Method for comprehensive identification and comprehensive risk assessment of phosphorus-containing organic compounds in environment

Abstract

The invention provides a method for comprehensive identification and comprehensive risk assessment of phosphorus-containing organic compounds in an environment, and belongs to the technical field of environmental analytical chemistry. The method for comprehensively identifying the phosphorus-containing organic compounds in the environment comprises the following steps: acquiring an initial exclusion list and an inclusion list; collecting mass spectrum data of the environmental sample to be detected through a data dependent collection mode; determining target mass spectrum data not in the exclusion list, and updating the exclusion list according to the target mass spectrum data; repeating the operation of collecting and determining the target mass spectrum data until a preset condition is met, and obtaining data dependent collection data; obtaining a suspected target analysis database of the phosphorus-containing organic compound; and performing suspected target analysis on the data dependent acquisition data based on the suspected target analysis database, determining a determined or possible structure of a first candidate compound matched with the suspected target compound in the environmental sample to be detected, and obtaining the phosphorus-containing organic compound contained in the environmental sample to be detected.

Description

Technical Field

[0001] The invention belongs to the technical field of environmental analytical chemistry, and specifically relates to a method for comprehensive identification and comprehensive risk assessment of phosphorus-containing organic compounds in the environment. Background Art

[0002] Organic phosphorus compounds (OPCs) refer to various organic analogs containing phosphorus in their molecular structures, including organophosphorus compounds, organophosphonium compounds, and organophosphine compounds. Among them, organophosphate esters (OPEs) are the most concerned environmental pollutants. Studies have found that organophosphate esters have a series of toxic effects on aquatic and terrestrial organisms, especially endocrine disruption, reproductive and developmental toxicity, and neurotoxicity. In addition to OPEs, other organic phosphorus compounds with different molecular structures may exist in the environment. For example, quaternary phosphonium salts (QPS), phosphine oxides (PO), organophosphonates (OPNs), and organothiophosphates (OTPEs) account for about 15% of the total production of organic phosphorus compounds in China. However, the limited understanding of new organic phosphorus compounds other than OPEs has hindered a thorough understanding of their environmental incidence and exposure risks.

[0003] The development of mass spectrometry acquisition methods is the key to comprehensively identifying phosphorus-containing organic compounds in the environment. Traditional methods usually use target analysis methods based on low-resolution mass spectrometry, which makes it difficult to achieve structural identification of unknown phosphorus-containing organic compounds in the environment. With the popularization and application of high-resolution mass spectrometry, analysis methods based on electrostatic field orbital trap mass spectrometry (Orbitrap MS) have been widely developed and have been applied to the identification of environmental pollutants. At present, the data acquisition modes of this high-resolution mass spectrometry can be divided into two types, one is data-dependent acquisition (DDA) and the other is data-independent acquisition (DIA). Among them, data-dependent acquisition (DDA) can capture high-abundance ions and provide high-quality primary mass spectra (MS 1 ) data and secondary mass spectrometry (MS 2 ) data, but its disadvantage is that it is easy to miss low-abundance compounds, resulting in information loss and low data coverage. Data-independent acquisition (DIA) can capture all ions in the sample at the same time, ensuring high coverage and detection of low-abundance compounds, but the resulting mass spectrum is highly complex, with multiple precursor ions and their fragment ions mixed in the same isolation window, making it difficult to interpret the spectrum.

[0004] In addition, it is also crucial to identify priority pollutants based on their residue levels and risk characteristics of organic phosphorus compounds. However, the risk characteristics of pollutants can vary greatly under different standards, which poses a challenge to determining pollutant priorities. Summary of the invention

[0005] In view of the above technical problems, the present invention provides a method for comprehensive identification and comprehensive risk assessment of phosphorus-containing organic compounds in the environment, in order to at least partially solve the above technical problems. The specific technical solutions provided by the present invention are as follows.

[0006] As a first aspect of the present invention, a method for comprehensively identifying phosphorus-containing organic compounds in an environment is provided, comprising: Obtaining an initial exclusion list and an inclusion list, wherein the exclusion list is applicable to storing the first mass spectrum data of a blank sample, and the inclusion list is applicable to storing the second mass spectrum data of an environmental sample to be tested; The third mass spectrum data after the first injection of the environmental sample to be tested is collected by a data-dependent collection mode; Determine target mass spectrum data not in the exclusion list according to the third mass spectrum data after one injection, and update the exclusion list according to the target mass spectrum data to obtain an updated exclusion list; Repeating the operations of collecting and determining target mass spectrum data until the target mass spectrum data meets a preset condition relative to the number of included lists, thereby obtaining data-dependent collected data, wherein the data-dependent collected data includes the target mass spectrum data of all injections; Obtaining a suspected target analysis database of phosphorus-containing organic compounds, wherein the suspected target analysis database stores structural information and mass spectrum information of the suspected target compounds; Based on the suspected target analysis database, suspected target analysis is performed on the data-dependent collected data to determine the definite or possible structure of the first candidate compound matching the suspected target compound in the environmental sample to be tested, and the phosphorus-containing organic compound contained in the environmental sample to be tested is obtained.

[0007] As a second aspect of the present invention, a method for comprehensive risk assessment of phosphorus-containing organic compounds in an environment is provided, comprising: The above-mentioned comprehensive identification method is used to identify the phosphorus-containing organic compounds contained in the environmental sample to be tested; The persistence, bioaccumulation, mobility, toxicity, quantitative concentration and detection frequency of the phosphorus-containing organic compounds are evaluated to determine the risk level of pollutants in the environmental samples to be tested.

[0008] Based on the above technical solution, the method for comprehensive identification and comprehensive risk assessment of phosphorus-containing organic compounds in the environment provided by the present invention has at least one of the following beneficial effects.

