CN115389690B - A comprehensive identification method for benzotriazole ultraviolet absorber pollutants in the environment - Google Patents

Abstract

A comprehensive identification method for benzotriazole ultraviolet absorber pollutants in the environment, including obtaining a suspected targeted analysis database of benzotriazole ultraviolet absorber pollutants; performing liquid chromatography-mass spectrometry analysis on environmental samples to be tested, Obtain data-dependent acquisition data and data-independent acquisition data; perform matching analysis on the data-dependent acquisition data based on the information of suspected target compounds, and determine the structure of the compound that matches the suspected target compound; from data-independent acquisition Extract the characteristic fragment ions of benzotriazole ultraviolet absorber pollutants from the data, so as to extract candidate compounds based on the characteristic fragment ions; analyze the chromatographic information and mass spectrum information related to the candidate compounds in the data-dependent acquisition data, and determine the candidate compounds Structure. The invention realizes the comprehensive recognition of the benzotriazole ultraviolet absorber pollutants in the environment, can be applied to various complex environmental media, and has wide application prospects.

Description

A comprehensive identification method for benzotriazole ultraviolet absorber pollutants in the environment

technical field

The invention relates to the field of environmental analytical chemistry, in particular to a comprehensive identification method for benzotriazole ultraviolet absorber pollutants in the environment.

Background technique

Benzotriazole UV absorbers (BZT-UVs) are a class of artificially synthesized organic compounds that are widely used as chemical additives in plastics, coatings, textiles, paints, printing and dyeing, and building materials because they can absorb full-spectrum ultraviolet rays in natural light. , cosmetics and other fields. In the process of industrial production and daily use, BZT-UVs will inevitably enter into the environmental medium and accumulate in organisms. The environmental distribution and toxicity effects of BZT-UVs have received extensive attention. The types of known BZT-UVs in the current environment are extremely limited, and a comprehensive identification of the occurrence of BZT-UVs in environmental media is crucial to accurately assessing the health hazards and ecological risks of BZT-UVs.

Analytical methods are key to the comprehensive identification of BZT-UVs. Traditionally, BZT-UVs environmental monitoring adopts a targeted analysis method based on low-resolution mass spectrometry, which relies on real standards, making it difficult to screen and identify unknown BZT-UVs. The popular application of high-resolution mass spectrometry provides a technical means for comprehensively identifying BZT-UVs in complex environmental media. The suspected targeted and non-targeted analysis methods based on high-resolution mass spectrometry are one of the effective strategies to guide the comprehensive identification of BZT-UVs homologues. However, how to screen and identify the structure of massive high-resolution mass spectrometry data based on suspected targeted and non-targeted analysis methods is a technical problem that needs to be solved urgently.

Contents of the invention

In view of this, the main purpose of the present invention is to provide a comprehensive identification method for BZT-UVs pollutants in the environment, in order to at least partially solve at least one of the above-mentioned technical problems.

To achieve the above object, the technical scheme of the present invention is as follows:

A method for comprehensive identification of BZT-UVs pollutants in the environment, comprising the following steps: obtaining a suspected targeted analysis database of BZT-UVs pollutants, wherein the suspected targeted analysis database is constructed to store suspected targeted compounds Structural information and mass spectrometry prediction information; liquid chromatography-mass spectrometry analysis is performed on the environmental samples to be tested, and the collection of fragment ions in the mass spectrometry analysis is carried out in the data-dependent acquisition mode and data-independent acquisition mode respectively, so as to obtain the measured samples. The data-dependent collection data and the data-independent collection data of the environmental samples; performing matching analysis on the data-dependent collection data based on the structural information and mass spectrometry prediction information of the suspected target compound, and determining the The definite or possible structure of the compound matching the suspected target compound; extracting characteristic fragment ions of BZT-UVs pollutants from the data-independent collection data, so as to extract candidate compounds based on the characteristic fragment ions, Wherein, the candidate compound is distinguished from the compound matching the suspected target compound; the chromatographic information and mass spectral information related to the candidate compound in the data-dependent collection data are analyzed to determine the identity of the candidate compound possible structure.

