A method for identifying organophosphates and its applications

Abstract

The present invention discloses the application of a peptide segment with an amino acid sequence as shown in SEQ ID NO: 1 in the recognition of organophosphates, and provides a method for recognizing organophosphates. After pre-treating a sample to be tested, the sample is subjected to high performance liquid chromatography and then mixed with a peptide segment with an amino acid sequence as shown in SEQ ID NO: 1, followed by mass spectrometry to obtain the mass spectrometry result of the sample to be tested. At the same time, a control group is set up to obtain the control mass spectrometry result of the sample to be tested. Based on the mass spectrometry results, analysis is carried out, and characteristic peaks that are significantly up-regulated and satisfy △m / z = 560.2918 in the mass spectrometry result of the sample to be tested are selected as preliminary screening characteristic peaks. The preliminary screening characteristic peaks are combined with a database for identification, thereby recognizing organophosphates. The method shown in the present invention can accurately recognize organophosphates in a sample to be tested, avoiding the interference of a large number of unknown substances in the sample to be tested.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and in particular, to a method for identifying organophosphates and its applications. Background Art

[0002] Organophosphate diesters (di-OPEs) have been widely detected in environmental media such as water bodies, sediments, the atmosphere, and biological samples. A major source of di-OPEs is the direct use of industrial products or impurities in the industrial production of tri-OPEs.

[0003] Diphenyl phosphate, dibutyl phosphate, bis(2-ethylhexyl) phosphate, etc. are all widely used flame retardants or plasticizers, with an annual output as high as 17,050 tons. In addition, with the gradual phasing out of toxic halogenated flame retardants such as polybrominated diphenyl ethers, the demand for organophosphate triesters (tri-OPEs) in global consumption and production has increased sharply, with a production volume exceeding 2.5 million tons. The extensive use of tri-OPEs has led to their widespread presence in environmental media, and the products formed through biological or abiotic transformation are another important source of di-OPEs in the environment. The ubiquitous di-OPEs' negative impact on organisms cannot be ignored.

[0004] Toxicological studies have shown that di-OPEs can cause toxic effects on aquatic organisms, including cytotoxicity, developmental toxicity, teratogenicity, reproductive toxicity, endocrine disruption effects, etc. Epidemiological studies have also revealed the potential adverse reactions of di-OPEs to humans. For example, the concentration level of di-OPEs in pregnant women's urine is related to abnormal growth and development of infants and symptoms such as neurodevelopmental damage. More worryingly, compared with the parent compound tri-OPEs, some di-OPEs can produce similar or even higher toxic effects. Therefore, identifying organophosphate diesters and their transformation products in the environment is of great significance.

[0005] Based on the screening technology of high-performance liquid chromatography-high-resolution mass spectrometry, it has become possible to simultaneously detect thousands of compounds in a single sample run. By establishing a compound library, the targeted screening technology can reveal the occurrence of di-OPEs of high concern in various environmental media. However, the types and quantities of di-OPEs covered by the compound library are relatively few and the coverage is limited. The non-targeted screening technology greatly improves the structural identification efficiency of unknown di-OPEs and their transformation products by mining molecular feature information such as retention time, quasi-molecular ion peak, characteristic fragment ions, and isotope peak distribution. However, the large number of coexisting interfering substances in the environment makes the structural analysis of compounds still a time-consuming and difficult task.

[0006] Therefore, at present, the identification of unknown organophosphoric diesters and their transformation products in the environment still poses certain challenges. At the same time, the large number of interfering molecules coexisting in complex matrices greatly increases the difficulty and time consumption of data processing. In view of the wide distribution, variety of types, and different structures of organophosphoric diesters existing in the environment, the development of a rapid, sensitive, economical, and high-throughput method for the identification and screening of organophosphoric diesters and their transformation products is conducive to improving the screening efficiency of high-risk substances. Summary of the Invention

[0007] The object of the present invention is to overcome the above-mentioned deficiencies of the prior art and provide a method for identifying organophosphates and its applications.

[0008] The first object of the present invention is to provide the application of a peptide segment with the amino acid sequence shown in SEQ ID NO: 1 in the identification of organophosphates.

[0009] The second object of the present invention is to provide a method for identifying organophosphates.

[0010] The third object of the present invention is to provide the application of the above method in the identification of organophosphates.

