Separation and purification and high-throughput target quantitative analysis method for multiple types of environmental pollutants in complex matrix biological sample

Abstract

The invention belongs to the technical field of environmental analytical chemistry, and particularly relates to a separation and purification and high-throughput target quantitative analysis method for multiple types of environmental pollutants in a complex matrix biological sample. The method comprises the following specific steps: S1, carrying out ultrasonic extraction on a biological sample to obtain an extracting solution; s2, performing dispersive solid-phase extraction and purification treatment on the extracting solution to obtain a solution to be detected; s3, determining the environmental pollutants in the liquid to be detected by adopting a selected ion scanning mode of the gas chromatograph-mass spectrometer. The pretreatment method disclosed by the invention can effectively reduce complex matrix interference of the biological sample, and can quickly and efficiently perform high-throughput target analysis on multiple types of environmental pollutants in the biological sample; the method disclosed by the invention is low in detection limit and capable of realizing trace detection of a biological sample; the invention provides a new method for researching the occurrence condition of the environmental pollutants in the 17 marine organisms in Liaoning Bay, and has important significance for the progress of environmental science.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of environmental analysis chemistry, and particularly relates to a separation and purification method for multiple types of environmental pollutants in a complex matrix biological sample and a high-throughput target quantitative analysis method and application thereof. BACKGROUND

[0002] Environmental pollutants widely exist in the environment, can enter organisms through biological enrichment and be transmitted to high-trophic-level organisms along the food chain, and many of the environmental pollutants pose potential threats to ecosystems and human health due to their biological toxicity, persistence and biological accumulation. Therefore, it is of great significance to accurately and sensitively analyze multiple types of environmental pollutants in biological samples for improving the ecological environment quality.

[0003] At present, the pretreatment methods for biological samples include Soxhlet extraction and liquid-liquid extraction, but there are problems of consumption of a large amount of organic solvents and time, and it is difficult to achieve the purpose of high-throughput target quantitative analysis of multiple types of environmental pollutants. In addition, the traditional method has a serious problem of matrix interference in a complex biological sample, and only a single type or similar physicochemical properties of environmental pollutants in the biological matrix can be effectively analyzed, and it is difficult to accurately quantify trace environmental pollutants. For example, when analyzing the content of organochlorine pesticides in fish tissues, the detection limit may be as high as tens of nanograms per gram, while the concentration of environmental pollutants in the actual environment may be as low as nanograms or even picograms. Therefore, it is urgent to develop a high-throughput target analysis method for environmental pollutants, improve the accuracy of identification and quantification of multiple types of environmental pollutants, and efficiently and cost-effectively analyze the exposure level and occurrence characteristics of environmental pollutants. SUMMARY

[0004] The application provides a high-throughput target quantitative analysis method for multiple types of environmental pollutants in a biological sample, which focuses on optimizing the sample pretreatment technology to effectively reduce the interference of the matrix in the biological sample, and simultaneously adopts a selected ion scanning mode to realize a high-throughput target quantitative analysis method for multiple types of environmental pollutants in a biological sample, which is accurate and reliable in determination result and low in detection limit.

[0005] The first aspect of the application provides a separation and purification method for multiple types of environmental pollutants in a complex matrix biological sample and a high-throughput target quantitative analysis method, and the steps are as follows:

[0006] The separation and purification method for environmental pollutants in a biological sample before gas chromatography-mass spectrometry analysis, characterized in that it comprises the following steps:

[0007] S1. An organic solvent is used as an extraction solvent to extract the biological sample, and after extraction, solid-liquid separation is performed to obtain an extraction liquid;

[0008] S2. The extraction solution is subjected to dispersive solid phase extraction purification treatment, and solid-liquid separation is performed after the purification treatment to obtain a to-be-tested solution that can be used for gas chromatography-mass spectrometry analysis;

[0009] The dispersive solid phase extraction purification agent is a mixture of PSA, C18 and Z-Sep, and the mass ratio of the three is 1:0.8-1.2:0.8-1.2, preferably 1:0.9-1.1:0.9-1.1, and more preferably 1:0.95-1.05:0.95-1.05.

[0010] The biological sample is a freeze-dried solid biological sample;

[0011] The extraction solvent is dichloromethane-n-hexane (1:0.8-1.2, preferably 1:0.9-1.1, and more preferably 0.95-1:1.05; v / v); the solid-liquid ratio of the freeze-dried biological sample and the extraction solvent is 1 (g): 15-25 (mL), preferably 1 (g): 18-22 (mL), and more preferably 1 (g): 19-21 (mL);

[0012] The environmental pollutants include at least one or a combination of multiple polycyclic aromatic hydrocarbons, polychlorinated biphenyls, chlorobenzenes, polybrominated diphenyl ethers, phenols, phthalates, nitrobenzenes, herbicides, insecticides and fungicides in Table 2-4, 7-8.

[0013] In S1:

[0014] The extraction is ultrasonic extraction, and the temperature and time of ultrasonic extraction are 20-30°C (preferably 22-28°C, and more preferably 25-27°C) and 20-40 min (preferably 25-35 min, and more preferably 30-35 min), respectively;

[0015] The solid-liquid separation is centrifugal separation, and the time and speed of centrifugal separation are 15-25 min (preferably 18-22 min, and more preferably 20-21 min) and 3000-4000 rpm (preferably 3300-3700 rpm, and more preferably 3500-3600 rpm), respectively.

[0016] In S2:

[0017] The liquid-solid ratio of the extraction solution and the dispersive solid phase extraction purification reagent is 1 (mL): 50-70 (mg), preferably 1 (mL): 55-65 (mg), and more preferably 1 (mL): 60-65 (mg);

[0018] The purification treatment is mixed mechanical stirring, and the time is 1-6 min (preferably 3-5 min, and more preferably 4-5 min) ;

[0019] The solid-liquid separation is centrifugal separation, and the time and speed of the centrifugal separation are 15-25 min (preferably 18-22 min, and more preferably 20 min-21) and 3000-4000 rpm (preferably 3300-3700 rpm, and more preferably 3500-3600 rpm), respectively.

[0020] In S2, the following steps are performed:

[0021] After the solid-liquid separation, the supernatant is obtained, the supernatant is concentrated by nitrogen blowing, the solvent is replaced by n-hexane, the nitrogen blowing is continued, the internal standard is added, the n-hexane is used to constant volume, the filter membrane with a pore size of 0.22-0.45 μm is used, and the to-be-tested liquid is obtained.

[0022] 6. The method of claim 5, wherein the internal standard comprises eight substances, which are 4-chlorotoluene-d4, 1,4-dichlorobenzene-d4, naphthalene-d8, acenaphthene-d10, phenanthrene-d10, fluoranthene-d10, -d12 and perylene-d12.

