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

Abstract

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

Description

Technical Field

[0001] The present invention belongs to the technical field of environmental analytical chemistry, and in particular 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) broadly refer to various organic analogs containing phosphorus in their molecular structures, including organophosphorus, organophosphonium, and organophosphine compounds. Organophosphate esters (OPEs) are of greatest concern as environmental pollutants. Studies have shown that organophosphates have a range of toxic effects on aquatic and terrestrial organisms, particularly endocrine disruption, reproductive and developmental toxicity, and neurotoxicity. In addition to OPEs, other organic phosphorus compounds with diverse molecular structures may be present in the environment. For example, quaternary phosphonium salts (QPSs), phosphine oxides (POs), organophosphonates (OPNs), and organothiophosphates (OTPEs) account for approximately 15% of China's total production of organic phosphorus compounds. However, limited knowledge of novel organic phosphorus compounds beyond OPEs has hindered a thorough understanding of their environmental occurrence 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 low-abundance compounds are easily missed, resulting in information loss and low data coverage. Data-independent acquisition (DIA) can simultaneously capture all ions in the sample, ensuring high coverage and detection of low-abundance compounds. However, the resulting mass spectra are highly complex, with multiple precursor ions and their fragment ions intermingled in the same isolation window, making interpretation more difficult.

[0004] In addition, it is crucial to identify priority pollutants based on their residue levels and risk characteristics. However, the risk characteristics of pollutants can vary significantly under different standards, which poses a challenge to prioritizing pollutants. Summary of the Invention

[0005] In response to 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:

[0007] Obtaining an initial exclusion list and an inclusion list, wherein the exclusion list is suitable for storing the first mass spectrum data of the blank sample, and the inclusion list is suitable for storing the second mass spectrum data of the environmental sample to be tested;

[0008] The third mass spectrum data after the single injection of the environmental sample to be tested is collected in a data-dependent acquisition mode;

[0009] determining target mass spectrum data not in the exclusion list according to the third mass spectrum data after the single injection, and updating the exclusion list according to the target mass spectrum data to obtain an updated exclusion list;

[0010] Repeating the operations of acquiring and determining target mass spectrum data until the target mass spectrum data satisfies a preset condition relative to the number of the inclusion list, thereby obtaining data-dependent acquired data, wherein the data-dependent acquired data includes the target mass spectrum data of all injections;

[0011] 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;

[0012] 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.

[0013] 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:

[0014] The above-mentioned comprehensive identification method is used to identify the phosphorus-containing organic compounds contained in the environmental sample to be tested;

[0015] 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.

[0016] Based on the above technical solution, the method for comprehensive identification and integrated risk assessment of phosphorus-containing organic compounds in the environment provided by the present invention has at least one of the following beneficial effects.

[0017] (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 as possible in the environmental sample to be tested, multiple data-dependent acquisitions are performed, and after each acquisition, the first mass spectrometry data in the exclusion list is updated according to the collected target mass spectrometry data. The acquisition 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 the 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. Through multiple data-dependent acquisitions, 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 is performed on phosphorus-containing organic compounds in the environment, thereby achieving accurate, rapid and high-throughput identification of phosphorus-containing organic compounds in complex environmental media.

[0018] (2) In embodiments of the present invention, the comprehensive risk assessment of identified phosphorus-containing organic compounds in environmental samples can be performed without relying on toxicity testing, which facilitates 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 prioritizing the risks of unknown phosphorus-containing organic compounds with existing analytical techniques and can provide technical support for environmental monitoring and chemical regulation. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] 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;

[0020] 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;

[0021] Figure 3 Flowchart of the comprehensive identification method of phosphorus-containing organic compounds in Example 1 of the present invention;

[0022] Figure 4 This is a workflow diagram of the optimized data-dependent acquisition mode in Example 1 of the present invention;

[0023] Figure 5 is the pretreatment recovery rate of the phosphorus-containing organic compound in the aqueous medium in Example 2 of the present invention;

[0024] 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;

[0025] Figure 7These are the 12 phosphorus-containing organic compounds with the highest risk priority in Example 4 of the present invention. DETAILED DESCRIPTION

[0026] 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.

