Non-targeted analysis method for identifying phthalic ester endocrine disrupter in underground water based on high-resolution mass spectrum high-throughput screening

Abstract

The invention discloses a non-targeted analysis method for identifying phthalic ester endocrine disruptors in underground water based on high-resolution mass spectrum high-throughput screening. The method comprises the following steps: collecting an underground water sample; the method comprises the following steps: pre-treating a collected underground water sample to obtain a to-be-analyzed and detected sample; and carrying out phthalate analysis and detection on a sample to be analyzed and detected by using high performance liquid chromatography-tandem mass spectrometry. The invention relates to a non-targeted analysis method for high-throughput screening and identification of phthalic ester endocrine disruptors in underground water based on high-resolution mass spectrum characteristic fragment search, which is full-flow identification from sampling to analysis and identification provided for phthalic ester pollutants in an underground water environment for the first time. On the basis of solid-phase extraction and liquid chromatography-tandem high-resolution mass spectrometry technologies, the problem of cross contamination in the pretreatment and detection process can be reduced, and the accuracy rate is high.

Description

Technical Field

[0001] The present invention relates to the technical field of environmental detection, and particularly relates to a non-targeted analysis method for high-throughput screening and identification of phthalate endocrine disruptors in groundwater based on high-resolution mass spectrometry. Background Art

[0002] Phthalic acid esters (PAEs), also known as phthalates, are common plasticizers widely used in life and production. They are one of the synthetic organic compounds with the largest production volume and the widest application scope in the world. With the extensive use of PAEs, they are also commonly found in the atmosphere, water bodies, soil, etc. PAEs can interfere with the endocrine systems of humans and animals at extremely low concentrations, and some also have carcinogenic, teratogenic, and mutagenic effects. They are typical endocrine disruptors. However, the detection and management of PAEs in groundwater have not been fully established yet. Therefore, detecting their residual levels in soil is of great significance for controlling soil pollution and protecting human health.

[0003] According to literature research, there are few reports on the screening of new pollutants of phthalates in groundwater at present. For the identification of phthalates, fluorescence methods and targeted analysis techniques are more commonly used. However, problems such as the instability of fluorescent substances, easy occurrence of self-absorption and inner filter effect, and not all substances can produce fluorescence restrict the development of fluorescence methods. Similarly, the current targeted screening of phthalates only screens and identifies a limited number of species, and it is impossible to obtain a comprehensive screening of the phthalate mass spectra. In addition, there is no report on the full-process analysis of non-targeted screening from sampling to identification. Summary of the Invention

[0004] The purpose of the present invention is to provide a non-targeted analysis method for high-throughput screening and identification of phthalate endocrine disruptors in groundwater based on high-resolution mass spectrometry. The present invention uses high-performance liquid chromatography-high-resolution mass spectrometry coupling technology to perform a full-process analysis of PAEs in groundwater from sampling to identification, with high precision, simplicity, and speed, and can provide an effective path for the detection and management of PAEs.

[0005] To achieve the above purpose, the technical solution provided by the present invention is:

[0006] A non-targeted analysis method for high-throughput screening and identification of phthalate endocrine disruptors in groundwater based on high-resolution mass spectrometry, comprising the following steps:

[0007] Collect groundwater samples;

[0008] Pretreat the collected groundwater samples to obtain samples to be analyzed and detected;

[0009] The detection sample to be analyzed is screened for phthalate endocrine disruptors using high performance liquid chromatography - tandem mass spectrometry, where:

[0010] The conditions for high performance liquid chromatography are as follows:

[0011] An octadecylsilane (C18) column is used, column temperature: 35 - 45 °C; an aqueous acetonitrile solution containing 4 - 6% by volume is used as mobile phase A, and methanol is used as mobile phase B; mobile phase flow rate: 0.3 - 0.5 mL / min, elution determination time: 50 - 60 min;

[0012] The conditions for mass spectrometry are as follows:

[0013] A Triple TOF5600 time - of - flight mass spectrometer is used, the ion source is an electrospray ionization source (ESI); the ionization mode is: positive ion mode + negative ion mode;

[0014] The screening analysis method for phthalate endocrine disruptors is as follows:

[0015] The mass spectrometry data after liquid chromatography - high resolution mass spectrometry detection is compared with the characteristic fragments of PAEs. If there are 2 or more fragments in the mass spectrometry data that are the same as the characteristic fragments of PAEs, it is determined that the sample is suspected of containing phthalate substances, and the suspected screening of PAEs is completed. The mass spectrometry data of the suspected screening is analyzed and confirmed.

