A method for synchronous extraction and detection of 59 endocrine disruptors in amniotic fluid

Abstract

The present invention discloses a method for synchronously extracting and detecting 59 endocrine disruptors in amniotic fluid, belonging to the technical field of substance detection. The method includes the following steps: 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 detected in step S1; S3, adding the amniotic fluid sample to be detected in step S2 to an activated HLB extraction column to remove impurities and extract the target analyte; S4, eluting the extraction column to obtain an eluate, blowing it to near dryness, and adding a reconstitution solvent to obtain a sample to be detected; S5, using a liquid chromatography-mass spectrometry instrument to detect the sample to be detected 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. This method realizes the synchronous extraction, detection and analysis of at least 59 typical environmental endocrine disruptors in amniotic fluid, which is simple and convenient, and solves the problems of large sample consumption and long time consumption.

Description

Technical Field

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

[0002] Environmental endocrine disruptors (EDCs), also known as environmental hormones, are chemicals that exist in the natural environment or are released into the environment due to human pollution and can interfere with the function of the endocrine system in organisms, affecting the normal hormone levels and internal environment homeostasis in organisms. They may further endanger human development, metabolism, reproduction, etc. In daily production and life, people 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 potential threats to the health of the body can be widely detected in various environmental media and biological matrices, such as hydroxy polycyclic aromatic hydrocarbons (OH-PAHs), phthalate metabolites (mPAEs), perfluorinated compounds (PFASs), parabens, organophosphate metabolites (mOPEs), bisphenols (BPs), etc.

[0003] Humans are exposed to endocrine disruptors in the environment through oral ingestion, inhalation, skin contact, etc. Since EDCs have a hormone-like structure, such substances will bind to normal hormone receptors in the body, enabling them to have estrogenic and anti-estrogenic activities, and at the same time also having anti-androgenic and thyroid hormone effects, etc. Research has shown that these endocrine disruptors play a role in the pathogenesis of various endocrine disorders, including reproductive and developmental disorders, polycystic ovary syndrome, neurodevelopmental disorders and other metabolic disorders. The exposure routes and sources of EDCs are extensive. Pregnant women and developing fetuses, as sensitive populations, are vulnerable to environmental exposure. Some studies have pointed out that amniotic fluid, as the cradle for nurturing life, not only provides a living environment for the fetus, but also can directly reflect the physiological state of the fetus. Amniotic fluid is an important medium for reflecting the exposure of various EDCs in the fetus. However, there is currently a lack of detection methods for endocrine disruptors in amniotic fluid. Given the widespread distribution of endocrine disruptors and the long-term low-dose mixed exposure of multiple endocrine disruptors in the population, most of the current related research focuses on the detection methods of a single or a certain type of endocrine disruptor, and there is a lack of comprehensive 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 with less amniotic fluid consumption, short time consumption, and the ability to simultaneously extract and detect multiple endocrine disruptors in amniotic fluid, so as to identify compounds with a wide range of polarities from complex biological matrices, evaluate the exposure levels of endocrine disruptors in the population, and provide a basic condition for further exploring the relationship between the mixed exposure of 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 has less amniotic fluid consumption, short time consumption, and can simultaneously extract and detect multiple endocrine disruptors, and has advantages such as high precision, high sensitivity, low blank interference, and high sample recovery rate, which is conducive to evaluating the exposure levels of mixed endocrine disruptors in pregnant women.

[0006] To achieve the above object, the present invention provides a method for simultaneously extracting and detecting 59 endocrine disruptors in amniotic fluid, including the following steps:

[0007] Sample pretreatment:

[0008] S1. Add ammonium acetate and β-glucuronidase to the amniotic fluid sample, vortex and incubate.

[0009] S2. Add a stable isotope-labeled internal standard to the amniotic fluid sample to be tested in step S1.

[0010] S3. Add the amniotic fluid sample to be tested in step S2 to the activated HLB extraction column to remove impurities and extract the target analyte.

[0011] S4. Elute the extraction column to obtain an eluate, blow it to near dryness, and then add a reconstitution solvent to obtain a sample to be tested.

