A screening method and a quantification method for dopamine-like active molecules in water bodies
Through the cavity size selectivity and hydrogen bonding of synthetic peptides combined with LC-MS, the problem of poor selectivity of dopamine-like active molecules in water bodies is solved, and high selectivity and efficient screening and quantitative detection are achieved.
Patent Information
- Application Number
- CN202310487013.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-04-28
- Publication Date
- 2025-08-01
- Estimated Expiration
- 2043-04-28
AI Technical Summary
The prior art has poor selectivity for dopamine-like active molecules in water bodies, which leads to difficulty in screening.
The LC-MS method was used to combine synthetic peptides (cyclic peptide 1, cyclic peptide 2 or linear peptide) for water concentrate analysis, and the cavity size selectivity and hydrogen bonding of the synthetic peptide were used to form a complex, improving selective screening and quantitative detection of dopamine-like active molecules.
Highly selective screening and quantitative detection of dopamine-like active molecules in complex matrix water bodies can be achieved, and dopamine-like active molecules in water bodies can be accurately identified and quantified.
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Figure CN118604149B_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of analytical detection, and specifically relates to a screening method and a quantification method for dopamine-like active molecules in water bodies. Background Art
[0002] In daily life and production, humans have discharged tens of thousands of chemicals and their related transformation products into water, causing serious pollution to water bodies and posing a serious threat to human health and the ecological environment. It is worth noting that the potential hazards caused by the discharge of small molecules with neuroactivity and their transformation products have not been deeply studied. Therefore, high attention needs to be paid to the distribution and content of small molecules with neuroactivity and their transformation products in water bodies.
[0003] Dopamine is an important catecholamine neurotransmitter in the mammalian nervous system and plays a key role in the learning and memory activities of mammals, and can affect the activities of neurons, the attention and motivation behaviors of mammals. Dopamine-like active molecules are molecules with neuroactivity similar to dopamine, such as: fenoldopam, apomorphine, isooctylamine, amantadine, pramipexole, etc., and dopamine-like active molecules may affect the behaviors of aquatic organisms, thus posing a threat to the ecological environment.
[0004] The complexity of the water environment increases the difficulty of screening and quantitative detection of dopamine-like active molecules in water bodies. When existing methods are used for the water environment, due to the poor selectivity for dopamine-like active molecules, it becomes difficult to screen dopamine-like active molecules in water bodies. Therefore, it is of great significance to develop a screening method and a quantification method for dopamine-like active molecules in water bodies with high selectivity for dopamine-like active molecules. Summary of the Invention
[0005] The primary object of the present invention is to solve the problem of poor selectivity of the prior art for dopamine-like active molecules in water bodies, and to provide a screening method for dopamine-like active molecules in water bodies.
[0006] Another object of the present invention is to provide a quantification method for dopamine-like active molecules in water bodies.
[0007] In order to achieve the above object, the present invention adopts the following technical solutions:
[0008] A screening method for dopamine-like active molecules in water bodies, comprising the following steps:
[0009] S1. Perform solid-phase extraction on the water body and concentrate to obtain a water body concentrate;
[0010] S2. Analyze the water body concentrate and the synthetic peptide solution with a concentration of 20 - 50 μM / L by LC-MS, and use the LC-MS spectrum to make a volcano plot. Conduct targeted secondary analysis on the suspicious signals with up-regulated signals, and match them with the mass spectrometry database to identify the types of dopamine-like active molecules in the water body;
[0011] The synthetic peptide described in step S2 is one of cyclic peptide 1, cyclic peptide 2 or linear peptide;
[0012] The cyclic peptide 1 has the molecular structure shown in formula (1):
[0013]
[0014] The cyclic peptide 2 has the molecular structure shown in formula (2):
[0015]
[0016] The linear peptide has the molecular structure shown in formula (3):
[0017]
[0018] The present invention utilizes the cavity size selectivity of the synthetic peptide and the hydrogen bond interaction between it and the dopamine-like active molecule, enabling the synthetic peptide to selectively interact with the dopamine-like active molecule to form a complex, improving the selectivity for the dopamine-like active molecule in the water body, and thus realizing the screening of dopamine-like active molecules in the water body with a complex matrix.
[0019] In the present invention, cyclic peptide 1, cyclic peptide 2 and linear peptide are all commercially available. Among them, cyclic peptide 1 is synthesized from cysteine, proline, glycine and ethylamine; cyclic peptide 2 is synthesized from cysteine, proline and glycine; linear peptide is synthesized from glycine, proline and ethylamine.
[0020] In the present invention, the solvent of the synthetic peptide solution is one or more of methanol, acetonitrile, acetonitrile solution with 0.1 - 5% (v / v) trifluoroacetic acid, acetonitrile solution with 0.1 - 5% (v / v) formic acid, acetonitrile solution with 0.1 - 5% (v / v) acetic acid, methanol solution with 0.1 - 5% (v / v) trifluoroacetic acid, methanol solution with 0.1 - 5% (v / v) formic acid or methanol solution with 0.1 - 5% (v / v) acetic acid.
[0021] The present invention draws a volcano plot using MetaboAnalyst software based on the retention time and ion abundance of the LC-MS spectrum.