[0016] To optimize the above technical solution, the specific measures taken also include:

[0017] After the groundwater sample is collected, it is placed in a cold storage box and transported to the determination laboratory at 4 °C.

[0018] Furthermore, the steps for pretreatment of the collected groundwater sample are as follows:

[0019] First, it passes through a glass fiber filter membrane to remove suspended solids;

[0020] It is extracted with n - hexane, and the organic phase is collected;

[0021] It is extracted with dichloromethane under acidic conditions, and the organic phase is collected;

[0022] It is extracted with dichloromethane under alkaline conditions, and the organic phase is collected;

[0023] The collected n - hexane organic phase, dichloromethane organic phase extracted under acidic conditions, and dichloromethane organic phase extracted under alkaline conditions are combined and dehydrated; first, it is dried with a rotary evaporator, and then dried with nitrogen;

[0024] It is made up to volume, filtered, and stored.

[0025] Among them, the n-hexane extraction is as follows: Add NaCl and shake well to dissolve it in the solution, add n-hexane and shake. Gas is released during shaking; let it stand until the organic phase and the aqueous phase are completely separated;

[0026] The dichloromethane extraction under acidic conditions is as follows: Adjust the pH of the aqueous phase to 2 with HCl, add dichloromethane; repeat the above extraction steps to complete the extraction under acidic conditions;

[0027] The dichloromethane extraction under basic conditions is as follows: Adjust the pH of the aqueous phase to 11 with NaOH, add dichloromethane; repeat the above steps to complete the extraction under basic conditions.

[0028] Furthermore, in high performance liquid chromatography, an aqueous solution of acetonitrile with a volume percentage of 5% is used as mobile phase A, and methanol is used as mobile phase B; elution determination time: 55 min; mobile phase flow rate: 0.4 mL / min; the injection volume for high performance liquid chromatography-tandem mass spectrometry is: 3 - 10 μm.

[0029] Furthermore, in high performance liquid chromatography, a Waters BEH C18 column is used, with specifications: 2.1 mm × 50 mm, 2.5 μm Waters.

[0030] Furthermore, in mass spectrometry, curtain gas: 25 psi; nebulizing gas: 45 psi; heating gas: 45 psi; ion source temperature: 550 °C; ion spray voltage: 4500 V / -4500 V positive / negative ion mode; declustering voltage: 80 V / -80 V positive / negative ion mode; collision voltage: 40 V / -40 V positive / negative ion mode; collision voltage range: 20 V.

[0031] Furthermore, the characteristic fragments of the PAEs are obtained from the MSDIAL database, and fragments with a mass error within 5 mDa are considered to be the same fragment.

[0032] Furthermore, the mass spectrometry data suspected of screening is analyzed and identified using the mass spectrometry molecular annotation software SIRIUS.

[0033] Compared with the prior art, the beneficial effects of the present invention are:

[0034] In view of the problems that in current fluorescence analysis and detection, due to the instability of fluorescent substances, easy occurrence of self-absorption and inner filter effect, and the fact that not all substances can produce fluorescence, the detection accuracy is not high, and traditional targeted analysis can only identify a limited number of substances and cannot conduct a wide and comprehensive screening of all PAEs, the present invention provides a non-targeted analysis method for high-throughput screening and identification of phthalate endocrine disruptors in groundwater based on the search for characteristic fragments of high-resolution mass spectrometry. This method realizes the full-process identification of PAEs in groundwater from sampling to analysis and identification, providing technical support for the investigation and control of PAEs in the groundwater environment.

