Method for synchronously extracting and detecting 59 endocrine disruptors in amniotic fluid

Abstract

The invention discloses a method for synchronously extracting and detecting 59 endocrine disruptors in amniotic fluid, and belongs to the technical field of substance detection. Comprising the following steps: S1, adding beta-glucuronidase into an amniotic fluid sample, and incubating; s2, adding a stable isotope labeled internal standard substance into the amniotic fluid sample to be detected in the step S1; s3, adding the amniotic fluid sample to be detected in the step S2 into the activated HLB extraction column, removing impurities and extracting a target detection object; s4, eluting the extraction column to obtain an eluent, blowing the eluent to nearly dry, and adding a redissolving solvent to obtain a to-be-detected sample; and S5, detecting the to-be-detected sample obtained in the step S4 by using a liquid chromatograph-mass spectrometer to complete detection and analysis of the endocrine disrupter. The positive ion mode and the negative ion mode are simultaneously carried out in the detection process. The method realizes synchronous extraction, detection and analysis of at least 59 typical environmental endocrine disruptors in amniotic fluid, is simple and convenient, and solves the problems of large sample consumption and long time consumption.

Description

Technical Field

[0001] The invention relates to the technical field of substance detection, and in particular to a method for synchronously extracting and detecting 59 endocrine disruptors in amniotic fluid. Background Art

[0002] Environmental endocrine disruptors (EDCs) refer to chemical substances that exist in the natural environment or are released into the environment through artificial pollution and can interfere with the endocrine system function of organisms, affect the normal hormone levels and internal environment homeostasis in organisms, and are also called environmental hormones. This may harm human development, metabolism, and reproduction. In daily production and life, we are easily exposed to many chemical substances or chemical mixtures that can interfere with normal hormone functions, such as flame retardants, plasticizers, pesticides, etc., and a large number of studies have shown that various endocrine disruptors that pose a potential threat to human health can be widely detected in a variety of environmental media and biological matrices, such as hydroxyl polycyclic aromatic hydrocarbons (OH-PAHs), phthalate metabolites (mPAEs), perfluorinated compounds (PFASs), parabens (Parabens), organophosphate metabolites (mOPEs), bisphenols (BPs), etc.

[0003] Humans are exposed to endocrine disruptors in the environment through oral, inhalation, and skin contact. Since EDCs have a hormone-like structure, such substances will bind to normal hormone receptors in the body, making them have estrogenic and anti-estrogen activities, and also have antagonistic androgenic and thyroid hormone effects. Studies have shown that these endocrine disruptors play a role in the pathogenesis of a variety of endocrine disorders, including metabolic disorders such as reproductive development disorders, polycystic ovary syndrome, and neurodevelopmental disorders. EDCs are exposed to a wide range of pathways and sources. Pregnant women and developing fetuses are sensitive populations and are susceptible to environmental exposure. Studies have pointed out that amniotic fluid, as the cradle of life, not only provides a living environment for the fetus, but also directly reflects the physiological state of the fetus. Amniotic fluid is an important medium that reflects the exposure of multiple EDCs in the fetus, but there is currently a lack of detection methods for endocrine disruptors in amniotic fluid. Given the wide distribution of endocrine disruptors, there has been a long-term low-dose mixed exposure of multiple endocrine disruptors in the population, and most of the current related research focuses on the detection methods of a certain type or a certain type of endocrine disruptors, and there is a lack of comprehensive and systematic research on the simultaneous detection and analysis methods of multiple endocrine disruptors. At the same time, the existing analysis methods still have problems such as long time consumption and large sample consumption that need to be solved.

[0004] Therefore, there is an urgent need to establish an analytical method that requires less amniotic fluid, takes less time, and can simultaneously extract and detect multiple endocrine disruptors in amniotic fluid, so as to simultaneously identify compounds with a wide range of polarities from complex biological matrices, assess the exposure level of endocrine disruptors in the population, and provide a basic condition for further exploring the relationship between mixed exposure to multiple endocrine disruptors and human health outcomes. Summary of the invention

[0005] The present invention aims to provide a method for simultaneously determining multiple endocrine disruptors in amniotic fluid. The method of the present invention consumes a small amount of amniotic fluid, takes a short time, can simultaneously extract and detect multiple endocrine disruptors, and has the advantages of high precision, high sensitivity, low blank interference, and high sample recovery rate, which is beneficial for evaluating the exposure level of mixed endocrine disruptors in pregnant and lying-in women.