[0022] Preferably, the synthetic peptide described in step S2 is cyclic peptide 1 or cyclic peptide 2.
[0023] Preferably, the dopamine-like active molecule is one or more of dopamine, fenoldopam, apomorphine, isooctylamine, amantadine, pramipexole, clozapine, imipramine, perphenazine, molindone, lamotrigine or amoxapine.
[0024] In the present invention, the water body is a water sample, which can be a river water sample or a sea water sample.
[0025] Preferably, the concentration multiple of the water body concentrate in step S1 is 250 to 2000 times.
[0026] Preferably, the water body concentrate in step S1 is stored at a temperature below 4°C.
[0027] Preferably, step S1 is specifically as follows: first, rinse the solid-phase extraction column with an organic solvent, then inject the water body into the solid-phase extraction column, and then elute with an organic solvent to obtain an eluate, dry it, then re-dissolve it with an organic solvent, and filter it to obtain a water body concentrate.
[0028] More preferably, the organic solvent is one or more of methanol, acetonitrile, carbon tetrachloride, toluene, ethyl acetate, ethanol or water.
[0029] More preferably, the solid-phase extraction column is one or more of a C18 solid-phase extraction cartridge, an HLB solid-phase extraction column, a C8 solid-phase extraction column or an SLC solid-phase extraction column.
[0030] In the present invention, the drying in step S1 can be to blow dry the eluate with an inert gas, or to dry the eluate by rotary evaporation, or to vacuum dry the eluate; the inert gas used is one or both of nitrogen or argon.
[0031] Preferably, the analysis of the water body concentrate and the synthetic peptide solution with a concentration of 20 to 50 μM / L by LC-MS means that the water body concentrate is first separated by LC, and then mixed with the synthetic peptide solution with a concentration of 20 to 50 μM / L through a three-way valve and enters the MS.
[0032] Preferably, the flow rate of the synthetic peptide solution with a concentration of 20 to 50 μM / L entering the LC-MS is 10 to 25 μL / min.
[0033] Conventional LC-MS in the art can be used for the screening method of dopamine-like active molecules in the water body of the present invention. For example, the LC-MS in step S2 can be UHPLC-QTOF / MS.
[0034] When the LC-MS in step S2 is UHPLC-QTOF / MS, its analysis conditions are as follows:
[0035] Chromatographic column: ZORBAX Eclipse Plus C18, ZORBAX-SB-C18 or Poroshell 120 EC-C18; Mobile phase A: pure aqueous phase, aqueous solution of isopropanol with a volume fraction of 0.1 - 5%, aqueous solution of methanol with a volume fraction of 0.1 - 20%, aqueous solution of acetonitrile with a volume fraction of 0.1 - 20%, or aqueous solution of formic acid with a volume fraction of 0.1 - 5%; Mobile phase B: pure methanol, pure acetonitrile, methanol solution of formic acid with a volume fraction of 0.1%, or acetonitrile solution of formic acid with a volume fraction of 0.1%; Flow rate of mobile phase A and B: 0.2 - 0.5 mL / min; Nozzle voltage: 0 - 1000 V; Nebulizer pressure: 30 - 50 psi; Drier flow rate: 8 - 11 L / min; Drier temperature: 300 - 350 °C; Capillary voltage: 3500 - 4000 V; Analyzed by SIM mode.
[0036] In the screening method of dopamine-like active molecules in water body of the present invention, the gradient changes of mobile phase A and mobile phase B are shown in the following table:
[0037] Table 1 Gradient changes of mobile phase A and mobile phase B in the screening method
[0038]
[0039] Note: In the above table, the volume fraction of mobile phase A refers to the percentage of the volume of mobile phase A in the total volume of mobile phase A and mobile phase B; the volume fraction of mobile phase B refers to the percentage of the volume of mobile phase B in the total volume of mobile phase A and mobile phase B.
[0040] As can be seen from Table 1, within 0.00 - 1.00 min, the volume fraction of mobile phase A remains 95% and the volume fraction of mobile phase B remains 5%, flowing to waste liquid; within 1.00 - 5.00 min, the volume fraction of mobile phase A decreases from 95% to 65% and the volume fraction of mobile phase B increases from 5% to 35%, flowing through the mass spectrometer; within 5.00 - 25.00 min, the volume fraction of mobile phase A decreases from 65% to 5% and the volume fraction of mobile phase B increases from 35% to 95%, flowing through the mass spectrometer; and so on.
[0041] A quantitative method for dopamine-like active molecules in water body, comprising the following steps:
[0042] Identify the types of dopamine-like active molecules in the water body using the screening method of dopamine-like active molecules in the water body to obtain the specific types of dopamine-like active molecules, and then establish a standard concentration curve of the specific types of dopamine-like active molecules using LC-MS technology, and the content of the specific types of dopamine-like active molecules in the water body can be obtained by calculation.
