Method for detecting perfluoroalkyl and polyfluoroalkyl substances in human early pregnancy villus tissue and application

Abstract

The invention provides a method for detecting perfluorinated and polyfluorinated alkyl substances in human early pregnancy villus tissues and application, and relates to the technical field of biological detection.The method comprises the following steps that a sample is obtained, digestive juice, a process monitoring standard solution and an internal standard mixed solution are added into the sample, then zirconium oxide grinding beads and methyl alcohol are sequentially added, homogenate, ultrasonic treatment and centrifugation are conducted, and the content of perfluorinated and polyfluorinated alkyl substances in the human early pregnancy villus tissues is obtained; sediment and supernate are obtained, and the supernate is collected; adding methanol into the sediment again, carrying out homogenization, ultrasonic treatment and centrifugation again to obtain supernate, and mixing the supernate obtained in the two steps to obtain an extracting solution; diluting the extracting solution, and carrying out WAX solid-phase extraction; and performing quantitative detection on the perfluoro and polyfluoroalkyl substances in the liquid to be detected in a multi-reaction monitoring mode by adopting a high performance liquid chromatography-tandem mass spectrometry method. The method can realize detection of human early pregnancy villus tissues, and has high sensitivity, high selectivity, high accuracy and strong anti-interference capability.

Description

Technical Field

[0001] This invention relates to the field of biodetection technology, and in particular to a method and application for detecting perfluorinated and polyfluoroalkyl substances in human early pregnancy chorionic villus tissue. Background Technology

[0002] Per- and polyfluoroalkyl substances (PFAS) are a class of stable synthetic organic compounds with hydrophobic and oleophobic properties, widely used in industrial fields such as textiles, papermaking, coatings, and fire-fighting materials, as well as in consumer products. However, PFAS exhibit environmental persistence, bioaccumulation, and potential biotoxicity, and are associated with various adverse health effects, such as immunotoxicity, developmental toxicity, endocrine disruption, and carcinogenic risks, making them a persistent organic pollutant of global concern.

[0003] The main routes of human exposure to PFAS include food, water, and inhalation. Once inside the body, PFAS bind to serum proteins and are distributed throughout organs and tissues via the bloodstream. Notably, PFAS can cross the placental barrier, causing direct exposure in early fetal development, potentially having a significant impact on fetal growth and development. Therefore, monitoring the level of PFAS exposure at the maternal-fetal interface during pregnancy is crucial for assessing the risk of early developmental exposure. Human early pregnancy chorionic villus tissue, as a precursor to placental formation and the initial interface for maternal-fetal material exchange, directly reflects the PFAS exposure load in early embryonic development and is an ideal biosample for such exposure assessment.

[0004] However, accurate quantitative analysis of trace PFAS in biological tissue samples faces significant challenges. First, biological sample matrices are complex, containing numerous endogenous interfering substances such as proteins and lipids, severely hindering the extraction and detection of target analytes. Second, PFAS themselves are diverse, with varying physicochemical properties, making it difficult for traditional methods to achieve simultaneous high-sensitivity detection of multiple PFAS. Third, PFAS are ubiquitous in laboratory environments (such as consumables and reagents), easily introducing background contamination and leading to biased results. Currently, while methods for detecting PFAS in biological fluids such as serum and urine have been reported, methods specifically for detecting PFAS in human early pregnancy chorionic villus tissue—a unique and valuable matrix—remain incomplete. Existing technologies may suffer from cumbersome pretreatment steps, unsatisfactory purification effects, inability to effectively cover novel PFAS compounds, and insufficient sensitivity to detect trace levels of residues.

[0005] Therefore, developing an analytical method specifically for human early pregnancy chorionic villus sampling that can simultaneously detect multiple traditional and novel PFAS, and that possesses high sensitivity, high selectivity, high accuracy, and strong anti-interference capabilities, is of urgent and important practical significance for advancing research on PFAS exposure and health risks during pregnancy.

[0006] Therefore, this invention is proposed. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides a method for detecting perfluorinated and polyfluoroalkyl substances (PFAS) in human early pregnancy chorionic villus tissue. This method enables the detection of PFAS in human early pregnancy chorionic villus tissue and possesses high sensitivity, high selectivity, high accuracy, and strong anti-interference capabilities. It has urgent and significant practical implications for advancing research on PFAS exposure and health risks during pregnancy.