[0027] During the implementation of this invention, it was discovered that improving the recognition efficiency of high-resolution mass spectrometry is key to comprehensively identifying phosphorus-containing organic compounds in the environment. The key to achieving this is improving the data coverage of the data-dependent acquisition (DDA) model to ensure the detection of low-abundance compounds. To address this challenge, the present invention utilizes multiple injections, continuously adjusting the primary mass spectrometric data from the exclusion list in the DDA method with each injection, significantly improving mass spectrometric data coverage. This optimized data-dependent acquisition model enables accurate, rapid, and high-throughput comprehensive identification of unknown phosphorus-containing organic compounds in complex environmental media.

[0028] Furthermore, in chemical management, multi-criteria decision analysis (MCDA) can be used to integrate various risk criteria to prioritize hazardous chemicals. Therefore, the present invention combines MCDA with mass spectrometry to relatively rank the risk levels of phosphorus-containing organic compounds present in the actual environment.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] According to an embodiment of the present invention, the first mass spectrometry data includes primary mass spectrometry data for all compounds in a blank sample; the second mass spectrometry data includes primary mass spectrometry data for all compounds in the environmental sample to be tested. The primary mass spectrometry data for all compounds in the blank sample and the environmental sample to be tested are first obtained. However, since determining the structure of the compounds requires matching the secondary mass spectrometry data of the compounds, the structural information of the compounds in the environmental sample to be tested cannot be determined at this stage.

[0033] S102: Collect the third mass spectrum data after the first injection of the environmental sample to be tested in a data-dependent acquisition mode.

[0034] According to an embodiment of the present invention, the third mass spectral data includes primary mass spectral data of the compound collected after a single injection and secondary mass spectral data corresponding to the primary mass spectral data. The mass spectral data collected during the data-dependent acquisition process is the primary mass spectral data and the corresponding secondary mass spectral data that are in the inclusion list but not in the exclusion list, thereby achieving comprehensive acquisition of compounds with different abundances in the environmental sample to be tested.

[0035] S103: Determine target mass spectrum data that is not in the exclusion list based on the third mass spectrum data after one injection, and update the exclusion list based on the target mass spectrum data to obtain an updated exclusion list.

[0036] According to an embodiment of the present invention, this step S103 specifically includes sub-steps S1031 - S1033 .

[0037] 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.

[0038] 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.

[0039] S1033: Add the primary mass spectrum data in the target mass spectrum data to the exclusion list to obtain an updated exclusion list.

[0040] 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 the inclusion list, thereby obtaining data-dependent collected data, wherein the data-dependent collected data includes the target mass spectrum data of all injections.

[0041] According to an embodiment of the present invention, the preset condition includes: if 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.

[0042] In an embodiment of the present invention, primary mass spectrometry data for all compounds in a test environmental sample is first acquired. Multiple data-dependent acquisitions are then performed on the test environmental sample. An exclusion list is updated based on the target mass spectrometry data, ensuring that the mass spectrometry data collected during each acquisition is for compounds with higher abundance that are not on the updated exclusion list. The acquisition of compounds in the test environmental sample is concluded when the number of collected mass spectrometry data for compounds with higher abundance falls below 10% of the inclusion list, and data-dependent acquisition ceases.

[0043] S105: 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.

[0044] According to an embodiment of the present invention, a suspected target analysis database includes a suspected list of suspected target compounds suspected of having a phosphorus-containing organic compound structural skeleton, screened from public chemical databases. The suspected target compounds have a mass spectrometry characteristic peak abundance greater than 10,000, and a mass deviation set to 0.005 Da. Exemplary public chemical databases include MassBank MoNA, MassBank EU, and mzCloud. The phosphorus-containing organic compound structural skeleton includes at least one of an organophosphate (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 organophosphonate (COP(C)(OC)=O, as shown in Formula 4), an organothiophosphate (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.

[0045] Formula 1, Formula 2, Formula 3, Formula 4, Formula 5, Formula 6.

[0046] 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.