[0035] Through the collection, preservation, transportation, pretreatment, instrument detection, and analysis and identification of groundwater samples, the present invention realizes the full-process high-throughput screening of new phthalate pollutants in groundwater; based on the characteristic fragments of phthalate substances in the MSDIAL software database, by comparing with the detection results, the suspected identification of phthalate substances in groundwater is realized, and the SIRIUS software is used to further identify PAEs; based on solid-phase extraction (SPE) and liquid chromatography-tandem high-resolution mass spectrometry (LC-MS) technologies, the problem of cross-contamination in the pretreatment and detection processes can be reduced, and the accuracy is high.

[0036] The present invention is a non-targeted analysis method for high-throughput screening and identification of phthalate endocrine disruptors in groundwater based on the search for characteristic fragments of high-resolution mass spectrometry. It is the first solution proposed for phthalate pollutants in the groundwater environment, and more suitable extraction solvents and extraction methods are selected according to the different medium characteristics of groundwater. The detection method of the present invention can identify and screen phthalate compounds from high-resolution full-scan data, and the screening targets are no longer limited to the more than twenty kinds that can be detected by targeted analysis and detection methods. Brief Description of the Drawings

[0037] Figure 1 : Schematic flow chart of the non-targeted analysis method for high-throughput screening and identification of phthalate endocrine disruptors in groundwater based on high-resolution mass spectrometry of the present invention. Detailed Embodiments

[0038] The above content of the present invention will be further described in detail below in the form of specific embodiments, but it should not be understood that the scope of the above subject matter of the present invention is limited to the following embodiments. All technologies implemented based on the above content of the present invention belong to the scope of the present invention.

[0039] The experimental methods used in the following embodiments are all conventional methods unless otherwise specified. The reagents, methods, and equipment used are all conventional reagents, methods, and equipment in the technical field unless otherwise specified.

[0040] For the sake of brevity, only some numerical values and ranges of optional items are specifically disclosed in this article. However, any lower limit can be combined with any upper limit to form a range not explicitly recorded; and any lower limit can be combined with other lower limits to form a range not explicitly recorded. Similarly, any upper limit can be combined with any other upper limit to form a range not explicitly recorded; the optional items within the range of optional items can also be combined arbitrarily.

[0041] The present invention provides a non-targeted analysis method for high-throughput screening and identification of phthalate endocrine disruptors in groundwater based on high-resolution mass spectrometry, including the following steps:

[0042] Collect groundwater samples;

[0043] Pretreat the collected groundwater samples to obtain samples to be analyzed and detected;

[0044] Use high-performance liquid chromatography-tandem mass spectrometry to screen and analyze phthalate endocrine disruptors in the samples to be analyzed and detected, where:

[0045] The conditions of high-performance liquid chromatography are as follows:

[0046] Use a C18 chromatographic column, column temperature: 35 - 45 °C; use an aqueous acetonitrile solution containing 4 - 6% by volume as mobile phase A, and methanol as mobile phase B; mobile phase flow rate: 0.3 - 0.5 mL / min, elution determination time: 50 - 60 min;

[0047] The conditions of mass spectrometry are as follows:

[0048] Use a Triple TOF5600 time-of-flight mass spectrometer, and the ion source is an electrospray ionization source ESI; the ionization mode is: positive ion mode + negative ion mode;

[0049] The screening and analysis method for phthalate endocrine disruptors is as follows:

[0050] Compare the mass spectrometry data after liquid phase-high resolution mass spectrometry detection with the characteristic fragments of PAEs. If there are 2 or more fragments in the mass spectrometry data that are the same as the characteristic fragments of PAEs, it is determined that the sample is suspected to contain phthalate substances, and the suspected screening of PAEs is completed. Analyze and confirm the mass spectrometry data of the suspected screening.

[0051] After the groundwater samples are collected, they are placed in a refrigerated box and transported to the determination laboratory at 4 °C.