[0043] Conventional LC-MS in this field can all be used in the quantitative method of dopamine-like active molecules in the water body of the present invention, and its LC-MS analysis conditions are as follows:
[0044] Chromatographic column: ZORBAX Eclipse Plus C18, ZORBAX-SB-C18 or Poroshell 120EC-C18; Mobile phase A: pure water phase, aqueous solution of isopropanol with a volume fraction of 0.1 - 5%, aqueous solution of methanol with a volume fraction of 0.1 - 20%, aqueous solution of acetonitrile with a volume fraction of 0.1 - 20% or aqueous solution of formic acid with a volume fraction of 0.1% - 5%; Mobile phase B: pure methanol, pure acetonitrile, methanol solution of formic acid with a volume fraction of 0.1% or acetonitrile solution of formic acid with a volume fraction of 0.1%; Flow rate of mobile phase A and B: 0.2 - 0.5 mL / min; Curtain gas: 20 - 40 psi; Ionization voltage: 5500 V; Ion source temperature: 200 - 550 °C; Nebulizer: 40 - 60 psi; Auxiliary heating gas: 40 - 60 psi; Quantitative analysis is carried out using the multiple reaction monitoring mode (MRM).
[0045] In the quantitative method of dopamine-like active molecules in the water body of the present invention, the gradient change of mobile phase A and mobile phase B is as shown in the following table:
[0046] Table 2 Gradient change of mobile phase A and mobile phase B in the quantitative method
[0047]
[0048] Note: In the above table, the volume fraction of mobile phase A refers to the percentage of the volume of mobile phase A in the total volume of mobile phase A and mobile phase B; the volume fraction of mobile phase B refers to the percentage of the volume of mobile phase B in the total volume of mobile phase A and mobile phase B.
[0049] As can be seen from Table 2, within 0.00 - 2.00 min, the volume fraction of mobile phase A decreases from 95% to 50%, and the volume fraction of mobile phase B increases from 5% to 50%; within 2.00 - 7.00 min, the volume fraction of mobile phase A decreases from 50% to 5%, and the volume fraction of mobile phase B increases from 50% to 95%; and so on.
[0050] The quantitative method established by the present invention using synthetic peptides can achieve the quantitative detection of dopamine-like active molecules in the water body.
[0051] Compared with the prior art, the beneficial effects of the present invention are:
[0052] The present invention utilizes the cavity size selectivity of synthetic peptides and the hydrogen bonding between them and dopamine-like active molecules, enabling the synthetic peptides to selectively interact with dopamine-like active molecules to form complexes, improving the selectivity for dopamine-like active molecules in water bodies, and thus realizing the screening and quantitative detection of dopamine-like active molecules in water bodies with complex matrices. Description of the Drawings
[0053] Figure 1 It is the chromatogram of cyclic peptide 1; the abscissa is time, with the unit of minutes (min); the ordinate is the response value.
[0054] Figure 2 It is the mass spectrum of cyclic peptide 1; Counts vs. Mass-to-Charge represents ion abundance vs. mass-to-charge ratio, the abscissa is the mass-to-charge ratio (Mass-to-Charge (m / z)); the ordinate is the ion abundance (Counts).
[0055] Figure 3 It is the mass spectrum of dopamine; Counts vs. Mass-to-Charge represents ion abundance vs. mass-to-charge ratio, the abscissa is the mass-to-charge ratio (Mass-to-Charge (m / z)); the ordinate is the ion abundance (Counts).
[0056] Figure 4 It is the mass spectrum of complex 1; Counts vs. Mass-to-Charge represents ion abundance vs. mass-to-charge ratio, the abscissa is the mass-to-charge ratio (Mass-to-Charge (m / z)); the ordinate is the ion abundance (Counts).
[0057] Figure 5 It is the chromatogram of cyclic peptide 2; the abscissa is time, with the unit of minutes (min); the ordinate is the response value.
[0058] Figure 6 It is the mass spectrum of cyclic peptide 2; Counts vs. Mass-to-Charge represents ion abundance vs. mass-to-charge ratio, the abscissa is the mass-to-charge ratio (Mass-to-Charge (m / z)); the ordinate is the ion abundance (Counts).
[0059] Figure 7 It is the mass spectrum of dopamine; Counts vs. Mass-to-Charge represents ion abundance vs. mass-to-charge ratio, the abscissa is the mass-to-charge ratio (Mass-to-Charge (m / z)); the ordinate is the ion abundance (Counts).
[0060] Figure 8Mass spectrum of Complex 2; Counts vs. Mass-to-Charge represents ion abundance vs. mass-to-charge ratio, with the horizontal axis being the mass-to-charge ratio (Mass-to-Charge (m / z)); the vertical axis being the ion abundance (Counts).
[0061] Figure 9 Chromatogram of linear peptide; the horizontal axis is time, with the unit of minutes; the vertical axis is the response value.
[0062] Figure 10 Mass spectrum of linear peptide; Counts vs. Mass-to-Charge represents ion abundance vs. mass-to-charge ratio, with the horizontal axis being the mass-to-charge ratio (Mass-to-Charge (m / z)); the vertical axis being the ion abundance (Counts).
[0063] Figure 11 Mass spectrum of dopamine; Counts vs. Mass-to-Charge represents ion abundance vs. mass-to-charge ratio, with the horizontal axis being the mass-to-charge ratio (Mass-to-Charge (m / z)); the vertical axis being the ion abundance (Counts).
[0064] Figure 12 Mass spectrum of Complex 3; Counts vs. Mass-to-Charge represents ion abundance vs. mass-to-charge ratio, with the horizontal axis being the mass-to-charge ratio (Mass-to-Charge (m / z)); the vertical axis being the ion abundance (Counts).