[0047] In an embodiment of the present invention, suspected target analysis of a phosphorus-containing organic compound includes collecting primary mass spectral data and secondary mass spectral data via high-resolution mass spectrometry. Specifically, collecting primary mass spectral data and secondary mass spectral data via high-resolution mass spectrometry includes performing suspected target analysis using an optimized data-dependent acquisition method. The secondary mass spectral data of the compound collected using the data-dependent acquisition method is compared with the structural information and mass spectral information of the suspected target compound in a suspected target analysis database to obtain a confirmed or probable structure of the first candidate compound.

[0048] In an embodiment of the present invention, the abundance of the compound in the environmental sample to be tested is different, and there will be a situation where low-abundance compounds are 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 level of the abundance of the compound in the environmental sample to be tested, until the preset conditions are met, so as to collect as much information as possible on the compound 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, a suspected target analysis database is established, and high-throughput suspected target analysis is performed on phosphorus-containing organic compounds in the environment, realizing accurate, rapid and high-throughput identification of phosphorus-containing organic compounds in complex environmental media.

[0049] Furthermore, in order to achieve more comprehensive identification of phosphorus-containing organic compounds in the environmental sample to be tested, the present invention also provides a non-target analysis method based on the above-mentioned suspected target analysis.

[0050] 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.

[0051] Specifically, according to other embodiments of the present invention, a non-target analysis method based on the above-mentioned suspected target analysis is provided, wherein 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.

[0052] S201: Acquire a database of characteristic fragment ions of phosphorus-containing organic compounds.

[0053] 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 the phosphorus-containing organic compound standards includes steps S2011-S2013.

[0054] S2011: Obtain a standard solution of a phosphorus-containing organic compound and prepare the standard solution using a polar organic solvent (e.g., methanol, acetonitrile, water, etc.).

[0055] S2012: Full scan and data-dependent acquisition modes were used to obtain mass spectrometric data of phosphorus-containing organic compound standards. 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.

[0056] S2013: Summarize the common fragment ions of phosphorus-containing organic compound standards as the characteristic fragment ions of phosphorus-containing organic compounds.

[0057] S202: Collect the environmental sample to be tested in a data-independent collection mode to obtain data-independent collection data.

[0058] According to an embodiment of the present invention, the data independent acquisition mode acquisition method is set to be 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 Scanning. With this acquisition method, precursor ions are labeled according to the liquid phase retention times of their corresponding characteristic fragment ions.

[0059] S203: performing non-target analysis on the data-independent collected 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.

[0060] In an embodiment of the present invention, non-targeted analysis of phosphorus-containing organic compounds includes acquiring secondary mass spectral data via high-resolution mass spectrometry and identifying compounds based on characteristic fragment ions. Specifically, acquiring secondary mass spectral data via high-resolution mass spectrometry includes performing non-targeted analysis using an optimized data-independent acquisition method. This non-targeted analysis involves labeling the liquid phase elution time of the precursor ion in a data-independent acquisition mode based on the mass spectral fragmentation characteristics of a standard and characteristic fragment ions summarized in the literature, based on the principle that the liquid phase elution time of the precursor ion of the phosphorus-containing organic compound and its characteristic fragment ions are identical. The molecular formula and structure of the phosphorus-containing organic compound are inferred using fragmentation prediction tools (such as MetFrag and SIRIUS-CSI:FingerID) and compound database searches (such as PubChem and ChemSpider). The secondary mass spectral data of the compound acquired using the data-independent acquisition method is analyzed based on the characteristic fragment ions to obtain the confirmed or possible structure of the second candidate compound.

[0061] In 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. Using this optimized data-independent acquisition mode, high-throughput, non-targeted analysis of phosphorus-containing organic compounds is possible, enabling accurate, rapid, and high-throughput identification of phosphorus-containing organic compounds in complex environmental media.

[0062] 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 confirmed or possible structures of the first candidate compound and the second candidate compound, and the first candidate compound and the second candidate compound are integrated to obtain more comprehensive structural information and mass spectrum information of the phosphorus-containing organic compounds in the environmental sample to be tested.

[0063] In an embodiment of the present invention, the aforementioned data-dependent and data-independent acquisition methods are illustratively performed using liquid chromatography-high-resolution mass spectrometry. Preferably, the determination is performed using an Ultimate-3000 ultra-performance liquid chromatography-Orbitrap Fusion high-resolution mass spectrometer (UPLC-HRMS). The ion source is an electrospray ionization (ESI) source, the spray voltage is 3000-3500 V, the ion transfer tube temperature is 300-350°C, the nebulization temperature is 300-350°C, and the mass m / z range is 100-800.

