B-type trichothecene toxin metabolite screening method and application

Abstract

The invention discloses a B-type trichothecene toxin metabolite screening method and application. The screening method comprises the following steps: 1) constructing a B-type trichothecin metabolite prediction database, and determining general characteristic structure diagnosis ions of B-type trichothecin; 2) performing first matching screening on the primary mass spectrum information of each compound in the sample to be detected in the B-type trichothecene toxin metabolite prediction database, and determining a compound molecular formula corresponding to each mass spectrum peak in the primary mass spectrum to obtain a first compound molecular formula set; performing matching screening on the secondary mass spectrum information of each compound in the sample to be detected and general characteristic structure diagnosis ions of the B-type trichothecene toxin, and collecting compound molecular formulas of which the matching number of characteristic structure fragment ions is greater than or equal to a preset threshold value to obtain a second compound molecular formula set; and 3) analyzing the molecular formula of the compound in the intersection of the two sets, and determining the candidate structure of the B-type trichothecene toxin metabolite in the sample to be detected.

Description

Technical Field

[0001] The present invention relates to the technical field of analysis and detection, and more specifically, to a screening method and application of type B trichothecene toxin metabolites. Background Art

[0002] Trichothecenes (TCTs) are sesquiterpenoid fungal toxins containing epoxy groups produced by Fusarium, which mainly cause neurological, gastrointestinal and immune disorders in animals. TCTs can be divided into four groups, namely A, B, C and D types. Among them, type B trichothecenes (TCT-Bs), such as deoxynivalenol (DON) and its derivatives, are widely contaminated in food and seriously threaten food security. Therefore, major countries and regions in the world attach great importance to its pollution problem and have successively formulated limit standards for DON.

[0003] Studies have shown that plants will activate defense mechanisms after being contaminated by TCT-Bs. Through the action of enzymes such as cytochrome P450, uridine diphosphate glucuronyl transferase (UGTs), sulfotransferase, and glutathione S-transferase, phase I and phase II metabolic reactions occur to form new metabolites. TCT-Bs also metabolize themselves in fungi to produce new metabolites. This joint metabolism of fungi and plants makes the types of TCT-Bs metabolites in grains more complex and diverse. Risk assessment of metabolites shows that these metabolites may be toxic. However, due to changes in their physical and chemical properties such as chemical structure, molecular weight, polarity and solubility, traditional analytical methods cannot detect them, resulting in a serious underestimation of TCT-Bs contamination. Therefore, it is urgent to develop efficient and accurate identification and quantitative analysis methods for TCT-Bs and their metabolites, and to systematically study and evaluate the food safety issues caused by the synergistic contamination of TCT-Bs and their metabolites.

[0004] With the development of high-resolution mass spectrometry, non-targeted screening has gradually become the main means of screening and identifying unknown metabolites. With its high resolution and high scanning rate, high-resolution mass spectrometry can simultaneously provide accurate mass numbers, isotope distribution and secondary fragmentation information of the parent and metabolites, thereby realizing the screening of unknown metabolites and the determination of candidate molecular formulas and chemical structures without repeated pretreatment and detection. Nevertheless, how to comprehensively, efficiently and accurately identify and identify unknown metabolites of interest from the huge compound spectrum information of LC-HRMS is still one of the main challenges facing the current identification of unknown substances. The most common method for metabolite identification is to match MS and MS / MS spectra with standards, but this method is heavily dependent on standards and has limited recognition capabilities for potential or new metabolites without standards. With the development of simulation computing technology, researchers have constructed metabolic pathway-driven metabolite prediction databases such as BioTransformer, Mycotoxin DB, and ToxinDB to expand the types of metabolites. These databases have greatly expanded the identification range of unknown metabolites, but the existing predictive metabolic databases are mainly suitable for the prediction of broad-spectrum metabolites, and the prediction coverage and accuracy of specific types of metabolites still need to be improved. In addition, the convenience of screening unknown metabolites also needs to be improved.

