A method for non-target screening of low-abundance pollutants in environmental water samples based on thin-film microextraction technology and application thereof

Abstract

The application discloses a non-target screening analysis method for low-abundance pollutants in environmental water samples based on a thin film micro-extraction technology and application thereof, and through preparation of a thin film micro-extraction coating material and control of extraction parameters, the number of mass spectrum characteristic signals obtained in subsequent substance analysis is improved, the screening coverage of pollutants in the sample in non-target screening analysis and the analysis sensitivity are improved, and therefore, pollutants with different properties and large concentration differences can be extracted and enriched from environmental water samples in a wide spectrum, especially low-abundance organic pollutants in the environmental water samples can also be realized in wide spectrum extraction. Further, liquid chromatography-high resolution mass spectrometry combined with the above-mentioned sampling method based on the thin film extraction technology can realize non-target screening analysis of organic pollutants in the environmental water samples, especially non-target screening and identification analysis of low-abundance organic pollutants in the environmental water samples.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of environmental analysis, and in particular, relates to a non-target screening analysis method for low-abundance pollutants in environmental water samples based on thin-film microextraction technology and application thereof. BACKGROUND

[0002] Trace pollutants in water samples include a variety of analytes with different chemical properties, polarity, solubility and chemical stability. Such pollution can have unexpected and extremely harmful consequences on the health of humans and wild animals. At present, there is no single method that can achieve non-target screening and identification analysis of low-abundance organic pollutants in environmental water samples.

[0003] The key and challenge of realizing non-target screening analysis is to avoid missing some pollutants as much as possible in the screening process. Given that the types of pollutants are unknown, the polarity range and concentration range they are in are also unknown, the sample pretreatment method for non-target screening needs to be able to extract and enrich pollutants with different properties and large concentration differences from environmental water samples. Studies have shown that the time spent on sample pretreatment accounts for more than 70% of the entire analysis process. At present, solid phase extraction (SPE) is widely used in non-target screening analysis of pollutants to reflect the overall pollution of the sample and find new pollutants and transformation products. The main process of SPE mainly includes three steps of sample loading, elution and elution. The impurities in the sample have weak interaction with the adsorbent, which are removed in the elution stage, and then the appropriate type of eluent is used to elute the target analyte, and finally the impurities are removed and concentrated. In addition, solid phase extraction and pressurized solvent extraction have been used for screening of organic pollutants in environmental water and water sediments, respectively. For the screening of organic pollutants in water samples, mixed extractants such as polar group-terminated octadecyl-modified silica gel particles are used to extract and enrich pollutants. For the screening of organic pollutants in water sediments, a variety of organic solvents with different polarity are used to extract weakly polar and strongly polar pollutants by pressurized solvent extraction. However, the existing traditional SPE technology for non-target screening analysis of organic pollutants is generally suitable for high-concentration substances, while trace pollutants are easily ignored due to low signal peaks. In addition, a few studies have also developed multi-layer SPE adsorbent materials to achieve efficient enrichment of organic pollutants with different physical and chemical properties in the environment. On the other hand, due to the complicated steps, both solid phase extraction and pressurized solvent extraction need to transfer the sample to the laboratory for sample pretreatment operation. Some substances will be adsorbed on the bottle wall during the sample transfer process, or some unstable substances will be lost during this transfer process. The storage and transportation of the sample greatly increase the cost of monitoring, and also increase the risk of sample contamination and loss of analytes in the sample.

[0004] Therefore, there is an urgent need for a sample pretreatment method that can fully reveal the profile of all analytes in a sample without loss of pollutants, accelerating the exploration of new pollutants and pollutant transformation products. SUMMARY

[0005] The present application aims to overcome the above-mentioned defects and deficiencies in the prior art, and provides a non-target screening analysis method for low-abundance pollutants in environmental water samples based on thin-film microextraction technology and its application.

[0006] The above-mentioned object of the present application is realized by the following technical solutions:

[0007] The present application first provides a method for sampling low-abundance organic pollutants in environmental water samples based on thin-film microextraction technology. The thin film is immersed in the water sample for 20-80 min, then taken out and dried, and immediately immersed in a desorption solution with a volume of 500-2000 μL for 5-15 min. After filtration, the sample solution to be tested is obtained. The thin film is a hydrophilic modified carbon cloth as a carrier, coated with HLB / PDMS or C18 / PDMS adsorbent material on the surface. The desorption solution is methanol, acetonitrile or a mixture of methanol / acetonitrile.

[0008] The present application extracts and enriches pollutants in water by preparing thin-film microextraction coating materials and applying thin-film microextraction (TFME) technology to water sampling. Specifically, the carbon cloth carrier is treated by hydrophilic modification, then loaded with HLB / PDMS or C18 / PDMS adsorbent material to prepare the thin film material, and the extraction parameters are controlled, so that pollutants with different properties and concentrations can be extracted and enriched from environmental water samples in a broad spectrum, especially low-abundance organic pollutants in environmental water samples can also be extracted in a broad spectrum, thereby used for the analysis of trace pollutants in subsequent water samples.

[0009] Preferably, the adsorbent material is HLB / PDMS. Polydimethylsiloxane (PDMS) has good adsorption effect on medium and low polarity substances, and is the most commonly used extraction phase component in thin-film microextraction. The present application shows that C18 coating material can detect more mass spectrum signal characteristics, and has preference for high-hydrophobic and low-abundance environmental pollutants in the extracted water sample, which can screen out organic pollutants missed by traditional solid-phase extraction technology.