[0006] To achieve the above object, the present invention provides a method for synchronously extracting and detecting 59 endocrine disruptors in amniotic fluid, comprising the following steps: Sample preparation: S1. Add ammonium acetate and β-glucuronidase to the amniotic fluid sample, vortex and incubate; S2, adding a stable isotope-labeled internal standard to the amniotic fluid sample to be tested in step S1; S3, adding the amniotic fluid sample to be tested in step S2 to the activated HLB extraction column to remove impurities and extract the target detection object; S4, washing the extraction column to obtain the eluent, blowing it to near dryness, and then adding a re-dissolving solvent to obtain a sample to be tested; In step S4, two extractions are introduced into the extraction column, and different amounts of extractants are used in the first extraction and the second extraction.

[0007] Testing of samples to be tested: S5, using a liquid chromatography-mass spectrometer to detect the sample obtained in step S3, and complete the detection and analysis of endocrine disruptors. The detection process is carried out simultaneously in positive ion mode and negative ion mode.

[0008] Preferably, the amniotic fluid sample to be tested is at least one of human or animal amniotic fluid, and the concentration of the matrix in the amniotic fluid to be tested is not less than 0.005 ng / mL.

[0009] Preferably, ammonium acetate and β-glucuronidase are added to the amniotic fluid sample, and the mixture is vortexed and then incubated, comprising the following steps: Take 500 μL of the amniotic fluid sample in a centrifuge tube, and add 200 μL of pre-prepared ammonium acetate and 10 μL of β-glucuronidase to the amniotic fluid sample to complete the enzymatic hydrolysis treatment; preferably, place it in a vortex oscillator for vortexing and then put it in a constant temperature water bath for incubation.

[0010] The concentration of ammonium acetate is 0.8-1 M. The concentration of β-glucuronidase is 80000-100000 unit\L. The vortex time is 0.1-0.3 min, the temperature of the constant temperature water bath is 36.5-37° C., and the enzymolysis time is 8-14 h.

[0011] Preferably, the extraction column is an HLB solid phase extraction column, and its activation solvent includes 4 ml of methanol and 4 ml of ultrapure water.

[0012] Preferably, after the HLB solid phase extraction column is activated, 2 ng of the stable isotope-labeled internal standard compound is added to the amniotic fluid sample in step S1, and the amniotic fluid sample with the internal standard added is added to the HLB solid phase extraction column.

[0013] Preferably, the extraction solvent is 4-5 ml of 5% ammonia methanol as a solvent to elute the target detection object; after eluting with 4-5 ml of 5% ammonia methanol, 2-3 ml of 5% ammonia methanol is used to elute the target object for the second time.

[0014] Preferably, the eluent is blown to near dryness by nitrogen blowing, and the solvent used for re-dissolution is methanol, and the amount of methanol used for re-dissolution is 0.18-0.22 ml.

[0015] Preferably, in step S4, after redissolution, the membrane used for membrane filtration is a nylon membrane with a pore size of 0.1-0.4 μm.

[0016] According to one embodiment of the present invention, the concentration of the aqueous solution of ammonium acetate is 5-10 mM.

[0017] According to one embodiment of the present invention, the concentration of the formic acid aqueous solution is 0.08-0.1%.

[0018] Preferably, according to one embodiment of the present invention, in the ultra-high performance liquid chromatography-tandem mass spectrometry, a Shimadzu liquid chromatography-mass spectrometer LCMS-8060NX is used, a HPLC column Shim-pack GIST C18 chromatographic column is used, the length is 100 mm, the inner diameter is 2.1 mm, the filler particle diameter is 2 μm, the column temperature is 30-40°C, the injection volume is 4-6 μL, and the flow rate is 0.2-0.3 mL / min.

[0019] According to one embodiment of the present invention, in ultra-high performance liquid chromatography-tandem mass spectrometry, the collection mode is a multiple reaction detection mode; an electrospray ion source ESI is used, including positive ion mode and negative ion mode, the ion source temperature is 200-300°C, and the ionization voltage is 4000-5000V.