[0065] Figure 13 Mass spectrum of fenoldopam; Counts vs. Mass-to-Charge represents ion abundance vs. mass-to-charge ratio, with the horizontal axis being the mass-to-charge ratio (Mass-to-Charge (m / z)); the vertical axis being the ion abundance (Counts).
[0066] Figure 14 Mass spectrum of Complex 4; Counts vs. Mass-to-Charge represents ion abundance vs. mass-to-charge ratio, with the horizontal axis being the mass-to-charge ratio (Mass-to-Charge (m / z)); the vertical axis being the ion abundance (Counts).
[0067] Figure 15 Volcano plot (FC > 2.0, p < 0.05) obtained after data processing of the LC-MS spectra of the cyclic peptide 1 experimental group and the methanol control group in Example 1. Detailed implementation mode
[0068] The present invention will be further described below in conjunction with embodiments. These embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. For the experimental methods without specific conditions in the following embodiments, they are generally carried out according to the conventional conditions in the art or according to the conditions recommended by the manufacturer; the raw materials, reagents, etc. used, unless otherwise specified, are all raw materials and reagents that can be obtained from commercial channels such as conventional markets. Any non-substantial changes and substitutions made by those skilled in the art based on the present invention fall within the scope of protection required by the present invention.
[0069] The reagents used in the present invention are as follows:
[0070] Cyclic peptide 1: P220810-T242 / SR0117, NH2-D-Cys-D-Pro-Gly-D-Cys-NHCH2CH3(C1-C4) hydrochloride, Hefei Sen'er Biotechnology Co., Ltd., molecular weight: 403.50, white powder, purity: >95%, having the molecular structure shown in formula (1):
[0071]
[0072] Cyclic peptide 2: P221129-PE0219 / SR0219, D-Cys-D-Pro-Gly-D-Cys-NH2(C1-C4) hydrochloride, Hefei Sen'er Biotechnology Co., Ltd., molecular weight: 375.48, white powder, purity: >95%, having the molecular structure shown in formula (2):
[0073]
[0074] Linear peptide: P221122-PE0214-1 / SR0214-1, Gly-D-Pro-Gly-Gly-NHCH2CH3 hydrochloride, Hefei Sen'er Biotechnology Co., Ltd., molecular weight: 313.36, white powder, purity: >95%, having the molecular structure shown in formula (3):
[0075]
[0076] In the screening method for dopamine-like active molecules in the water body of the present invention, the LC-MS in step S2 is UHPLC-QTOF / MS (Agilent, LC1290-G6545), and its analysis conditions are:
[0077] Chromatographic column: ZORBAX Eclipse Plus C18; Mobile phase A: Aqueous solution of 0.1% formic acid by volume; Mobile phase B: Methanol solution of 0.1% formic acid by volume; Flow rate: 0.2 mL / min; Nozzle voltage: 500 V; Nebulizer pressure: 35 psi; Drier flow rate: 11 L / min; Drier temperature: 320 °C; Capillary voltage: 3500 V; Analyzed in SIM mode.
[0078] In the screening method for dopamine-like active molecules in water bodies of the present invention, the gradient changes of mobile phase A and mobile phase B are shown in the following table:
[0079] Table 1 Gradient changes of mobile phase A and mobile phase B in the screening method
[0080]
[0081] Note: In the above table, the volume fraction of mobile phase A refers to the percentage of the volume of mobile phase A in the total volume of mobile phase A and mobile phase B; the volume fraction of mobile phase B refers to the percentage of the volume of mobile phase B in the total volume of mobile phase A and mobile phase B.
[0082] As can be seen from Table 1, within 0.00 - 1.00 min, the volume fraction of mobile phase A remains at 95% and the volume fraction of mobile phase B remains at 5%, flowing to the waste liquid; within 1.00 - 5.00 min, the volume fraction of mobile phase A decreases from 95% to 65% and the volume fraction of mobile phase B increases from 5% to 35%, flowing through the mass spectrometer; within 5.00 - 25.00 min, the volume fraction of mobile phase A decreases from 65% to 5% and the volume fraction of mobile phase B increases from 35% to 95%, flowing through the mass spectrometer; and so on.
[0083] In the quantification method for dopamine-like active molecules in water bodies of the present invention, the LC-MS analysis conditions are as follows:
[0084] Chromatographic column: ZORBAX Eclipse Plus C18; Mobile phase A: Aqueous solution of 0.1% formic acid by volume; Mobile phase B: Methanol solution of 0.1% formic acid by volume; Flow rate: 0.3 mL / min; Curtain gas: 35 psi; Ionization voltage: 5500 V; Ion source temperature: 450 °C; Nebulizer: 40 psi; Auxiliary heating gas: 40 psi; Quantitative analysis is carried out using the multiple reaction monitoring mode (MRM).
[0085] In the quantification method for dopamine-like active molecules in water bodies of the present invention, the gradient changes of mobile phase A and mobile phase B are shown in the following table:
[0086] Table 2 Gradient Changes of Mobile Phase A and Mobile Phase B in the Quantitative Method
[0087]
[0088] Note: In the above table, the volume fraction of mobile phase A refers to the percentage of the volume of mobile phase A in the total volume of mobile phase A and mobile phase B; the volume fraction of mobile phase B refers to the percentage of the volume of mobile phase B in the total volume of mobile phase A and mobile phase B.
