A screening method and a quantification method for active molecules of 5-HT receptor modulators in water bodies

Abstract

The present invention discloses a screening method and a quantification method for 5-HT receptor modulator-like active molecules in water bodies. The screening method comprises the following steps: S1. Solid-phase extraction is carried out on the water body, and the water body concentrate is obtained through concentration; S2. The water body concentrate and a hydroxy synthetic peptide solution with a concentration of 20-50 μM / L are analyzed by LC-MS, a volcano plot is made by using the LC-MS spectrum, targeted secondary analysis is carried out on the suspicious signals with up-regulated signals, and the signals are matched with a mass spectrometry database to identify the types of 5-HT receptor modulator-like active molecules in the water body; the hydroxy synthetic peptide in step S2 is a hydroxy cyclic peptide or a hydroxy linear peptide; the hydroxy cyclic peptide is the hydroxy linear peptide. The present invention utilizes the interaction between the hydroxy synthetic peptide and the 5-HT receptor modulator-like active molecule to form a complex, thereby realizing the screening and quantitative detection of the 5-HT receptor modulator-like active molecule in a complex matrix water body.

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 active molecules of 5-HT receptor modulators in water bodies. Background Art

[0002] 5-HT receptors, also known as 5-hydroxytryptamine receptors or serotonin receptors, are G protein-coupled receptors (GPCRs) and ligand-gated ion channels (LGICs) located in the central and peripheral nervous systems of animals. They regulate the transmission of both excitatory and inhibitory neurotransmitters, participate in regulating mood, body temperature balance, sleep, and the response levels of psychological stress, and also regulate self-control and cognitive functions. They are not only closely related to neurological diseases such as Parkinson's disease, schizophrenia, Alzheimer's disease, and bipolar disorder, but also related to the important mechanisms of drug addiction. 5-HT receptor modulators refer to substances that bind to 5-HT receptors and regulate the functions of 5-HT receptors. Active molecules of 5-HT receptor modulator analogs refer to molecules with neuroactivities similar to those of 5-HT receptor modulators, such as nicotine, tryptamine, 5-hydroxytryptamine, rizatriptan, fluvoxamine, maprotiline, nortriptyline, amisulpride, epinastine, duloxetine, fluoxetine, etc.

[0003] With the progress of human production and life, active molecules of 5-HT receptor modulator analogs have long been discharged into the water environment, causing serious pollution to the water bodies and posing a serious threat to human health and the ecological environment. Therefore, high attention needs to be paid to the distribution and content of active molecules of 5-HT receptor modulator analogs in water bodies. However, the complexity of the water environment increases the difficulty of screening and quantitative detection of active molecules of 5-HT receptor modulator analogs in water bodies. When existing methods are used in the water environment, due to the poor selectivity for active molecules of 5-HT receptor modulator analogs, it becomes difficult to screen active molecules of 5-HT receptor modulator analogs in water bodies. Therefore, it is of great significance to develop a screening method and a quantification method for active molecules of 5-HT receptor modulator analogs with high selectivity. Summary of the Invention

[0004] The primary objective of the present invention is to solve the problem of poor selectivity of the existing technology for active molecules of 5-HT receptor modulator analogs in water bodies, and to provide a screening method for active molecules of 5-HT receptor modulator analogs in water bodies.

[0005] Another objective of the present invention is to provide a quantification method for active molecules of 5-HT receptor modulator analogs in water bodies.

[0006] To achieve the above objectives, the present invention adopts the following technical solutions:

[0007] A screening method for 5-HT receptor modulator-like active molecules in water bodies, comprising the following steps:

[0008] S1. Perform solid-phase extraction on the water body and concentrate to obtain a water body concentrate;

[0009] S2. Analyze the water body concentrate and a hydroxy 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 a mass spectrometry database to identify the types of 5-HT receptor modulator-like active molecules in the water body;

[0010] The hydroxy synthetic peptide described in step S2 is one of a hydroxy cyclic peptide or a hydroxy linear peptide;