[0023] In S3, the environmental pollutants in the to-be-tested liquid are determined by using a gas chromatograph-mass spectrometer in a selected ion scanning mode.

[0024] The gas chromatograph-tandem mass spectrometry is in a SIM mode.

[0025] The chromatographic column of the chromatographic separation instrument of the gas chromatograph-tandem mass spectrometry is selected as an HP-5MS.

[0026] The detection data are chromatograms and mass spectra of the environmental pollutants.

[0027] The qualitative analysis is to qualitatively analyze the environmental pollutants by comparing the retention time in the chromatogram and comparing the characteristic ion fragments in the mass spectrum.

[0028] The quantitative analysis is to integrate the chromatograms of the series of environmental pollutant mixed standards (mixed standard environmental pollutants) to obtain a linear equation Y=aX+b of each environmental pollutant, Y represents the response peak area ratio of the environmental pollutant to the corresponding internal standard, X represents the concentration ratio of the environmental pollutant to the corresponding internal standard, the to-be-tested sample is detected by using the gas chromatograph-tandem mass spectrometry, the response peak area of the to-be-tested sample is obtained, and the concentration of the environmental pollutant in the to-be-tested sample can be calculated according to the linear equation.

[0029] In the SIM mode, 156 standard curves, the detection limit and the quantitative limit are obtained by one needle injection.

[0030] In a preferred embodiment, the environmental pollutants include 156 kinds of polycyclic aromatic hydrocarbons, polychlorinated biphenyls, chlorobenzenes, polybrominated diphenyl ethers, phenols, phthalates, nitrobenzenes, herbicides, insecticides and fungicides.

[0031] In a preferred embodiment, the S1 step specifically comprises: weighing 0.3 g of the freeze-dried biological sample into a centrifuge tube, adding 6 mL of dichloromethane-n-hexane (1:1, v / v) as an extraction solvent, vortexing for 1 min, and then ultrasonic extraction at 25°C for 30 min. Subsequently, centrifugation is performed at 3900 r / min for 15 min, and the supernatant is collected. The above extraction step is repeated once and the supernatants are combined.

[0032] In a preferred embodiment, the S2 step specifically comprises: taking the supernatant into a centrifuge tube, adding a dispersive solid-phase extraction purification reagent, vortexing for 1 min to mix uniformly, centrifuging at 3900 r / min for 15 min, and taking the supernatant. The supernatant is concentrated to near dryness by nitrogen blowing, replaced with 2 mL of n-hexane once, and then concentrated to 0.5 mL by nitrogen blowing. After adding 100 μL of an internal standard (1 μg / mL), the volume is made up to 1 mL with n-hexane, and then filtered through a 0.22 μm polytetrafluoroethylene filter membrane to obtain a test solution.

[0033] In a preferred embodiment, the dispersive solid-phase extraction purification reagent comprises primary and secondary amine-bonded silica gel (PSA), octadecyl-bonded silica gel (C18), and zirconium-based adsorbent (Z-Sep).

[0034] In a preferred embodiment, the internal standard comprises 4-chlorotoluene-d4, 1,4-dichlorobenzene-d4, naphthalene-d8, acenaphthene-d10, phenanthrene-d10, fluoranthene-d10, d12, and perylene-d12.

[0035] In a preferred embodiment, in the selected ion scanning mode, the S3 step specifically comprises: the working parameters of the chromatograph in the gas chromatograph-mass spectrometer are as follows: chromatographic column: HP-5MS (30 m x 0.25 mm x 0.25 μm); injection mode: splitless injection; injection volume: 1 μL; temperature program: initial temperature 40°C, holding for 2 min, increasing to 310°C at a rate of 8°C / min, holding for 5 min; carrier gas: helium; carrier gas flow rate: 1.2 mL / min.

[0036] In a preferred embodiment, in the selected ion scanning mode, the S3 step specifically comprises: the working parameters of the mass spectrometer in the gas chromatograph-mass spectrometer are as follows: ion source: EI source; electron energy: 70 eV; ion source temperature: 320°C.

[0037] The second aspect of the application provides application of a method for separating and purifying multi-class environmental pollutants in a complex matrix biological sample and high-throughput target quantitative analysis.

[0038] The application has the following advantages:

[0039] The application provides a method for high-throughput target quantitative analysis of multi-class environmental pollutants in a biological sample, which has the following advantages:

[0040] (1) The application can efficiently and accurately detect the environmental pollutant level of a biological sample (especially marine organisms), and the detection result is accurate and reliable, and the dispersion degree of parallel samples is small.

[0041] (2) The application selects PSA, C18 and Z-Sep as the purification agent for dispersive solid phase extraction, which can effectively remove fatty acids, lipids and proteins in the biological sample, and will not have adsorption effect on the target pollutants.

[0042] (3) The application has strong feasibility in testing multiple target pollutants, and the span of each physicochemical parameter is significant, log K ow covers the range from strong hydrophilicity to strong hydrophobicity (0.6-10.7), and the molecular weight distribution is extensive (123.1-643.6 g / mol), each chromatographic peak can be effectively separated, and the peak shape is good, the correlation coefficient of the chromatographic peak and the target compound is high, which is beneficial to accurate quantitative analysis of the target;

[0043] (4) The application has high instrument response degree, low detection limit, can detect low content biological samples, and the testing steps are simple, the detection efficiency is high, and it is easy to popularize in the field of analysis and detection;

[0044] (5) The application provides a new idea and method for the research of trace detection of environmental pollutants in marine organisms, which has important significance for the progress of environmental science.

[0045] The pretreatment method of the application can effectively reduce the interference of the complex matrix of the biological sample, and can quickly and efficiently perform high-throughput target analysis of multi-class environmental pollutants in the biological sample; the application has low detection limit and can realize trace detection of the biological sample; the application provides a new method for the research of environmental pollutants in 17 kinds of marine organisms in Liaodong Bay, which has important significance for the progress of environmental science. BRIEF DESCRIPTION OF DRAWINGS

[0046] Figure 1 is a flow chart for preparing 156 kinds of environmental pollutant standard solutions.

[0047] Figure 2is the concentration (a) and the proportion (b) (selected ion scan) of 17 marine organisms in environmental pollutants.

[0048] Figure 3 is the RSD distribution diagram of the recovery rate of 156 environmental pollutants at low, medium and high three different standard addition levels (n = 5).

[0049] Figure 4 is the influence diagram of 7 different dispersive solid phase extraction purifiers on the recovery rate of environmental pollutants involved in the present application.

[0050] Figure 5 is the matrix effect of 156 environmental pollutants.

[0051] Figure 6 is the processing and analysis flow chart of biological samples. DETAILED DESCRIPTION

[0052] The present application can be more easily understood in view of the following detailed description of the preferred method of practicing the present application and the examples included. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. If there is a conflict between the definitions in the specification and those in the patent, the definitions in the specification are intended to prevail.