[0089] As can be seen from Table 2, within 0.00 - 2.00 min, the volume fraction of mobile phase A decreases from 95% to 50%, and the volume fraction of mobile phase B increases from 5% to 50%; within 2.00 - 7.00 min, the volume fraction of mobile phase A decreases from 50% to 5%, and the volume fraction of mobile phase B increases from 50% to 95%; and so on.
[0090] (1) Verification of the Formation of a Complex between Cyclopeptide 1 and Dopamine-like Active Molecules
[0091] Prepare a mixed solution of cyclopeptide 1 and dopamine at a molar ratio of 1:1, and then analyze it by mass spectrometry;
[0092] This mixed solution is a methanol solution of complex 1 formed by cyclopeptide 1 and dopamine.
[0093] Figure 1 is the chromatogram of cyclopeptide 1; the abscissa is time, with the unit of minutes (min); the ordinate is the response value. Figure 2 is the mass spectrum of cyclopeptide 1; Counts vs. Mass-to-Charge represents ion abundance vs. mass-to-charge ratio, the abscissa is the mass-to-charge ratio (Mass-to-Charge (m / z)); the ordinate is the ion abundance (Counts). Figure 3 is the mass spectrum of dopamine; Counts vs. Mass-to-Charge represents ion abundance vs. mass-to-charge ratio, the abscissa is the mass-to-charge ratio (Mass-to-Charge (m / z)); the ordinate is the ion abundance (Counts). Figure 4 is the mass spectrum of complex 1; Counts vs. Mass-to-Charge represents ion abundance vs. mass-to-charge ratio, the abscissa is the mass-to-charge ratio (Mass-to-Charge (m / z)); the ordinate is the ion abundance (Counts).
[0094] Table 3 Mass Spectrometry Information of Cyclopeptide 1, Dopamine, and Complex 1
[0095]
[0096] FromFigures 1 - 4 As can be seen from Table 3, cyclo - peptide 1 can form complex 1 with dopamine, indicating that by using the cavity size selectivity of the synthetic peptide (cyclo - peptide 1) and the hydrogen - bond interaction between the synthetic peptide and dopamine - like active molecules, the present invention successfully enables the synthetic peptide to selectively interact with dopamine - like active molecules to form a complex, laying a foundation for the screening and quantitative detection of dopamine - like active molecules in complex - matrix water bodies.
[0097] (2) Verification of the formation of complex between cyclo - peptide 2 and dopamine - like active molecules
[0098] Prepare a mixed solution of cyclo - peptide 2 and dopamine at a molar ratio of 1:1, and then analyze it by mass spectrometry;
[0099] This mixed solution is a methanol solution of complex 2 formed by cyclo - peptide 2 and dopamine.
[0100] Figure 5 is the chromatogram of cyclo - peptide 2; the abscissa is time, with the unit of minute (min); the ordinate is the response value. Figure 6 is the mass spectrum of cyclo - peptide 2; Counts vs. Mass - to - Charge represents ion abundance vs. mass - to - charge ratio, the abscissa is the mass - to - charge ratio (Mass - to - Charge (m / z)); the ordinate is the ion abundance (Counts). Figure 7 is the mass spectrum of dopamine; Counts vs. Mass - to - Charge represents ion abundance vs. mass - to - charge ratio, the abscissa is the mass - to - charge ratio (Mass - to - Charge (m / z)); the ordinate is the ion abundance (Counts). Figure 8 is the mass spectrum of complex 2; Counts vs. Mass - to - Charge represents ion abundance vs. mass - to - charge ratio, the abscissa is the mass - to - charge ratio (Mass - to - Charge (m / z)); the ordinate is the ion abundance (Counts).
[0101] Table 4 Mass - spectrometry information of cyclo - peptide 2, dopamine and complex 2
[0102]
[0103]
[0104] From Figures 4 - 8 and Table 4, it can be seen that cyclo - peptide 2 can form complex 2 with dopamine, indicating that by using the cavity size selectivity of the synthetic peptide (cyclo - peptide 2) and the hydrogen - bond interaction between the synthetic peptide and dopamine - like active molecules, the present invention successfully enables the synthetic peptide to selectively interact with dopamine - like active molecules to form a complex, laying a foundation for the screening and quantitative detection of dopamine - like active molecules in complex - matrix water bodies.
[0105] (3) Verification of the formation of a complex between the linear peptide and the dopamine-like active molecule
[0106] A. Prepare a mixed solution by mixing the linear peptide and dopamine at a molar ratio of 1:1, and then analyze it by mass spectrometry;
[0107] This mixed solution is a methanol solution of complex 3 formed by the linear peptide and dopamine.
