A method for simultaneous detection of 16 endocrine disruptors in environmental water

Abstract

The invention discloses a method for simultaneously detecting 16 endocrine disruptors in environmental water, and belongs to the field of environmental detection. The method includes: sample collection and preservation; sample pretreatment; establishment of a standard curve; and determination of the concentration of endocrine disruptors in water samples by high performance liquid chromatography-tandem mass spectrometry. The present invention can simultaneously detect trace amounts of bisphenol A, 4-tert-butylphenol, 4-butylphenol, 4-pentylphenol, 4-hexylphenol, 4-tert-octylphenol, 4-heptylphenol, 4-nonylphenol, 4-n-octylphenol, 4-n-nonylphenol, estriol, diethylstilbestrol, 17β-estradiol, 17α-ethinylestradiol, estrone, and 16 endocrine disruptors in various environmental waters such as surface water, groundwater, sewage, and seawater, thereby reducing the detection limit.

Description

Technical Field

[0001] The present invention relates to the field of environmental detection, and in particular to a method for simultaneously detecting 16 endocrine disruptors in environmental water. Background Art

[0002] Environmental endocrine disruptors are a new type of pollutants, and there are many types, such as bisphenol A, alkylphenols, and estrogens such as estrone, estrol, and estradiol. A large number of research reports show that when these environmental endocrine disruptors enter the body through certain pathways, such as contaminated water, food, or absorption through the skin, they can trigger different molecular pathways and interfere with the synthesis, secretion, transport, binding and metabolism of endogenous hormones in the human body, leading to damage to the endocrine functions of the reproductive, nervous, immune and other systems, affecting the body's homeostasis and self-regulation ability, thereby causing potential harm to the health of the organism or its offspring.

[0003] Most environmental endocrine disruptors have high boiling points, and the main detection instruments are liquid chromatography (LC) and high-performance liquid chromatography-tandem mass spectrometry (LC-MS / MS). However, the simple LC detection method often has problems such as large interference from the sample matrix and easy false positives. Since the LC-MS / MS method combines the advantages of the liquid chromatography separation system and the mass spectrometry detection system, especially the application of multi-stage mass spectrometry tandem, it can provide richer and more accurate compound structure information. For the analysis of complex samples, even when it is difficult to completely separate by liquid chromatography, the multiple reaction monitoring mode (MRM) is used to perform multi-stage fragment ion scanning on the target analyte, which can still effectively qualitatively and quantitatively analyze the compound, becoming the main analytical method for the detection of environmental endocrine disruptors; in terms of enrichment methods, the enrichment technologies of endocrine disruptors in water mainly include liquid-liquid extraction and solid phase extraction. Among them, the solid phase extraction (SPE) method is simple to operate, consumes less solvent, can be automated, and can simultaneously complete the enrichment, purification, concentration and other steps. It is a mature pretreatment method.

[0004] At present, there is no technical method that can accurately determine the trace amounts of 16 endocrine disruptors in various environmental water bodies at the same time. The main detection methods that can be used are as follows:

[0005] 1) "Determination of Environmental Hormone Compounds in Water Quality - Solid Phase Extraction-Liquid Chromatography-Tandem Mass Spectrometry (DB37 / T4158-2020)" is only applicable to the determination of 11 target substances such as 17β-estradiol in drinking water and its source water, and does not consider the adsorption of 4-nonylphenol in the sample bottle.

[0006] 2) "Determination of Six Environmental Estrogen Compounds in Water by Solid Phase Extraction-High Performance Liquid Chromatography-Tandem Mass Spectrometry (DB44 / T 2016-2017)" is only applicable to the determination of six target substances such as ethinyl estradiol in surface water, groundwater and sewage, and does not consider the adsorption of 4-nonylphenol in the sample bottle.

[0007] 3) "Determination of 9 alkylphenol compounds and bisphenol A in water quality - Solid phase extraction high performance liquid chromatography (HJ 1192-2021)" uses liquid chromatography for detection, and the detection limit is too high to meet the current requirements for the detection of trace endocrine disruptors in environmental water bodies. It does not take into account the loss of 4-tert-butylphenol and 4-butylphenol during the nitrogen concentration stage and the adsorption of 4-nonylphenol, 4-n-octylphenol, and 4-n-nonylphenol in the sample bottle, and the determination range is only bisphenol A and 9 alkylphenols.

