CN111413444B - Method for identifying androgen active substance in environmental sample based on characteristic liquid fragment matching - Google Patents
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Abstract
The invention provides a method for identifying androgen active substances in an environmental sample based on characteristic liquid mass fragment matching, which comprises the steps of selecting an activity warning structure related to androgen activity; classifying the 'activity warning structure' and establishing a correlation between the 'activity warning structure' and a mass spectrum characteristic fragment structure; extracting characteristic fragments capable of representing activity, and verifying through a standard substance and a mass spectrum database; constructing a screening method of androgen active substances based on the characteristic fragments and applying the screening method. Compared with the traditional method, the method has higher flux, wider coverage range of compounds and more types, establishes direct connection between toxicity characteristics and mass spectrum fragments, can carry out high-flux screening on a large number of unknown compounds, does not need to determine the complete structure of the compounds, can quickly screen out possible androgen active substances according to the matching of secondary fragments, greatly improves the non-target screening efficiency, and further provides a new technical scheme for identifying key toxic substances in the environment.
Description
The technical field is as follows:
the invention belongs to the technical field of environmental detection, and particularly relates to a method for identifying androgen active substances in an environmental sample based on characteristic liquid fragment matching.
Background art:
in recent years, the number of chemical synthesis and use has been increased sharply, and they enter the environment through various routes, resulting in some detection of endocrine disrupting effects in various environmental media around the world, and therefore, there is an urgent need to identify the main toxic substances causing endocrine disrupting effects. In endocrine disrupting effects, the androgenic effect is relatively poorly studied compared to the estrogenic effect, and the mode of action of androgenic active substances is also complex, may be both mimetic and resistant, and may also be both mimetic and resistant. Previous studies have demonstrated that the compounds responsible for androgenic activity are mostly natural and synthetic steroids, whereas for antiandrogenic effects, which may be caused by multiple classes of compounds, low-throughput chemical analysis often fails to identify the major toxic substances. However, in current Effect-Directed Analysis (EDA) studies, the success rate of identifying toxic substances based on a given compound list is low, rather than the time and effort required for identifying non-target compounds, and the difficulty of identifying toxic substances is high due to the lack of spectral libraries and toxicity data and the small number of compounds with mass spectra and toxicity information. Specifically, 2237 compounds with androgen activity were tested in the ToxCast database, but only 529 compounds with high-resolution liquid mass spectra were tested in the mzCloud database, and the ratio was only 23.65%, so if suspected screening was performed on the basis of the toxicity list compound, most of the compounds with matching primary mass spectra information would be unable to determine structure due to lack of spectral information, making the toxicity identification process time-consuming and labor-consuming. Therefore, the method for directly matching the characteristic liquid fragment with the toxicity information of the liquid fragment can greatly improve the efficiency of identifying the toxicant.
In view of the fact that the structure of the compound determines the property, studies have shown that the two compounds have similar structures and similar activities, and the secondary mass spectrum fragments also have certain similarity, for example, the study of Shen et al (Nature Communication,2019,10(1):151-6.) found that, in a metabolic reaction, a specific reaction scores more than 50% of the similarity of the mass spectra of the reactant and the product at both ends (full score of 1), and a method for deriving the mass spectra of the compounds without standards in the metabolic network by using the mass spectra of the known compounds is established, which can be used for identifying the compounds. MS-FINDER establishes a feature fragment library of metabolites (Nature Methods,2019,16(4):295-8.), and can be used for metabolite class matching in non-target structure identification; NIST developed a new "hybrid search" approach (Analytical Chemistry,2019,91(21): 13924-32) that can identify metabolites that are not present in tandem mass spectral libraries by using a combination of direct peak matching and neutral loss matching. This new search method successfully finds structurally similar compounds in the library by matching peaks within a certain neutral loss range to increase the score of similar compounds.
Based on the theory that structurally similar compounds have some similarity in their secondary mass spectral fragments, research has been successful in identifying contaminants of interest as well as their metabolic and conversion products. First, ionic fragments are utilized which certain homologous compounds contain characteristics, such as chloro-and bromo-groups, iodo-groups, perfluoro/polyfluoro compounds, sulfonic acids, organophosphates, etc., which often contain characteristic elements (e.g., halogens, elemental sulfur, elemental phosphorus, etc.). In recent years, there have also been some studies that have begun to use feature fragment matching strategies for screening certain types of compounds in liquid mass analysis. For example, in the field of identification of risk substances in food, the prior art publication No. CN109781915A discloses a rapid screening lockout method for risk-adding substances in food. By researching the mass spectrum fragmentation mechanism of 4 antibiotic substances, a method for rapidly screening unknown risk substances is successfully established by means of characteristic fragment matching rules, average deviation ratio and the like, and sulfamethazine and metabolic products thereof in pork samples are successfully identified. The method has obvious advantages in screening and analyzing risk-added compounds randomly collected without grouping food samples, has the advantages of quickly locking potential risk compounds and quickly and accurately determining the nature, only aims at four compounds of sulfonamides, quinolones, tetracyclines and beta-receptor inhibitors, and has a limited coverage range.