[0052] In some embodiments, the steps for pretreating the collected groundwater samples are as follows:

[0053] First, pass through a glass fiber filter membrane to remove suspended solids;

[0054] Extract with n-hexane and collect the organic phase;

[0055] Extract with dichloromethane under acidic conditions and collect the organic phase;

[0056] Extract with dichloromethane under basic conditions and collect the organic phase;

[0057] Combine the collected n - hexane organic phase, the dichloromethane organic phase extracted under acidic conditions, and the dichloromethane organic phase extracted under basic conditions and remove water; first dry with a rotary evaporator and then dry with nitrogen;

[0058] Make up the volume, filter, and store.

[0059] Among them, the n - hexane extraction is as follows: Add NaCl and shake well to dissolve it in the solution, add n - hexane and shake. Gas is released during shaking; let it stand until the organic phase and the aqueous phase are completely separated;

[0060] The dichloromethane extraction under acidic conditions is as follows: Adjust the pH of the aqueous phase to 2 with HCl, add dichloromethane; repeat the above extraction steps to complete the extraction under acidic conditions;

[0061] The dichloromethane extraction under basic conditions is as follows: Adjust the pH of the aqueous phase to 11 with NaOH, add dichloromethane; repeat the above steps to complete the extraction under basic conditions.

[0062] In some embodiments, in high - performance liquid chromatography, the mobile - phase flow rate is 0.3 - 0.5 mL / min; the injection volume for high - performance liquid chromatography - tandem mass spectrometry is 3 - 10 μm.

[0063] In some embodiments, in high - performance liquid chromatography, the chromatographic column uses a Waters BEH C18 column, with specifications: 2.1 mm×50 mm, 2.5 μm Waters.

[0064] In some embodiments, in mass spectrometry, curtain gas: 25 psi; nebulizing gas: 45 psi; heating gas: 45 psi; ion - source temperature: 550 °C; ion - spray voltage: 4500 V / -4500 V in positive / negative ion mode; declustering voltage: 80 V / -80 V in positive / negative ion mode; collision voltage: 40 V / -40 V in positive / negative ion mode; collision - voltage range: 20 V.

[0065] In some embodiments, the characteristic fragments of PAEs are obtained from the MSDIAL database, and fragments with a mass error within 5 mDa are considered to be the same fragment.

[0066] In some embodiments, the mass - spectrometry data suspected of screening are analyzed and identified using the mass - spectrometry molecular annotation software SIRIUS.

[0067] The technical solutions of the present invention will be further described in detail below in conjunction with specific embodiments:

[0068] Example

[0069] A non-targeted analysis method for high-throughput screening and identification of phthalate endocrine disruptors in groundwater based on the search for characteristic fragments of high-resolution mass spectrometry, comprising the following steps:

[0070] (1) Sample collection, preservation and transportation:

[0071] Select representative points in the plot for sample collection. For water wells, use a DP-50 diesel core drill rig to drill wells. After the monitoring well drilling is completed, install a hard PVC well pipe with a bottom seal and an inner diameter of 63 mm. After the monitoring well is built, it is necessary to backfill the outside of the screen pipe with clean quartz sand with a particle size ≥ 0.25 mm as the water filter layer. The quartz sand is backfilled to the groundwater level, and then impermeable bentonite is backfilled on its upper part. Finally, use cement mortar to backfill to the natural ground level at the wellhead. After the groundwater well is built, wash the well and then collect the water sample using a bailer. Before sampling, the sampling equipment must be thoroughly cleaned and disinfected to avoid cross-contamination. The interval between well washing and sampling of the groundwater well should not exceed 2 h. After the sample is collected, send the sample to the designated laboratory within the specified time according to the sampling plan. The groundwater sample should be stored in a sample bottle at 4 °C and transported in a refrigerated box. Before transportation, it should be packaged and the label should be firmly attached to the outside of the pipe, indicating the project name, borehole number, sampling number, depth, date, pipe number and soil type.