[0011] In order to achieve the above objects, the present invention is realized through the following solutions:

[0012] The present invention claims the application of a peptide segment with the amino acid sequence shown in SEQ ID NO: 1 in the identification of organophosphates.

[0013] Peptide segment (SEQ ID NO: 1): Ac-SGAGKT-NH2;

[0014] Among them, the N-terminus of the peptide segment with the amino acid sequence shown in SEQ ID NO: 1 has an acetylation modification, and the C-terminus has an amino modification.

[0015] Preferably, the organophosphate is organophosphoric monoester and / or organophosphoric diester.

[0016] More preferably, the organic phosphoric acid monoester is 2-ethylhexyl phosphate (EHP), 2-[1-[(4-amino-2-methylpyrimidin-5-yl)methyl]-2-methylpyridin-1-ium-3-yl]ethyl dihydrogen phosphate (PTP), 4-chloro-3-fluoro-4-oxobutyl dihydrogen phosphate (CFOBP), 3-(allyloxy)-2-phenoxyphenyl dihydrogen phosphate (APDP), and 2-(fluoromethyl)-5-(4-methylimidazo[2,1-f][1,2,4]triazin-7-yl)pentyl dihydrogen phosphate (FMPDP); the organic phosphoric acid diester is dibutyl phosphate (DBP), diphenyl phosphate (DPP), bis(2-ethylhexyl) phosphate (DEHP), bis-(1-chloro-2-propyl) phosphate (BCPP), 1-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (PE(18:1(9Z)0:0)), 1-(9Z-hexadecenoyl)-sn-glycero-3-phosphoethanolamine PE(16:1(9Z) / 0:0), 1-hexadecanoyl-sn-glycero-3-phosphoethanolamine Lyso(PE(0:0 / 16:0)), phenylethylene hydrogen phosphate (EPP), and bis(5-hydroxy-4-oxohex-6-en-1-yl) hydrogen phosphate (BHOHP).

[0017] The present invention also claims protection for a method for identifying organic phosphates, comprising the following steps:

[0018] S1. Pretreatment of the sample to be tested: The sample to be tested is successively extracted and eluted, the eluate is collected and dried, and then dissolved in a control solution to obtain an extract of the sample to be tested; the control solution is methanol or acetonitrile;

[0019] S2. Inject the extract of the sample to be tested obtained in step S1 into a high-performance liquid chromatograph for high-performance liquid chromatography. The substance output by the high-performance liquid chromatograph is mixed with a peptide solution having an amino acid sequence as shown in SEQ ID NO: 1 through a three-way joint and then injected into a mass spectrometer for mass spectrometry analysis to obtain a mass spectrometry result of the sample to be tested;

[0020] Inject the extract of the sample to be tested obtained in step S1 into a high-performance liquid chromatograph for high-performance liquid chromatography. The substance output by the high-performance liquid chromatograph is mixed with the control solution shown in step S1 through a three-way joint and then injected into a mass spectrometer for mass spectrometry analysis to obtain a control mass spectrometry result of the sample to be tested;

[0021] S3. Select the characteristic peaks that are significantly up-regulated in the mass spectrometry result of the sample to be tested obtained in step S2 compared to the control mass spectrometry result of the sample to be tested, and select the characteristic peak with Δm / z = 560.2918 as the preliminary screening characteristic peak;

[0022] S4. Identify the structures of the initially screened characteristic peaks obtained in step S3, and then identify the organophosphates in the sample to be tested.

[0023] Preferably, in step S1, the extraction is performed on the sample to be tested using an HLB solid-phase extraction column.

[0024] More preferably, the HLB solid-phase extraction column is an HLB solid-phase extraction column rinsed with a control solution and balanced with deionized water; the control solution is methanol or acetonitrile.

[0025] Preferably, in step S1, the elution is performed using a control solution; the control solution is methanol or acetonitrile.

[0026] Preferably, the chromatographic conditions for the high-performance liquid chromatography are as follows: the flow rate is 200 - 400 μL / min; the column temperature is 25 - 40 °C; the mobile phase gradient is: the initial gradient of mobile phase B is set at 10%, then increased to 50% within 10 min, then increased to 90% at 12 min and maintained for 3 min, then decreased to 10% at 15.1 min and maintained for 5 min, where mobile phase A is water and mobile phase B is acetonitrile.

