A pretreatment method suitable for simultaneous extraction of perfluoro and polyfluoroalkyl substances, organophosphates and fluorine-containing liquid crystals in biological samples

Abstract

The application provides a pretreatment method suitable for simultaneous extraction of perfluoro and polyfluoro alkyl substances, organophosphates and fluorine-containing liquid crystals in a biological sample. The method comprises: adding acetonitrile and an internal standard to the biological sample, vortexing and centrifuging to extract the supernatant, repeating the extraction and combining the supernatants; adding isohexane to the combined acetonitrile supernatant to perform liquid-liquid extraction, separating the isohexane solution and the acetonitrile solution after static layering, repeating the extraction and combining the isohexane solutions; purifying the acetonitrile solution through a dephosphorization column; nitrogen gas concentrating the acetonitrile solution and the isohexane solution respectively; redissolving the concentrated acetonitrile solution sample with methanol and the concentrated isohexane solution sample with isohexane to obtain a sample to be analyzed. The application can simultaneously extract three types of target substances from one biological sample, simplifies the operation, saves the sample, the recovery rates of the three types of target substances all reach the ideal range, and has reliability and effectiveness.

Description

Technical Field

[0001] This invention relates to the field of environmental pollutant detection technology, and more specifically, to a pretreatment method suitable for the simultaneous extraction of perfluorinated and polyfluoroalkyl substances, organophosphates and fluorinated liquid crystals from biological samples. Background Technology

[0002] Perfluorinated and polyfluoroalkyl substances (PFAS) are a class of organic compounds in which all or part of the hydrogen atoms in the carbon chain are replaced by fluorine atoms. They possess extremely high physicochemical stability and persistence, and exhibit bioaccumulation. These substances are widely used in many areas of industrial production and daily life, such as surface treatment of textiles, fire-fighting foam, and food packaging materials. Due to their widespread presence in the environment and their recalcitrant nature, PFAS have become a new class of environmental pollutants of global concern. Studies have shown that these substances can enter the human body through the food chain and may induce various health hazards, including cardiovascular toxicity, hepatotoxicity, metabolic disorders, carcinogenicity, endocrine disruption, immunosuppression, and reproductive and developmental toxicity. Accurate monitoring of PFAS exposure levels in organisms and the human body is of great significance for assessing their environmental and health risks.

[0003] Organophosphates are a class of organic compounds containing carbon-phosphorus bonds, primarily used as flame retardants and plasticizers in foam materials, plastic products, textiles, hydraulic fluids, and various building materials. Unlike polybrominated diphenyl ether (PBDE) flame retardants, which were once widely used but are now phased out, organophosphates are typically added to products physically rather than through chemical bonding. Therefore, they are more easily released into the environment during use through wear and volatilization. Humans can be exposed to organophosphates through various routes, including indoor dust ingestion, inhalation, and food chain transmission. Toxicological studies have confirmed that organophosphates have various toxic effects, including endocrine disruption, neurotoxicity, and reproductive and developmental toxicity; their potential health risks should not be ignored.

[0004] Fluorinated liquid crystals (FLLCDs) are liquid crystal compounds whose molecular structures contain fluorine atoms or fluorine-containing groups. Due to their ability to effectively improve display quality and response speed, FLLCDs are widely used as common materials in high-end LCD displays such as those for televisions, computer monitors, and mobile phone screens. With the increasing amount of electronic waste, FLLCDs, as a new type of organic pollutant, have attracted scientific attention regarding their release, migration, transformation, and ultimate fate in the environment. Preliminary studies have revealed that FLLCDs, once introduced into organisms or the human body, may cause long-term exposure risks such as endocrine disruption, reproductive and developmental toxicity, cytotoxicity, and oxidative stress. Therefore, developing reliable analytical methods to monitor the presence levels of FLLCDs in biological samples is the scientific basis for assessing their ecological and environmental risks to human health. In existing technologies, the extraction and detection of perfluorinated and polyfluoroalkyl substances, organophosphates, and FLLCDs in biological samples generally employ a single-target-oriented pretreatment strategy. Specifically, the extraction of perfluorinated and polyfluoroalkyl substances often employs mixed-type weak anion exchange solid-phase extraction columns; the extraction of organophosphates commonly uses amino solid-phase extraction columns; while for fluorinated liquid crystals, existing methods typically only use simple solvent extraction or purification with Florisil solid-phase extraction columns. This parallel processing mode of "one target, one method, one sample" results in the analysis of the above three types of pollutants requiring three biological samples and three sets of sample pretreatment steps with different operational procedures. This not only greatly increases the complexity of experimental operations and prolongs the processing cycle, but more importantly, when the biological samples themselves are very precious, difficult to obtain, or have extremely limited sample quantities (e.g., human placenta, tissue biopsy samples, etc.), existing single-target-oriented methods will face the dilemma of being unfeasible or having poor representative results. Therefore, there is an urgent need to develop a pretreatment method that can simultaneously and efficiently extract perfluorinated and polyfluoroalkyl substances, organophosphates, and fluorinated liquid crystals from a single biological sample to solve the technical problems of complex operation, slow processing speed, and large sample consumption in existing technologies. Summary of the Invention