[0009] (1) In the embodiment of the present invention, the abundance of compounds in the environmental sample to be tested is different, and low-abundance compounds may be missed. In order to collect as many compounds in the environmental sample to be tested as possible, multiple data-dependent collections are performed, and after each collection, the first mass spectrometry data in the exclusion list is updated according to the collected target mass spectrometry data. Collection is performed according to the abundance of compounds in the environmental sample to be tested until the preset conditions are met, so as to collect as much information as possible on the compounds in the environmental sample to be tested, avoid missing low-abundance compounds in the environmental sample to be tested, and significantly improve the coverage of mass spectrometry data. The method for comprehensive identification of phosphorus-containing organic compounds in the environment provided by the present invention has simple operation steps. Through multiple data-dependent collections, phosphorus-containing compounds in the environment can be comprehensively identified without relying on commercial standards. A suspected target analysis database is established, and high-throughput suspected target analysis of phosphorus-containing organic compounds in the environment is performed, so as to achieve accurate, rapid and high-throughput identification of phosphorus-containing organic compounds in complex environmental media.

[0010] (2) In the embodiments of the present invention, the comprehensive risk assessment of the identified phosphorus-containing organic compounds in the environmental samples to be tested can be performed without relying on toxicity experiments, which is helpful for subsequent chemical regulation and control of priority pollutants. The method for comprehensive risk assessment of phosphorus-containing organic compounds in the environment provided by the present invention overcomes the technical difficulties of prior analytical technology in prioritizing the risks of unknown phosphorus-containing organic compounds, and can provide technical support for environmental monitoring and chemical regulation. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 A flow chart of a method for comprehensive identification of phosphorus-containing organic compounds in an environment according to an embodiment of the present invention;

[0012] Figure 2 A flow chart of a method for comprehensive identification of phosphorus-containing organic compounds in an environment provided in another embodiment of the present invention;

[0013] Figure 3 is a flow chart of a comprehensive identification method of phosphorus-containing organic compounds in Example 1 of the present invention;

[0014] Figure 4 This is a workflow diagram of the optimized data-dependent acquisition mode in Example 1 of the present invention;

[0015] Figure 5 is the pretreatment recovery rate of the phosphorus-containing organic compound in the aqueous medium in Example 2 of the present invention;

[0016] Figure 6 This is a flow chart of high-resolution mass spectrometry analysis of unknown phosphorus-containing organic compounds in Example 3 of the present invention;

[0017] Figure 7These are the 12 phosphorus-containing organic compounds with the highest risk priority in Example 4 of the present invention. DETAILED DESCRIPTION

[0018] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings.

[0019] In the process of realizing the present invention, it was found that how to improve the recognition efficiency of high-resolution mass spectrometry is the key to comprehensively identifying phosphorus-containing organic compounds in the environment. Among them, the breakthrough of the data-dependent acquisition (DDA) mode lies in how to improve its data coverage and ensure the detection of low-abundance compounds. To solve this problem, the present invention uses multiple injections to continuously adjust the primary mass spectrometry data of the exclusion list in the DDA method for each injection, thereby significantly improving the coverage of the mass spectrometry data. Using the optimized data-dependent acquisition mode, unknown phosphorus-containing organic compounds in complex environmental media can be comprehensively identified accurately, quickly and with high throughput.

[0020] Furthermore, in terms of chemical management, various risk criteria can be integrated through multi-criteria decision analysis (MCDA) to determine the priority of hazardous chemicals. Therefore, the present invention combines multi-criteria decision analysis with mass spectrometry to relatively rank the risk levels of phosphorus-containing organic compounds present in the actual environment.

[0021] Figure 1 This is a flow chart of a method for comprehensive identification of phosphorus-containing organic compounds in an environment according to an embodiment of the present invention.

[0022] Specifically, according to some embodiments of the present invention, a method for comprehensively identifying phosphorus-containing organic compounds in an environment is provided, such as Figure 1 As shown, steps S101-S106 are included.

[0023] S101: Acquire an initial exclusion list and an inclusion list, wherein the exclusion list is suitable for storing the first mass spectrum data of a blank sample, and the inclusion list is suitable for storing the second mass spectrum data of an environmental sample to be tested.

[0024] According to an embodiment of the present invention, the first mass spectrum data includes the primary mass spectrum data of all compounds in the blank sample; the second mass spectrum data includes the primary mass spectrum data of all compounds in the environmental sample to be tested. First, the primary mass spectrum data of all compounds in the blank sample and the environmental sample to be tested are obtained respectively. Since the determination of the structure of the compound also requires the matching of the secondary mass spectrum data of the compound, the structural information of the compound in the environmental sample to be tested cannot be determined at this time.

[0025] S102: Collect the third mass spectrum data after the first injection of the environmental sample to be tested in a data-dependent acquisition mode.

[0026] According to an embodiment of the present invention, the third mass spectrum data includes the primary mass spectrum data of the compound collected after one injection and the secondary mass spectrum data corresponding to the primary mass spectrum data. The mass spectrum data collected during the data-dependent acquisition process is the primary mass spectrum data and the corresponding secondary mass spectrum data in the inclusion list but not in the exclusion list, so as to achieve comprehensive acquisition of compounds with different abundances in the environmental sample to be tested.

[0027] S103: Determine target mass spectrum data that is not in the exclusion list according to the third mass spectrum data after one injection, and update the exclusion list according to the target mass spectrum data to obtain an updated exclusion list.

[0028] According to an embodiment of the present invention, this step S103 specifically includes sub-steps S1031 - S1033 .