Based on the above-mentioned technical scheme, the comprehensive identification method of BZT-UVs pollutants in the environment of the present invention has at least one or part of the following beneficial effects:

Based on the constructed suspected targeted analysis database of BZT-UVs pollutants, the present invention conducts suspected targeted analysis on the data-dependent collection data in the liquid chromatography-mass spectrometry analysis data of the environmental samples to be tested, and obtains the suspected target compound. The definite or probable structure of the matched compound, and non-targeted analysis of the data-independent acquisition data, to obtain the probable structure of the candidate compound that is different from the suspected target compound, through the two methods of suspected targeted analysis and non-targeted analysis The complementarity of these two identification methods has realized the comprehensive identification and structural identification of BZT-UVs that may exist in the environmental samples to be tested, does not rely on real standards, and can also be implemented in the absence of any reference compound information, and can be applied to complex environments In the medium, provide technical support for environmental monitoring.

Description of drawings

Fig. 1 is the flowchart of the comprehensive identification method of BZT-UVs pollutants in the environment in the present invention.

Fig. 2 is a detailed flow chart of the comprehensive identification method of BZT-UVs pollutants in the environment in Example 1 of the present invention.

Detailed ways

In order to make the object, technical solution and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings.

In the process of realizing the present invention, it is found that in the process of suspected targeted analysis, how to establish a suspected targeted analysis database is a technical difficulty in using the suspected targeted analysis strategy to identify BZT-UVs; there is no reference for non-targeted analysis The difficulty in the application of compound information lies in the screening and structure identification of the massive high-resolution mass spectrometry data collected. The suspected targeted analysis database constructed based on the structural characteristics of BZT-UVs in the present invention combines the complementary identification methods of suspected targeted analysis and non-targeted analysis to realize comprehensive identification of BZT-UVs homologues in complex environmental media. Among them, it needs to be explained in advance that the suspected target database refers to the database containing known BZT-UVs compounds that may exist in the environment, and this known BZT-UVs compounds that may exist in the environment It is a suspected target compound.

Specifically, according to some embodiments of the present invention, a method for comprehensively identifying BZT-UVs pollutants in the environment is provided, including the following steps A to E ( FIG. 1 ).

Step A: Obtain a suspected targeted analysis database of BZT-UVs pollutants, wherein the suspected targeted analysis database is constructed to store structural information and mass spectrum prediction information of suspected targeted compounds.

According to an embodiment of the present invention, in this step, the suspected target analysis database is constructed through the following steps A1-A3.

In step A1, all compounds with 2-hydroxybenzotriazole structural fragments were screened from public chemical databases.

According to the embodiment of the present invention, the public chemical data includes but not limited to the Chinese Existing Chemical Substances Inventory (IECSC), the US Toxic Substances Control Act Inventory (TSCA), the EU Chemicals Registration, Evaluation, Authorization and Restriction List (REACH) and Canadian Domestic Substance List (DSL), etc.

According to an embodiment of the present invention, in order to facilitate accurate screening of compounds, this step A1 specifically includes: calculating the matching degree between the compound to be screened in the public chemical database and the SMILES formula of 2-hydroxybenzotriazole based on the Tanimoto coefficient algorithm, that is, the similarity degree; and when the matching degree satisfies the preset condition, the compound to be screened is determined as a compound having a 2-hydroxybenzotriazole structural fragment.

A further option is to use the Python platform and the Tanimoto coefficient algorithm to calculate the matching degree. The molecular structural formula of 2-hydroxybenzotriazole C ₁₂ H ₉ N ₃ O, and its corresponding SMILES formula is C1=CC=C(C( =C1)N2N=C3C=CC=CC3=N2)O. In the field, the SMILES formula is a character string to describe the chemical structure, thereby converting the complex chemical structure formula into a computer-recognizable character string form.

As a preferred embodiment, when the matching degree is not lower than 0.7, the compound to be screened corresponding to the matching degree is determined to be a compound having a 2-hydroxybenzotriazole structural fragment. Further preferably, after the computer screening based on the aforementioned matching degree calculation, manual inspection is further performed to increase the correct rate.

In step A2, the biotransformation products of the screened compound in vivo are predicted based on the metabolic transformation pathways of phase I, phase II and phase III in vivo.

According to an embodiment of the present invention, the metabolic transformation pathway is determined based on the reported pollutants in vivo metabolic transformation pathway and BZT-UVs molecular structure characteristics, such as involving oxidation, hydrolysis, reduction, methylation, sulfation, acetylation and various a binding reaction, etc.