[0005] This invention aims to provide a pretreatment method for simultaneously extracting perfluorinated and polyfluoroalkyl substances, organophosphates, and fluorinated liquid crystals from biological matrix samples. This addresses the technical problems of existing technologies that require separate processing of these three types of contaminants, are complex to operate, and require large sample volumes. The core of this invention lies in achieving simultaneous purification, separation, enrichment, and concentration of these three target substances in a single biological sample through a streamlined pretreatment process, resulting in smaller sample volumes, better impurity removal, and faster processing speed.

[0006] To solve the above-mentioned technical problems, the technical solution proposed in this application is as follows:

[0007] This invention provides a pretreatment method suitable for the simultaneous extraction of perfluorinated and polyfluoroalkyl substances, organophosphates, and fluorinated liquid crystals from biological samples, comprising the following steps: Add acetonitrile and internal standard to the biological sample, vortex to mix evenly, centrifuge, and extract the supernatant; add acetonitrile to the residue again, repeat the vortexing, centrifugation and supernatant extraction operation, and combine the supernatants obtained from the two extractions; Isohexane was added to the combined acetonitrile supernatant for liquid-liquid extraction. After standing and separating the layers, an isohexane solution and an acetonitrile solution were obtained. Isohexane was added to the acetonitrile solution again for liquid-liquid extraction. After standing and separating the layers, the isohexane solutions obtained from the two liquid-liquid extractions were combined. The acetonitrile solution obtained after liquid-liquid extraction is passed through a dephospholipid column, and the effluent is collected. The acetonitrile solution and the isohexane solution were concentrated under nitrogen atmosphere, respectively. The concentrated acetonitrile solution sample was reconstituted with methanol, and the concentrated isohexane solution sample was reconstituted with isohexane to obtain the sample to be analyzed.

[0008] Furthermore, the sample to be analyzed is detected by high performance liquid chromatography in tandem with Orbitrap mass spectrometry to achieve simultaneous qualitative and quantitative analysis of three target substances: perfluorinated and polyfluorinated alkyl substances, organophosphates, and fluorinated liquid crystals.

[0009] Furthermore, the biological sample is a human placenta sample.

[0010] Furthermore, the products from each operation unit in the acetonitrile extraction step and the isohexane liquid-liquid extraction step are retained and enter into the subsequent analysis process, and no phase is discarded.

[0011] Furthermore, in the liquid-liquid extraction step, isohexane is used as a selective extractant for fluorinated liquid crystals, extracting the fluorinated liquid crystals from the acetonitrile phase to the isohexane phase, thereby achieving inter-group separation of the fluorinated liquid crystals from perfluorinated and polyfluoroalkyl substances and organophosphates.

[0012] Furthermore, the dephospholipid column is CNWBOND Dephospholipid PPR Pro, with a specification of 300mg / 3mL, used to specifically adsorb phospholipid interfering substances in the acetonitrile solution.

[0013] Furthermore, the internal standard includes stable isotopes. 13 C-labeled perfluorinated and polyfluoroalkyl substances, stable isotope D-labeled organophosphates, and 13 C 12 -PCB-52.

[0014] Furthermore, when using acetonitrile solution samples that have been redissolved and concentrated in methanol, internal standards of perfluorinated and polyfluoroalkyl substances and organophosphate esters are added; when using isohexane solution samples that have been redissolved and concentrated in isohexane, internal standards of fluorinated liquid crystals are added.

[0015] Furthermore, the ratio of the amount of biological sample to the amount of acetonitrile used in a single extraction is 0.5 g: 6 mL; the nitrogen concentration temperature is 30 °C.

[0016] Furthermore, the vortex operation time is 1 minute; the centrifugation conditions are a rotation speed of 5000 rpm and a time of 20 minutes.