[0072] (2) Pretreatment in the laboratory:

[0073] Before extracting the water sample, first pass it through a glass fiber filter membrane to remove suspended solids. After filtration, weigh about 500 mL of the water sample, adjust the pH to 7 with hydrochloric acid, pour it into a 1 L separating funnel, and add an internal standard. Add 60 g of NaCl and shake well to dissolve it in the solution. Add 80 mL of n-hexane to the separating funnel and shake for 5 - 10 minutes, releasing gas during shaking. Let it stand until the organic phase and the aqueous phase are completely separated, and then open the knob to collect the organic phase. Secondly, adjust the pH of the extract to 2 with HCl and add 80 mL of dichloromethane; repeat the above extraction steps to complete the extraction under acidic conditions. Then, adjust the pH to 11 with NaOH and add 80 mL of dichloromethane. Repeat the above steps to complete the extraction under alkaline conditions. Combine the three collected organic phases, use anhydrous sodium sulfate to remove water, evaporate to almost dryness with a rotary evaporator, and then dry with nitrogen. Finally, add n-hexane to make the volume up to 1 mL. Pass through a 0.45 μm PTFE filter membrane and transfer it to a chromatographic injection vial, and store it in a -20 °C refrigerator for later measurement.

[0074] (3) Instrumentation analysis:

[0075] The phthalate analysis and detection of the sample were carried out using high performance liquid chromatography - tandem mass spectrometry. The liquid chromatography system was the UltiMate 3000 Series, and the high - resolution mass spectrometry system was the Triple TOF 5600 time - of - flight mass spectrometer. For liquid chromatography, the chromatographic column was a Waters BEH C18 column, and the column temperature was set at 40 °C. Phase A was an aqueous solution (containing 5% acetonitrile by volume), and Phase B was methanol. The flow rate of the mobile phase was 0.4 mL / min, the injection volume was 5 μm, and the gradient elution was controlled within 55 min and a two - phase mobile phase was used for gradient elution to achieve better separation. For high - resolution mass spectrometry, the ionization source was an electrospray ionization (ESI) source, the spray voltage was at - 4500 V / +4500 V, and the ion source temperature was 550 °C. The ion spray voltage was at 4500 V / -4500 V; the declustering voltage was at 80 V / -80 V; the collision voltage was at 40 V / -40 V; the collision voltage range was 20 V.

[0076] (4) Suspected screening:

[0077] The mass spectrometry data of the sample after liquid - high - resolution mass spectrometry detection was compared with the characteristic fragments of PAEs. If there were 2 or more fragments in the mass spectrometry data of the sample that were the same as the characteristic fragments of PAEs, it was determined that the sample was suspected of containing phthalate substances, and the suspected screening of PAEs was completed.

[0078] Table 1: Characteristic fragments of PAEs

[0079]

[0080] (5) Analysis and identification:

[0081] For the mass spectrometry data that met the suspected screening, the SIRIUS software was used to further identify PAEs.

[0082] The above - mentioned are only the preferred embodiments of the present invention and do not impose any form of limitation on the present invention. Any person skilled in the relevant art, without departing from the scope of the technical solution of the present invention and based on the technical essence of the present invention, any simple modification, equivalent replacement, and improvement made to the above embodiments still fall within the protection scope of the technical solution of the present invention.

Claims

1. A non-targeted analysis method for high-resolution mass spectrometry based high-throughput screening and identification of phthalate endocrine disruptors in groundwater, characterized in that: The following steps are involved: Conduct groundwater sampling; Pre-processing the collected groundwater samples to obtain samples to be analyzed; The samples to be analyzed were screened for phthalate endocrine disruptors using high performance liquid chromatography-tandem mass spectrometry, where: The conditions for HPLC were: A C18 chromatographic column was used, the column temperature was 35-45°C; an acetonitrile aqueous solution containing 4-6% by volume was used as mobile phase A, and methanol was used as mobile phase B; the mobile phase flow rate was 0.3-0.5 mL / min, and the elution measurement time was 50-60 min; The conditions for mass spectrometry were: A Triple TOF5600 time-of-flight mass spectrometer was used, and the ion source was an electrospray source ESI; the ionization mode was: positive ion mode + negative ion mode; The screening analysis method for phthalate endocrine disruptors is: The mass spectrometry data after liquid chromatography-high-resolution mass spectrometry detection is compared with the characteristic fragments of PAEs. If two or more fragments in the mass spectrometry data are the same as the characteristic fragments of PAEs, it is determined that the sample is suspected of containing phthalates, and the suspected screening of PAEs is completed. The mass spectrometry data of the suspected screening is analyzed and confirmed.