For example, Thermo Fisher's Compound Discoverer (version 3.3) software can be used to predict the metabolic transformation products of BZT-UVs in phase I and phase II in vivo.

In step A3, a suspected targeted analysis database is constructed based on the screened compounds and corresponding biotransformation products.

It can be understood that the screened compound and the corresponding biotransformation product are regarded as suspected target compounds, and the suspected target analysis database includes structural information and mass spectrum prediction information of the screened compound and the corresponding biotransformation product.

According to an embodiment of the present invention, the structural information includes compound names and chemical formulas, and the mass spectrum prediction information includes accurate mass numbers of adducted ions and predicted secondary fragment ion information. Optionally, the name of the compound can be, for example, the full name of the chemical name, the abbreviation of the chemical name, etc.; the chemical formula can be, for example, a molecular formula, such as C ₁₃ H ₁₁ N ₃ O, etc.; Proton accurate mass number, etc., and proton accurate mass number is more commonly used; predicting secondary fragment ion information can be predicted by MetFrag software, for example.

Step B: Perform liquid chromatography-mass spectrometry analysis on the environmental sample to be tested. Fragment ions are collected in the data-dependent acquisition mode and data-independent acquisition mode in the mass spectrometry analysis, so as to obtain the data-dependent acquisition of the environmental sample to be tested. Data and data-independent acquisition data.

According to an embodiment of the present invention, in this step B, an ultra-high liquid chromatograph and a high-resolution mass spectrometry system can be used to perform liquid chromatography-mass spectrometry analysis on the environmental samples to be tested, so as to facilitate the analysis and detection of complex environmental samples .

According to an embodiment of the present invention, the liquid chromatography conditions are configured as a gradient elution procedure, so as to better separate the environmental samples to be tested.

According to an embodiment of the present invention, the data-dependent acquisition mode of mass spectrometry is a primary ion scan and a secondary ion scan mode under positive ion conditions using an atmospheric pressure chemical ionization source, an electrospray ionization source, or an atmospheric pressure optical ionization source; The typical acquisition mode is the first-order ion scanning mode under the positive ion condition of the atmospheric pressure chemical ionization source, electrospray ionization source or atmospheric pressure optical ionization source in the mass spectrometry mode of in-source collision-induced dissociation (abbreviated as IS-CID). Among them, the ionization sources used in the data-independent acquisition mode and the data-dependent acquisition mode can be the same or different. Compared with the data-dependent acquisition data, the data in the data-independent acquisition mode can be collected by breaking the whole spectrum in the source. All compound fragment information is conducive to the comprehensive identification of BZT-UVs pollutants.

Further, the data-dependent acquisition data include primary mass spectrogram (MS ¹ ) and secondary mass spectrogram (MS ² ), MS ¹ is obtained by electrostatic field orbitrap, and the scanning range is mass-to-charge ratio m/z=100-1000, MS ¹ is used to obtain information such as the mass number of adducted ions, and MS ² is obtained through high-energy collision dissociation (HCD) mode. The scanning range depends on the mass-to-charge ratio m/z of the parent ion of MS ¹ , and is mainly used to obtain secondary fragment ions information.

According to an embodiment of the present invention, data-dependent acquisition data needs to be deconvoluted and analyzed with high-resolution mass spectrum data, and the signal intensity threshold may be, for example, 10e4.

Further, data-independent acquisition data includes MS ¹ containing all fragment ion information, MS ¹ is obtained by electrostatic field orbitrap, and the scanning range is m/z=100-1000, which is mainly used to obtain characteristic fragment ions Signal intensities and molecular formulas of all candidate compounds.

Step C: Perform matching analysis on the data-dependent collected data based on the structural information of the suspected target compound and the mass spectrometry prediction information, and determine the definite or possible structure of the compound in the environmental sample to be tested that matches the suspected target compound.

According to the embodiment of the present invention, this step C is suspected targeting analysis, by analyzing whether there is a precursor compound matching the suspected targeting compound in the environmental sample to be tested, and then obtaining the definite or possible structure of the precursor compound, the specific Substeps C1 to C3 are included.

In sub-step C1, the mass of the adducted ion in the data-dependent collection data is matched with the accurate mass of the adducted ion of the suspected target compound, and the matched suspected precursor compound is screened from the suspected target compound;

In sub-step C2, the secondary fragment ions in the data-dependent collection data are matched with the predicted secondary fragment ion information of the suspected precursor compound, and the matched precursor compound is screened from the suspected precursor compound;

In sub-step C3, the definite or possible structure of the precursor compound is determined based on the chromatographic retention behavior of the precursor compound and the secondary fragment ions, and the precursor compound is a compound that matches the suspected target compound.

