Method and equipment for rapid screening and targeted quantification of perfluoroalkyl and polyfluoroalkyl compounds in serum

Abstract

The invention discloses a method and equipment for rapid screening and targeted quantification of perfluoroalkyl compounds and polyfluoroalkyl compounds in serum, and belongs to the field of detection of perfluoroalkyl compounds and polyfluoroalkyl compounds. The method comprises the following steps: (1) preparing a standard solution and a standard curve for screening library construction and quantitative analysis; (2) screening the standard solution by using an electrostatic field orbitrap high-resolution mass spectrum and constructing a PFAS secondary screening mass spectrum database; (3) enriching and concentrating the serum sample by using small-volume liquid-liquid extraction to obtain a detection sample; (4) carrying out non-targeted screening on the detection sample by adopting the constructed PFAS secondary screening mass spectrum database, and providing semi-quantitative PFAS concentration estimation in combination with a reference substance and an alternative internal standard; and (5) constructing a UPLC-MS / MS quantitative analysis method based on a semi-quantitative screening result, and accurately determining the specific concentration of the PFAS in the detection sample.

Description

Technical Field

[0001] The present invention relates to the field of detection of perfluoroalkyl and polyfluoroalkyl compounds, and in particular to a method and device for rapid screening and targeted quantification of perfluoroalkyl and polyfluoroalkyl compounds in serum. Background Art

[0002] Perfluoroalkyl and polyfluoroalkyl substances (PFAS) are a class of chemicals first synthesized in the 1940s. Due to their exceptional resistance to degradation and widespread persistence in the environment, PFAS are widely used in a variety of industries and consumer products, including metal plating, electronics, paints, medical devices, waterproof fabrics, and non-stick cookware. However, these properties also make them difficult to remove and persist in organisms for long periods of time, particularly in humans exposed through food, water, air, and consumer products. In recent years, scientific attention has increased significantly regarding PFAS, particularly their potential health risks. Studies have shown that long-term exposure to PFAS may lead to health problems such as immune system abnormalities, endocrine disruption, cancer, kidney disease, and low birth weight. Long-chain PFAS, due to their longer biological half-lives, remain in the human body longer than short-chain PFAS, posing potentially greater health risks. In response, the Stockholm Convention on the Prevention and Control of POPs (POPs) was first adopted in 2009 for international control, and my country has also gradually strengthened its control over these chemicals. As industry regulation increases, it becomes crucial to conduct efficient biomonitoring to assess the level of PFAS exposure in the population and provide a scientific basis for policy making.

[0003] At present, the main challenges faced by the biomonitoring of serum PFAS include: first, there are many types of PFAS and a lack of complete standard substances, which leads to limited coverage of different PFAS types by existing detection technologies; second, although traditional targeted detection is sensitive, it is limited to PFAS with standard substances, and it is difficult to fully reflect the diverse exposure pathways. In order to overcome these limitations, a better biomonitoring strategy needs to combine PFAS screening with targeted quantitative detection. Based on this, the present invention has developed a serum analysis method that combines ultra-high performance liquid chromatography-electrostatic field orbital trap high-resolution mass spectrometry (UPLC-Q-Orbitrap) with ultra-high performance liquid chromatography-triple quadrupole mass spectrometry (UPLC-TQ-MS). This method first uses high-resolution mass spectrometry technology to widely screen PFAS in serum samples to identify the main PFAS pollutants; then, based on a targeted method, the selected PFAS are accurately quantitatively analyzed. This method can effectively identify and quantify a variety of PFAS in serum, provide scientific support for the occurrence characteristics, retention patterns and health risk assessment of perfluorinated compounds in organisms, and thus promote the optimization of public health monitoring and related decision-making. Summary of the Invention

[0004] In view of the shortcomings of the existing technology, the present invention proposes a method and equipment for rapid screening and targeted quantification of perfluoroalkyl and polyfluoroalkyl compounds in serum.

[0005] The purpose of the present invention can be achieved through the following technical solutions:

[0006] The first aspect of the present invention relates to a method for rapid screening and targeted quantification of per- and polyfluoroalkyl compounds in serum for non-diagnostic purposes, comprising the following steps:

[0007] Preparation of standard solutions and standard curves for screening library construction and quantitative analysis;

[0008] Electrostatic field orbital trap high-resolution mass spectrometry was used to screen standard solutions and construct a PFAS secondary screening mass spectrum database;

[0009] Serum samples were enriched and concentrated using small-volume liquid-liquid extraction to obtain test samples;

[0010] The constructed PFAS secondary screening mass spectrometry database is used to perform non-targeted screening on the test samples, and a semi-quantitative PFAS concentration estimate is provided in combination with reference substances and alternative internal standards;

[0011] Based on the semi-quantitative screening results, a UPLC-MS / MS quantitative analysis method was constructed to accurately determine the specific concentration of PFAS in the test samples.

[0012] Optionally, the reference comprises a NIST standard reference.

[0013] Optionally, the method for preparing the standard solution comprises the following steps:

[0014] Pipette the mixed standard of perfluorinated and polyfluoroalkyl compounds into a sampling vial, dilute to volume with methanol to obtain a PFAS standard solution; Pipette the PFAS standard solution into a sampling vial, dilute to volume with methanol to prepare a mixed standard solution, and add the internal standard.