[0064] In an embodiment of the present invention, prior to identifying phosphorus-containing organic compounds in the environment, 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; and sewage includes one or more of domestic sewage, agricultural sewage, and industrial wastewater.

[0065] Specifically, the enrichment and extraction of phosphorus-containing organic compounds from environmental media involved pretreatment using an Oasis HLB column stacked with a Sep-Pak AC2 Plus column. Specifically, the environmental media was first filtered through a 0.7 μm glass fiber filter, and the pH was adjusted to approximately 6.5 to ensure adequate adsorption of neutral and ionizable analytes. Extraction and concentration were then performed using a 24-well solid-phase extraction device (Visiprep, 24-well, Sigma Aldrich). Prior to sample loading, the column was conditioned with 5 mL of methanol / dichloromethane (v / v = 1:1), 5 mL of methanol, and 10 mL of ultrapure water. Sample was loaded at a rate of 3-5 mL / min. After loading, the column was completely dried under nitrogen. Elution was performed six times with 6 mL of methanol and 6 mL of methanol / dichloromethane. The eluates were combined, evaporated to near dryness under nitrogen, and then reconstituted with 200 μL of methanol. Optimally, when loading, the water sample should be passed through the Oasis HLB column first, followed by the Sep-Pak AC2 Plus column. During elution, the eluent should be passed through the Sep-Pak AC2 Plus column first, followed by the Oasis HLB column, to maximize the removal of contaminants accumulated in the column. Compared to using only HLB and Multilayer columns, this method offers the best recovery and enhances adsorption of polar compounds (such as quaternary phosphonium salts).

[0066] 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 the pollutants in the environmental sample to be tested.

[0067] In embodiments of the present invention, the comprehensive risk assessment of identified phosphorus-containing organic compounds in environmental samples can be performed independently of toxicity testing, which facilitates subsequent chemical regulation and the management of priority pollutants. The method provided by the present invention for comprehensive risk assessment of phosphorus-containing organic compounds in the environment overcomes the technical difficulties of prioritizing the risks of unknown phosphorus-containing organic compounds with existing analytical techniques, and can provide technical support for environmental monitoring and chemical regulation.

[0068] According to an embodiment of the present invention, determining the risk level of pollutants in the environmental sample to be tested includes: assigning equal weights 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 the pollutants in the environmental sample to be tested based on the size of the score.

[0069] In an embodiment of the present invention, six indicators measuring the risk level of pollutants—persistence (P), bioaccumulation (B), mobility (M), toxicity (T), quantitative concentration (Q), and detection frequency (DF)—are evaluated using predictive tools. This is primarily because many phosphorus-containing organic compounds are new pollutants and experimental data are unavailable. Each risk criterion is calculated using multiple predictive tools, and the predicted value for each risk criterion is obtained by inputting the structural information of the identified phosphorus-containing compounds. The toxicity criterion includes multidimensional data on aquatic ecotoxicity, endocrine disruption effects, mutagenicity, carcinogenicity, and developmental toxicity. It is worth noting that when multiple predictive tools calculate the same risk criterion, the consensus value (i.e., the arithmetic mean) is used for risk assessment, as different model combinations cover a wider range of application areas.

[0070] The present invention provides a method for comprehensive identification and comprehensive risk assessment of phosphorus-containing organic compounds in the environment. By optimizing a high-resolution mass spectrometry analysis system and multi-criteria decision analysis, high-throughput identification and risk prioritization 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, the risks of phosphorus-containing organic compounds identified in the environment can be predicted directly through open source prediction tools without relying on toxicity assessment experiments and commercial standards, thereby achieving structural analysis and risk prioritization 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.

[0071] The present invention is further illustrated 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 can also be implemented without these specific details. Moreover, in the case of no conflict, the details in the following embodiments can be arbitrarily combined into other feasible embodiments. All instruments, consumables and reagents in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0072] In the following examples, some reagents and detection instruments are described as follows.

