A kit and detection method for synchronous detection of catecholamines and their metabolites

Abstract

The present invention provides a kit and a detection method for synchronously detecting catecholamines and their metabolites, which relates to the technical field of analytical detection. The kit contains a diluent, a solid-phase extraction reagent, an LC-MS / MS detection reagent, a solid-phase extraction device, and a solid-phase extraction packing material. Among them, the solid-phase extraction packing material creatively adopts zwitterionic exchange microspheres containing both carboxyl and quaternary ammonium groups. The kit can not only synchronously and efficiently separate and detect 8 kinds of catecholamines and their metabolites in urine samples, but also separate and detect 6 kinds of amino-containing catecholamines and their metabolites in blood samples. When the above kit is used to separate and detect catecholamines and their metabolites, the extraction recovery rate is relatively high, and the linear range covers the reference value range of normal detection, which can significantly shorten the detection time of urine samples, improve the detection efficiency of mass spectrometry, and is conducive to popularization and application in clinical detection.

Description

Technical Field

[0001] The present invention relates to the technical field of analytical detection, and particularly relates to a kit and a detection method for synchronously detecting catecholamines and their metabolites. Background Art

[0002] Catecholamine CA is a class of nerve substances containing catechol and amino groups, which are important neurotransmitters and hormones in the human body and have strong physiological activities. The detection of catecholamine and metabolite concentrations mainly measures epinephrine E, norepinephrine NE, dopamine DA, and their intermediate metabolites metanephrine MN, normetanephrine NMN, 3-methoxytyramine 3-MT, and the terminal metabolites vanillylmandelic acid VMA, homovanillic acid HVA. Catecholamines and their metabolites have large polarities, similar structures, low contents and large interferences in biological samples, making the detection difficult. Liquid chromatography-tandem mass spectrometry LC-MS / MS has become the main method for detecting CA and metabolites due to its high sensitivity, strong specificity and ability to rapidly screen a large number of samples, and is currently recommended by guidelines and consensuses.

[0003] For liquid chromatography-tandem mass spectrometry, a suitable pretreatment method is crucial. Since the concentrations of catecholamines and their metabolites in plasma are extremely low, solid-phase extraction is usually used for sample concentration during pretreatment. The core of solid-phase extraction technology is the packing material. Selecting an SPE packing material with moderate adsorption to the target substance is the premise to ensure accurate detection. Catecholamines and their metabolites all belong to monoamine substances and have amino groups, while the terminal metabolites vanillylmandelic acid and homovanillic acid of catecholamines in urine have strong carboxyl groups. Currently, commercially available solid-phase extraction packings for catecholamines can only adsorb a class of compounds with the same properties. At least two different types of kits are required for pretreatment to completely measure 8 catecholamines and their metabolites on the market, which is time-consuming and laborious. There is no suitable kit on the market that can perform one-time separation and extraction of 8 catecholamines and their metabolites, which severely restricts the efficiency of detecting catecholamines and their metabolites by liquid chromatography-tandem mass spectrometry. Therefore, it has very important application value to develop a kit that can not only synchronously separate and detect 8 catecholamines and their metabolites in urine at one time, but also detect 6 catecholamines and their metabolites in blood. Summary of the Invention

[0004] The purpose of the present invention is to provide a kit and a detection method for synchronously detecting catecholamines and their metabolites, so as to solve the technical problems of low detection efficiency and low accuracy of catecholamines and their metabolites in urine samples in the prior art.

[0005] To solve the above technical problems, a kit for synchronously detecting catecholamines and their metabolites provided by the present invention contains a diluent, a solid-phase extraction reagent, an LC-MS / MS detection reagent, a solid-phase extraction device, and a solid-phase extraction packing material;

[0006] The solid-phase extraction packing material is zwitterionic exchange microspheres;

[0007] The zwitterionic exchange microspheres contain carboxyl functional groups and quaternary ammonium functional groups;

[0008] Because the zwitterionic exchange microspheres contain both carboxyl functional groups and quaternary ammonium functional groups, they have the performance of simultaneously separating acidic analytes and basic analytes, and can simultaneously separate six basic catecholamines and their metabolites and two terminal metabolites of acidic catecholamines contained in urine at one time, which is beneficial to simplifying the separation and detection steps and improving the detection efficiency;

[0009] The solid-phase extraction reagent includes an activation solution, a balance solution, a washing solution, an elution solution, and a reconstitution solution;

[0010] The LC-MS / MS detection reagent includes a standard product, a quality control product, an internal standard working solution, and a mobile phase.