[0011] The hydroxy cyclic peptide has a molecular structure shown in formula (1):

[0012]

[0013] The hydroxy linear peptide has a molecular structure shown in formula (2):

[0014]

[0015] The present invention utilizes the cavity size selectivity of the hydroxy synthetic peptide and the hydrogen bond interaction between the hydroxy synthetic peptide and the 5-HT receptor modulator-like active molecule, enabling the hydroxy synthetic peptide to selectively interact with the 5-HT receptor modulator-like active molecule to form a complex, improving the selectivity for the 5-HT receptor modulator-like active molecule in the water body, and thus realizing the screening of the 5-HT receptor modulator-like active molecule in a water body with a complex matrix.

[0016] The inventors found in previous studies that the selectivity of the synthetic peptides without hydroxy, such as cyclic peptide 1 and linear peptide, for binding to dopamine-like active molecules is much stronger than that for binding to 5-HT receptor modulator-like active molecules. To better screen the 5-HT receptor modulator-like active molecules in the water body and improve the selectivity of the synthetic peptide for the 5-HT receptor modulator-like active molecule, the inventors prepared the hydroxy synthetic peptide through computational rational design. The hydroxy -OH in the hydroxy synthetic peptide can affect the cavity of the polypeptide and the hydrogen bond sites for interacting with small molecules, thereby improving the selectivity of the synthetic peptide for the 5-HT receptor modulator-like active molecule, and further facilitating the screening of the 5-HT receptor modulator-like active molecule in a water body with a complex matrix.

[0017] Cyclic peptide 1 has a molecular structure shown in formula (3):

[0018]

[0019] Linear peptide has a molecular structure shown in formula (4):

[0020]

[0021] In the present invention, both the hydroxycyclopeptide and the hydroxylateptide are commercially available.

[0022] In the present invention, the solvent of the hydroxy synthetic peptide solution is one or more of methanol, acetonitrile, an acetonitrile solution of trifluoroacetic acid with a volume fraction of 0.1 - 5%, an acetonitrile solution of formic acid with a volume fraction of 0.1 - 5%, an acetonitrile solution of acetic acid with a volume fraction of 0.1 - 5%, a methanol solution of trifluoroacetic acid with a volume fraction of 0.1 - 5%, a methanol solution of formic acid with a volume fraction of 0.1 - 5%, or a methanol solution of acetic acid with a volume fraction of 0.1 - 5%.

[0023] In the present invention, a volcano plot is drawn using MetaboAnalyst software based on the retention time and ion abundance of the LC-MS spectrum.

[0024] Preferably, the hydroxy synthetic peptide in step S2 is a hydroxycyclopeptide.

[0025] Preferably, the 5-HT receptor modulator active molecule is one or more of nicotine, tryptamine, 5-hydroxytryptamine, rizatriptan, fluvoxamine, maprotiline, nortriptyline, amisulpride, epinastine, duloxetine, or fluoxetine.

[0026] In the present invention, the water body is a water sample, which can be a river water sample or a sea water sample.

[0027] Preferably, the concentration multiple of the water body concentrate in step S1 is 250 - 2000 times.

[0028] Preferably, the water body concentrate in step S1 is stored at a temperature below 4°C.

[0029] 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, 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 the water body concentrate.

[0030] More preferably, the organic solvent is one or more of methanol, acetonitrile, carbon tetrachloride, toluene, ethyl acetate, ethanol, or water.

[0031] 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.

[0032] In the present invention, the drying in step S1 may be blowing the eluent with an inert gas, or drying the eluent by rotary evaporation, or drying the eluent under vacuum; the inert gas used is one or both of nitrogen or argon.

[0033] Preferably, the analysis of the water concentrate and the hydroxy synthetic peptide solution with a concentration of 20 - 50 μM / L by LC-MS means that the water concentrate is first separated by LC, and then mixed with the hydroxy synthetic peptide solution with a concentration of 20 - 50 μM / L through a three-way valve and enters the MS.