[0020] According to one embodiment of the present invention, in the ultra-high performance liquid chromatography-tandem mass spectrometry, the elution method used is gradient elution, and the elution procedures of the gradient elution are shown in Table 1 below; Table 1: Elution procedures for positive and negative ion modes

[0021] The 59 different types of typical environmental endocrine disruptors include at least one of hydroxylated polycyclic aromatic hydrocarbons, phthalate metabolites, perfluorinated compounds, parabens, organophosphate metabolites, and bisphenols.

[0022] Phenols include bisphenol-A, bisphenol-S, bisphenol-F, bisphenol-AF, bisphenol-E, bisphenol-B, bisphenol-AP, bisphenol-Z, bisphenol-P, tetrachlorobisphenol A, tetrabromobisphenol A ; Parabens include methylparaben, ethylparaben, butylparaben, propylparaben; organophosphate metabolites include dibutyl phosphate, diphenyl phosphate, di-o-cresyl phosphate, p-cresyl phosphate, bis-1-chloro-2-propyl phosphate, bis-2-butoxyethyl phosphate, bis-2-chloroethyl phosphate; phthalate metabolites include mono-2-ethyl-5-carboxypentyl phthalate, mono-2-isobutyl phthalate, mono-n-butyl phthalate, mono-2-ethylhexyl phthalate, mono-ethyl phthalate, mono-methyl phthalate, mono-benzyl phthalate, mono-3-carboxypropyl phthalate, mono-2-ethyl -5-oxohexyl phthalate, 5-mono 2-ethyl-5-hydroxyhexyl phthalate, mono [2-carboxymethylhexyl] phthalate; perfluorinated compounds include perfluorooctanoic acid, perfluorononanoic acid, perfluorohexanoic acid, perfluorooctane sulfonic acid, perfluorohexane sulfonic acid, perfluorooctane sulfonamide, perfluorobutyric acid, perfluoropentanoic acid, perfluoroheptanoic acid, perfluorodecanoic acid, perfluoroundecanoic acid, perfluorododecanoic acid, perfluorotridecanoic acid, perfluorotetradecanoic acid, perfluoroheptane sulfonic acid, perfluorobutane sulfonic acid, 6:2 chloropolyfluoroalkyl ether sulfonate, 8:2 chloropolyfluoroalkyl ether sulfonate; hydroxy polycyclic aromatic hydrocarbons include at least one of 1-hydroxyphenanthrene, 2-hydroxyphenanthrene, 3-hydroxyphenanthrene, 1-hydroxynaphthalene, 2-hydroxynaphthalene, 2-hydroxyfluorene, 3-hydroxyfluorene and 1-hydroxypyrene.

[0023] Therefore, the present invention adopts a method for synchronously extracting and detecting 59 endocrine disruptors in amniotic fluid using the above structure, which has the following beneficial effects: The present invention provides a method for the simultaneous extraction and detection of 59 different types of typical environmental endocrine disruptors in amniotic fluid, which overcomes many deficiencies of existing detection technologies, especially the problems of large sample consumption, long time consumption, and single substance detection in existing detection methods. The present invention achieves the simultaneous extraction, detection, and analysis of at least 59 typical environmental endocrine disruptors in amniotic fluid by improving sample pretreatment and liquid phase analysis methods. It is simple and convenient, and solves the problems of large sample consumption and long time consumption. At the same time, the average recovery rate of the method of the present invention ranges from 65% to 141%; the linearity is good, r 2 All were greater than 0.99; the sensitivity was high, and the LODs and LOQs of the method were 0.02 ng / mL~0.2 ng / mL and 0.07 ng / mL~0.66 ng / mL.

[0024] As for reducing sample consumption, the method of the present invention only requires 0.5 mL of amniotic fluid sample, which greatly reduces sample demand. This invention helps solve the problem of difficulty in collecting amniotic fluid and limited sample size. As for solving the problem of time-consuming detection process, the method of the present invention can complete the detection of 59 endocrine disrupting substances within 12 minutes through a two-step method combined with positive and negative ion dual modes, which greatly shortens the detection time and significantly improves efficiency.