[0053] The terms "comprise", "comprising", "have", "having", "include", "including", or any other variation thereof, used in this document, are intended to cover a non-exclusive inclusion. For example, a process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements on the list, but can include other elements not expressly listed or inherent to such process, method, article, or apparatus.

[0054] The numerical ranges used herein should be understood to include any and all combinations of any upper limit and any lower limit, including isolated integer values. For example, when a range of "1-4" is disclosed, the range should be interpreted to include "1", "2", "3", "4", "1-2", "1-3", "1-4", "2-3", "2-4", and "3-4", etc. Unless otherwise stated, the range includes its endpoints and all integers and combinations thereof within the range.

[0055] To solve the above problems, the first aspect of the present application provides a high-throughput target quantitative analysis method for environmental pollutants in biological samples, and the specific steps include:

[0056] S1. Ultrasonic extraction of biological samples to obtain an extraction solution;

[0057] S2. Dispersive solid phase extraction purification treatment of the extraction solution to obtain a test solution;

[0058] S3. The environmental pollutants in the test solution are determined by using a gas chromatograph-mass spectrometer in a selected ion scan mode.

[0059] In some preferred embodiments, the environmental pollutants include 156 kinds of polycyclic aromatic hydrocarbons, polychlorinated biphenyls, chlorobenzenes, polybrominated biphenyl ethers, phenols, phthalates, nitrobenzenes, herbicides, insecticides and fungicides.

[0060] In some preferred embodiments, the biological sample is stored in a -80℃ refrigerator under light-avoiding and refrigeration before analysis.

[0061] In some preferred embodiments, the S1 step specifically comprises: adding the biological sample into a centrifugal tube, adding an extraction solvent, vortexing to mix uniformly, ultrasonic extraction, centrifugation, and collecting the supernatant as the extraction solution.

[0062] Further preferably, the S1 step specifically comprises: adding the biological sample into a centrifugal tube, adding an extraction solvent, vortexing for 1 min to mix uniformly, ultrasonic extraction at 25℃ for 30 min. Subsequently, centrifugation is performed at 3900 r / min for 15 min, and the supernatant is collected. The above extraction step is repeated once, and the supernatants are combined.

[0063] Further preferably, the extraction solvent is dichloromethane-n-hexane.

[0064] More preferably, the extraction solvent is dichloromethane-n-hexane (1:1, v / v).

[0065] More preferably, the solid-liquid ratio of the biological sample and the first extraction solvent is 1:20.

[0066] The environmental pollutants in the biological sample have low content and are difficult to be detected by instruments, and thus need to be accurately and rapidly analyzed in actual detection. Prolonged pretreatment and upper detection time can lead to unstable detection results, while too short pretreatment time cannot effectively extract the target compounds in the biological sample. Meanwhile, different types of biological samples have dynamic and complex characteristics, and the existing analysis methods are difficult to accurately, rapidly and comprehensively detect the environmental pollutant levels of various biological samples. It is found through a large number of methodological experiments that when a dichloromethane-n-hexane mixed solution is used to extract the biological sample and a specific vortex-ultrasonic-centrifugation operation is used, the leaching of the environmental pollutants in the biological sample can be promoted. Since the detection limit of the target compounds in the biological sample is low, the temperature of vortexing, ultrasonic extraction and centrifugation can significantly inhibit the loss of the environmental pollutants in the biological sample during the extraction process, thereby achieving effective detection.

[0067] In some preferred embodiments, the S2 step is specifically: taking the supernatant into a centrifugal tube, adding a dispersive solid-phase extraction purification reagent, vortexing for 1 min to mix uniformly, centrifuging at 3900 r / min for 15 min, and taking the supernatant. The supernatant is concentrated to near dryness by nitrogen blowing, 2 mL of n-hexane is used to replace the solvent once, the nitrogen blowing is continued to concentrate to 0.5 mL, 100 μL of an internal standard (1 μg / mL) is added, and then n-hexane is used to dilute to 1 mL, and then the mixture is passed through a 0.22 μm polytetrafluoroethylene filter membrane to obtain a to-be-tested solution.

[0068] In some preferred embodiments, the dispersive solid-phase extraction purification reagent comprises a combination of one or more of PSA, C18 and Z-Sep.

[0069] Further preferably, the dispersive solid-phase extraction purification reagent is a mixture of PSA, C18 and Z-Sep. The mass ratio of the three is 1:1:1.

[0070] In some preferred embodiments, the volume ratio of the extraction solution and the dispersive solid-phase extraction purification reagent is 40:1.

[0071] In some preferred embodiments, the internal standard comprises 4-chlorotoluene-d4, 1,4-dichlorobenzene-d4, naphthalene-d8, acenaphthene-d10, phenanthrene-d10, fluoranthene-d10, -d12 and perylene-d12.

[0072] In a preferred embodiment, in the selected ion scanning mode, the working parameters of the chromatography in the gas chromatograph-mass spectrometer are as follows: chromatographic column: HP-5MS (30 m x 0.25 mm x 0.25 μm); injection mode: splitless injection; injection volume: 1 μL; temperature program: initial temperature 40℃, holding for 2 min, increasing the temperature to 310℃ at a rate of 8℃ / min, holding for 5 min; carrier gas: helium; carrier gas flow rate is 1.2 mL / min.

[0073] In a preferred embodiment, in the selected ion scanning mode, the working parameters of the mass spectrometry in the gas chromatograph-mass spectrometer are as follows: ion source: EI source; electron energy: 70 eV; ion source temperature: 320℃.

[0074] There are various methods for separating and detecting environmental pollutants in biological samples. Liquid chromatography has flexible mobile phase adjustment, but the analysis speed is slow, the equipment cost is high, and it is difficult to popularize. Gas chromatography has the advantages of high sensitivity of detector, fast analysis speed, etc., and becomes an effective means for analyzing and detecting environmental pollutants.

[0075] However, the matrix in the biological sample is complex, and ordinary pretreatment methods are difficult to realize effective separation of each target analyte. Further research of the present application found that when a mixture of three dispersive solid phase extraction purification reagents, primary and secondary amine bonded silica gel (PSA), octadecyl bonded silica gel (C18) and zirconium-based adsorbent (Z-Sep), is used to purify the extraction liquid of the biological sample, and then the target substances are separated by an HP-5MS chromatographic column (30 m x 0.25 mm x 0.25 μm), and then the specific elution program described above is used, the effective separation of 156 target substances of environmental pollutants can be realized, and the test effect has good reproducibility.

[0076] In some preferred embodiments, the internal standard method is used for qualitative and quantitative analysis of the biological sample, that is, a standard solution with gradient concentration is configured, the peak area ratio of the environmental pollutants in the standard solution is obtained by machine, and a standard curve is drawn; the measured data of the biological sample are substituted into the standard curve to obtain the high-throughput target detection result of the environmental pollutants of the biological sample.

