Analytical methods for compounds that cause oxidative stress

Abstract

The present disclosure provides an analysis method for compounds with oxidative stress effects, comprising: pre-treating a sample to be analyzed to obtain a mixture to be analyzed; measuring the mixture to be analyzed by liquid chromatography-high-resolution mass spectrometry to obtain primary mass number data and secondary fragment structure data; determining a target compound from the mixture to be analyzed by using the primary mass number data, the secondary fragment structure data and a preset chemical data set, wherein the preset chemical data set contains information on compounds with oxidative stress effects; screening target compounds that meet a first preset condition from the target compounds by using a bioanalysis equivalent concentration to obtain a main contributing compound; and performing oxidative stress compound effect analysis on the main contributing compound to obtain an analysis result.

Description

Technical Field

[0001] The present disclosure relates to the technical field of environmental sample pollutant screening, and in particular to an analysis method for oxidative stress effect compounds. Background Art

[0002] Exposure to environmental pollutants can lead to an increase in reactive oxygen species and electrophilic reagents in the body, resulting in oxidative stress, which in turn causes oxidative damage to biological macromolecules such as lipids, proteins, and nucleic acids. The body's antioxidant defense system can respond adaptively to this, producing a variety of antioxidant and detoxification-related substances and enzymes. This process is called oxidative stress. Many studies have shown that oxidative stress plays an important role in adverse health outcomes (inflammation, cardiovascular and cerebrovascular diseases, respiratory diseases, etc.) caused by exposure to environmental pollutants. Therefore, oxidative stress has become an important indicator commonly used in the study of pollutant toxicity.

[0003] At present, the oxidative stress effect components identified in environmental media such as atmospheric particulate matter, dust, surface water, sewage, etc. are relatively limited, and there is a large knowledge gap. In addition, the relevant technologies for the identification and analysis of oxidative stress effect compounds in environmental media such as atmospheric particulate matter, dust, surface water, sewage, etc. generally use targeted methods, and the identification efficiency is low. Summary of the invention

[0004] In view of the above technical problems, the present disclosure provides an analysis method for oxidative stress effect compounds to at least partially solve at least one of the above technical problems.

[0005] In order to solve the above technical problems, the present disclosure provides an analysis method for oxidative stress effect compounds, comprising:

[0006] Pre-treating the sample to be analyzed to obtain a mixture to be analyzed;

[0007] Liquid chromatography-high resolution mass spectrometry is used to measure the mixture to be analyzed, and primary mass number data and secondary fragment structure data are obtained;

[0008] Determining a target compound from the mixture to be analyzed using the primary mass data, the secondary fragment structure data, and a preset chemical data set, wherein the preset chemical data set includes information on compounds having an oxidative stress effect;

[0009] Using the bioanalysis equivalent concentration to screen the target compounds that meet the first preset condition from the target compounds to obtain the main contributing compounds; and

[0010] The compound effect analysis of oxidative stress was performed on the main contributing compounds to obtain the analysis results.

[0011] According to an embodiment of the present disclosure, the sample to be analyzed includes one of a solid sample or a liquid sample;

[0012] The sample to be analyzed is pre-treated to obtain a mixture to be analyzed including:

[0013] Using the QuEChERS method to process the solid sample to obtain a mixture to be analyzed; or

[0014] The liquid sample is enriched and purified by solid phase extraction to obtain the mixture to be analyzed.

[0015] According to an embodiment of the present disclosure, the solid sample is processed using the QuEChERS method to obtain a mixture to be analyzed, including:

[0016] Using acetonitrile or acidified acetonitrile to shake and vortex to extract organic matter in the solid sample to obtain an organic supernatant;

[0017] The organic supernatant is treated to remove water and impurities, and then concentrated and redissolved to obtain a mixture to be analyzed.

[0018] According to an embodiment of the present disclosure, determining a target compound from a mixture to be analyzed using primary mass number data, secondary fragment structure data, and a preset chemical data set includes:

[0019] Identify candidate compounds from the mixture to be analyzed based on primary mass data and a preset chemical data set;

[0020] Based on the reference spectra and secondary fragment structure data of the candidate compounds, compounds that meet the second preset condition are screened from the candidate compounds to obtain the target compound.

[0021] According to an embodiment of the present disclosure, the compound information in the preset chemical data set includes the accurate mass number of the precursor ion of the compound;

[0022] Based on primary mass data and a pre-set chemical data set, candidate compounds are identified from the mixture to be analyzed including:

[0023] For each mass number in the primary mass number data, matching the accurate mass number of the precursor ion that meets the third preset condition from the preset chemical data set to obtain the accurate mass number of the candidate precursor ion, wherein the third preset condition includes that the difference between the mass number and the accurate mass number of the precursor ion is within a preset range;

[0024] The compound corresponding to the accurate mass of the candidate precursor ion is determined as the candidate compound.

[0025] According to an embodiment of the present disclosure, for each target compound in the target compounds, a target standard corresponding to the target compound is obtained;

[0026] Compare the retention time and secondary fragment structure data of the target compound with the retention time and secondary fragment structure data of the target standard to obtain a comparison result;

[0027] When the comparison result shows that the retention time and secondary fragment structure of the target compound and the target standard satisfy the fourth preset condition, the target compound is determined to be a confirmed compound;

[0028] Among them, the target compounds that meet the first preset condition are screened from the target compounds using the bioanalysis equivalent concentration, and the main contributing compounds include:

[0029] The compounds meeting the first preset condition are screened from the confirmed compounds using the bioanalysis equivalent concentration to obtain the main contributing compounds.