[0010] Further, the preparation method of the thin film comprises the following steps:

[0011] S1. Carbon cloth pretreatment: after the carbon cloth is cleaned by ultrasonic treatment in a solution, the carbon cloth is placed in a mixed solution of concentrated sulfuric acid and nitric acid, and is subjected to hydrothermal reaction treatment for 3-4 h, and is rinsed clean to obtain a modified carbon cloth;

[0012] S2. Coating material preparation: the adsorption material is ultrasonically dispersed in cyclohexane, polydimethylsiloxane is added, and the mixture is again ultrasonically dispersed, the cyclohexane is volatilized by nitrogen blowing, the polydimethylsiloxane curing agent is added, and the mixture is uniformly mixed to obtain the coating material; the adsorption material is HLB or C18.

[0013] S3. The coating material prepared in S2 is coated on the modified carbon cloth prepared in S1, and is cured at 90-95℃ for 4-5 h in a nitrogen atmosphere, and the other side of the carbon cloth is coated once.

[0014] The carbon cloth preparation method is a modification treatment of the carbon cloth, and the carbon cloth after the modification treatment can improve the surface hydrophilicity and the affinity to the adsorption material.

[0015] Preferably, the ultrasonic cleaning in S1 is ultrasonic cleaning of the carbon cloth in acetone, ethanol and deionized water for 30 minutes each.

[0016] Preferably, the hydrothermal reaction in S2 is a hydrothermal reaction at 95℃.

[0017] Preferably, the hydrothermal reaction in S2 is hydrothermal reaction for 3 h.

[0018] Preferably, the curing in S3 is curing at 90℃ for 4 h in a nitrogen atmosphere.

[0019] Further, the extraction is extraction for 60-80 min.

[0020] Preferably, the extraction is extraction for 60 min.

[0021] Further, the volume of the desorption liquid is 1000-2000 μL.

[0022] Preferably, the volume of the desorption liquid is 1000-1500 μL.

[0023] Preferably, the volume of the desorption liquid is 1000 μL.

[0024] Further, the desorption solution is methanol or a mixed solution thereof.

[0025] Preferably, the desorption solution is methanol.

[0026] Further, the desorption is desorption for 10-15 min.

[0027] Preferably, the desorption is desorption for 10 min.

[0028] The method can extract and enrich substances in a broad spectrum, and is suitable for non-target screening analysis.

[0029] Therefore, the application provides application of the sampling method in environmental water sample sampling analysis.

[0030] Further, the environmental water sample sampling analysis includes on-site sampling analysis and water sample collection.

[0031] Preferably, the environmental water sample sampling analysis is on-site sampling analysis.

[0032] The application also provides application of the sampling method in non-target screening analysis of low-abundance pollutants in water samples.

[0033] A non-target screening analysis method for low-abundance pollutants in water, comprising the following steps:

[0034] S1. Preprocessing an environmental water sample according to any of the above sampling methods to obtain a sample solution to be tested;

[0035] S2. Non-target scanning analysis of the sample solution to be tested obtained in step S1 is performed in positive and negative modes respectively using liquid chromatography-mass spectrometry to obtain raw data;

[0036] S3. Data preprocessing is performed on the raw data obtained in step S2 to obtain a CSV file containing the accurate mass-to-charge ratio, retention time and peak response value of the characteristic peaks in the mass spectrum data in each component;

[0037] S4. The CSV file obtained in step S3 is used to establish a target-msms method according to the retention time and mass-to-charge ratio, and the secondary mass spectrum signal spectrum is obtained under a collision energy of 10-40V, and the compound is confirmed by database comparison.

[0038] Further, the data preprocessing in S3 includes converting the raw data into an mzXML format file using ProteoWizard MSconvert software, filtering the raw data through a Peaking Picking filter, importing the file data into an MZmine 3.3.0 data processing platform, and performing steps such as mass detection, ADAP chromatography reconstruction, isotope removal, peak alignment, peak filtering and peak deduplication.

[0039] Preferably, the mass detection processing condition is an allowed time error of 0.2 min, and the peak intensity minimum is 1000, and the allowed mass error is 20ppm.

[0040] Preferably, the ADAP chromatography reconstruction processing condition is a peak intensity minimum of 1000, and the allowed mass error is 20ppm.

[0041] Preferably, the deisotoping processing condition is most intense mode, allowing a mass error of 20ppm.

[0042] Preferably, the peak alignment processing condition is RANSAC aligner mode, allowing a time error of 0.2min, and allowing a mass error of 20ppm.

[0043] Preferably, the peak filtering processing condition allows a time error of 0.2min, and allows a mass error of 20ppm.

[0044] Preferably, the peak deduplication processing condition is New Average mode, allowing a time error of 0.2min, and allowing a mass error of 20ppm.

[0045] Further, the method further comprises repeating steps S2-S4 using a standard sample of the compound confirmed to match the fragments in step S4, obtaining the primary mass spectrum and secondary mass spectrum results, and comparing the retention time and mass spectrum fragment ions in the mass spectrum data obtained from the test sample, to finally determine the screened compound at Level 1; the Level 1 level is matched with the secondary mass spectrum and retention time of the standard sample.