[0073] Reagents: Commercial standards of 24 phosphorus-containing organic compounds.

[0074] Liquid chromatography-mass spectrometry: Ultimate-3000 ultra-high performance liquid chromatography coupled with Orbitrap Fusion high-resolution mass spectrometry system (Thermo Fisher Scientific, USA); chromatographic column: ACQUITY UPLC® BEH C18 column, 1.7 μm, 2.1 mm id × 100 mm length (Waters Corporation, USA).

[0075] Example 1

[0076] This Example 1 is a laboratory test example, focusing on identifying phosphorus-containing organic compounds present in spiked samples using an optimized high-resolution mass spectrometry method.

[0077] Figure 3 Flowchart of the comprehensive identification method of phosphorus-containing organic compounds in Example 1 of the present invention.

[0078] like Figure 3 As shown, Example 1 of the present invention achieves comprehensive identification of phosphorus-containing organic compounds in a sample through steps 1 to 6.

[0079] 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).

[0080] Table 1

[0081]

[0082] Step 2: Based on the structural skeleton of phosphorus-containing organic compounds, search from the China Existing Chemical Substances Inventory and the U.S. Distributed Structure Searchable Toxicity Database. The structural skeletons of phosphorus-containing organic compounds include organophosphates (COP(O)(OC)=O), quaternary phosphonium salts (C[P+](C)(C)C), phosphine oxides (CP(C)(C)=O), organophosphonates (COP(C)(OC)=O), and organothiophosphates (COP(OC)(O)=S or COP(SC)(O)=O).

[0083] The specific steps are as follows: First, CSV files from the Chinese and US chemical databases are obtained and read using the Pandas library in Python. Next, the RDKit library is used to normalize the chemical molecular structures and perform substructure searches. Each molecule is normalized using RDKit, retaining only the largest molecular fragments. Molecular weights are calculated for these fragments, and molecules with molecular weights between 100 and 800 are selected. Next, a substructure search is performed using the structural skeletons of the aforementioned phosphorus-containing organic compounds (represented in SMILES format), selecting molecules containing these substructures. The final results are saved as a new CSV file as a list of suspected phosphorus-containing organic compounds.

[0084] Step 3: Use the suspected list established in step 2 as the 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.

[0085] Figure 4 This is a workflow diagram of the optimized data-dependent acquisition mode in Example 1 of the present invention.

[0086] like Figure 4 As shown, the mixed standard solution was analyzed by optimized data-dependent acquisition (DDA) mode, with three injections and the exclusion list adjusted each time to collect as much mass spectrometric data as possible. The high-resolution mass spectrometric data were then processed using the patRoon 2.3.1 platform. Specifically, the mass spectrometric 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 spectral data from 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 using characteristic fragment ions.

[0087] Step 4: Targeted analysis was performed on the standards to summarize the fragmentation patterns of phosphorus-containing organic compounds. Published fragment ions were summarized by searching core collections such as Web of Science, resulting in a total of 36 characteristic fragment ions, as shown in Table 2. In mass spectrometry-independent acquisition (DIA) mode, non-targeted analysis based on characteristic fragment markers was used to identify homologues of phosphorus-containing organic compounds to address omissions from the list of suspected target chemicals.

[0088] Table 2

[0089]

[0090]

[0091] Step 5: Non-target analysis based on characteristic fragment markers can be performed in MS 1 and MS 2 Scans alternate 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).

[0092] Step 6: Prepare 200 μL of a solution containing the 24 phosphorus-containing organic compound standards listed in Table 1, using the same mobile phase as the initial injection (e.g., methanol, acetonitrile, or water). Analyze the solution using an Ultimate-3000 ultra-high performance liquid chromatography coupled to an Orbitrap Fusion high-resolution mass spectrometry system.

[0093] Liquid chromatography conditions: Column temperature 35°C; mobile phase consisting of methanol (A) and water (B). To enhance the ionization of phosphorus-containing organic compounds in mass spectrometry, 0.1% formic acid or 1 mM ammonium acetate can be added to either solution, depending on their properties. The mobile phase gradient elution procedure was as follows: first, 5% methanol, 95% water for 1 minute; then, increasing methanol to 100% over 2 minutes; then, maintaining 100% methanol for 5 minutes; then, decreasing methanol to 5% over 0.1 minutes; and finally, maintaining 5% methanol, 95% water for 5 minutes. The mobile phase flow rate was 0.3 mL / min; the injection volume was 5–10 μL.