[0034] Preferably, the flow rate of the hydroxy synthetic peptide solution with a concentration of 20 - 50 μM / L entering the LC-MS is 10 - 25 μL / min.

[0035] Conventional LC-MS in the art can be used for the screening method of the 5-HT receptor modulator active molecules in the water body of the present invention. For example, the LC-MS in step S2 can be UHPLC-QTOF / MS.

[0036] When the LC-MS in step S2 is UHPLC-QTOF / MS, its analysis conditions are as follows:

[0037] 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%, aqueous solution of acetic acid with a volume fraction of 0.1 - 5% or aqueous solution of formic acid with a volume fraction of 0.1%; Mobile phase B: pure methanol, pure acetonitrile, methanol solution of formic acid with a volume fraction of 0.1%, acetonitrile solution of formic acid with a volume fraction of 0.1 - 5%, methanol solution of acetic acid with a volume fraction of 0.1 - 5% or acetonitrile solution of acetic acid with a volume fraction of 0.1 - 5%; Flow rates 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 in SIM mode.

[0038] In the screening method of the 5-HT receptor modulator active molecules in the water body of the present invention, the gradient changes of mobile phase A and mobile phase B are shown in the following table:

[0039] Table 1 Gradient changes of mobile phase A and mobile phase B in the screening method

[0040]

[0041]

[0042] 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.

[0043] 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 - 20.00 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.

[0044] A method for quantifying active molecules of 5-HT receptor modulator-like in water bodies, comprising the following steps:

[0045] Identifying the types of active molecules of 5-HT receptor modulator-like in water bodies by using a screening method for active molecules of 5-HT receptor modulator-like in water bodies to obtain the specific types of active molecules of 5-HT receptor modulator-like, and then establishing a standard concentration curve for the specific types of active molecules of 5-HT receptor modulator-like by using LC-MS or GC-MS technology, and calculating the content of the specific types of active molecules of 5-HT receptor modulator-like in water bodies.

[0046] Conventional GC-MS or LC-MS in the art can be used in the method for quantifying active molecules of 5-HT receptor modulator-like in water bodies of the present invention. When using GC-MS, its GC-MS analysis conditions are:

[0047] Mass spectrometry ionization source: EI source; carrier gas: high-purity helium gas; injection is carried out in splitless mode; gas-phase capillary column: HP-5MS, HP-5 or DB-5MS; injection port temperature is 200 - 300 °C; transfer line temperature is 230 - 250 °C.

[0048] When using LC-MS, its LC-MS analysis conditions are:

[0049] Chromatographic column: ZORBAX Eclipse Plus C18, ZORBAX-SB-C18 or Poroshell 120 EC-C18; Mobile phase, 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%, aqueous solution of acetic acid with a volume fraction of 0.1 - 5% or aqueous solution of formic acid with a volume fraction of 0.1%; Mobile phase, phase B: pure methanol, pure acetonitrile, methanol solution of formic acid with a volume fraction of 0.1%, acetonitrile solution of formic acid with a volume fraction of 0.1%, methanol solution of acetic acid with a volume fraction of 0.1 - 5% or acetonitrile solution of acetic acid with a volume fraction of 0.1 - 5%; Flow rate of mobile phase, phase A and phase 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).

[0050] In the quantitative method for active molecules of 5-HT receptor modulator-like in water bodies of the present invention, the gradient changes of mobile phase, phase A and mobile phase, phase B are shown in the following table:

[0051] Table 2 Gradient changes of mobile phase, phase A and mobile phase, phase B in the quantitative method

[0052]

[0053] Note: In the above table, the volume fraction of mobile phase, phase A refers to the percentage of the volume of mobile phase, phase A in the total volume of mobile phase, phase A and mobile phase, phase B; the volume fraction of mobile phase, phase B refers to the percentage of the volume of mobile phase, phase B in the total volume of mobile phase, phase A and mobile phase, phase B.