Jang et al established three types of illicit drugs and classification methods thereof by using a machine learning method (Analytical Chemistry,2019,91(14):9119-28), and scored the score criteria of the structure score values to facilitate screening of partial transformation products thereof; Campos-Manas et al used a characteristic debris matching method (Science of Total Environment,2019,664:874-84) in the detection of opioids in wastewater and surface water, successfully identified conversion products of the opioid drug, and validated by standards. In recent years, research has been carried out on combination of a method for using characteristic fragment ions and EDA, Muz, after the teratogenic effect of surface water affected by industrial wastewater discharge is tested, the method for using EDA to identify that aromatic amine substances are main teratogenic substances is developed, and a method for screening the characteristic fragment ions of aromatic amine substances (Chemosphere,2017,166:300-10) is developed on the basis of the method, and the method based on derivatization successfully improves the response of the aromatic amine substances on liquid substances and identifies unknown non-target aromatic amine substances on the basis of the method.
However, the above invention and research still have the following technical problems to be solved: firstly, the compounds are only aimed at a selected part of compounds with similar structures, and although the mass spectrum fragmentation laws of the compounds are well researched, the compounds have a definite molecular formula range and low flux when being screened; secondly, although the selected target substance has a part of function modes based on risks or specificities, the structure is fixed and cannot be explained by the action on the mechanism, so that direct connection with certain toxicity characteristics cannot be established; and thirdly, only the conversion products of the large compound with the substituted individual positions on the framework can be identified.
The invention content is as follows:
the invention aims to provide a method for identifying androgen active substances in an environmental sample based on characteristic liquid substance fragment matching aiming at the problems of low success rate of identifying toxic substances by a given compound list, low efficiency of identifying non-target toxic substances, time consumption and labor consumption in the process of identifying the androgen active substances.
The invention adopts the following technical scheme:
a method for identifying an androgen active substance in an environmental sample based on characteristic liquid mass fragment matching, comprising the steps of:
s1, selecting a high-throughput screening model of the androgen active substance, wherein the model comprises the correlation between the characteristic structure of the compound and whether the compound has androgen activity, and selecting an 'activity warning structure' related to the androgen activity;
s2, classifying the categories of functional groups contained in the 'activity warning structure' in the S1 through functional group classification standards, inquiring characteristic secondary fragments corresponding to each category in the existing characteristic fragment library corresponding to compound classification, sorting and summarizing to form secondary fragment information corresponding to each 'activity warning structure', establishing a correlation between the 'activity warning structure' and the characteristic secondary fragment structure, and forming a 'activity warning structure' secondary fragment library;
s3, verifying the characteristic secondary fragments summarized in the S2 by a spectrogram obtained by a methanol solution of a standard substance on a liquid chromatogram-high resolution mass spectrum, and if the selected standard substance does not contain a compound containing an 'activity warning structure', verifying the spectrogram of the compound containing the 'activity warning structure' in a mass spectrum database to establish a standard substance characteristic secondary fragment database;
s4, performing on-machine test on the methanol solution of the environmental sample extract according to a liquid chromatography-high resolution mass spectrometry method in S3, extracting non-target compounds, selecting a certain concerned non-target peak, comparing and screening the molecular formula and the secondary fragment with a standard substance characteristic secondary fragment database established in S3, and if the molecular formula and the secondary fragment do not meet the standard substance characteristic secondary fragment database, judging that the molecular formula and the secondary fragment do not have androgen activity; if the two types of the compounds are matched, the compounds can be judged to have the androgen activity, and subsequent poison identification processes such as structure calculation, toxicity confirmation and the like are carried out.
Further, the selected "activity warning structure" related to androgen activity is an androgen active substance secondary warning structure in an endocrine disruptor high-throughput screening model and a screening method established in two patents of publication numbers CN109815532A and CN109545289A, and is specifically shown in table 1.
Further, in S2, the functional group classification criterion is Classyfire Compound classification criterion (http:// classsyfire. wishartlab. com).
Further, in S2, the feature fragment library is the self-contained feature fragment library of the MS-FINDER3.0 Software (http:// prime.psc.riken.jp/Metabolomics _ Software/MS-FINDER/index.html).
Further, in S3, the standard was selected as a chemical commonly found in 428 environmental samples, and the concentration of the methanol solution was 50. mu.g/L.
Further, in S3, the liquid chromatography conditions are: ultra-high performance liquid chromatograph: using Ultimate 3000UHPLC, Thermo ScientificTMThe united states of america; mobile phase: phase A: fisher water, phase B: methanol; a chromatographic column: waters ACQUITY UPLC BEH C18,1.7 μm,2.1mm × 150 mm; sample introduction amount: 10 mu L of the solution; column temperature: 40 ℃; gradient elution method: 0min 5% B, 1min 5% B, 36min 5% B, 50min 100% B, 50.1min 5% B.