[0008] 4) "Standard Test Methods for Drinking Water Part 8: Organic Matter Index (GB / T 5750.875.1 / 75.2)" is only applicable to the determination of bisphenol A, nonylphenol and other five compounds in drinking water, and does not take into account the adsorption of 4-octylphenol and 4-nonylphenol in the sample bottle.

[0009] 5) "Determination of 22 compounds including bisphenol A, nonylphenol and estradiol in water by online solid phase extraction liquid chromatography-triple quadrupole mass spectrometry (T / CAQI 382-2024)" is only applicable to clean surface water and groundwater, and the instrument is expensive and has a low penetration rate.

[0010] During the use of the above-mentioned detection methods, firstly, the scope of application is incomplete. Some of them can only measure clean surface water and groundwater, and do not include seawater. Secondly, the detection limit is too high and cannot meet the current requirements for the detection of trace endocrine disruptors in environmental water bodies. Thirdly, the recovery rate is too low in actual operation. Fourth, there is insufficient discussion on blank interference, which causes troubles to actual users. Summary of the invention

[0011] The invention aims to provide a method for simultaneously detecting 16 endocrine disruptors including trace amounts of bisphenol A, 4-tert-butylphenol, 4-butylphenol, 4-pentylphenol, 4-hexylphenol, 4-tert-octylphenol, 4-heptylphenol, 4-nonylphenol, 4-n-octylphenol, 4-n-nonylphenol, estriol, diethylstilbestrol, 17β-estradiol, 17α-ethinylestradiol, estrone and hexylestradiol in various environmental water bodies such as surface water, groundwater, sewage and seawater, with a low detection limit.

[0012] To achieve the above object, the technical solution adopted by the present invention is as follows: a method for simultaneously detecting 16 endocrine disruptors in environmental water, comprising the following steps:

[0013] Step 1: Sample collection and storage

[0014] Collect water samples in clean brown glass bottles, adjust the pH to between 1 and 2, and store at 4°C away from light;

[0015] Step 2: Sample pretreatment

[0016] Activate the HLB solid phase extraction column with 5 mL of n-hexane, 5 mL of dichloromethane, 5 mL of methanol and 10 mL of experimental water in sequence; measure the volume of all collected samples with a measuring cylinder and pass them through the solid phase extraction column at a flow rate of 10 mL / min; after the loading is completed, rinse the measuring cylinder and sample bottle with 10 mL of methanol in sequence, add 30 mL of experimental water, continue to load the solid phase extraction column, and then rinse the solid phase extraction column with 10 mL of experimental water; after the elution is completed, blow dry the solid phase extraction column with nitrogen, and then elute with 1.5 mL of methanol and 5 mL of dichloromethane in sequence at a flow rate of 1 mL / min to 2 mL / min, and collect the methanol eluate and the dichloromethane eluate into a concentration tube respectively;

[0017] At 35°C, concentrate the dichloromethane elution with liquid nitrogen to 0.3mL-0.4mL, then transfer to the methanol elution, and wash the concentrator tube with methanol twice and transfer it together; continue to concentrate with nitrogen to 0.5mL-0.8mL, add internal standard solution, make up to 1mL with methanol, vortex mix, pass through a glass fiber or nylon 0.22μm filter membrane, and then put it into a brown injection bottle, store at 4°C for testing;

[0018] Step 3: Establishment of standard curve

[0019] Prepare standard working solutions with at least 5 concentration points. The reference concentrations of bisphenol A, 4-butylphenol, 4-pentylphenol, 4-hexylphenol, 4-tert-octylphenol, 4-heptylphenol, 4-n-octylphenol, 4-n-nonylphenol, estriol, diethylstilbestrol, 17β-estradiol, 17α-ethinylestradiol, estrone, and hexylestradiol are 0.5 μg / L, 1 μg / L, 2 μg / L, 4 μg / L, 8 μg / L, 20 μg / L, and 50 μg / L, respectively. The reference concentration of 4-tert-butylphenol is 2 .5μg / L, 5μg / L, 10μg / L, 20μg / L, 40μg / L, 100μg / L, 250μg / L, the reference concentrations of 4-nonylphenol (isomer mixture) were 1μg / L, 2μg / L, 4μg / L, 8μg / L, 16μg / L, 40μg / L, 100μg / L, respectively; the internal standard concentration was 20μg / L, and a standard curve was established with the concentration ratio of the target compound to the corresponding internal standard as the abscissa and the ratio of its corresponding peak area to the internal standard peak area as the ordinate;

