CN115524427B - Detection method of catecholamine hormones - Google Patents
Detection method of catecholamine hormones Download PDFInfo
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- CN115524427B CN115524427B CN202211496145.6A CN202211496145A CN115524427B CN 115524427 B CN115524427 B CN 115524427B CN 202211496145 A CN202211496145 A CN 202211496145A CN 115524427 B CN115524427 B CN 115524427B
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- functional monomer
- catecholamine
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- imprinted polymer
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- 238000000034 method Methods 0.000 claims abstract description 16
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- 206010020772 Hypertension Diseases 0.000 description 2
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F292/00—Macromolecular compounds obtained by polymerising monomers on to inorganic materials
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J9/00—Working-up of macromolecular substances to porous or cellular articles or materials; After-treatment thereof
- C08J9/26—Working-up of macromolecular substances to porous or cellular articles or materials; After-treatment thereof by elimination of a solid phase from a macromolecular composition or article, e.g. leaching out
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N30/08—Preparation using an enricher
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J2351/00—Characterised by the use of graft polymers in which the grafted component is obtained by reactions only involving carbon-to-carbon unsaturated bonds; Derivatives of such polymers
- C08J2351/10—Characterised by the use of graft polymers in which the grafted component is obtained by reactions only involving carbon-to-carbon unsaturated bonds; Derivatives of such polymers grafted on to inorganic materials
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Abstract
The invention relates to the technical field of synthesis and application of catechol amine molecularly imprinted polymer, and discloses a detection method of catechol amine hormone, which comprises the following steps: pretreating a sample to obtain a vacuum concentrated sample; performing specific binding, enrichment and separation operations on a vacuum concentrated sample by using magnetic nano microspheres coated with a catecholamine molecularly imprinted polymer shell layer, and performing liquid chromatography tandem mass spectrometry; the method for preparing the magnetic nano-microspheres comprises the following steps: the magnetic nano-microsphere coated with the catechol amine molecularly imprinted polymer shell layer is prepared by using a surface molecular imprinting technology, taking the magnetic nano-microsphere as a matrix material, catechol amine hormone as a template molecule and a functional monomer Mc containing alkenyl and boric acid functional groups as a functional monomer through covalent bonding of the functional monomer Mc and hydroxyl groups at the ortho positions of the template molecule through boron affinity reaction. The invention improves the specificity, sensitivity and detection flux of catecholamine hormone detection.
Description
Technical Field
The invention relates to the technical field of synthesis and application of catechol amine molecularly imprinted polymers, and particularly relates to a detection method of catechol amine hormones.
Background
Catecholamine is a nerve substance containing catechol and amine groups, and comprises various substances such as norepinephrine (E), epinephrine (NE) and Dopamine (DA), and the main physiological effects of the catecholamine are that alpha receptors of blood vessels are excited, blood vessels are contracted, the heart is excited, the heart rate is accelerated, and the systolic pressure is increased. Excessive catecholamine secretion may lead to hypertension and myocardial infarction. Methoxy adrenaline (MN), methoxy noradrenaline (NMN) and 3-methoxy tyramine (3-MT), which are metabolites of adrenaline, noradrenaline and dopamine, respectively. MN and NMN are called MNs together and are specific markers of pheochromocytoma and paraganglioma; 3-MT in the blood may be a marker for malignant metastasis of PPGLs.
Pheochromocytoma (PCC) and Paraganglioma (PGL) are tumors originating from adrenal medulla or extraadrenal sympathetic nerve chain, respectively, and mainly synthesize and secrete a large amount of catecholamine and its metabolites, causing a series of clinical symptoms such as blood pressure rise of patients. PCC and PGL are called PPGLs together, are one of important causes of secondary hypertension, and have important significance for clinically screening the causes of hypertension patients. At present, the screening index commonly used by the PPGLs in clinic is catecholamine and metabolites thereof.
The 'influence of hemolysis on the detection of catecholamine and its metabolites in plasma by liquid chromatography tandem mass spectrometry' (examination of expert consensus on diagnosis and treatment of pheochromocytoma and paraganglioma, vol.33, p.12, p.1103 in 12.8.12.2018.) discloses that the use of LC-MS/MS is recommended to determine MN and NMN levels, however, due to the low content of catecholamine and its metabolites in plasma, the traditional LC-MS/MS has the disadvantage of low sensitivity and even no measurement when measuring low-content components.