Further, in S3, the mass spectrometry conditions are: a mass spectrometer is adopted: q activeTMFocus combined quadrupole OrbitrapTM，Thermo ScientificTMThe united states of america; the collection mode is as follows: full MS-ddMS 2; polarity: sampling samples once in a positive ion/negative ion mode; ddMS2 mode: discovery: full MS parameters: resolution ratio: 35000(m/z200), scanning range: 80-1000m/z, AGC target value: 1e 6; maximum IT: automatic; spectrogram data type: a contour map; ddMS2 Discovery parameter: resolution 17500(m/z200), isolation window; 1.0 m/z;step NCE: 20,50 and 80/110,140,170 are injected once respectively; AGC target value: 5e 4; maximum IT: automatic; and (3) cycle counting: 3; and (4) triggering a vertex: 2-6 s; and (3) dynamic exclusion: 6.0 s; charge number exclusion: 2-8,>8, exclusion of isotopes: opening; spectrogram data type: and (5) contour drawing.
Further, in S3, the method for screening the first-order mass spectrum peak of the compound in the standard validation comprises: using the Target Screening function in the TraceFinder 5.0SP1(Thermo Fisher, USA) software, based on the list of compounds predicted to have androgenic effects in the standards, a Screening method was established in positive and negative ion mode, with the adduct mode [ M + H [ ]]+Or [ M-H]-Mass number error range 5 ppm; and the isotope error is 20%, the detected standard sample is matched, and the accurate mass number and retention time of the standard sample are derived.
Further, in S3, the method for extracting the secondary fragment corresponding to the primary mass spectrum peak of the compound in the standard verification includes: converting the raw file obtained by collection into ABF format through ABF Converter 3.0.0(Reifycs, Japan) software, and then performing data processing by using MS-DIAL 4.0 software; the parameters to be set are: minimum peak height: 1000, parts by weight; consider the chloro-bromo isotope: is that; the other parameters are default parameters; after the Peak extraction is finished, clicking in a Peak spot viewer based on the accurate mass number and retention time of the compound, displaying a secondary spectrogram on the right side, leading out secondary fragments of the secondary spectrograms into MS-FINDER3.0 software, and calculating the molecular formula of each fragment ion; then, the fragments of the compound can be matched with potential characteristic fragment ions contained in the 'activity warning structure' of the corresponding classification, and the error range is 2 mDa.
Further, in S3, the method for verifying the standard product includes: using the Search-MS/MS Fragment Search in MS-DIAL software, input mass number of feature Fragment, Fragment type: daughter ions, error range: 0.002Da, ion abundance%: 1 e-5; after the search is finished, clicking the Unique Ion and the Show Ion Table in sequence, deriving a matching list, and judging whether the corresponding compound can be screened through the process.
Further, in S3, the mass spectrum database is an mzCloud high resolution liquid mass spectrum database (https:// www.mzcloud.org).
Further, in S3, the mass spectrometry spectrum verification method includes: grouping the compounds in the mzCloud database according to the contained 'activity warning structure' in S1, and screening out the corresponding compounds; then, for each potential secondary fragment, searching a compound containing corresponding characteristic ions by using a Peak Search function in an mzCloud database, and judging whether the compound is contained in a liquid mass spectrum of the compound of the corresponding classification; the search setting parameters are specifically: the search type is MSn; a database: reference; the search range is as follows: a re-corrected spectrogram; m/z precision: 0.002Th (Da); lowest relative response: 1 percent; the highest relative response: 100 percent; ionization mode: ESI; a mass analyzer: FT; ion fragmentation mode: HCD.
Further, in S4, the non-target compound extraction method includes: converting the raw file obtained by collection into ABF format through ABF Converter 3.0.0(Reifycs, Japan) software, and then performing data processing by using MS-DIAL 4.0 software; the parameters to be set are: minimum peak height: 1000, parts by weight; consider the chloro-bromo isotope: is that; the other parameters are default parameters; subsequently, MS-DIAL 4.0 software is used to perform peak alignment processing on the two samples under the collision energy respectively, and the specific parameters are as follows: mass number error: 0.01 Da; retention time error: 0.1 min.
Further, in S4, the rule followed for non-target compound screening is: the first rule is that fragments containing characteristic elements are preferred, the characteristic elements being oxygen, nitrogen or fluorine; the second rule is that the greater the number of carbon atoms contained in the fragment, the higher the probability that a parent ion containing a higher priority daughter ion will contain a characteristic structure.
The invention has the beneficial effects that:
(1) the invention innovatively combines an activity warning structure representing the activity effect of the compound with a characteristic mass spectrum fragment, and the warning fragment is used for non-target and high-resolution mass spectrum identification of an active substance, thereby providing a new thought for a toxicity identification part in an EDA method;
(2) based on 29 'activity warning structures' related to androgen activity, compared with mass spectrum fragmentation characteristics of only some compounds with similar structures in the existing research, the method disclosed by the invention is higher in flux, wider in range of covered compounds and more in category, and can be used for high-flux screening of a large number of unknown compounds;
(3) the database established by the invention covers the mass spectrum fragmentation characteristics of the compound which binds to androgen receptor and generates a quasi-antagonistic effect, compared with the existing research, the database can be explained by the action on the mechanism and establishes direct connection with certain toxicity characteristics;
(4) the method established by the invention is successfully applied to the actual environmental water body, overcomes the defects of time and labor consumption, high structural identification difficulty and lack of standard substance spectrogram and toxicity data of the traditional non-target compound identification, can quickly screen out possible androgen active substances according to the matching of secondary fragments under the condition of not determining the complete structure of the compound, greatly improves the screening efficiency of the non-target compound, and further provides a new technical scheme for identifying key toxic substances in the environment.