[0020] Step 4: Determine the concentration of endocrine disruptors in water samples using high performance liquid chromatography-tandem mass spectrometry, where:

[0021] Liquid chromatography conditions were as follows: liquid chromatography column: C18 reverse phase chromatography column, 3.0 mm*150 mm, 2.7 μm; mobile phase A: 0.1 mmol / L ammonium fluoride aqueous solution; mobile phase B: 0.1 mmol / L ammonium fluoride methanol solution; mobile phase gradient elution program: 0 min, 40% mobile phase A, 60% mobile phase B; 4 min, 5% mobile phase A, 95% mobile phase B; 10 min, 5% mobile phase A, 95% mobile phase B; 10.10 min, 40% mobile phase A, 60% mobile phase B; 12 min, 40% mobile phase A, 60% mobile phase B; running time: 12 min; flow rate: 0.4 mL / min; column temperature: 40 °C; injection volume: 2 μL;

[0022] The mass spectrometry conditions were as follows: ion source: electrospray ion source (ESI), negative ion mode; monitoring mode: multiple reaction monitoring (MRM); drying gas temperature: 275°C; drying gas flow rate: 6 L / min; sheath gas temperature: 350°C; sheath gas flow rate: 11 L / min; nebulizer pressure: 40 psi; capillary voltage: 3500 V.

[0023] Preferably, in step one, the water sample is one of surface water, groundwater, sewage and seawater.

[0024] Furthermore, the detection method also includes a blank check: the blank check concentration multiple of the methanol reagent is not less than 15 times, and the blank check concentration multiple of the dichloromethane reagent is not less than 5 times; a glass syringe, glass fiber or nylon material 0.22μm needle filter is used to filter the concentrated sample.

[0025] Furthermore, a delay column is added between the liquid chromatography solvent mixing pump and the injector to eliminate the background interference of the instrument system. The delay column is a C18 reverse phase chromatography column with a filler particle size of 5 μm, a column length of 50 mm, and an inner diameter of 4.6 mm.

[0026] Preferably, in step 2, the internal standard solution has a concentration of 1 mg / L and is prepared by diluting the volume with methanol.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] 1) By optimizing the type of liquid chromatography mobile phase and the concentration of ammonium fluoride, the response value of the target compound was increased, thereby reducing the detection limit.

[0029] 2) By changing the pretreatment process: the methanol eluate and the dichloromethane eluate are collected separately, the dichloromethane eluate is concentrated first, and then the methanol eluate is added to continue concentrating, thereby simplifying the solvent conversion step, reducing the loss of low-boiling point components 4-tert-butylphenol and 4-butylphenol in the solvent conversion and concentration stages, improving the recovery rate, and shortening the solvent conversion and concentration time; adding a bottle washing step after the sample loading is completed solves the problem that 4-nonylphenol, 4-n-octylphenol, 4-n-nonylphenol and other highly hydrophobic carbon long-chain alkylphenols are easily adsorbed on the bottle wall, resulting in a low recovery rate.

[0030] 3) Through technical blank testing of mainstream reagents and filter membranes, it is preferred to use cleaned glass fiber filter membranes or nylon filter membranes, providing a selection reference for analysts in practical applications and reducing test blank interference. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 It is the standard curve graph of 16 endocrine disruptors;

[0032] Figure 2 It is the MRM chromatogram of the endocrine disruptors (internal standards) with sequence numbers 1 to 4 in the detection injection;

[0033] Figure 3 It is the MRM chromatogram of endocrine disruptors (internal standards) with sequence numbers 5-8 in the detection injection;

[0034] Figure 4 It is the MRM chromatogram of the endocrine disruptors (internal standards) with sequence numbers 9-12 in the detection injection;

[0035] Figure 5 It is the MRM chromatogram of the endocrine disruptors (internal standards) with sequence numbers 13-16 in the detection injection;

[0036] Figure 6 It is the MRM chromatogram of the endocrine disruptors (internal standards) with sequence numbers 17-20 in the detection injection;

[0037] Figure 7 It is the MRM chromatogram of the endocrine disrupting chemicals (internal standards) detected in the injection numbers 21-25. DETAILED DESCRIPTION

[0038] The present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0039] 1. Reagents and Materials

[0040] Unless otherwise specified, all reagents of analytical grade or above that meet national standards were used in the analysis, and the experimental water was pure water that did not contain the target compound.