[0012] In step S4, two extractions are introduced in the extraction column, and the amounts of the extraction agent used in the first extraction and the second extraction are different.

[0013] Detection of the sample to be tested:

[0014] S5. Use a liquid chromatography-mass spectrometry instrument to detect the sample to be tested obtained in step S3 to complete the detection and analysis of endocrine disruptors. The detection process is carried out simultaneously in the positive ion mode and the negative ion mode.

[0015] 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.

[0016] Preferably, adding ammonium acetate and β-glucuronidase to the amniotic fluid sample, vortexing and incubating, includes the following steps:

[0017] Take 500 μL of amniotic fluid sample into a centrifuge tube, and simultaneously add 200 μL of pre-prepared ammonium acetate and 10 μL of β-glucuronidase to the amniotic fluid sample to be tested to complete the enzymatic hydrolysis treatment; preferably, place it in a vortex oscillator for vortex oscillation and then put it into a constant temperature water bath for incubation.

[0018] The concentration of the prepared ammonium acetate is 0.8 - 1 M. The concentration of the prepared β-glucuronidase is 80,000 - 100,000 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 enzymatic hydrolysis time is 8 - 14 h.

[0019] 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.

[0020] Preferably, after the HLB solid phase extraction column is activated, add 2 ng of a stable isotope-labeled internal standard compound to the amniotic fluid sample in step S1, and add the amniotic fluid sample with the internal standard added to the HLB solid phase extraction column.

[0021] Preferably, the extraction solvent is 4 - 5 ml of 5% ammonia methanol as a washing agent to elute the target analyte; after eluting with 4 - 5 ml of 5% ammonia methanol, use 2 - 3 ml of 5% ammonia methanol to elute the target again.

[0022] Preferably, the eluate is blown to near dryness by the nitrogen blowing method, and the solvent used for reconstitution is methanol, and the amount of methanol used for reconstitution is 0.18 - 0.22 ml.

[0023] Preferably, in step S4, after reconstitution, the membrane used for membrane filtration is a nylon membrane with a pore size of 0.1 - 0.4 μm.

[0024] According to an embodiment of the present invention, the concentration of the ammonium acetate aqueous solution is 5 - 10 mM.

[0025] According to an embodiment of the present invention, the concentration of the formic acid aqueous solution is 0.08 - 0.1%.

[0026] Preferably, according to an embodiment of the present invention, in ultra-high performance liquid chromatography-tandem mass spectrometry, a Shimadzu LCMS-8060NX liquid chromatography-mass spectrometry instrument is used, an HPLCcolumn Shim-packGIST C18 chromatographic column with a length of 100 mm, an inner diameter of 2.1 mm, and a filler particle diameter of 2 μm, a column temperature of 30 - 40 °C, an injection volume of 4 - 6 μL, and a flow rate of 0.2 - 0.3 mL / min.

[0027] According to an embodiment of the present invention, in ultra-high performance liquid chromatography-tandem mass spectrometry, the acquisition mode is multiple reaction monitoring mode; an electrospray ionization 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 4000-5000 V.

[0028] According to an embodiment of the present invention, in ultra-high performance liquid chromatography-tandem mass spectrometry, the elution method used is gradient elution. The elution program of gradient elution is shown in Table 1 below, and the elution program of gradient elution is shown in Table 1 below;

[0029] Table 1: Elution program for positive and negative ion modes

[0030]

[0031] The different types of typical environmental endocrine disruptors in 59 include at least one of hydroxy polycyclic aromatic hydrocarbons, phthalate metabolites, perfluorinated compounds, p-hydroxybenzoates, organophosphate metabolites, and bisphenols.