[0027] Further preferably, the chromatographic column is a Poroshell 120 EC-C18 chromatographic column; the specifications of the chromatographic column are 3.0 × 150 mm, 1.9 μm.

[0028] Preferably, the peptide solution with the amino acid sequence as shown in SEQ ID NO: 1 in step S2 is mixed with the substance output from the high-performance liquid chromatograph at a flow rate of 20 μL / min through a three-way joint.

[0029] More preferably, the concentration of the peptide solution is 50 μmol / L.

[0030] Preferably, the conditions for the mass spectrometry analysis in step S2 are as follows:

[0031] Detection is performed in the negative ion mode, the sheath gas temperature is 100 - 400 °C, the sheath gas flow rate is 5 - 11 L / min, the nozzle voltage is 1000 - 4000 V, the nebulizer gas pressure is 5 - 50 psi, the drying gas temperature is 100 - 400 °C, the drying gas flow rate is 5 - 11 L / min, the capillary voltage is 2000 - 4000 V, the cone voltage is 65 V, the octopole voltage is 750 V, and the fragmentation voltage is 100 - 200 V.

[0032] More preferably, the conditions for the mass spectrometry analysis are as follows:

[0033] Detection was carried out in the negative ion mode. The sheath gas temperature was 400 °C, the sheath gas flow rate was 11 L / min, the nozzle voltage was 1800 V, the nebulizer gas pressure was 10 psi, the drying gas temperature was 250 °C, the drying gas flow rate was 11 L / min, the capillary voltage was 4000 V, the cone voltage was 65 V, the octapole voltage was 750 V, and the fragmentation voltage was 125 V.

[0034] Further preferably, the sheath gas is nitrogen.

[0035] Preferably, in step S2, the mass spectrometry analysis is carried out using a quadrupole time-of-flight mass spectrometer equipped with an ESI source.

[0036] Preferably, before selecting the significantly up-regulated characteristic peaks in step S3, the missing values in the mass spectrometry results are filled using MetaboAnalyst.

[0037] More preferably, the filling of the missing values is specifically: replacing the missing values of the sample with 1 / 5 of the minimum positive value of the variable corresponding to the missing value.

[0038] Preferably, the significantly up-regulated characteristic peaks in step S3 are the characteristic peaks with up-regulation, Fold change > 5.0 and p-value < 0.05.

[0039] Preferably, in step S4, the structure identification is specifically carried out by searching the secondary spectrum database, predicting using sirius software and verifying with standards; the standards are analytical pure grade reagents of dibutyl phosphate (DBP), diphenyl phosphate (DPP), bis(2-ethylhexyl) phosphate (DEHP), bis-(1-chloro-2-propyl) phosphate (BCPP), 1-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (PE(18:1(9Z)0:0)), and 2-ethylhexyl phosphate (EHP).

[0040] The present invention also claims the application of any of the above-mentioned methods in the identification of organophosphates.

[0041] Preferably, the organophosphate is organophosphate monoester and / or organophosphate diester.

[0042] More preferably, the organic phosphoric acid monoester is 2-ethylhexyl phosphate (EHP), 2-[1-[(4-amino-2-methylpyrimidin-5-yl)methyl]-2-methylpyridin-1-ium-3-yl]ethyl dihydrogen phosphate (PTP), 4-chloro-3-fluoro-4-oxobutyl dihydrogen phosphate (CFOBP), 3-(allyloxy)-2-phenoxyphenyl dihydrogen phosphate (APDP), and 2-(fluoromethyl)-5-(4-methylimidazo[2,1-f][1,2,4]triazin-7-yl)pentyl dihydrogen phosphate (FMPDP); the organic phosphoric acid diester is dibutyl phosphate (DBP), diphenyl phosphate (DPP), bis(2-ethylhexyl) phosphate (DEHP), bis-(1-chloro-2-propyl) phosphate (BCPP), 1-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (PE(18:1(9Z)0:0)), 1-(9Z-hexadecenoyl)-sn-glycero-3-phosphoethanolamine PE(16:1(9Z) / 0:0), 1-hexadecanoyl-sn-glycero-3-phosphoethanolamine Lyso(PE(0:0 / 16:0)), phenylethylene hydrogen phosphate (EPP), and bis(5-hydroxy-4-oxohex-6-en-1-yl) hydrogen phosphate (BHOHP).