[0030] According to an embodiment of the present disclosure, the target compound includes n, n≥1;

[0031] Using bioanalysis equivalent concentrations, the target compounds were screened for compounds that met the first preset condition, and the main contributing compounds included:

[0032] For the i-th target compound among the target compounds, determine the i-th bioanalytical equivalent concentration corresponding to the i-th target compound, and finally obtain n bioanalytical equivalent concentrations;

[0033] The n bioassay equivalent concentrations were summed to obtain the cumulative bioassay equivalent concentration;

[0034] Calculate the ratio of the ith bioanalytical equivalent concentration to the cumulative bioanalytical equivalent concentration to obtain the ith relative effect contribution value corresponding to the ith target compound;

[0035] When it is determined that the ith relative effect contribution value satisfies the first preset condition, the ith target compound is determined to be the main contributing compound.

[0036] According to an embodiment of the present disclosure, the first preset condition includes that a single relative effect contribution value is greater than a first threshold, and the sum of the relative effect contribution values ​​of all major contributing compounds is greater than a second threshold.

[0037] According to an embodiment of the present disclosure, the main contributing compounds include m, m≥1;

[0038] Analysis of the compound effects of oxidative stress on the main contributing compounds included:

[0039] For the jth main contributing compound among the m main contributing compounds, determine the jth single effect concentration of the jth main contributing compound that induces the oxidative stress effect, and finally obtain m single effect concentrations;

[0040] Determine the effect concentration of the mixed main contribution compound that induces the oxidative stress effect to obtain a composite effect concentration, wherein the mixed main contribution compound is obtained by mixing the concentration ratios of m main contribution compounds in the sample to be analyzed;

[0041] The combination index was calculated using m single effect concentrations and combined effect concentrations;

[0042] The combined index was used to analyze the compound effects of oxidative stress on the main contributing compounds.

[0043] According to an embodiment of the present disclosure, the above-mentioned method for analyzing the oxidative stress effect of a compound further includes:

[0044] Obtaining identified compounds with oxidative stress effects corresponding to the type of sample to be analyzed;

[0045] For each of the identified compounds, when it is determined that the target compound does not contain the identified compound, the identified compound is determined as the target compound.

[0046] According to the embodiments of the present disclosure, the mixture to be analyzed is measured by using liquid chromatography-high-resolution mass spectrometry, the target compound is determined using the measurement data and the preset chemical data set, and then the main contributing compounds are screened using the bioanalysis equivalent concentration, and the main contributing compounds are analyzed for the oxidative stress compound effect. Since the suspected target screening method that uses the measurement data and the preset chemical data set to determine the target compound effectively improves the analysis throughput, overcomes the limitations of the existing targeted identification technology, enriches the understanding of oxidative stress effect pollutants in environmental samples, and provides the main contributing compounds at the same time, the compound effect analysis of the main contributing compounds can reduce the workload caused by a large number of low-contribution non-critical compounds in the traditional process. The overall process is easy to operate and promote, and can provide technical support for the discovery of oxidative stress effect pollutants. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 The flowchart of the method for analyzing the oxidative stress effect compounds according to the embodiment of the present disclosure is schematically shown;

[0048] Figure 2 is a partial schematic diagram of a preset chemical data set established in Example 1 of the present disclosure;

[0049] Figure 3 is a schematic diagram of the oxidative stress effect pollutants with a confidence level of 1 identified in Example 1 of the present disclosure;

[0050] Figure 4 is the relative effect contribution value of the main effect contributing compound identified in Example 1 of the present disclosure;

[0051] Figure 5 is a dose-effect curve diagram of the main effect contributing compound of Example 1 of the present disclosure;

[0052] Figure 6 These are the primary spectrum and secondary fragment spectrum of piperine identified in Example 2 of the present disclosure. DETAILED DESCRIPTION

[0053] In order to make the objectives, technical solutions and advantages of the present disclosure more clearly understood, the present disclosure is further described in detail below in combination with specific embodiments and with reference to the accompanying drawings.

[0054] The present invention utilizes liquid chromatography-high resolution mass spectrometry to measure the mixture to be analyzed, determines the target compound using the measurement data and a preset chemical data set, and then uses bioanalysis equivalent concentrations to screen the main contributing compounds, and performs oxidative stress compound effect analysis on the main contributing compounds.

[0055] Figure 1 The flowchart of the method for analyzing the oxidative stress effect compounds according to the embodiment of the present disclosure is schematically shown.

[0056] like Figure 1 As shown, the analysis method of oxidative stress effect compounds of this embodiment includes operations S110 to S150.

[0057] In operation S110 : pre-processing the sample to be analyzed to obtain a mixture to be analyzed.

[0058] According to an embodiment of the present disclosure, the sample to be analyzed includes a solid sample or a liquid sample. The pre-treatment of the sample to be analyzed to obtain the mixture to be analyzed includes: using the QuEChERS method to treat the solid sample to obtain the mixture to be analyzed; or using a solid phase extraction method to enrich and purify the liquid sample to obtain the mixture to be analyzed.

[0059] According to an embodiment of the present disclosure, solid samples may include, for example, soil, sediment, atmospheric particulate matter, etc.; liquid samples may include, for example, surface water, groundwater, sewage, etc.

[0060] According to the embodiments of the present disclosure, solid samples are prepared as follows: soil and sediment are dried, ground, and sieved in advance; dust is dried and sieved in advance; and the collection membrane of atmospheric particulate matter samples is cut into pieces in advance. Liquid samples are prepared as follows: liquid samples such as surface water, groundwater, and sewage are centrifuged or filtered to remove insoluble substances in advance.

[0061] According to an embodiment of the present disclosure, pre-processing the sample to be analyzed to obtain the mixture to be analyzed includes: using the QuEChERS method to process the solid sample to obtain the mixture to be analyzed; or using the solid phase extraction method to enrich and purify the liquid sample, and obtaining the mixture to be analyzed after purification using a solid phase extraction column.

[0062] According to the embodiments of the present disclosure, QuEChERS is the abbreviation of Quick, Easy, Cheap, Effective, Rugged, and Safe, and is a method for rapid sample pretreatment, which utilizes the interaction between an adsorbent and impurities in a sample matrix to adsorb the impurities and thereby achieve purification.