[0015] Optionally, the mixed standard product of perfluoro and polyfluoroalkyl compounds includes one or more of perfluorobutanecarboxylic acid, perfluoropentanecarboxylic acid, perfluorobutane sulfonic acid, perfluorohexanecarboxylic acid, perfluoropentane sulfonic acid, perfluorobutane sulfonamide, 2,3,3,3-trifluoro-2-(1,1,2,2,3,3-trifluoropropane)-propane acid perfluoro(2-methyl-3-oxahexanoic acid), perfluoroheptanecarboxylic acid, 4.8-dioxa-3H-perfluorononanoic acid, perfluorohexane sulfonic acid, perfluorooctanecarboxylic acid, perfluoroheptane sulfonic acid, telomer sulfonic acid, perfluorononane carboxylic acid, perfluorooctane sulfonic acid, chlorinated polyfluoroalkyl ether sulfonate, perfluorononane sulfonic acid, perfluorodecane carboxylic acid, perfluoroundecane carboxylic acid, perfluorodecane sulfonic acid, chlorinated polyfluoroalkyl ether sulfonate, perfluorododecane carboxylic acid, perfluorotridecane carboxylic acid, perfluorotetradecane carboxylic acid, perfluorohexadecane carboxylic acid, and perfluorooctadecane carboxylic acid;

[0016] The internal standards include: 13C4-PFBA, 13C5-PFPeA, 13C5-PFHxA, 13C4-PFHpA, 13C8-PFOA, 13C9-PFNA, 13C6-PFDA, 13C7-PFUdA, 13C2-PFDoA, 13C2-PFTeDA, 13C3-PFBS, 13C3-PFHxS and 13C8-PFOS.

[0017] Optionally, the small volume liquid-liquid extraction is an N-aminoethyl-aminopropyl group adsorbent liquid-liquid extraction method;

[0018] The N-aminoethyl-aminopropyl group adsorbent liquid-liquid extraction method includes the following steps: using tetrabutylammonium hydrogen sulfate as an ion pair reagent to generate a cationic complex of the target substance, and extracting it with acetonitrile; taking 0.2 mL of serum sample, adding 2.5 ng (5 μL, 100 ng / mL) of mixed (13 types) internal standards, transferring to a 15 mL polypropylene (PP) centrifuge tube, adding 0.4 mL of 0.25 ng / mL Na2CO3 buffer solution, 0.2 mL of 0.5 ng / mL TBAHS, and vortexing for 5 minutes; then adding 1 mL of acetonitrile and vortexing for 5 minutes, and ultrasonically extracting for 10 minutes; then centrifuging at 12000 rpm for 10 minutes, transferring the organic layer to another PP centrifuge tube, and then repeating the extraction twice. The organic layer and the previous one are mixed and added to an N-aminoethyl-aminopropyl group adsorbent (40-60 μm, 60A) to remove impurities. The obtained organic solvent was evaporated and the volume was made up to 200 μL with methanol: water (1:1); the sample was centrifuged and the supernatant was taken and stored in a sampling vial containing a 250 μL glass-lined tube for testing.

[0019] Optionally, the mass spectrometry conditions for the electrostatic field orbital trap high-resolution mass spectrometry and non-targeted screening are set as follows:

[0020] Acquisition mode Full MS / ddMS2 , electrospray ion source negative ion mode; ion spray voltage, -4500 V; declustering voltage, 55 V; inlet voltage, 10 V; collision cell outlet voltage, 12 V; temperature, 500 ° C; air curtain, 35.0 psi; collision gas, 8 psi; spray gas, 50 psi; drying gas, 50 psi; mass range: 70-1000 m / z; sheath gas flow rate: 60; auxiliary gas flow rate: 25; scan gas flow rate: 2; spray voltage: 3.6; capillary temperature: 300 ° C; S- Lens RF level: 50; auxiliary gas heater temperature: 370°C; full scan MS parameters: resolution, 70,000-140,000; AGC target value, 1e6; maximum IT, 100 ms; spectrum data type, center point; MS2 parameters related to secondary data: resolution, 17,500; AGC target, 1e5; maximum IT, 50 ms; cycle number, 20; TopN, 20; isolation window, 1.5 m / z; fixed first mass, 50.0 m / z;

[0021] The mass spectrometry conditions in the UPLC-MS / MS quantitative analysis method are set as follows:

[0022] Electrospray ion source negative ion mode; ion spray voltage, -4500 V; declustering voltage, 55 V; inlet voltage, 10 V; collision cell outlet voltage, 12 V; temperature, 500°C; gas curtain, 35.0 psi; collision gas, 8 psi; spray gas, 50 psi; drying gas, 50 psi.

[0023] Optionally, the mode is Full MS / ddMS 2 ddMS 2 The mode is used to perform fragment ion analysis on unknown substances and provide structural information; by optimizing acquisition parameters, constructing screening processes, optimizing screening parameters, and optimizing collision energy, useful fragment ions are generated to obtain high-quality product ion information in the full scan, and accurate fragment analysis is performed in the PFAS secondary screening mass spectrum database; chemical structure matching is performed in combination with the PFAS secondary screening mass spectrum library.

[0024] Optionally, the non-diagnostic purposes include: the impact of the external environment on the content of perfluorinated and polyfluorinated alkyl compounds in the human or animal body or food chain for scientific research purposes, assisting in the formulation of environmental protection standards or environmental assessment.

[0025] The second aspect of the present invention relates to a device for rapid screening and targeted quantification of per- and polyfluoroalkyl compounds in serum, comprising:

[0026] The standard configuration module can prepare standard solutions by mixing standard samples of perfluoroalkyl and polyfluoroalkyl compounds and obtain standard curves;

[0027] The PFAS secondary screening mass spectrometry database was constructed by screening standard solutions using electrostatic field orbital trap high-resolution mass spectrometry.