[0077] Further preferably, the configuration method of the standard solution is that the environmental pollutant standard and the isotope internal standard are respectively prepared into a standard stock solution with a concentration of 1 μg / mL using n-hexane, and stored at-20℃. An appropriate amount of stock solution is taken and gradient diluted using a 2 ml volumetric flask to prepare a standard solution with gradient concentration.

[0078] Further preferably, the concentration of each standard with gradient concentration is 0.1, 0.4, 0.8, 1, 4, 8, 10, 40, 80, 100, 400 and 800 ng / mL.

[0079] In some preferred embodiments, after obtaining the standard curve of the target environmental pollutant standard, a selected ion scanning mode is used for high-throughput target quantitative analysis of multiple categories of environmental pollutants in the biological sample.

[0080] The second aspect of the present application provides an application of a separation and purification method and a high-throughput target quantitative analysis method of multiple categories of environmental pollutants in a complex matrix biological sample. The method is applied to the research on the occurrence of environmental pollutants in 17 marine organisms in Liaodong Bay.

[0081] The specific embodiments of the present application are further described below in combination with the drawings and technical solutions.

[0082] Example 1

[0083] The present embodiment provides a separation and purification method and a high-throughput target quantitative analysis method of multiple categories of environmental pollutants in a complex matrix biological sample, and the specific steps include:

[0084] S1. Ultrasonic extraction is performed on the freeze-dried solid biological sample to obtain an extraction liquid;

[0085] S2. The extraction solution is subjected to a dispersive solid phase extraction purification treatment to obtain a test solution;

[0086] S3. The environmental pollutants in the test solution are determined by using a gas chromatograph-mass spectrometer in a selected ion scan mode.

[0087] The environmental pollutants include 156 kinds of polycyclic aromatic hydrocarbons, polychlorinated biphenyls, chlorobenzenes, polybrominated diphenyl ethers, phenols, phthalates, nitrobenzenes, herbicides, insecticides and fungicides.

[0088] The biological samples are 17 kinds of marine biological samples, including 7 kinds of fish, Acanthogobius ommaturus, Sardinela sindensis, Platycephalus indicus, Thamnaconus septentrionalis, Acanthopagrus schlegeli, Lateolabrax maculates and Nibea albiflora; 3 kinds of bivalves, Mytilus galoprovincialis, Crassostrea gigas and Tegilarca granosa; 2 kinds of molluscs, Portunus trituberculatus and Oratosquila oratoria; 2 kinds of starfish, Archaster typicus and Asterias amurensis; 1 kind of gastropod, Neverita didyma; 1 kind of cephalopod, Octopus ocelatus; 1 kind of brown algae, Undaria pinnatifida Suringar, which is derived from the sea area near Yingkou Port in Liaodong Bay, and the specific information is shown in Table 1.

[0089] Table 1 Name and quantity of 17 kinds of marine organisms in Liaodong Bay

[0090]

[0091]

[0092] The S1 step is specifically: taking the freeze-dried biological sample into a centrifuge tube, adding an extraction solvent, vortexing for 1 min to mix evenly, then ultrasonic extraction at 25℃ for 30 min, ultrasonic frequency is 28 kHz, power is 150 W. Subsequently, centrifugation at 3900 r / min for 15 min, and collecting the supernatant of the first extraction;

[0093] The solid after the first extraction centrifugation is replaced with the above-mentioned biological sample to repeat the above extraction process once to obtain the supernatant of the second extraction; and the supernatants of the two times are combined as the extraction liquid.

[0094] The dry weight of the freeze-dried biological sample is 0.3 g.

[0095] The biological sample is specifically: fish back meat for fish; inner shell, inner shell and inner spiral meat for bivalves, mollusks and gastropods, respectively; thick meat part of the middle disc area for starfish; biological head meat for cephalopods; frond part for Undaria pinnatifida. The biological sample is freeze-dried.

[0096] The freeze-dried biological sample is freeze-dried at a cold trap temperature of -80℃ for 24 h. The freeze-drying machine model is FD-1A-50, Beijing Boyikang Experimental Instrument Co., Ltd.

[0097] The extraction solvent is dichloromethane-n-hexane (1:1, v / v).

[0098] The solid-liquid ratio of the freeze-dried biological sample and the extraction solvent is 1 (g):20 (mL).

[0099] The S2 step is specifically: taking 5 mL of the extraction liquid obtained in the S1 step into a centrifuge tube, adding a dispersive solid-phase extraction purification reagent (100 mg C18+100 mg PSA+100 mg Z-Sep), vortexing for 1 min to mix evenly, centrifuging at 3900 r / min for 15 min, and taking the supernatant. The supernatant is concentrated to 0.5 mL by nitrogen blowing, replaced with 2 mL of n-hexane once, continuously concentrated to 0.5 mL by nitrogen blowing, added with 100 μL of an internal standard (1 μg / mL), then diluted to 1 mL with n-hexane, passed through a pore size 0.22 μm polytetrafluoroethylene filter membrane, and the membrane permeate is the obtained test liquid (i.e. the sample loading liquid).

[0100] The dichloromethane and n-hexane (both are HPLC grade) are purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd., USA.

[0101] In the dispersive solid-phase extraction purification reagent, C18 and PSA are purchased from Beijing Nage Technology Co., Ltd., and Z-Sep is purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.

[0102] The dispersive solid phase extraction purification reagent comprises a mixture of C18, PSA and Z-Sep in a mass ratio of 1:1:1.

[0103] The liquid-solid ratio of the supernatant and the dispersive solid phase extraction purification reagent is 1 (mL):60 (mg).

[0104] In the S3 step, the gas chromatograph-mass spectrometer (GC-MS, Shimadzu, Japan) is used in the selected ion scanning mode, and the working parameters of the mass spectrometer are as follows: ion source: EI source; electron energy: 70 eV; transfer line temperature: 320 DEG C; ion source temperature: 320 DEG C; mass-to-charge ratio range: 45 amu-600 amu; and collision energy optimization range: 3-60 V.

[0105] In the S3 step, the gas chromatograph-mass spectrometer (GC-MS, Shimadzu, Japan) is used in the selected ion scanning mode, and the working parameters of the mass spectrometer are as follows: ion source: EI source; electron energy: 70 eV; transfer line temperature: 320 DEG C; ion source temperature: 320 DEG C; mass-to-charge ratio range: 45 amu-600 amu; and collision energy optimization range: 3-60 V.