[0032] Phenolic substances include bisphenol-A, bisphenol-S, bisphenol-F, bisphenol-AF, bisphenol-E, bisphenol-B, bisphenol-AP, bisphenol-Z, bisphenol-P, tetrachlorobisphenol A, and tetrabromobisphenol A; p-hydroxybenzoates include methyl p-hydroxybenzoate, ethyl p-hydroxybenzoate, butyl p-hydroxybenzoate, and propyl p-hydroxybenzoate; organophosphate metabolites include dibutyl phosphate, diphenyl phosphate, di-o-tolyl phosphate, p-tolyl phosphate, bis(1-chloro-2-propyl) phosphate, bis(2-butoxyethyl) phosphate, and 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, monoethyl phthalate, monomethyl phthalate, monobenzyl phthalate, mono(3-carboxypropyl) phthalate, mono(2-ethyl-5-oxohexyl) phthalate, 5-mono(2-ethyl-5-hydroxyhexyl) phthalate, and mono[2-carboxymethylhexyl] phthalate; perfluorinated compounds include perfluorooctanoic acid, perfluorononanoic acid, perfluorohexanoic acid, perfluorooctane sulfonic acid, perfluorohexane sulfonic acid, perfluorooctane sulfonamide, perfluorobutyric acid, perfluorovaleric acid, perfluoroheptanoic acid, perfluorodecanoic acid, perfluoroundecanoic acid, perfluorododecanoic acid, perfluorotridecanoic acid, perfluorotetradecanoic acid, perfluoroheptane sulfonic acid, perfluorobutane sulfonic acid, 6:2 chlorinated polyfluoroalkyl ether sulfonate, and 8:2 chlorinated polyfluoroalkyl 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.

[0033] Therefore, the method for synchronously extracting and detecting 59 endocrine disruptors in amniotic fluid with the above structure of the present invention has the following beneficial effects:

[0034] The present invention provides a method for synchronously extracting and detecting 59 different types of typical environmental endocrine disruptors in amniotic fluid, overcoming many deficiencies of the existing detection technologies, especially problems such as large sample consumption, long time consumption, and single substance detection in the existing detection methods. By improving the sample pretreatment and liquid phase analysis methods, the present invention realizes the synchronous extraction, detection, and analysis of at least 59 typical environmental endocrine disruptors in amniotic fluid, which is both simple and convenient, and solves the problems of large sample consumption and long time consumption. At the same time, the average recovery rate range of the method of the present invention is 65% - 141%; the linearity is good, and r 2 is greater than 0.99 on average; the sensitivity is high, and the LODs and LOQs of the method are 0.02 ng / mL - 0.2 ng / mL and 0.07 ng / mL - 0.66 ng / mL.

[0035] For reducing the sample consumption, the method of the present invention only requires 0.5 mL of amniotic fluid sample, greatly reducing the sample demand. This invention helps to solve the problems of difficult collection of amniotic fluid and limited sample volume. For solving the problem of long time consumption in the detection process, the method of the present invention can complete the detection of 59 endocrine disruptors within 12 minutes through a two-step method combined with positive and negative ion dual modes, greatly shortening the detection time and significantly improving the efficiency.

[0036] The technical solution of the present invention will be further described in detail below through the drawings and embodiments. Description of the Drawings

[0037] Figure 1 It is a schematic diagram of the step flow of the method for synchronously extracting and detecting 59 different types of typical environmental endocrine disruptors in amniotic fluid in the embodiment;

[0038] Figure 2 It is the extraction chromatogram of hydroxy polycyclic aromatic hydrocarbons and their analogs in the target analytes of the embodiment;

[0039] Figure 3 It is the extraction chromatogram of bisphenol compounds and their analogs in the target analytes of the embodiment;

[0040] Figure 4 It is the extraction chromatogram of organophosphate flame retardant metabolites and their analogs in the target analytes of the embodiment;

[0041] Figure 5 It is the extraction chromatogram of perfluorinated compounds and their analogs in the target analytes of the embodiment;

[0042] Figure 6Extraction chromatogram of phthalate metabolites and their analogs in the target analyte of the example;

[0043] Figure 7 Extraction chromatogram of parabens and their analogs in the target analyte of the example. Detailed implementation mode

[0044] The technical solution of the present invention will be further described below with reference to the accompanying drawings and examples.