[0008] In order to achieve the objective of this invention, the following technical solution is adopted: This invention provides a method for detecting perfluorinated and polyfluoroalkyl substances in human early pregnancy chorionic villus tissue, comprising the following steps: S1. Take placental villus tissue from early pregnancy and process it to obtain villus samples; S2. Add a digestive solution containing collagenase and trypsin to the sample and incubate together. Filter, centrifuge, discard the supernatant, and obtain cell clusters. S3. Add process monitoring standard solution to the cell cluster. The process monitoring standard is a perfluorinated compound analog that does not exist in the natural environment. Then add internal standard mixture. Then add zirconium oxide grinding beads and methanol in sequence. Homogenize, sonicate, and centrifuge to obtain sediment and supernatant. Collect the supernatant. S4. Add methanol to the sediment again, homogenize, sonicate, and centrifuge again to obtain supernatant. Mix the supernatant obtained in step S3 with the supernatant obtained in step S4 to obtain extract. S5. Dilute the extract and perform WAX solid-phase extraction; then use high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) in multiple reaction monitoring (MRM) mode to quantitatively detect perfluorinated and polyfluoroalkyl substances in the test solution. The MRM mode monitors ion pairs including quantitative ion pairs of perfluorinated and polyfluoroalkyl substances, corresponding isotope internal standard ion pairs, quantitative ion pairs of the process monitoring target, and at least one pair of characteristic ion pairs for indicating endogenous matrix interference.

[0009] Furthermore, the internal standard mixture comprises C13-labeled perfluorobutane sulfonic acid, C13-labeled perfluorohexane sulfonic acid, C13-labeled perfluorooctane sulfonic acid, C13-labeled 1H,1H,2H,2H-perfluorohexane sulfonate sodium, C13-labeled 1H,1H,2H,2H-perfluorooctane sulfonate sodium, C13-labeled 1H,1H,2H,2H-perfluorodecane sulfonate sodium, C13-labeled perfluorobutyric acid, and C13-labeled... It is composed of perfluorovaleric acid, C13-labeled perfluorohexanoic acid, C13-labeled perfluoroheptanoic acid, C13-labeled perfluorooctanoic acid, C13-labeled perfluorononanoic acid, C13-labeled perfluorodecanoic acid, C13-labeled perfluoroundecanoic acid, C13-labeled perfluorododecanoic acid, C13-labeled perfluorotetradecanoic acid, C13-labeled perfluoro-1-octanesulfonamide, deuterated N-methylperfluorooctylsulfonamide acetic acid, and deuterated N-ethylperfluorooctylsulfonamide acetic acid.

[0010] Furthermore, the concentration of any one of the substances constituting the internal standard mixture is 1 mg / L.

[0011] Furthermore, in step S3, which involves adding zirconia grinding beads, 4-6 3mm grinding beads and 2-4 5mm grinding beads are added.

[0012] Furthermore, in step S3, which involves adding zirconia grinding beads, five 3mm grinding beads and three 5mm grinding beads are added.

[0013] Furthermore, the specific steps for homogenization in steps S3 and S4 are as follows: The sample containing the internal standard mixture, methanol, and zirconium oxide grinding beads was stirred at a speed of 6.5 m / s, then shaken for 1 min and rested for 1 min. Repeat the above steps twice.

[0014] Furthermore, the specific steps of the WAX ​​solid-phase extraction are as follows: S401. The diluted extract was activated using a WAX column with 2% ammonia, pure methanol and water in sequence to obtain an activated sample. S402. Wash the activated sample, vacuum it, elute it, blow it with nitrogen, and reconstitute it.

[0015] Furthermore, the nitrogen blowing temperature is 40°C; Furthermore, the reagent used in the resolution step of S402 is a mixture of methanol and water in a volume ratio of 8:2.

[0016] Furthermore, the process monitoring label is one of perfluoro-1,4-succinic acid, perfluoro-1,6-adipic acid, or perfluoro-1,8-octanoic acid.

[0017] Furthermore, the collagenase is type II collagenase; Furthermore, in the digestive fluid, the concentration of type II collagenase is 0.5-2.0 mg / mL, and the concentration of trypsin is 0.1-0.5 mg / mL.

[0018] Furthermore, the concentration of type II collagenase was 1.0 mg / mL, and the concentration of trypsin was 0.25 mg / mL.

[0019] Furthermore, based on the ratio of the measured concentration of the process monitoring target in the final test solution to its theoretical added concentration, the measured concentrations of the target perfluorinated and polyfluoroalkyl substances in the same batch of samples are corrected. When the ratio exceeds a preset threshold, the batch of sample data is determined to be significantly contaminated by the process and is removed or marked.

[0020] Furthermore, based on the ratio of the measured concentration of the process monitoring target in the final test solution to its theoretical added concentration, the measured concentrations of the target perfluorinated and polyfluoroalkyl substances in the same batch of samples are corrected. When the ratio exceeds a preset threshold, the batch of sample data is determined to be significantly contaminated by the process and is removed or marked.

[0021] Furthermore, the chromatographic column used in the high-performance chromatography-tandem mass spectrometry method is a C18 column.

[0022] Furthermore, the method also includes quality control using any one of matrix curves, full-process blank, spiked recovery, or parallel samples.

[0023] The present invention also provides a method for detecting perfluorinated and polyfluoroalkyl substances in human early pregnancy chorionic villus tissue, and its application in the detection of perfluorinated and polyfluoroalkyl substances in human early pregnancy chorionic villus tissue.