[0029] S1031: Filter the primary mass spectrum data that is not in the exclusion list from the third mass spectrum data to obtain fourth mass spectrum data.

[0030] S1032: Determine target mass spectrum data according to the fourth mass spectrum data, where the target mass spectrum data includes the primary mass spectrum data in the fourth mass spectrum data and the secondary mass spectrum data corresponding to the primary mass spectrum data in the fourth mass spectrum data.

[0031] S1033: Add the primary mass spectrum data in the target mass spectrum data to the exclusion list to obtain an updated exclusion list.

[0032] S104: Repeat the operations of collecting and determining target mass spectrum data until the target mass spectrum data meets a preset condition relative to the number of included lists, thereby obtaining data-dependent collected data, wherein the data-dependent collected data includes the target mass spectrum data of all injections.

[0033] According to an embodiment of the present invention, the above-mentioned preset conditions include: when the amount of primary mass spectrometry data in the target mass spectrometry data is less than 10% of the inclusion list, data-dependent acquisition is stopped.

[0034] In an embodiment of the present invention, the primary mass spectrometry data of all compounds in the environmental sample to be tested is first obtained, and then the environmental sample to be tested is subjected to multiple data-dependent acquisitions, and the exclusion list is updated according to the target mass spectrometry data, so that the mass spectrometry data collected each time is the mass spectrometry data of the higher abundance compounds outside the updated exclusion list. When the mass spectrometry data of the higher abundance compounds collected is less than 10% of the inclusion list, it is determined that the acquisition of the compounds in the environmental sample to be tested is completed, and the data-dependent acquisition is stopped.

[0035] S105: Acquire a suspected target analysis database of phosphorus-containing organic compounds, wherein the suspected target analysis database stores structural information and mass spectrum information of the suspected target compounds.

[0036] According to an embodiment of the present invention, the suspected target analysis database includes a suspected list of suspected target compounds suspected of having a phosphorus-containing organic compound structural skeleton screened from a public chemical database. Wherein, the mass spectrometry characteristic peak abundance of the suspected target compound is greater than 10,000, and the mass deviation is set to 0.005Da. Exemplarily, the public chemical database includes MassBank MoNA, MassBank EU and mzCloud. The phosphorus-containing organic compound structural skeleton includes at least one of an organic phosphate (COP(O)(OC)=O, as shown in Formula 1), a quaternary phosphonium salt (C[P+](C)(C)C, as shown in Formula 2), a phosphine oxide (CP(C)(C)=O, as shown in Formula 3), an organic phosphonate (COP(C)(OC)=O, as shown in Formula 4), and an organic thiophosphate (COP(OC)(O)=S, as shown in Formula 5, or COP(SC)(O)=O, as shown in Formula 6). There is no restriction on the types of R1, R2, R3 and R4. As long as the screened compound contains the structural skeleton shown in Formula 1 to Formula 6, it will be included in the suspected target analysis database. Formula 1, Formula 2: Formula 3, Formula 4: Formula 5, Formula 6.

[0037] S106: Perform suspected target analysis on the data-dependent collected data based on the suspected target analysis database to determine the definite or possible structure of the first candidate compound matching the suspected target compound in the environmental sample to be tested, and obtain the phosphorus-containing organic compound contained in the environmental sample to be tested.

[0038] In an embodiment of the present invention, the suspected target analysis of the phosphorus-containing organic compound includes collecting primary mass spectrometry data and secondary mass spectrometry data by high-resolution mass spectrometry. Specifically, collecting primary mass spectrometry data and secondary mass spectrometry data by high-resolution mass spectrometry includes using an optimized data-dependent acquisition method to perform suspected target analysis. The secondary mass spectrometry data of the compound collected by the data-dependent acquisition mode is compared with the structural information and mass spectrometry information of the suspected target compound in the suspected target analysis database, thereby obtaining a confirmed or possible structure of the first candidate compound.

[0039] In an embodiment of the present invention, the abundance of compounds in the environmental sample to be tested is different, and low-abundance compounds may be missed. In order to collect as many compounds as possible in the environmental sample to be tested, multiple data-dependent collections are performed, and after each collection, the first mass spectrometry data in the exclusion list is updated according to the collected target mass spectrometry data, and the collection is performed according to the abundance of the compounds in the environmental sample to be tested until the preset conditions are met, so as to collect as much information as possible on the compounds in the environmental sample to be tested, avoid missing the detection of low-abundance compounds in the environmental sample to be tested, and significantly improve the coverage of mass spectrometry data. The method for comprehensive identification of phosphorus-containing organic compounds in the environment provided by the present invention has simple operating steps, and through multiple data-dependent collections, phosphorus-containing compounds in the environment can be comprehensively identified, independent of commercial standards, and a suspected target analysis database is established. High-throughput suspected target analysis of phosphorus-containing organic compounds in the environment is performed, and accurate, rapid and high-throughput identification of phosphorus-containing organic compounds in complex environmental media is achieved.

[0040] Furthermore, in order to achieve more comprehensive identification of phosphorus-containing organic compounds in the environmental samples to be tested, the present invention also provides a non-target analysis method based on the above-mentioned suspected target analysis.

[0041] Figure 2 This is a flow chart of a method for comprehensive identification of phosphorus-containing organic compounds in an environment provided in another embodiment of the present invention.

[0042] Specifically, according to some other embodiments of the present invention, a non-target analysis method based on the above-mentioned suspected target analysis is provided, and after completing the above-mentioned suspected target analysis, a non-target analysis is performed on the environmental sample to be tested, such as Figure 2 As shown, steps S201-S203 are included.

[0043] S201: Acquire a database of characteristic fragment ions of phosphorus-containing organic compounds.