[0043] Compared with the prior art, the present invention has the following beneficial effects:

[0044] The present invention provides the use of a peptide segment with the amino acid sequence shown in SEQ ID NO: 1 in the recognition of organic phosphates, and provides a method for recognizing organic phosphates. After pre-treating a sample to be tested, it is mixed with a peptide segment with the amino acid sequence shown in SEQ ID NO: 1 after high performance liquid chromatography and then subjected to mass spectrometry to obtain the mass spectrometry result of the sample to be tested. At the same time, a control group is set up and the control mass spectrometry result of the sample to be tested is obtained. Based on the mass spectrometry result, analysis is carried out and characteristic peaks that are significantly up-regulated in the mass spectrometry result of the sample to be tested and satisfy △m / z = 560.2918 are selected as the preliminary screening characteristic peaks. The preliminary screening characteristic peaks are combined with a database for identification, and then organic phosphates are recognized. Using the method shown in the present invention, the organic phosphates in the sample to be tested can be accurately recognized, avoiding the interference of a large number of unknown substances in the sample to be tested. Description of the Drawings

[0045] Figure 1 It is a schematic flow chart of a method for recognizing organic phosphates in Example 1;

[0046] Figure 2 It is a collision-induced dissociation curve diagram of the labeled group in Example 2;

[0047] Figure 3 It is a principal component analysis result diagram in Example 2;

[0048] Figure 4 A volcano plot drawn for step S3 when the marker group in Example 2 is processed according to the equation shown in Example 1;

[0049] Figure 5 A distribution diagram of the m / z signal versus retention time during the mass spectrometry process of the marker group in Example 2;

[0050] Figure 6 A volcano plot drawn for step S3 when processed according to the method shown in Example 1 in Example 3;

[0051] Figure 7 A change diagram for screening the number of characteristic peaks in Example 3. Detailed implementation manners

[0052] The present invention will be further elaborated in detail below in conjunction with the accompanying drawings of the specification and specific embodiments. The embodiments are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following embodiments are all conventional methods unless otherwise specified; the materials, reagents, etc. used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0053] Example 1 A method for identifying organophosphates

[0054] A schematic flow diagram of a method for identifying organophosphates is as Figure 1 shown.

[0055] 1. Peptide synthesis

[0056] Based on the solid-phase peptide synthesis method, a peptide segment (6P) with the amino acid sequence shown in SEQ ID NO: 1 is synthesized;

[0057] 6P (SEQ ID NO: 1): Ac-SGAGKT-NH2.

[0058] 2. Method for identifying organophosphates

[0059] 6P with the amino acid sequence shown in SEQ ID NO: 1 is combined to identify organophosphates, and the binding-guided analysis technique is used to identify organophosphates in the sample to be tested, which specifically includes the following steps:

[0060] S1. Place the HLB solid-phase extraction column on the solid-phase extraction device. After rinsing it 2 times with 5 mL of methanol to remove the impurities in the HLB solid-phase extraction column itself, and then balancing it 2 times with 5 mL of deionized water, the balanced HLB solid-phase extraction column is obtained;

[0061] After extracting 500 mL of the sample to be tested using a balanced HLB solid-phase extraction column, the HLB solid-phase extraction column was then eluted three times with 5 mL of methanol. The eluates were collected and combined. After drying the eluates under nitrogen, they were redissolved in 500 μL of methanol, and then filtered using a 0.22 μm organic nylon filter head. The filtrate was collected to obtain the extract of the sample to be tested.

[0062] S2. Dissolve 6P with the amino acid sequence shown in SEQ ID NO: 1 in methanol to prepare a 25 mM 6P stock solution. Then, dilute the 6P stock solution with methanol to a 50 μM 6P solution.