[0063] According to an embodiment of the present disclosure, the QuEChERS method is used to process the solid sample to obtain the mixture to be analyzed, including: using acetonitrile or acidified acetonitrile to oscillate and vortex to extract organic matter in the solid sample to obtain an organic supernatant; dewatering and removing impurities from the organic supernatant, and then concentrating and re-dissolving to obtain the mixture to be analyzed.

[0064] According to an embodiment of the present disclosure, in the process of using acetonitrile or acidified acetonitrile to oscillate and vortex to extract organic matter in a solid sample, magnesium sulfate and sodium chloride are added to remove water and promote phase separation. Magnesium sulfate and an adsorbent are then added to the organic supernatant to remove water and impurities such as pigments and metal ions, and then the supernatant is centrifuged and concentrated by nitrogen blowing, and redissolved using an organic solvent. Among them, the adsorbent can be N-propylethylenediamine or C18, and the organic solvent can be methanol.

[0065] According to an embodiment of the present disclosure, a liquid sample is processed using a solid phase extraction (SPE) method. For example, the method may include activating an HLB solid phase extraction column with deionized water and then loading the sample, controlling the flow rate, eluting with methanol for two rounds, and then eluting with methanol: dichloromethane (1:1) for two rounds, and then re-dissolving with methanol after the eluted liquid nitrogen is concentrated.

[0066] The QuEChERS method or solid phase extraction method is used to perform non-specific pretreatment on the samples to be analyzed, which helps to extract organic pollutants of different polarities from the samples to be analyzed, improves the screening range of pollutants, and helps to achieve high-throughput analysis.

[0067] In operation S120 , the mixture to be analyzed is measured by liquid chromatography-high resolution mass spectrometry to obtain primary mass number data and secondary fragment structure data.

[0068] According to an embodiment of the present disclosure, high performance liquid chromatography-orbitrap mass spectrometry can be used to collect data on the mixture to be analyzed, so as to facilitate the application in the analysis and detection of complex environmental samples.

[0069] According to an embodiment of the present disclosure, the liquid chromatography condition is configured as a gradient elution program to better separate the mixture to be analyzed.

[0070] According to the embodiments of the present disclosure, in the high performance liquid chromatography-orbitrap mass spectrometry (HPLC-Orbitrap MS, ThermoFisher Scientific Inc., Waltham, MA) determination, the chromatographic column selected was an ACQUITY UPLC C18 column (Waters, 1.7 μm, 2.1 mm id×100 mm), the column temperature was 35° C., and the separation time was 35 min. 1 mM ammonium acetate was added to the mobile phases methanol (A) and water (B), respectively. The flow rate was constant at 0.3 mL·min -1 . The gradient elution program is as follows: phase A is maintained at 5% for 1 minute, then linearly increased to 100% within 24 minutes and maintained for 5 minutes; then A is immediately reduced to 5% and maintained for 5 minutes. Electrospray ionization (ESI) positive and negative modes are used for determination. Mixed samples are prepared for qualitative screening of compounds. Among them, mixed samples refer to samples collected once at fixed intervals within a certain period of time, and multiple samples are finally obtained, and multiple samples are mixed to obtain a mixed sample.

[0071] According to an embodiment of the present disclosure, a full scan plus data-dependent analysis (DDA) is performed on the mixture to be analyzed to obtain primary mass data and secondary fragment structure data. For the full scan, the scan range is set to m / z 100–800, and the resolution is 120,000 (m / z 200). For DDA, the top ten precursor ions in the full scan are fragmented using HCD, with a collision energy of 40±20% and a resolution of 30,000 (m / z 200).

[0072] In operation S130 : a target compound is determined from the mixture to be analyzed by using the primary mass data, the secondary fragment structure data and the preset chemical data set, wherein the preset chemical data set contains information on compounds having oxidative stress effects.

[0073] According to an embodiment of the present disclosure, the method for establishing a preset chemical data set includes, because the antioxidant response element (ARE) pathway is the main pathway for the human body to make an adaptive response to oxidative stress, using the activation degree of the anti-ARE pathway as an indicator of the oxidative stress effect, and then using one or more means such as public toxicity data sets, literature search and text mining, machine learning prediction, etc. to collect target oxidative stress effect compounds and establish a preset chemical data set. Among them, the information of compounds with oxidative stress effects contained in the preset chemical data set specifically includes: chemical name, CAS number, Pubchem CID number, molecular formula, and the exact mass number of the precursor ion of the compound.

[0074] It should be noted that the rules for establishing the preset chemical data set include: (a) Since most compounds will generate [M+H] under ESI mode measurement + and [MH] - , so the precursor ion only uses [M+H] + and [MH] - Due to the special mass spectrometry ionization mode of quinone substances, it is necessary to use [M+H] in negative mode. - ; (b) Remove substances containing metal and semi-metal elements to avoid interference with detection; (c) + / K + / Cl - / Br - Ions are treated as [M-Na] - / [MK] - / [M-Cl] + / [M-Br] + ; (d) Removal of divalent and multivalent ionic compounds; (e) Treatment of additive substances [M·X] (X=H2O, HCl, etc.) to [M+H] + and [MH] - .

[0075] According to an embodiment of the present disclosure, the preset chemical dataset may be defined as an “MS-ready” chemical dataset.

[0076] According to an embodiment of the present disclosure, the above-mentioned determination of the target compound from the above-mentioned mixture to be analyzed using the above-mentioned primary mass number data, the above-mentioned secondary fragment structure data and the preset chemical data set includes: determining candidate compounds from the above-mentioned mixture to be analyzed according to the above-mentioned primary mass number data and the above-mentioned preset chemical data set; and screening compounds that meet the second preset condition from the above-mentioned candidate compounds according to the reference spectrum of the above-mentioned candidate compounds and the above-mentioned secondary fragment structure data to obtain the above-mentioned target compound.