[0044] According to an embodiment of the present invention, the characteristic fragment ion database is obtained by performing target analysis on phosphorus-containing organic compound standards and screening a public chemical database, wherein the target analysis on phosphorus-containing organic compound standards includes steps S2011-S2013.

[0045] S2011: Obtain standard solution of phosphorus-containing organic compounds and prepare the standard solution using polar organic solvents (e.g. methanol, acetonitrile, water, etc.).

[0046] S2012: The mass spectrometry data of the phosphorus-containing organic compound standards were obtained using full scan and data-dependent acquisition modes, and the fragment ions of the phosphorus-containing organic compound standards were obtained using high energy collision dissociation (HCD) mode; the ion source used was an electrospray ionization (ESI) source.

[0047] S2013: Summarize the common fragment ions of phosphorus-containing organic compound standards as characteristic fragment ions of phosphorus-containing organic compounds.

[0048] S202: Collect the environmental samples to be tested in a data-independent collection mode to obtain data-independent collection data.

[0049] According to an embodiment of the present invention, the data independent acquisition mode acquisition method is set to be in the MS 1 Scan and MS 2 The acquisition is cycled back and forth between scans, and each cycle includes an MS 1 Scan and five MS 2 Scan. With this acquisition method, precursor ions are labeled according to the liquid phase retention times of their corresponding characteristic fragment ions.

[0050] S203: performing non-target analysis on the data-independent acquisition data based on the characteristic fragment ion database to determine the definite or possible structure of the second candidate compound in the environmental sample to be tested that is different from the first candidate compound, and obtaining the phosphorus-containing organic compound contained in the environmental sample to be tested.

[0051] In an embodiment of the present invention, non-target analysis of phosphorus-containing organic compounds includes collecting secondary mass spectrometry data by high-resolution mass spectrometry and identifying compounds according to characteristic fragment ions. Specifically, collecting secondary mass spectrometry data by high-resolution mass spectrometry includes using an optimized data-independent acquisition method to perform non-target analysis. The above-mentioned non-target analysis refers to marking the liquid phase elution time of the precursor ion based on the same liquid phase elution time of the precursor ion of the phosphorus-containing organic compound and its characteristic fragment ion in the data-independent acquisition mode according to the mass spectrometry fragmentation characteristics of the standard and the characteristic fragment ions summarized in the literature, and inferring the molecular formula and structure of the phosphorus-containing organic compound through fragmentation prediction tools (such as MetFrag and SIRIUS-CSI: FingerID) and compound database searches (such as PubChem and Chemspider). The secondary mass spectrometry data of the compound collected by the data-independent acquisition data is analyzed based on the characteristic fragment ions, thereby obtaining the confirmed or possible structure of the second candidate compound.

[0052] In the embodiments of the present invention, by summarizing the characteristic fragment ions of phosphorus-containing organic compounds, the precursor ions corresponding to the fragment ions can be efficiently identified in a data-independent acquisition mode. By using the optimized data-independent acquisition mode, high-throughput non-target analysis of phosphorus-containing organic compounds can be performed, and phosphorus-containing organic compounds in complex environmental media can be accurately, quickly and highly identified.

[0053] In an embodiment of the present invention, a data-dependent acquisition mode and a data-independent acquisition mode are combined to identify phosphorus-containing organic compounds in the environmental sample to be tested through suspected target analysis and non-target analysis, to obtain the definite or possible structures of the first candidate compound and the second candidate compound, and to integrate the first candidate compound and the second candidate compound to obtain more comprehensive structural information and mass spectrometry information of the phosphorus-containing organic compounds in the environmental sample to be tested.

[0054] In an embodiment of the present invention, illustratively, the above-mentioned data-dependent acquisition method and data-independent acquisition method are performed by liquid chromatography-high resolution mass spectrometry. Preferably, the Ultimate-3000 ultra-high performance liquid chromatography-Orbitrap Fusion high-resolution mass spectrometer (UPLC-HRMS) is used for determination. The ion source is an electrospray ionization (ESI) source, the spray voltage is 3000-3500V, the ion transfer tube temperature is 300-350°C, the atomization temperature is 300-350°C, and the mass number m / z ranges from 100-800.

[0055] In an embodiment of the present invention, before identifying phosphorus-containing organic compounds in the environment, the phosphorus-containing organic compounds in the environmental medium are enriched and extracted. The environmental medium includes one or more of surface water, groundwater, rainwater, and sewage; surface water includes one or more of fresh water, seawater, and salt lakes; groundwater includes one or more of shallow groundwater and deep groundwater; sewage includes one or more of domestic sewage, agricultural sewage, and industrial wastewater.

[0056] Specifically, the enrichment and extraction of phosphorus-containing organic compounds in environmental media include: pre-treatment of environmental media by stacking Oasis HLB columns with Sep-Pak AC2 Plus columns. Specifically, the environmental media was first filtered using a 0.7 μm glass fiber filter, and then the pH was adjusted to about 6.5 to ensure sufficient adsorption capacity for neutral and ionizable analytes. Then, a 24-well solid phase extraction device (Visiprep, 24-well, Sigma Aldrich) was used for extraction and concentration. Before loading, the column was activated with 5 mL of methanol / dichloromethane (v=1:1), 5 mL of methanol, and 10 mL of ultrapure water in sequence. The sample was loaded at a rate of 3-5 mL per minute. After loading, the column was completely dried under nitrogen. Elution was performed six times using 6 mL of methanol and 6 mL of methanol / dichloromethane. After the eluates were combined, they were evaporated to near dryness under nitrogen and then re-dissolved with 200 μL of methanol. Preferably, when loading, the water sample should first pass through the Oasis HLB column, then through the Sep-Pak AC2 Plus column; and when eluting, the eluent should first pass through the Sep-Pak AC2 Plus column, then through the Oasis HLB column, to ensure that the pollutants enriched in the column can be eluted to the greatest extent. Compared with the method of using only HLB columns and Multilayer columns, this method has the best recovery rate and can increase the adsorption of polar compounds (such as quaternary phosphonium salts).