[0106] The biological sample is subjected to qualitative and quantitative analysis by using an internal standard method, wherein the qualitative analysis is confirmed by the characteristic ions of the mass spectrum and the retention time of the chromatogram; the quantitative analysis is performed by using the chromatographic peak area ratio of the target environmental pollutants to the corresponding internal standard, drawing a standard curve, and calculating the pollutant concentration by using the internal standard grouping method. The X axis of the standard curve is the concentration ratio of each pollutant in the 156 target environmental pollutant standard solutions in the sample solution to the corresponding internal standard; the Y axis is the chromatographic peak area ratio of each pollutant in the 156 target environmental pollutant standard solutions in the sample solution to the corresponding internal standard. The actual concentration of the environmental pollutants in the biological sample is obtained by substituting the measured peak area of the biological sample into the standard curve, that is, the high-throughput target detection result.

[0107] The internal standard comprises eight substances (i.e., eight single standards), which are 4-chlorotoluene-d4, 1,4-dichlorobenzene-d4, naphthalene-d8, acenaphthene-d10, phenanthrene-d10, fluoranthene-d10, -d12 and perylene-d12. In the internal standard grouping, the order of the eight substances corresponds to groups 1-8 in Table 2, respectively.

[0108] The configuration method of the eight internal standard substance solution is: the concentration of each internal standard substance single standard is 10 μg / mL (the solvent is HEX n-hexane), 12.5 μL of each internal standard substance single standard solution is taken, a total of 100 μL, and then 900 μL of HEX is added to configure an internal standard mixed standard solution with a concentration of 1 μg / mL, wherein the final concentration of the single standard in the eight internal standard solutions is 0.125 μg / mL.

[0109] The configuration method of the 156 standard solutions of environmental pollutants is: eight bottles of mixed standard solutions purchased are configured from 10 μg / mL to 1 μg / mL.

[0110] The eight bottles of mixed standard (mixed standard environmental pollutants) solutions are as follows: 15 PAHs mixed standard solutions (each standard environmental pollutant has a concentration of 10 μg / mL), 27 PCBs mixed standard solutions (each standard environmental pollutant has a concentration of 10 μg / mL), 10 chlorobenzene mixed standard solutions (each standard environmental pollutant has a concentration of 10 μg / mL), 5 PBDEs mixed standard solutions (each standard environmental pollutant has a concentration of 10 μg / mL), 6 phenol mixed standard solutions (each standard environmental pollutant has a concentration of 10 μg / mL), 12 PAEs mixed standard solutions (each standard environmental pollutant has a concentration of 10 μg / mL), 10 nitrobenzene mixed standard solutions (each standard environmental pollutant has a concentration of 10 μg / mL), and 71 pesticide mixed standard solutions (each standard environmental pollutant has a concentration of 10 μg / mL), a total of eight bottles of 156 environmental pollutant mixed standard solutions. 12.5 μL of each bottle of mixed standard solution is taken, a total of 100 μL, and then 900 μL of n-hexane (HEX) is added to configure a standard stock solution containing 156 environmental pollutants with a concentration of 1 μg / mL, which is stored in a-20℃ refrigerator.

[0111] An appropriate amount of the stock solution is taken and gradient dilution is performed using a 1 ml volumetric flask to prepare standard solutions with gradient concentrations. The gradient concentrations of each standard environmental pollutant in the 156 mixed standard solutions with internal standards are 0.1, 0.4, 0.8, 1, 4, 8, 10, 40, 80, 100, 400 and 800 ng / mL, and the corresponding internal standard concentrations (the sum of the concentrations of the eight internal standards) are all 0.05 μg / mL. The specific configuration method is shown in Figure 1 The gradient concentration standard solutions are determined by gas chromatography-mass spectrometry in the selected ion scanning mode, and the 156 standard curves are shown in Table 2. The 156 target environmental pollutants show good linear relationships in the range of 0.1-800 ng / mL, and the correlation coefficients (r 2 ) are all greater than 0.99, and the fitting degree is high.

[0112] After obtaining the sample solution with concentration gradient, the machine analysis is performed.

[0113] Table 2 156 standard curve information

[0114]

[0115]

[0116]

[0117]

[0118]

[0119]

[0120] The 17 biological samples, each sample is three parallel samples. At the same time, in order to ensure the quality of data, 1 program blank sample and 1 matrix standard sample are added every 5 samples. The sample is 51 biological samples, 4 program blanks, 5 matrix standards and 1 matrix blank.

[0121] The program blank sample uses the same quality of ultrapure water instead of the freeze-dried biological sample, and the same pretreatment process as the biological sample (same as the above S1, S2, S3 process and condition). It is used to detect whether the target environmental pollutants are introduced in the whole experimental process (reagents, containers, environment);

[0122] The matrix standard sample is a laboratory freshwater cultured Ptereleotris grammica. The juvenile fish is 2.5 cm long, cultured for 45 days, and grown into adult fish 20 cm long. At this time, the back meat of the fish is taken as a matrix standard sample, and the same pretreatment process (same as the above S1 and S2 process and condition) is performed. After obtaining the test solution (1 mL), 1 μg / mL of standard stock solution containing 156 environmental pollutants and 1 μg / mL of internal standard mixed standard solution (the final concentration of single standard in eight internal standard solutions is 0.125 μg / mL) are added. The final concentration of each standard environmental pollutant is 1 ng / mL, and the corresponding internal standard concentration (the sum of the concentrations of the same eight internal standards) is 0.05 μg / mL. It is used to evaluate whether the error caused by the matrix effect.

[0123] The matrix blank also uses a laboratory freshwater cultured Ptereleotris grammica. The same pretreatment process (same as the above S1, S2, S3 process and condition) is performed, and no target environmental pollutants are added. It is used to detect whether the matrix itself contains target environmental pollutants.

[0124] Matrix effect experiment: Matrix effect refers to the influence of non-target components in the sample on the analyte to be tested, resulting in systematic deviation of the measured value from the true value. The matrix effect is evaluated by determining the slope of the matrix standard curve (Matrix) and the slope of the solvent standard curve (Solvent).

[0125] The matrix standard curve refers to the sample of the striped tail goby raised in laboratory water, from the length of the juvenile fish of 2.5 cm, raised for 45 days, and grown into adult fish of 20 cm. At this time, the fish back meat is taken as the matrix for the standard sample, and the same pretreatment process (as described in S1 and S2 above) is performed. After obtaining the test solution (1 mL), 1 μg / mL of the standard stock solution containing 156 environmental pollutants and 1 μg / mL of the internal standard mixed standard solution (0.125 μg / mL of single standard in eight internal standard solutions) are added. The final concentration of each standard environmental pollutant is 0.1, 0.4, 0.8, 1, 4, 8, 10, 40, 80, 100, 400 and 800 ng / mL, and the corresponding internal standard concentration (the sum of the same concentration of eight internal standards) is 0.05 μg / mL. As the sample solution, the process and conditions of the above S3 step are used to determine the matrix standard curve by using the selected ion scanning mode of the gas chromatography-mass spectrometry instrument. The solvent standard curve is the standard curve of the above-mentioned 156 target pollutants (Table 2). The test results are shown in Figure 5 and Tables 3 and 4, after extraction and purification, using formula 1, 74.4% of the target environmental pollutants are weak matrix effect (|matrix effect|<20%), 23.1% of the target environmental pollutants are moderate matrix effect (|matrix effect|<50%), and only 2.5% of the target environmental pollutants are strong matrix effect (|matrix effect|>50%), indicating that the pretreatment method has good effect on removing matrix. Among them, only methoxychlor and phenyl phosphorothioate have high matrix effect, which is 96% and 124% respectively, which is significantly higher than other target environmental pollutants, indicating that they are seriously interfered by matrix in biological samples.