[0045] Unless otherwise defined, the technical terms or scientific terms used in the present invention should have the ordinary meaning understood by those of ordinary skill in the field to which the present invention belongs. The "first", "second" and similar terms used in the present invention do not denote any order, quantity or importance, but are only used to distinguish different components. The terms such as "comprising" or "including" mean that the elements or objects appearing before this term cover the elements or objects listed after this term and their equivalents, without excluding other elements or objects. The terms such as "connected" or "coupled" are not limited to physical or mechanical connections, but may include electrical connections, whether direct or indirect. The terms such as "upper", "lower", "left", "right" are only used to represent relative positional relationships, and when the absolute position of the object to be described changes, the relative positional relationship may also change accordingly.

[0046] Example

[0047] As Figure 1 shown, the present invention provides a method for synchronous extraction and detection of 59 endocrine disruptors in amniotic fluid. The specific steps are as follows:

[0048] Sample pretreatment:

[0049] S1. Take the amniotic fluid sample to be tested and thaw it at 4°C, place it in a 10 mL centrifuge tube, then add 200 μL of 1 mol / L ammonium acetate and 10 μL of 100000 unit / L β-glucuronidase, vortex and then place it in a water bath at 37°C for overnight incubation;

[0050] S2. Add 4 ml of methanol + 4 ml of ultrapure water to the HLB solid phase extraction column to activate it;

[0051] S3. Add 2 ng of the target analyte mixed standard solution to the amniotic fluid sample treated in step S1, and set 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 wash the extraction column;

[0052] S4. Use 5 ml of 5% ammonia in methanol to elute the target compound. After completion, use 2 ml of 5% ammonia in methanol to elute again. Subsequently, place the eluate in a nitrogen evaporator and evaporate to near dryness. Add 200 μL of methanol to the sample for reconstitution. After reconstitution, filter through a 0.22 μm nylon membrane and then vortex for 0.2 min. Transfer the sample to a 9 mm threaded brown glass injection vial equipped with a glass liner tube to obtain the sample to be tested;

[0053] Sample detection:

[0054] The sample to be tested obtained in step S4 was analyzed by liquid chromatography-tandem mass spectrometry (LC-MS / MS). A 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 range was 70% - 120% and the relative standard deviation was less than 20%, the accuracy of the method was considered good.

[0055] The analytes in the amniotic fluid sample 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), methyl paraben (MP), ethyl paraben (EP), butyl paraben (BP), propyl paraben (PP), dibutyl phosphate (DBP), diphenyl phosphate (DPHP), di-o-tolyl phosphate (DoCP), p-tolyl 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), mono(ethyl) phthalate (MEP), mono(methyl) phthalate (MMP), mono(benzyl) phthalate (MBzP), mono(3-carboxypropyl) phthalate (MCPP), mono(2-ethyl-5-oxohexyl) phthalate (MEOHP), 5-mono(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP), mono[2-carboxymethylhexyl] phthalate (MCMHP), perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), perfluorohexanoic acid (PFHxA), perfluorooctanesulfonic acid (PFOS), perfluorohexanesulfonic acid (PFHxS), perfluorooctanesulfonamide (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), perfluoroheptanesulfonic acid (PFHpS), perfluorobutanesulfonic acid (PFBS), 6:2 chlorinated polyfluoroalkyl ether sulfonate (6:2 Cl-PFESA), 8:2 chlorinated polyfluoroalkyl ether sulfonate (8:2 Cl-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).

[0056] Specifically, Figure 2Extraction 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.

[0057] Specifically, Figure 3 Extraction chromatograms of TCBPA, TBBPA, BPAF, BPS, BPA, BPF, BPB, BPE, BPP, BPZ, BPAP, and 13C12-BPS.

[0058] Specifically, Figure 4 Extraction chromatograms of DBP, DPHP, BCEP, BCIPP, BBOEP, DoCP, DpCP, DBP-D18, BBOEP-D8, and DpCP-D14.

[0059] Specifically, Figure 5 Extraction chromatograms of 8:2 Cl-PFESA, 6:2 Cl-PFESA, PFOSA, PFOS, PFHxS, PFNA, PFOA, PFHxA, PFBA, PFPeA, PFHpA, PFDA, PFUnDA, PFDoDA, PFTrDA, PFTeDA, PFHpS, PFBS, 13C8-PFOS, 13C7-PFUnDA, 13C8-PFOA, and 13C5-PFPeA.