[0025] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Schematic diagram of the steps of the method for simultaneous extraction and detection of 59 different types of typical environmental endocrine disruptors in amniotic fluid in the embodiment; Figure 2 is the extraction chromatogram of hydroxyl polycyclic aromatic hydrocarbons and their analogs in the target analytes of the embodiment; Figure 3 is the extraction chromatogram of bisphenol compounds and their analogs in the target analytes of the embodiment; Figure 4 is an extraction chromatogram of organophosphate flame retardant metabolites and their analogs in the target analytes of the embodiment; Figure 5 The extraction chromatogram of perfluorinated compounds and their analogs in the target analytes of the embodiment; Figure 6 is the extraction chromatogram of phthalate metabolites and their analogs in the target analytes of the embodiment; Figure 7 The following is the extraction chromatogram of parabens and their analogs in the target analytes of the example. DETAILED DESCRIPTION

[0027] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0028] Unless otherwise defined, the technical terms or scientific terms used in the present invention should be understood by people with ordinary skills in the field to which the present invention belongs. The words "first", "second" and similar words used in the present invention do not indicate any order, quantity or importance, but are only used to distinguish different components. "Include" or "comprise" and similar words mean that the elements or objects appearing before the word include the elements or objects listed after the word and their equivalents, without excluding other elements or objects. "Connect" or "connected" and similar words are not limited to physical or mechanical connections, but may include electrical connections, whether direct or indirect. "Up", "down", "left", "right" and the like are only used to indicate relative positional relationships. When the absolute position of the described object changes, the relative positional relationship may also change accordingly.

[0029] Example like Figure 1 As shown, the present invention provides a method for synchronously extracting and detecting 59 endocrine disruptors in amniotic fluid, and the specific steps include the following: Sample pretreatment: S1. Thaw the amniotic fluid sample at 4°C and place it in a 10 mL centrifuge tube. Then add 200 μL of 1 mol / L ammonium acetate and 10 μL of 100,000 unit / L β-glucuronidase. Vortex and incubate in a water bath at 37°C overnight. S2. Add 4 ml of methanol + 4 ml of ultrapure water to the HLB solid phase extraction column to activate it; S3, add 2 ng of the target analyte mixed standard solution to the amniotic fluid sample treated in step S1, set up a blank sample, add 710 μL of ultrapure water to the centrifuge tube, and add 2 ng of the target analyte mixed standard solution. Then load the amniotic fluid sample and the blank sample onto the HLB solid phase extraction column. Then use 4 ml of 2% formic acid water to elute the extraction column; S4. Use 5 ml of 5% ammonia methanol to elute the target. After completion, use 2 ml of 5% ammonia methanol to elute again. Then place the eluate in a nitrogen blower and blow it until it is almost dry. Take 200 μL of methanol and add it to the sample for re-dissolution. After re-dissolution, filter through a 0.22 μm nylon membrane and vortex for 0.2 min. Transfer the sample to a 9 mm threaded brown glass injection vial equipped with a glass liner to obtain the sample to be tested; Sample testing: Liquid chromatography-tandem mass spectrometry (LC-MS / MS) was used to detect and analyze the test samples obtained in step S4, and the standard curve of each substance was established by the 10-point method. The average recovery rate of 59 typical environmental endocrine disruptors in amniotic fluid was calculated. When the recovery rate ranged from 70% to 120% and the relative standard deviation was less than 20%, the accuracy of the method was considered to be good.