Disclosure of Invention
The invention provides a method for detecting catecholamine hormones, which aims to solve the technical problems that the sensitivity is low and even the determination cannot be carried out when LC-MS/MS determination is adopted due to the fact that the concentration of the catecholamine hormones in a sample to be determined is low.
A method for detecting catecholamine hormones, the method comprising the steps of:
firstly, preprocessing a sample to obtain a vacuum concentrated sample;
performing specific binding, enrichment and separation operations on a vacuum concentrated sample by using magnetic nano microspheres coated with a catechol amine molecularly imprinted polymer shell layer, and performing liquid chromatography tandem mass spectrometry;
the preparation method of the magnetic nano-microsphere comprises the following steps: the magnetic nano-microsphere coated with the catechol amine molecularly imprinted polymer shell layer is prepared by using a surface molecular imprinting technology, taking the magnetic nano-microsphere as a matrix material, catechol amine hormone as a template molecule and a functional monomer Mc containing alkenyl and boric acid functional groups as a functional monomer through covalent bonding of the functional monomer Mc and hydroxyl groups at the ortho positions of the template molecule through boron affinity reaction.
Preferably, the preparation method of the functional monomer Mc comprises: beta-bromophenylethane and bromine generate electrophilic substitution reaction on a benzene ring to generate a functional monomer Ma, the functional monomer Ma eliminates hydrogen bromide under the action of alkali to obtain a functional monomer Mb, and the functional monomer Mb firstly generates lithium-bromine exchange with n-butyl lithium and then reacts with boric acid ester to obtain the functional monomer Mc.
Preferably, the template molecule is one or a combination of several of dopamine, epinephrine and norepinephrine.
Preferably, the molar mass ratio of dopamine, epinephrine and norepinephrine in the template molecule is (0-10): (1-10): (0-10).
Preferably, the method detects epinephrine, norepinephrine, dopamine, homovanillic acid, vanillylmandelic acid in the biological sample.
Preferably, in the second step, the vacuum concentrated sample is taken and is subjected to constant volume by using a potassium dihydrogen phosphate buffer solution, the magnetic nano-microspheres coated with the catecholamine type molecularly imprinted polymer shell layer are added for oscillation adsorption, then the magnetic nano-microspheres are enriched under the action of an external magnetic field, then a mixed solvent composed of absolute ethyl alcohol, purified water and acetic acid is adopted as a desorption solution for oscillation desorption, and the mixture is dried by nitrogen and then redissolved by using an initial mobile phase.
Preferably, in the second step, the volume ratio of the absolute ethyl alcohol to the purified water to the acetic acid is (6-10): (0.5-2): (0.5-2).
Compared with the prior art, the invention has the following beneficial technical effects:
the invention utilizes a surface molecular imprinting technology, takes magnetic nano-microspheres as a matrix material, takes catecholamine hormones (dopamine, adrenaline and noradrenaline) as a template molecule, and takes a functional monomer Mc containing alkenyl and boric acid functional groups as a functional monomer, the functional monomer Mc and the ortho hydroxyl of the catecholamine hormones of the template molecule are covalently combined through boron affinity reaction to prepare the magnetic nano-microspheres coated with the shells of the catecholamine molecular imprinting polymer, the catecholamine molecular imprinting polymer has binding sites which are complementary to the template molecule in size, structure and functional groups, can specifically bind the dopamine, adrenaline and noradrenaline in the catecholamine hormones, is used for high-selection and rapid enrichment and separation of the catecholamine hormones in samples, and improves the specificity, sensitivity and detection flux of the catecholamine hormones.
Drawings
FIG. 1 is a chemical structural formula of a functional monomer Ma;
FIG. 2 is a chemical structural formula of a functional monomer Mb;
FIG. 3 is a chemical structural formula of the functional monomer Mc.