[0077] According to an embodiment of the present disclosure, the reference spectrum can be a spectrum in the mzCloud spectrum library, the MoNA spectrum library, or a self-built predicted spectrum library, wherein both the mzCloud and MoNA spectrum libraries are mass spectrometry databases; the self-built predicted spectrum library refers to generating predicted spectra using CFM-ID for compounds that do not have spectra in the mzCloud and MoNA spectrum libraries.

[0078] For example, Compound Discover (CD) software can be used to match the primary mass data, secondary fragment structure data, and preset chemical data set of the mixture sample to be analyzed, and determine the target compound from the mixture to be analyzed.

[0079] According to an embodiment of the present disclosure, the compound information in the preset chemical data set includes the precise mass number of the precursor ion of the compound; the determining of the candidate compound from the mixture to be analyzed based on the primary mass number data and the preset chemical data set includes: for each mass number in the primary mass number data, matching the precise mass number of the precursor ion that meets a third preset condition from the preset chemical data set to obtain the precise mass number of the candidate precursor ion, wherein the third preset condition includes that the difference between the mass number and the precise mass number of the precursor ion is within a preset range; determining that the compound corresponding to the precise mass number of the candidate precursor ion is the candidate compound.

[0080] According to an embodiment of the present disclosure, a method for determining candidate compounds from a mixture to be analyzed based on primary mass data and a preset chemical data set may be: (a) using forward CD software to import a preset chemical data set, a MoNA spectral library, or a self-built predicted spectrum library; (b) deconvolution, i.e., extracting characteristic peaks; (c) peak processing, including retention time alignment, isotope and adduct merging, and compound merging between samples; (d) feature matching, matching each mass number in the primary mass data with the accurate mass number of the precursor ion in the preset chemical data set, and obtaining candidate compounds according to a third preset condition. The third preset condition may be that the difference between the mass number and the accurate mass number of the above-mentioned precursor ion is ±5ppm.

[0081] According to an embodiment of the present disclosure, the fragment structure data of the candidate compound is the secondary fragment spectrum data of the candidate compound. The CD software is used to perform structural identification on the candidate compound through secondary fragment spectrum matching, and the compound that meets the second preset condition is screened from the candidate compound to obtain the target compound. The second preset condition includes that the compound matching score is higher than 50 points.

[0082] According to an embodiment of the present disclosure, for each target compound among the above-mentioned target compounds, a target standard substance corresponding to the above-mentioned target compound is obtained; the retention time and secondary fragment structure data of the above-mentioned target compound are compared with the retention time and secondary fragment structure data of the above-mentioned target standard substance to obtain a comparison result; when the above-mentioned comparison result indicates that the retention time and secondary fragment structure of the above-mentioned target compound and the above-mentioned target standard substance meet the fourth preset condition, the above-mentioned target compound is determined to be a confirmed compound; wherein the above-mentioned use of the biological analysis equivalent concentration to screen the target compounds that meet the first preset condition from the above-mentioned target compounds to obtain the main contributing compound includes: using the above-mentioned biological analysis equivalent concentration to screen the compounds that meet the above-mentioned first preset condition from the above-mentioned confirmed compounds to obtain the above-mentioned main contributing compound.

[0083] According to the embodiments of the present disclosure, only the standard of the target compound needs to be obtained, which reduces the demand for the standard, reduces the workload, and speeds up the analysis.

[0084] According to an embodiment of the present disclosure, when the comparison results show that the retention time of the target compound and the target standard substance and the secondary fragment structure meet the fourth preset condition, the target compound is determined to be a confirmed compound. The fourth preset condition includes that the retention time deviation between the target compound and the target standard substance is less than 0.3min, and at least 2 fragments in the secondary fragment structure spectrum can match.

[0085] It should be noted that the confidence level of the target compound that meets the fourth preset condition is 1.

[0086] According to the embodiments of the present disclosure, the following parameters may also be manually checked for the target compound: confirming whether the fragment structure data of the target compound has a good peak shape (a single peak with a normal distribution), whether the ionization mode of the target compound is reasonable (such as amino and carbonyl compounds are ionized in a positive mode and hydroxyl and carboxyl compounds are ionized in a negative mode), whether there are two or more secondary fragment matches or only one secondary fragment matches but its signal intensity accounts for more than 70% of the cumulative signal intensity of all fragments.

[0087] According to an embodiment of the present disclosure, the target compound also includes obtaining an identified compound with an oxidative stress effect corresponding to the type of the sample to be analyzed; for each identified compound in the identified compounds, if it is determined that the target compound does not contain the identified compound, the identified compound is determined as the target compound. The identified compound that is not detected by this method is added to the target compound to improve the accuracy of subsequent analysis of the main contributing compounds and the composite effect.

[0088] According to the embodiments of the present disclosure, since the screening scope of the present method flow is small molecule compounds that can be determined using LC-HRMS, some compounds with lower polarity (such as polycyclic aromatic hydrocarbons) are not within the analysis scope. For solid samples, since some polycyclic aromatic hydrocarbons with 4 rings or more are widely present and may make important contributions to the oxidative stress effect, gas chromatography-mass spectrometry can be used for targeted determination. The occurrence concentration of polycyclic aromatic hydrocarbons in liquid samples is generally low and need not be determined. The above-mentioned identified compounds that have not been measured by this method are added to the target compounds in order to improve the accuracy of subsequent analysis of the main contributing compounds and compound effects.

[0089] According to the embodiments of the present disclosure, by utilizing primary mass number data, secondary fragment structure data, and a preset chemical data set to perform suspected target screening, it is beneficial to improve the analysis throughput and enrich the understanding of pollutants with oxidative stress effects in environmental samples.

[0090] In operation S140 : target compounds that meet the first preset condition are screened from the target compounds using the biological analysis equivalent concentration to obtain the main contributing compounds.

[0091] According to an embodiment of the present disclosure, the bioanalytical equivalent concentration (BEQ) is the conversion of the dose of compound i into the dose of a reference compound that produces an equivalent effect, wherein the reference compound may be tert-butylhydroquinone (tBHQ).