[0126] Matrix effect (%) = ((S matrix / S solvent )-1)×100% Formula 1

[0127] In the formula, Matrix effect represents the matrix effect, S matrix and S solvent respectively refer to the slope of the matrix standard curve and the slope of the solvent standard curve of the corresponding target pollutant.

[0128] Table 3 Matrix standard curve information of 156 items

[0129]

[0130]

[0131]

[0132]

[0133]

[0134]

[0135] Determination of the limit of detection and the limit of quantification: laboratory freshwater aquaculture of O. maculatus, from the juvenile fish body length 2.5 cm, cultured for 45 days, and grown into adult fish 20 cm, at this time the fish back meat is taken, and used as a substrate to add standard samples, and the same pretreatment process (as described above S1 and S2 process and conditions) is performed, and after obtaining the test solution (1 mL), 1 pg / mL of a standard stock solution containing 156 environmental pollutants and 1 pg / mL of an internal standard mixed standard solution (in which the final concentration of single standard in eight internal standard solutions is 0.125 pg / mL) are added, and the final concentration of each standard environmental pollutant is 0.1, 0.4, 0.8, 1, 4, 8, 10, 40, 80, 100, 400 and 800 ng / mL, and the corresponding internal standard concentration (the sum of the same concentration of 8 internal standards) is 0.05 pg / mL. As the sample loading solution, the above S3 step process and conditions are used to determine the limit of detection and the limit of quantification of the method by gas chromatography-mass spectrometry in selected ion scan mode, and the concentrations corresponding to the signal-to-noise ratio of 3 and the signal-to-noise ratio of 10 are determined as the limit of detection and the limit of quantification of the method for pretreatment and on-machine detection, and the specific method is as described above. The results are shown in Table 4, and the method detection limit of 80% and 98% of the target environmental pollutants is less than 10 ng / g and 20 ng / g, respectively; the method quantification limit of 80% and 94% of the target environmental pollutants is less than 20 ng / g and 100 ng / g, respectively.

[0136] Table 4 Limit of detection, limit of quantification and matrix effect of 156 environmental pollutants

[0137]

[0138]

[0139]

[0140]

[0141]

[0142]

[0143] The concentration data of the environmental pollutants detected in the 17 biological samples were obtained by the instrument analysis. In the first step, the concentration data of each detected environmental pollutant were subtracted by the average value of the concentration of the environmental pollutants detected in the double procedural blank samples (according to the EPA standard, subtracting the double blank samples can reduce the false positive rate to about 5%, which meets the majority of scientific research standards), and then it was determined whether the value was greater than 0. If it was greater than 0, it was added to the average value of the concentration of the environmental pollutants detected in the double procedural blank samples to correct the error. If it was less than zero, it was directly retained as 0 (since negative values are often unreasonable in biology, such as concentration cannot be negative. This data processing method is a quality control means, which eliminates untrustworthy negative data, thereby ensuring that the final data are all valid); in the second step, the arithmetic mean of each detected environmental pollutant concentration data of the three parallel samples of each biological sample was obtained, and the data are shown in Table 5; in the third step, since the water content of fish and other biological samples is high, which is 50%, the data of the second step were multiplied by 0.5 to obtain the dry weight of the biological sample, with the unit of ng; in the fourth step, the data were divided by the initial dry weight of 0.3 g to obtain the dry weight concentration, with the unit of ng / g. The final data are shown in Tables 5 and 6.

[0144] Table 5: Arithmetic mean of the environmental pollutants detected in 17 biological samples

[0145]

[0146]

[0147]

[0148]

[0149] Table 6: Types and concentrations of environmental pollutants detected in 17 biological samples, ng / g.

[0150]

[0151]

[0152]

[0153] Among the 42 environmental pollutants detected in the marine organisms in Liaodong Bay, there were 16 pesticides (omethoate, phorate, cadusafos, fonofos, acetochlor, alachlor, phorate, imidacloprid, allethrin II, tebuconazole, disulfoton, butonate, imidacloprid, phorate sulfoxide, fenpropathrin II, phenothrin II), 10 phthalates (DEEP, DEP, DBP, BMOP, DiPP, DPNP, DHP, BBP, DCHP, DINP), 7 polycyclic aromatic hydrocarbons (naphthalene, acenaphthene, fluorene, phenanthrene, chrysen, dibenzo[a,h]anthracene, benzo[ghi]perylene), 3 nitrobenzene (2,4-dinitrotoluene, nitrobenzene and 1,2-dinitrobenzene), 3 phenol (4-hexylphenol, 4-tert-octylphenol, 4-octylphenol), 2 polychlorinated biphenyls (PCB-169, PCB-189) and 1 chlorobenzene (1,2-dichlorobenzene).

[0154] As shown in Figure 2 (a), the total environmental pollutant content in the 17 marine organism samples in Liaodong Bay ranged from 2.00×10 1 to 4.98×10 3 ng / g dw, with an average of 1.39×10 3 ng / g dw. There were large differences in the concentrations among marine organisms, with the highest total environmental pollutant concentration in Mytilus edulis (4.98×10 3 ng / g dw), followed by Oratosquilla oratoria (3.44×10 3 ng / g dw) and Crassostrea gigas (2.74×10 3 ng / g dw), and the relatively low concentration in Eleotris fusca (2.00×10 1 ng / g dw). The reason for not detecting environmental pollutants was that the 17 biological samples did not contain or contained less than the detection limit.

[0155] The composition of various environmental pollutants in the 17 marine organisms is shown in Figure 2 (b). The concentration of pesticides in marine organisms accounted for 0-80%, phthalates were detected in marine organisms (concentration ratio: 12-82%), and the concentration of nitrobenzene in marine organisms accounted for 0-28%. Pesticides, phthalates and nitrobenzene were the main detected substances in marine organisms, with an average total concentration of 7.20×10 2 , 5.40×10 2 and 1.10×10 2 ng / g dw, respectively. Among them, only phthalates and polycyclic aromatic hydrocarbons were detected in Eleotris fusca, accounting for 20% and 80%, respectively.