[0017] Compared with the prior art, the present invention achieves the following beneficial technical effects: This invention utilizes acetonitrile extraction to remove proteins, followed by liquid-liquid extraction with isohexane and acetonitrile to separate compounds. Further purification of the sample using a dephospholipid column ensures the target analytes remain undisturbed. Nitrogen concentration and subsequent reconstitution with methanol and isohexane further enhance sample purity and detection sensitivity. Test results demonstrate that the proposed method achieves ideal recoveries of perfluorinated and polyfluoroalkyl substances, organophosphates, and fluorinated liquid crystals, proving its reliability and effectiveness. Its most significant contribution lies in achieving simultaneous extraction of three target compounds from a single biological sample, greatly simplifying the operation, significantly reducing processing time, and conserving valuable biological samples. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 The graph shows the spiked recovery results of perfluorinated and polyfluoroalkyl substances (PFASs) provided in the embodiments of the present invention.

[0020] Figure 2 The graph shows the spiked recovery results of organic phosphate esters (OPEs) provided in the embodiments of the present invention.

[0021] Figure 3 The graph shows the spiked recovery rate of fluorinated liquid crystals (FLCMs) provided in the embodiments of the present invention. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] In one embodiment of this application, a pretreatment method is provided for simultaneously extracting perfluorinated and polyfluorinated alkyl substances (PFASs), organophosphates (OPEs), and fluorinated liquid crystals (FLCMs) from biological samples. The method specifically includes the following steps: First, a certain amount of biological sample, such as human placental tissue, is weighed and placed in a glass centrifuge tube. Acetonitrile and a specific type of internal standard are added to the tube. The internal standard includes stable isotopes. 13 C-labeled PFASs, stable isotope D-labeled OPEs, and 13 C 12-PCB-52. Vigorously vortex the mixture to ensure thorough mixing, allowing acetonitrile to act as a protein precipitant, denaturing and precipitating proteins in the biological sample while simultaneously releasing the target analyte from the biological matrix and dissolving it in the acetonitrile. Subsequently, centrifuge the tube, allowing the precipitated protein and other solid residues to settle to the bottom. The supernatant is the acetonitrile extract containing the target analyte. Carefully transfer this supernatant to a new glass centrifuge tube. To extract the target analyte as completely as possible, add acetonitrile again to the remaining solid sample, repeating the vortexing, centrifugation, and supernatant transfer process. Combine the acetonitrile supernatants from both extractions to obtain the total acetonitrile extract. Next, add isohexane to the combined acetonitrile extract and vortex again to ensure sufficient contact between the two solvents. Allow the mixture to stand to allow natural stratification. Since isohexane is less dense than acetonitrile, the upper layer is the isohexane phase, and the lower layer is the acetonitrile phase. The upper isohexane solution was separated into a new glass tube by liquid-liquid extraction or pipetting. To more fully extract the target analytes partitioned into the isohexane phase, especially the weakly polar fluorinated liquid crystals, fresh isohexane was added again to the lower acetonitrile solution, and the above liquid-liquid extraction and separation process was repeated. The isohexane solutions obtained from the two liquid-liquid extractions were combined to obtain the total isohexane extract. At this point, after liquid-liquid extraction, the weakly polar fluorinated liquid crystals were selectively extracted and enriched in the isohexane phase, while the relatively more polar PFASs and OPEs remained in the acetonitrile phase. Subsequently, the separated acetonitrile solution was passed through a dephospholipid column. This column was filled with an adsorbent that specifically adsorbs phospholipids. When the acetonitrile solution flowed through the column, residual phospholipid interferences in the sample were adsorbed and retained, while the target analytes flowed out with the solvent. This eluent was collected, thus achieving further purification of the acetonitrile phase sample. The purified acetonitrile solution and the combined isohexane solution were placed in a nitrogen evaporator and concentrated under a gentle nitrogen flow to remove most of the organic solvent, reducing the sample volume to near dryness. Finally, the nearly dry concentrated acetonitrile solution was redissolved with methanol to redissolve the PFASs and OPEs; simultaneously, the nearly dry concentrated isohexane solution was redissolved with isohexane to redissolve the fluorinated liquid crystals. These two redissolved solutions were transferred to separate vials to obtain the analyte samples ready for subsequent instrumental analysis. Through these steps, two analyte solutions enriched with different target analytes were successfully obtained from a single biological sample.