2. According to claim 1, a non-targeted analysis method for high-resolution mass spectrometry-based high-throughput screening and identification of phthalate endocrine disruptors in groundwater, characterized in that: After groundwater samples were collected, they were placed in a refrigerator at 4°C and transported to the testing laboratory.

3. The non-targeted analysis method for high-resolution mass spectrometry-based high-throughput screening and identification of phthalate endocrine disruptors in groundwater according to claim 1, characterized in that: The steps for pre-treatment of collected groundwater samples are as follows: First pass through a glass fiber filter to remove suspended matter; Extract with n-hexane and collect the organic phase; Extract with dichloromethane under acidic conditions and collect the organic phase; Extract with dichloromethane under alkaline conditions and collect the organic phase; The collected n-hexane organic phase, the dichloromethane organic phase extracted under acidic conditions, and the dichloromethane organic phase extracted under alkaline conditions are combined to remove water; first dried using a rotary evaporator, and then dried using nitrogen; Set volume, filter and save.

4. The non-targeted analysis method for high-resolution mass spectrometry-based high-throughput screening and identification of phthalate endocrine disruptors in groundwater according to claim 3, characterized in that: The n-hexane extraction is as follows: add NaCl to the sample solution and shake it to dissolve it in the solution, add n-hexane and shake it, and release gas during shaking; let it stand until the organic phase and the aqueous phase are completely separated; The dichloromethane extraction under acidic conditions is as follows: adjusting the pH of the aqueous phase to 2 with HCl, adding dichloromethane; repeating the above extraction steps to complete the extraction under acidic conditions; The dichloromethane extraction under alkaline conditions is as follows: adjusting the pH of the aqueous phase to 11 with NaOH, adding dichloromethane; and repeating the above steps to complete the extraction under alkaline conditions.

5. The non-targeted analysis method for high-resolution mass spectrometry-based high-throughput screening and identification of phthalate endocrine disruptors in groundwater according to claim 1, characterized in that: In the high performance liquid chromatography, an aqueous solution containing 5% by volume of acetonitrile was used as mobile phase A, and methanol was used as mobile phase B; the elution measurement time was 55 min. 6.1 The non-targeted analysis method for high-resolution mass spectrometry-based high-throughput screening and identification of phthalate endocrine disruptors in groundwater according to claim 1, characterized in that: In high performance liquid chromatography, the mobile phase flow rate is: 0.4 mL / min; the injection volume of high performance liquid chromatography-tandem mass spectrometry is: 3 to 10 μm.

7. The non-targeted analysis method for high-resolution mass spectrometry-based high-throughput screening and identification of phthalate endocrine disruptors in groundwater according to claim 1, characterized in that: In the high performance liquid chromatography, the chromatographic column used was a Waters BEH C18 column with the specifications of 2.1 mm×50 mm, 2.5 μm Waters.

8. The non-targeted analysis method for high-resolution mass spectrometry-based high-throughput screening and identification of phthalate endocrine disruptors in groundwater according to claim 1, characterized in that: In mass spectrometry, curtain gas: 25 psi; Atomizing gas: 45psi; Heating gas: 45psi; ion source temperature: 550℃; ion spray voltage: 4500V / -4500V positive / negative ion mode; declustering voltage: 80V / -80V positive / negative ion mode; collision voltage: 40V / -40V positive / negative ion mode; collision voltage range: 20V.

9. The non-targeted analysis method for high-resolution mass spectrometry-based high-throughput screening and identification of phthalate endocrine disruptors in groundwater according to claim 1, characterized in that: The PAEs characteristic fragments were obtained from the MSDIAL database, and fragments with a mass error within 5 mDa were considered to be the same fragment.

10. The non-targeted analysis method for high-resolution mass spectrometry-based high-throughput screening and identification of phthalate endocrine disruptors in groundwater according to claim 1, characterized in that: The mass spectrometry data of suspected screening were analyzed and identified using the mass spectrometry molecular annotation software SIRIUS.

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