[0041] (1) Reagents

[0042] Sodium thiosulfate (Na 2 S 2 O 3 ): Superior purity.

[0043] High purity nitrogen: >99.999%.

[0044] Hexane (C 6 H 14 ): Pesticide residue level.

[0045] Dichloromethane (CH 2 Cl 2 ): Pesticide residue level.

[0046] Methanol (CH 3 OH): Pesticide residue level

[0047] Acetone (CH 3 COCH 3 ): Pesticide residue level.

[0048] Hydrochloric acid: high purity, ρ=1.19 g / mL.

[0049] Hydrochloric acid solution: Measure 100 mL of hydrochloric acid, slowly add it into 100 mL of experimental water, and mix well.

[0050] Ammonium fluoride: >99.99%.

[0051] Ammonium fluoride aqueous solution I: ρ = 1 mol / L; weigh 3.70 g of ammonium fluoride, add it to 100 mL of experimental water, and mix well.

[0052] Ammonium fluoride aqueous solution II: ρ = 0.1 mmol / L; take 50 μL of ammonium fluoride aqueous solution I, add it to 500 mL of experimental water, and mix well.

[0053] Ammonium fluoride methanol solution: ρ = 0.1 mmol / L; take 50 μL of ammonium fluoride aqueous solution I, add it to 500 mL of methanol, and mix well.

[0054] Endocrine disruptor standard stock solution: ρ = 100 mg / L, purchased from commercial stores, stored below -20°C in the dark or stored according to the instructions, returned to room temperature before use, and shaken well.

[0055] Intermediate working solution of endocrine disruptors: take a certain amount of standard stock solution of endocrine disruptors with a microsyringe, dilute it with methanol, and prepare the intermediate working solution so that the concentration of bisphenol A, 4-butylphenol, 4-pentylphenol, 4-hexylphenol, 4-tert-octylphenol, 4-heptylphenol, 4-n-octylphenol, 4-n-nonylphenol, estriol, diethylstilbestrol, 17β-estradiol, 17α-ethinylestradiol, estrone, and hexylestradiol is 10 mg / L, the concentration of 4-tert-butylphenol is 50 mg / L, and the concentration of 4-nonylphenol (isomer mixture) is 20 mg / L.

[0056] Standard working solution of endocrine disruptors: Take a certain amount of standard intermediate working solution of endocrine disruptors with a microsyringe, dilute it with methanol, and prepare a 1mg / L standard working solution (including 5mg / L of 4-tert-butylphenol and 2mg / L of 4-nonylphenol). Store in a sealed container at 4℃ away from light, with a shelf life of three months.

[0057] Internal standard stock solution: ρ = 100 mg / L. The internal standards are estriol-d 3 , bisphenol A 16 , diethylstilbestrol-d 8 , 17α-ethinylestradiol-d 4 , 17β-estradiol-d 2 , estrone-d 2 , 4-tert-octylphenol- 13 C 6 , hexaneestrol-d 6 , 4-n-nonylphenol-d 4 , you can directly purchase a certified standard solution, or you can prepare it using a standard substance. The stock solution should be stored below 4°C away from light or refer to the manufacturer's product instructions. It should be returned to room temperature and shaken before use.

[0058] Internal standard solution: ρ = 1 mg / L (reference concentration). Dilute the internal standard stock solution with methanol. Refrigerate the internal standard solution at 4°C and keep it away from light. The shelf life is three months. Return to room temperature and shake well before use.

[0059] Table 1 Names and CAS numbers of 16 endocrine disruptors and 9 quantitative internal standards

[0060]

[0061] (2) Materials

[0062] Syringe filter: 0.22μm pore size, glass fiber or nylon material.

[0063] Sampling bottle: 250mL, brown narrow-mouth glass bottle with glass stopper. Wash once with laboratory water before use, and rinse thoroughly with methanol or acetone 2 to 3 times. Avoid using surfactant detergents to clean the sample bottle.

[0064] Solid phase extraction cartridge: HLB (6 mL, 200 mg).

[0065] Microsyringes: 10μL, 50μL and 100μL.

[0066] Injection bottle: 2mL, brown glass bottle.

[0067] Experimental syringe: 5mL, made of glass.

[0068] Measuring cylinder: 50mL, 100mL, 250mL.

[0069] 2. Instruments and equipment

[0070] High performance liquid chromatography-tandem mass spectrometry: equipped with an electrospray ionization source (ESI) with gradient elution and multiple reaction monitoring capabilities.