[0092] It should be noted that before using the bioanalysis equivalent concentration, the confirmed compound obtained in operation S130 is first quantified by the external standard method using the full scan mode for a single sample. In addition, the quantitative results are also conducive to analyzing the environmental occurrence characteristics of the identified compounds, such as geographical distribution, time trend, etc. Among them, within a certain period of time, a sample is collected once at a fixed interval, and finally multiple samples are obtained, and the sample collected each time is defined as a single sample.

[0093] According to an embodiment of the present disclosure, the target compounds include n, where n≥1. The process of using the bioanalysis equivalent concentration to screen the target compounds that meet the first preset condition from the target compounds to obtain the main contribution compound includes: using the bioanalysis equivalent concentration to screen the compounds that meet the first preset condition from the target compounds, and obtaining the main contribution compound includes: for the i-th target compound in the target compounds, determining the i-th bioanalysis equivalent concentration corresponding to the i-th target compound, and finally obtaining n bioanalysis equivalent concentrations; adding the n bioanalysis equivalent concentrations to obtain the cumulative bioanalysis equivalent concentration; calculating the ratio of the i-th bioanalysis equivalent concentration to the cumulative bioanalysis equivalent concentration to obtain the i-th relative effect contribution value corresponding to the i-th target compound; and determining the i-th target compound as the above-mentioned main contribution compound when it is determined that the above-mentioned i-th relative effect contribution value meets the first preset condition.

[0094] According to an embodiment of the present disclosure, the bioassay equivalent concentration (BEQ) is calculated using the following formula.

[0095]

[0096] BEQ i =∑ j BEQ i,j (1-2)

[0097] BEQ cumulative =∑ i BEQ i (1-3)

[0098]

[0099] in:

[0100] SC i,j (Sample Concentration) is the concentration of target compound i in sample j (μM, μ·mol·g -1 , μ·mol·m -3 );

[0101] EC 50 is the half-maximal effect concentration (μM).

[0102] According to the embodiments of the present disclosure, the EC 50 The values ​​can be obtained from public toxicity datasets (such as the Tox21 qHTS dataset), literature, machine learning predictions, etc.

[0103] The Tox21 qHTS dataset is a collection of toxicity data obtained through quantitative high-throughput screening based on the Toxicology in the 21st Century (Tox21) project.

[0104] It should be noted that the relative effect contribution ratio is the ratio of the target compound BEQ to the cumulative BEQ. The importance of the compounds can be ranked according to the relative effect contribution ratio, and then the main contributing compounds can be selected. Among them, the cumulative BEQ is obtained by adding up the BEQs of all target compounds.

[0105] According to an embodiment of the present disclosure, the first preset condition includes that a single relative effect contribution value is greater than a first threshold, and the sum of the relative effect contribution values ​​of all major contributing compounds is greater than a second threshold.

[0106] According to an embodiment of the present disclosure, the first threshold may be 5%, and the second threshold may be 60%.

[0107] In operation S150 : performing oxidative stress compound effect analysis on the main contributing compounds to obtain analysis results.

[0108] According to the embodiments of the present disclosure, conducting a composite effect analysis is beneficial for analyzing whether there is a synergistic or antagonistic effect between compounds and analyzing their reaction mechanisms, thereby facilitating the design of an appropriate pollutant control method, thereby providing a theoretical basis for controlling pollutants with oxidative stress effects in complex environments.

[0109] According to an embodiment of the present disclosure, the main contributing compounds include m, m≥1. The process of performing oxidative stress compound effect analysis on the main contributing compounds includes: for the jth main contributing compound among the m main contributing compounds, determining the jth single effect concentration of the jth main contributing compound that induces oxidative stress effect, and finally obtaining m single effect concentrations, determining the effect concentration of the mixed main contributing compound that induces oxidative stress effect, and obtaining the compound effect concentration, wherein the mixed main contributing compound is obtained by mixing the concentration ratios of the above m main contributing compounds in the sample to be analyzed, calculating the combination index using the m single effect concentrations and the compound effect concentration, and performing oxidative stress compound effect analysis on the main contributing compounds using the combination index.

[0110] According to the embodiments of the present disclosure, the concentration ratio of the mixture is designed according to the actual environmental conditions, which can reduce the redundant workload of the concentration ratio design that is not related to the environmental conditions in the traditional process. This process design overcomes the huge workload caused by the large number of compounds involved in the evaluation of the combined effects of environmental compounds and the need to design a series of concentration ratios, making it feasible, cost-effective, and able to obtain relatively important information.

[0111] According to an embodiment of the present disclosure, the method for analyzing the compound effect of oxidative stress on the main contributing compound includes using a HepG2 cell line stably transfected with an ARE luciferase reporter gene to evaluate the compound effect of the target compound that makes the main contribution in operation step S140. The method calculates the bioanalytical equivalent concentration to give the main contributing compound, and then calculates the compound index to evaluate its compound effect, which can reduce the workload caused by a large number of low-contribution non-critical compounds in the traditional process.

[0112] According to an embodiment of the present disclosure, the method can be specifically as follows: using a white 96-well plate, seeding each well with 1.2 e 5 After 24 hours of growth, the dimethyl sulfoxide (DMSO) solution of the main contributing compound was added for pollutant exposure. The volume of the DMSO solution did not exceed 1% of the volume of the culture medium, and the exposure time was 20 hours. 8-12 concentration gradients of the test compound were prepared, and the dilution multiple was 2-3 times. DMSO and tBHQ were used as blank and positive controls, respectively. The cells were lysed using lysis buffer, the fluorescent substrate diphenylterazine was added, and the fluorescence signal was measured using an enzyme reader. Single compounds and mixtures were measured, where the mixture was prepared according to the average concentration ratio of the environmental samples. Prism (GraphPad, San Diego, CA) software was used to fit the dose-effect curve based on the four-parameter equation describing the symmetrical "S" curve. The software can calculate different effect concentrations and their confidence intervals. The combination index (CI) was then calculated to analyze the combined oxidative stress effects of the main contributing compounds.