[0011] Furthermore, the carboxyl functional group density of the zwitterionic exchange microspheres is 1 mmol / g - 5 mmol / g; the quaternary ammonium functional group density is 0.5 mmol / g - 2 mmol / g; the pore size is 1 nm - 50 nm; the particle size is 10 μm - 300 μm, preferably 30 μm - 60 μm;

[0012] By selecting zwitterionic exchange microspheres with appropriate carboxyl functional group density, quaternary ammonium functional group density, pore size, and particle size, the detection sensitivity of the kit can be more targeted improved. Since the concentration range of catecholamines and their metabolites in urine is usually in the μg level, while the concentration range of catecholamines and their metabolites in blood is generally lower, only in the pg level, selecting appropriate zwitterionic exchange microspheres as the solid-phase extraction packing material is of more important significance for improving the detection sensitivity of blood samples.

[0013] Furthermore, the preparation method of the zwitterionic exchange microspheres is as follows:

[0014] Step M1, adding a dispersant to distilled water and stirring well to obtain a dispersant aqueous solution with a concentration of 0.1% - 20%; preferably 3% - 10%;

[0015] The dispersant is hydroxypropyl methylcellulose, and / or polyvinyl alcohol;

[0016] Step M2, weighing 50-90 parts of the polymer monomer mixture, 1-90 parts of the porogen, and 0.003-1 parts of the initiator according to their mass fractions, and stirring them thoroughly to obtain 100 parts of the oil phase mixture;

[0017] The polymer monomer mixture includes divinylbenzene and chlorine-containing aromatic compounds; the mass ratio of divinylbenzene to the chlorine-containing aromatic compound is 1:(1-3);

[0018] The chlorine-containing aromatic compound is selected from p-chloromethylstyrene and 4-chlorostyrene; the introduction of the chlorine-containing aromatic compound can subsequently undergo a grafting reaction with a tertiary amine monomer, thereby providing active sites for smoothly grafting quaternary ammonium functional groups on the microspheres.

[0019] The porogen is a mixture of one or more of dichloromethane, n-dodecane, toluene, o-dichlorobenzene, and liquid paraffin;

[0020] The initiator is azobisisobutyronitrile and / or benzoyl peroxide;

[0021] Appropriate addition of porogens and initiators during the preparation of the zwitterionic ion exchange microspheres is beneficial to adjusting the pore size and particle size of the microspheres to meet the needs of different tests.

[0022] Step M3, fully mixing the acrylic acid ester monomer with the oil phase mixture and the dispersant aqueous solution, and reacting under condensation reflux at 50-80° C. for 4-20 hours; after the reaction, cooling to room temperature, washing with water and ethanol in turn, and drying at 60° C. for 10-12 hours to obtain a first reaction product;

[0023] The acrylic acid ester monomer is selected from methyl acrylate, methyl methacrylate, and ethyl methacrylate;

[0024] The added mass of the acrylic ester monomer is 1%-20% of the polymer monomer mixture;

[0025] The mass ratio of the oil phase mixture to the dispersant aqueous solution is (30-60):100;

[0026] Step M4, taking the first reaction product and placing it in a toluene solution to swell for 0.5-1.5 hours to obtain a swollen mixture; adding a tertiary amine monomer to the swollen mixture, and continuing to stir and react at 80-90° C. for 4-20 hours. After the reaction is completed, a second reaction product is obtained;

[0027] The tertiary amine monomer is selected from N,N-dimethylbutylamine, trimethylamine, and triethylamine;

[0028] The amount of the tertiary amine monomer added is 1-10 times the mass of the chlorine-containing aromatic compound;

[0029] Step M5, add an alkaline methanol solution to the second reaction product, stir at a low speed at 65 - 75 °C, and carry out an ester hydrolysis reaction for 20 - 30 h; after cooling to room temperature, wash successively with ethanol, sodium bicarbonate solution, and water, and then dry to obtain the zwitterionic exchange microsphere adsorption material.

[0030] Further, the diluent is a blood test diluent and / or a urine test diluent;

[0031] The blood test diluent is a 10% - 15% methanol solution;

[0032] The urine test diluent is a 0.02 mol / L sodium carbonate - sodium bicarbonate solution; preferably, the mass ratio of sodium carbonate to sodium bicarbonate is (1 - 3):(7 - 9).