[0030] The target substances in the amniotic fluid samples to be tested include: bisphenol-A (BPA), bisphenol-S (BPS), bisphenol-F (BPF), bisphenol-AF (BPAF), bisphenol-E (BPE), bisphenol-B (BPB), bisphenol-AP (BPAP), bisphenol-Z (BPZ), bisphenol-P (BPP), tetrachlorobisphenol A (TCBPA), tetrabromobisphenol A (TBBPA), methylparaben (MP), ethylparaben (EP), butylparaben (BP), propylparaben (PP), dibutyl phosphate (DBP), diphenyl phosphate (DPHP), di-o-cresyl phosphate (DoCP), p-cresyl phosphate (DpCP), bis-1-chloro-2-propyl phosphate (BCIPP), bis-2-butoxyethyl phosphate (BBOEP), bis-2-chloroethyl phosphate (BCEP), mono-2-ethyl-5-carboxypentyl phthalate (MECPP), mono-2-isobutyl phthalate (MiBP), mono-n-butyl phthalate (MBP), mono-2-ethylhexyl phthalate (MEHP), monoethyl phthalate (MEP), monomethyl phthalate (MMP), monobenzyl phthalate (MBzP), mono-3-carboxypropyl phthalate (MBZP), perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), perfluorohexanoic acid (PFHxA), perfluorooctane sulfonic acid (PFOS), perfluorohexane sulfonic acid (PFHxS), perfluorooctane sulfonamide (PFOSA), perfluorobutyric acid (PFBA), perfluoropentanoic acid (PFPeA), perfluoroheptanoic acid (PFHpA), perfluorodecanoic acid (PFDA), perfluoroundecanoic acid (PFUnDA), perfluorododecanoic acid (PFDoDA), perfluorotridecanoic acid (PFTrDA), perfluorotetradecanoic acid (PFTeDA), perfluoroheptane sulfonic acid (PFHpS), perfluorobutane sulfonic acid (PFBS), 6:2 chloropolyfluoroalkyl ether sulfonate (6:2 Cl-PFESA), 8:2 chloropolyfluoroalkyl ether sulfonate (8:2Cl-PFESA), 1-hydroxyphenanthrene (1OH-PHE), 2-hydroxyphenanthrene (2OH-PHE), 3-hydroxyphenanthrene (3OH-PHE), 1-hydroxynaphthalene (1OH-NAP), 2-hydroxynaphthalene (2OH-NAP), 2-hydroxyfluorene (2OH-FLU), 3-hydroxyfluorene (3OH-FLU) and 1-hydroxypyrene (1OH-PYR).

[0031] Specifically, Figure 2These are the extraction chromatograms of 1OH-PHE, 2OH-PHE, 3OH-PHE, 1OH-NAP, 2OH-NAP, 2OH-FLU, 3OH-FLU, 1OH-PYR, 1OH-NAP-D8, 2OH-PHE-D9, and 1OH-PYR-D9.

[0032] Specifically, Figure 3 These are the extraction chromatograms of TCBPA, TBBPA, BPAF, BPS, BPA, BPF, BPB, BPE, BPP, BPZ, BPAP, and 13C12-BPS.

[0033] Specifically, Figure 4 These are the extraction chromatograms of DBP, DPHP, BCEP, BCIPP, BBOEP, DoCP, DpCP, DBP-D18, BBOEP-D8, and DpCP-D14.

[0034] Specifically, Figure 5 For 8:2 Cl-PFESA, 6:2 Extraction chromatograms of Cl-PFESA, PFOSA, PFOS, PFHxS, PFNA, PFOA, PFHxA, PFBA, PFPeA, PFHpA, PFDA, PFUnDA, PFDoDA, PFTrDA, PFTeDA, PFHpS, PFBS, 13C8-PFOS, 13C7-PFUnDA, 13C8-PFOA, 13C5-PFPeA.

[0035] Specifically, Figure 6 These are the extraction chromatograms of MECPP, MEHP, MBP, MiBP, MEP, MMP, MBzP, MCPP, MEOHP, MEHHP, MCMHP, MBP-D4, 13C4-MEP, and 13C4-MECPP.

[0036] Specifically, Figure 7 These are the extraction chromatograms of MP, EP, PP, BP, and MP-D4.

[0037] The mass spectrometry parameters of the above-mentioned method for simultaneous extraction and detection of 59 typical environmental endocrine disruptors in amniotic fluid are shown in Table 2.

[0038] Table 2. Target mass spectrometry parameter settings

[0039] In the above-mentioned method for simultaneous extraction and detection of 59 typical environmental endocrine disruptors in amniotic fluid, the basic information of the target analytes is shown in Table 3.