[0024] The present invention has the following technical effects: The detection method provided by this invention can achieve highly sensitive detection of trace levels of PFAS, accurately determining the PFAS content in human early pregnancy chorionic villus tissue as low as 0.2 ng / g, meeting the requirements of trace analysis for biomonitoring; at the same time, it has good accuracy and precision, ensuring the reliability and repeatability of the detection results.

[0025] Furthermore, this detection method can effectively remove matrix interference from complex tissues and significantly reduce background noise, demonstrating high stability and efficiency in practical applications. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0027] In a first aspect, the present invention provides a method for detecting perfluorinated and polyfluoroalkyl substances in human early pregnancy chorionic villus tissue, comprising the following steps: S1. Take placental villus tissue from early pregnancy and process it to obtain villus samples; S2. Add a digestive solution containing collagenase and trypsin to the sample and incubate together. Filter, centrifuge, discard the supernatant, and obtain cell clusters. S3. Add process monitoring standard solution to the cell cluster. The process monitoring standard is a perfluorinated compound analog that does not exist in the natural environment. Then add internal standard mixture. Then add zirconium oxide grinding beads and methanol in sequence. Homogenize, sonicate, and centrifuge to obtain sediment and supernatant. Collect the supernatant. S4. Add methanol to the sediment again, homogenize, sonicate, and centrifuge again to obtain supernatant. Mix the supernatant obtained in step S3 with the supernatant obtained in step S4 to obtain extract. S5. Dilute the extract and perform WAX solid-phase extraction; then use high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) in multiple reaction monitoring (MRM) mode to quantitatively detect perfluorinated and polyfluoroalkyl substances in the test solution. The MRM mode monitors ion pairs including quantitative ion pairs of perfluorinated and polyfluoroalkyl substances, corresponding isotope internal standard ion pairs, quantitative ion pairs of the process monitoring target, and at least one pair of characteristic ion pairs for indicating endogenous matrix interference.

[0028] Early pregnancy chorionic villus tissue is a highly heterogeneous complex containing various components such as trophoblast cells, mesenchymal cells, and blood vessels. PFAS distribution may be uneven across different cell types. Traditional whole-cell grinding methods result in indiscriminate fragmentation, failing to guarantee the representativeness of the analyzed samples. This invention utilizes a digestive solution containing collagenase and trypsin for gentle and controlled digestion of the chorionic villus tissue. The two work synergistically to achieve efficient and specific release of trophoblast cells. Subsequent filtration and centrifugation yield enriched, biologically representative cell clusters, serving as the starting point for subsequent analyses.

[0029] This processing method focuses the analysis from disorganized tissue homogenates to functionally relevant cell populations, enabling the test results to more accurately reflect the PFAS load exposed to the embryo, thus enhancing the biological significance and accuracy of the data. Simultaneously, because villous tissue is scarce and difficult to obtain, enzymatic hydrolysis makes the cell clusters easier to break down completely by subsequent mechanical grinding, avoiding incomplete extraction caused by tissue fiber entanglement, improving PFAS recovery rate and reproducibility between different samples. Cell enrichment also reduces irrelevant mechanisms introduced by target cells, lessening the burden on subsequent purification.

[0030] In some embodiments, the internal standard mixture comprises C13-labeled perfluorobutane sulfonic acid, C13-labeled perfluorohexane sulfonic acid, C13-labeled perfluorooctane sulfonic acid, C13-labeled 1H,1H,2H,2H-perfluorohexane sulfonate sodium salt, C13-labeled 1H,1H,2H,2H-perfluorooctane sulfonate sodium salt, C13-labeled 1H,1H,2H,2H-perfluorodecane sulfonate sodium salt, C13-labeled perfluorobutyric acid, and C13-labeled... It is composed of perfluorovaleric acid, C13-labeled perfluorohexanoic acid, C13-labeled perfluoroheptanoic acid, C13-labeled perfluorooctanoic acid, C13-labeled perfluorononanoic acid, C13-labeled perfluorodecanoic acid, C13-labeled perfluoroundecanoic acid, C13-labeled perfluorododecanoic acid, C13-labeled perfluorotetradecanoic acid, C13-labeled perfluoro-1-octanesulfonamide, deuterated N-methylperfluorooctylsulfonamide acetic acid and deuterated N-ethylperfluorooctylsulfonamide acetic acid.

[0031] Based on the specific exposure profiles of PFAS in biological samples and the complex matrix interference characteristics, a homogeneous mixture of 19 isotopic internal standards was designed. This internal standard system effectively overcomes the severe matrix effect in biological samples through comprehensive coverage and full-process correction. Quantification is achieved by measuring the signal ratio of the target analyte to the internal standard, effectively correcting for errors throughout the analytical process. By using 19 isotopic internal standards covering different chain lengths and functional groups, accurate quantification of multiple PFAS was achieved. This significantly improves the accuracy and precision of the method and reduces the impact of matrix effects and operational fluctuations.