Through the above sub-steps C1 to C3, the mass numbers of the adducted ions and the secondary fragment ions are matched sequentially, which is conducive to more accurately determining whether the suspected target compound in the suspected target database is contained in the sample to be tested, and then through the analysis of the characteristics Match the chromatographic retention behavior (such as chromatographic retention time, etc.) of the successful compound with the fragmentation law of the mass spectrum determined by the secondary fragment ions, and propose the definite or possible structure of the precursor compound.

Step D: Extracting characteristic fragment ions of BZT-UVs pollutants from the data-independent acquisition data, so as to extract candidate compounds based on the characteristic fragment ions, wherein the candidate compounds are distinguished from compounds matching the suspected target compounds.

According to an embodiment of the present invention, this step D is a non-targeted analysis, and the characteristic fragment ions of BZT-UVs pollutants are usually secondary fragment ions shared by BZT-UVs pollutants, thus theoretically based on the characteristic fragment ions It can be reversed to determine all BZT-UVs pollutants in the environmental sample to be tested. At the same time, in this step, only the candidate compounds that are different from the precursor compounds in step C are extracted and analyzed in subsequent steps, and the suspected targeted analysis Coupling with non-targeted analysis enables comprehensive identification of BZT-UVs pollutants while simplifying the analysis process.

According to an embodiment of the present invention, this step D specifically includes sub-steps D1 to D3.

In sub-step D1, the ion chromatogram (EIC) of characteristic fragment ions containing BZT-UVs pollutants is extracted from the data-independent acquisition data.

In sub-step D2, candidate molecular formulas corresponding to characteristic fragment ions are extracted from MS ¹ corresponding to the EIC retention time.

In sub-step D3, the candidate molecular formula which is distinguished from the precursor compound is determined as the molecular formula of the candidate compound.

Through the above sub-steps D1 to D3, based on the extracted characteristic fragment ions, find the molecular formula of its possible corresponding candidate compound, such as C ₂₀ H ₂₅ N ₃ O ₂ , and then proceed to step E to infer its possible structure based on mass spectrometry data.

According to an embodiment of the present invention, the characteristic fragment ions of BZT-UVs pollutants may include known widely detected secondary fragment ions of BZT-UVs, such as [C ₆ H ₆ N ₃ ] ⁺ (m/z= 120.0562) and [C ₁₂ H ₁₀ N ₃ O] ⁺ (m/z=212.0824), may also include characteristic fragment ions obtained through experimental analysis in the present invention, such as [C ₁₃ H ₁₀ N ₃ O] ⁺ (m/ z=224.0824) and [C ₁₅ H ₁₄ N ₃ O] ⁺ (m/z=252.1137).

Step E: Analyzing the chromatographic information and mass spectral information related to the candidate compound in the data-dependent collection data to determine the possible structure of the candidate compound.

According to an embodiment of the present invention, this step E specifically includes obtaining the chromatographic retention behavior and secondary fragment ions of the candidate compound from the data-dependent collection data to determine the possible structure of the candidate compound.

More specifically, based on the molecular formula of the candidate compound in step D, the chromatographic retention behavior and secondary fragment ions of the candidate compound are obtained from the data-dependent acquisition data, and the chromatographic retention behavior and the mass spectrometry fragmentation law based on the secondary fragment ions are analyzed. Identify plausible candidate compounds and deduce their possible structures.

According to an embodiment of the present invention, the method of the present invention further includes determining the characteristic fragment ions of BZT-UVs pollutants, specifically including the following steps F and G.

Step F: performing liquid chromatography-mass spectrometry analysis in the IS-CID mass spectrometry mode on a plurality of authentic standard substances present in the environment in the suspected targeted analysis database. As a preferred embodiment, the real standard can be selected from the BZT-UVs found in the environment through text mining. The text mining method includes but not limited to keyword extraction and other methods.

Step G: Adjust different cone voltages and analyze the characteristic fragment ion signals of multiple real standards to determine the optimal cone voltage and the corresponding characteristic fragment ions of BZT-UVs pollutants. It can be understood that the characteristic fragment ions of BZT-UVs pollutants are secondary fragment ions shared by multiple authentic standards.