[0113] According to an embodiment of the present disclosure, the calculation formula of CI is shown in 1-5.

[0114]

[0115] in:

[0116] x is the effect value, and its value range is (0, 100);

[0117] EC x,mix is: the concentration of a mixture of multiple major contributing compounds that triggers effect x;

[0118] EC x,j is: the concentration of the jth main contributing compound when it triggers effect x;

[0119] CI less than, equal to, and greater than 1 represent synergistic, additive, and antagonistic effects, respectively.

[0120] It should be noted that in order to consider the uncertainty of the CI value, it is assumed that EC x,mix and EC x,j The data were subjected to log-normal distribution, and 10,000 Monte Carlo simulations were performed. Random values ​​were selected within the confidence interval for calculation. The median and upper and lower 2.5% quantiles of all Monte Carlo simulation results at different effect concentrations were used as the reported values ​​of CI. Among them, Monte Carlo simulation is based on a probability model. According to the process described by this model, the results of the simulated experiment are used as an approximate solution to the problem.

[0121] According to an embodiment of the present disclosure, operation S150 performs compound effect analysis on the main contributing compounds given by operation S140 to reduce the workload caused by a large number of low-contribution non-critical compounds in the traditional process. The overall process is easy to operate and promote, and can provide technical support for the discovery of pollutants with oxidative stress effects.

[0122] According to an embodiment of the present disclosure, the method of the present disclosure also includes performing exposure risk assessment for environmental media to which the human body is directly exposed, such as atmospheric fine particulate matter, and specifically includes the following operations:

[0123] Replace SC in formula (1-1) i,j Replace with the equivalent concentration C in the biological test exposure system used in the toxicity data source i,j (μM) and then perform BEQ calculation to complete the exposure risk assessment of environmental media to which the human body is directly exposed.

[0124] For example, using the atmospheric fine particulate matter and toxicity data from the Tox21 qHTS dataset as an example, the pollutant concentration (μg·m -3) is converted to the equivalent concentration C in the biological test exposure system i,j (μM). Equivalent concentration C i,j (μM) The calculation formula is shown in 1-6. It can be calculated by MPPD v.2.1 (Applied Research Associates Inc., Raleigh, NC); and the risk quotient (HQ) (Formula 1-7) is calculated. When the HQ value is >1 and <1, it indicates whether lifetime exposure has the risk of causing adverse effects.

[0125]

[0126] in:

[0127] SC i,j is the measured pollutant concentration (μg·m -3 );

[0128] AB is the respiratory rate (m 3 ·d -1 );

[0129] f is the alveolar deposition ratio of fine particles;

[0130] PSA is the bottom area of ​​each well of a 1536-well plate (0.023 cm 2 );

[0131] SA is the alveolar surface area (m 2 );

[0132] MW i is the molecular weight;

[0133] V is the volume of solution in each well of a 1536-well plate (5 μL).

[0134] It should be noted that AB and SA were determined by consulting relevant literature, and the alveolar surface area of ​​adults can be taken as the literature value of 75m 2 The respiratory rate can be divided into different regions by checking the exposure manual. For example, the average respiratory rate of adults in Beijing is 16.1m / s. 3 / d.PSA(0.023cm 2 ) and V (5 μL) are the bottom area and solution volume of each well of a 1536-well plate, respectively, which are the exposure conditions used in the Tox21 qHTS bioassay.

[0135]

[0136] in:

[0137] EC 1,tBHQ For the least observable adverse effect level (LOAEL), EC 5,tBHQor EC 10,tBHQ represent;

[0138] MF is the correction factor;

[0139] HQ values ​​>1 and <1 indicate whether lifetime exposure poses a risk of causing adverse effects.

[0140] It should be noted that the correction factor can be assigned according to the actual scenario, such as uncertainties ranging from healthy population to sensitive population, from observed adverse effect level (LOAEL) to no observed adverse effect level (NOAEL), from incomplete toxicity data to complete toxicity data, from cell tests to humans, from short-term exposure to lifetime exposure. Each item can be assigned a value of 3 or 10, and the correction factor is the product of various uncertainty factors.

[0141] According to the embodiments of the present disclosure, the screening scope of the method of the present disclosure is small molecule compounds that can be measured using LC-HRMS, and some compounds with lower polarity (such as polycyclic aromatic hydrocarbons) are not within the scope of analysis. For solid samples, since some polycyclic aromatic hydrocarbons with four rings or more are widely present and may have an important contribution to the oxidative stress effect, gas chromatography-mass spectrometry can be used for targeted determination. The occurrence concentration of polycyclic aromatic hydrocarbons in liquid samples is generally low and can be omitted.

[0142] Example 1

[0143] This example is a laboratory test example, focusing on realizing the analysis of the oxidative stress effect of compounds by establishing a method system, and its specific steps include:

[0144] (1) Sample extraction: From January 2019 to January 2020, an atmospheric fine particulate matter sample was collected every six days, for a total of 59 samples, using a quartz membrane with a diameter of 9 cm. Use ceramic scissors to cut the quartz membrane into square fragments with a side length of about 5 mm. Use 15 mL of acidified acetonitrile (containing 1% acetic acid) to extract organic matter, to which 2 g of magnesium sulfate and 0.5 g of sodium chloride are added. After centrifugation to obtain the supernatant, 0.3 g of magnesium sulfate and 0.05 g of N-propylethylenediamine are added. After centrifugation to obtain the supernatant, it is concentrated by nitrogen blowing and redissolved in 100 μL of methanol. In qualitative screening, 1 cm is taken from each sample. 2 , make a mixed sample and then carry out pretreatment; in quantitative analysis, cut 1 / 4 of each sample for pretreatment.