[0028] The sample pretreatment module can enrich and concentrate serum samples through small-volume liquid-liquid extraction to obtain test samples;

[0029] A semi-quantitative detection module capable of performing non-targeted screening of the test sample and providing a semi-quantitative PFAS concentration estimate in combination with reference substances and alternative internal standards;

[0030] Furthermore, the quantitative detection module can accurately measure the specific concentration of PFAS in the test sample through the UPLC-MS / MS quantitative analysis method.

[0031] The third aspect of the present invention relates to the application of the method for rapid screening and targeted quantification of perfluoroalkyl and polyfluoroalkyl compounds in serum for non-diagnostic purposes or the device for rapid screening and targeted quantification of perfluoroalkyl and polyfluoroalkyl compounds in serum, including: the impact of the external environment on the content of perfluoroalkyl and polyfluoroalkyl compounds in the human body or animal body or food chain for scientific research purposes, or assisting in the formulation of environmental protection standards or environmental assessment.

[0032] Beneficial effects of the present invention:

[0033] Due to the adoption of the above technical solution, the present invention has the following advantages compared with the prior art: the present invention establishes a method for rapid screening and targeted quantification of per- and polyfluoroalkyl compounds (PFAS) in serum. By constructing a high-resolution screening database based on UPLC-Q-Orbitrap technology, PFAS in serum are accurately qualitatively analyzed in multiple dimensions such as secondary characteristic fragments, isotope distribution and retention time; at the same time, based on the multiple reaction monitoring (MRM) parameters optimized by UPLC-MS / MS, the PFAS confirmed by the screening are quantitatively analyzed using a precise internal standard method. During the analysis process, the present invention ensures the sensitivity and accuracy of the analysis. Through a single liquid-liquid extraction sample pretreatment, comprehensive non-targeted screening of PFAS in serum and quantitative detection of characteristic PFAS can be completed. The perfluorinated compounds in the sample are separated, qualitatively screened and quantitatively analyzed, and the entire process is coherent and efficient, which significantly improves the efficiency of detection and analysis. In addition, the present invention focuses on the study of the occurrence characteristics of PFAS in serum, and has the characteristics of wide coverage, simple operation, good repeatability and high sensitivity. It provides an innovative method for comprehensive monitoring of per- and polyfluoroalkyl compounds in serum, which is suitable for the field of biological monitoring. This method provides effective technical support for the detection of PFAS pollution in biological monitoring and provides a data basis for further health risk assessment. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The present invention will be further described below with reference to the accompanying drawings.

[0035] Figure 1 This is the total ion current TIC diagram of PFAS substances after improving the peak shape and mobile phase in the example.

[0036] Figure 2 Extracted ion current (XIC) graphs for library construction and screening of various substances in the examples.

[0037] Figure 3 The absolute recovery results of PFAS extracted by different pretreatment methods are compared in the examples.

[0038] Figure 4 The relative recovery results of PFAS extracted by different pretreatment methods after optimizing the pH value in the examples are compared.

[0039] Figure 5 NIST standard reference materials were used to continuously evaluate the method performance results in the examples.

[0040] Figure 6 This is the result of screening PFAS characteristics of serum samples using a self-built library in the example.

[0041] Figure 7 This is a box plot of the quantitative results of PFAS biomonitoring of 100 serum samples in the example.

[0042] Figure 8 The Full MS / ddMS was constructed for the UPLC-Q-Orbitrap method in this study. 2 Diagram of the secondary library screening workflow. DETAILED DESCRIPTION

[0043] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts shall fall within the scope of protection of the present invention.

[0044] The following description is used to refine the present invention so that those skilled in the art can reproduce the present invention.

[0045] The technical solution employed in the present invention is a method for rapid screening and targeted quantification of per- and polyfluoroalkyl compounds in serum. This method uses UPLC-Q-Orbitrap technology to construct a secondary high-resolution mass spectrometry library for PFAS. Serum samples are pretreated using a small-volume liquid-liquid extraction method. Based on the PFAS screening results, a UPLC-MS / MS quantitative analysis method is constructed and validated. Specifically, the method comprises the following steps:

[0046] Step (1): Preparation of standard solution and standard curve for screening library construction and quantitative analysis;

[0047] Step (2): Screen the standard solution using an electrostatic field orbital trap high-resolution mass spectrometer (Orbitrap) and construct a high-resolution PFAS secondary screening mass spectrometry database;

[0048] Step (3): using small volume liquid-liquid extraction to enrich and concentrate the serum sample;

[0049] Step (4): using the constructed high-resolution PFAS secondary screening mass spectrometry database, perform non-targeted screening on the sample of step (3), and provide a semi-quantitative PFAS concentration estimate in combination with reference substances and alternative internal standards;

[0050] Step (5): Based on the screening results, a UPLC-MS / MS quantitative analysis method is constructed to accurately determine the specific concentration of PFAS in the sample described in step (3) to complete the quantitative analysis.

[0051] As a preferred embodiment, the chromatographic conditions for the Orbitrap screening and the quantitative analysis of PFAS in serum using UPLC-MS / MS are consistent. The present invention makes the separation, qualitative screening, and quantitative analysis of perfluorinated compounds in samples a continuous process, shortens working time, and greatly improves detection and analysis efficiency. Under the premise of ensuring analytical sensitivity and accuracy, the present invention can complete comprehensive qualitative screening of PFAS in serum and characteristic PFAS quantification using a single liquid-liquid extraction sample pretreatment, making the separation, qualitative screening, and quantitative analysis of perfluorinated compounds in samples a continuous process, greatly improving detection and analysis efficiency.