According to an embodiment of the present invention, the data-independent acquisition mode in step B is preferably performed under the optimum cone voltage condition.

According to the embodiments of the present invention, the acquisition of real standards can be used to accurately determine the characteristic fragment ions of BZT-UVs pollutants on the one hand, so that non-targeted analysis can be performed more accurately, and on the other hand, it can also be treated based on real standards. Targeted analysis of compounds matched to authentic standards in the test environment.

Further, the method of the present invention also includes targeted analysis of the environmental samples to be tested, specifically including step H: performing matching analysis on the data-dependent collection data based on the chromatographic retention behavior and mass spectrum information of multiple authentic standards, and determining the Targeted compounds and amounts matched to authentic standards. Quantitative analysis of targeted compounds in environmental samples to be tested can be achieved through targeted analysis.

According to the embodiments of the present invention, the method of the present invention can be applied in a variety of complex environmental media, and the environmental samples to be tested include water samples, solid samples and biological samples containing BZT-UVs; wherein, the water samples can be industrial sewage, sewage Treatment plant influent water, river water, surface water, sea water, drinking water, ground water, etc. Solid samples can be sewage treatment plant sediment, water sludge, sediment, soil, indoor dust, atmospheric particles, etc. Biological samples can be human breast milk , urine, serum, animal organs, fish, birds, sharks, molluscs, plants, etc.

A number of specific embodiments are listed below to describe the technical solution of the present invention in detail. It should be noted that the following specific embodiments are only for illustration, and are not intended to limit the present invention.

In the following examples, some reagents and detection instruments are described as follows:

Reagents: 12 authentic standards of BZT-UVs listed in Table 1 below.

Liquid chromatography-mass spectrometry: ultra-high performance liquid chromatography and high-resolution mass spectrometry system (Ultimate-3000 liquid chromatography-Orbitrap high resolution mass spectrometry, Thermo Fisher, USA); chromatographic column: Waters ACQUITY C18 (1.7 μm , 2.1×100mm).

Example 1

This embodiment is a test embodiment in the laboratory, focusing on realizing the identification of BZT-UVs present in the spiked sample by establishing a method system, and its specific steps include (Fig. 2):

Step 1: Summarize the 16 types of BZT-UVs that have been found in the environment as shown in Table 1 through text mining, and select 12 types of BZT-UVs with higher detection rates and detection concentrations to purchase real standards.

Table 1

Step 2: Based on the public chemical databases including IECSC, TSCA, REACH and DSL, use the Python platform and the Tanimoto coefficient algorithm to calculate the structural similarity between the compound to be screened in the public chemical database and 2-hydroxybenzotriazole, and the score threshold It is set to 0.7, that is, the compounds to be screened with a similarity greater than or equal to 0.7 are determined to be compounds with 2-hydroxybenzotriazole structural fragments. Since the real standard substance falls within the scope of the compounds screened in this step, this step additionally screens to obtain 21 BZT-UVs homologues that have not purchased the real standard substance.

Step 3: Based on the known metabolic transformation pathways of 30 pollutants in organisms as shown in Table 2, use the Compound Discoverer (version 3.3) software of ThermoFisher to predict the metabolic transformation products of BZT-UVs in phase I and phase II in vivo. The established pathway covers all 20 known biotransformation products of BZT-UVs.

Table 2

Step 4: Construct the BZT-UVs homologues obtained in steps 1 to 3 as a suspected targeted analysis database (i.e. in-house database), including compound names, chemical formulas, accurate mass numbers of adducted ions, and secondary fragment ion information predicted by MetFrag software .

Step 5: Continuously inject the 12 BZT-UVs with real standards selected in step 1 into the IS-CID mass spectrometry mode, and adjust the cone voltage to 10, 20, 30, 40, 50, 60 and 70eV respectively , to compare the characteristic fragment ion signal intensities of different BZT-UVs under a series of cone voltages.

Preferably, 30eV is the optimal cone voltage for scanning characteristic fragment ions in IS-CID mass spectrometry mode, so as to satisfy all characteristic fragment ions with appropriate signal intensity.

Step 6: Prepare 1 mL of methanol solution containing 12 kinds of authentic standards, all of which have a concentration of 100 μg/L, and then inject and analyze by liquid chromatography-mass spectrometer.