The specific implementation mode is as follows:
in order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.
Example 1
The method comprises the following steps: selecting an androgen active substance high-throughput screening model, and selecting 29 'activity warning structures' related to androgen activity in the endocrine disruptor high-throughput screening models and screening methods established in two patents of publication numbers CN109815532A and CN109545289A, as shown in Table 1:
TABLE 1 Secondary Warning Structure for androgen actives
Step two: matching and classifying the 29 active warning structures in the step one based on Classyfire functional group classification standard (http:// Classyfire. wishardlab. com), as shown in Table 2;
extracting each group of characteristic secondary fragments corresponding to each classification in a characteristic fragment library, wherein the characteristic fragment library is a self-contained characteristic fragment library of MS-FILDER 3.0 Software (http:// prime.psc.riken.jp/Metabolomics _ Software/MS-FILTER/index.html); and (3) sorting and summarizing to form secondary fragment information corresponding to each 'active warning structure', and finally screening out secondary fragments not exceeding the corresponding atomic number contained in the 'active warning structure' according to the limitation of the element composition (the number of carbon, nitrogen and oxygen atoms) of the 'active warning structure', so as to form a 'active warning structure' secondary fragment library.
TABLE 2 matching results of "active alert Structure" with Classyfire database
Step three: verifying the characteristic secondary fragments summarized in the step two by using a spectrogram obtained by a methanol solution of a standard substance on a liquid chromatogram-high resolution mass spectrum, and if the selected standard substance does not contain a compound containing an 'activity warning structure', verifying the spectrogram of the compound containing the 'activity warning structure' in an mzCloud high resolution liquid mass spectrum database (https:// www.mzcloud.org) to establish a standard substance characteristic secondary fragment database; wherein the standard is selected from chemicals common in 428 environmental samples, see table 3; the concentration of the methanol solution was 50. mu.g/L.
(I) Standard substance verification
1. Test method
A methanol solution with the concentration of 50 mu g/L of chemicals which are commonly detected in 428 environmental samples in the table 3 is prepared, and the test method is as follows:
(1) the liquid phase method comprises the following steps:
ultra-high performance liquid chromatograph: ultimate 3000UHPLC, Thermo scientific, USA;
mobile phase: phase A: fisher mass spectrum ultrapure water, phase B: methanol;
a chromatographic column: waters ACQUITY UPLC BEH C18,1.7 μm,2.1mm × 150 mm;
sample introduction amount: 10 mu L of the solution; column temperature: 40 ℃;
gradient elution method: 0min 5% B, 1min 5% B, 36min 100% B, 50min 100% B, 50.1min 5% B, 55min 5% B.
(2) The mass spectrometry method comprises the following steps:
mass spectrometry: q activeTMFocus combined quadrupole OrbitrapTM，Thermo ScientificTMThe united states of america;
the collection mode is as follows: full MS-ddMS 2; polarity: sampling samples once in a positive ion/negative ion mode; ddMS2 mode: discovery:
full MS parameters: resolution ratio: 35000(m/z200), scanning range: 80-1000m/z, AGC target value: 1e6(ii) a Maximum IT: automatic; spectrogram data type: and (5) contour drawing.
ddMS2 Discovery parameter: resolution 17500(m/z200), isolation window; 1.0 m/z; step NCE: 20,50 and 80/110,140,170 are injected once respectively; AGC target value: 5e4(ii) a Maximum IT: automatic; and (3) cycle counting: 3; and (4) triggering a vertex: 2-6 s; and (3) dynamic exclusion: 6.0 s; charge number exclusion: 2-8,>8, exclusion of isotopes: opening; spectrogram data type: and (5) contour drawing.
2. Data processing method
(1) Screening of first order mass spectra peaks
Using the Target Screening function in the TraceFinder 5.0SP1(Thermo Fisher, USA) software, based on the list of 57 compounds predicted to have androgenic effects, a Screening method was established in positive and negative ion mode, with the adduct mode [ M + H [ ]]+Or [ M-H]-(ii) a Mass number error range 5 ppm; and the isotope error is 20%, the detected standard sample is matched, and the accurate mass number and retention time of the standard sample are derived.
(2) Extraction of secondary fragments corresponding to primary mass spectrum peaks
The raw file obtained by collection is converted into ABF format by ABF Converter 3.0.0 (Reifyccs, Japan) software, and then data processing is carried out by using MS-DIAL 4.0 software. The parameters to be set are: minimum peak height: 1000, parts by weight; consider the chloro-bromo isotope: is that; the other parameters are default parameters.
After the Peak extraction is finished, clicking in a Peak spot viewer based on the accurate mass number and retention time of the compound, displaying a secondary spectrogram on the right side, leading out secondary fragments of the secondary spectrograms into MS-FINDER3.0 software, and calculating the molecular formula of each fragment ion; then, the fragments of the compound can be matched with potential characteristic fragment ions contained in the 'activity warning structure' of the corresponding classification, and the error range is 2 mDa.