Detailed Description
Example 1:
a method for detecting catecholamine hormones, the method comprising the steps of:
step one, pretreating a sample to obtain a vacuum concentrated sample, and the specific process comprises the following steps:
step1-1, taking 250 mu L of a plasma sample to be detected by using a 0.05 percent VC acetonitrile solution as a substitute substrate for the standard curve, and adding 750 mu L of diluted isotope internal standard working solution;
step1-2, firstly, vortexing the sample for 5min, and then centrifuging the sample for 15min at the temperature of 4 ℃ and the rotating speed of 14000rpm to obtain supernatant;
step1-3, putting 700 mu L of supernatant into a vacuum concentrator for concentration for 3.5 hours to obtain a vacuum concentrated sample;
step two, performing specific binding and enrichment separation operation on the vacuum concentrated sample by using the magnetic nano microspheres coated with the catecholamine molecularly imprinted polymer shell layer, wherein the specific implementation steps are as follows:
step2-1, taking the vacuum concentrated sample prepared in step1-3, diluting to 10.00mL with 20mmol/L potassium dihydrogen phosphate buffer solution, adding 10mg of magnetic nano microspheres coated with a catecholamine molecularly imprinted polymer shell layer, oscillating and adsorbing for 1h, and enriching the magnetic nano microspheres under the action of an external magnetic field;
step2-2, adopting absolute ethyl alcohol, purified water and acetic acid according to the volume ratio of 8:1: the mixed solvent composed of 1 is used as desorption solution, is vibrated and desorbed for 2h, and is re-dissolved to 100 mu L by using an initial mobile phase after being dried by nitrogen;
step2-3, taking 20 mu L of the mixture to carry out liquid chromatography tandem mass spectrometry, wherein the used analytical instruments and experimental parameters are as follows:
(1) Performing analysis and detection by using an Shimadzu LC-30A series 8050 instrument;
(2) The chromatographic column was Waters BEHC18 (2.1 × 50mm), the mobile phase aqueous phase was an aqueous solution containing 20mM ammonium acetate and 0.1% formic acid, the organic phase was acetonitrile, gradient elution was performed for 9min;
(3) Mass spectrometry was performed in electrospray ionization (ESI) mode, positive ion scan, wherein ion pairs are shown in table 1 below;
(4) The linear range of the measurements is shown in table 2 below.
TABLE 1 ion to proton/nucleus ratios for the respective components of catecholamine hormones
TABLE 2 detection Range of the content of each component in catecholamine hormones
Example 2:
the preparation method of the magnetic nano-microsphere coated with the catecholamine molecularly imprinted polymer shell layer comprises the following steps:
step one, synthesizing a functional monomer Mc containing alkenyl and boric acid functional groups, which comprises the following steps:
step1-1, functionSynthesis of monomer Ma: the functional monomer Ma is generated by electrophilic substitution reaction of beta-bromophenylethane and bromine on a benzene ring, and the specific synthetic steps are as follows: in a four-neck flask reactor, 555mg of beta-bromophenylethane and 20mg of antimony trioxide (Sb) are weighed 2 O 3 ) Stirring for 0.5h, keeping the temperature of the reaction system at 10 ℃, and dropwise adding 1.91g of bromine (Br) in 4h 2 ) Adding into a reactor, reacting at 20 deg.C for 10h, adding sulfuric acid solution to stop reaction, washing with EDTA and sodium sulfite solution, and washing to obtain functional monomer Ma with chemical structural formula shown in FIG. 1; the nuclear magnetic resonance spectrum characterization result of the functional monomer Ma is as follows: 1 H NMR(300MHz，DMSO)，δ：7.95(s，2H)，3.63(t，2H)，3.05(t，2H)； 13 c NMR (75MHz, DMSO), delta: 127.1 (s, 2C), 116.6 (s, C), 141.1 (s, C), 132.2 (d, 2 CH), 30.2 (t, CH) 2 )，29.6(t，CH 2 )；