[0046] Further, the method is step S1, using solid phase extraction technology or liquid-liquid extraction technology and any of the above-mentioned sampling methods to pretreat the water sample to obtain a test sample, and performing steps S2 and S3 on the test sample, performing single factor analysis, data filtering, data standardization and normalization, principal component analysis, and differential fold analysis on the obtained CSV file, drawing a volcano plot, screening for higher expressed mass spectrum characteristic peaks, establishing a target-msms method according to the retention time and mass-to-charge ratio, obtaining a secondary mass spectrum signal spectrum under a collision energy of 10-40V, comparing with a database to confirm the compound matching the fragments, further purchasing a standard sample of the compound confirmed to match the fragments in step S4, repeating steps S2-S4, obtaining the primary mass spectrum and secondary mass spectrum results, and comparing the retention time and mass spectrum fragment ions in the mass spectrum data obtained from the test sample, to finally determine the screened compound at Level 1; the Level 1 level is matched with the secondary mass spectrum and retention time of the standard sample.

[0047] Preferably, step S1 is to pretreat the environmental water sample to obtain a test sample solution using solid phase extraction technology and any of the above-mentioned sampling methods.

[0048] Preferably, the single factor analysis, data filtering, data standardization and normalization are performed on the MetaboAnalyst5.0 (https: / / www.metaboanalystca) website.

[0049] Preferably, the data filtering is to set the relative standard deviation (RSD) of the data as 40% as a threshold for data filtering.

[0050] Preferably, the data standardization is to standardize the sample based on the sum of the data in the sample.

[0051] The present application greatly improves the analysis sensitivity of the developed method by preparing a thin film solid phase microextraction material and applying it to the non-target screening analysis of various organic pollutants in water, using the large coating volume of the thin film solid phase microextraction to greatly increase the effective surface area of the extraction phase in contact with the sample; on the other hand, the fast extraction kinetics and flexible shape ensure that it can be used for in-situ sampling analysis, avoiding the risk of sample contamination and loss of analytes in the sample during water sample preservation and transportation, and improving the efficiency of sample analysis. Further statistical analysis of the results of non-target screening of various organic pollutants in water by different extraction techniques and the thin film microextraction technology developed in the present application reveals that the thin film microextraction technology using C18 coating material can detect more mass spectrum signal characteristics, and has a preference for high hydrophobicity and low abundance environmental pollutants in the extracted water sample.

[0052] Compared with the prior art, the present application has the following beneficial effects:

[0053] The present application provides an environmental water sample low-abundance pollutant sampling method based on thin film microextraction technology, which improves the number of mass spectrum characteristic signals obtained in subsequent substance analysis by preparing a thin film microextraction coating material and controlling the extraction parameters, improves the screening coverage rate and analysis sensitivity of pollutants in the sample in non-target screening analysis, so as to be able to extract and enrich pollutants with different properties and large concentration differences from environmental water samples in a broad spectrum, especially low-abundance organic pollutants in environmental water samples. Further, the combination of liquid chromatography-high resolution mass spectrometry and the above-mentioned sampling method based on thin film extraction technology can realize non-target screening analysis of organic pollutants in environmental water samples, especially non-target screening and identification analysis of low-abundance organic pollutants in environmental water samples. BRIEF DESCRIPTION OF DRAWINGS

[0054] Figure 1 It is a flowchart of the sampling and non-target screening analysis method.

[0055] Figure 2 It is a scanning electron microscope image of a commercial C18 particle coated solid phase microextraction thin film.

[0056] Figure 3 It is a scanning electron microscope image of a commercial HLB particle coated solid phase microextraction thin film.

[0057] Figure 4Principal component analysis of different sample pre-treatment methods for different coating materials; a is the principal component analysis plot between SPE treatment group, SPME treatment group and quality control group using C18 as coating material (i.e. POS-C18) in positive ion mode; b is the principal component analysis plot between SPE treatment group, SPME treatment group and quality control group using HLB as coating material (i.e. POS-HLB) in positive ion mode; c is the principal component analysis plot between SPE treatment group, SPME treatment group and quality control group using C18 as coating material (i.e. NEG-C18) in negative ion mode; d is the principal component analysis plot between SPE treatment group, SPME treatment group and quality control group using HLB as coating material (i.e. NEG-HLB) in negative ion mode.

[0058] Figure 5 Orthogonal partial least squares discriminant analysis of different sample pre-treatment methods for different coating materials; a is the orthogonal partial least squares discriminant analysis plot between SPE treatment group and SPME treatment group using C18 as coating material (i.e. POS-C18) in positive ion mode; b is the orthogonal partial least squares discriminant analysis plot between SPE treatment group and SPME treatment group using HLB as coating material (i.e. POS-HLB) in positive ion mode; c is the orthogonal partial least squares discriminant analysis plot between SPE treatment group and SPME treatment group using C18 as coating material (i.e. NEG-C18) in negative ion mode; d is the orthogonal partial least squares discriminant analysis plot between SPE treatment group and SPME treatment group using HLB as coating material (i.e. NEG-HLB) in negative ion mode.

[0059] Figure 6 Model validation of orthogonal partial least squares discriminant analysis; a is the model validation plot of orthogonal partial least squares discriminant analysis between SPE treatment group and SPME treatment group using C18 as coating material (i.e. POS-C18) in positive ion mode; b is the model validation plot of orthogonal partial least squares discriminant analysis between SPE treatment group and SPME treatment group using HLB as coating material (i.e. POS-HLB) in positive ion mode; c is the model validation plot of orthogonal partial least squares discriminant analysis between SPE treatment group and SPME treatment group using C18 as coating material (i.e. NEG-C18) in negative ion mode; d is the model validation plot of orthogonal partial least squares discriminant analysis between SPE treatment group and SPME treatment group using HLB as coating material (i.e. NEG-HLB) in negative ion mode. The abscissa represents the similarity with the original model, and the ordinate represents the value of R 2 and Q 2 The intercepts of Q 2 are all below 0.05.