(3) Authentication
After peak extraction was complete, the mass number of the characteristic Fragment, Fragment type: daughter ions, error range: 0.002Da, ion abundance%: 1 e-5; after the search is finished, clicking the Unique Ion and the Show Ion Table in sequence, deriving a matching list, and judging whether the corresponding compound can be screened through the process.
From the tests, 57 compounds among 428 common standard compounds in the environment were predicted to have androgen activity, and could be detected by liquid quality and secondary fragment information could be obtained. They covered 16 "activity alert structures" in which 49 androgen actives were validated (compound information and groupings are shown in table 4), with a pass rate of 85.96%.
Unmatched compounds are shown in table 5, and include compounds with low response in ESI sources, such as 17- α -estradiol, 17- β -estradiol, mestranol, three compounds containing "activity alert structure" 2-3, which, due to low response, fail to detect secondary fragments containing 8 and less carbon atoms associated with formula C8 of "activity alert structure" 2-3; the compound diethanolamine lauric acid containing the "Activity Warning Structure" 3-2 measured only C4H10NO2 +、C12H21O+、C12H23O2 +Molecular formula C of three secondary fragments, 3-2' activity warning structure11NO is associated with secondary fragments containing 11 and less carbon atoms, 1 or 0 nitrogen atoms, whereas diethanolamine lauric acid cannot be measured for its associated secondary fragments.
Furthermore, in the negative ion mode, the generated fragments of halogen-containing compounds, which are high in response and tend to be halogen-containing ions, including the compounds 4-hydroxychlorothalonil and bromoxynil of the "activity alert structure" 2-1, can only detect secondary fragments containing chlorine and bromine, which cannot be detected from the molecular formula C of the "activity alert structure" 2-17N-related secondary fragments containing 7 and less carbon atoms, 1 nitrogen atom; the compound tetrabromobisphenol A containing 'activity warning structure' 2-6 can only detect Br-And (4) fragmenting. Therefore, in the conventional studies, the halogenated compound is often based on a halogen ion (Cl)-，Br-，I-) Non-target halide screening is performed. The fragment ions in the invention mainly contain characteristic elements such as nitrogen, oxygen and the like, most of compounds containing the two elements are detected in a positive ion mode, and 38 standard substances are detectedThe detection is only carried out in a positive ion mode, 5 compounds can be detected in both positive and negative ions, and only 6 compounds can be detected in a negative ion mode, which indicates that the standard substance containing the 'activity warning structure' is mainly detected in the positive ion mode, and some compounds, particularly halogen-containing compounds, which are detected in the negative ion mode are probably out of the applicable range of the established method.
Finally, the fragment ions obtained from the standard were matched with the fragment database to determine the characteristic fragment ions for standard validation, covering 16 "active alert structures", as shown in table 6.
TABLE 3-428 Standard lists
TABLE 4 information on Compounds containing "Activity alert Structure" verified by standards
TABLE 5 Compound containing "Activity alert Structure" and its measured fragmentation information not validated by Standard sample
TABLE 6 characteristic fragment ions corresponding to "active alert Structure" verified by Standard
(II) Mass Spectroscopy database verification
Mass Spectrometry spectrogram verification is performed by using a high-resolution liquid mass spectrogram (https:// www.mzcloud.org) of a compound in an mzCloud database; information of 9002 compounds (as long as 2019,10 and 14 days) are obtained from Reference Library of an mzCloud high-resolution liquid mass spectrometry database website, including mzCloud, name, Inchi number, molecular formula, InChIKey and the like, and since SMILE numbers provided on the website are not complete and the format is problematic, and cannot be directly introduced into PaDEL-Descriptor software, each compound contains the Inchi number information, the Inchi number is uniformly converted into a standard SMILE number by using Open Babel 2.4.1 software for activity prediction.
Dividing the compounds in the mzCloud database into 29 second-level active warning structure groups according to a hierarchical warning model, and grouping to screen out corresponding compounds; then, for each potential secondary fragment, searching a compound containing corresponding characteristic ions by using a Peak Search function in an mzCloud database, and judging whether the compound is contained in a liquid mass spectrum of the compound of the corresponding classification; the search setting parameters are specifically: the search type is as follows: MSn; a database: reference; the search range is as follows: a re-corrected spectrogram; m/z precision: 0.002Th (Da); lowest relative response: 1 percent; the highest relative response: 100 percent; ionization mode: ESI; a mass analyzer: FT; ion fragmentation mode: HCD.
73 compounds in the mzCloud spectrogram database cover 7 fragments of 'activity warning structures' (1-1, 1-4, 1-5, 1-9, 2-2, 2-5, 3-3), cover 67 compounds, and the proportion reaches 91.78%; there were 6 compounds not covered, see table 7; the molecular formulas of 1-4 and 3-3 'activity warning structures' contained in the compounds are respectively C5NO and C6NO, the number of atoms is small, and secondary fragments containing 5 or 6 or less carbon atoms, 1 or 0 nitrogen atom or oxygen atom are generated, so that the compound is required to be fragmented to a high degree, and 6 substances can not generate related fragments; of these compounds, compounds with mzcloudd 4529 have only a spectrum from APCI source, and not within the test range of the present method, compounds with mzcloudd 4443, 5271 and 7005 have more halogen atoms and thus may not produce fragments directly related to the "active alert structure", while compounds with mzcloudd 3766 and 6366 have 4 oxygen atoms and are more difficult to punch fragments containing only 1 or 0 oxygen atoms.