[0108] Figure 9 is the chromatogram of the linear peptide; the abscissa is time, in minutes (minutes); the ordinate is the response value. Figure 10 is the mass spectrum of the linear peptide; Counts vs. Mass-to-Charge represents ion abundance vs. mass-to-charge ratio, the abscissa is the mass-to-charge ratio (Mass-to-Charge (m / z)); the ordinate is the ion abundance (Counts). Figure 11 is the mass spectrum of dopamine; Counts vs. Mass-to-Charge represents ion abundance vs. mass-to-charge ratio, the abscissa is the mass-to-charge ratio (Mass-to-Charge (m / z)); the ordinate is the ion abundance (Counts). Figure 12 is the mass spectrum of complex 3; Counts vs. Mass-to-Charge represents ion abundance vs. mass-to-charge ratio, the abscissa is the mass-to-charge ratio (Mass-to-Charge (m / z)); the ordinate is the ion abundance (Counts).
[0109] Table 5 Mass spectrometry information of the linear peptide, dopamine, and complex 3
[0110]
[0111] From Figures 9 - 12 and Table 5, it can be seen that the linear peptide can form complex 3 with dopamine, indicating that the present invention successfully enables the synthetic peptide to selectively interact with the dopamine-like active molecule to form a complex by utilizing the cavity size selectivity of the synthetic peptide (linear peptide) and the hydrogen bond interaction between the synthetic peptide and the dopamine-like active molecule, laying a foundation for the screening and quantitative detection of dopamine-like active molecules in complex matrix water bodies.
[0112] B. Prepare a mixed solution by mixing the linear peptide and fenoldopam at a molar ratio of 1:1, and then analyze it by mass spectrometry;
[0113] This mixed solution is a methanol solution of complex 4 formed by the linear peptide and fenoldopam.
[0114] Figure 13Mass spectrum of fenoldopam; Counts vs. Mass-to-Charge represents ion abundance vs. mass-to-charge ratio, with the abscissa being the mass-to-charge ratio (Mass-to-Charge (m / z)); the ordinate being the ion abundance (Counts). Figure 14 Mass spectrum of complex 4; Counts vs. Mass-to-Charge represents ion abundance vs. mass-to-charge ratio, with the abscissa being the mass-to-charge ratio (Mass-to-Charge (m / z)); the ordinate being the ion abundance (Counts).
[0115] Table 6 Mass spectrometry information of linear peptide, fenoldopam and complex 4
[0116]
[0117] From Figure 10 、 Figures 13 - 14 and Table 6, it can be seen that the linear peptide can form complex 4 with fenoldopam, indicating that the present invention utilizes the cavity size selectivity of the synthetic peptide (linear peptide) and the hydrogen bond interaction between the synthetic peptide and the dopamine-like active molecule, and successfully enables the synthetic peptide to selectively interact with the dopamine-like active molecule to form a complex, laying a foundation for the screening and quantitative detection of dopamine-like active molecules in complex matrix water bodies.
[0118] In addition, by comparing Figure 4 、 8 and 12, it can be seen that the ion abundance of complex 1 is 4.8×10 6 and the ion abundance of complex 2 is 7×10 4 are both higher than the ion abundance of complex 3, which is 7.5×10 3 , indicating that the interaction between dopamine and cyclic peptide 1 or cyclic peptide 2 is stronger than the interaction between dopamine and the linear peptide. That is, compared with the linear peptide, cyclic peptide 1 and cyclic peptide 2 have stronger selectivity for dopamine-like active molecules, can better form complexes with dopamine-like active molecules, and can more sensitively screen out dopamine-like active molecules from complex matrix water bodies.
[0119] Example 1
[0120] A screening method for dopamine-like active molecules in water bodies in this example includes the following steps:
[0121] S1. The river water sample collected on the same day is immediately filtered through a 0.22 μm PVDF membrane, placed in a refrigerator at 4°C for storage, and restored to room temperature in advance before solid-phase extraction;
[0122] First, rinse and activate the C18 solid-phase extraction column with 20 mL of methanol, then equilibrate the C18 solid-phase extraction column with 20 mL of pure water. Next, inject 500 mL of river water sample into the C18 solid-phase extraction column, and then elute with 20 mL of methanol to obtain the eluate. Blow-dry the eluate with nitrogen, then re-dissolve it with 500 μL of methanol and filter it through a 0.22 μm filter head to obtain a water body concentrate with a concentration factor of 1000 times, and store it at -20 °C;
[0123] S2. Use the cyclic peptide 1 solution (with methanol as the solvent) as the experimental group and methanol as the control group;
[0124] First, separate 5 μL of the water body concentrate by LC at a flow rate of 200 μL / min, and then mix it with the cyclic peptide 1 solution with a concentration of 50 μM / L at a flow rate of 20 μL / min through a three-way valve and enter the MS to obtain the LC-MS spectrum of the experimental group;
[0125] First, separate 5 μL of the water body concentrate by LC at a flow rate of 200 μL / min, and then mix it with methanol at a flow rate of 20 μL / min through a three-way valve and enter the MS to obtain the LC-MS spectrum of the control group;
[0126] Use the LC-MS spectra of the experimental group and the control group to create volcano plots for the experimental group and the control group. As Figure 15 shown, perform targeted secondary analysis on the suspicious signals with up-regulated signals and match them with the mass spectrometry database to identify the types of dopamine-like active molecules in the water body.