[0032] PFAS background contamination is a primary challenge in trace analysis. Traditional full-process blanks are independent of sample quality control and cannot reflect the actual contamination experienced by each sample during processing. This invention adds a known amount of a "process monitoring standard" (such as perfluoro-1,4-succinic acid) to each sample at the very beginning of sample processing (step S3). This substance is a perfluorinated compound analog that does not exist in the natural environment, and its chemical properties are highly similar to the target PFAS. Therefore, it will experience the same contamination, adsorption, and loss as the target PFAS throughout the entire sample pretreatment process (extraction, transfer, purification, concentration, and instrumentation). During final detection, its ion pairs are monitored synchronously using MRM mode to accurately determine its final concentration in each sample. This elevates contamination monitoring from the "batch level" to the "individual sample level," providing personalized evidence of data reliability for each sample. By calculating the recovery rate of the process monitoring standard (measured value / added value), the degree of process contamination or abnormal loss experienced by the sample can be quantified. Based on this, the measured values ​​of samples in the same batch can be systematically corrected, or unreliable data can be decisively removed or marked when the recovery rate exceeds a preset threshold, greatly improving the overall reliability of the data. If the recovery rate of the process monitoring standard for a certain batch of samples is generally abnormal, the problem can be quickly located in a specific step of the pretreatment process, enabling efficient troubleshooting.

[0033] Even after WAX purification, biological samples may still contain endogenous substances (such as specific phospholipids) that co-elute with the target PFAS, producing a matrix effect and leading to inaccurate quantification. In the MRM method, this invention creatively adds at least one pair of "endogenous interference characteristic ion pairs" in addition to the target analyte, internal standard, and process monitoring target ion pairs.

[0034] This channel is not used for quantification, but only for diagnosis. When analyzing a chromatogram, if a high-intensity peak of an "interfering ion" appears near the retention time of a certain PFAS chromatographic peak, it indicates the presence of co-elution interference.

[0035] In some embodiments, the concentration of any one of the substances constituting the internal standard mixture is 1 mg / L.

[0036] In some embodiments, in step S2 of adding zirconia grinding beads, 4-6 3mm grinding beads and 2-4 5mm grinding beads are added.

[0037] In some embodiments, in step S2 of adding zirconia grinding beads, five 3mm grinding beads and three 5mm grinding beads are added.

[0038] This invention optimizes the size and quantity ratio of the grinding beads, utilizing the synergistic effect of zirconia grinding beads of different sizes during homogenization. Smaller beads provide a larger contact area and finer fragmentation, while larger beads provide stronger impact force. Together, they achieve efficient and thorough fragmentation of villous tissue cells, allowing for more complete release of intracellular PFAS into the extraction solvent. This avoids lower detection results due to incomplete tissue fragmentation and enhances the reproducibility of the method.

[0039] In some embodiments, the specific steps of homogenization in steps S3 and S4 are as follows: The sample containing the internal standard mixture, methanol, and zirconium oxide grinding beads was stirred at a speed of 6.5 m / s, then shaken for 1 min and rested for 1 min. Repeat the above steps twice.

[0040] The homogenization process ensures thorough tissue disruption while avoiding degradation of the target material or instrument wear due to excessive heat generation, achieving a controllable, efficient, and low-temperature homogenization process. This further ensures stable extraction efficiency, reduces intra-batch and inter-batch variations, and improves the precision and reliability of the method.

[0041] In some embodiments, the specific steps of the WAX ​​solid-phase extraction are as follows: S401. The diluted extract was activated using a WAX column with 2% ammonia, pure methanol and water in sequence to obtain an activated sample. S402. Wash the activated sample, vacuum it, elute it, blow it with nitrogen, and reconstitute it.

[0042] WAX (weak anion exchange) packing material specifically adsorbs negatively charged PFAS molecules through ion exchange. A specific activation sequence ensures the packing material is in the appropriate ionization state and removes impurities. Elution with methanol containing ammonia neutralizes the packing material's charge, thereby efficiently releasing the PFAS. This achieves effective sample purification and concentration, significantly removing major matrix interferences such as phospholipids and proteins, reducing background noise, and ensuring high PFAS recovery, thus creating conditions for high-sensitivity detection.

[0043] Furthermore, the nitrogen blowing temperature is 40°C; The reagent used in the resolution step of S402 is a mixture of methanol and water in a volume ratio of 8:2.

[0044] In some embodiments, the process monitoring label is one of perfluoro-1,4-succinic acid, perfluoro-1,6-adipic acid, or perfluoro-1,8-octanoic acid.

[0045] In some embodiments, the collagenase is type II collagenase; Furthermore, in the digestive fluid, the concentration of type II collagenase is 0.5-2.0 mg / mL, and the concentration of trypsin is 0.1-0.5 mg / mL.

[0046] In some embodiments, the concentration of type II collagenase is 1.0 mg / mL and the concentration of trypsin is 0.25 mg / mL.

[0047] In some embodiments, the multi-reaction monitoring mode targets all internal standard components in the internal standard solution and monitors specific precursor ions and their corresponding characteristic ions.