[0005] Diagnostic ions are fragment ions with common characteristics generated by the mass spectrometry fragmentation behavior of a certain type of compound. Diagnostic ion filtering technology driven by compound characteristic structure has become an important means to screen specific categories of unknown metabolites. Cui et al. developed an unknown identification strategy based on diagnostic ion filtering technology, and successfully identified four TCT-B metabolites in food through four diagnostic ions. González-Jartín et al. applied diagnostic ion filtering technology to the identification of unknown type A trichothecenes toxin (TCT-A) metabolites, and successfully used three diagnostic ions to identify seven NEO and T-2 metabolites in the extract of F. sporotrichioides. However, conventional diagnostic ion filtering technology selects 3-7 common characteristic ions for screening based on the common characteristic structure of the compound, which is easily affected by factors such as different adduct forms, source cleavage fragments, isotopes and neutral losses, resulting in the generation of a large number of candidate metabolites, huge amount of redundant information, easy to produce false positives, and heavily dependent on expert experience, which is time-consuming and laborious. Summary of the invention

[0006] Based on this, an object of the present invention is to provide a method for screening type B trichothecene toxin metabolites. The type B trichothecene toxin metabolite prediction database constructed in the screening method basically covers all type B trichothecene toxin metabolites that may be produced in currently known metabolic reactions, and after combined analysis with the common characteristic structural diagnostic ions of type B trichothecene toxins, the molecular formula of the type B trichothecene toxin metabolites in the sample to be tested can be inferred relatively quickly and accurately, and then the metabolic pathway provided by the type B trichothecene toxin metabolite prediction database is combined to eliminate the interference of false positives, and finally the candidate structure of the type B trichothecene toxin metabolite is inferred.

[0007] Another object of the present invention is to provide an application of the above screening method in simulating the analysis of the types of mycotoxins in food.

[0008] In order to achieve the above first object, the present invention adopts the following technical scheme:

[0009] The present invention discloses a method for screening type B trichothecene toxin metabolites, comprising the following steps:

[0010] 1) Construct a prediction database for type B trichothecenes metabolites and identify the common characteristic structural diagnostic ions of type B trichothecenes;

[0011] 2) performing a first matching screening on the primary mass spectrum information of each compound in the sample to be tested in the type B trichothecene toxin metabolite prediction database to determine the compound molecular formula corresponding to each mass spectrum peak in the primary mass spectrum, and obtaining a first compound molecular formula set;

[0012] Matching and screening the secondary mass spectrometry information of each compound in the sample to be tested with the common characteristic structure diagnostic ions of type B trichothecenes, collecting the molecular formulas of compounds whose matching number of characteristic structure fragment ions is greater than or equal to a preset threshold, and obtaining a second compound molecular formula set;

[0013] 3) Analyzing the compound molecular formulas in the intersection of the first compound molecular formula set and the second compound molecular formula set to determine the candidate structures of the type B trichothecene toxin metabolites in the sample to be tested.

[0014] Further, the construction of a type B trichothecene toxin metabolite prediction database includes:

[0015] Simulate all metabolic reactions that type B trichothecenes may undergo, where the total number of metabolic steps is 1-3 steps, summarize the potential metabolite structures and metabolic pathways of type B trichothecenes after metabolism, and construct a prediction database for type B trichothecenes metabolites;

[0016] Wherein, the metabolic reaction includes phase I metabolic reaction and / or phase II metabolic reaction.

[0017] Further, the phase I metabolic reaction includes but is not limited to one of a dehydration reaction, a desaturation reaction, a hydration reaction, a nitro reduction reaction, an oxidation reaction, and a reduction reaction;

[0018] The phase II metabolic reactions include, but are not limited to, one or more of acetylation, arginine substitution, cysteine ​​substitution, glucoside substitution, glucuronide substitution, glutamine substitution, glycine substitution, glutathione substitution, methylation, ornithine substitution, hexadecyl substitution, octadecyl substitution, sulfonation, taurination, diglucoside substitution, triglucoside substitution, tetraglucoside substitution, xylan-glucose substitution, malonyl-glucoside substitution, feruloyl glucose substitution, N-acetylcysteine ​​substitution, N-acetyl substitution, benzoyl substitution, hexitol substitution, disoxihydride substitution, and de-epoxy reaction.

[0019] Furthermore, the number of reaction steps of the phase I metabolic reaction is 0 and the number of reaction steps of the phase II metabolic reaction is 1-3, or the number of reaction steps of the phase I metabolic reaction is 1 and the number of reaction steps of the phase II metabolic reaction is 0-2.