[0145] (2) LC-HRMS determination: The instrument parameters are shown in Table 1.

[0146] Table 1. LC-HRMS determination related parameter settings

[0147]

[0148]

[0149] (3) Screening and quantification of suspected targets:

[0150] Figure 2 It is a partial schematic diagram of the preset chemical data set established in Example 1 of the present disclosure.

[0151] like Figure 2 As shown in the figure, based on the 995 oxidative stress effect pollutants, i.e., ARE pathway agonists, screened by Tox21 qHTS, an "MS-ready" chemical data set, i.e., a preset chemical data set, was established, and a total of 945 substances were included in the data set. The full scan plus DDA data of the mixed sample was screened for suspected targets using CD v.3.1 according to the process and parameters in Table 2.

[0152] Table 2. CD v.3.1 test process and parameter settings

[0153]

[0154]

[0155] Figure 3 It is a schematic diagram of the oxidative stress effect pollutants with a confidence level of 1 identified in Example 1 of the present disclosure.

[0156] Candidate compounds were obtained by matching 248 and 186 features with the exact mass of precursor ions in the "MS-ready" chemical data set in the positive and negative modes of ESI, respectively. Secondly, 47 target compounds were identified through secondary fragment structure spectrum matching and manual inspection. Finally, 23 confirmed compounds were obtained from the comparison of the 47 target compounds with the target standards, with a retention time deviation of less than 0.3min and at least 2 fragments of the secondary fragment structure spectrum being able to match, reaching a confidence level of 1.

[0157] like Figure 3 As shown, among them, 23 confirmed compounds reached a confidence level of 1 after comparison with standard samples, including 6 phytochemicals (linolenic acid, caffeine, isatin, piperine, apigenin, coine), 5 pesticides (8-hydroxyquinoline, pyraclostrobin, acetochlor, dichlorvos, malaxon), 6 synthetic phenolic additives (bisphenol A, 3,5-di-tert-butyl-4-hydroxybenzyl alcohol, 1-naphthol, 4-phenylphenol, 2,4-dihydroxybenzophenone, 2-naphthol), 2 organophosphorus flame retardants (tri-o-cresyl phosphate, triphenyl phosphate), 2 personal care products (12-hydroxystearic acid, octocrylene), 1 guanidine rubber accelerator (1,3-diphenylguanidine), and 1 oxidized polycyclic aromatic hydrocarbon (2-hydroxyanthraquinone).

[0158] In addition, since PAHs are known to be important oxidative stress effect compounds in atmospheric fine particulate matter, this implementation case additionally measured the concentrations of 8 PAHs, including benzo[a]anthracene, chrysene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, benzo[e]pyrene, dibenzo[a,h]anthracene, and benzo[ghi]perylene.

[0159] (4) Relative effect contribution assessment:

[0160] Figure 4 is the relative effect contribution value of the main effect contributing compound identified in Example 1 of the present disclosure.

[0161] like Figure 4 As shown in the figure, by calculating the relative effect contribution values ​​of the compounds, it was found that the two PAHs, benzo[b]fluoranthene and benzo[k]fluoranthene, and the two organic nitrogen pesticides, pyridaben and acetochlor, had the highest contribution ratios of 22%, 14%, 32%, and 7%, respectively. The contribution ratio of each compound to the cumulative BEQ was calculated based on the toxicity data provided by the 21st Century Toxicology (Tox21) project using quantitative high throughput screening (qHTS) and the concentration data obtained by instrumental measurement.

[0162] In addition, since the human body is directly exposed to atmospheric fine particles, an exposure risk assessment was conducted. The results showed that pyridabenz, benzo[b]fluoranthene, benzo[k]fluoranthene, and acetochlor had HQ greater than 1 in 15%, 15%, 3%, and 3% of the samples, respectively, which could cause health risks, while the remaining compounds had HQ less than 1 in all samples and could be considered safe.

[0163] (5) Assessment of compound effects:

[0164] Figure 5 It is a dose-effect curve diagram of the main effect contributing compound in Example 1 of the present disclosure.

[0165] like Figure 5 As shown in the figure, the oxidative stress effects of benzo[b]fluoranthene, acetochlor, benzo[k]fluoranthene, and pyridabenz) alone and in combination increased with the increase in the logarithm of concentration (i.e., the logarithm of dose), and all of them, except pyridabenz, could reach a high efficiency level (53%–101%).

[0166] The dose-effect relationships of the main contributing compounds (benzo[b]fluoranthene, acetochlor, benzo[k]fluoranthene, and pyraclostrobin) alone and in mixtures were determined using a HepG2 cell line stably transfected with the ARE luciferase reporter gene, and the mixtures were prepared according to the average concentration ratio of environmental samples (100:61:33:2). EC 10The corresponding CI values ​​had a median of 0.09, and the upper and lower 2.5% quantiles were 0.7 and 0.01, respectively, indicating that the four compounds exhibited a strong synergistic effect at the above concentration ratio.

[0167] This result further emphasizes the importance of the major contributing compounds, which should attract attention from research and management fields.

[0168] Example 2: Taking piperine as an example, the identification process is explained

[0169] Figure 6 It is the primary mass number spectrum and secondary fragment structure spectrum of piperine identified in Example 1 of the present disclosure, wherein Figure (A) is the primary mass number spectrum of piperine, and Figure (B) is the secondary fragment structure spectrum of piperine.

[0170] like Figure 6 As shown in Figure (A), in the primary mass number spectrum of piperine in the sample of Example 1, m / z = 286.1438 ± 5 ppm ([M+H] + ) chromatographic peak. Its secondary fragment structure spectrum (MS 2 ) matches the piperine standard in the mzCloud library with a score of 90. Figure 6 As shown in Figure (B), the matching secondary fragment ions m / z = 286.1442, 201.0550, 171.0446, 143.0494, 135.0444, 115.0545 are the following fragment ions: [C 17 H 20 O3N] + , [C 12 H9O3] + , [C 11 H7O2] + , [C 10 H7O] + 、[C8H7O2] + 、[C9H7] + , the mass deviation is in the range of 1.4-3.0ppm. After analysis, the retention time of the pepper salt identified in Example 1 and the piperine standard product only deviated by 0.07min, and both of them matched the secondary fragments of the standard product, so it was confirmed to be piperine.