Step1-2, synthesis of a functional monomer Mb: eliminating hydrogen bromide by using the functional monomer Ma under the action of alkali to obtain a functional monomer Mb, wherein the specific synthesis steps are as follows: adding 423mg of functional monomer Ma into a four-neck flask, then adding 6mg of tetramethylammonium bromide and 100mL of absolute ethyl alcohol, keeping the temperature of a reaction system at 30 ℃, dropwise adding 50g of 40% sodium hydroxide solution into the reactor within 4h, keeping the temperature for 4h after dropwise adding, dissolving a reaction product in n-hexane, adding deionized water for washing, drying with magnesium sulfate, and distilling under reduced pressure to obtain functional monomer Mb, wherein the chemical structural formula of the functional monomer Mb is shown in figure 2; the result of the nuclear magnetic resonance spectrum characterization of the functional monomer Mb is as follows: 1 H NMR(300MHz，DMSO)，δ：7.87(s，2H)，7.37(dd，1H)，5.72(dd，1H)，5.60(dd，1H)； 13 c NMR (75MHz, DMSO), delta: 123.2 (s, 2C), 118.6 (s, C), 140.5 (s, C), 133.6 (d, 2 CH), 134.5 (d, CH), 114.3 (t, CH) 2 )；
Step1-3, synthesis of a functional monomer Mc: firstly, lithium bromine exchange is carried out on a functional monomer Mb and n-butyllithium, and then the functional monomer Mc is prepared by reacting with boric acid ester, wherein the specific synthesis steps are as follows: 255mg of tetramethylethylenediamine and 20mL of anhydrous ether are stirred and cooled to-10 ℃, and 140mg of the solution is dropwise added in 0.5hCooling 9.6mL of anhydrous diethyl ether of n-butyllithium to-60 ℃, dropwise adding 50mL of anhydrous diethyl ether dissolved with 337mg of functional monomer Mb, keeping the reaction at-60 ℃ for 0.5h, dropwise adding 150mL of anhydrous diethyl ether dissolved with 1.35g of triisopropyl borate within 20min, heating to 25 ℃, reacting at 25 ℃ for 1h, quenching the reaction by adopting 4% sodium hydroxide, adjusting the pH to 6, extracting, and drying to obtain a functional monomer Mc, wherein the chemical structural formula of the functional monomer Mc is shown in figure 3; the nuclear magnetic resonance spectrum characterization result of the functional monomer Mc is as follows: 1 H NMR(300MHz，DMSO)，δ：7.28(s，2H)，7.37(dd，1H)，5.72(dd，1H)，5.60(dd，1H)，4.2(s，6H)； 13 c NMR (75MHz, DMSO), delta: 134.0 (s, C), 136.2 (s, 2C), 130.7 (s, C), 128.6 (d, 2 CH), 134.5 (d, CH), 114.3 (t, CH) 2 )；
Step two, synthesizing a templated functional monomer, which comprises the following specific steps: adding 233mg of functional monomer Mc, 189mg of dopamine hydrochloride, 220mg of epinephrine hydrochloride and 206mg of norepinephrine hydrochloride into a mixed solution consisting of 9.5mL of methanol and 0.5mL of piperidine, adding 5g of molecular sieve, stirring and reacting for 24 hours at room temperature, and carrying out reduced pressure distillation to obtain a templated functional monomer;
step three, synthesizing alkenyl ferroferric oxide magnetic nano microspheres: adding 270mg of ferric trichloride hexahydrate and 80mg of sodium citrate dihydrate into 20mL of ethylene glycol, stirring for reaction for 1h, adding 480mg of sodium acetate, continuously stirring for reaction at room temperature until a brownish red colloid is generated, then reacting for 20h at 200 ℃, adding 10mL of absolute ethyl alcohol dissolved with 2mL of vinyl triethoxysilane, then adding 0.8mL of acetic acid buffer solution, vacuumizing for 1h, washing with absolute ethyl alcohol, and vacuum-drying at 60 ℃ to constant weight to obtain the alkenyl ferroferric oxide magnetic nano microsphere;
step four, synthesizing the magnetic nano-microspheres IM coated with the catecholamine molecularly imprinted polymer shell layer: dissolving 18mg of templated functional monomer, 388mg of ethylene glycol dimethacrylate and 33mg of azobisisobutyronitrile into 15mLN, N-dimethylformamide, ultrasonically dispersing for 10min, adding 100mg of alkenylated ferroferric oxide magnetic nano-microspheres, vacuumizing for 1h, reacting for 12h at 60 ℃, and reacting by adopting absolute ethyl alcohol, purified water and acetic acid according to a volume ratio of 8:1:1, and washing the mixed solvent to remove the template molecules, and drying the mixed solvent at 50 ℃ in vacuum to constant weight to obtain the magnetic nanoparticle IM coated with the catecholamine molecularly imprinted polymer shell.