Finally, the fragment ions in the mzCloud database are matched with the fragment database, and the characteristic fragment ions verified by the mass spectrometry spectrogram database are determined, which comprise 7 'activity warning structures', and are shown in table 8.
Through the screening of a standard substance and a mass spectrum spectrogram database, 6 'activity warning structures' (1-6, 1-7, 1-8, 1-10, 2-4 and 2-8) still exist, but cannot be verified due to the fact that the structures are complex and mass spectrum data of the standard substance are lacked, but the number of the 'activity warning structures' in 845 compounds used for establishing corresponding models is only 59, and the 'activity warning structures' contained in the other 786 compounds can extract characteristic secondary fragments verified by the spectrogram library and account for 93.02% of all the compounds, so that the relation between the 'activity warning structures' and the secondary fragments of the compounds established by the method can cover most androgen active compounds; the 59 compounds without spectrogram information are compounds in a ChemBL database, none of which has a CAS number, the compounds may not be common in the environment, or the compounds are not suitable for compounds capable of being detected by liquid quality, such as some endocrine disruptors like organochlorine pesticides, polychlorinated biphenyl, polybrominated diphenyl ethers, and the like, the detection limit of the compounds is higher when the compounds are detected by the liquid quality, the substances are detected by the gas quality to obtain the lower detection limit, and estrogenic compounds like 17-beta-estradiol and the like can obtain higher response in the liquid quality of an APCI source. Therefore, for compounds not covered by the method, the range of compounds that can be detected should be widened by combining gas chromatography or different ion sources such as APCI and APPI in liquid.
TABLE 7 information on Compounds containing "Activity alert Structure" not validated by spectral library
TABLE 8 characteristic fragment ions corresponding to "Activity alert Structure" validated library
Step four, collecting actual environment samples with different pollution degrees, including industrial wastewater and surface water; the method disclosed by the invention with the publication number of CN109682897A is used for pretreatment, a methanol solution is prepared, the concentration multiple of a wastewater sample is 1000 times, the concentration multiple of surface water is 4000 times, an on-machine test is carried out according to a liquid chromatography-high resolution mass spectrometry method in the third step, non-target compounds are extracted, and data analysis is carried out. The extraction method of the non-target compound comprises the following steps: converting the raw file obtained by collection into ABF format through ABF Converter 3.0.0(Reifycs, Japan) software; then using MS-DIAL 4.0 software to process data; the parameters to be set are: minimum peak height: 1000, parts by weight; consider the chloro-bromo isotope: is that; the other parameters are default parameters. Subsequently, MS-DIAL 4.0 software is used to perform peak alignment processing on the two samples under the collision energy respectively, and the specific parameters are as follows: mass number error: 0.01 Da; retention time error: 0.1 min. Then comparing and screening the molecular formula and the secondary fragments with the characteristics verified in the third step, and if the molecular formula and the secondary fragments are not matched with the characteristics verified in the third step, judging that the fragments do not have androgen activity; if so, it can be judged that it is likely to have androgenic activity; the rules followed by the screening are: the first rule is that fragments containing characteristic elements are preferred, the characteristic elements being oxygen, nitrogen or fluorine; the second rule is that the number of carbon atoms contained in the fragment is high in priority, and the parent ion containing a daughter ion with high priority is preferentially selected, and the probability that the parent ion contains a characteristic structure is higher; in the screening process, the characteristic fragments input are the corresponding fragments of each "activity alert structure" verified in step three, and the exact mass number and retention time of the possible androgen active substance can be derived.
Comparative example 1
Collecting actual environmental samples with different pollution degrees, including industrial wastewater and surface water; the method disclosed by the invention with the publication number of CN109682897A is used for pretreatment, a methanol solution is prepared, the concentration multiple of a wastewater sample is 1000 times, the concentration multiple of surface water is 4000 times, and an on-machine test is carried out according to a liquid chromatography-high resolution mass spectrometry method in the third step.
The difference from example 1 is that the target compound was selected and the list of suspect screens was 914 compounds from the mzCloud Reference database 8778 that were predicted to have androgenic activity. Suspected screening was performed using TraceFinder 5.0SP1(Thermo Fisher, USA) software, database in Library Search function using db format database of mzVault Reference May 2019 from Compound discover 3.0SP1(Thermo Fisher, USA) software, Library check Identify, database Search type: mzVault, MS Order: MS2, mzVault Setting: search type: positive ion: classic, negative ion: high Chem; prefilter Type: compound; fragment Tolerence: 2 Mmu; score Threshold: 60, adding a solvent to the mixture; 60 for the paging Value; selecting Reverse Search; ignore Precursor is selected. The remaining parameter settings were the same as in example 1. After the detected compounds are determined, their names, molecular formulas and retention times in the detected samples are introduced into TraceFinder software, and the Target Screening function is used to detect the corresponding compounds in all samples, with the parameters set: checking identity by RT; peak Detection selects Neoarest RT.