[0052] As a preferred embodiment, the self-built secondary database information of the standard products (mixed standard products of 26 perfluoroalkyl and polyfluoroalkyl compounds) (see Table 1) is as follows:

[0053] Table 1. Optimization of fragment ions for high-resolution Orbitrap self-built secondary fragment screening of per- and polyfluoroalkyl compounds

[0054]

[0055] The optimized acquisition parameters are as follows: Q Exactive MS instrument using electrospray (ESI-) mode ionization, mass range: 70-1500 m / z; sheath gas flow rate: 60; auxiliary gas flow rate: 25; scan gas flow rate: 2; spray voltage (kV):

[0056] 3.6; capillary temperature: 300°C; S-lens RF level: 50; auxiliary gas heater temperature: 370°C. Full-scan MS parameters: resolution, 70,000 (maximum 140,000); AGC target value, 1e6; maximum time interval, 100 ms; spectrum data type, center point. MS2 parameters related to secondary data: resolution, 17,500; AGC target, 1e5; maximum time interval, 50 ms; cycle number, 20; TopN, 20; isolation window, 1.5 m / z; fixed first mass, 50.0 m / z; (N)CE, optimized for PFAS secondary fragment response and prioritized for acquisition in the inclusion list (see Table 2).

[0057] Table 2. Self-built secondary fragment screening (N)CE optimization of per- and polyfluoroalkyl compounds

[0058]

[0059] The pretreatment step includes adjusting the pH of the serum sample to 4 or 5 using acetic acid or ammonium acetate-acetic acid buffer solution, removing serum protein using acetonitrile, enriching the fluorine-containing pollutants in the serum three times, balancing at low temperature for 1 hour to remove basal lipids, removing impurities using N-aminoethyl-aminopropyl group adsorbent (40-60 μm, 60A), blowing to near dryness with nitrogen, re-dissolving and waiting for loading on the machine;

[0060] The UPLC-MS / MS method includes a chromatographic column: an X-Bridge™ BEH C18 chromatographic column (100 mm×2.1 mm, 1.7 μm).

[0061] Preferably, the high performance liquid chromatography-tandem mass spectrometry (UPLC-Q-Orbitrap / UPLC-MS / MS) includes a chromatographic column flow rate of 0.3 mL / min; an injection volume of 10 μL; and a mobile phase of 2 mmol / L ammonium formate-water solution phase A and methanol solution phase B.

[0062] Preferably, the UPLC-Q-Orbitrap / UPLC-MS / MS gradient elution program is as follows: 0–0.5 min, 20% B; 0.5–7 min, 20–100% B; 7–10 min, 100% B; 10–10.5 min, 20% B; 10.5–15 min, 20% B.

[0063] Preferably, the high performance liquid chromatography-tandem mass spectrometry (UPLC-Q-Orbitrap / UPLC-MS / MS) includes mass spectrometry conditions of electrospray ionization source negative ion mode (ESI-); ion spray voltage (IS), +4500V; declustering voltage (DP), 55V; entrance voltage (EP), 10V; collision chamber exit voltage (CXP), 12V; temperature (TEM), 500°C; air curtain (CUR), 35.0psi; collision gas (CAD), 8psi; spray gas (GS1), 50psi; drying gas (GS2), 50psi. Compared with previous inventions, the present invention can complete the comprehensive screening of PFAS and characteristic PFAS quantification in serum samples using a single liquid-liquid extraction sample pretreatment under the premise of ensuring analytical sensitivity and accuracy, making the separation, qualitative and quantitative analysis of perfluorinated compounds in samples a continuous process, greatly improving the efficiency of detection and analysis.

[0064] As a preferred technical solution, the high performance liquid chromatography-tandem mass spectrometry (UPLC-Q-Orbitrap) constructs Full MS / ddMS 2 Secondary library screening workflow Figure 8 As shown:

[0065] As a preferred technical solution, the Full MS / ddMS 2 The specific implementation parameters of the secondary library screening workflow are as follows (see Table 3).

[0066] Table 3. Full MS / ddMS 2 Secondary library screening working parameters

[0067]

[0068] As a preferred technical solution, the secondary screening mass spectrometry database PFAS screening non-target screening results uses corresponding reference calibrants and alternative internal standards to provide semi-quantitative concentration estimates. The 7 non-target PFAS reference standards are as follows (see Table 4).

[0069]

[0070] As a preferred technical solution, the high performance liquid chromatography-tandem mass spectrometry (UPLC-LC-MS / MS) method was constructed to quantitatively analyze PFAS, and the MRM parameters of 26 perfluorinated compounds and 13 perfluorinated isotope internal standards were optimized as follows (see Table 5).

[0071] Table 5. Optimization of MRM quantitative parameters for per- and polyfluoroalkyl compounds

[0072]

[0073] An application of the method of step (1) to a blood sample: the sample is 100 serum samples from a cohort of the Elderly Environment and Health Study.

[0074] Preferably, the method is used to screen serum PFAS, perform non-target semi-quantification and quantitative analysis of 26 PFAS.

[0075] Preferably, 17 PFAS, including PFBS, PFHxA, HFPO-DA, PFHpA, PFOA, PFNA, PFOS, FSTA (6 / 2), and DONA, were significantly detected in the serum samples (with a detection rate greater than 70%). Descriptive statistics showed that the median level of PFOS was the highest, reaching 11.08 ng / L, with a range of 1.87 to 68.78 ng / mL.