[0040] Table 3: Basic characteristics of target analytes

[0041] Table 4 Test correlation coefficient R 2 , LOD and LOQ

[0042] As shown in Table 4, the standard curve of this method has good linearity, R 2 All were greater than or equal to 0.99; the method was highly sensitive, with LODs and LOQs of 0.02-0.2 ng / mL and 0.07-0.66 ng / mL, respectively.

[0043] 59 compounds in the blank amniotic fluid sample were quantitatively spiked with high, medium and low concentrations, and according to the set instrument conditions (the test conditions were the same as those in the example and measured three times in parallel), the recovery rate was high, and the relative standard deviation (RSD) of the measured value was 1%~34%. The detailed results are shown in Table 5. It shows that the above method has good stability and high accuracy.

[0044] Table 5: Recovery and precision of target analytes

[0045] The present invention was used in actual amniotic fluid samples to measure the concentration of target compounds in 5 amniotic fluid samples. The results are shown in the following table; Table 6: Detection levels of 59 target analytes in amniotic fluid samples

[0046] Note: nd means not detected Except for the 24 targets in Table 6, the remaining targets BPF, BPE, BPB, BPAP, BPZ, BPP, TCBPA, TBBPA, BP, DoCP, DpCP, BCIPP, BBOEP, BCEP, MEP, MMP, MBzP, MCPP, MEOHP, MEHHP, MCMHP, PFPeA, PFHpA, PFDoDA, PFTeDA, PFBS, 8:2 Cl-PFESA, 1OH-PHE, 2OH-PHE, 3OH-PHE, 1OH-NAP, 2OH-NAP, 2OH-FLU, 3OH-FLU and 1OH-PYR were not detected.

[0047] In summary, the method for simultaneous extraction and detection of 59 different types of typical environmental endocrine disruptors in amniotic fluid provided by the present invention overcomes many shortcomings of existing detection technologies, especially the problems of large sample consumption, long time consumption and single detection of existing detection methods. By improving the instrumental analysis method, the simultaneous extraction and detection of 59 different types of typical environmental endocrine disruptors in amniotic fluid is realized, which is simple, fast, economical and environmentally friendly. At the same time, the average recovery rate of this method ranges from 65% to 141%; in addition, the standard curve of this method has good linearity, R 2 All of them were greater than or equal to 0.99; the method had high sensitivity, and its LODs and LOQs were 0.02-0.2 ng / mL and 0.07-0.66 ng / mL, respectively.

[0048] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. A method for synchronous extraction and detection of 59 endocrine disruptors in amniotic fluid, characterized in that: The following steps are involved: Sample preparation: S1, adding β-glucuronidase to the amniotic fluid sample and incubating; S2, adding a stable isotope-labeled internal standard to the amniotic fluid sample to be tested in step S1; S3, adding the amniotic fluid sample to be tested in step S2 to the activated HLB extraction column to remove impurities and extract the target detection object; S4, washing the extraction column to obtain the eluent, blowing it to near dryness, and then adding a re-dissolving solvent to obtain a sample to be tested; In step S4, two extractions are introduced into the extraction column, and different amounts of extractants are used in the first extraction and the second extraction; Testing of samples to be tested: S5. Using a liquid chromatography-mass spectrometer to detect the sample obtained in step S4, to complete the detection and analysis of endocrine disruptors, the detection process is carried out simultaneously in positive ion mode and negative ion mode.

2. A method for synchronous extraction and detection of 59 endocrine disruptors in amniotic fluid according to claim 1, characterized in that: The concentration of the matrix in the amniotic fluid to be tested in step S2 and step S3 is not less than 0.005 ng / mL.

3. A method for synchronous extraction and detection of 59 endocrine disruptors in amniotic fluid according to claim 1, characterized in that: In step S2, adding a stable isotope-labeled internal standard to the amniotic fluid sample to be tested comprises the following steps: Take 500 μL of amniotic fluid sample in a centrifuge tube, add 200 μL of pre-prepared ammonium acetate and 10 μL of β-glucuronidase to the amniotic fluid sample to be tested, and after the enzymatic hydrolysis is completed, add 2 ng of mixed internal standard to 710 μL of the mixed sample.

4. The method for synchronously extracting and detecting 59 endocrine disruptors in amniotic fluid according to claim 1, characterized in that: In step S3, the extraction column is an HLB column.