Example 3:
the preparation method of the magnetic nano-microsphere without the catecholamine molecular engram in the polymer shell layer comprises the following steps: dissolving 10mg of the functional monomer Mc, 388mg of ethylene glycol dimethacrylate and 33mg of azobisisobutyronitrile prepared in the first step of the embodiment 2 in 15mLN, N-dimethylformamide, ultrasonically dispersing for 10min, adding 100mg of the alkenylated ferroferric oxide magnetic nanosphere prepared in the third step of the embodiment 2, vacuumizing for 1h, reacting for 12h at 60 ℃, and vacuum drying at 50 ℃ to constant weight to obtain the catechol amine molecularly imprinted-free magnetic nanosphere which is a non-imprinted polymer shell magnetic nanosphere NIM;
wherein, the preparation raw material of the magnetic nano microsphere NIM is a functional monomer Mc;
the preparation raw material of the magnetic nanoparticle IM is a templated functional monomer, and the templated functional monomer is prepared by covalently bonding a functional monomer Mc and catecholamine molecules (dopamine hydrochloride, epinephrine hydrochloride and norepinephrine hydrochloride) through boron affinity reaction.
Example 4:
in order to investigate the adsorption behavior of the magnetic nano-microsphere coated with the catecholamine molecularly imprinted polymer shell layer, an adsorption experiment is carried out on the magnetic nano-microsphere, and the specific experimental process is as follows:
step one, preparing catecholamine solution with the concentration of 0, 50, 100, 150, 200 and 250 μmol/L (the molar mass ratio of dopamine hydrochloride, epinephrine hydrochloride and norepinephrine hydrochloride is 1;
weighing six equal parts of the magnetic nano microspheres IM coated with the catecholamine molecularly imprinted polymer shell layer in the embodiment 2, wherein each part is 10mg in mass;
measuring catecholamine solutions with the concentrations of 0, 50, 100, 150, 200 and 250 mu mol/L, wherein the volume of each concentration is 5mL;
putting a magnetic nano microsphere IM into the catecholamine solution with each concentration, oscillating and adsorbing for 1h at the rotating speed of 200r/min at room temperature, measuring the concentration change of the catecholamine in the solution, and calculating the adsorption amount of the magnetic nano microsphere IM to the catecholamine;
weighing six equal parts of the magnetic nano microspheres NIM with the non-imprinted polymer shell layer in the embodiment 3, wherein each part is 10mg in mass;
measuring catecholamine solutions with the concentrations of 0, 50, 100, 150, 200 and 250 mu mol/L, wherein the volume of each concentration is 5mL;
putting a magnetic nanoparticle NIM into the catecholamine solution with each concentration, oscillating and adsorbing for 1h at the rotation speed of 200r/min at room temperature, measuring the concentration change of the catecholamine in the solution, and calculating the adsorption amount of the magnetic nanoparticle NIM on the catecholamine;
wherein, the calculation formula of the catecholamine adsorption quantity Q is as follows:
Q=[(C 0 -C e )×V]/W
in the formula, C 0 The concentration of catecholamine in the initial solution is expressed in μmol/L;
C e the concentration of catecholamine in the equilibrium solution is expressed in the unit of mu mol/L;
v is the initial volume of the adsorption solution in L;
w is the mass of the magnetic nano microsphere, and the unit is g.