[0063] Experimental group: Inject 3 μL of the extract of the sample to be tested obtained in step S1 into a high-performance liquid chromatograph (Agilent, 1290) for high-performance liquid chromatography. The substances output by the high-performance liquid chromatograph are mixed with a 50 μM 6P solution through a three-way joint and then injected into a quadrupole time-of-flight mass spectrometer (Agilent, 6545) equipped with an ESI source for mass spectrometry analysis to obtain the mass spectrometry results (spectra) of the sample to be tested;

[0064] The conditions for high-performance liquid chromatography are as follows: The chromatographic column is a Poroshell 120 EC-C18 chromatographic column (3.0×150 mm, 1.9 μm); the flow rate is 200 μL / min; the column temperature is 35 °C; the mobile phase gradient is: the initial gradient of mobile phase B is set to 10%, then increased to 50% within 10 min, then increased to 90% at 12 min and maintained for 3 min, then decreased to 10% at 15.1 min and maintained for 5 min, where mobile phase A is water and mobile phase B is acetonitrile;

[0065] The 6P solution is mixed with the substances output by the high-performance liquid chromatograph at a flow rate of 20 μL / min through a micro-injection pump;

[0066] The conditions for mass spectrometry analysis are as follows: An electrospray ionization source is used, and gas-phase complexes are detected in the negative ion mode. The sheath gas temperature is 400 °C, the sheath gas flow rate is 11 L / min, the nozzle voltage is 1800 V, the nebulizing gas pressure is 10 psi, the drying gas temperature is 250 °C, the drying gas flow rate is 11 L / min, the capillary voltage is 4000 V, the cone voltage is 65 V, the octopole voltage is 750 V, the fragmentation voltage is 125 V, and the sheath gas is nitrogen.

[0067] The only difference between the control group and the experimental group is that the 50 μM 6P solution is replaced with methanol, and the rest of the treatments are exactly the same, obtaining the control mass spectrometry results (spectra) of the sample to be tested;

[0068] S3. Use the ProteoWizard MSconvert software to convert the mass spectrometry results of the test samples obtained in step S2 into mass spectrometry results in mzML format respectively. Then import the mass spectrometry results in mzML format into the Mzmine 2.53 software, and perform mass feature detection, ADAP chromatographic reconstruction, isotope feature detection, peak filtering and peak alignment based on the default parameters to obtain a CSV file of the mass spectrometry results of the test samples recording retention time, mass-to-charge ratio and abundance;

[0069] Perform the same processing on the control mass spectrometry results of the test samples obtained in step S2 to obtain a CSV file of the control mass spectrometry results of the test samples recording retention time, mass-to-charge ratio and abundance.

[0070] Import the CSV file of the mass spectrometry results of the test samples and the CSV file of the control mass spectrometry results of the test samples into the MetaboAnalyst data processing platform (https: / / www.metaboanalyst.ca), and use 1 / 5 of the minimum positive value of the missing value corresponding variable in the two CSV files of the mass spectrometry results to fill in the missing values. Then perform statistical analysis and draw a volcano plot based on the preset parameters of the MetaboAnalyst data processing platform. Select the characteristic peaks that are significantly up-regulated in the CSV file of the mass spectrometry results of the test samples compared with the CSV file of the control mass spectrometry results of the test samples (Fold change > 5 and p-value < 0.05), and select the characteristic peak with △m / z = 560.2918 as the candidate characteristic peak;

[0071] S4. Based on the candidate characteristic peaks obtained in step S3, set the mass-to-charge ratio deviation △ppm = 20, retrieve through the secondary spectrum database (https: / / mona.fiehnlab.ucdavis.edu / downloads), predict using the sirius software and verify with standards to identify the information of the candidate characteristic peaks, and then identify the organophosphates in the test samples;

[0072] The standards are analytical pure grade reagents of dibutyl phosphate (DBP), diphenyl phosphate (DPP), bis(2-ethylhexyl) phosphate (DEHP), bis-(1-chloro-2-propyl) phosphate (BCPP), 1-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (PE(18:1(9Z)0:0)), 2-ethylhexyl phosphate (EHP).

[0073] Example 2 Verification of a method for identifying organophosphates

[0074] I. Experimental method

[0075] Take 300 mL of river water from the Pearl River in Guangzhou, add 8 organophosphorus ester compounds (diphenyl phosphate, dibutyl phosphate, diethyl phosphate, dimethyl phosphate, bis(2,3-dibromopropyl) phosphate, bis(1,3-dichloro-2-propyl) phosphate, monophenyl phosphate, and monomethyl phosphate) to a final concentration of 2 ng / L for each organophosphorus ester compound to obtain spiked river water. After filtering the spiked river water through a medium-speed qualitative filter paper (30 - 50 μm), sonicate it for 10 min to obtain pretreated spiked river water.