[0071] Chromatographic column: C18 reverse phase column with a filler particle size of 2.7 μm, a column length of 150 mm, and an inner diameter of 3.0 mm, or other chromatographic columns with similar performance.

[0072] Delay column: C18 reverse phase column with a filler particle size of 5 μm, a column length of 50 mm, and an inner diameter of 4.6 mm.

[0073] Solid phase extraction device: manual or automatic, with adjustable flow rate.

[0074] Nitrogen blowing concentrator.

[0075] 3. Sample collection and preservation

[0076] After collecting the sample in a 250mL brown glass bottle that has been cleaned with pure water and methanol, adjust the pH to between 1-2 with 6mol / L hydrochloric acid. Store at 4°C away from light and pre-treat within 7 days. If the sample contains residual chlorine (the residual chlorine in the sample can be detected on-site using a residual chlorine meter or residual chlorine test paper), sodium thiosulfate needs to be added to the sample to make the concentration of sodium thiosulfate in the sample 80mg / L. Collect at least one full-procedure blank sample for each batch of samples. Fill the clean sampling bottle with experimental water, bring it to the sampling site, open and seal it at the same time as the sampling sample bottle, and then transport it back to the laboratory with the sample.

[0077] 4. Sample pretreatment

[0078] Activate the HLB solid phase extraction column with 5mL of n-hexane, 5mL of dichloromethane, 5mL of methanol and 10mL of experimental water in turn. During the activation process, the surface of the filler in the solid phase extraction column does not expose the liquid surface. After measuring the volume of all the collected samples with a measuring cylinder, pass them through the solid phase extraction column at a flow rate of 10mL / min. After the loading is completed, rinse the measuring cylinder and sample bottle with 10mL of methanol in turn, add 30mL of experimental water, continue to load the solid phase extraction column, and then rinse the solid phase extraction column with 10mL of experimental water. After the elution is completed, blow dry the solid phase extraction column with nitrogen, and then elute with 1.5mL of methanol and 5mL of dichloromethane in turn at a flow rate of 1mL / min to 2mL / min, and collect the methanol eluate and dichloromethane eluate into a concentration tube respectively.

[0079] Concentrate the dichloromethane eluate to 0.3mL-0.4mL by nitrogen blowing at 35℃, then transfer the methanol eluate into the tube with a Pasteur glass pipette, and wash the concentrator tube twice with about 1mL of methanol and transfer it together. Continue to concentrate the eluate to 0.5mL-0.8mL by nitrogen blowing, add the internal standard solution, make up to 1mL with methanol, vortex mix, pass through a 0.22μm filter membrane made of glass fiber or nylon, and place it in a 2mL brown injection bottle, store at 4℃ for testing.

[0080] 5. Sources and elimination of blank interference

[0081] Bisphenol A and 4-nonylphenol are easily introduced into consumables, reagents, and pretreatment instrument systems. Avoid using plastic products. Perform a blank check before testing. The blank check concentration multiple of methanol reagent should be no less than 15 times, and the blank check concentration multiple of dichloromethane reagent should be no less than 5 times. Use a glass syringe, glass fiber or nylon 0.22μm needle filter to filter the concentrated sample, and wash it with methanol before use if necessary. A delay column needs to be added between the liquid chromatography solvent mixing pump and the injector to eliminate background interference from the instrument system.

[0082] 6. Establishment of standard curve

[0083] Prepare standard working solutions of at least 5 concentration points. The reference concentrations of bisphenol A, 4-butylphenol, 4-pentylphenol, 4-hexylphenol, 4-tert-octylphenol, 4-heptylphenol, 4-n-octylphenol, 4-n-nonylphenol, estriol, diethylstilbestrol, 17β-estradiol, 17α-ethinylestradiol, estrone, and hexylestradiol are 0.5 μg / L, 1 μg / L, 2 μg / L, 4 μg / L, 8 μg / L, respectively. The reference concentrations of 4-tert-butylphenol were 2.5 μg / L, 5 μg / L, 10 μg / L, 20 μg / L, 40 μg / L, 100 μg / L, 250 μg / L, and the reference concentrations of 4-nonylphenol (isomer mixture) were 1 μg / L, 2 μg / L, 4 μg / L, 8 μg / L, 16 μg / L, 40 μg / L, and 100 μg / L. The internal standard concentration was 20 μg / L, and the standard curve was established with the concentration ratio of the target compound to the corresponding internal standard as the horizontal axis and the ratio of its corresponding peak area to the internal standard peak area as the vertical axis.