[0076] Example

[0077] 1.1 Reagents and Materials

[0078] In the step (1), the materials and reagents used are as follows: 26 standards are perfluorobutanecarboxylic acid (PFBA), perfluoropentanecarboxylic acid (PFPeA), perfluorobutanesulfonic acid (PFBS), perfluorohexanecarboxylic acid (PFHxA), perfluoropentanesulfonic acid (PFPeS), perfluorobutanesulfonamide (FBSA), 2,3,3,3-trifluoro-2-(1,1,2,2,3,3-trifluoropropane)-propane acid perfluoro(2-methyl-3-oxahexanoic acid) (HFPO-DA (GenX)), perfluoroheptane carboxylic acid (PFHpA), 4.8-dioxa-3H-perfluorononanoic acid (DONA), perfluorohexane sulfonic acid (PFHxS), perfluorooctane carboxylic acid (PFOA), perfluoroheptane sulfonic acid (PFHpS), 6:2 fluorotelomer sulfonic acid (6:2FTSA), perfluorononane carboxylic acid (PFNA), perfluorooctane sulfonic acid (PFOS), 6:2 chlorinated polyfluoroalkyl ether sulfonate (F-53B (6 / 2)), perfluorononane sulfonic acid (PFNS), perfluorodecane carboxylic acid (PFDA), perfluoroundecanecarboxylic acid (PFUdA), perfluorodecanesulfonic acid (PFDS), 8:2 chloropolyfluoroalkyl ether sulfonate (F-53B (8 / 2)), perfluorododecanecarboxylic acid (PFDoA), perfluorotridecanecarboxylic acid (PFTrDA), perfluorotetradecanecarboxylic acid (PFTeDA), perfluorohexadecanecarboxylic acid (PFHxDA), perfluorooctadecanecarboxylic acid (PFOdA, HPLC grade, Dr. Ehrenstorfer, Germany); 13 mixed The internal standards included (13C4-PFBA, 13C5-PFPeA, 13C5-PFHxA, 13C4-PFHpA, 13C8-PFOA, 13C9-PFNA, 13C6-PFDA, 13C7-PFUdA, 13C2-PFDoA, 13C2-PFTeDA, 13C3-PFBS, 13C3-PFHxS, 13C8-PFOS, TRC, Canada); methanol and acetonitrile (HPLC grade, Fisher Scientific, USA); methyl tert-butyl ether (MBTE), tetrabutylammonium hydrogen sulfate (HPLC grade, ROE Scientific, USA); ammonium formate (HPLC grade, ROE Scientific, USA), and sodium bicarbonate (analytical grade, Huadong Medicine Company).

[0079] 1.2 Instruments and Equipment

[0080] The instruments used in steps (2), (4) and (5) are as follows: UPLC-Q-Orbitrap analysis using Thermo Fisher Q Exactive TM Combined quadrupole Orbitrap TMMass spectrometer, data acquisition and processing used TraceFinder5.1 and Compound Discover 3.3 software; UPLC-TQ-MS analysis used the UPLC-AB Sciex Triple Quad 5500MS system, which consists of a Triple Quad 5500 mass spectrometer (AB Sciex, USA) and an ExionUPLC system (AB Sciex, USA), and data acquisition and processing used SCIEX Anaylst software; ultrapure water equipment (Millipore, USA).

[0081] 1.3 Preparation of standard solution and standard curve

[0082] In the step (1), the standard curve is configured as follows: accurately pipette 100 μL of a mixed standard of perfluoroalkyl and polyfluoroalkyl compounds into a 1.5 mL injection vial, dilute to 1 mL with methanol, and obtain a 100 μg / L standard solution containing 26 PFAS. Accurately pipette 100 μL of the 100 μg / L PFAS standard solution into a 1.5 mL injection vial, dilute to 1 mL with methanol, and configure a 10 μg / L mixed standard solution. Before use, use a methanol-water (1:3, v / v) solution to configure mixed standard solutions of 0.1 μg / L, 0.2 μg / L, 1 μg / L, 5 μg / L, 10 μg / L, 20 μg / L, and 100 μg / L, and add an internal standard (200 μg / L, 25 μL, final concentration 5 μg / L).

[0083] 1.4 Pretreatment methods for extraction of serum perfluoroalkyl and polyfluoroalkyl compounds

[0084] In the step (3), the serum sample pretreatment method is as follows: ion pair extraction technology is used to extract PFASs from human serum samples, mainly using tetrabutylammonium hydrogen sulfate (TBAHS) as an ion pair reagent to form a cationic complex of the target substance, and acetonitrile is used for extraction. Take 0.2mL of serum sample, add 2.5ng (5μL, 100ng / mL) mixed (13 types) of internal standards, transfer to a 15mL polypropylene (PP) centrifuge tube, 0.4mL 0.25ng / mL Na2CO3 buffer solution, 0.2mL 0.5ng / mL TBAHS, and vortex for 5min. Then add 1mL acetonitrile and vortex for 5min, and ultrasonically extract for 10min. Then centrifuge at 12000rpm for 10min, transfer the organic layer to another PP centrifuge tube, and repeat the extraction twice. The organic layer is mixed with the previous one and added with N-aminoethyl-aminopropyl group adsorbent (40-60μm, 60A) to remove impurities. The organic solvent was evaporated and the volume was adjusted to 200 μL with methanol: water (1:1). The sample was centrifuged and the supernatant was collected and stored in a vial containing a 250 μL glass-lined tube for testing.