[0076] Place the HLB solid-phase extraction column on the solid-phase extraction device. After rinsing it twice with 5 mL of methanol to remove impurities in the HLB solid-phase extraction column itself, balance it twice with 5 mL of deionized water to obtain a balanced HLB solid-phase extraction column.

[0077] After extracting 300 mL of the pretreated spiked river water using the balanced HLB solid-phase extraction column, then elute the HLB solid-phase extraction column three times with 5 mL of methanol, collect and combine the eluate. After drying the eluate under nitrogen, dissolve it in 300 μL of methanol and filter it using a 0.22 μm organic nylon filter head. Collect the filtrate to obtain the extract of the river water to be tested. Prepare a 50 μM 6P solution as shown in Example 1.

[0078] Labeling group (6P): Inject the extract of the river water to be tested into a high-performance liquid chromatograph (Agilent, 1290) with an injection volume of 3 μL for high-performance liquid chromatography. The substances output by the high-performance liquid chromatograph are mixed with a 50 μM 6P solution through a three-way joint and then injected into a quadrupole time-of-flight mass spectrometer (Agilent, 6545) equipped with an ESI source for mass spectrometry analysis to obtain the mass spectrometry results (spectra) of the extract of the river water to be tested. At the same time, perform collision-induced dissociation (CID) scanning during the mass spectrometry process to obtain the collision-induced dissociation curve of the labeling group, and perform Boltzmann function fitting on the collision-induced dissociation curve to obtain the centroid collision energy (E cm，1 / 2 ) when the dissociation ratio of 6P to each organophosphorus ester is 50%.

[0079] The conditions for high-performance liquid chromatography are as follows: The chromatographic column is a Poroshell 120 EC-C18 chromatographic column (3.0×150 mm, 1.9 μm); the flow rate is 200 μL / min; the column temperature is 35 °C; the mobile phase gradient is: the initial gradient of mobile phase B is set to 10%, then it is increased to 50% within 10 min, then it is increased to 90% at 12 min and maintained for 3 min, then it is decreased to 10% at 15.1 min and maintained for 5 min, where mobile phase A is water and mobile phase B is acetonitrile.

[0080] The 6P solution was mixed with the substance output from the high-performance liquid chromatograph at a flow rate of 20 μL / min by a micro-injection pump;

[0081] The conditions for mass spectrometry analysis were as follows: an electrospray ionization source was used, the gas-phase complex was detected in the negative ion mode, the sheath gas temperature was 400 °C, the sheath gas flow rate was 11 L / min, the nozzle voltage was 1800 V, the nebulizing gas pressure was 10 psi, the drying gas temperature was 250 °C, the drying gas flow rate was 11 L / min, the capillary voltage was 4000 V, the cone voltage was 65 V, the octupole voltage was 750 V, the fragmentation voltage was 125 V, and the sheath gas was nitrogen.

[0082] The only difference between Control Group 1 (Control) and the labeled group was that the 50 μM 6P solution was replaced with methanol, and the rest of the treatments were exactly the same, obtaining the control mass spectrometry results (spectra) of the river water extract to be tested.

[0083] Then, according to the methods shown in steps S3 and S4 in Example 1, the organophosphates in the river water extract to be tested were identified.

[0084] At the same time, the mixture of the substance output from the high-performance liquid chromatograph and the 6P solution during the treatment of the labeled group (at the three-way joint), and the mixture 1 of the substance output from the high-performance liquid chromatograph and methanol during the treatment of the control group (at the three-way joint) were obtained for principal component analysis.

[0085] II. Experimental Results

[0086] The collision-induced dissociation curve diagram of the labeled group was as Figure 2 shown, and the centroid collision energy when the dissociation ratio of the peptide 6P to the organophosphate was 50% was shown in Table 1.

[0087] Table 1 Centroid collision energy when the dissociation ratio of the peptide 6P to the organophosphate was 50%

[0088]

[0089] The results showed that in the negative ion mode, the peptide 6P with the amino acid sequence shown in SEQ ID NO: 1 could form complexes with 8 different organophosphate compounds and generate stable composite signals, and all 8 organophosphate compounds added to the sample could be accurately identified.