[0057] As a second aspect of the present invention, a method for comprehensive risk assessment of phosphorus-containing organic compounds in the environment is provided, comprising: using the above-mentioned comprehensive identification method to identify the phosphorus-containing organic compounds contained in the environmental sample to be tested; evaluating the persistence, bioaccumulation, mobility, toxicity, quantitative concentration, and detection frequency of the phosphorus-containing organic compounds contained therein to determine the risk level of pollutants in the environmental sample to be tested.

[0058] In an embodiment of the present invention, the comprehensive risk of the identified phosphorus-containing organic compounds in the environmental samples to be tested can be assessed without relying on toxicity experiments, which is helpful for subsequent chemical supervision and control of priority pollutants. The method for comprehensive risk assessment of phosphorus-containing organic compounds in the environment provided by the present invention overcomes the technical difficulties of prior analysis technology in prioritizing the risks of unknown phosphorus-containing organic compounds, and can provide technical support for environmental monitoring and chemical supervision.

[0059] According to an embodiment of the present invention, determining the risk level of pollutants in the environmental sample to be tested includes: assigning the same weight to persistence, bioaccumulation, mobility, toxicity, quantitative concentration, and detection frequency to calculate the final score of each pollutant in the environmental sample to be tested, and determining the risk level of pollutants in the environmental sample to be tested based on the size of the score.

[0060] In an embodiment of the present invention, six indicators for measuring the risk level of pollutants, namely persistence (P), bioaccumulation (B), mobility (M), toxicity (T), quantitative concentration (Q), and detection frequency (DF), are evaluated by prediction tools, mainly because many phosphorus-containing organic compounds are new pollutants and experimental data are not available. Each risk criterion is calculated using multiple prediction tools, and the predicted value of each risk criterion is obtained by inputting the structural information of the identified phosphorus-containing compounds. Among them, the toxicity criterion includes multidimensional data on aquatic ecotoxicity, endocrine disruption effects, mutagenicity, carcinogenicity, and developmental toxicity. It is worth noting that when there are calculation results of multiple prediction tools for the same risk criterion, the consensus value (i.e., the arithmetic mean) is used for risk assessment because different model combinations cover a wider range of application areas.

[0061] The present invention provides a method for comprehensive identification and comprehensive risk assessment of phosphorus-containing organic compounds in the environment. By optimizing the high-resolution mass spectrometry analysis system and multi-criteria decision analysis, high-throughput identification and risk priority ranking of phosphorus-containing organic compounds in environmental media are achieved. The present invention overcomes the omission of unknown phosphorus-containing organic compounds by existing analytical technologies. By combining high-resolution mass spectrometry analysis and multi-criteria decision analysis, it is possible to predict the risks of phosphorus-containing organic compounds identified in the environment directly through open source prediction tools without relying on toxicity assessment experiments and commercial standards, thereby achieving structural analysis and risk priority ranking of unknown phosphorus-containing organic compounds in environmental media, and achieving high-throughput comprehensive identification of phosphorus-containing organic compounds in environmental media, which can provide technical support for environmental monitoring of phosphorus-containing organic compounds and chemical regulation.

[0062] The present invention is further described below by examples and related test experiments. In the detailed description below, for ease of explanation, many specific details are set forth to provide a comprehensive understanding of the embodiments of the present invention. However, it is obvious that one or more embodiments may also be implemented without these specific details. And, in the absence of conflict, the details in the following embodiments may be combined into other feasible embodiments at will. All instruments, consumables and reagents in the following examples, if not otherwise specified, may be obtained from commercial sources.

[0063] In the following examples, some reagents and detection instruments are described as follows.

[0064] Reagents: Commercial standards of 24 organic phosphorus-containing compounds.

[0065] Liquid chromatography-mass spectrometry: Ultimate-3000 ultra-high performance liquid chromatography and Orbitrap Fusion high-resolution mass spectrometry system (Thermo Fisher Scientific, USA); Chromatographic column: ACQUITY UPLC® BEH C18 column, 1.7μm, 2.1mm id×100 mm length (Waters Corporation, USA).

[0066] Example 1

[0067] This Example 1 is a laboratory test example, which focuses on identifying phosphorus-containing organic compounds present in spiked samples using an optimized high-resolution mass spectrometry analysis method.

[0068] Figure 3 This is a flow chart of the comprehensive identification method of phosphorus-containing organic compounds in Example 1 of the present invention.

[0069] like Figure 3 As shown, Example 1 of the present invention achieves comprehensive identification of phosphorus-containing organic compounds in a sample through steps one to six.

[0070] Step 1: Summarize the 24 traditional organic phosphorus compounds (OPCs) found in the environment through text mining as shown in Table 1. These 24 traditional organic phosphorus compounds (OPCs) cover the molecular structures of three types of organic phosphorus compounds, namely 16 triesters of phosphorus (tri-OPEs), 5 quaternary phosphonium salts (QPSs) and 3 phosphine oxides (POs).

[0071] Table 1

[0072] Step 2: According to the structural skeleton of phosphorus-containing organic compounds, search from the China Existing Chemical Substances List and the US Distributed Structure Searchable Toxicity Database. Among them, the structural skeleton of phosphorus-containing organic compounds includes organic phosphates (COP(O)(OC)=O), quaternary phosphonium salts (C[P+](C)(C)C), phosphine oxides (CP(C)(C)=O), organic phosphonates (COP(C)(OC)=O) and organic thiophosphates (COP(OC)(O)=S or COP(SC)(O)=O).