Example 5:
in order to investigate the specific selectivity of the magnetic nano-microsphere coated with the catecholamine molecularly imprinted polymer shell, pyrocatechol and ractopamine are selected as references to carry out a selectivity experiment, and the specific experimental process is as follows:
firstly, preparing catechol solution with the concentration of 0, 50, 100, 150, 200 and 250 mu mol/L, and then measuring the solution, wherein the volume of each concentration is 5mL;
weighing six equal parts of the magnetic nano microspheres IM coated with the catecholamine molecularly imprinted polymer shell layer in the embodiment 2, wherein each part is 10mg in mass;
putting a magnetic nano microsphere IM into the catechol solution with each concentration, oscillating and adsorbing for 1h at the rotating speed of 200r/min at room temperature, measuring the change of the catechol concentration in the solution, and calculating the adsorption amount of the magnetic nano microsphere IM on catechol;
step two, measuring catechol solutions with the concentrations of 0, 50, 100, 150, 200 and 250 mu mol/L, wherein the volume of each concentration is 5mL;
weighing six equal parts of the magnetic nanospheres NIM with the non-imprinted polymer shell layer in the embodiment 3, wherein the mass of each part is 10mg;
putting a magnetic nano microsphere NIM into the catechol solution with each concentration, oscillating and adsorbing for 1h at the rotation speed of 200r/min at room temperature, measuring the change of the catechol concentration in the solution, and calculating the adsorption amount of the magnetic nano microsphere NIM on catechol;
step three, firstly preparing ractopamine solution with the concentration of 0, 50, 100, 150, 200 and 250 mu mol/L, and then measuring the solution, wherein the volume of each concentration is 5mL;
weighing six equal parts of the magnetic nano microspheres IM coated with the catecholamine molecularly imprinted polymer shell layer in the embodiment 2, wherein each part is 10mg in mass;
putting a magnetic nano microsphere IM into the ractopamine solution with each concentration, oscillating and adsorbing for 1h at the room temperature at the rotating speed of 200r/min, measuring the change of the ractopamine concentration in the solution, and calculating the adsorption amount of the magnetic nano microsphere IM on ractopamine;
measuring ractopamine solutions with the concentrations of 0, 50, 100, 150, 200 and 250 mu mol/L, wherein the volume of each concentration is 5mL;
putting a magnetic nano microsphere NIM into the ractopamine solution with each concentration, oscillating and adsorbing for 1h at the rotating speed of 200r/min at room temperature, measuring the change of the ractopamine concentration in the solution, and calculating the adsorption amount of the magnetic nano microsphere NIM on ractopamine;
wherein, blotting factor IF (IF = Q) is adopted IM /Q NIM ，Q IM For a target object, namely magnetic nano microspheres IM coated with a catecholamine molecularly imprinted polymer shellAmount of adsorption, Q NIM The adsorption capacity of the magnetic nanosphere NIM which is a non-imprinted polymer shell layer to the target substance) and the selection coefficient SC (SC = IF) t /IF c ，IF t As an imprinting factor, IF, of catecholamines template molecules c As an imprinting factor of a reference substance) to evaluate the specific selectivity of the magnetic nanospheres IM coated with the catecholamine molecularly imprinted polymer shell layer and the magnetic nanospheres NIM of the non-imprinted polymer shell layer to the catecholamine and the reference substance thereof.
Example 6:
in example 4, the adsorption amounts and the imprinting factors of the magnetic nanospheres IM and the magnetic nanospheres NIM in the catecholamine solution are shown in table 3 below.
TABLE 3 adsorption experiment results of magnetic nanoparticles IM and NIM in catecholamine solution
As can be seen from table 3, the molecular imprinting cavities of the magnetic nanoparticle IM coated with the catecholamine molecular imprinting polymer shell are distributed on the surface of the magnetic nanoparticle IM, so that the mass transfer resistance is low, and the target molecules of the catecholamine molecules can rapidly enter the cavities, thereby achieving a good adsorption effect.
The magnetic nano-microsphere NIM without the imprinted polymer shell layer contains boron hydroxyl which can selectively identify dopamine molecules containing ortho-hydroxyl, but the adsorption capacity of the magnetic nano-microsphere NIM to catecholamine molecules is not as good as that of the magnetic nano-microsphere IM coated with the catecholamine molecularly imprinted polymer shell layer due to the lack of holes matched with the structure of the dopamine molecules.
Example 7:
in example 5, the adsorption amounts and imprinting factors of the magnetic nanospheres IM and the magnetic nanospheres NIM in the catechol solution are shown in table 4 below.