[0048] Utilizing a triple quadrupole mass analyzer in tandem mass spectrometry, the first stage selects the precursor ion of the target analyte, the second stage breaks it down by collision, and the third stage selects its characteristic daughter ions. This dual mass screening mechanism provides extremely high selectivity. This mode can greatly improve the selectivity (anti-interference capability) of detection, effectively distinguishing the target PFAS from the co-eluted matrix components, thereby ensuring the accuracy and reliability of qualitative and quantitative analysis, especially at trace levels.

[0049] In some embodiments, the chromatographic column used in the high-performance chromatography-tandem mass spectrometry is a C18 column.

[0050] In some embodiments, the method further includes quality control using any one of matrix profiling, full-process blank, spiked recovery, or parallel samples.

[0051] This invention enables effective real-time monitoring and evaluation of the accuracy, precision, and contamination levels throughout the entire analytical process, ensuring the validity of each batch of experimental data. The quality assurance system established by this invention ensures the traceability and reliability of the test results, meeting the requirements of bioanalytical standards and providing a solid guarantee for the scientific interpretation and application of the data.

[0052] The present invention also provides a method for detecting perfluorinated and polyfluoroalkyl substances in human early pregnancy chorionic villus tissue, and its application in the detection of perfluorinated and polyfluoroalkyl substances in human early pregnancy chorionic villus tissue.

[0053] The following is a detailed explanation using specific embodiments: Example 1 1. Sample collection and preservation On the day of the abortion procedure, fasting peripheral blood and early pregnancy placental villus tissue were collected from the study subjects by medical personnel trained by the project team. All sampling instruments were professionally sterilized to avoid cross-contamination.

[0054] Villus collection: The villous tissue aspirated during surgery is placed into a negative pressure bottle and directly introduced into a medical kidney dish through a suction tube.

[0055] Rinsing and drying: Rinse the villi repeatedly with sterile saline until there is no obvious bloodstain, then place them on filter paper to absorb the dryness.

[0056] Aliquoting and labelling: Weigh eight 1.8 mL cryovials using an electronic balance and label them as “sample number + a–h”. The first four tubes (a–d): each contain approximately 150 mg of villi for testing.

[0057] The last four tubes (e–h): Divide the remaining fluff into four equal parts for later use, and record the mass of each tube.

[0058] Storage: Seal tightly with sealing film, place in a cryopreservation box, and immediately transfer to -80 ℃ for storage. Only one freeze-thaw cycle is allowed before testing.

[0059] 2. Sample pretreatment Take approximately 100–150 mg of frozen chorionic villi sample.

[0060] S1. Targeted Enzymatic Digestion and Cell Enrichment: Transfer samples to pre-chilled centrifuge tubes and add 1 mL of Hanks' Balanced Salt Solution containing type II collagenase (1 mg / mL) and trypsin (0.25 mg / mL). Incubate gently at 37°C with a shaker for 20 minutes. Subsequently, filter the digest mixture through a 40 μm cell sieve to remove undigested tissue fragments. Collect the filtrate, centrifuge at 4°C and 1500 rpm for 5 minutes, discard the supernatant, and obtain enriched cell clusters.

[0061] S2. Add process monitoring standard and internal standard: Add 20 μL of process monitoring standard working solution (process monitoring standard is perfluoro-1,4-succinic acid, concentration is 50 ng / mL) to the enriched cell clusters, followed by 20 μL of internal standard mixture. The internal standard information is shown in Table 1.

[0062] Table 1: Composition and content of perfluorinated internal standard stock solutions labeled with 19 isotopes Add zirconia grinding beads to the tube: 5 beads of 3 mm and 3 beads of 5 mm. Then add 800 μL of methanol and homogenize at 4 °C (speed 6.5 m / s, shake for 1 min, rest for 1 min, repeat 2 times). Sonicate at room temperature for 10 min and centrifuge (14,000 rpm, 10 min, 4 °C). Collect the supernatant. Add 800 μL of methanol to the sediment and repeat the homogenization-sonication-centrifugation process. Collect the supernatant. Combine the two supernatants, totaling about 1600 μL, to obtain the extract.

[0063] 3. Purification and Concentration (WAX Solid Phase Extraction) Dilution: Take the combined extract and add ultrapure water to a total volume of 10 mL (organic phase volume fraction ≤30%); Column activation: Use a WAX column (specification 150 mg / 6 mL) and activate it sequentially with 5 mL of 2% (v / v) ammonia / methanol → 5 mL of methanol → 10 mL of water.

[0064] Sample loading: Slowly load the sample solution (let it flow down naturally); Washing: Wash with 5mL of water to remove impurities; Dry column: Evacuate or apply positive pressure for about 10 minutes; Elution: Elute slowly with 7 mL of 2% (v / v) ammonia / methanol and collect in a polypropylene tube; then drain the residual liquid after a few minutes. Concentration: Nitrogen blown at 40°C until nearly dry; Reconstitution: Add 150 μL of methanol:water = 8:2 (v / v), vortex thoroughly, and then centrifuge. Transfer: Transfer the supernatant into a vial for UHPLC-MS / MS detection.