[0073] The specific operation is as follows: first, obtain the CSV files of the Chinese and American chemical databases, and use the Pandas library in python to read the CSV files. Then, use the RDKit library to perform standardization and substructure search of chemical molecular structures. Each molecule is standardized by RDKit, only the largest molecular fragment is retained, and the molecular weight is calculated to screen out molecules with a molecular weight between 100 and 800. Next, use the structural skeleton of the above-mentioned phosphorus-containing organic compounds (represented in SMILES format) to perform a substructure search, screen out molecules containing these substructures, and save the final results as a new CSV file as a suspected list of phosphorus-containing organic compounds.

[0074] Step 3: Use the suspected list established in step 2 as a suspected target analysis database, and perform “MS-Ready” standardization on the phosphorus-containing organic compounds in the suspected list to obtain their theoretical monoisotopic masses.

[0075] Figure 4 This is a workflow diagram of the optimized data-dependent acquisition mode in Example 1 of the present invention.

[0076] like Figure 4 As shown, the mixed standard solution was analyzed by optimized data-dependent acquisition (DDA) mode, and three injections were performed, each time the exclusion list was adjusted to collect as much mass spectrometric data as possible. Then, the high-resolution mass spectrometric data were processed using the patRoon 2.3.1 platform. Specifically, the mass spectrometry raw files were converted to mzML format by ProteoWizard, and the OpenMS algorithm was used for feature detection, grouping, and alignment. In this process, the suspected features were obtained by matching the exact mass of the measured features with the theoretical monoisotopic masses (MIMs) in the suspected list. The noise intensity threshold was set to 10,000 and the mass deviation was set to 0.005 Da. The MS 2 The data were compared with mass spectrometry data in public mass spectral libraries (i.e., MassBank MoNA, MassBank EU, and mzCloud) to annotate the structures of the suspected compounds. For the remaining suspected compounds not found in the public mass spectral libraries, their structures were elucidated with the help of characteristic fragment ions.

[0077] Step 4: Target analysis was performed on the standard and the fragmentation patterns of phosphorus-containing organic compounds were summarized. The published fragment ions were summarized by searching core collections such as Web of Science, and a total of 36 characteristic fragment ions were summarized as shown in Table 2. In the mass spectrometry-independent acquisition mode (DIA), non-target analysis based on characteristic fragment markers was used to identify homologues of phosphorus-containing organic compounds to make up for the omissions in the list of suspected target chemicals.

[0078] Table 2

[0079] Step 5: Non-target analysis based on characteristic fragment markers can be performed in MS 1 and MS 2 Scans are alternated between cycles, each cycle consisting of one MS 1 scan, followed by five MS 2 Scan. Therefore, the precursor ions of phosphorus-containing organic compounds can be labeled with characteristic fragment ions according to the liquid phase retention time. The candidate molecular formula of the precursor ion is calculated using the Genform algorithm, and the elemental composition range is limited to C 0-100 H 0-200 O 0-10 N 0-10 P 0-3 S 0-3 F 0-10 Cl 0-10 Br 0-10 The molecular formula and structure were inferred by fragmentation prediction tools (such as MetFrag and SIRIUS-CSI:FingerID) and compound database searches (such as PubChem and ChemSpider).

[0080] Step 6: Prepare 200 μL of a solution containing 24 phosphorus-containing organic compound standards in Table 1, where the solution is consistent with the initial mobile phase during injection (for example, methanol, acetonitrile, and water), and then analyze it on an Ultimate-3000 ultra-high performance liquid chromatography and Orbitrap Fusion high-resolution mass spectrometry system.

[0081] Liquid chromatography conditions: chromatographic column temperature is 35°C; the mobile phase consists of methanol solution (A) and aqueous solution (B). In order to enhance the ionization ability of phosphorus-containing organic compounds in mass spectrometry, 0.1% formic acid or 1mM ammonium acetate can be added to the two solutions according to their properties. The mobile phase gradient elution program is: first, 5% methanol and 95% water are maintained for 1 minute; methanol is increased to 100% within 2 minutes; then 100% methanol is maintained for 5 minutes; then methanol is reduced to 5% within 0.1 minutes; finally, 5% methanol and 95% water are maintained for 5 minutes; the mobile phase flow rate is 0.3mL / min; the injection volume is 5~10μL.

[0082] Mass spectrometry conditions: the ion source adopted electrospray ionization mode (ESI); the positive ion spray voltage was 3500 V, and the negative ion spray voltage was −2500 V; the ion transfer tube temperature was 350 °C; the nebulization temperature was 350 °C; the sheath gas and auxiliary gas pressures were 35 Arb and 10 Arb, respectively.

[0083] Optimize Data Dependent Acquisition (DDA) Parameters: The initial exclusion list was based on the MS 1 The inclusion list is based on the MS of a mixed solution of 24 phosphorus-containing organic compound standards. 1 The data information is established. Then, DDA is repeated three times, that is, the sample is injected three times. After each DDA acquisition, the MS in the inclusion list and exclusion list are adjusted. 1 Data information, that is, the MS collected in the previous data-dependent manner 1 The data information should be included in the exclusion list of the next acquisition to exhaustively collect the MS 2 Data information. Among them, MS 1 The data were acquired by an electrostatic field orbital trap with a resolution of 120,000 FWHM and a scan range of m / z = 100-800; MS 2 Data were acquired by high energy collision dissociation (HCD) with the resolution set to 30,000 FWHM and the collision energy set to gradient energy.