[0090] The principal component analysis result diagram was as Figure 3 shown, and the results showed that: the labeled group and the control group overlapped on the PCA2 axis and could not be distinguished; while the labeled group was significantly separated from the control group on the PCA1 axis.

[0091] Moreover, methanol is a common component in both the labeled group and the control group, while peptide 6P is the single variable in the labeled group; therefore, the PCA1 axis is used to explain the contribution of introducing 6P to the change in signal abundance, and the PCA2 axis is used to explain the contribution of the solvent effect of introducing methanol to the change in signal abundance; in addition, since the contribution of the PCA1 axis to data analysis is 70.6% and the contribution of the PCA2 axis is 11.5%, it shows that the contribution of peptide 6P to the change in signal abundance is dominant. Combining with the peptide 6P with the amino acid sequence shown in SEQ ID NO: 1 can achieve more accurate identification of organophosphates.

[0092] When the labeled group was processed according to steps S3 and S4 of Example 1, the volcano plot drawn in step S3 was as Figure 4 shown, and the distribution map of m / z signals during the mass spectrometry process of the labeled group with respect to retention time was as Figure 5 shown.

[0093] The results showed that: based on the Figure 4 shown volcano plot, it was possible to determine that after introducing peptide 6P with the amino acid sequence shown in SEQ ID NO: 1 into the labeled group, 981 significantly up-regulated characteristic peak signals were generated, and the types of 8 spiked organophosphate compounds also appeared in the significantly up-regulated characteristic peak signals; at the same time, in the distribution map of m / z with respect to retention time, peptide 6P with the amino acid sequence shown in SEQ ID NO: 1 showed good anti-interference ability. Specifically, within a 0.2 min change in retention time in the presence of interference signals, peptide 6P could non-covalently label organophosphates (phosphodiesters and monoester compounds) and did not form gas-phase composite ions with other interfering components.

[0094] Based on the above results, peptide 6P with the amino acid sequence shown in SEQ ID NO: 1 can accurately bind to organophosphates in the sample to form a complex and is not affected by other interfering components in the sample.

[0095] Example 3 Application of a method for identifying organophosphates

[0096] I. Experimental method

[0097] Taking 500 mL of underground wastewater from Yancheng Industrial Park in Jiangsu as the sample to be tested, identifying organophosphates according to the method shown in Example 1, and recording the number of characteristic peak signals collected during the identification of organophosphates;

[0098] In the process of identifying organophosphates, the reference standards selected are dibutyl phosphate (DBP), diphenyl phosphate (DPP), bis(2-ethylhexyl) phosphate (DEHP), 2-ethylhexyl phosphate (EHP), bis(1-chloro-2-propyl) phosphate (BCPP), and 1-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (PE(18:1(9Z)0:0)).

[0099] II. Experimental Results

[0100] When identifying organophosphates in underground wastewater according to the method shown in Example 1, the volcano plot drawn in step S3 is as shown in Figure 6 shown, and the change diagram of the number of characteristic peaks screened is as shown in Figure 7 shown.

[0101] A total of 9346 characteristic peak signals were collected in the mass spectrometry results of the sample to be tested obtained in step S2; based on the volcano plot shown in Figure 6 shown, 816 characteristic peaks that were significantly up-regulated compared to the CSV file of the mass spectrometry results of the control sample to be tested were collected from the CSV file of the mass spectrometry results of the sample to be tested, and 215 characteristic peaks were determined as candidate characteristic peaks from these 816 significantly up-regulated characteristic peaks (simultaneously meeting the conditions of significant up-regulation and Δm / z = 560.2918).