[0065] 4. Chromatographic-mass spectrometry conditions Chromatographic column: C18 column (Waters BEHC18, 2.1×50mm, 1.7μm); Mobile phases: Phase A = 2–5 mM ammonium acetate aqueous solution, Phase B = methanol; Flow rate / temperature: 0.3 mL / min; 40℃; injection volume: 5–10 μL; Liquid chromatography employs gradient elution to sequentially separate different target analytes. The gradient elution program is shown in Table 2.

[0066] Table 2: Gradient elution program Mass spectrometry conditions: Ion source: ESI negative ion mode; Dry gas flow rate: 12 L / min, temperature: 320℃; Capillary voltage: 3.5 kV; Detection mode: Multiple reaction monitoring (MRM) acquisition. Specific MRM conditions for the target compound and internal standard are shown in Table 3.

[0067] In addition to monitoring the quantitative ion pairs of all target PFAS and their corresponding isotopic internal standards, the following two types of characteristic ion pairs are also monitored simultaneously: A. Process monitoring standard ion pair: A specific parent ion-daughter ion pair is set for the perfluoro-1,4-succinic acid, which is the standard for monitoring the entire process.

[0068] B. Characteristic ion pairs of endogenous interfering substances: Set at least one pair of characteristic ion pairs to indicate phospholipid interference.

[0069] Table 3: Multiple reaction monitoring conditions for target compounds and internal standards System control: Delay column and PEEK piping are used to avoid background peaks.

[0070] 5. Quality Control Design Process contamination assessment: In each batch of sample testing, process contamination is assessed by calculating the recovery rate of the process monitoring standard (perfluoro-1,4-succinic acid). If the recovery rate exceeds the range of 70%-130%, it indicates that there may be abnormal contamination or loss in the processing of the sample or batch of samples, and the relevant data should be used with caution or rejected.

[0071] Real-time diagnosis of endogenous interference: When analyzing chromatograms, check the signal intensity of characteristic ion pairs of endogenous interfering substances near the elution time of each target PFAS peak. If a high-intensity interference peak appears at this retention time, it is determined that the quantitative result of the PFAS may be affected by co-eluting matrix interference, reducing data reliability. Confirmation is required by optimizing chromatographic conditions or using the standard addition method.

[0072] Matrix curves: Prepare matrix curves at least five concentration points, ranging from 0.05 to 50 ng / mL, using weighted 1 / x regression, R0. 2 ≥0.995.

[0073] Completely blank process: at least one per batch (using empty tubes, without organization), interspersed throughout the testing process.

[0074] Spiked recovery: Set 3 levels such as 0.5, 5, and 50 ng / g, n≥6, recovery rate 60-130%, RSD≤15%.

[0075] Parallel samples: At least 10% of the samples in each batch are parallel.

[0076] Quality control: Each batch includes 1–2 low / medium / high concentration QC samples.

[0077] 1. The specific steps for quality control in serum matrix are as follows: Quantification was performed in serum matrix using matrix-matched external standards. Standard working solutions containing isotopic internal standards were prepared using blank serum, with five concentration points set at 0.5, 2, 5, 25, and 50 ng / mL. A standard curve was established using 1 / x weighted least squares regression. Both compounds showed good linearity in the 0.5–50 ng / mL range. The correlation coefficients between PFOA and PFOS reached 0.999 and 0.998, respectively. The back-calculated concentration deviations, except for the lowest point, were all within ±15%, with the lowest point controlled within ±20%.

[0078] Sensitivity assessment showed that the signal-to-noise ratio (SNR) for PFOA was approximately 3 at 0.03 ng / mL, which was determined to be the limit of detection; the limit of detection for PFOS was approximately 0.05 ng / mL. Accuracy and precision were validated using a three-level spiked recovery assay, with low, medium, and high levels set at 0.5, 5, and 50 ng / mL, and six replicates for each level. The recoveries of PFOA were 95.4%, 101.2%, and 98.3%, with intra-day relative standard deviations (RSDs) of 6.8%, 5.1%, and 4.7%, respectively. The recoveries of PFOS were 90.6%, 97.5%, and 102.1%, with RSDs less than 8% for all levels. Matrix effects were assessed using post-extraction spiked loading, and the relative response after internal standard correction was within the range of 96%–108%. Stability testing showed that the deviation of samples placed in an autosampler at 10°C for 24 hours was less than 10%, and the deviation remained within a certain range after three freeze-thaw cycles. The values ​​ranged from 11% to +9%, all of which met the requirements of bioanalytical methodologies.