[0060] Figure 7 Figure 2 is a Venn diagram between different sample pretreatment methods; a is a Venn diagram between the SPE treatment group, SPME treatment group and quality control group using C18 as the coating material (i.e., POS-C18) in positive ion mode; b is a Venn diagram between the SPE treatment group, SPME treatment group and quality control group using C18 as the coating material (i.e., NEG-C18) in negative ion mode; c is a Venn diagram between the SPE treatment group, SPME treatment group and quality control group using HLB as the coating material (i.e., POS-HLB) in positive ion mode; d is a Venn diagram between the SPE treatment group, SPME treatment group and quality control group using HLB as the coating material (i.e., NEG-HLB) in negative ion mode.

[0061] Figure 8 This is a heat map of different sample pretreatment methods for C18 coating materials.

[0062] Figure 9 This is a heat map of different sample pretreatment methods for HLB coating materials.

[0063] Figure 10 Figure 1 shows a volcano plot comparing primary mass spectrometry signals detected by SPME and SPE for C18-coated materials. (a) shows the volcano plot for the SPE and SPME groups in positive ion mode using C18 as the coating material (i.e., POS-C18). (b) shows the SPE group in negative ion mode using C18 as the coating material (i.e., NEG-C18). Red and blue dots in the volcano plot for the SPME groups indicate features significantly upregulated by SPME and SPE, respectively, while gray dots indicate features with no significant changes. Larger dots indicate features of contaminants identified by the standards.

[0064] Figure 11 Figure 1. Volcano plots comparing primary mass spectrometry signals detected by SPME and SPE for HLB-coated materials. (a) Volcano plot comparing SPE and SPME treatments in positive ion mode using HLB as the coating material (i.e., POS-HLB). (b) Volcano plot comparing SPE and SPME treatments in negative ion mode using HLB as the coating material (i.e., NEG-HLB). Red and blue dots indicate features significantly upregulated by SPME and SPE, respectively. Gray dots indicate features with no significant changes. Larger dots indicate features identified as contaminants by the standards.

[0065] Figure 12 Kow values ​​of organic pollutants identified in the SPME and SPE treatment groups of C18 coating materials.

[0066] Figure 13 Kowvalues of the organic pollutants identified for the HLB-coated SPME and SPE treatment groups.

[0067] Figure 14 Concentrations of the organic pollutants identified for the C18-coated SPME and SPE treatment groups.

[0068] Figure 15 Concentrations of the organic pollutants identified for the HLB-coated SPME and SPE treatment groups. DETAILED DESCRIPTION

[0069] The present application is further illustrated by the following description with reference to the accompanying drawings and specific examples. The reagents, methods, and equipment employed in the present application are those conventionally used in the art unless otherwise specified.

[0070] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0071] Carbon cloth was purchased from Beijing An Nai Ji Energy Engineering Technology Co., Ltd. (Beijing, China); commercial C18 particles and commercial HLB particles were purchased from Shanghai An Pu Experimental Science and Technology Co., Ltd. (Shanghai, China).

[0072] Example 1. Preparation of thin film microextraction coating materials

[0073] Pretreatment of carbon cloth: Commercially available carbon cloth was cut into the desired size and cleaned by ultrasonic treatment in acetone, ethanol, and deionized water for 30 minutes each. Subsequently, the cleaned carbon cloth was treated by hydrothermal reaction with a mixture of concentrated sulfuric acid and nitric acid (v:v = 1:1) at 95 °C for 3 hours to improve the surface hydrophilicity and affinity for adsorbent materials. After treatment, the obtained modified carbon cloth was rinsed clean in deionized water.

[0074] Preparation of thin film solid phase microextraction coating: 500 mg of adsorbent material was ultrasonically dispersed in 10 mL of cyclohexane, then 2 g of high-viscosity polydimethylsiloxane was weighed and added to the above dispersed particles in cyclohexane, and ultrasonically dispersed again. The adsorbent material was any one of HLB, C18. Then most of the cyclohexane was volatilized from the mixture by nitrogen blowing, and the nitrogen blowing was stopped when there was almost no flow when the mixture was inverted in a vial. Finally, 200 mg of polydimethylsiloxane curing agent (peroxide-based catalyst) was added and mixed by hand.

[0075] Subsequently, the pretreated carbon cloth was laid on a horizontal table top, and the adsorbent mixture was extruded onto the top of the carbon cloth using a syringe. Then, the gap between the coating rod and the carbon cloth was adjusted to 100 μm, and the adsorbent mixture was slowly coated on the surface of the carbon cloth manually. Finally, the coating was cured at 90°C for 4 hours in a nitrogen atmosphere. Since the carbon cloth is double-sided, the entire process needs to be repeated for the second time to complete the preparation of the solid-phase microextraction film. After the film is prepared, the whole film is manually cut into the required size for subsequent sampling and detection analysis.

[0076] From the scanning electron microscope image (Figure 2), it can be seen that the coating material is uniformly distributed on the film and has a rich pore structure. Figure 2 3 Compared with the conventional solid-phase microextraction fiber, the solid-phase microextraction film prepared in the present application has a larger coating volume, good adsorption performance, higher sensitivity, and is suitable for non-target screening analysis of various pollutants in real water samples.