Through matching with a compound predicted to have androgen in an mzCloud library, 64 Level2 a-grade compounds containing an 'activity warning structure' are detected in all samples, and through comparison of accurate mass number and retention time, the total 56 compounds are found to be screened by the method of example 1, the coverage rate of the compounds containing the 'activity warning structure' reaches 87.50 percent, so that the substances which can be screened by the method can cover the compounds containing the 'activity warning structure', and the established connection between mass spectrum fragments and the compound 'activity warning structure' can be applied to poison screening of actual environmental samples.
Comparative example 2
Collecting actual environmental samples with different pollution degrees, including industrial wastewater and surface water; the method disclosed by the invention with the publication number of CN109682897A is used for pretreatment, a methanol solution is prepared, the concentration multiple of a wastewater sample is 1000 times, the concentration multiple of surface water is 4000 times, and an on-machine test is carried out according to a liquid chromatography-high resolution mass spectrometry method in the third step.
The difference from example 1 is that the target compound was selected and the list of suspected screenings for 2237 compounds tested in the ToxCast database to have androgenic activity, the parameter settings were the same as in step three of example 1.
After screening of a suspicious list, 95 compounds which are matched in the first stage and contain the secondary fragments exist in all samples, wherein 50 compounds which have liquid mass spectrograms exist in an mzCloud database, and only 23 compounds with identification confidence Level2a exist after matching of the secondary fragments; while 56 compounds with Level2a grades can be screened out in example 1, and the method provided by the invention is proved to be capable of predicting through the correlation of an activity warning structure model, so that a large number of compounds without toxicity information can be identified. In the compounds with undeterminable structures, 45 liquid mass spectrograms are not available, and compared with the matched peaks in the example 1, 20 of the compounds contain characteristic fragments corresponding to the contained 'activity warning structures', which indicates that the compounds possibly contain 'activity warning structures', and are possible androgen active substances; the 27 compounds with mismatched spectra were compared to the matched peaks in example 1, and 14 contained characteristic fragments corresponding to the "active alert structure" contained therein, indicating that the peaks detected, although not the compounds of the target list, may be isomers containing the "active alert structure". Thus, the methods developed by the present invention allow for the determination that suspect screening methods are unable to determine whether a compound of structure is likely to have androgenic activity, thereby reducing the amount of work required for subsequent structure identification and toxicity confirmation.
The method of the invention innovatively combines an activity warning structure representing the activity effect of the compound with the characteristic mass spectrum fragments, has higher flux, wider range of covered compounds and more categories compared with the traditional method, establishes direct connection between toxicity characteristics and mass spectrum fragments, can carry out high-flux screening on a large number of unknown compounds, can quickly screen out possible androgen active substances according to the matching of secondary fragments under the condition of not determining the complete structure of the compound, greatly improves the non-target screening efficiency, and further provides a new technical scheme for identifying key toxic substances in the environment.
The above is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above embodiments, and all technical solutions belonging to the idea of the present invention belong to the protection scope of the present invention, it should be noted that, for those skilled in the art, several modifications and decorations without departing from the principle of the present invention should be regarded as the protection scope of the present invention.
Claims (5)
1. A method for identifying an androgen active substance in an environmental sample based on characteristic liquid mass fragment matching, comprising the steps of:
s1, selecting a high-throughput screening model of the androgen active substance, wherein the model comprises the correlation between the characteristic structure of the compound and whether the compound has androgen activity, and selecting an 'activity warning structure' related to the androgen activity;
s2, classifying the categories of functional groups contained in the 'activity warning structure' in the S1 through functional group classification standards, inquiring characteristic secondary fragments corresponding to each category in the existing characteristic fragment library corresponding to compound classification, sorting and summarizing to form secondary fragment information corresponding to each 'activity warning structure', establishing a correlation between the 'activity warning structure' and the characteristic secondary fragment structure, and forming a 'activity warning structure' secondary fragment library;
s3, verifying the characteristic secondary fragments summarized in the S2 by a spectrogram obtained by a methanol solution of a standard substance on a liquid chromatogram-high resolution mass spectrum, and if the selected standard substance does not contain a compound containing an 'activity warning structure', verifying the spectrogram of the compound containing the 'activity warning structure' in a mass spectrum database to establish a standard substance characteristic secondary fragment database;
the standard substance is selected from chemicals which are commonly found in 428 environmental samples, and the concentration of a methanol solution is 50 mug/L;
the liquid chromatography conditions were: ultra-high performance liquid chromatograph: an Ultimate 3000UHPLC, Thermo Scientific, U.S.A.was used; mobile phase: phase A: fisher water, phase B: methanol; a chromatographic column: waters ACQUITY UPLC BEH C18,1.7 μm,2.1mm × 150 mm; sample introduction amount: 10 mu L of the solution; column temperature: 40 ℃; gradient elution method: 0min 5% B, 1min 5% B, 36min 100% B, 50min 100% B, 50.1min 5% B, 55min 5% B;