[0024] In one embodiment of this application, the sample to be analyzed prepared by the above pretreatment method is further used for high-performance liquid chromatography-tandem Orbitrap mass spectrometry (HPLC-Orbitrap-MS / MS). Specifically, the reconstituted acetonitrile phase sample and isohexane phase sample are injected into the HPLC-Orbitrap-MS / MS instrument, respectively, and analyzed using chromatographic and mass spectrometric conditions suitable for PFASs, OPEs, and FLCMs. This method enables simultaneous qualitative identification and quantitative determination of three target groups: perfluorinated and polyfluoroalkyl substances, organophosphates, and fluorinated liquid crystals.

[0025] In one embodiment of this application, the biological sample is specifically a human placental sample. Due to the difficulty in obtaining human placental samples and the limited sample quantity, the method for simultaneously extracting three types of substances from a single sample provided in this application is particularly suitable for the analysis of such precious biological samples, minimizing sample consumption and obtaining more comprehensive exposure information.

[0026] In one embodiment of this application, a key feature of the pretreatment method is that, from the initial acetonitrile extraction to the subsequent isohexane liquid-liquid extraction, the products obtained from each operating unit—including the supernatant from the first acetonitrile extraction, the supernatant from the second acetonitrile extraction, the isohexane and acetonitrile solutions obtained from the first liquid-liquid extraction, and the isohexane solution obtained from the second liquid-liquid extraction—are all properly retained and incorporated into the subsequent analytical process; no phase is discarded. This contrasts sharply with the prior art practice of discarding a phase (usually the phase containing impurities) after liquid-liquid extraction. By retaining all phases and processing and detecting the target analytes in different phases separately, this application achieves the goal of simultaneously obtaining three types of target analytes of different polarities from a single biological sample.

[0027] In one embodiment of this application, the role of isohexane in the liquid-liquid extraction step is clearly defined. In this step, isohexane is not used as a purifying agent to remove lipid impurities, but rather as a selective extractant for fluorinated liquid crystals. This is because the solubility of fluorinated liquid crystals in isohexane is much higher than their solubility in acetonitrile, while under the same liquid-liquid extraction conditions, PFASs and OPEs tend to remain in the acetonitrile phase. Therefore, by performing liquid-liquid extraction of the acetonitrile extract with isohexane, fluorinated liquid crystals can be efficiently extracted from the acetonitrile phase to the isohexane phase, thereby achieving inter-group separation of fluorinated liquid crystals from perfluorinated and polyfluoroalkyl substances and organophosphates.

[0028] In one embodiment of this application, the specific model of the dephospholipid column is CNWBOND Dephospholipid PPRPro, with a specification of 300 mg / 3 mL. This column is specifically designed for the adsorption of phospholipid interferences in acetonitrile solutions. After acetonitrile extraction and isohexane liquid-liquid extraction, a small amount of phospholipids may still remain in the acetonitrile phase. If these phospholipids subsequently enter the liquid chromatography-mass spectrometry system, they will produce a severe matrix effect, inhibiting the ionization of the target analyte, reducing detection sensitivity, and even contaminating the chromatographic column and mass spectrometry ion source. Using this dephospholipid column for solid-phase extraction of the acetonitrile phase can efficiently remove these phospholipid interferences, thereby significantly improving the accuracy and stability of subsequent analyses.

[0029] In one embodiment of this application, the internal standard substance is specifically composed of: stable isotopes. 13 C-labeled perfluorinated and polyfluoroalkyl substances, stable isotope D-labeled organophosphates, and 13 C 12 -PCB-52. These internal standards are highly similar to their corresponding target analytes in terms of physicochemical properties, and can undergo all steps such as extraction, purification and concentration simultaneously during the pretreatment process, thereby correcting for target analyte loss caused by the operation process and improving the accuracy and precision of quantitative analysis.

[0030] In one embodiment of this application, when reconstituted a concentrated acetonitrile solution sample, in addition to adding methanol as a reconstitution solvent, perfluorinated and polyfluoroalkyl substances and organophosphate internal standards were further added. These internal standards are used to correct for signal fluctuations and matrix effects during instrument analysis, ensuring accurate quantification of PFASs and OPEs. Similarly, when using a concentrated isohexane solution sample reconstituted with isohexane, a fluorinated liquid crystal internal standard was further added for accurate quantification of FLCMs.