[0020] Furthermore, the common characteristic structural diagnostic ions of type B trichothecenes are determined to include:

[0021] Analyze the secondary mass spectra of known type B trichothecene toxin metabolite standards, determine fragment ions with common characteristic structures, and obtain diagnostic ions with common characteristic structures of type B trichothecene toxins;

[0022] Among them, the common characteristic structure diagnostic ions include characteristic fragments with m / z of 279.1227, 277.1071, 249.1121, 247.0965, 231.1016, 229.0859, 203.1067, 201.0910, 191.1067, 189.0910, 177.0910, 175.0754, 125.0597, 123.0441, 109.0648, 107.0491, and 137.0597.

[0023] Furthermore, the molecular weight of each metabolite in the type B trichothecene toxin metabolite prediction database is greater than or equal to 200 Da.

[0024] Furthermore, the preset threshold is 4.

[0025] Furthermore, the screening method is performed by the data processing software Compound Discover 3.3.

[0026] Furthermore, matching and screening the primary mass spectrometry information of each compound in the sample to be tested in the type B trichothecene toxin metabolite prediction database includes:

[0027] A mass spectrometer is used to collect data for each compound in the sample to be tested, and data processing software is used to perform peak extraction, alignment and integration on the collected raw data. The primary mass spectrometry information of each compound in the sample to be tested is compared with the data information of the type B trichothecene toxin metabolite prediction database to determine the molecular formula of the compound corresponding to each mass spectrometry peak in the primary mass spectrum.

[0028] Furthermore, the comparison is to identify the structure and metabolic pathway of the metabolite by combining information such as molecular formula, isotope distribution peak, accurate molecular weight, retention time, metabolic reaction, etc., and control the mass accuracy Δm≤5ppm, the isotope distribution peak matching degree (IP)≥70 points, and the response intensity≥e 5 , the expected retention time deviation is <2min.

[0029] Further, step 3) includes:

[0030] According to the compound molecular formula and the corresponding metabolic pathway, a false positive interference subtraction operation is performed on the compound molecular formula in the intersection of the first compound molecular formula set and the second compound molecular formula set to determine the candidate structure of the type B trichothecene toxin metabolite in the sample to be tested.

[0031] In order to achieve the above second purpose, the present invention adopts the following technical solutions:

[0032] The present invention discloses an application of the above-mentioned screening method in simulating the analysis of types of mycotoxins in grains.

[0033] The beneficial effects of the present invention are as follows:

[0034] 1. The present invention constructs a relatively complete prediction database of type B trichothecenes metabolites based on the potential metabolic reactions and metabolic pathways of type B trichothecenes. In this prediction database, the metabolic pathway of each metabolite can be learned, which is conducive to eliminating the interference of false positives.

[0035] 2. The purpose of determining the common characteristic structure diagnostic ions is to use the structure of the known metabolite standard to obtain fragment ions with common characteristic structures, so as to analyze the mass spectrometry fragmentation behavior of type B trichothecenes and improve the accuracy of judging the metabolite structure in the sample to be tested.

[0036] 3. The present invention proposes a comprehensive screening and identification method for TCT-Bs metabolites based on the dual drive of metabolic pathways and core skeleton structures. The prediction database of type B trichothecenes metabolites and the common characteristic structural diagnostic ions of type B trichothecenes are used to match the primary mass spectrometry information and the secondary mass spectrometry information of each compound in the sample to be tested to screen the results. Then, the candidate structures of type B trichothecenes metabolites in the sample to be tested can be obtained relatively quickly and accurately based on the metabolic reactions and metabolic pathways, thereby eliminating false positives and improving the accuracy of screening.

[0037] 4. Compared with traditional screening and identification methods, the screening method provided by the present invention is not limited to the screening of existing metabolite standards, but is also effective in screening unknown metabolite structures of type B trichothecenes. In addition, while maintaining a high screening coverage, it can effectively reduce the number of candidate metabolites, thereby improving the screening and identification efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] The specific implementation modes of the present invention are further described in detail below in conjunction with the accompanying drawings.

[0039] Figure 1 A flowchart showing the operation of the screening method of the present invention.

[0040] Figure 2 Shown are secondary mass spectra of NIV, DON, DOM, DON-3G, 3-AcDON standards and wheat samples in a specific embodiment.