the mass spectrum conditions are as follows: a mass spectrometer is adopted: q exact Focus combination quadrupole Orbitrap ™, Thermo Scientific ™ chamber, USA; the collection mode is as follows: full MS-ddMS 2; polarity: sampling samples once in a positive ion/negative ion mode; ddMS2 mode: discovery: full MS parameters: resolution ratio: at m/z200 resolution of 35000, scan range: 80-1000m/z, AGC target value: 1e 6; ddMS2 Discovery parameter: isolating the window at a resolution of 17500 m/z 200; 1.0 m/z; step NCE: 20,50 and 80/110,140,170 are injected once respectively; AGC target value: 5e 4; maximum IT: automatic; and (3) cycle counting: 3; and (4) triggering a vertex: 2-6 s; and (3) dynamic exclusion: 6.0 s; charge number exclusion: 2-8, >8, excluding isotopes: opening; spectrogram data type: a contour map;
the screening method of the first-order mass spectrum peak of the compound in the standard product verification comprises the following steps: establishing a Screening method under a positive and negative ion mode by using a Target Screening function in TraceFinder 5.0SP1 software and based on a list of compounds predicted to have an androgen effect in a standard substance, wherein an adduct mode is [ M + H ]]+Or [ M-H]-Mass number error range 5 ppm; matching the detected standard sample with isotope error of 20 percent, and deriving the accurate mass number and retention time of the standard sample;
the extraction method of the secondary fragment corresponding to the primary mass spectrum peak of the compound in the standard product verification comprises the following steps: converting the raw file obtained by collection into ABF format through ABF Converter 3.0.0 software, and then performing data processing by using MS-DIAL 4.0 software; the parameters to be set are: minimum peak height: 1000, parts by weight; consider the chloro-bromo isotope: is that; the other parameters are default parameters; after the Peak extraction is finished, clicking in a Peak spot viewer based on the accurate mass number and retention time of the compound, displaying a secondary spectrogram on the right side, leading out secondary fragments of the secondary spectrograms into MS-FINDER3.0 software, and calculating the molecular formula of each fragment ion; then, the fragments of the compound are matched with potential characteristic fragment ions contained in an 'activity warning structure' corresponding to the fragment of the compound, and the error range is 2 mDa;
s4, performing on-machine test on the methanol solution of the environmental sample extract according to a liquid chromatography-high resolution mass spectrometry method in S3, extracting non-target compounds, selecting a certain concerned non-target peak, comparing and screening the molecular formula and the secondary fragment with a standard substance characteristic secondary fragment database established in S3, and if the molecular formula and the secondary fragment do not meet the standard substance characteristic secondary fragment database, judging that the molecular formula and the secondary fragment do not have androgen activity; if so, judging that the protein possibly has androgen activity, and carrying out a subsequent poison identification process;
non-target compound screening follows the rules: the first rule is that fragments containing characteristic elements are preferred, the characteristic elements being oxygen, nitrogen or fluorine; the second rule is that the greater the number of carbon atoms contained in the fragment, the higher the probability that a parent ion containing a higher priority daughter ion will contain a characteristic structure.
2. The method of claim 1, wherein in step S2, the functional group classification criteria is classsyfire compound classification criteria, and the signature fragment library is a self-contained signature fragment library of MS-folder 3.0 software; in S3, the mass spectrum database is an mzCloud high resolution liquid mass spectrum database.
3. The method for identifying an androgen active substance in an environmental sample based on characteristic liquid-mass fragment matching of claim 1, wherein in S3, the method for standard validation is: using the Search-MS/MS Fragment Search in MS-DIAL software, input mass number of feature Fragment, Fragment type: daughter ions, error range: 0.002Da, ion abundance%: 1 e-5; after the search is finished, clicking the Unique Ion and the Show Ion Table in sequence, deriving a matching list, and judging whether the corresponding compound can be screened through the process.
4. The method for identifying an androgen active substance in an environmental sample based on feature liquid mass fragment matching of claim 1, wherein in S3, the mass spectrogram verification method is: grouping the compounds in the mzCloud database according to the contained 'activity warning structure' in S1, and screening out the corresponding compounds; then, for each potential secondary fragment, searching a compound containing corresponding characteristic ions by using a Peak Search function in an mzCloud database, and judging whether the compound is contained in a liquid mass spectrum of the compound of the corresponding classification; the search setting parameters are specifically: the search type is as follows: MSn; a database: reference; the search range is as follows: a re-corrected spectrogram; m/z precision: 0.002Th, i.e. when the number of charges carried by the ion is 1, the accuracy of m is 0.002 Da; lowest relative response: 1 percent; the highest relative response: 100 percent; ionization mode: ESI; a mass analyzer: FT; ion fragmentation mode: HCD.
5. The method for identifying an androgen active substance in an environmental sample based on characteristic liquid-mass fragment matching according to claim 1, wherein in S4, the non-target compound extraction method is: converting the raw file obtained by collection into ABF format through ABF Converter 3.0.0 software, and then performing data processing by using MS-DIAL 4.0 software; the parameters to be set are: minimum peak height: 1000, parts by weight; consider the chloro-bromo isotope: is that; the other parameters are default parameters; subsequently, MS-DIAL 4.0 software is used to perform peak alignment processing on the two samples under the collision energy respectively, and the specific parameters are as follows: mass number error: 0.01 Da; retention time error: 0.1 min.
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