[0102] Based on the 215 candidate characteristic peaks, 14 organophosphates were identified and recognized from the underground wastewater, including 9 organophosphate diesters and 5 organophosphate monoesters. The 14 organophosphates are shown in Table 2 and are respectively: dibutyl phosphate (DBP), diphenyl phosphate (DPP), bis(2-ethylhexyl) phosphate (DEHP), 2-ethylhexyl phosphate (EHP), bis(1-chloro-2-propyl) phosphate (BCPP), 1-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (PE(18:1(9Z)0:0)), 1-(9Z-hexadecenoyl)-sn-glycero-3-phosphoethanolamine PE(16:1(9Z) / 0:0), 1-hexadecanoyl-sn-glycero-3-phosphoethanolamine Lyso(PE(0:0 / 16:0)), phenylvinyl hydrogen phosphate (EPP), bis(5-hydroxy-4-oxohex-6-en-1-yl) hydrogen phosphate (BHOHP), 2-[1-[(4-amino-2-methylpyrimidin-5-yl)methyl]-2-methylpyridin-1-ium-3-yl]ethyl dihydrogen phosphate (PTP), 4-chloro-3-fluoro-4-oxobutyl dihydrogen phosphate (CFOBP), 3-(allyloxy)-2-phenoxyphenyl dihydrogen phosphate (APDP), and 2-(fluoromethyl)-5-(4-methylimidazo[2,1-f][1,2,4]triazin-7-yl)pentyl dihydrogen phosphate (FMPDP).

[0103] Table 2 14 kinds of organophosphates

[0104]

[0105] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than limiting the protection scope of the present invention. For those of ordinary skill in the art, other different forms of changes or modifications can be made on the basis of the above description and ideas. It is not necessary and impossible to enumerate all implementation manners here. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the claims of the present invention.

Claims

1. A method for identifying organophosphates, characterized in that, It includes the following steps: S1. Pretreatment of the sample to be tested: The sample to be tested is successively subjected to extraction and elution, the eluate is collected and dried, and then dissolved in a control solution to obtain an extract of the sample to be tested; the control solution is methanol or acetonitrile; S2. Inject the extract of the sample to be tested obtained in step S1 into a high-performance liquid chromatograph for high-performance liquid chromatography. The substance output by the high-performance liquid chromatograph is mixed with the peptide solution shown in the amino acid sequence SEQ ID NO: 1 through a three-way joint and then injected into a mass spectrometer for mass spectrometry analysis to obtain the mass spectrometry result of the sample to be tested; Among them, the peptide shown in SEQ ID NO: 1 is: Ac-SGAGKT-NH2; Inject the extract of the sample to be tested obtained in step S1 into a high-performance liquid chromatograph for high-performance liquid chromatography. The substance output by the high-performance liquid chromatograph is mixed with the control solution shown in step S1 through a three-way joint and then injected into a mass spectrometer for mass spectrometry analysis to obtain the control mass spectrometry result of the sample to be tested; S3. Select the characteristic peaks that are significantly up-regulated in the mass spectrometry result of the sample to be tested obtained in step S2 compared with the control mass spectrometry result of the sample to be tested, and select the characteristic peak with △m / z = 560.2918 as the preliminary screening characteristic peak; S4. Perform structure identification on the preliminary screening characteristic peak obtained in step S3, and then identify the organophosphate in the sample to be tested.

2. The method according to claim 1, wherein The chromatographic conditions of the high-performance liquid chromatography in step S2 are: the flow rate is 200-400 μL / min; the column temperature is 25-40 °C; the mobile phase gradient is: the initial gradient of mobile phase B is set to 10%, then increased to 50% within 10 min, then increased to 90% at 12 min and maintained for 3 min, then decreased to 10% at 15.1 min and maintained for 5 min, where mobile phase A is water and mobile phase B is acetonitrile.

3. The method according to claim 1, characterized in that, The peptide solution shown in the amino acid sequence SEQ ID NO: 1 in step S2 is mixed with the substance output by the high-performance liquid chromatograph through a three-way joint at a flow rate of 5-50 μL / min.

4. The method according to claim 3, characterized in that The concentration of the peptide solution is 1-100 μmol / L.

5. The method according to claim 1, wherein The conditions of the mass spectrometry analysis in step S2 are: Detected in the anion mode, the sheath gas temperature is 100-400 °C, the sheath gas flow rate is 5-11 L / min, the nozzle voltage is 1000-4000 V, the nebulizing gas pressure is 5-50 psi, the drying gas temperature is 100-400 °C, the drying gas flow rate is 5-11 L / min, the capillary voltage is 2000-4000 V, the cone voltage is 65 V, the octapole voltage is 750 V, and the fragmentation voltage is 100-200 V.

6. The method according to claim 1, wherein Before step S3 selects the significantly up-regulated characteristic peaks, MetaboAnalyst is used to fill in the missing values of the mass spectrometry results.

7. Use of the method according to any one of claims 1 to 6 in identifying organophosphates.

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