[0041] Figure 3 The secondary mass spectrum of unknown metabolites in a wheat sample according to a specific embodiment is shown. DETAILED DESCRIPTION

[0042] In order to more clearly illustrate the present invention, the present invention is further described below in conjunction with preferred embodiments and accompanying drawings. Similar components in the accompanying drawings are represented by the same reference numerals. It should be understood by those skilled in the art that the content specifically described below is illustrative rather than restrictive, and should not be used to limit the scope of protection of the present invention.

[0043] In order to improve the screening efficiency and accuracy of type B trichothecene toxin metabolites in food, the present invention provides a method for screening type B trichothecene toxin metabolites, which specifically comprises the following steps:

[0044] 1) Construction of a prediction database for type B trichothecenes metabolites

[0045] Efficient prediction of TCT-Bs potential metabolites is one of the important means to identify unknown metabolites. The construction of the type B trichothecenes metabolite prediction database is based on the possible metabolic reactions of type B trichothecenes that have been reported. Since the metabolites of type B trichothecenes are mainly deoxynivalenol (DON) or its derivatives, the following study takes DON as an example for detailed description.

[0046] To date, there are 34 metabolic reactions that can occur in DON (see Table 1), among which numbers 1-6 belong to phase I metabolic reactions, and numbers 7-34 belong to phase II metabolic reactions.

[0047] Table 1

[0048]

[0049]

[0050] Generally, DON undergoes up to three metabolic reactions. The possible metabolic pathways and the number of metabolite molecular formulas produced are as follows:

[0051] (1) When the number of phase I metabolic reaction steps is 0 and the number of phase II metabolic reaction steps is 1, 28 DON metabolite molecular formulas M1 can be generated through mathematical statistics;

[0052] (2) When the number of phase I metabolic reaction steps is 0 and the number of phase II metabolic reaction steps is 2, 406 DON metabolite molecular formulas M2 can be generated through mathematical statistics;

[0053] (3) When the number of metabolic reaction steps in phase I is 0 and the number of metabolic reaction steps in phase II is 3, the number of DON metabolite molecular formulas M3 that can be generated through mathematical statistical calculations is 4060;

[0054] (4) When the number of phase I metabolic reaction steps is 1 and the number of phase II metabolic reaction steps is 0, six DON metabolite molecular formulas M4 can be generated through mathematical statistics;

[0055] (5) When the number of phase I metabolic reaction steps is 1 and the number of phase II metabolic reaction steps is 1, 168 DON metabolite molecular formulas M5 can be generated through mathematical statistics;

[0056] (6) When the number of phase I metabolic reaction steps is 1 and the number of phase II metabolic reaction steps is 2, mathematical statistics show that there are 2436 DON metabolite molecular formulas M6 that can be generated.

[0057] After calculation, a total of 10,657 metabolite formulas may be obtained. After deleting metabolite formulas with a molecular weight less than 200 Da and repeated metabolite formulas, the final predicted database contains 4,321 metabolite formulas and information on various metabolic transfer groups.

[0058] In order to further evaluate the performance of the above-mentioned prediction database, the coverage of the prediction database constructed by the present invention and the BioTransformer metabolic prediction tool was compared. The prediction database constructed by the present invention and BioTransformer predicted 4321 and 26 TCT-Bs metabolite formulas, respectively, and the number of matches with the 41 TCT-Bs metabolite formulas reported in the literature was 41 and 14, respectively, with coverage rates of 100% and 33.3%, respectively. The results show that the prediction database constructed by the present invention performs best in terms of both the number of predicted metabolites and coverage. In addition, newly reported metabolic pathways can be added to the prediction database at any time to continuously improve its coverage. The prediction database covers the metabolic conversion process, which is convenient for quickly inferring the chemical transformation process of candidate compounds and their possible chemical structures.