[0077] Examples 2-15

[0078] ​The solid phase microextraction film prepared in Example 1 was used to perform different film microextraction pretreatment processes according to the conditions shown in Table 1. The film was immersed in a river water sample for a certain extraction time, the film surface was wiped dry, and then the film was immediately immersed in a certain volume of desorption solution for desorption. The eluent was filtered through a 0.22 μm nylon membrane to remove insoluble impurities, and was stored in a -20 °C refrigerator for subsequent instrumental analysis. The used film was washed with methanol three times to remove residual analytes, and then was dried in air for standby. Subsequently, HPLC-QTOF-MS analysis was performed. Agilent Zorbax Eclipse Plus C18 liquid chromatography (2.1 mm x 150 mm, 1.8 μm) and gradient elution mode were used to separate the analytes. In the positive ion mode, mobile phase A was a water solution containing 0.1% formic acid, and mobile phase B was a methanol solution containing 0.1% formic acid; in the negative ion mode, mobile phase A was a water solution containing 0.1% formic acid, and mobile phase B was pure methanol solution. The injection volume was set to 5 μL, and the flow rate was 200 μL / min. TOF-MS full scan was performed in the mass range of m / z 50 to 1250, and MS / MS was performed in the mass range of m / z 50 to 1250 in high resolution mode. Fragment ions were generated by collision-induced dissociation with nitrogen at standard collision energies (CE) of 10, 20 and 40 V, respectively. Other experimental parameters were as follows: nebulizer gas pressure, 50 psi; dry gas flow rate, 8 L / min; sheath gas flow rate, 11 L / min; gas temperature, 320 °C; sheath gas temperature, 400 °C; capillary voltage, 3500 V; nozzle voltage, 1000 V; in-source voltage, 175 V. All the gases used were nitrogen. Non-target data processing was performed using Agilent Masshunter software (TOF Qualitative Analysis B.07.00). For the negative ion mode, the capillary voltage was set to -3000 V, and the mobile phases used were a 0.1% formic acid water solution (A) and methanol (B), and other settings were consistent with those of the positive ion mode.

[0079] The number of mass spectral characteristics of different pretreatment methods measured in the positive and negative ion modes is shown in Table 1.

[0080] When the extraction time is 60 to 80 min, the desorption time is 5 to 15 min, the desorption solution is methanol or a methanol / acetonitrile mixed solution, and the volume of the desorption solution is 1000 to 2000 μL, the number of mass spectral signal characteristics obtained is higher, i.e., the extraction parameters are feasible. The optimal extraction conditions are an extraction time of 60 min, a desorption time of 10 min, a methanol desorption solution, and a desorption solution volume of 1000 μL. The number of mass spectral characteristic signals obtained by C18 coating material is 15882, and the number of mass spectral characteristic signals obtained by HLB coating material is 13734.

[0081] Table 1. Specific example of screening river water samples using TFME technology

[0082]

[0083]

[0084] Comparative Example

[0085] River water collected on the same day was immediately filtered with a 0.22 μιη nylon membrane filter to remove insoluble impurities and stored in a 4°C refrigerator. The river water was restored to room temperature before solid phase extraction. The C18 or HLB solid phase extraction column was placed in a solid phase extraction device, repeatedly wetted with deionized water to remove air bubbles, and then activated with methanol. 500 mL of filtered river water was enriched through the extraction column, so that most of the substances in the river water were retained in the filler. Subsequently, 40 mL of methanol was used for elution, and the collected eluent was concentrated and evaporated to dryness using a rotary evaporator. Then, 500 μί of methanol was added for dissolution and constant volume. After filtration through a 0.22 μιη nylon 66 filter head, the filtrate was transferred to a 2 mL liquid chromatography sample bottle containing a 250 μί internal cannula and stored in a -20°C refrigerator.

[0086] Test Example

[0087] I. Experimental Methods

[0088] In order to illustrate the application of thin film microextraction technology to non-target screening and identification analysis of low-abundance organic pollutants in environmental water samples, Example 14 is selected below for specific characterization and application, and is compared with the comparative example.

[0089] The implementation process of Example 14 is shown in Figure 1 The steps of the developed method include sample preparation, sample analysis, data processing, identification and quantification. Except for the sample preparation method, the sample analysis, data processing, and identification and quantification of the comparative example are consistent with Example 14. The processing group of Example 14 is named as the SPME processing group, and the processing group of the comparative example is named as the SPE processing group. The developed method is specifically as follows:

[0090] (1) Sample preparation: The prepared C18-coated film was immersed in the river water sample, and after extraction for 60 min, the surface of the film was wiped dry with a dust-free paper, and immediately immersed in 1500 μί of methanol desorption solution for 10 min. The eluent was filtered with a 0.22 μιη nylon membrane filter to remove insoluble impurities, and then used for HPLC-QTOF-MS analysis.

[0091] (2) Loading analysis: The experiment used Agilent Zorbax Eclipse Plus C18 liquid chromatography (2.1 mm x 150 mm, 1.8 μm) and gradient elution mode to realize the separation of analytes. Before the experiment, 50 μL was taken from each bottle of desorption solution, which was made into a mixed standard sample for use as a quality control (QC) sample during instrument detection. During sample analysis, every 5 samples were inserted with two QC samples to observe and correct the instrument state. Suspicious and non-target screening was performed on high performance liquid chromatography, which was combined with high resolution hybrid quadrupole time-of-flight mass spectrometer with electrospray ionization (ESI) source operating in positive / negative ion mode. A Zorbax Eclipse Plus C18 column (2.1 mm x 150 mm, 1.8 μm, Agilent Technologies, USA) maintained at 30°C was used for separation of chemicals. For positive ion mode, the mobile phase used was 0.1% formic acid in water (A) and 0.1% formic acid in methanol (B). The gradient was set as follows: 5 μL of sample was injected into LC-Q-TOF-MS, the flow rate was set to 200 μL / min, starting with 95% solvent A for 1 min, then decreasing to 65% until 5 min, then decreasing to 5% until 20 min and maintaining for 19 min, then increasing to 95% until 40 min, and maintaining 95% until 45 min for equilibration. For negative ion mode, the mobile phase used was 0.1% formic acid in water (A) and methanol (B). The gradient was set as the same as the positive ion mode.