[0054] As can be seen from Table 2, within 0.00 - 2.00 min, the volume fraction of mobile phase, phase A decreases from 90% to 10%, and the volume fraction of mobile phase, phase B increases from 10% to 90% and is maintained for 2 min; within 4.00 - 4.10 min, the volume fraction of mobile phase, phase A increases from 10% to 90%, and the volume fraction of mobile phase, phase B decreases from 90% to 10%; and so on.

[0055] The quantitative method established by the present invention using hydroxy synthetic peptides can achieve quantitative detection of active molecules of 5-HT receptor modulator-like in water bodies.

[0056] Compared with the prior art, the beneficial effects of the present invention are:

[0057] The present invention utilizes the cavity size selectivity of hydroxy synthetic peptides and the hydrogen bonding between them and active molecules of 5-HT receptor modulators, enabling the hydroxy synthetic peptides to selectively interact with active molecules of 5-HT receptor modulators to form complexes, improving the selectivity for active molecules of 5-HT receptor modulators in water bodies, and thus realizing the screening and quantitative detection of active molecules of 5-HT receptor modulators in water bodies with complex matrices.

[0058] Compared with synthetic peptides without hydroxyl groups (cyclic peptide 1, linear peptide), the hydroxy synthetic peptides of the present invention have stronger selectivity for active molecules of 5-HT receptor modulators and can better realize the screening of active molecules of 5-HT receptor modulators in water bodies with complex matrices. Description of the Drawings

[0059] Figure 1 It is the chromatogram of hydroxy cyclic peptide; the abscissa is time, with the unit of minutes (min); the ordinate is the response value.

[0060] Figure 2 It is the mass spectrum of hydroxy cyclic peptide; Counts vs. mass-to-charge ratio represents ion abundance vs. mass-to-charge ratio, the abscissa is the mass-to-charge ratio (m / z); the ordinate is the ion abundance (Counts).

[0061] Figure 3 It is the mass spectrum of 5-HT; Counts vs. mass-to-charge ratio represents ion abundance vs. mass-to-charge ratio, the abscissa is the mass-to-charge ratio (m / z); the ordinate is the ion abundance (Counts).

[0062] Figure 4 It is the mass spectrum of hydroxy complex 1; Counts vs. mass-to-charge ratio represents ion abundance vs. mass-to-charge ratio, the abscissa is the mass-to-charge ratio (m / z); the ordinate is the ion abundance (Counts).

[0063] Figure 5 It is the chromatogram of hydroxy linear peptide; the abscissa is time, with the unit of minutes (min); the ordinate is the response value.

[0064] Figure 6 It is the mass spectrum of hydroxy linear peptide; Counts vs. mass-to-charge ratio represents ion abundance vs. mass-to-charge ratio, the abscissa is the mass-to-charge ratio (m / z); the ordinate is the ion abundance (Counts).

[0065] Figure 7 It is the mass spectrum of 5-HT; Counts vs. mass-to-charge ratio represents ion abundance vs. mass-to-charge ratio, the abscissa is the mass-to-charge ratio (m / z); the ordinate is the ion abundance (Counts).

[0066] Figure 8Mass spectrum of hydroxy complex 2; Counts vs. mass-to-charge ratio represents ion abundance vs. mass-to-charge ratio, with the abscissa being the mass-to-charge ratio (m / z); the ordinate being the ion abundance (Counts).

[0067] Figure 9 It is a volcano plot (FC > 2.0, p < 0.05) obtained after data processing of the LC-MS spectra of the hydroxycyclopeptide experimental group and the methanol control group in Example 1. Specific implementation manners

[0068] The present invention will be further elaborated below in conjunction with examples. These examples 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 noted in the following examples, 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 the conventional market. Any non-substantive 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] Hydroxycyclopeptide: P221122-PE0214-2 / SR0214-2, D-Cys-D-Pro-Ser-D-Cys-NHCH2CH3(C1-C4) hydrochloride, Hefei Sen'er Biotechnology Co., Ltd., molecular weight: 433.54, white powder, purity: >95%, having the molecular structure shown in formula (1):

[0071]