[0085] 1.5 Chromatographic and mass spectrometry conditions

[0086] In the steps (1) and (5), the chromatographic conditions (UPLC) used are as follows:

[0087]

[0088] 1.5.2 Mass spectrometry conditions (Q-Orbitrap / TQ-MS)

[0089] In the steps (2) and (4), the high-resolution mass spectrometry (Q-Orbitrap) conditions used are as follows: library construction and screening conditions, acquisition mode Full MS / ddMS 2Electrospray ionization source, negative ion mode (ESI-); ion spray voltage (IS), -4500 V; declustering voltage (DP), 55 V; entrance voltage (EP), 10 V; collision cell exit voltage (CXP), 12 V; temperature (TEM), 500°C; curtain gas (CUR), 35.0 psi; collision gas (CAD), 8 psi; spray gas (GS1), 50 psi; drying gas (GS2), 50 psi. Mass range: 70–1000 m / z; sheath gas flow rate: 60; auxiliary gas flow rate: 25; scan gas flow rate: 2; spray voltage (kV): 3.6; capillary temperature: 300°C; S-lens RF level: 50; auxiliary gas heater temperature: 370°C. Full-scan MS parameters: resolution, 70,000 (maximum 140,000); AGC target value, 1e6; maximum IT, 100 ms; spectrum data type, center point. MS2 parameters related to the secondary data: resolution, 17,500; AGC target, 1e5; maximum IT, 50 ms; number of cycles, 20; TopN, 20; isolation window, 1.5 m / z; fixed first mass, 50.0 m / z.

[0090] In the step (5), the mass spectrometry (TQ-MS) conditions used are as follows: electrospray ionization source negative ion mode (ESI-); ion injection voltage (IS), -4500 V; declustering voltage (DP), 55 V; entrance voltage (EP), 10 V; collision cell exit voltage (CXP), 12 V; temperature (TEM), 500°C; gas curtain (CUR), 35.0 psi; collision gas (CAD), 8 psi; spray gas (GS1), 50 psi; and drying gas (GS2), 50 psi.

[0091] 2 Results and Discussion

[0092] 2.1 Construction of PFAS reference material library and optimization of quantitative chromatographic conditions

[0093] In order to improve the peak shape and better achieve compound separation, step (1) of this embodiment uses Shim-packGIST C18 chromatographic column (100×2.1mm, 2.0μm), CORTECS UPLC C18 chromatographic column (2.1×100mm, 1.6μm), X-BridgeTM BEH C18 chromatographic column (100mm×2.1mm, 1.7μm) and CORTECS UPLC C18 chromatographic column (2.1×100mm, 1.7μm, Waters) for separation experiments. It was found that the X-BridgeTM BEH C18 chromatographic column (100mm×2.1mm, 1.7μm) had better peak shape and response value, so the X-BridgeTM BEH C18 chromatographic column was selected to separate the target compound. The three mobile phase systems of 0.1% formic acid-water solution, 2mmol / L ammonium formate-water solution and 2mmol / L ammonium formate-0.01% ammonia-water solution were compared respectively. It was found that when 2mmol / L ammonium formate-water solution was used in phase A and methanol solution was used in phase B, the separation effect of 26 PFAS was the best. Under this mobile phase system, the peak shape obtained was more complete and the sensitivity was the highest. Therefore, this example selected 2mmol / L ammonium formate-water solution and methanol as the chromatographic mobile phase. The chromatograms of the optimized 26 PFAS pollutants are shown in Figure 2. Figure 1 .

[0094] 2.2 Optimization of sample pretreatment for PFAS extraction

[0095] To optimize the above step (3), the serum spike recovery rate of hydrophilic-lipophilic balance column (HLB), carbon 18 solid phase extraction column (C18) and weak anion exchange (WAX) solid phase extraction column, as well as N-aminoethyl-aminopropyl group adsorbent (PSA) liquid-liquid extraction method were investigated to test the enrichment effect of 26 perfluorinated compounds. The experiment showed that when using the PSA liquid-liquid extraction method, the absolute recovery rate of all compounds was qualified and the matrix effect was small. Therefore, the PSA liquid-liquid extraction method was selected to extract perfluorinated and polyfluorinated alkyl compounds, see Figure 3 .

[0096] By comparing the effects of different pH values ​​on the recovery rate, acetic acid and ammonium acetate-acetic acid buffer solution were used to adjust the pH of serum samples to 4 or 5; acetonitrile was used to remove serum proteins, and fluorine-containing pollutants in the serum were enriched three times. The basal lipids were removed by low temperature equilibrium for 1 hour, and the relative recovery rate and substance peak specificity were the best after PSA vortex adsorption and impurity removal. Figure 4 To reduce matrix effects after serum PFAS extraction, the optimal solvent ratio was reconstituted with methanol:water (1:3, v / v) after near-drying with nitrogen. In summary, the pretreatment method described above was subsequently optimized for qualitative screening and quantitative detection of PFAS in samples.

[0097] In addition to the recovery, to clarify the optimization results of step (3), the NIST Standard Reference Material (SRM) was used to continuously evaluate the performance of the method optimization. Replicate samples of unspiked human serum NIST AM-S-Y2304 were extracted with each batch of samples to ensure consistent and accurate results. This reference material contains thirteen PFAS (both linear and branched) naturally present in mixed human serum with concentration certification values ​​ranging from 0.111 to 10.5 μg / L. Because this NIST standard covers a concentration range of one part per trillion (ppt) to one part per billion (ppb), it is very durable in method performance testing. The accuracy and robustness results of the method are shown in Figure 2. Figure 5 The bar graph compares the experimentally calculated concentrations for fifteen replicates of AM-S-Y2304 extraction and analysis with the NIST certified values ​​(both linear and branched). The scatter plot shows the percentage difference between the experimental results and the certified range, with all measured values ​​falling within ±10% of the certified value. Only PFOS showed consistently higher experimental results than the certified value, likely due to differences in the treatment of branched and linear isomers during data processing. Furthermore, the RSD percentages for all six replicates were all below 4%, demonstrating very stable sample extraction and analysis.