[0092] TOF-MS full scan was performed in the mass range of m / z 50 to 1250, while MS / MS was performed in the mass range of m / z 50-1250 in high resolution mode. Fragment ions were generated by collision-induced dissociation with nitrogen at standard collision energies (CE) of 10, 20 and 40 V, respectively. Other experimental parameters were: nebulizer gas pressure, 50 psi; dry gas flow, 8 L / min; sheath gas flow, 11 L / min; gas temperature, 320°C; sheath gas temperature, 400°C; capillary voltage, 3500 V; nozzle voltage, 1000 V; in-source voltage, 175 V. All the gases used were nitrogen. Agilent Masshunter software (TOF Qualitative Analysis B.07.00) was used for non-target data processing. For negative ion mode, the capillary voltage was set to -3000 V, the mobile phase used was 0.1% formic acid in water (A) and methanol (B), and other settings were consistent with the positive ion mode.

[0093] (3) Data processing: The raw data was converted into mzXML format file by ProteoWizard MSconvert software through Peaking Picking filter, and the data of these files was imported into MZmine3.3.0 data processing platform. After quality detection (allowable time error 0.2 min, peak intensity minimum 1000, allowable mass error 20 ppm), ADAP chromatogram reconstruction (peak intensity minimum 1000, allowable mass error 20 ppm), deisotoping (most intense mode, allowable mass error 20 ppm), peak alignment (RANSAC aligner mode, allowable time error 0.2 min, allowable mass error 20 ppm), peak filtering (allowable time error 0.2 min, allowable mass error 20 ppm) and peak de-redundancy (New Average mode, allowable time error 0.2 min, allowable mass error 20 ppm), etc. steps, the CSV file of accurate mass-to-charge ratio, retention time and peak response value of characteristic peaks in mass spectrum data in each component was integrated. The exported CSV file was uploaded to MetaboAnalyst 5.0 (https: / / www.metaboanalyst.ca) website after format adjustment for single factor analysis, and mass spectrum peak intensity was used as data analysis type, and the threshold value of relative standard deviation (RSD) 40% of data was set for data filtering; the samples were normalized by sum of data within the sample (normalization by sum), and then the auto scalling of data was selected in automatic mode. The characteristic peaks obtained by SPME and SPE methods in positive and negative ion modes were subjected to principal component analysis, fold change analysis and heat map analysis by applying Statistical Analysis function, respectively, to judge the difference of data obtained by different sample pretreatment forms and its coverage range of organic pollutants.

[0094] (4) Identification and quantification: The water sample screening data obtained by the SPME group method was compared with the data obtained by the SPE group method to investigate the application prospect of the SPME group method in non-targeted screening analysis. The p-value (p-value) and fold change (FC) of the characteristic peaks obtained by t-test were plotted into a volcano plot, and the characteristic peaks with higher expression in the TFME and SPE methods (p < 0.01) were screened out, respectively. The target-msms method was established according to the retention time and mass-to-charge ratio of the primary mass spectrum signal, and the secondary mass spectrum signal spectrum was obtained under different collision energies (10, 20, 40 V). The results were analyzed by Agilent software and compared with the database to confirm the compounds with matching fragments. Finally, the standard compounds of the compounds identified by database comparison above were purchased back, and the same method was used for experiment to obtain the primary mass spectrum and secondary mass spectrum results, which were compared with the retention time and mass spectrum fragment ions in the actual sample, and finally the screened compounds were determined at Level 1 level. In the whole process, the reliability of the screened compounds rises from Level 5 (primary mass spectrum confirms mass-to-charge ratio), to Level 2 (secondary mass spectrum is consistent with the spectrum library), and finally rises to the highest level of Level 1 (matches with the secondary mass spectrum and retention time of the standard). The identified organic pollutants were quantitatively analyzed by using ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry (HPLC-QQQ-MS) (ESI source) (AB Sciex Triple Quad4500, USA).

[0095] (5) Quantitative analysis: The identified pollutants were determined by tandem mass spectrometry with a high performance liquid chromatography-electrospray ionization (ESI) source. The target analytes were separated using a Zorbax Eclipse Plus C18 column (2.1 mm × 150 mm, 1.8 μm, Agilent Technologies, USA) maintained at 30°C. For the positive ion mode, the mobile phases used were 0.1% formic acid in water (A) and 0.1% formic acid in methanol (B). The gradient was set as follows: 5 μL of sample was injected into the HPLC-MS / MS, the flow rate was set to 300 μL / min, starting from 95% solvent A and held for 1 minute, then reduced to 5% solvent A for 4 minutes and held for 12 minutes, then increased to 95% solvent A for 19 minutes, and then held at 95% solvent A for 20 minutes for equilibrium. For the negative ion mode, the mobile phases used were 0.1% formic acid in water (A) and methanol (B). The gradient was set the same as for the positive ion mode. The mass spectrometer was operated in positive ion mode with multiple reaction monitoring (MRM). The gas temperature and sheath gas temperature were set to 320 and 270°C, respectively. The capillary voltage and nozzle voltage were set to 4000 and 1500 V, respectively. The nebulizer gas was 45 psi. The gas flow rate and sheath gas flow rate were set to 5 and 12 L / min, respectively. LC-MS data acquisition was performed using MassHunter software version B.07.00 (Agilent). For negative ion mode, the capillary voltage and nozzle voltage were set to -3500 and -1000 V, respectively. Other settings were consistent with the positive ion mode.