[0059] 2) Determine the common characteristic structural diagnostic ions of type B trichothecenes

[0060] The typical characteristics of TCT-Bs are a sesquiterpene skeleton structure with C9,10-double bonds and C12,13-epoxidation. NIV and Fus-X have four substituents at C3, C4, C15 and C7, while DON, DON-3G and 3-AcDON have an H atom at the C4 position, so there are only three substituents in the structure. With the breakage and ring opening of the parent core structural group, fragment ions with common characteristic structures can be generated. By analyzing the secondary high-resolution mass spectra of existing TCT-Bs metabolite standards, the mass spectral fragmentation behavior of TCT-Bs metabolites was analyzed. The characteristic structural fragment ions of TCT-Bs metabolites with different numbers of substituents are different. Among them, the characteristic structural fragment ions of DON, DON-3G and 3-AcDON with three substituents are mainly 279.1227, 249.1121, 231.1016, 203.1067, 1 91.1067, 177.0910, 125.0597, 109.0648, and 137.0597, and the response is relatively high. The characteristic structural fragment ions of NIV and Fus-X with tetrasubstituted groups are mainly 277.1071, 247.0965, 229.0859, 201.0910, 189.0910, 175.0754, 123.0441, 107.0491, and 137.0597. There are 2 H atoms difference between the characteristic structural fragment ions of TCT-Bs with tri-substitution and tetra-substitution. These characteristic structural fragments are unique structural fragments of TCT-Bs. Therefore, these structural fragments with common characteristics are selected as diagnostic ions for screening of unknown TCT-Bs. The diagnostic ions are 279.1227, 277.1071, 249.1121, 247.0965, 231.1016, 229.0859, 203.1067, 201.0910, 191.1067, 189.0910, 177.0910, 175.0754, 125.0597, 123.0441, 109.0648, 107.0491, and 137.0597. The common characteristic structural fragment diagnostic ions proposed in the present invention are significantly more than the traditional diagnostic ions (usually 3-7), thereby improving the accuracy of screening. In addition, the number of substituents can be analyzed through the response intensity of different diagnostic ions, thereby facilitating the rapid classification of identified TCT-Bs.

[0061] The following steps are combined Figure 1 Provide explanation.

[0062] 3) Data collection of samples to be tested

[0063] Thirty wheat samples collected from high-pollution risk areas in central my country were screened, and data were collected using a high-resolution mass spectrometer. The steps are as follows:

[0064] After the sample was crushed (90% passed through a 40-mesh sieve) and mixed, 5.00 g was accurately weighed, and 20 mL of acetonitrile-water-acetic acid (70:29:1, volume ratio) extract was added, vortexed for 1 min, and after oscillation for 30 min, the solid-liquid separation was performed at 4000 r / min for 10 min. 0.5 mL of the supernatant was accurately transferred to a 1.5 mL centrifuge tube, diluted with 0.5 mL of water, vortexed for 1 min, and then centrifuged at 12000 r / min for 10 min. The supernatant was filtered with a 0.22 μm PTFE filter membrane, mixed and tested. The primary and secondary mass spectrometry simultaneous acquisition method of ultra-high performance liquid chromatography (UltiMate 3000, Thermo Fisher Scientific, Rockford, IL, USA)-high-resolution mass spectrometry (QExactive, Thermo Fisher Scientific, Rockford, IL, USA) was used for analysis to obtain the primary and secondary mass spectrometry information of each compound in the sample to be tested.

[0065] Compound Discover 3.3 was used to perform peak extraction, alignment, integration, background subtraction and other operations on the collected raw data.

[0066] The high-resolution mass spectrometer is a quadrupole / electrostatic field orbital trap high-resolution mass spectrometer Q-Exactive, purchased from ThermoFisher Scientific. The mass spectrometry conditions are: the temperature of the heated electrospray ion source is 300°C, the capillary voltage is 3.2 kV, the temperature of the ion transfer tube is 320°C, the sheath gas is 35 units, and the auxiliary gas is 10 units. Full scan / ddms2 scanning mode: the acquisition range is 100-1200Da, ESI+ positive ion mode acquisition, the primary mass spectrometry resolution is 70000FWHM, the secondary mass spectrometry resolution is 35000FWHM, and the collision energy is 20eV, 40eV, and 60eV.

[0067] 4) Screening and analysis

[0068] Compare the primary mass spectrometry information in the sample to be tested with the data information in the prediction database, combine the molecular formula, isotope distribution peak, accurate molecular weight, retention time, metabolic reaction and other information to identify the structure and metabolic pathway of the metabolite, control the mass accuracy Δm≤5ppm, the isotope distribution peak matching score (IP)≥70 points, and the response intensity≥e 5 , the expected retention time deviation is <2min, and the first compound molecular formula set is obtained.