[0072] Hydroxylinear peptide: P220722-T205 / SR0100, NH2-Gly-D-Pro-L-Ser-Gly-NHCH2CH3 hydrochloride, Hefei Sen'er Biotechnology Co., Ltd., molecular weight: 344.31, white powder, purity: >95%, having the molecular structure shown in formula (2):

[0073]

[0074] In the screening method for active molecules of 5-HT receptor modulators in the water body of the present invention, the LC-MS described in step S2 is UHPLC-QTOF / MS (Agilent, LC1290-G6545), and its analysis conditions are:

[0075] Chromatographic column: ZORBAX Eclipse Plus C18; Mobile phase A: Aqueous solution with 0.1% formic acid by volume; Mobile phase B: Methanol solution with 0.1% formic acid by volume; Flow rate: 0.2 mL / min; Nozzle voltage: 1000 V; Nebulizer pressure: 50 psi; Drying gas flow rate: 8 L / min; Drying gas temperature: 320 °C; Capillary voltage: 3500 V; Analyzed by SIM mode.

[0076] In the screening method for 5-HT receptor modulator-like active molecules in the water body of the present invention, the gradient changes of mobile phase A and mobile phase B are shown in the following table:

[0077] Table 1 Gradient changes of mobile phase A and mobile phase B in the screening method

[0078]

[0079]

[0080] 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.

[0081] 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.

[0082] In the quantification method for 5-HT receptor modulator-like active molecules in the water body of the present invention, when using LC-MS, the LC-MS analysis conditions are as follows:

[0083] Chromatographic column: ZORBAX Eclipse Plus C18; Mobile phase A: Aqueous solution with 0.1% formic acid by volume; Mobile phase B: Methanol solution with 0.1% formic acid by volume; Flow rate of mobile phase A and B: 0.3 mL / min; Curtain gas: 20 psi; Ionization voltage: 5500 V; Ion source temperature: 200 °C; Sprayer: 60 psi; Auxiliary heating gas: 60 psi; Quantification is carried out using the multiple reaction monitoring mode (MRM).

[0084] In the method for quantifying active molecules of 5-HT receptor modulator-like in the water body of the present invention, the gradient changes of mobile phase A and mobile phase B are shown in the following table:

[0085] Table 2 Gradient changes of mobile phase A and mobile phase B in the quantification method

[0086]

[0087] 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.

[0088] As can be seen from Table 2, within 0.00 - 2.00 min, the volume fraction of mobile phase A decreases from 90% to 10%, and the volume fraction of mobile phase B increases from 10% to 90%, and it is maintained for 2 min; within 4.00 - 4.10 min, the volume fraction of mobile phase A increases from 10% to 90%, and the volume fraction of mobile phase B decreases from 90% to 10%; and so on.

[0089] In the method for quantifying active molecules of 5-HT receptor modulator-like in the water body of the present invention, when using GC-MS, the GC-MS analysis conditions are as follows: the mass spectrometry ionization source is an EI source, the carrier gas is high-purity helium, injection is carried out in a splitless mode, the gas-phase capillary column is HP-5MS, the injection port temperature is 270 °C, the column oven temperature programming is: after maintaining at 50 °C for 2 min, it is heated to 160 °C at a rate of 30 °C / min, then heated to 180 °C at a rate of 20 °C / min and maintained for 1 min, the total time is 8 min, and the transfer line temperature is 250 °C.

[0090] (1) Verification of the formation of hydroxy complexes between hydroxycyclopeptides and active molecules of 5-HT receptor modulator-like

[0091] A mixed solution is prepared by mixing hydroxycyclopeptide and 5-HT at a molar ratio of 1:1, and then analyzed by mass spectrometry;

[0092] This mixed solution is a methanol solution of hydroxy complex 1 formed by hydroxycyclopeptide and 5-HT.