[0084] Data Independent Acquisition (DIA) Parameters: Set the acquisition method to 1 and MS 2 Scans are alternated between cycles, each cycle consisting of one MS 1 scan, followed by five MS 2 A total of 7 DIA methods were established, each with a mass range of approximately 100 m / z (i.e., 100-200 m / z, 200-300 m / z, 300-400 m / z, 400-500 m / z, 500-600 m / z, 600-700 m / z, 700-800 m / z). Each method contained 20 isolation windows of 5 m / z width, and the window overlap was set to 1 m / z to prevent the omission of compounds at the edge of the isolation window.

[0085] Through the above specific steps, all 24 phosphorus-containing organic compounds in the methanol standard solution were fully identified, with an identification rate of 100%. This shows that the comprehensive identification method can accurately identify the phosphorus-containing organic compounds present in the sample. Among them, step three suspected target analysis and step five non-target analysis both achieved the correct identification of phosphorus-containing organic compounds in the sample. The two comprehensive identification methods complement each other and can ensure the comprehensive identification of phosphorus-containing organic compounds in the environmental medium.

[0086] Example 2

[0087] Similar to the implementation process of Example 1, the spiked methanol standard solution was replaced with a spiked 500 mL pure water sample. The purpose of this Example 2 is to test the enrichment ability of the sample pretreatment process for phosphorus-containing organic compounds in the aqueous environmental medium.

[0088] The operation steps are basically the same as those in Example 1, except that in step 6, 500 mL of water sample is filtered using a 0.7 μm glass fiber filter, and then the pH is adjusted to about 6.5 to ensure that neutral and ionizable analytes have sufficient adsorption capacity. Then, a 24-well solid phase extraction device is used for extraction and concentration. Before loading, the column is activated with 5 mL of methanol / dichloromethane (v=1:1), 5 mL of methanol, and 10 mL of ultrapure water in sequence. Load the sample at a rate of 3-5 mL per minute. After loading, the column is completely dried under nitrogen. Elution is performed six times using 6 mL of methanol and 6 mL of methanol / dichloromethane. After the eluate is combined, it is evaporated to near dryness under nitrogen, and then re-dissolved with 200 μL of methanol for subsequent instrumental analysis.

[0089] Figure 5 It is the pretreatment recovery rate of the phosphorus-containing organic compound in the aqueous medium in Example 2 of the present invention.

[0090] like Figure 5 As shown in the figure, the analysis results show that all 24 phosphorus-containing organic compounds were identified, indicating that this comprehensive identification method is suitable for complex environmental media and is less affected by matrix interference. In addition, compared with the method of using only HLB columns and Multilayer columns, this method has the best recovery rate and can significantly increase the adsorption of polar compounds (such as quaternary phosphonium salts).

[0091] Example 3

[0092] The purpose of this Example 3 is to test the recognition capability of the comprehensive recognition method of the present invention for unknown phosphorus-containing organic compounds in environmental samples. The collected environmental samples are surface water samples from Chinese estuaries.

[0093] The operation steps are basically the same as those in Example 1. The difference from Example 1 is that in step 6, the surface water sample is pre-treated, including water sample filtration, pH adjustment, HLB superimposed Sep AC2 column solid phase extraction and rotary evaporation nitrogen blowing re-dissolution procedure. The results showed that a total of 64 phosphorus-containing organic compounds were identified, including 13 new quaternary phosphonium salts and 4 new phosphine oxides. (2-Carboxyethyl)triphenylphosphonium (abbreviated as CarbEtPh3P + ) as an example.

[0094] Figure 6 This is a flow chart of high-resolution mass spectrometry analysis of unknown phosphorus-containing organic compounds in Example 3 of the present invention.

[0095] like Figure 6 As shown, four characteristic fragment ions (i.e. [C6H5P] + , [C 12 H8P] + , [C 12 H 10 P] + and [C 18 H 15 P] + ) was used to label the precursor ions of quaternary phosphonium analogs, thereby identifying a new quaternary phosphonium salt (i.e., PubChem CID 2733850) at a retention time (RT) of 10.75 minutes, which was the first compound detected in environmental media using this comprehensive identification method. This shows that this method can efficiently identify unknown phosphorus-containing organic compounds and fill the gaps in the existing pollutant monitoring list.

[0096] Example 4

[0097] The purpose of this Example 4 is to test the risk priority ranking of identified phosphorus-containing organic compounds using multi-criteria decision analysis.

[0098] The risk properties of the 64 organic phosphorus compounds identified in Example 3 were calculated using an open source prediction tool, including their persistence (P), bioaccumulation (B), mobility (M), and toxicity (T). In addition, the quantitative concentration (Q) and detection rate (DF) of the pollutants were included as indicators of exposure intensity and spatial distribution behavior, respectively. The P, B, M, T, Q, and DF criteria were given equal weights to calculate the final score for each compound. The lower the score, the higher the risk priority.

[0099] Among them, the toxicity (T) standard includes multi-dimensional data on aquatic ecotoxicity (i.e. acute toxicity to algae, daphnia and fish), endocrine disruption effects (i.e. estrogen, androgen and thyroid hormone receptor binding activity), mutagenicity, carcinogenicity, and developmental toxicity. It is worth noting that when there are calculation results of multiple prediction tools for the same risk criterion, the consensus value (i.e. arithmetic mean) is used for risk assessment.

[0100] Figure 7 These are the 12 phosphorus-containing organic compounds with the highest risk priority in Example 4 of the present invention.

[0101] like Figure 7 As shown in the results, the ranked arylphosphine oxides and arylquaternary phosphoniums have a high risk level similar to that of organophosphates, and therefore require more attention. This shows that this method can quickly screen out potential risk priority pollutants in the environment through calculations without relying on experimental data, which is beneficial to the regulation of chemicals and environmental protection.