[0069] The secondary mass spectrometry information in the sample to be tested is matched and screened with the common characteristic structure diagnostic ions obtained above, and the compound molecular formulas with the characteristic structure fragment ion matching number greater than or equal to 4 are obtained, and the mass accuracy Δm≤5ppm is controlled to obtain a second compound molecular formula set.

[0070] The compound formulas in the intersection of the first compound formula set and the second compound formula set were subjected to a false positive interference subtraction operation. The basis was that the technicians finally obtained 9 TCT-Bs metabolite formulas based on the compound formulas and the corresponding metabolic pathways, among which compounds 1-5 were confirmed to be NIV, DON, DON-3G, 3-AcDON, DOM (mass spectra see Figure 2 ).

[0071] The parent ion peak of compound 6 detected by high-resolution mass spectrometry was 492.2066 ([M+NH4]+), and the predicted molecular formula provided by the prediction database was C 21 H 30 O 12 (Δm = -1.89ppm), the isotope matching score is 84 points. From the high-resolution secondary mass spectrum ( Figure 3 ) It can be seen that after the compound is cleaved, there are 13 secondary mass spectrometry fragment ions that match the common characteristic structural fragment diagnostic ions, meeting the requirement that the number of characteristic fragment ion matches is ≥ 4. Among them, the fragment ions with m / z in the range of 100-320 are highly similar to the NIV fragment ions, and it is speculated that it is a NIV metabolite. Further analysis shows that the parent ion 492.2066 of this compound loses Δ179.0772 (C6H 11 O6+NH4) to form a fragment ion of 313.1267, which is the [M+H]+ peak of NIV. Combined with the metabolic pathways of candidate compounds matching this molecular formula in the prediction database, it is speculated that this compound is a product of the hydroxyl group (OH) being replaced by glucoside (C6H 11 O6) substitution, confirming that it is glucosidylated NIV.

[0072] The parent ion peak of compound 7 detected by high-resolution mass spectrometry was 534.2172 ([M+NH4]+), and the predicted molecular formula provided by the prediction database was C 23 H 32 O 13 (Δm = -1.58ppm), the isotope matching score is 74 points. From the high-resolution mass spectrum ( Figure 3 ) It can be seen that after the cleavage of the compound, there are 12 secondary fragment ions that match the common characteristic structure fragment diagnostic ions, which meets the requirement of characteristic fragment ion matching number ≥ 4, and it is judged to be a NIV metabolite. The metabolic pathway of the candidate compound matching the molecular formula in the prediction database is that the two hydroxyl groups (OH) are respectively replaced by glucosides (C6H11 O6), acetyl (C2H3O2), so the compound may be acetyl and glucoside disubstituted NIV (Ac-NIV-Glu). Further combined with MS / MS spectrum analysis, the parent ion of the compound lost the glucoside group after fragmentation to form 337.1265, and continued to lose an OH to form 319.1163. In addition, the parent ion of the compound can also lose the glucoside group and acetyl group to form 313.1267, which is the [M+H]+ peak of NIV. The fragment ions with m / z in the range of 100-320 are highly similar to the NIV fragment ions, proving that the compound is acetyl and glucoside disubstituted NIV (Ac-NIV-Glu).

[0073] The parent ion peak of compound 8 detected by high-resolution mass spectrometry was 562.2111 ([M+NH4]+), and the predicted molecular formula provided by the prediction database was C 24 H 32 O 14 (Δm = -2.15ppm), the isotope matching score is 76 points. From the high-resolution mass spectrum ( Figure 3 ) It can be seen that after the compound is cleaved, there are 9 secondary fragment ions that match the mass spectrometry characteristic structure fragment diagnostic ions, meeting the requirement that the number of characteristic fragment ion matches is ≥ 4. After the parent ion of the compound is cleaved, the malonyl glucoside group (C9H 12 O8) forms 297.1333, which is the [M+H]+ of DON. Among them, the fragment ions with m / z in the range of 100-300 are similar to the fragment ions of DON, and it is speculated that they are DON metabolites. Combined with the metabolic pathways of candidate compounds matching this molecular formula in the TCT-Bs metabolite prediction database, it is speculated that this compound is malonyl glucoside DON (DON-MalGlu), that is, the hydroxyl group (OH) is replaced by malonyl glucoside.