[0156] The further high-throughput target detection results showed that 16 kinds of pesticides were detected in marine organisms in Liaodong Bay, including 9 organophosphorus pesticides, 4 amide pesticides, 2 pyrethroid pesticides, and 1 fungicide. The highest total concentration of pesticides in marine organisms was as high as 2.71×10 3 ng / g dw (mouth shrimp), and the average total concentration was 7.20×10 2 ng / g dw. In 17 kinds of marine organisms, the detection rates of heptenophos, methyl isothion, cyphenothrin, and sulfiram (all insecticides) were the highest, reaching 58.8%, 52.9%, 35.3%, and 29.4%, respectively. The detection rates of land insect phosphorus, ethidimphos, dimethen, allyl chrysanthemum, ethidimphos, and butylamine were all less than 10%;

[0157] The total concentration of phthalate esters in marine organisms in Liaodong Bay ranged from 3.50-4.10×10 3 ng / g dw (with an average of 5.40×10 2 ng / g dw), and DEEP and DnHP were the most commonly detected phthalate esters in marine organisms (with detection rates of 52.9% and 47.1%, respectively), and the average total concentrations were 0.76 and 0.04 μg / g dw. Phthalate esters also had high concentrations in the bodies of Strongylocentrotus intermedius and Mytilus edulis, reaching 2.70×10 2 and 1.30×10 2 ng / g dw, respectively.

[0158] Three kinds of nitrobenzene environmental pollutants were detected in marine organisms in Liaodong Bay, namely 2,4-dinitrotoluene, nitrobenzene, and 1,2-dinitrobenzene. The highest concentration of NBs in marine organisms was as high as 4.00×10 2 ng / g dw (long oyster). Among them, the detection rate of 1,2-dinitrobenzene was less than 10%. 2,4-dinitrotoluene and nitrobenzene were the most commonly detected nitrobenzenes, with detection rates of 76.5% and 64.7%, respectively.

[0159] Methodology verification: The accuracy and precision of the analysis method were verified by the recovery rate and RSD of the standard addition experiment.

[0160] The sample of the O. maculatus was bred in the laboratory with clean water. The sample was taken from the back of the fish after the fish grew to 20 cm in length from 2.5 cm in length after 45 days of breeding. The sample was used as a matrix for a standard sample. The same pretreatment process (as described in S1 and S2 above) was performed. After the sample was obtained (1 mL), a standard stock solution containing 156 environmental pollutants at a concentration of 1 μg / mL and an internal standard mixed standard solution at a concentration of 1 μg / mL (in which the final concentration of a single standard in the eight internal standard solutions was 0.125 μg / mL) were added. The final concentration of each standard environmental pollutant was 20, 50, and 100 ng / mL, and the corresponding internal standard concentration (the sum of the concentrations of the eight internal standards) was 0.05 μg / mL. The sample was used as a sample. The sample was determined by gas chromatography-mass spectrometry in selected ion scan mode according to the process and conditions of S3 described above.

[0161] The 156 target environmental pollutants were added at low (20 ng), medium (50 ng), and high (100 ng) levels. Five parallel experiments were continuously performed at different levels. The results showed that, in the low concentration addition experiment, 83% (130 kinds) of the environmental pollutants had an average recovery rate of 60-140%. Among them, at different concentrations, the substances with a recovery rate of less than 60% were mainly phenols and pesticides. When the addition concentration was medium and high, 90% (141 kinds) of the environmental pollutants had a recovery rate in the range of 60-140%. Five parallel experiments (taking the arithmetic mean) were continuously performed at different levels. The test results are shown in Table 7 and Figure 3 At low, medium, and high concentrations, the recovery rate RSD of more than 93% of the target environmental pollutants was less than 30%, indicating that the analysis method has good stability.

[0162] Table 7 Low, medium, and high concentration addition experiment results of 156 environmental pollutants

[0163]

[0164]

[0165]

[0166]

[0167]

[0168]

[0169] Comparative Example 1

[0170] The comparative example provides a method for separating and purifying multi-class environmental pollutants in a complex matrix biological sample and high-throughput target quantitative analysis, the specific implementation process and conditions are the same as those of example 1 (the same as the above S1, S2, S3 process and condition), and the difference is that the fish is bred in laboratory water, and the fish is taken from the back meat when the fish grows to 20 cm from the juvenile fish with a body length of 2.5 cm and is bred for 45 days. The fish is used as a matrix for sample addition, and the same pretreatment process (the same as the above S1 and S2 process and condition) is performed. After the sample is obtained (1 mL), 1 μg / mL of a standard stock solution containing 156 environmental pollutants and 1 μg / mL of an internal standard mixed standard solution (the final concentration of a single standard in eight internal standard solutions is 0.125 μg / mL) are added. The final concentration of each standard environmental pollutant is 1 μg / mL, and the corresponding internal standard concentration (the sum of the concentrations of the eight internal standards) is 0.05 μg / mL. As the sample liquid; the process and conditions of the above S3 step are used to determine the sample liquid by using a gas chromatograph-mass spectrometer in a selected ion scanning mode; and the dispersive solid phase extraction purifier is only 300 mg PSA.

[0171] Comparative example 2

[0172] The comparative example provides a method for separating and purifying multi-class environmental pollutants in a complex matrix biological sample and high-throughput target quantitative analysis, the specific implementation process and conditions are the same as those of comparative example 1, and the difference is that the dispersive solid phase extraction purifier is only 300 mg C18.

[0173] Comparative example 3

[0174] The comparative example provides a method for separating and purifying multi-class environmental pollutants in a complex matrix biological sample and high-throughput target quantitative analysis, the specific implementation process and conditions are the same as those of comparative example 1, and the difference is that the dispersive solid phase extraction purifier is only 300 mg Z-Sep.

[0175] Comparative example 4

[0176] The comparative example provides a method for separating and purifying multi-class environmental pollutants in a complex matrix biological sample and high-throughput target quantitative analysis, the specific implementation process and conditions are the same as those of comparative example 1, and the difference is that the dispersive solid phase extraction purifier is only 300 mg Z-Sep.

[0177] Comparative example 5

[0178] The comparative example provides a method for separating and purifying multi-class environmental pollutants in a complex matrix biological sample and high-throughput target quantitative analysis, the specific implementation process and conditions are the same as those of comparative example 1, and the difference is that the dispersive solid phase extraction purifier is only 300 mg Z-Sep.

[0179] Comparative Example 6

[0180] The present comparative example provides a method for separation and purification of multi-class environmental pollutants in complex matrix biological samples and high-throughput target quantitative analysis. The specific implementation process and conditions are the same as those of Comparative Example 1, except that the dispersive solid-phase extraction purifier is 150 mg C18+150 mg Z-Sep.