[0093] Figure 1 is the chromatogram of hydroxycyclopeptide; the abscissa is time, in minutes (minutes); the ordinate is the response value. Figure 2 is the mass spectrum of hydroxycyclopeptide; Counts vs. mass-to-charge ratio represents ion abundance vs. mass-to-charge ratio, the abscissa is the mass-to-charge ratio (m / z); the ordinate is the ion abundance (Counts). Figure 3It is the mass spectrum of 5-HT; Counts vs. mass-to-charge ratio represents ion abundance vs. mass-to-charge ratio. The abscissa is the mass-to-charge ratio (m / z); the ordinate is the ion abundance (Counts). Figure 4 It is the mass spectrum of hydroxy complex 1; Counts vs. mass-to-charge ratio represents ion abundance vs. mass-to-charge ratio. The abscissa is the mass-to-charge ratio (m / z); the ordinate is the ion abundance (Counts).

[0094] Table 3 Mass spectrometry information of hydroxycyclopeptide, 5-HT and hydroxy complex 1

[0095]

[0096] From Figures 1 to 4 and Table 3, it can be seen that hydroxycyclopeptide can form hydroxy complex 1 with 5-HT, indicating that the present invention utilizes the cavity size selectivity of hydroxy synthetic peptide (hydroxycyclopeptide) and the hydrogen bond interaction between it and the 5-HT receptor modulator active molecule-like, and successfully enables the hydroxy synthetic peptide to selectively interact with the 5-HT receptor modulator active molecule-like to form a complex, laying a foundation for the screening and quantitative detection of the 5-HT receptor modulator active molecule-like in complex matrix water bodies.

[0097] (2) Verification of the formation of hydroxy complex between hydroxy linear peptide and 5-HT receptor modulator active molecule

[0098] Prepare a mixed solution of hydroxy linear peptide and 5-HT at a molar ratio of 1:1, and then analyze it by mass spectrometry;

[0099] This mixed solution is a methanol solution of hydroxy complex 2 formed by hydroxy linear peptide and 5-HT.

[0100] Figure 5 It is the chromatogram of hydroxy linear peptide; the abscissa is time, in minutes (min); the ordinate is the response value. Figure 6 It is the mass spectrum of hydroxy linear peptide; Counts vs. mass-to-charge ratio represents ion abundance vs. mass-to-charge ratio. The abscissa is the mass-to-charge ratio (m / z); the ordinate is the ion abundance (Counts). Figure 7 It is the mass spectrum of 5-HT; Counts vs. mass-to-charge ratio represents ion abundance vs. mass-to-charge ratio. The abscissa is the mass-to-charge ratio (m / z); the ordinate is the ion abundance (Counts). Figure 8 It is the mass spectrum of hydroxy complex 2; Counts vs. mass-to-charge ratio represents ion abundance vs. mass-to-charge ratio. The abscissa is the mass-to-charge ratio (m / z); the ordinate is the ion abundance (Counts).

[0101] Table 4 Mass spectrometry information of hydroxy linear peptide, 5-HT and hydroxy complex 2

[0102]

[0103] From Figures 4 to 8 As can be seen from Table 4, hydroxy linear peptide can form hydroxy complex 2 with 5-HT, indicating that the present invention utilizes the cavity size selectivity of hydroxy synthetic peptide (hydroxy linear peptide) and the hydrogen bond interaction between the hydroxy synthetic peptide and the 5-HT receptor modulator-like active molecule, and successfully enables the hydroxy synthetic peptide to selectively interact with the 5-HT receptor modulator-like active molecule to form a complex, laying a foundation for the screening and quantitative detection of the 5-HT receptor modulator-like active molecule in complex matrix water bodies.

[0104] In addition, by comparing Figure 4 and 8 it can be known that the ion abundance of hydroxy complex 1 is 2.4×10 6 which is much higher than the ion abundance of hydroxy complex 2, 3.3×10 4 , indicating that the interaction between hydroxy cyclic peptide and the 5-HT receptor modulator-like active molecule is stronger than the interaction between hydroxy linear peptide and the 5-HT receptor modulator-like active molecule, that is, hydroxy cyclic peptide has stronger selectivity for the 5-HT receptor modulator-like active molecule, can better form a complex with the 5-HT receptor modulator-like active molecule, and can more sensitively screen out the 5-HT receptor modulator-like active molecule from complex matrix water bodies.