[0127] Figure 15 The volcano plot (FC > 2.0, p < 0.05) obtained after data processing of the LC-MS spectra of the cyclic peptide 1 experimental group and the methanol control group in Example 1. From Figure 15 it can be seen that there are a total of 2499 suspicious signals in the river water samples in the experimental group (the right side of the volcano plot) and the control group (the left side of the volcano plot). After using cyclic peptide 1, 321 suspicious signals in the river water samples showed up-regulated signals, that is, the use of cyclic peptide 1 reduced the suspicious signals in the river water samples to 321, indicating that cyclic peptide 1 has high selectivity for dopamine-like active molecules in the water body, which is beneficial to improving the screening level of dopamine-like active molecules in the water body.
[0128] Through targeted secondary analysis of 321 suspicious signals with up-regulated signals and combining fragment ions and parent ions for mass spectrometry database retrieval, 3 types of dopamine-like active molecules were successfully identified, as shown in the following table:
[0129] Table 7 LC-MS information of 3 types of dopamine-like active molecules in river water samples
[0130]
[0131] As can be seen from Table 7, the river water sample contains 3 types of dopamine-like active molecules, namely isooctylamine, amantadine, and pramipexole, indicating that the screening method for dopamine-like active molecules in the water body of the present invention has high selectivity for dopamine-like active molecules in the water body and can accurately and efficiently screen out dopamine-like active molecules from the water body with complex matrix.
[0132] Example 2
[0133] A screening method for dopamine-like active molecules in a water body in this example is only different from that in Example 1 in that a 50 μM / L cyclopeptide 2 solution is used instead of a 50 μM / L cyclopeptide 1 solution, and specifically includes the following steps:
[0134] S1. The river water sample collected on the same day is immediately filtered through a 0.22 μm PVDF membrane, placed in a refrigerator at 4°C for storage, and restored to room temperature in advance before solid-phase extraction.
[0135] First, rinse and activate the C18 solid-phase extraction small column with 20 mL of methanol, balance the C18 solid-phase extraction small column with 20 mL of pure water, then inject 500 mL of river water sample into the C18 solid-phase extraction small column, and then elute with 20 mL of methanol to obtain an eluate. Blow dry the eluate with nitrogen, then re-dissolve it with 500 μL of methanol, and filter it with a 0.22 μm filter head to obtain a water body concentrate with a concentration factor of 1000 times, and store it at -20°C.
[0136] S2. Use the cyclopeptide 2 solution (solvent is methanol) as the experimental group and methanol as the control group;
[0137] First, separate 5 μL of the water body concentrate through LC at a flow rate of 200 μL / min, and then mix it with a 50 μM / L cyclopeptide 2 solution at a flow rate of 20 μL / min through a three-way valve and enter the MS to obtain the LC-MS spectrum of the experimental group;
[0138] First, separate 5 μL of the water body concentrate through LC at a flow rate of 200 μL / min, and then mix it with methanol at a flow rate of 20 μL / min through a three-way valve and enter the MS to obtain the LC-MS spectrum of the control group;
[0139] Use the LC-MS spectra of the experimental group and the control group to make volcano plots of the experimental group and the control group, perform targeted secondary analysis on the suspicious signals with up-regulated signals, and match them with the mass spectrometry database to identify the types of dopamine-like active molecules in the water body.
[0140] The volcano plots obtained after data processing of the LC-MS spectra of the cyclopeptide 2 experimental group and the methanol control group in Example 2 are the same as those of Figure 15Similarly, three types of dopamine-like active molecules, namely isooctylamine, amantadine, and pramipexole, can still be identified from the river water samples. That is, cyclic peptide 2 also has high selectivity for dopamine-like active molecules in water bodies and can accurately and efficiently screen out dopamine-like active molecules from complex matrix water bodies.
[0141] Example 3
[0142] The screening method for dopamine-like active molecules in water bodies in this example is only different from that in Example 1 in that a 50 μM / L linear peptide solution is used instead of a 50 μM / L cyclic peptide 1 solution, and specifically includes the following steps:
[0143] S1. The river water samples collected on the same day are immediately filtered through a 0.22 μm PVDF membrane, stored in a refrigerator at 4°C, and restored to room temperature in advance before solid-phase extraction.
[0144] First, activate the C18 solid-phase extraction small column with 20 mL of methanol, balance the C18 solid-phase extraction small column with 20 mL of pure water, then inject 500 mL of river water samples into the C18 solid-phase extraction small column, and then elute with 20 mL of methanol to obtain an eluate. Blow dry the eluate with nitrogen, then re-dissolve it with 500 μL of methanol, and filter it with a 0.22 μm filter head to obtain a water body concentrate with a concentration factor of 1000 times, and store it at -20°C.
[0145] S2. Use the linear peptide solution (with methanol as the solvent) as the experimental group and methanol as the control group;
[0146] First, separate 5 μL of the water body concentrate through LC at a flow rate of 200 μL / min, and then mix it with a 50 μM / L linear peptide solution at a flow rate of 20 μL / min through a three-way valve and enter the MS to obtain the LC-MS spectrum of the experimental group.
[0147] First, separate 5 μL of the water body concentrate through LC at a flow rate of 200 μL / min, and then mix it with methanol at a flow rate of 20 μL / min through a three-way valve and enter the MS to obtain the LC-MS spectrum of the control group.