[0094] Mass spectrometry conditions: the ion source was in electrospray ionization (ESI) mode; 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 nebulizer temperature was 350°C; and the sheath gas and auxiliary gas pressures were 35 Arb and 10 Arb, respectively.

[0095] Optimize Data Dependent Acquisition (DDA) Parameters: The initial exclusion list is based on the MS in blank samples (methanol in this example). 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 collect the MS in the sample in detail. 2 Data information. Among them, MS 1 The data were acquired by 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 a resolution of 30,000 FWHM and a collision energy gradient.

[0096] Data Independent Acquisition (DIA) Parameters: Set the acquisition method to 1 and MS 2 Scans alternate between cycles, each cycle consisting of one MS 1 scan, followed by five MS 2 Seven DIA methods were developed, each covering 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, and 700-800 m / z). Each method contained 20 isolation windows of 5 m / z width, with a window overlap of 1 m / z to prevent the omission of compounds at the edges of the isolation windows.

[0097] Through the above-mentioned specific steps, all 24 phosphorus-containing organic compounds in the methanol standard solution were fully identified, with an identification rate of 100%. This demonstrates that the comprehensive identification method can accurately identify phosphorus-containing organic compounds present in the sample. Both the suspected target analysis in step 3 and the non-target analysis in step 5 correctly identified the 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 environmental media.

[0098] Example 2

[0099] 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 was to test the enrichment ability of the sample pretreatment process for phosphorus-containing organic compounds in the aqueous environmental medium.

[0100] The procedure was essentially the same as in Example 1, except that, in step 6, 500 mL of water sample was filtered through a 0.7 μm glass fiber filter and the pH was adjusted to approximately 6.5 to ensure adequate adsorption of neutral and ionizable analytes. Extraction and concentration were then performed using a 24-well solid-phase extraction device. Prior to sample loading, the column was primed with 5 mL of methanol / dichloromethane (v / v = 1:1), 5 mL of methanol, and 10 mL of ultrapure water. Sample was loaded at a rate of 3-5 mL / min. After loading, the column was completely dried under nitrogen. Elution was performed in six steps using 6 mL of methanol and 6 mL of methanol / dichloromethane. The eluates were combined and evaporated to near dryness under nitrogen, then reconstituted with 200 μL of methanol for subsequent instrumental analysis.

[0101] Figure 5 is the pretreatment recovery rate of the phosphorus-containing organic compound in the aqueous medium in Example 2 of the present invention.

[0102] like Figure 5 As shown in the analysis results, all 24 phosphorus-containing organic compounds were identified, demonstrating that this comprehensive identification method is suitable for complex environmental media and is less susceptible to matrix interference. Furthermore, compared to methods using only HLB cartridges and Multilayer cartridges, this method offers the best recovery rates and significantly increases the adsorption of polar compounds, such as quaternary phosphonium salts.

[0103] Example 3

[0104] The purpose of Example 3 is to test the recognition capability of the comprehensive identification method of the present invention for unknown phosphorus-containing organic compounds in environmental samples. The environmental samples collected are surface water samples from estuaries in China.

[0105] The operation steps were basically the same as those in Example 1. The difference from Example 1 was that in step 6, the surface water sample was pretreated, including water sample filtration, pH adjustment, HLB superimposed Sep AC2 column solid phase extraction, and rotary evaporation nitrogen blowdown re-dissolution. 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.

[0106] Figure 6This is a flow chart of the high-resolution mass spectrometric analysis of unknown phosphorus-containing organic compounds in Example 3 of the present invention.

[0107] like Figure 6 As shown, the 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 ion of a quaternary phosphonium analog, leading to the identification of a novel quaternary phosphonium salt (PubChem CID 2733850) at a retention time (RT) of 10.75 minutes. This was the first compound detected in environmental media using this comprehensive identification method. This demonstrates the method's high efficiency in identifying unknown phosphorus-containing organic compounds, filling a gap in existing pollutant monitoring inventories.

