was added after the solvent mixer. Mobile phase: A is 4 mmol / L ammonium formate aqueous solution, B is acetonitrile; Flow rate: 0.4 mL / min; Column temperature: 35℃-40℃; Sample cell temperature: 4℃-10℃; Injection volume: 1μL-5μL; Gradient elution program: 0-1.5 min, 10% B; 1.5-8.0 min, 10%→99% B; 8.0-12.0 min, 99% B; 12.0-12.5 min, 99%→10% B; 12.5-15.0 min, 10% B.

3. The analytical method for high-throughput screening of 82 perfluorinated and polyfluoroalkyl substances in serum based on UHPLC-MS / MS according to claim 1, characterized in that, The mass spectrometry conditions in step S3 are as follows: Ionization method: Electrospray ESI ion source; MRM scanning with switching between positive and negative ions; Collision gas CAD: 9 psi-10 psi; Curtain gas CUR: 35 psi-40 psi; Atomizing gas GS1: 30 psi-40 psi; Heating gas GS2: 65 psi-70 psi; Spray voltage IS: +1300 V positive mode and -1800 V negative mode; Desolvent temperature TEM: 350 °C; The collision energies of ion pairs are shown in the table below: Table 1. Retention times and mass spectrometry parameters of 82 PFASs and isotopic internal standards Note: ∗ Quantitative ion pairs; CE: Collision Energy.

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