[0079] 2. The specific steps for quality control in villous tissue are as follows: Curve configuration was performed using tissue homogenate as the matrix in villous tissue. Approximately 150 mg of blank villous tissue was extracted and processed using the same WAX ​​purification process as the real sample before being spiked. Matrix-matched external standard curves were established at five concentration points: 0.2, 1, 5, 25, and 50 ng / ml, and fitted using 1 / x weighted regression. The linear correlation coefficients of PFNA, PFDA, and 9Cl-PF3ONS reconstituted solutions reached 0.997, 0.996, and 0.995, respectively, in the range of 0.2–50 ng / ml, with overall concentration deviations not exceeding ±15%. Sensitivity was assessed using the signal-to-noise ratio method, and the limits of detection for PFNA, PFDA, and 9Cl-PF3ONS reconstituted solutions were approximately 0.03 ng / ml. Accuracy and precision were assessed through matrix spiked recovery, with six parallel replicates at three levels: 0.5, 5, and 50 ng / ml. The recoveries of PFNA were 93.1%–103.4%, PFDA 90.2%–108.5%, and 9Cl-PF3ONS 84.7%–95.8%, with intra-day and inter-day relative standard deviations (RSDs) all not exceeding 13%. Procedure blanks and carryover effects were assessed simultaneously. A matrix blank was injected after high-concentration samples, and the residual signals of the target ion pairs were below 20% of their respective LOQs. No significant interference peaks were observed in the procedure blank. Subtraction of the overall method blank had no substantial impact on quantification.

[0080] 6. Methodological validation metrics Limit of detection and limit of quantitation (LOD / LOQ): Using signal-to-noise ratio (S / N) as the criterion, LOD was defined as S / N=3 and LOQ as S / N=10. The LOD of this method for 31 PFAS ranged from 0.03 to 0.10 ng / mL, and the experimental results are shown in Table 4.

[0081] Table 4: LOD detection results of 31 PFAS The LOD of PFOA, PFHxS, PFNA, and PFDA was 0.03 ng / mL. The LOD of PFOS, PHOSA, PFHpS, etc. is 0.05 ng / mL; The LOD of PFBA, HFPO-DA, PFNS, and PFTeDA is 0.10ng / mL.

[0082] The LOQ is approximately three times that of the LOD, i.e., 0.09–0.30 ng / mL.

[0083] Precision: Assessed by intra-day and inter-day repeatability tests, with relative standard deviation (RSD) ≤15%.

[0084] Matrix effect: The results were assessed using the post-column injection method, and after internal standard correction, the results were in the range of 90%-110%.

[0085] stability: Three freeze-thaw cycles: recovery rate 85–115%; Placed in the injector for 24–48 hours: deviation ≤15%; Store at -80℃ for 3–6 months: No significant decrease in the concentration of each PFAS.

[0086] Memory effect: When a blank is injected after a high concentration sample, the blank response is less than 20% of the LOQ.

[0087] 7. Result Calculation The sample concentration is calculated using the following formula: in: Quantitative results from the instrument (ng / mL); : Reconstitution volume (mL), which is 0.150 mL in this method; : Villous tissue mass (g); If an external standard triple gradient is used, the regression result of the standard curve should be taken as the standard. .

[0088] If necessary, the mean or median of the entire program blanks should be deducted.

[0089] The experimental results are shown below: High detection rate compounds PFOA and PFOS were detected in all samples, with a detection rate of 100%. The detection rate of 9Cl-PF3ONS was 93.2%; The PFDA detection rate was 90.0%; The detection rate of PFNA was 87.3%; The detection rate of PFUnDA was 86.4%; The detection rate of PFTrDA was 80.1%.

[0090] low detection rate compounds The detection rates of PFBA, NaDONA, 11Cl-PF3OUdS, PFDoA, PFTeDA, PFHxS, etc. ranged from 2% to 16%. The detection rates of PFBS, 4:2FTS, and PFHpA were all below 2%.

[0091] No compound detected Some compounds (such as HFPO-DA, N-EtFOSAA, N-MeFOSAA, PHOSA, PFDS, PFHxA, etc.) were not detected in this batch of samples.

[0092] Process monitoring performance verification The recovery rate of the process monitoring standard (perfluoro-1,4-succinic acid) was statistically analyzed for 10 batches, totaling 120 samples. The experimental results are shown in Table 5.

[0093] Table 5: Performance Verification Results of Process Monitoring Standards Abnormal recovery rate of process monitoring targets: The recovery rates of the process monitoring standards in batches 4 and 6 were abnormal, with 2 and 3 samples, respectively, exceeding the preset threshold.

[0094] Troubleshooting for the anomaly: After investigation, the abnormality of the fourth batch was found to be due to batch differences in the solid phase extraction column, and the abnormality of the sixth batch was found to be due to temporary contamination of the laboratory's pure water system.

[0095] Data processing measures: Based on process monitoring data, seven unreliable samples were promptly removed, preventing the generation of erroneous data.

[0096] 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 or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the technical solutions of the embodiments of the present invention.