[0108] Example 4

[0109] The purpose of this Example 4 is to test the risk priority ranking of identified phosphorus-containing organic compounds using multi-criteria decision analysis.

[0110] An open-source prediction tool was used to calculate the risk properties of the 64 phosphorus-containing organic compounds identified in Example 3, including their persistence (P), bioaccumulation (B), mobility (M), and toxicity (T). Additionally, 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 assigned equal weights to calculate the final score for each compound. Lower scores indicate higher risk priorities.

[0111] The toxicity (T) criterion includes multidimensional data on aquatic ecotoxicity (i.e., acute toxicity to algae, daphnia, and fish), endocrine disruption effects (i.e., estrogen, and androgen, and thyroid hormone receptor binding activity), mutagenicity, carcinogenicity, and developmental toxicity. It is worth noting that when multiple prediction tools calculate the same risk criterion, the consensus value (i.e., the arithmetic mean) is used for risk assessment.

[0112] Figure 7 These are the 12 phosphorus-containing organic compounds with the highest risk priority in Example 4 of the present invention.

[0113] like Figure 7As shown, the ranked arylphosphine oxides and arylquaternary phosphoniums have a similarly high risk level as organophosphates and therefore require greater attention. This demonstrates that this method can quickly screen potential risk-priority pollutants in the environment through calculations without relying on experimental data, which is beneficial for chemical regulation and environmental protection.

[0114] 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 to 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 period of time; and it can obtain priority pollutants that need to be focused on supervision based on 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.

[0115] 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 are only specific embodiments of the present invention and are 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: Obtaining an initial exclusion list and an inclusion list, wherein the exclusion list is applicable to storing first mass spectrum data of a blank sample, the first mass spectrum data including primary mass spectrum data of all compounds in the blank sample, and the inclusion list is applicable to storing second mass spectrum data of an environmental sample to be tested, the second mass spectrum data including primary mass spectrum data of all compounds in the environmental sample to be tested; Acquiring third mass spectrum data after a single injection of the environmental sample to be tested in a data-dependent acquisition mode, wherein the third mass spectrum data includes primary mass spectrum data of the compound acquired after the single injection and secondary mass spectrum data corresponding to the primary mass spectrum data; filtering the primary mass spectrum data not in the exclusion list from the third mass spectrum data to obtain fourth mass spectrum data; determining target mass spectrum data according to the fourth mass spectrum data, wherein 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; updating the exclusion list according to the target mass spectrum data, adding the primary mass spectrum data in the target mass spectrum data to the exclusion list to obtain an updated exclusion list; Repeating the operations of acquiring and determining target mass spectrum data until the amount of primary mass spectrum data in the target mass spectrum data is less than 10% of the inclusion list, stopping the data-dependent acquisition, and obtaining data-dependent acquired data, wherein the data-dependent acquired data includes 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 in the environmental sample to be tested that matches the suspected target compound, and obtain the phosphorus-containing organic compound contained in the environmental sample to be tested.

2. The method according to claim 1, further comprising: Obtain a database of characteristic fragment ions of phosphorus-containing organic compounds; Collecting the environmental sample to be tested 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 obtain the phosphorus-containing organic compound contained in the environmental sample to be tested.

3. The method according to claim 2, wherein: The characteristic fragment ion database is obtained by performing target analysis on phosphorus-containing organic compound standards and screening a public chemical database.

4. The method according to claim 3, wherein: The target analysis of the phosphorus-containing organic compound standard comprises: Obtain standard solutions 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.

5. 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 structure 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.

6. A method for comprehensive risk assessment of phosphorus-containing organic compounds in the environment, comprising: Identify the phosphorus-containing organic compounds contained in the environmental sample to be tested using the comprehensive identification method described in any one of claims 1 to 5; 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.

7. The method for comprehensive risk assessment according to claim 6, wherein: Determining the risk level of pollutants in the environmental sample to be tested includes: Persistence, bioaccumulation, mobility, toxicity, quantitative concentration, and detection frequency are given equal weights to calculate the final score of each pollutant in the environmental sample to be tested. The risk level of the pollutant in the environmental sample to be tested is determined by the size of the score.

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