[0148] Use the LC-MS spectra of the experimental group and the control group to make volcano plots of the experimental group and the control group, perform targeted secondary analysis on the suspicious signals with up-regulated signals, and match them with the mass spectrometry database to identify the types of dopamine-like active molecules in the water body.
[0149] The volcano plots obtained after data processing of the LC-MS spectra of the linear peptide experimental group and the methanol control group in Example 3 are the same as Figure 15Similarly, three types of dopamine-like active molecules, namely isooctylamine, amantadine, and pramipexole, can still be identified from the river water sample. That is, the linear peptide also has high selectivity for dopamine-like active molecules in water bodies and can accurately and efficiently screen out dopamine-like active molecules from complex matrix water bodies.
[0150] Example 4
[0151] A method for quantifying dopamine-like active molecules in water bodies in this example includes the following steps:
[0152] Using the screening method for dopamine-like active molecules in water bodies in Example 1, the types of dopamine-like active molecules in the water body (river water sample) were identified as three, namely isooctylamine, amantadine, and pramipexole. Then, a series of standard concentrations of isooctylamine, amantadine, and pramipexole were prepared respectively, and then LC-MS (Agilent 1260 Infinity-ABSciex Triple Quad TM 4500) was used to establish the standard concentration curves (standard curves of peak area versus concentration) of these three types of dopamine-like active molecules. By calculation, the contents of these three types of dopamine-like active molecules in the water body (river water sample) were obtained. The experimental results are shown in the following table:
[0153] Table 8 Quantitative results of three types of dopamine-like active molecules in river water samples
[0154]
[0155] As can be seen from Table 8, through the method for quantifying dopamine-like active molecules in water bodies of the present invention, the concentration of isooctylamine in the river water sample was measured to be approximately 0.78 μg / L, the concentration of amantadine was approximately 0.23 μg / L, and the concentration of pramipexole was approximately 0.013 μg / L. This indicates that the method for quantifying dopamine-like active molecules in water bodies of the present invention can be used for quantitative analysis of dopamine-like active molecules in complex matrix water bodies.
[0156] Obviously, the above-mentioned embodiments of the present invention are merely examples for clearly illustrating the present invention and are not intended to limit the implementation manners of the present invention. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation manners here. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included within the protection scope of the claims of the present invention.
Claims
1. A screening method for dopamine-like active molecules in water bodies, characterized in that, It includes the following steps: S1. Perform solid-phase extraction on the water body and concentrate to obtain a concentrated water body solution; S2. Analyze the concentrated water body solution and a synthetic peptide solution with a concentration of 20 - 50 μM / L by LC-MS, make a volcano plot using the LC-MS spectrum, conduct targeted secondary analysis on the suspicious signals with up-regulated signals, and match with the mass spectrometry database to identify the types of dopamine-like active molecules in the water body; The synthetic peptide described in step S2 is one of cyclic peptide 1, cyclic peptide 2, or linear peptide; The cyclic peptide 1 has the molecular structure shown in formula 1: Formula 1; The cyclic peptide 2 has the molecular structure shown in formula 2: Formula 2; The linear peptide has the molecular structure shown in formula 3: Formula 3; The dopamine-like active molecule is one or more of dopamine, fenoldopam, isooctylamine, amantadine, pramipexole.
2. The screening method according to claim 1, wherein The synthetic peptide described in step S2 is cyclic peptide 1 or cyclic peptide 2.
3. The screening method according to claim 1, wherein The concentration multiple of the concentrated water body solution described in step S1 is 250 - 2000 times.
4. The screening method according to claim 1, characterized in that, Step S1 specifically means first rinsing the solid-phase extraction column with an organic solvent, then injecting the water body into the solid-phase extraction column, then eluting with an organic solvent to obtain an eluate, drying, then redissolving with an organic solvent, and filtering to obtain a concentrated water body solution.
5. The screening method according to claim 4, characterized in that, The organic solvent is one or more of methanol, acetonitrile, carbon tetrachloride, toluene, ethyl acetate, ethanol, or water.
6. The screening method according to claim 4, wherein The solid-phase extraction column is one or more of a C18 solid-phase extraction cartridge, an HLB solid-phase extraction column, a C8 solid-phase extraction column, or an SLC solid-phase extraction column.
7. The screening method according to claim 1, wherein The analysis of the concentrated water body solution and a synthetic peptide solution with a concentration of 20 - 50 μM / L by LC-MS means that the concentrated water body solution is first separated by LC, and then mixed with a synthetic peptide solution with a concentration of 20 - 50 μM / L through a three-way valve and enters the MS.
8. The screening method according to claim 1, wherein The flow rate of the synthetic peptide solution with a concentration of 20 - 50 μM / L entering the LC-MS is 10 - 25 μL / min.
9. A method for quantifying dopamine-like active molecules in water bodies, characterized in that, It includes the following steps: Use the screening method for dopamine-like active molecules in the water body described in any one of claims 1 - 8 to identify the types of dopamine-like active molecules in the water body, obtain the specific types of dopamine-like active molecules, and then use LC-MS technology to establish a standard concentration curve for the specific types of dopamine-like active molecules, and the content of the specific types of dopamine-like active molecules in the water body can be obtained by calculation.