[0084] The standard curves of 16 endocrine disruptors including bisphenol A, 4-tert-butylphenol, 4-butylphenol, 4-pentylphenol, 4-hexylphenol, 4-tert-octylphenol, 4-heptylphenol, 4-nonylphenol, 4-n-octylphenol, 4-n-nonylphenol, estriol, diethylstilbestrol, 17β-estradiol, 17α-ethinylestradiol, estrone, and hexanestradiol are shown in Figure 2. Figure 1 As shown, it can be seen that the linear correlation coefficient of each standard curve equation is >0.995, which meets the quantitative requirements.

[0085] 7. Test the sample liquid on the machine

[0086] The liquid chromatography conditions were:

[0087] Liquid chromatography column: C18 column, 3.0mm*150mm, 2.7μm;

[0088] Mobile phase A: 0.1mmol / L ammonium fluoride aqueous solution;

[0089] Mobile phase B: 0.1mmol / L ammonium fluoride methanol solution;

[0090] Mobile phase gradient elution program: 0 min, 40% mobile phase A, 60% mobile phase B; 4 min, 5% mobile phase A, 95% mobile phase B; 10 min, 5% mobile phase A, 95% mobile phase B; 10.10 min, 40% mobile phase A, 60% mobile phase B; 12 min, 40% mobile phase A, 60% mobile phase B;

[0091] Running time: 11 minutes;

[0092] Flow rate: 0.4 mL / min;

[0093] Column temperature: 40°C;

[0094] Injection volume: 2 μL;

[0095] The mass spectrometry conditions were:

[0096] Ion source: electrospray ionization (ESI), negative ion mode;

[0097] Monitoring method: multiple reaction monitoring (MRM);

[0098] Drying gas temperature: 275℃;

[0099] Drying gas flow: 6L / min;

[0100] Atomizer pressure: 40psi;

[0101] Capillary voltage: 3500V;

[0102] Sheath gas temperature: 350℃;

[0103] Sheath gas flow rate: 11L / min;

[0104] The multiple reaction monitoring (MRM) parameters of the target compounds are shown in Table 2:

[0105] Table 2 Mass spectrometry parameters of 16 endocrine disruptors and quantitative internal standards

[0106]

[0107]

[0108] The MRM spectra of 16 endocrine disruptors and 9 internal standards (20 μg / L) were obtained according to the above method. Figure 2 to Figure 7 As shown. Figure 2 to Figure 7 As shown in the figure, the separation of 16 endocrine disruptors is good, and the peaks are symmetrical and sharp. Using ion pair quantification, 16 endocrine disruptors can be measured simultaneously.

[0109] When the sampling volume is 250mL, the fixed volume is 1.0mL, and the injection volume is 2μL, the detection limit of the method is 0.1ng / L~3ng / L, and the lower limit of determination is 0.4ng / L~12ng / L. See Table 3 for details.

[0110] Table 3 Method detection limit and determination lower limit

[0111]

[0112]

[0113] 8. Precision and accuracy

[0114] Six repeated measurements were carried out on spiked samples of pure water, surface water, groundwater, sewage and seawater with spiked concentrations of 0.025 μg / L, 0.10 μg / L and 0.25 μg / L, respectively: the spiked recoveries ranged from 72.1% to 120%, 71.0% to 128%, 71.9% to 126%, 81.0% to 130% and 70.0% to 111%, respectively, and the within-laboratory relative standard deviations were 0.9% to 9.8%, 1.1% to 19%, 0.8% to 9.8%, 0.8% to 17% and 0.5% to 9.2%, respectively.

[0115] The above description is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by any technician familiar with the technical field within the technical scope disclosed by the present invention and within the spirit and principle of the present invention should be covered by the protection scope of the present invention.