[0098] 2.3FullMS / ddMS 2 Non-target PFAS screening and semi-quantitative analysis optimization

[0099] To clarify the optimization results of step (4) of the embodiment, the FullMS mode can be used for non-target screening, and the ddMS 2 The (data-dependent secondary mass spectrometry) mode is used to analyze fragment ions of unknown substances and provide structural information. Experiments have shown that by optimizing acquisition parameters, constructing a screening process, optimizing screening parameters (see Table 3), and optimizing collision energy (see Table 2) to generate useful fragment ions, it is ensured that PFAS of different chain lengths and functional groups can be fully fragmented to obtain high-quality parent ion information in the full scan (see Figure 2 ) can be used for accurate fragment analysis in secondary mass spectrometry. Combined with the PFAS secondary screening mass spectrometry database and the public mass spectrometry database for chemical structure matching, the confidence level of identification of unknown PFAS is improved. The screening results are shown in Figure 6 .

[0100] In the above embodiment, the specific information of the public database used is shown in the following table:

[0101]

[0102] 2.4 PFAS Quantification Methodology Optimization and Validation

[0103] To clarify the optimization results of step (5), UPLC-MS / MS analysis was performed using a mixed standard working solution of 0.1 to 20 μg / L. A standard curve was drawn with the injected mass concentration as the horizontal axis (x) and the quantitative ion pair peak area as the vertical axis (y). The results showed that the 26 PFAS pollutant standard solutions had a good linear relationship in the range of 0.1 to 20 μg / L, as shown in Table 6. The signal-to-noise ratio S / N = 3 was used as the detection limit (LOD), and S / N = 10 was used as the quantification limit (LOQ), as shown in Table 7.

[0104] Table 6. Standard curves and correlation coefficients for quantification ions of 26 PFAS pollutants

[0105]

[0106]

[0107]

[0108] Table 7. Detection limits, quantification limits, relative recoveries, and relative standard deviations of the monitoring methods for 26 PFAS pollutants

[0109]

[0110] 2.5 Application of rapid screening and targeted quantification methods for per- and polyfluoroalkyl compounds in serum

[0111] The established method was used to extract, rapidly screen and quantitatively detect PFAS in serum samples from 100 elderly healthy cohort subjects.

[0112] High-resolution UPLC-Q-Orbitrap screening results are as follows Figure 6 As shown in the figure, a total of 17 PFAS were identified in the self-built secondary library in the serum high-resolution spectrum, and more than 20 were confirmed in the other secondary libraries. The results of quantitative detection are shown in the figure. Figure 7 As shown, 17 PFAS, including PFBS, PFHxA, HFPO-DA, PFHpA, PFOA, PFNA, PFOS, FSTA (6 / 2), and DONA, were significantly detected in serum samples (detection rate greater than 70%). Descriptive statistics showed that PFOS had the highest median concentration, reaching 11.08 ng / L, with a range of 1.87 to 68.78 ng / mL.

[0113] The above embodiments are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary engineering technicians in this field should fall within the scope of protection determined by the claims of the present invention.

[0114] Throughout this specification, references to terms such as "one embodiment," "example," or "specific example" indicate that the specific features, structures, materials, or characteristics described in conjunction with that embodiment or example are included in at least one embodiment or example of the present invention. In this specification, schematic representations of these terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

[0115] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention, and such changes and modifications fall within the scope of the invention as claimed.

Claims

1. A method for rapid screening and targeted quantification of per- and polyfluoroalkyl compounds in serum for non-diagnostic purposes, characterized in that: The following steps are involved: Preparation of standard solutions and standard curves for screening library construction and quantitative analysis; Electrostatic field orbital trap high-resolution mass spectrometry was used to screen standard solutions and construct a PFAS secondary screening mass spectrum database; Serum samples were enriched and concentrated using small-volume liquid-liquid extraction to obtain test samples; The constructed PFAS secondary screening mass spectrometry database is used to perform non-targeted screening on the test samples, and a semi-quantitative PFAS concentration estimate is provided in combination with reference substances and alternative internal standards; Based on the semi-quantitative screening results, a UPLC-MS / MS quantitative analysis method was constructed to accurately determine the specific concentration of PFAS in the test samples.

2. The method for rapid screening and targeted quantification of perfluoroalkyl and polyfluoroalkyl compounds in serum for non-diagnostic purposes according to claim 1, characterized in that: The reference materials include NIST standard reference materials.

3. The method for rapid screening and targeted quantification of per- and polyfluoroalkyl compounds in serum for non-diagnostic purposes according to claim 1, characterized in that: The method for preparing the standard solution comprises the following steps: Pipette the mixed standard of perfluorinated and polyfluoroalkyl compounds into a sampling vial, dilute to volume with methanol to obtain a PFAS standard solution; Pipette the PFAS standard solution into a sampling vial, dilute to volume with methanol to prepare a mixed standard solution, and add the internal standard.

4. The method for rapid screening and targeted quantification of perfluoroalkyl and polyfluoroalkyl compounds in serum for non-diagnostic purposes according to claim 3, characterized in that: The mixed standard product of perfluoro and polyfluoroalkyl compounds includes one or more of perfluorobutanecarboxylic acid, perfluoropentanecarboxylic acid, perfluorobutane sulfonic acid, perfluorohexanecarboxylic acid, perfluoropentane sulfonic acid, perfluorobutane sulfonamide, 2,3,3,3-trifluoro-2-(1,1,2,2,3,3-trifluoropropane)-propane acid perfluoro(2-methyl-3-oxahexanoic acid), perfluoroheptanecarboxylic acid, 4.8-dioxa-3H-perfluorononanoic acid, perfluorohexane sulfonic acid, perfluorooctanecarboxylic acid, perfluoroheptane sulfonic acid, telomer sulfonic acid, perfluorononanecarboxylic acid, perfluorooctane sulfonic acid, chlorinated polyfluoroalkyl ether sulfonate, perfluorononane sulfonic acid, perfluorodecanecarboxylic acid, perfluoroundecanecarboxylic acid, perfluorodecane sulfonic acid, chlorinated polyfluoroalkyl ether sulfonate, perfluorododecanecarboxylic acid, perfluorotridecanecarboxylic acid, perfluorotetradecanecarboxylic acid, perfluorohexadecanecarboxylic acid, and perfluorooctadecanecarboxylic acid; The internal standard includes one or more of 13C4-PFBA, 13C5-PFPeA, 13C5-PFHxA, 13C4-PFHpA, 13C8-PFOA, 13C9-PFNA, 13C6-PFDA, 13C7-PFUdA, 13C2-PFDoA, 13C2-PFTeDA, 13C3-PFBS, 13C3-PFHxS and 13C8-PFOS.