[0096] 2. Data Analysis

[0097] (1) Principal Component Analysis (PCA) diagram

[0098] From the score plot of multivariate unsupervised PCA ( Figure 4 ) obtained significant mass spectrometry signal responses. Under different sample pretreatment conditions, both the C18 and HLB coating materials exhibited clear separation of the three mass spectrometry signal responses. The results demonstrated significant differences in mass spectrometry signal responses between the SPME and SPE treatment groups. Furthermore, PCA analysis revealed close distances between points within each group, demonstrating good intra-group reproducibility.

[0099] (2) OPLS-DA (Orthogonal Partial Least Squares Discriminant Analysis, OPLS-DA) diagram and its verification

[0100] In addition, the supervised analysis OPLS-DA model was fitted to distinguish the mass spectrometry signal responses between the SPME treated group and the SPE control group, showing good model prediction ability. OPLS-DA score plot ( Figure 5 ) showed that for C18 and HLB coating materials, the mass spectrometry signal characteristics enriched by the SPME treatment group and the SPE control group in positive and negative ion modes were clearly separated. In addition, the internal cross-validation of the OPLS-DA model showed that according to the results of the permutation test ( Figure 6 ), Q 2 The intercept value is <0.05, the slope of the fitting curve is positive, and there is no overfitting phenomenon in the OPLS-DA model.

[0101] (3) Wayne Diagram

[0102] In order to understand the mass spectrometry signal responses between the SPME treated group and the SPE control group in positive and negative ion modes with different coating materials, the Venn diagram ( Figure 7 We found that the number of mass spectrometry signals under positive ions was greater than that under negative ions. For the C18-coated material, the number of mass spectrometry signals in the SPME-treated group was greater than that in the SPE-treated group. For the HLB-coated material, the number of mass spectrometry signals in the SPE-treated group was greater than that in the SPME-treated group. Furthermore, when the sample pretreatment method was SPME, the number of characteristic signals obtained using the C18-coated material was greater than that obtained using the HLB-coated material. Therefore, using the C18-coated material has advantages in thin-film microextraction for non-target screening analysis.

[0103] (4) Heat map analysis

[0104] In order to illustrate the differences in screening signals obtained by different sample pretreatment methods, a hierarchical cluster analysis was performed on the obtained mass spectrometry signals. We can see that the number of mass spectrometry peaks in the positive ion mode is more than that in the negative ion mode. For the C18 coating material ( Figure 8 ), the number of mass spectrometry signals in the SPME treatment group was greater than that in the SPE control group, and the signal preferences obtained by different pre-treatment methods were different (the redder the color, the greater the signal intensity). However, for HLB coating materials ( Figure 9 ), the number of mass spectrometry signals in the SPME treatment group was less than that in the SPE control group, and there was no significant difference in the signals obtained by different pretreatment methods.

[0105] (5) Volcano chart analysis

[0106] The primary signals with large inter-group difference values ​​enriched in the volcano map of C18 coating materials are marked as secondary signals ( Figure 10), and then matched with the existing database, 12 compounds were matched in the cation mode, 8 of which were detected by SPME, and 4 were detected by SPE; 8 compounds were matched in the anion mode, 4 of which were detected by SPME, and 4 were detected by SPE. The first signal of the inter-group difference value enriched in the HLB coating material volcano plot was punched into a second signal Figure 11 ), and then matched with the existing database, 18 compounds were matched in the cation mode, 12 of which were detected by SPME, and 6 were detected by SPE; 9 compounds were matched in the anion mode, 4 of which were detected by SPME, and 5 were detected by SPE.

[0107] (6) Targeted quantification

[0108] The Kow value of the identified organic pollutants: The Kow value of the compounds identified by the C18 coating was checked, and it was found that the Kow value of the compounds identified by the SPME treatment group was higher than that of the compounds identified by the SPE treatment group Figure 12 ). However, the Kow value of the compounds identified by the HLB coating was checked Figure 13 ), it was found that the Kow value of the compounds identified by the two pretreatment methods spanned a large range and had no obvious difference. Therefore, the C18 coating material is more suitable for screening compounds missed by the SPE technique, and realizing more comprehensive non-target screening and identification analysis of organic pollutants in environmental water samples.

[0109] The concentration of the identified analytes: The compounds identified by the C18 coating were quantified Figure 14 ), we found that the concentration of the compounds identified by the SPME treatment group was one order of magnitude lower than that of the compounds identified by the SPE treatment group, that is, the thin film microextraction method based on the C18 coating material provided by the present application has more advantages in the analysis and identification of low-concentration compounds in environmental water samples. Similarly, the organic pollutants identified by the HLB coating were quantitatively analyzed Figure 15 ), it was found that the concentration of the organic pollutants identified by the SPME treatment group and the SPE treatment group was widely distributed, but when using the HLB coating, the distribution of the organic pollutants identified by the SPME treatment group was more extensive at low concentrations. In summary, the thin film microextraction method based on two different coating materials provided by the present application has more advantages in the analysis and identification of low-concentration compounds in environmental water samples, and the thin film microextraction method based on the C18 coating material is more suitable for the extraction and analysis of low-abundance pollutants.