Claims

1. A method for simultaneously detecting 16 endocrine disruptors in environmental water, characterized in that: The following steps are involved: Step 1: Sample collection and storage Collect water samples in clean brown glass bottles, adjust the pH to between 1 and 2, and store at 4°C away from light; Step 2: Sample pretreatment Activate the HLB solid phase extraction column with 5 mL of n-hexane, 5 mL of dichloromethane, 5 mL of methanol and 10 mL of experimental water in turn; measure the volume of all collected samples with a measuring cylinder and pass them through the solid phase extraction column at a flow rate of 10 mL / min; after the loading is completed, rinse the measuring cylinder and sample bottle with 10 mL of methanol in turn, add 30 mL of experimental water, continue to load the solid phase extraction column, and then rinse the solid phase extraction column with 10 mL of experimental water; after the elution is completed, blow dry the solid phase extraction column with nitrogen, and then elute with 1.5 mL of methanol and 5 mL of dichloromethane in turn at a flow rate of 1 mL / min~2 mL / min, and collect the methanol eluate and the dichloromethane eluate into a concentration tube respectively; At 35°C, concentrate the dichloromethane elution with liquid nitrogen to 0.3mL~0.4mL, then transfer to the methanol eluent, and wash the concentrator tube with methanol twice and transfer it together; continue to concentrate with nitrogen to 0.5mL~0.8mL, add internal standard solution, make up to 1mL with methanol, vortex mix, pass through a glass fiber or nylon 0.22μm filter membrane, and then put it into a brown injection bottle, store at 4°C for testing; Step 3: Establishment of standard curve Prepare standard working solutions with at least 5 concentration points. The reference concentrations of bisphenol A, 4-butylphenol, 4-pentylphenol, 4-hexylphenol, 4-tert-octylphenol, 4-heptylphenol, 4-n-octylphenol, 4-n-nonylphenol, estriol, diethylstilbestrol, 17β-estradiol, 17α-ethinylestradiol, estrone, and hexylestradiol are 0.5 μg / L, 1 μg / L, 2 μg / L, 4 μg / L, 8 μg / L, 20 μg / L, and 50 μg / L, respectively. The reference concentration of 4-tert-butylphenol is 2 .5μg / L, 5μg / L, 10μg / L, 20μg / L, 40μg / L, 100μg / L, 250μg / L, the reference concentrations of 4-nonylphenol (isomer mixture) were 1μg / L, 2μg / L, 4μg / L, 8μg / L, 16μg / L, 40μg / L, 100μg / L; the internal standard concentration was 20μg / L, and the standard curve was established with the concentration ratio of the target compound to the corresponding internal standard as the abscissa and the ratio of its corresponding peak area to the internal standard peak area as the ordinate; Step 4: Determine the concentration of endocrine disruptors in water samples using high performance liquid chromatography-tandem mass spectrometry, where: Liquid chromatography conditions were as follows: liquid chromatography column: C18 reverse phase column, 3.0 mm*150 mm, 2.7 μm; mobile phase A: 0.1 mmol / L ammonium fluoride aqueous solution; mobile phase B: 0.1 mmol / L ammonium fluoride methanol solution; mobile phase gradient elution program: 0 min, 40% mobile phase A, 60% mobile phase B; 4 min, 5% mobile phase A, 95% mobile phase B; 10 min, 5% mobile phase A, 95% mobile phase B; 10.10 min, 40% mobile phase A, 60% mobile phase B; 12 min, 40% mobile phase A, 60% mobile phase B; running time: 12 min; flow rate: 0.4 mL / min; Column temperature: 40°C; injection volume: 2 μL; The mass spectrometry conditions were as follows: ion source: electrospray ion source ESI, negative ion mode; monitoring mode: multiple reaction monitoring MRM; drying gas temperature: 275°C; drying gas flow rate: 6 L / min; sheath gas temperature: 350°C; sheath gas flow rate: 11 L / min; nebulizer pressure: 40 psi; capillary voltage: 3500 V; A delay column was added between the liquid chromatography solvent mixing pump and the injector to eliminate background interference of the instrument system. The delay column was a C18 reverse phase chromatography column with a filler particle size of 5 μm, a column length of 50 mm, and an inner diameter of 4.6 mm.

2. The method for simultaneously detecting 16 endocrine disruptors in environmental water according to claim 1, characterized in that: In step 1, the water sample is one of surface water, groundwater, sewage and seawater.

3. The method for simultaneously detecting 16 endocrine disruptors in environmental water according to claim 1, characterized in that: It also includes a blank check: the blank check concentration of methanol reagent is not less than 15 times, and the blank check concentration of dichloromethane reagent is not less than 5 times; use a glass syringe, glass fiber or nylon 0.22μm needle filter to filter the concentrated sample.

4. The method for simultaneously detecting 16 endocrine disruptors in environmental water according to claim 1, characterized in that: In step 2, the internal standard solution has a concentration of 1 mg / L and is prepared by diluting the volume with methanol.

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