5. The method for rapid screening and targeted quantification of per- and polyfluoroalkyl compounds in serum for non-diagnostic purposes according to claim 1, characterized in that: The small volume liquid-liquid extraction is an N-aminoethyl-aminopropyl group adsorbent liquid-liquid extraction method; The N-aminoethyl-aminopropyl group adsorbent liquid-liquid extraction method The method comprises the following steps: using tetrabutylammonium hydrogen sulfate as an ion pair reagent to generate a cationic complex of the target substance, and extracting the target substance with acetonitrile; taking 0.2 mL of serum sample, adding a mixed internal standard, transferring the sample to a centrifuge tube, vortexing and ultrasonically extracting the sample, transferring the organic layer, and then repeating the extraction twice; mixing the organic layer with the previous one, and adding an N-aminoethyl-aminopropyl group adsorbent to remove impurities; evaporating the obtained organic solvent, and fixing the volume with methanol:water (1:1); and taking the supernatant after centrifuging the sample.

6. The method for rapid screening and targeted quantification of per- and polyfluoroalkyl compounds in serum for non-diagnostic purposes according to claim 1, characterized in that: The mass spectrometry conditions for the electrostatic field orbital trap high-resolution mass spectrometry and non-targeted screening are set as follows: Acquisition mode Full MS / ddMS 2 , electrospray ion source negative ion mode; ion spray voltage, -4500 V; declustering voltage, 55 V; entrance voltage, 10 V; collision cell exit voltage, 12 V; Temperature, 500°C; curtain gas, 35.0 psi; collision gas, 8 psi; spray gas, 50 psi; Drying gas, 50 psi; mass range: 70–1000 m / z; sheath gas flow: 60; auxiliary gas flow: 25; scan gas flow: 2; spray voltage: 3.6; capillary temperature: 300°C; S-lens RF level: 50; auxiliary gas heater temperature: 370°C; full-scan MS parameters: resolution, 70,000–140,000; AGC target value, 1e6; maximum IT, 100 ms; spectrum data type, center point; MS2 parameters related to secondary data: resolution, 17,500; AGC target, 1e5; maximum IT, 50 ms; cycle number, 20; TopN, 20; isolation window, 1.5 m / z; fixed first mass, 50.0 m / z; The mass spectrometry conditions in the UPLC-MS / MS quantitative analysis method are set as follows: Electrospray ionization source negative ion mode; ion spray voltage, -4500 V; declustering voltage, 55 V; entrance voltage, 10 V; collision cell exit voltage, 12 V; Temperature, 500°C; curtain gas, 35.0 psi; collision gas, 8 psi; spray gas, 50 psi; drying gas, 50 psi.

7. The method for rapid screening and targeted quantification of per- and polyfluoroalkyl compounds in serum for non-diagnostic purposes according to claim 6, characterized in that: The described mode is Full MS / ddMS 2 ddMS 2 The mode is used to perform fragment ion analysis on unknown substances and provide structural information. By optimizing acquisition parameters, constructed screening processes, optimized screening parameters, and optimized collision energy, useful fragment ions are generated to obtain high-quality product ion information in the full scan, and accurate fragment analysis is performed in the PFAS secondary screening mass spectrum database. Combined with the PFAS secondary screening mass spectrometry library for chemical structure matching.

8. The method for rapid screening and targeted quantification of per- and polyfluoroalkyl compounds in serum for non-diagnostic purposes according to claim 1, characterized in that: The non-diagnostic purposes include: the impact of the external environment on the content of perfluorinated and polyfluorinated alkyl compounds in the human or animal body or food chain for scientific research purposes, assisting in the formulation of environmental protection standards or environmental assessment.

9. A device for rapid screening and targeted quantification of per- and polyfluoroalkyl compounds in serum, characterized in that: include: The standard configuration module can prepare standard solutions by mixing standard samples of perfluoroalkyl and polyfluoroalkyl compounds and obtain standard curves; The PFAS secondary screening mass spectrometry database was constructed by screening standard solutions using electrostatic field orbital trap high-resolution mass spectrometry. The sample pretreatment module can enrich and concentrate serum samples through small-volume liquid-liquid extraction to obtain test samples; A semi-quantitative detection module capable of performing non-targeted screening of the test sample and providing a semi-quantitative PFAS concentration estimate in combination with reference substances and alternative internal standards; Furthermore, the quantitative detection module can accurately measure the specific concentration of PFAS in the test sample through the UPLC-MS / MS quantitative analysis method.

10. Use of the method for rapid screening and targeted quantification of per- and polyfluoroalkyl compounds in serum for non-diagnostic purposes according to any one of claims 1 to 8 or the device for rapid screening and targeted quantification of per- and polyfluoroalkyl compounds in serum according to claim 9, characterized in that: include: The impact of the external environment on the content of per- and polyfluoroalkyl compounds in humans or animals or in the food chain for scientific research purposes, or to assist in the formulation of environmental protection standards or environmental assessments.

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