[0060] Specifically, Figure 6 Extraction chromatograms of MECPP, MEHP, MBP, MiBP, MEP, MMP, MBzP, MCPP, MEOHP, MEHHP, MCMHP, MBP-D4, 13C4-MEP, and 13C4-MECPP.

[0061] Specifically, Figure 7 Extraction chromatograms of MP, EP, PP, BP, and MP-D4.

[0062] The method for the simultaneous extraction and detection of 59 typical environmental endocrine disruptors in amniotic fluid is shown in Table 2 for the mass spectrometry parameters.

[0063] Table 2. Target substance mass spectrometry parameter settings

[0064]

[0065] In the above method for the 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.

[0066] Table 3: Basic characteristics of target analytes

[0067]

[0068] Table 4: Test correlation coefficient R 2 , LOD and LOQ

[0069]

[0070] As can be seen from Table 4, the standard curve of this method has good linearity, and R 2 are all greater than or equal to 0.99; this method has high sensitivity, and its LODs and LOQs are 0.02 - 0.2 ng / mL and 0.07 - 0.66 ng / mL respectively.

[0071] For the 59 compounds in the amniotic fluid blank sample, high, medium, and low concentrations were quantitatively spiked, and according to the set instrument conditions (the test conditions are the same as those in the example and were measured in parallel 3 times), the recovery rate is high, and the relative standard deviation (RSD) of the measured values is 1% - 34%. The detailed results are shown in Table 5. It shows that the above method has good stability and high precision.

[0072] Table 5: Recovery rate and precision of target analytes

[0073]

[0074] The present invention was used in actual amniotic fluid samples to determine the concentrations of the target compounds in 5 amniotic fluid samples, and the results are shown in the following table;

[0075] Table 6: Detected contents of 59 target analytes in amniotic fluid samples

[0076]

[0077] Note: nd indicates not detected

[0078] Except for the 24 target substances in Table 6, the remaining target substances 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.

[0079] In summary, the method for synchronous extraction and detection of 59 different types of typical environmental endocrine disruptors in amniotic fluid provided by the present invention overcomes many deficiencies of the existing detection technologies, especially the problems of large sample consumption, long time consumption and single detection of the existing detection methods. By improving the instrumental analysis method, the synchronous extraction and detection of 59 different types of typical environmental endocrine disruptors in amniotic fluid are realized, which is simple, fast, economical and environmentally friendly. At the same time, the average recovery rate range of this method is 65% - 141%; in addition, the standard curve of this method has good linearity, and the R 2 is greater than or equal to 0.99; this method has high sensitivity, and its LODs and LOQs are 0.02 - 0.2 ng / mL and 0.07 - 0.66 ng / mL respectively.