[0031] In one embodiment of this application, key operating parameters were optimized to achieve the best extraction efficiency and purification effect. Specifically, the preferred ratio of the biological sample to the acetonitrile used in a single extraction is 0.5 g: 6 mL. At this ratio, acetonitrile can fully penetrate the biological sample, effectively precipitating proteins and dissolving the target analyte. Simultaneously, the nitrogen concentration temperature is preferably set to 30°C. This temperature condition is mild and allows for rapid evaporation of the organic solvent without causing thermal degradation of the target analyte.

[0032] Example This embodiment uses human placental samples as the specific biological matrix to detail the pretreatment method of the present invention for the simultaneous extraction of perfluorinated and polyfluoroalkyl substances, organophosphates, and fluorinated liquid crystals. This embodiment aims to verify the application effect of this method in actual biological samples, including its purification capacity, target analyte separation effect, and recovery rate. The pretreatment method in this embodiment was applied to placental samples for analysis. The spiked recoveries of PFASs, OPEs, and FLCMs were quantitatively determined using the internal standard method. In one embodiment of this application, the specific conditions for vortex mixing and centrifugation were optimized. The vortexing time is preferably 1 min, which is sufficient to allow the sample and solvent to mix thoroughly and uniformly. The centrifugation conditions are preferably 5000 rpm for 20 min. Under these centrifugation conditions, the precipitated proteins and solid residues can be fully settled, obtaining a clear supernatant, which facilitates subsequent transfer operations.

[0033] The pretreatment method for simultaneously extracting perfluorinated and polyfluoroalkyl substances, organophosphates, and fluorinated liquid crystals from biological matrix samples according to embodiments of this application includes the following steps: (1) Take 0.5 g of placental sample and stable isotope 13 C-labeled PFASs, stable isotope D-labeled OPEs, 13 C 12 - PCB-52 and acetonitrile solution were mixed using a vortex mixer for 1 min; (2) Centrifuge the sample at 5000 rpm for 20 min, separate the supernatant, add acetonitrile to the lower solid sample, and repeat the above operation; (3) Combine the two supernatants, add isohexane, mix with a vortex shaker for 1 min, let stand to separate the layers, add isohexane to the lower acetonitrile solution, mix with a vortex shaker for 1 min, and let stand to separate the layers. (4) The acetonitrile solution after the two separations was dephospholipid PPR Pro, 300 mg 3 mL; (5) The obtained acetonitrile solution and isohexane solution were concentrated to near dryness under nitrogen atmosphere; (6) The nearly dry acetonitrile solution was reconstituted with 200 μL of methanol, and the nearly dry isohexane solution was reconstituted with 200 μL of isohexane, and then injected for analysis.

[0034] The following examples further illustrate this application; Sample blank: Take 0.5 g of sample, add 6 mL of acetonitrile, vortex mix for 1 min, centrifuge at 5000 rpm for 20 min, extract the supernatant, and repeat the above operation once. Place the two supernatants in a new PP tube, add 6 mL of isohexane, vortex for 1 min, let stand and separate the layers, place the upper liquid in a new PP tube, and add 6 mL of isohexane to the acetonitrile and repeat the above operation. The lower acetonitrile solution was treated with a dephospholipid PPR Pro column (300 mg, 3 mL) to remove fat. The resulting isohexane and acetonitrile solutions were concentrated to near dryness under nitrogen at 30 °C. The near-dry acetonitrile solution was reconstituted with 170 μL of methanol, 1.0 ng (0.01 mL, 100 ng / mL) of PFASs internal standard, and 2.0 ng (0.02 mL, 100 ng / mL) of OPEs internal standard. The near-dry isohexane solution was reconstituted with 180 μL of isohexane and 2.0 ng (0.02 mL, 100 ng / mL) of FLCMs internal standard. The solutions were then transferred to vials for analysis.

[0035] Sample spiking: Take 0.5 g of sample and add 2.0 ng (0.02 mL, 100 ng / mL) of PFASs standard, 2.0 ng (0.02 mL, 100 ng / mL) of OPEs standard, 2.0 ng (0.02 mL, 100 ng / mL) of FLCMs standard and 6 mL of acetonitrile. Vortex for 1 min, centrifuge at 5000 rpm for 20 min, extract the supernatant, and repeat the above operation once. Place the two supernatants in a new PP tube, add 6 mL of isohexane, vortex for 1 min, and allow to stand for separation. Place the upper liquid in a new PP tube, add 6 mL of isohexane to the acetonitrile, and repeat the above operation. The lower acetonitrile solution was treated with a dephospholipid PPR Pro column (300 mg, 3 mL) to remove fat. The resulting isohexane and acetonitrile solutions were concentrated to near dryness under nitrogen at 30 °C. The near-dry acetonitrile solution was reconstituted with 170 μL of methanol, 1.0 ng (0.01 mL, 100 ng / mL) of PFASs internal standard, and 2.0 ng (0.02 mL, 100 ng / mL) of OPEs internal standard. The near-dry isohexane solution was reconstituted with 180 μL of isohexane and 2.0 ng (0.02 mL, 100 ng / mL) of FLCMs internal standard. The solutions were then transferred to vials for analysis.