Liquid chromatography conditions: the temperature of the chromatographic column is 35° C.; the mobile phase consists of methanol solution (A) containing 0.5 mM ammonium acetate and aqueous solution (B) containing 0.5 mM ammonium acetate. The mobile phase gradient elution program is as follows: firstly keep 70% A for 1 min; increase A to 100% within 14 min; then keep 100% A for 5 min; then decrease A to 70% within 0.1 min; finally keep 70% A for 4.9 min; The flow rate of the mobile phase was 0.3 mL/min; the injection volume was 5 μL.

Mass spectrometry conditions: The ion source adopts the positive ion mode of the atmospheric pressure chemical ionization source; the spray voltage is 3500V; the ion source temperature is 200°C, the ion transfer tube temperature is 350°C, and the atomization temperature is 400°C; , 1 and 10Arb. Data-dependent acquisition mode parameters: MS ¹ is acquired by electrostatic field orbitrap, the resolution is 120000 (m/z=200), the scanning range is m/z=100-1000, the maximum injection time is 100ms, and the automatic gain control target is 3e6, S-lensRF is 60%. MS ² is acquired by HCD mode, resolution is 60000 (m/z=200), collision energy is set at 10, 30, 50%, MS ¹ parent ion is isolated by quadrupole, isolation window width m/z=1, fragment Ions are detected by an electrostatic field orbitrap with a scan range dependent on the parent ion m/z. The data-independent acquisition mode adopts the IS-CID mass spectrometry mode, and the parameters are as follows: MS ¹ is obtained by electrostatic field orbitrap, the resolution is 120000 (m/z=200), the scanning range is m/z=100-1000, and the maximum injection The time is 100ms, the AGC target is 3e6, the S-lens RF is 60%, the mass range is normal, and the cone voltage is 30eV.

Step 7: Perform suspected target analysis on the data in the data-dependent acquisition mode of step 6 in Compound Discoverer (version 3.3), identify and record BZT-UVs and predict biotransformation products in the list. The process is as follows: 1. Deconvolution of high-resolution mass spectrometry data, with a signal intensity threshold of 10e4; 2. Suspected target database matching, including mass spectrometry data matching of adducted ion mass matching and secondary fragment ion matching of primary mass spectrometry, In the first-level mass spectrum matching, the mass spectrum deviation is 5ppm, the isotope threshold is 75%, and the signal-to-noise ratio is 5; 3. Analyze the chromatographic retention behavior and mass spectrum fragmentation law of the precursor compound whose characteristics are successfully matched, and propose a definite or possible structure.

Step 8: Perform non-targeted analysis on the data in the data-independent acquisition mode of step 6 in the Xcalibur Qual Browser (version 4.0) software to identify those that are not recorded in the chemical industry list and do not belong to the predicted biotransformation products, industrial intermediates or impurities The BZT-UVs. The process is as follows: 1. Extract four kinds of characteristic fragment ions, namely [C ₆ H ₆ N ₃ ] ⁺ (m/z=120.0562), [C ₁₂ H ₁₀ N ₃ O] ⁺ (m/z=212.0824), [C ₁₃ H ₁₀ N ₃ O] ⁺ (m/2=224.0824) and [C ₁₅ H ₁₄ N ₃ O] ⁺ (m/z=252.1137) EIC; 2, according to the retention time of the EIC diagram, extracted from the corresponding MS ¹ The characteristic fragment ions correspond to the molecular formula of the candidate compound, which is different from the successfully matched precursor compound in step 7; 3. Based on the molecular formula, analyze the chromatographic retention behavior and mass spectral fragmentation law of the candidate compound in the data-dependent acquisition mode, based on the mass spectral data Match plausible candidates and propose definite or probable structures.

It can be understood that, further, the BZT-UVs pollutants identified in steps 7 and 8 can also be semi-quantitatively analyzed with the help of structurally similar standards; Specifically, in this embodiment, since the sample to be tested is the real standard, the targeted analysis process will not be described in detail.

Through the above specific steps, all 12 kinds of BZT-UVs in the standard solution have been identified, with a correct rate of 100%, which shows the reliability of the identification method and can accurately identify the BZT-UVs existing in the sample. Among them, both the suspected targeted analysis in step 7 and the non-targeted analysis in step 8 have realized the correct identification of BZT-UVs in the sample. The two identification methods are complementary and can ensure the comprehensive identification of BZT-UVs in the environment.