5. The method for synchronously extracting and detecting 59 endocrine disruptors in amniotic fluid according to claim 1, characterized in that: In step S3, the eluent added to the extraction column is 4 mL of 2% formic acid water.

6. The method for synchronously extracting and detecting 59 endocrine disruptors in amniotic fluid according to claim 1, characterized in that: In step S3, impurities are removed by filtering through a membrane, which is a nylon membrane with a pore size of 0.1 μm-0.4 μm.

7. The method for synchronously extracting and detecting 59 endocrine disruptors in amniotic fluid according to claim 1, characterized in that: In step S4, the extractant introduced into the extraction column is 7 ml of 5% ammonia methanol, 5 ml of 5% ammonia methanol is used for the first extraction, and 2 ml of 5% ammonia methanol is used for the second extraction.

8. A method for synchronously extracting and detecting 59 endocrine disruptors in amniotic fluid according to any one of claims 1 to 7, characterized in that: The mobile phases used in liquid chromatography-tandem mass spectrometry include mobile phase A and mobile phase B. In the positive and negative ion modes of hydroxypolycyclic aromatic hydrocarbons, perfluorinated compounds, parabens, organophosphate metabolites, and bisphenols, mobile phase A is 5mM-10mM ammonium acetate aqueous solution, and mobile phase B is methanol; in the positive and negative ion modes of phthalate metabolites, mobile phase A is 0.08%-0.1% mass fraction formic acid aqueous solution, and mobile phase B is methanol; in liquid chromatography-tandem mass spectrometry, the injection volume is 4-6μL.

9. The method for synchronously extracting and detecting 59 endocrine disruptors in amniotic fluid according to claim 8, characterized in that: The 59 different types of typical environmental endocrine disruptors include at least one of hydroxylated polycyclic aromatic hydrocarbons, phthalate metabolites, perfluorinated compounds, parabens, organophosphate metabolites, and bisphenols.

10. The method for synchronously extracting and detecting 59 endocrine disruptors in amniotic fluid according to claim 9, characterized in that: Phenols include bisphenol-A, bisphenol-S, bisphenol-F, bisphenol-AF, bisphenol-E, bisphenol-B, bisphenol-AP, bisphenol-Z, bisphenol-P, tetrachlorobisphenol A, tetrabromobisphenol A ; Parabens include methylparaben, ethylparaben, butylparaben, propylparaben; organophosphate metabolites include dibutyl phosphate, diphenyl phosphate, di-o-cresyl phosphate, p-cresyl phosphate, bis-1-chloro-2-propyl phosphate, bis-2-butoxyethyl phosphate, bis-2-chloroethyl phosphate; phthalate metabolites include mono-2-ethyl-5-carboxypentyl phthalate, mono-2-isobutyl phthalate, mono-n-butyl phthalate, mono-2-ethylhexyl phthalate, mono-ethyl phthalate, mono-methyl phthalate, mono-benzyl phthalate, mono-3-carboxypropyl phthalate, mono-2-ethyl -5-oxohexyl phthalate, 5-mono 2-ethyl-5-hydroxyhexyl phthalate, mono [2-carboxymethylhexyl] phthalate; perfluorinated compounds include perfluorooctanoic acid, perfluorononanoic acid, perfluorohexanoic acid, perfluorooctane sulfonic acid, perfluorohexane sulfonic acid, perfluorooctane sulfonamide, perfluorobutyric acid, perfluoropentanoic acid, perfluoroheptanoic acid, perfluorodecanoic acid, perfluoroundecanoic acid, perfluorododecanoic acid, perfluorotridecanoic acid, perfluorotetradecanoic acid, perfluoroheptane sulfonic acid, perfluorobutane sulfonic acid, 6:2 chloropolyfluoroalkyl ether sulfonate, 8:2 chloropolyfluoroalkyl ether sulfonate; hydroxy polycyclic aromatic hydrocarbons include at least one of 1-hydroxyphenanthrene, 2-hydroxyphenanthrene, 3-hydroxyphenanthrene, 1-hydroxynaphthalene, 2-hydroxynaphthalene, 2-hydroxyfluorene, 3-hydroxyfluorene and 1-hydroxypyrene.

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