[0105] Example 1

[0106] A screening method for 5-HT receptor modulator-like active molecules in water bodies in this example includes the following steps:

[0107] S1. The river water sample collected on the same day is 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.

[0108] First, rinse and activate the C18 solid-phase extraction column with 20 mL of methanol, balance the C18 solid-phase extraction column with 20 mL of pure water, then inject 500 mL of the river water sample into the C18 solid-phase extraction 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.

[0109] S2. Use the hydroxy cyclic peptide solution (solvent is methanol) as the experimental group and methanol as the control group;

[0110] First, separate 5 μL of the water body concentrate through LC at a flow rate of 200 μL / min, and then mix it with the hydroxy cyclic peptide 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.

[0111] 5 μL of the water body concentrate was first separated by LC at a flow rate of 200 μL / min, and then mixed with methanol at a flow rate of 20 μL / min through a three-way valve and entered the MS to obtain the LC-MS spectrum of the control group;

[0112] Using the LC-MS spectra of the experimental group and the control group to make the volcano plots of the experimental group and the control group, as Figure 9 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 5-HT receptor modulator active molecules in the water body.

[0113] Figure 9 It is the volcano plot (FC > 2.0, p < 0.05) obtained after data processing of the LC-MS spectra of the hydroxycyclopeptide experimental group and the methanol control group in Example 1. From Figure 9 it can be seen that there are a total of 671 suspicious signals in the river water samples in the experimental group (on the right side of the volcano plot) and the control group (on the left side of the volcano plot). After using the hydroxycyclopeptide, 396 suspicious signals in the river water samples showed up-regulated signals, that is, the use of hydroxycyclopeptide reduced the suspicious signals in the river water samples to 396, indicating that hydroxycyclopeptide has high selectivity for 5-HT receptor modulator active molecules in the water body and is beneficial to improving the screening level of 5-HT receptor modulator active molecules in the water body.

[0114] By performing targeted secondary analysis on 396 suspicious signals with up-regulated signals and combining fragment ions and parent ions for mass spectrometry database retrieval, 2 types of 5-HT receptor modulator active molecules were successfully identified, as shown in the following table:

[0115] Table 5 LC-MS information of 2 types of 5-HT receptor modulator active molecules in river water samples

[0116]

[0117] As can be seen from Table 5, the river water samples contain 2 types of 5-HT receptor modulator active molecules, namely nicotine and tryptamine, indicating that the screening method of 5-HT receptor modulator active molecules in the water body of the present invention has high selectivity for 5-HT receptor modulator active molecules in the water body and can accurately and efficiently screen out 5-HT receptor modulator active molecules from complex matrix water bodies.

[0118] Example 2

[0119] A screening method for 5-HT receptor modulator active molecules in a water body in this example is only different from that in Example 1 in that a 50 μM / L hydroxyline peptide solution is used instead of a 50 μM / L hydroxycyclopeptide solution, and specifically includes the following steps:

[0120] S1. The river water samples collected on the same day were immediately filtered through a 0.22 μm PVDF membrane, stored at 4 °C in a refrigerator, and restored to room temperature in advance before solid-phase extraction.

[0121] First, rinse and activate the C18 solid-phase extraction column with 20 mL of methanol, balance the C18 solid-phase extraction column with 20 mL of pure water, then inject 500 mL of river water sample into the C18 solid-phase extraction 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.

[0122] S2. Use the hydroxyline peptide solution (with methanol as the solvent) as the experimental group and methanol as the control group.

[0123] First, separate 5 μL of the water body concentrate through LC at a flow rate of 200 μL / min, and then mix it with the hydroxyline peptide solution with a concentration of 50 μM / L at a flow rate of 20 μL / min through a three-way valve and enter MS to obtain the LC-MS spectrum of the experimental group.

[0124] 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 MS to obtain the LC-MS spectrum of the control group.

[0125] 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, 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 5-HT receptor modulator active molecules in the water body.