[0036] The recovery rate is calculated based on the above process: Recovery rate (%) = (Spiked concentration of sample - Measured concentration of blank sample) / Measured concentration of standard sample × 100%.

[0037] Figure 1-3 The recoveries of PFASs, OPEs, and FLCMs are respectively.

[0038] Compared with existing technologies, the advantages and positive effects of this method are: three types of substances can be extracted simultaneously from biological samples using a single sample. It also boasts advantages such as low sample volume, excellent impurity removal, and rapid processing speed.

[0039] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A pretreatment method suitable for the simultaneous extraction of perfluorinated and polyfluoroalkyl substances, organophosphates, and fluorinated liquid crystals from biological samples, characterized in that, Includes the following steps: Add acetonitrile and internal standard to the biological sample, vortex to mix evenly, centrifuge, and extract the supernatant; add acetonitrile to the residue again, repeat the vortexing, centrifugation and supernatant extraction operation, and combine the supernatants obtained from the two extractions; Isohexane was added to the combined acetonitrile supernatant for liquid-liquid extraction. After standing and separating the layers, an isohexane solution and an acetonitrile solution were obtained. Isohexane was added to the acetonitrile solution again for liquid-liquid extraction. After standing and separating the layers, the isohexane solutions obtained from the two liquid-liquid extractions were combined. The acetonitrile solution obtained after liquid-liquid extraction is passed through a dephospholipid column, and the effluent is collected. The acetonitrile solution and the isohexane solution were concentrated under nitrogen atmosphere, respectively. The concentrated acetonitrile solution sample was reconstituted with methanol, and the concentrated isohexane solution sample was reconstituted with isohexane to obtain the sample to be analyzed.

2. The pretreatment method according to claim 1, characterized in that, The samples to be analyzed are detected by high performance liquid chromatography in tandem with Orbitrap mass spectrometry to achieve simultaneous qualitative and quantitative analysis of three types of target substances: perfluorinated and polyfluorinated alkyl substances, organophosphates, and fluorinated liquid crystals.

3. The pretreatment method according to claim 1, characterized in that, The biological sample was a human placenta sample.

4. The pretreatment method according to claim 1, characterized in that, The products from each operation unit in the acetonitrile extraction step and the isohexane liquid-liquid extraction step are retained and enter into the subsequent analysis process; no phase is discarded.

5. The pretreatment method according to claim 1, characterized in that, In the liquid-liquid extraction step, isohexane is used as a selective extractant for fluorinated liquid crystals, extracting the fluorinated liquid crystals from the acetonitrile phase to the isohexane phase, thereby achieving inter-group separation of the fluorinated liquid crystals from perfluorinated and polyfluoroalkyl substances and organophosphates.

6. The pretreatment method according to claim 1, characterized in that, The dephospholipid column is CNWBOND Dephospholipid PPR Pro, with a specification of 300mg / 3mL, used to specifically adsorb phospholipid interfering substances in the acetonitrile solution.

7. The pretreatment method according to claim 1, characterized in that, The internal standard comprises stable isotopes 13 C-labeled perfluoro and polyfluoroalkyl substances, stable isotope D-labeled organophosphates, and 13 C 12 - PCB-52.

8. The pretreatment method according to claim 1, characterized in that, When using acetonitrile solution samples that have been redissolved and concentrated in methanol, internal standards of perfluorinated and polyfluoroalkyl substances and organophosphates are further added; when using isohexane solution samples that have been redissolved and concentrated in isohexane, internal standards of fluorinated liquid crystals are further added.

9. The pretreatment method according to claim 1, characterized in that, The ratio of the amount of biological sample to the amount of acetonitrile used in a single extraction is 0.5 g: 6 mL; the temperature for nitrogen concentration is 30 °C.

10. The pretreatment method according to claim 1, characterized in that, The vortex operation time is 1 minute; the centrifugation conditions are a rotation speed of 5000 rpm and a time of 20 minutes.

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