[0110] Non-target screening aims to qualitatively and quantitatively analyze all pollutants in environmental samples, and systematically reflects the overall situation of the sample. It is of great significance for early detection of new pollutants, mining of transformation products, exploration of transformation mechanism, development and evaluation of control methods. Suitable sample pretreatment step is one of the necessary conditions to obtain high-quality screening data. However, the traditional pretreatment method is complex in operation, time-consuming and labor-intensive, has serious matrix effect, and limited extraction and enrichment capacity, which hinders the further development of non-target screening research. Solid phase microextraction technology is a new sample pretreatment technology, which integrates sampling, enrichment, separation and impurity removal. Its extraction phase can be designed and adjusted according to the analysis purpose, and at the same time, the in-situ extraction volume is large, which can enrich and analyze some trace pollutants. It has a very high application prospect in the analysis of complex samples, but its application in non-target screening is still in the initial stage. In view of this, the application optimizes the application of thin film microextraction technology in non-target screening research. The results show that the thin film microextraction technology provided by the application has extraction preference for high hydrophobic and low concentration organic pollutants, which can make up for the omission of traditional solid phase extraction in non-target screening.

Claims

1. A method for sampling low-abundance organic pollutants in environmental water samples based on thin film microextraction technology, characterized in that: The film is immersed in the water sample for extraction for 20 to 80 minutes, taken out and wiped dry, and immediately immersed in a desorption solution with a volume of 500 to 2000 μL for desorption for 5 to 15 minutes, and the sample solution to be tested is obtained after filtration; the film is a hydrophilic modified carbon cloth as a carrier, and the surface is coated with C18 / PDMS adsorption material; The desorption solution is methanol, acetonitrile or a methanol / acetonitrile mixed solution; The method for preparing the film comprises the following steps: S1. Carbon cloth pretreatment: After ultrasonic cleaning in a solution, the carbon cloth is placed in a mixed solution of concentrated sulfuric acid and nitric acid for a hydrothermal reaction for 3-4 hours. The modified carbon cloth is then rinsed. S2 coating material preparation: the adsorption material was ultrasonically dispersed in cyclohexane, polydimethylsiloxane was added, ultrasonically dispersed again, the cyclohexane was volatilized by nitrogen blowing, polydimethylsiloxane curing agent was added, and mixed uniformly to obtain; the adsorption material was C18; S3. Apply the coating material prepared in S2 to the modified carbon cloth prepared in S1, and cure it at 90-95°C in a nitrogen atmosphere for 4-5 hours. Repeat the process to coat the other side of the carbon cloth.

2. The sampling method according to claim 1, characterized in that: The volume of the desorption solution is 1000-1500 μL; the desorption solution is methanol or a methanol / acetonitrile mixed solution.

3. The sampling method according to claim 1, characterized in that: The extraction is performed for 60 to 80 minutes.

4. The sampling method according to claim 1, characterized in that: The desorption process is carried out for 10 to 15 minutes.

5. Use of the sampling method according to any one of claims 1 to 4 in on-site sampling and analysis of environmental water samples.

6. Use of the sampling method according to any one of claims 1 to 4 in non-target screening analysis of low-abundance pollutants in environmental water samples.

7. A non-target screening and analysis method for low-abundance pollutants in water, characterized in that: The following steps are involved: S1. Pre-treating the environmental water sample according to any one of the sampling methods of claims 1 to 4 to obtain a sample solution to be tested; S2. The test sample solution obtained in step S1 was subjected to non-targeted scanning analysis of the test sample by liquid chromatography-mass spectrometry in positive and negative modes to obtain raw data; S3. The raw data obtained in step S2 is preprocessed to obtain a CSV file containing the accurate mass-to-charge ratio of the characteristic peaks in the mass spectrometry data, the retention time and the peak response value in each component; S4. For the CSV file obtained in step S3, a target-MS / MS method was established based on the retention time and mass-to-charge ratio. Secondary mass spectrometry signal spectra were obtained at a collision energy of 10-40 V and compared with the database to confirm the compounds with the fragments that matched.

8. The method according to claim 7, characterized in that: The method further includes using a standard of the compound whose fragments are confirmed to match in step S4, repeating steps S2 to S4, obtaining primary mass spectrometry and secondary mass spectrometry results, comparing them with the retention time and mass spectrometry fragment ions in the mass spectrometry data obtained from the sample to be tested, and ultimately determining the screened compound at the Level 1 level; the Level 1 level is matching the secondary mass spectrum and retention time of the standard.

9. The method according to claim 7, characterized in that: The step S1 is to pre-treat the environmental water samples using any sampling method described in claims 1 to 4 to obtain a sample solution to be tested, and the sample solution to be tested is subjected to steps S2 and S3, and the obtained CSV file is subjected to single factor analysis, data filtering, data standardization and normalization, principal component analysis, and difference fold analysis, and a volcano plot is drawn to screen and obtain mass spectrometry characteristic peaks with higher expression, establish a target-MSMS method for the mass spectrometry characteristic peaks according to retention time and mass-to-charge ratio, obtain a secondary mass spectrometry signal spectrum at a collision energy of 10 to 40 V, compare with the database to confirm the compound with the fragment matching, and identify and confirm the compound according to the method described in claim 8.

Images