[0126] The volcano plots obtained after data processing of the LC-MS spectra of the hydroxyline peptide experimental group and the methanol control group in Example 2 are similar to Figure 9 and still can identify 2 types of 5-HT receptor modulator active molecules, nicotine and tryptamine, from the river water samples. That is, hydroxyline peptide also has high selectivity for 5-HT receptor modulator active molecules in the water body and can accurately and efficiently screen out 5-HT receptor modulator active molecules from complex matrix water bodies.

[0127] Example 3

[0128] A method for quantifying 5-HT receptor modulator active molecules in a water body in this example includes the following steps:

[0129] Using the screening method for 5-HT receptor modulator active molecules in the water body of Example 1, two types of 5-HT receptor modulator active molecules were identified in the water body (river water sample), namely nicotine and tryptamine. Then, a series of standard concentrations of nicotine and tryptamine were prepared respectively, and LC-MS (Agilent 1260 Infinity-AB Sciex Triple Quad TM 4500) was used to establish the standard concentration curve of tryptamine (the standard curve of peak area vs. concentration), and GC-MS (Agilent 7890B-5977B) was used to establish the standard concentration curve of nicotine (the standard curve of peak area vs. concentration). By calculation, the contents of these two types of 5-HT receptor modulator active molecules in the water body (river water sample) were obtained. The experimental results are shown in the following table:

[0130] Table 6 Quantitative results of two types of 5-HT receptor modulator active molecules in river water samples

[0131]

[0132] As can be seen from Table 6, by using the quantitative method for 5-HT receptor modulator active molecules in the water body of the present invention, the concentration of nicotine in the river water sample was measured to be 4.15 μg / L, and the concentration of tryptamine was 0.22 μg / L; it shows that the quantitative method for 5-HT receptor modulator active molecules in the water body of the present invention can be used for the quantitative analysis of 5-HT receptor modulator active molecules in water bodies with complex matrices.

[0133] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, rather than limiting 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 enumerate all the implementation manners here. Any modifications, equivalent replacements, and improvements 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 screening method for active molecules of 5-HT receptor modulators 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 hydroxy 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 5-HT receptor modulator active molecules in the water body; The hydroxy synthetic peptide described in step S2 is one of hydroxy cyclic peptide or hydroxy linear peptide; The hydroxy cyclic peptide has the molecular structure shown in formula 1: Formula 1; The hydroxy linear peptide has the molecular structure shown in formula 2: Formula 2; The 5-HT receptor modulator active molecule is one or more of nicotine, tryptamine, and 5-hydroxytryptamine.

2. The screening method according to claim 1, wherein The hydroxy synthetic peptide described in step S2 is hydroxy cyclic peptide.

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, wherein Step S1 specifically is to first rinse the solid-phase extraction column with an organic solvent, then inject the water body into the solid-phase extraction column, then elute with an organic solvent to obtain an eluate, dry it, then re-dissolve it with an organic solvent, and filter to obtain the concentrated water body solution.

5. The screening method according to claim 4, wherein 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 C18 solid-phase extraction cartridge, HLB solid-phase extraction column, C8 solid-phase extraction column, or SLC solid-phase extraction column.

7. The screening method according to claim 1, wherein The analysis of the concentrated water body solution and the hydroxy 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 the hydroxy 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 hydroxy synthetic peptide solution with a concentration of 20 - 50 μM / L entering the LC-MS is 10 - 25 μL / min.

9. A quantitative method for active molecules of 5-HT receptor modulators in water bodies, characterized in that, It includes the following steps: Use the screening method for 5-HT receptor modulator active molecules in the water body described in any one of claims 1 - 8 to identify the types of 5-HT receptor modulator active molecules in the water body, obtain the specific types of 5-HT receptor modulator active molecules, and then use LC-MS or GC-MS technology to establish a standard concentration curve for the specific types of 5-HT receptor modulator active molecules, and the content of the specific types of 5-HT receptor modulator active molecules in the water body can be obtained by calculation.

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