TABLE 4 adsorption test results of magnetic nanoparticles IM and NIM in catechol solution
Example 8:
in example 5, the adsorption amounts and the imprinting factors of the magnetic nanospheres IM and the magnetic nanospheres NIM in the ractopamine solution are shown in table 5 below.
TABLE 5 adsorption test results of magnetic nanoparticles IM and NIM in ractopamine solution
As can be seen from tables 3, 4 and 5, the adsorption capacity of the magnetic nanoparticle IM coated with the catecholamine molecularly imprinted polymer shell to catechol that is not a similar substance but contains ortho-hydroxyl is equivalent to that of the catecholamine molecules, but the adsorption effect to ractopamine that does not contain ortho-hydroxyl is poor, which indicates that the selectivity of the magnetic nanoparticle IM coated with the catecholamine molecularly imprinted polymer shell to catecholamine hormones and structural analogues thereof is mainly the covalent condensation of the ortho-hydroxyl of the analyte and the functional monomer Mc containing alkenyl and boronic acid functional groups, while the adsorption selectivity of the magnetic nanoparticle IM coated with the catecholamine molecularly imprinted polymer shell to the catecholamine hormones and structural analogues thereof is poor because the magnetic nanoparticle NIM coated with the catecholamine and the non-imprinted polymer shell does not have such an action site, so the adsorption selectivity is poor.
Claims (6)
1. A detection method of catecholamine hormones is characterized by comprising the following steps of:
step one, pretreating a sample to obtain a vacuum concentrated sample;
performing specific binding, enrichment and separation operation on a vacuum concentrated sample by using ferroferric oxide magnetic nano microspheres coated with catecholamine molecular imprinting polymer shells, and then performing liquid chromatography tandem mass spectrometry;
the preparation method of the magnetic nano-microsphere comprises the following steps: by utilizing a surface molecular imprinting technology, taking ferroferric oxide magnetic nano microspheres as a matrix material, catechol amine hormone as a template molecule, and a functional monomer Mc containing alkenyl and boric acid functional groups as a functional monomer, and covalently combining the functional monomer Mc and hydroxyl groups at the ortho positions of the template molecule through boron affinity reaction to prepare the ferroferric oxide magnetic nano microspheres coated with the catechol amine molecular imprinting polymer shell;
the preparation method of the functional monomer Mc comprises the following steps: performing electrophilic substitution reaction on beta-bromophenylethane and bromine on a benzene ring to generate a functional monomer Ma, eliminating hydrogen bromide of the functional monomer Ma under the action of alkali to obtain a functional monomer Mb, performing lithium-bromine exchange on the functional monomer Mb and n-butyllithium, and reacting the functional monomer Mb with boric acid ester to obtain the functional monomer Mc;
the chemical structural formula of the functional monomer Ma is
；
The chemical structural formula of the functional monomer Mb is
；
The chemical structural formula of the functional monomer Mc is shown as
。
2. The method for detecting catecholamine hormones as claimed in claim 1, wherein the template molecule is one or a combination of dopamine, epinephrine and norepinephrine.
3. The method for detecting catecholamine hormones according to claim 2, wherein the molar mass ratio of dopamine, epinephrine and norepinephrine in the template molecule is (0-10): (1-10): (0-10).
4. The method according to claim 1, wherein the method can detect epinephrine, norepinephrine, metaepinephrine, norepinephrine, dopamine, homovanillic acid, and vanillylmandelic acid in a biological sample.
5. The method for detecting catecholamine hormones as claimed in claim 1, wherein in the second step, the vacuum concentrated sample is taken and is subjected to constant volume with potassium dihydrogen phosphate buffer solution, the magnetic nano-microspheres coated with the catecholamine molecularly imprinted polymer shell layer are added for oscillation and adsorption, then under the action of an external magnetic field, the magnetic nano-microspheres are enriched, then a mixed solvent composed of absolute ethyl alcohol, purified water and acetic acid is adopted as desorption solution for oscillation and desorption, and the mixed solvent is dried by nitrogen and then re-dissolved by using an initial mobile phase.
6. The method for detecting catecholamine hormones as claimed in claim 5, wherein in the second step, the volume ratio of absolute ethyl alcohol, purified water and acetic acid is (6-10): (0.5-2): (0.5-2).
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