[0074] Compound 9 was detected by high-resolution mass spectrometry and its parent ion peak was 518.2225 ([M+NH4]+). The predicted molecular formula provided by the prediction database was C 23 H 32 O 12 (Δm = -2.23ppm), the isotope matching score is 81 points. From the high-resolution mass spectrum ( Figure 3 ) It can be seen that after the cleavage of the compound, there are 6 secondary fragment ions that match the mass spectrometry characteristic structure fragment diagnostic ions, meeting the requirement that the number of characteristic fragment ion matches is ≥ 4. The parent ion of the compound loses the glucosidic group (C6H 11O6) to form 356.1163, 356.1163 loses hydroxyl (OH) to form 321.1333, and 356.1163 loses acetyl (C2H2O) to form 297.1333, which is the [M+H]+ peak of DON. Among them, the fragment ions with m / z in the range of 100-300 are similar to the DON fragment ions, and it is speculated that they are DON metabolites. Combined with the metabolic pathways of candidate compounds matching this molecular formula in the TCT-Bs metabolite prediction database, it is speculated that this compound is acetyl and glucoside disubstituted DON (Ac-DON-Glu), that is, the two hydroxyl groups (OH) are replaced by glucoside (C6H 11 O6) and acetyl (C2H3O2) substituted.

[0075] The screening results of type B trichothecenes in wheat samples are summarized in Table 2. Therefore, the molecular formula of unknown metabolites can be inferred through the prediction database and the common characteristic structural fragment diagnostic ions, and the structure of unknown metabolites can be finally determined through the analysis of the molecular formula and metabolic pathway of unknown metabolites by technicians.

[0076] Table 2 Screening results of type B trichothecenes in wheat samples

[0077]

[0078] In order to further verify the feasibility of the screening method provided by the present invention in screening and identifying TCT-B in actual contaminated samples, the screening method of the present invention is compared with the currently disclosed method. The applicant screened 30 wheat samples from the above-mentioned high-pollution risk area in central my country according to the screening characteristic fragment ions and screening conditions of the literature and patents. See Table 3. The number of candidate compounds screened out by literature 1, patent 1 and this patent are 29, 49, and 39, respectively, and the number of TCT-Bs finally preliminarily identified is 7, 8, and 9, respectively. Although the number of candidate compounds screened out by the method established in literature 1 (29) is lower than that of the screening method of the present invention (39), and the screening workload is the lowest, the metabolites DOM and DON-MalGlu are not screened out, and there is leakage. The method established in patent 1 also did not screen out DON-MalGlu, and the number of candidate compounds screened out was 49, the number of interfering compounds was large, and the screening workload was greater than that of the present invention. Due to changes in the structure of the metabolite, its optimal collision energy changes compared with the parent compound, and the secondary fragment ions generated will also change with the collision energy. High collision energy mainly produces low- and medium-quality end fragments, and low collision energy mainly produces medium- and high-quality end fragments. After analysis, the number of characteristic fragment ions reported in the literature is less than 7. The main characteristic fragment ions of literature 1 are high-quality end fragment ions (m / z>200), and patent 1 is mainly low-quality end fragment ions (m / z<137), which does not cover all quality end fragments (m / z 137-200 missing). In actual samples, the collision energy is high when collecting the secondary spectrum of DON-MalGlu, mainly fragment ions with m / z between 150-200, which leads to the leakage of DON-MalGlu. In general, the screening strategy of the present invention can effectively reduce the number of false positive compounds and the accuracy of the method, and combined with the metabolic prediction database, the metabolic mode and structural information of the candidate compound can be quickly inferred, which is helpful for the structural identification of metabolites.

[0079] Table 3

[0080]

[0081]

[0082] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not limitations on the implementation methods of the present invention. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made based on the above description. It is impossible to list all the implementation methods here. All obvious changes or modifications derived from the technical solution of the present invention are still within the protection scope of the present invention.