Example 2

Similar to the implementation process of Example 1, the spiked methanol solution was replaced with spiked real environmental sediment samples. The purpose of this example is to test the ability of the method to identify BZT-UVs in environmental samples under the condition of matrix interference.

The operation steps are basically the same as those in Example 1. The difference from Example 1 is that in step 6, the actual environmental samples are pretreated, including accelerated solvent extraction, gel permeation chromatography purification, silica gel column purification, and reconstitution by swirling nitrogen blowing. program. The results showed that all 12 spiked BZT-UVs were identified, indicating that the method is suitable for complex environmental media and is less affected by matrix interference.

Example 3

The purpose of this example is to test the overall ability of the method to identify BZT-UVs in environmental samples. The collected environmental samples were biological samples of Chlamys farreri.

The operating steps are basically the same as in Example 1, except that in Step 6, the pretreatment of the Chlamys farreri sample includes accelerated solvent extraction, gel permeation chromatography purification, silica gel column purification and rotary steam nitrogen blowing. dissolve program. The results showed that a total of 21 BZT-UVs were identified, including 10 targeted BZT-UVs (UV-P, UV-PS, UV-234, UV-320, UV-326, UV-327, UV-328, UV -329, UV-350 and UV-360), 5 kinds of biotransformation products (structures shown in formula I to formula V) and 4 kinds of impurity BZT-UVs (structures shown in formula 1 to formula 7). Among them, dechlorinated and methylated BZT-UVs biotransformation products (UV-326-H and UV-327-CH ₃ ) and impurity BZT-UVs were found in environmental media for the first time through this identification method, indicating that this method can Comprehensively identify BZT-UVs in the environment, filling the technical gap in the field of BZT-UVs environmental analytical chemistry.

Formula I～Formula V: Definite or possible structures of identified BZT-UVs biotransformation products

BZT@m/z=322:

BZT@m/z=330:

BZT@m/z=340:

BZT@m/z=410:

Formulas 1 to 7: Definite or possible structures of identified impurities BZT-UVs

Example 4

The purpose of this example is to test the high-throughput ability of this method to identify BZT-UVs in large-scale environmental samples. The environmental samples collected were 129 mollusk samples covering 9 cities in China's Bohai rim region (Dalian, Yingkou, Huludao, Beidaihe, Tianjin, Shouguang, Penglai, Yantai and Weihai).

The operation steps are basically the same as those in Example 1. The difference from Example 1 is that in Step 6, the mollusk sample is pretreated, including accelerated solvent extraction, gel permeation chromatography purification, silica gel column purification, and reconstitution by rotary evaporation and nitrogen blowing. program. The entire identification system completed the analysis of all environmental samples within 24 hours, and the identified BZT-UVs with real standards were verified by standards, indicating that this method can accurately, quickly and high-throughput the identification of BZT-UVs in the environment. Comprehensive identification.

The results of the above-mentioned embodiments comprehensively show that the comprehensive identification method of BZT-UVs pollutants in the environment involved in the present invention has high accuracy, and the suspected targeted and non-targeted analysis methods are complementary; it can be applied in a variety of complex environmental media, It is less affected by matrix interference; the types of identified compounds are comprehensive, and known, unknown, and transformation products can be efficiently identified; it has fast and high-throughput characteristics, and can complete large-scale environmental sample identification in a short time. Therefore, the method of the present invention has universal applicability and wide application prospect.

The specific embodiments described above have further described the purpose, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above descriptions are only specific embodiments of the present invention, and are not intended to limit the present invention. Within the spirit and principles of the present invention, any modifications, equivalent replacements, improvements, etc., shall be included in the protection scope of the present invention.

Claims (9)