[0102] The results of the above embodiments show that the method for comprehensive identification and comprehensive risk assessment of phosphorus-containing organic compounds in the environment of the present invention combines the advantages of high-resolution mass spectrometry and multi-criteria decision analysis, and can be applied in a variety of complex environmental media; it can accurately identify and analyze the structure of phosphorus-containing organic compounds in the environment; it has fast and high-throughput characteristics, and can complete the comprehensive identification of large-scale environmental samples in a short time; and it can obtain priority pollutants that need to be focused on according to the risk properties of phosphorus-containing organic compounds. Therefore, the method for comprehensive identification and comprehensive risk assessment of phosphorus-containing organic compounds in the environment of the present invention has universal applicability and broad application prospects.

[0103] The specific embodiments described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for comprehensively identifying phosphorus-containing organic compounds in the environment, comprising: Acquire an initial exclusion list and an inclusion list, wherein the exclusion list is suitable for storing the first mass spectrum data of a blank sample, and the inclusion list is suitable for storing the second mass spectrum data of an environmental sample to be tested; Collecting the third mass spectrum data after the first injection of the environmental sample to be tested in a data-dependent acquisition mode; Determine target mass spectrum data not in the exclusion list according to the third mass spectrum data after the one injection, and update the exclusion list according to the target mass spectrum data to obtain an updated exclusion list; Repeating the operations of collecting and determining target mass spectrum data until the target mass spectrum data meets a preset condition relative to the number of the inclusion list, thereby obtaining data-dependent collected data, wherein the data-dependent collected data includes the target mass spectrum data of all injections; Acquiring a suspected target analysis database of phosphorus-containing organic compounds, wherein the suspected target analysis database stores structural information and mass spectrum information of the suspected target compounds; Based on the suspected target analysis database, the data-dependent collected data is subjected to suspected target analysis to determine the definite or possible structure of the first candidate compound matching the suspected target compound in the environmental sample to be tested, and obtain the phosphorus-containing organic compound contained in the environmental sample to be tested.

2. The method according to claim 1, wherein: The first mass spectrum data includes primary mass spectrum data of all compounds in the blank sample; The second mass spectrum data includes primary mass spectrum data of all compounds in the environmental sample to be tested; The third mass spectrum data includes the primary mass spectrum data of the compound collected after the one injection and the secondary mass spectrum data corresponding to the primary mass spectrum data.

3. The method according to claim 2, wherein: The step of determining target mass spectrum data not in the exclusion list according to the third mass spectrum data after the one injection, and updating the exclusion list according to the target mass spectrum data to obtain an updated exclusion list comprises: Filtering the primary mass spectrum data not in the exclusion list from the third mass spectrum data to obtain fourth mass spectrum data; Determine target mass spectrum data according to the fourth mass spectrum data, wherein the target mass spectrum data includes primary mass spectrum data in the fourth mass spectrum data and secondary mass spectrum data corresponding to the primary mass spectrum data in the fourth mass spectrum data; The primary mass spectrum data in the target mass spectrum data is added to the exclusion list to obtain an updated exclusion list.

4. The method according to claim 1 or 3, wherein: The preset conditions include: If the amount of primary mass spectrometry data in the target mass spectrometry data is less than 10% of the inclusion list, the data-dependent acquisition is stopped.

5. The method according to claim 1, further comprising: Obtain the characteristic fragment ion database of phosphorus-containing organic compounds; The environmental sample to be tested is collected through a data-independent collection mode to obtain data-independent collection data; Based on the characteristic fragment ion database, non-target analysis is performed on the data-independent acquisition data to determine the definite or possible structure of the second candidate compound in the environmental sample to be tested that is different from the first candidate compound, and the phosphorus-containing organic compound contained in the environmental sample to be tested is obtained.

6. The method according to claim 5, wherein: The characteristic fragment ion database is obtained by performing target analysis on phosphorus-containing organic compound standards and screening a public chemical database.

7. The method according to claim 6, wherein: The target analysis of the phosphorus-containing organic compound standard comprises: Obtain standard solution of phosphorus-containing organic compounds; Obtaining a mass spectrum of the phosphorus-containing organic compound standard using a full scan and data-dependent acquisition mode, and obtaining fragment ions of the phosphorus-containing organic compound standard using a high-energy collision dissociation mode; The common fragment ions of the phosphorus-containing organic compound standards are summarized as characteristic fragment ions of phosphorus-containing organic compounds.

8. The method according to claim 1, wherein: The suspected target analysis database includes: A suspected list consisting of suspected target compounds suspected of having a phosphorus-containing organic compound structural skeleton screened from a public chemical database; The phosphorus-containing organic compound structural skeleton includes at least one of organic phosphates, quaternary phosphonium salts, phosphine oxides, organic phosphonates, and organic thiophosphates.

9. A method for comprehensive risk assessment of phosphorus-containing organic compounds in the environment, comprising: Using the comprehensive identification method described in any one of claims 1 to 8 to identify the phosphorus-containing organic compounds contained in the environmental sample to be tested; The persistence, bioaccumulation, mobility, toxicity, quantitative concentration and detection frequency of the phosphorus-containing organic compounds are evaluated to determine the risk level of the pollutants in the environmental sample to be tested.

10. The method for comprehensive risk assessment according to claim 9, wherein: Determining the risk level of pollutants in the environmental sample to be tested includes: The same weight is given to persistence, bioaccumulation, mobility, toxicity, quantitative concentration, and detection frequency to calculate the final score of each pollutant in the environmental sample to be tested, and the risk level of the pollutants in the environmental sample to be tested is determined by the size of the score.

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