[0080] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that they can still modify or equivalently replace the technical solutions of the present invention, and these modifications or equivalent replacements cannot make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for synchronous extraction and detection of 59 endocrine disruptors in amniotic fluid, characterized in that, It includes the following steps: Sample pretreatment: S1. Add β-glucuronidase to the amniotic fluid sample and then incubate; S2. Add a stable isotope-labeled internal standard to the amniotic fluid sample to be tested in step S1. Take 500 μL of the amniotic fluid sample in a centrifuge tube, and simultaneously add 200 μL of pre-prepared ammonium acetate and 10 μL of β-glucuronidase to the amniotic fluid sample to be tested; S3. Add the amniotic fluid sample to be tested in step S2 to the activated HLB extraction column to remove impurities and extract the target analyte; In step S3, the extraction column is an HLB column; the eluent added to the extraction column is 4 mL of 2% formic acid in water; S4. Elute the extraction column to obtain an eluate, blow it to near dryness, and then add a reconstitution solvent to obtain a sample to be tested; In step S4, two extractions are introduced in the extraction column, and the amounts of the extraction agent used in the first extraction and the second extraction are different; In step S4, the extraction agent introduced in the extraction column is 7 mL of 5% ammonia in methanol. 5 mL of 5% ammonia in methanol is used for the first extraction, and 2 mL of 5% ammonia in methanol is used for the second extraction; Detection of the sample to be tested: S5. Use a liquid chromatography-mass spectrometry (LC-MS) instrument to detect the sample to be tested obtained in step S4, complete the detection and analysis of endocrine disruptors, and the detection process is carried out simultaneously in the positive ion mode and the negative ion mode; In step S5, in liquid chromatography-tandem mass spectrometry, a C18 chromatographic column is used, with a gradient elution program. The mobile phase includes mobile phase A and mobile phase B. In the positive and negative ion modes of hydroxy polycyclic aromatic hydrocarbons, perfluorinated compounds, p-hydroxybenzoic acid esters, organophosphate metabolites, and bisphenols, mobile phase A is an aqueous solution of 5 mM - 10 mM ammonium acetate, and mobile phase B is methanol; in the positive and negative ion modes of phthalate metabolites, mobile phase A is an aqueous solution of formic acid with a mass fraction of 0.08% - 0.1%, and mobile phase B is methanol; the acquisition mode is the multiple reaction monitoring mode; an electrospray ionization source ESI is used, including the positive ion mode and the negative ion mode. The ion source temperature is 200 - 300 °C, and the ionization voltage is 4000 - 5000 V. And the 59 endocrine disruptors are specifically six categories including hydroxy polycyclic aromatic hydrocarbons, phthalate metabolites, perfluorinated compounds, p-hydroxybenzoic acid esters, organophosphate metabolites, and bisphenols.

2. The method for synchronous extraction and detection of 59 endocrine disruptors in amniotic fluid according to claim 1, wherein: In the amniotic fluid to be tested in steps S2 and S3, the concentration of the matrix 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, after the enzymatic hydrolysis treatment is completed, add 2 ng of the mixed internal standard to 710 μL of the mixed sample.

4. A method for synchronous extraction and detection of 59 endocrine disruptors in amniotic fluid according to claim 1, characterized in that: In step S3, the removal of impurities is achieved through a filtration membrane, and the filtration membrane is a nylon membrane with a filtration pore size of 0.1 μm - 0.4 μm.

5. A method for synchronous extraction and detection of 59 endocrine disruptors in amniotic fluid according to claim 1, characterized in that: In liquid chromatography-tandem mass spectrometry, the injection volume is 4 - 6 μL.

6. A method for synchronous extraction and detection of 59 endocrine disruptors in amniotic fluid according to claim 1, characterized in that: Phenolic substances include bisphenol-A, bisphenol-S, bisphenol-F, bisphenol-AF, bisphenol-E, bisphenol-B, bisphenol-AP, bisphenol-Z, bisphenol-P, tetrachlorobisphenol A, tetrabromobisphenol A; p-hydroxybenzoic acid esters include methyl p-hydroxybenzoate, ethyl p-hydroxybenzoate, butyl p-hydroxybenzoate, propyl p-hydroxybenzoate; organophosphate metabolites include dibutyl phosphate, diphenyl phosphate, di-o-tolyl phosphate, p-tolyl 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, monoethyl phthalate, monomethyl phthalate, monobenzyl 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, perfluorooctanesulfonic acid, perfluorohexanesulfonic acid, perfluorooctanesulfonamide, perfluorobutanoic acid, perfluoropentanoic acid, perfluoroheptanoic acid, perfluorodecanoic acid, perfluoroundecanoic acid, perfluorododecanoic acid, perfluorotridecanoic acid, perfluorotetradecanoic acid, perfluoroheptanesulfonic acid, perfluorobutanesulfonic acid, 6:2 chlorinated polyfluoroalkyl ether sulfonate, 8:2 chlorinated polyfluoroalkyl ether sulfonate; hydroxy polycyclic aromatic hydrocarbons include 1-hydroxyphenanthrene, 2-hydroxyphenanthrene, 3-hydroxyphenanthrene, 1-hydroxynaphthalene, 2-hydroxynaphthalene, 2-hydroxyfluorene, 3-hydroxyfluorene, and 1-hydroxypyrene.

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