[0171] The results of the above embodiments show that the analytical method for the oxidative stress effect of a compound of the present application can be applied to actual environmental samples, demonstrating accurate and high-throughput identification capabilities and simple and effective evaluation capabilities. Therefore, the method flow of the present disclosure has practical applicability and broad application prospects.

[0172] The specific embodiments described above further illustrate the purpose, technical solutions and beneficial effects of the present disclosure. It should be understood that the above description is only a specific embodiment of the present disclosure and is not intended to limit the present disclosure. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present disclosure should be included in the protection scope of the present disclosure.

Claims

1. A method for analyzing compounds with oxidative stress effects, comprising: Pre-treating the sample to be analyzed to obtain a mixture to be analyzed; The mixture to be analyzed is measured by liquid chromatography-high resolution mass spectrometry to obtain primary mass number data and secondary fragment structure data; Determining a target compound from the mixture to be analyzed using the primary mass number data, the secondary fragment structure data, and a preset chemical data set, wherein the preset chemical data set contains information on compounds having an oxidative stress effect; Wherein, the determining the target compound from the mixture to be analyzed by using the primary mass number data, the secondary fragment structure data and the preset chemical data set comprises: Determining candidate compounds from the mixture to be analyzed according to the primary mass number data and the preset chemical data set; According to the reference spectrum of the candidate compound and the secondary fragment structure data, a compound satisfying a second preset condition is screened from the candidate compound to obtain the target compound, wherein the second preset condition includes a compound matching score higher than 50 points; Wherein, the compound information in the preset chemical data set includes the accurate mass number of the precursor ion of the compound; Determining the candidate compound from the mixture to be analyzed according to the primary mass number data and the preset chemical data set includes: For each mass number in the primary mass number data, matching the accurate mass number of the precursor ion that meets the third preset condition from the preset chemical data set to obtain the accurate mass number of the candidate precursor ion, wherein the third preset condition includes that the difference between the mass number and the accurate mass number of the precursor ion is within a preset range; Determining a compound corresponding to the accurate mass number of the candidate precursor ion as the candidate compound; wherein, for each target compound in the target compounds, a target standard substance corresponding to the target compound is obtained; Comparing the retention time and secondary fragment structure data of the target compound with the retention time and secondary fragment structure data of the target standard to obtain a comparison result; If the comparison result shows that the retention time and the secondary fragment structure of the target compound and the target standard satisfy the fourth preset condition, the target compound is determined to be a confirmed compound, wherein the fourth preset condition includes that the retention time deviation between the target compound and the target standard is less than 0.3 min, and at least 2 fragments in the secondary fragment structure spectrum can match; Using the biological analysis equivalent concentration, screening compounds that meet the first preset condition from the confirmed compounds to obtain the main contributing compounds, and performing oxidative stress compound effect analysis on the main contributing compounds to obtain analysis results; Wherein, the confirmed compounds include n, n≥1; The method of screening compounds satisfying the first preset condition from the confirmed compounds by using the biological analysis equivalent concentration to obtain the main contributing compounds includes: For the i-th confirmed compound among the confirmed compounds, determine the i-th bioanalytical equivalent concentration corresponding to the i-th confirmed compound, and finally obtain n bioanalytical equivalent concentrations; Adding the n bioassay equivalent concentrations to obtain a cumulative bioassay equivalent concentration; Calculating the ratio of the i-th bioanalytical equivalent concentration to the cumulative bioanalytical equivalent concentration to obtain the i-th relative effect contribution value corresponding to the i-th confirmed compound; When it is determined that the i-th relative effect contribution value satisfies the first preset condition, the i-th confirmed compound is determined to be the main contributing compound; wherein the first preset condition includes that a single relative effect contribution value is greater than a first threshold value, and the sum of the relative effect contribution values ​​of all main contributing compounds is greater than a second threshold value.

2. The analysis method according to claim 1, wherein The sample to be analyzed includes one of a solid sample or a liquid sample; The pre-processing of the sample to be analyzed to obtain the mixture to be analyzed comprises: Processing the solid sample using the QuEChERS method to obtain the mixture to be analyzed; or The liquid sample is enriched and purified by a solid phase extraction method to obtain the mixture to be analyzed.

3. The analysis method according to claim 2, wherein: The using the QuEChERS method to process the solid sample to obtain the mixture to be analyzed comprises: Using acetonitrile or acidified acetonitrile to shake and vortex to extract organic matter in the solid sample to obtain an organic supernatant; The organic supernatant is treated to remove water and impurities, and then concentrated and redissolved to obtain the mixture to be analyzed.

4. The analysis method according to claim 1, wherein The main contributing compounds include m, m≥1; The oxidative stress compound effect analysis of the main contributing compounds comprises: For the jth main contributing compound among the m main contributing compounds, determining the jth single effect concentration of the jth main contributing compound that induces the oxidative stress effect, and finally obtaining m single effect concentrations; Determine the effect concentration of the mixed main contribution compound that induces the oxidative stress effect to obtain a composite effect concentration, wherein the mixed main contribution compound is obtained by mixing m main contribution compounds in a concentration ratio in the sample to be analyzed; Calculating a combination index using the m single effect concentrations and the composite effect concentration; The combined index is used to analyze the compound effects of oxidative stress on the main contributing compounds.

5. The analysis method according to claim 1, further comprising: Obtaining identified compounds with oxidative stress effects corresponding to the type of the sample to be analyzed; For each of the identified compounds, if it is determined that the identified compound is not included in the confirmation compound, the identified compound is determined as a confirmation compound.

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