Claims

1. A method for detecting perfluoro- and polyfluoroalkyl substances in human first trimester chorionic tissue, characterized in that, Includes the following steps: S1. Take placental villus tissue from early pregnancy and process it to obtain villus samples; S2. Add a digestive solution containing collagenase and trypsin to the sample and incubate together. Filter, centrifuge, discard the supernatant, and obtain cell clusters. S3. Add process monitoring standard solution to the cell cluster. The process monitoring standard is a perfluorinated compound analog that does not exist in the natural environment. Then add internal standard mixture. Then add zirconium oxide grinding beads and methanol in sequence. Homogenize, sonicate, and centrifuge to obtain sediment and supernatant. Collect the supernatant. S4. Add methanol to the sediment again, homogenize, sonicate, and centrifuge again to obtain supernatant. Mix the supernatant obtained in step S3 with the supernatant obtained in step S4 to obtain extract. S5. Dilute the extract and perform WAX solid-phase extraction; then use high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) in multiple reaction monitoring (MRM) mode to quantitatively detect perfluorinated and polyfluoroalkyl substances in the test solution. The MRM mode monitors ion pairs including quantitative ion pairs of perfluorinated and polyfluoroalkyl substances, corresponding isotope internal standard ion pairs, quantitative ion pairs of the process monitoring target, and at least one pair of characteristic ion pairs for indicating endogenous matrix interference.

2. The method of detecting perfluoroalkyl and polyfluoroalkyl substances in human early gestational villous tissue according to claim 1, wherein, The internal standard mixture consists of C13-labeled perfluorobutane sulfonic acid, C13-labeled perfluorohexane sulfonic acid, C13-labeled perfluorooctane sulfonic acid, C13-labeled 1H,1H,2H,2H-perfluorohexane sulfonate sodium salt, C13-labeled 1H,1H,2H,2H-perfluorooctane sulfonate sodium salt, C13-labeled 1H,1H,2H,2H-perfluorodecane sulfonate sodium salt, C13-labeled perfluorobutyric acid, and C13-labeled perfluoro... It is composed of valeric acid, C13-labeled perfluorohexanoic acid, C13-labeled perfluoroheptanoic acid, C13-labeled perfluorooctanoic acid, C13-labeled perfluorononanoic acid, C13-labeled perfluorodecanoic acid, C13-labeled perfluoroundecanoic acid, C13-labeled perfluorododecanoic acid, C13-labeled perfluorotetradecanoic acid, C13-labeled perfluoro-1-octanesulfonamide, deuterated N-methylperfluorooctylsulfonamide acetic acid, and deuterated N-ethylperfluorooctylsulfonamide acetic acid.

3. The method of detecting perfluoroalkyl and polyfluoroalkyl substances in human first trimester villous tissue according to claim 1, wherein, In step S3, 4-6 3mm grinding beads and 2-4 5mm grinding beads are added.

4. The method of detecting perfluoroalkyl and polyfluoroalkyl substances in human early gestational villous tissue according to claim 1, wherein, The specific steps for homogenization in steps S3 and S4 are as follows: The sample containing the internal standard mixture, methanol, and zirconium oxide grinding beads was stirred at a speed of 6.5 m / s, then shaken for 1 min and rested for 1 min. Repeat the above steps twice.

5. The method of detecting perfluoroalkyl and polyfluoroalkyl substances in human first trimester villous tissue according to claim 1, wherein, The process monitoring label is any one of perfluoro-1,4-succinic acid, perfluoro-1,6-adipic acid, or perfluoro-1,8-octanoic acid.

6. The method of detecting perfluoroalkyl and polyfluoroalkyl substances in human early gestational villous tissue according to claim 1, wherein, The collagenase is type II collagenase; Furthermore, in the digestive fluid, the concentration of type II collagenase is 0.5-2.0 mg / mL, and the concentration of trypsin is 0.1-0.5 mg / mL.

7. The method of detecting perfluoroalkyl and polyfluoroalkyl substances in human early gestational villous tissue according to claim 6, wherein, The concentration of type II collagenase was 1.0 mg / mL, and the concentration of trypsin was 0.25 mg / mL.

8. The method of detecting perfluoroalkyl and polyfluoroalkyl substances in human first trimester villous tissue according to claim 1, wherein, The measured concentrations of target perfluorinated and polyfluoroalkyl substances in the same batch of samples are corrected based on the ratio of the measured concentration of the process monitoring target in the final test solution to its theoretical added concentration. When the ratio exceeds a preset threshold, the batch of sample data is determined to be significantly contaminated by the process and is removed or marked.

9. The method of claim 1, wherein the perfluoroalkyl and polyfluoroalkyl substances are selected from the group consisting of PFOS, PFOA, PFHxS, PFBA, PFHP, PFHxA, PFBS, PFNA, and ETPA. The method also includes quality control using any one of matrix curves, full-process blanks, spiked recovery, or parallel samples.

10. The application of the method for detecting perfluorinated and polyfluoroalkyl substances in human early pregnancy villi tissue as described in any one of claims 1-9 in the detection of perfluorinated and polyfluoroalkyl substances in human early pregnancy villi tissue.

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