1. A comprehensive identification method for benzotriazole ultraviolet absorber class pollutants in the environment, comprising the following steps: Obtaining a suspected targeted analysis database of benzotriazole ultraviolet absorber pollutants, wherein the suspected targeted analysis database is constructed to store structural information and mass spectrum prediction information of suspected targeted compounds; Performing liquid chromatography-mass spectrometry analysis on the environmental sample to be tested, the collection of fragment ions in the mass spectrometry analysis is carried out in a data-dependent acquisition mode and a data-independent acquisition mode, so as to obtain the data-dependent acquisition data of the environmental sample to be tested and data-independent acquisition data; Perform matching analysis on the data-dependent collection data based on the structural information and mass spectrometry prediction information of the suspected targeting compound, and determine the definite or possible structure of the compound in the environmental sample to be tested that matches the suspected targeting compound ; Extract characteristic fragment ions of benzotriazole ultraviolet absorber pollutants from the data-independent collection data, so as to extract candidate compounds based on the characteristic fragment ions, wherein the candidate compounds are different from the suspected target Compounds matched to compounds; Analyzing the chromatographic information and mass spectral information related to the candidate compound in the data-dependent collection data to determine the possible structure of the candidate compound; Wherein, the suspected targeted analysis database is constructed through the following steps: Screen all compounds with 2-hydroxybenzotriazole structural fragments from public chemical databases; Predict the biotransformation products of screened compounds in vivo based on phase I, phase II and phase III metabolic transformation pathways in vivo; The suspected targeted analysis database is constructed based on the screened compounds and corresponding biotransformation products. 2. The comprehensive identification method according to claim 1, wherein the screening of all compounds with 2-hydroxybenzotriazole structural fragments from the public chemical database comprises: Calculate the matching degree of the SMILES formula between the compound to be screened and 2-hydroxybenzotriazole in the public chemical database based on the Tanimoto coefficient algorithm; When the matching degree satisfies the preset condition, the compound to be screened is determined to be a compound having a 2-hydroxybenzotriazole structural fragment. 3. The comprehensive identification method according to claim 1, characterized in that, the data-dependent acquisition mode is the primary ion scanning and Secondary ion scan mode; The data-independent acquisition mode is a primary ion scanning mode under the positive ion condition of the in-source collision-induced dissociation mass spectrometry mode. 4. The comprehensive identification method according to claim 1, wherein the structural information includes compound names and chemical formulas, and the mass spectrum prediction information includes accurate mass numbers of adducted ions and predicted secondary fragment ion information. 5. The comprehensive identification method according to claim 4, characterized in that, the data-dependent collection data is matched and analyzed based on the structural information and mass spectrum prediction information of the suspected target compound to determine the Confirmed or possible structures of compounds in environmental samples that match the suspected targeted compounds include: Matching the mass number of adducted ions in the data-dependent collection data with the accurate mass number of adducted ions of the suspected target compound, and screening the suspected targeted compound to obtain a matched suspected precursor compound; matching the secondary fragment ions in the data-dependent collection data with the predicted secondary fragment ion information of the suspected precursor compound, and screening the suspected precursor compound to obtain a matched precursor compound; The definite or possible structure of the precursor compound is determined based on the chromatographic retention behavior and secondary fragment ions of the precursor compound, and the precursor compound is a compound matching the suspected target compound. 6. The comprehensive identification method according to claim 5, wherein the characteristic fragment ions of the benzotriazole ultraviolet absorber pollutants are extracted from the data-independent collection data, so that based on the characteristic Candidate compounds for fragment ion extraction include: Extracting the ion chromatogram of characteristic fragment ions comprising benzotriazole ultraviolet absorber pollutants from the data-independent collection data; extracting candidate molecular formulas corresponding to the characteristic fragment ions from the primary mass spectrum corresponding to the retention time of the ion current diagram; A candidate molecular formula distinguishable from the precursor compound is determined as the molecular formula of the candidate compound. 7. The comprehensive identification method according to claim 1, wherein the chromatographic information and mass spectral information related to the candidate compound in the data-dependent collection data are analyzed to determine the possibility of the candidate compound Structures include: The chromatographic retention behavior and secondary fragment ions of the candidate compound are obtained from the data-dependent acquisition data to determine the possible structure of the candidate compound. 8. The comprehensive identification method according to claim 1, further comprising: performing liquid chromatography-mass spectrometry analysis in the source collision-induced dissociation mass spectrometry mode on a plurality of authentic standards present in the environment in the suspected targeted analysis database; Adjusting different cone voltages and analyzing the characteristic fragment ion signals of the plurality of true standards to determine the optimal cone voltage and the corresponding characteristic fragment ions of the benzotriazole ultraviolet absorber pollutants. 9. The comprehensive identification method according to claim 8, further comprising: Matching analysis is performed on the data-dependent collected data based on the chromatographic retention behavior and mass spectrometry information of the plurality of authentic standards, and the target compound and its content matching the plurality of authentic standards are determined.