Claims

1. A method for screening type B trichothecene toxin metabolites, characterized in that: The steps include: 1) Construct a prediction database for type B trichothecenes metabolites and identify the common characteristic structural diagnostic ions of type B trichothecenes; 2) performing a first matching screening on the primary mass spectrum information of each compound in the sample to be tested in the type B trichothecene toxin metabolite prediction database to determine the compound molecular formula corresponding to each mass spectrum peak in the primary mass spectrum, and obtaining a first compound molecular formula set; Matching and screening the secondary mass spectrometry information of each compound in the sample to be tested with the common characteristic structure diagnostic ions of type B trichothecenes, collecting the molecular formulas of compounds whose matching number of characteristic structure fragment ions is greater than or equal to a preset threshold, and obtaining a second compound molecular formula set; 3) Analyzing the compound molecular formulas in the intersection of the first compound molecular formula set and the second compound molecular formula set to determine the candidate structures of the type B trichothecene toxin metabolites in the sample to be tested.

2. The screening method according to claim 1, characterized in that The construction of a type B trichothecene toxin metabolite prediction database comprises: Simulate all metabolic reactions that type B trichothecenes may undergo, where the total number of metabolic steps is 1-3 steps, summarize the potential metabolite structures and metabolic pathways of type B trichothecenes after metabolism, and construct a prediction database for type B trichothecenes metabolites; Wherein, the metabolic reaction includes phase I metabolic reaction and / or phase II metabolic reaction.

3. The screening method according to claim 2, characterized in that The phase I metabolic reaction includes one of a dehydration reaction, a desaturation reaction, a hydration reaction, a nitro reduction reaction, an oxidation reaction, and a reduction reaction; The phase II metabolic reactions include one or more of acetylation, arginine substitution, cysteine ​​substitution, glucoside substitution, glucuronide substitution, glutamine substitution, glycine substitution, glutathione substitution, methylation, ornithine substitution, hexadecyl substitution, octadecyl substitution, sulfonation, taurination, diglucoside substitution, triglucoside substitution, tetraglucoside substitution, xylan-glucose substitution, malonyl-glucoside substitution, feruloyl glucose substitution, N-acetylcysteine ​​substitution, N-acetyl substitution, benzoyl substitution, hexitol substitution, disodium anhydride substitution, and de-epoxy reaction.

4. The screening method according to claim 2, characterized in that The number of reaction steps of the phase I metabolic reaction is 0 and the number of reaction steps of the phase II metabolic reaction is 1-3, or the number of reaction steps of the phase I metabolic reaction is 1 and the number of reaction steps of the phase II metabolic reaction is 0-2.

5. The screening method according to claim 1, characterized in that: The common characteristic structure diagnostic ions for determining type B trichothecenes include: Analyze the secondary mass spectra of known type B trichothecene toxin metabolite standards, determine fragment ions with common characteristic structures, and obtain diagnostic ions with common characteristic structures of type B trichothecene toxins; Among them, the common characteristic structure diagnostic ions include characteristic fragments with m / z of 279.1227, 277.1071, 249.1121, 247.0965, 231.1016, 229.0859, 203.1067, 201.0910, 191.1067, 189.0910, 177.0910, 175.0754, 125.0597, 123.0441, 109.0648, 107.0491, and 137.0597.

6. The screening method according to claim 1, characterized in that: The molecular weight of each metabolite in the predicted database of type B trichothecenes is greater than or equal to 200 Da.

7. The screening method according to claim 1, characterized in that: The preset threshold is 4.

8. The screening method according to claim 1, characterized in that: The matching screening of the primary mass spectrometry information of each compound in the sample to be tested in the type B trichothecene toxin metabolite prediction database includes: A mass spectrometer is used to collect data for each compound in the sample to be tested, and data processing software is used to perform peak extraction, alignment and integration on the collected raw data. The primary mass spectrometry information of each compound in the sample to be tested is compared with the data information of the type B trichothecene toxin metabolite prediction database to determine the molecular formula of the compound corresponding to each mass spectrometry peak in the primary mass spectrum.

9. The screening method according to claim 1, characterized in that: Step 3) includes: According to the compound molecular formula and the corresponding metabolic pathway, a false positive interference subtraction operation is performed on the compound molecular formula in the intersection of the first compound molecular formula set and the second compound molecular formula set to determine the candidate structure of the type B trichothecene toxin metabolite in the sample to be tested.

10. Application of the screening method according to any one of claims 1 to 9 in simulated analysis of mycotoxin types in food.

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