[0181] Verification Example

[0182] The specific implementation process and conditions are the same as those of Comparative Example 1, except that the dispersive solid-phase extraction purifier is a mixed adsorbent (100 mg PSA+100 mg C18+100 mg Z-Sep).

[0183] The comparative results of the above six comparative examples and verification examples are shown in Table 7 and Table 8, which compare the effects of 300 mg PSA, 300 mg Z-Sep, 300 mg C18, 150 mg PSA+150 mg C18, 150 mg PSA+150 mg Z-Sep, 150 mg C18+150 mg Z-Sep and their mixed adsorbent (100 mg PSA+100 mg C18+100 mg Z-Sep) on the recovery rates of 156 environmental pollutants. Figure 4 Figure 4 The final recovery rate in each column is averaged. As shown in Table 7 and Table 8, the mixed adsorbent (100 mg PSA+100 mg C18+100 mg Z-Sep) can obtain the best recovery rate results. Figure 4 Table 8 Recovery rate comparison of dispersive solid-phase extraction purifiers for 156 environmental pollutants, %.

[0184]

[0185]

[0186]

[0187]

[0188]

[0189]

[0190]

[0191] ​​Based on the above, the present application adopts a kind of separation and purification of multi-class environmental pollutants in complex matrix biological sample and high-throughput target quantitative analysis method to carry out analysis and detection to 17 kinds of marine organisms in Liaodong Bay.The method is accurate, fast, and the detection limit is low;156 kinds of target environmental pollutants in the range of 0.1-800ng / mL, show good linear relationship, the correlation coefficient (r 2 )All greater than 0.99;The accuracy and precision of the method meet the detection requirements, and provide an efficient and rapid analysis method for the detection of multi-class environmental pollutants in biological samples.

[0192] Finally, it is pointed out that the foregoing examples are merely exemplary for explaining some features of the method according to the present application. The appended claims can envisage as broad as possible, and are not limited to the listed embodiments. Any technical modification made according to the technical solution of the present application falls within the scope of the present application claimed.

Claims

1. A method for the separation, purification, and high-throughput target quantitative analysis of multiple environmental pollutants in complex matrix biological samples, characterized in that, Includes the following steps: S1. Organic solvents are used as extraction solvents to extract biological samples. After extraction, solid-liquid separation is performed to obtain the extract. S2. The extract is purified by dispersive solid-phase extraction, and then solid-liquid separation is performed to obtain the test solution that can be used for gas chromatography-mass spectrometry analysis. The dispersed solid-phase extraction purifying agent is a mixture of PSA, C18 and Z-Sep, with a mass ratio of 1:0.8-1.2:0.8-1.2, a preferred mass ratio of 1:0.9-1.1:0.9-1.1, and a more preferred mass ratio of 1:0.95-1.05:0.95-1.

05.

2. The method according to claim 1, characterized in that: The biological sample is a freeze-dried solid biological sample; The extraction solvent is dichloromethane-n-hexane (1:0.8-1.2, preferably 1:0.9-1.1, more preferably 0.95-1:1.05; v / v); the solid-liquid ratio of the freeze-dried biological sample to the extraction solvent is 1 (g):15-25 (mL), preferably 1 (g):18-22 (mL), and more preferably 1 (g):19-21 (mL); The environmental pollutants include at least one or a combination of 156 types of substances listed in Tables 2-4 and 7-8, including polycyclic aromatic hydrocarbons, polychlorinated biphenyls, chlorobenzenes, polybrominated diphenyl ethers, phenols, phthalates, nitrobenzenes, herbicides, insecticides, and fungicides.

3. The method according to claim 1, characterized in that: In S1: The extraction is ultrasonic extraction, with the temperature and time being 20-30℃ (preferably 22-28℃, more preferably 25-27℃) and 20-40min (preferably 25-35min, more preferably 30-35min), respectively. The solid-liquid separation is centrifugal separation, with a centrifugation time of 15-25 min (preferably 18-22 min, more preferably 20-21 min) and a centrifugation speed of 3000-4000 rpm (preferably 3300-3700 rpm, more preferably 3500-3600 rpm).

4. The method according to claim 1, characterized in that: In S2: The solid-liquid ratio of the extract to the dispersed solid-phase extraction purification reagent is 1 (mL): 50-70 (mg), preferably 1 (mL): 55-65 (mg), and more preferably 1 (mL): 60-65 (mg). The purification process involves mechanical mixing for 1-6 minutes (preferably 3-5 minutes, more preferably 4-5 minutes). The solid-liquid separation is centrifugal separation, with a centrifugation time of 15-25 min (preferably 18-22 min, more preferably 20-21 min) and a centrifugation speed of 3000-4000 rpm (preferably 3300-3700 rpm, more preferably 3500-3600 rpm).

5. The method according to claim 1 or 4, characterized in that: In S2: After solid-liquid separation, the supernatant was collected, concentrated by nitrogen blowing, and then the solvent was replaced with n-hexane. Nitrogen blowing was continued to concentrate the supernatant. After adding the internal standard, the solution was diluted to volume with n-hexane and filtered through a 0.22-0.45 μm filter membrane to obtain the test solution.

6. The method according to claim 5, characterized in that: The internal standard includes eight substances: 4-chlorotoluene-d4, 1,4-dichlorobenzene-d4, naphthalene-d8, acenaphthene-d10, phenanthrene-d10, and fluoranthene-d10. -d12 and perylene-d12.

7. The method according to any one of claims 1-6, characterized in that: S3. The environmental pollutants in the test solution were determined using the selected ion scanning mode of a gas chromatography-mass spectrometry system.

8. The method according to claim 7, characterized in that, The gas chromatography-tandem mass spectrometry method is selected in SIM mode; The chromatographic column used in the gas chromatography-tandem mass spectrometry (GC-MS) separation method is selected as HP-5MS.

9. The method according to claim 7 or 8, characterized in that, The detection data are the chromatograms and mass spectra of the environmental pollutants; The qualitative analysis is performed by comparing retention times in the chromatograms and comparing characteristic ion fragments in the mass spectrometry to identify environmental pollutants. The quantitative analysis involves integrating the chromatograms of the series of environmental pollutant mixed standards (mixed standard environmental pollutants) to obtain a linear equation for each environmental pollutant: Y = aX + b, where Y represents the ratio of the response peak area of ​​the environmental pollutant to the corresponding internal standard, and X represents the ratio of the concentration of the environmental pollutant to the corresponding internal standard. The sample to be tested is then detected by gas chromatography-tandem mass spectrometry to obtain the response peak area of ​​the sample. The concentration of the environmental pollutant in the sample to be tested can be calculated based on the linear equation.

10. The method according to claim 9, characterized in that, In SIM mode, a single injection yields 156 standard curves, along with their limits of detection and limits of quantitation.

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