[0033] Further, the activation solution is a 0 - 50% methanol solution;

[0034] The equilibration solution is deionized water;

[0035] The eluent includes a first eluent and a second eluent; the first eluent is deionized water; the second eluent is selected from methanol and acetonitrile;

[0036] The elution solution is a methanol solution containing 0.1% - 4% formic acid;

[0037] The reconstitution solution is deionized water;

[0038] The standard is a blood standard and / or a urine standard;

[0039] The quality control product is a blood quality control product and / or a urine quality control product;

[0040] The internal standard working solution is a blood internal standard working solution and / or a urine internal standard working solution;

[0041] The mobile phase includes mobile phase A and mobile phase B; mobile phase A is a 0.1% formic acid aqueous solution; mobile phase B is methanol.

[0042] Further, both the blood standard and the blood quality control product include adrenaline, noradrenaline, dopamine, metanephrine, normetanephrine, 3 - methoxytyramine, and the balance is a blood simulation matrix solution;

[0043] The standard curve concentration ranges of the component compounds in the blood standard product are as follows: epinephrine: 23.3 pg / ml - 1800 pg / ml, norepinephrine: 58.31 pg / ml - 4500 pg / ml, dopamine: 17.5 pg / ml - 1350 pg / ml, metanephrine: 11.67 pg / ml - 900 pg / ml, normetanephrine: 23.3 pg / ml - 1800 pg / ml, 3-methoxytyramine: 11.67 pg / ml - 900 pg / ml;

[0044] The blood quality control product includes a low blood quality control product and a high blood quality control product;

[0045] The target concentrations of the component compounds in the low blood quality control product are as follows: epinephrine: 120 pg / ml, norepinephrine: 300 pg / ml, dopamine: 90 pg / ml, metanephrine: 60 pg / ml, normetanephrine: 120 pg / ml, 3-methoxytyramine: 60 pg / ml;

[0046] The target concentrations of the component compounds in the high blood quality control product are as follows: epinephrine: 900 pg / ml, norepinephrine: 2250 pg / ml, dopamine: 675 pg / ml, metanephrine: 450 pg / ml, normetanephrine: 900 pg / ml, 3-methoxytyramine: 450 pg / ml;

[0047] The blood internal standard working solution includes epinephrine-d6: 40 ng / ml, norepinephrine-d6: 100 ng / ml, dopamine-d4: 40 ng / ml, metanephrine-d3: 20 ng / ml, normetanephrine-d3: 40 ng / ml, 3-methoxytyramine-d4: 20 ng / ml;

[0048] The blood simulated matrix solution includes a 4% bovine serum albumin solution and 0.1% - 0.5% ascorbic acid;

[0049] Preferably, the concentration of ascorbic acid in the blood simulated matrix solution is 0.3% - 0.5%.

[0050] Furthermore, both the urine standard product and the urine quality control product include epinephrine, norepinephrine, dopamine, metanephrine, normetanephrine, 3-methoxytyramine, homovanillic acid HVA, and vanillylmandelic acid VMA, and the balance is a simulated urine matrix solution;

[0051] The standard curve concentration ranges of the component compounds in the urine standard are as follows: epinephrine: 0.4 ng / ml - 80 ng / ml, norepinephrine: 6 ng / ml - 1200 ng / ml, dopamine: 10 ng / ml - 2000 ng / ml, metanephrine: 0.5 ng / ml - 100 ng / ml, normetanephrine: 0.8 ng / ml - 160 ng / ml, 3-methoxytyramine: 0.8 ng / ml - 160 ng / ml, homovanillic acid: 125 ng / ml - 25000 ng / ml, vanillylmandelic acid: 125 ng / ml - 25000 ng / ml;

[0052] The urine quality control products include urine low quality control products and urine high quality control products;

[0053] The target concentrations of the component compounds in the urine low quality control product are as follows: epinephrine: 4 ng / ml, norepinephrine: 60 ng / ml, dopamine: 100 ng / ml, metanephrine: 5 ng / ml, normetanephrine: 8 ng / ml, 3-methoxytyramine: 8 ng / ml, homovanillic acid: 1250 ng / ml, vanillylmandelic acid: 1250 ng / ml;

[0054] The target concentrations of the component compounds in the urine high quality control product are as follows: epinephrine: 32 ng / ml, norepinephrine: 480 ng / ml, dopamine: 800 ng / ml, metanephrine: 40 ng / ml, normetanephrine: 64 ng / ml, 3-methoxytyramine: 64 ng / ml, homovanillic acid: 10000 ng / ml, vanillylmandelic acid: 10000 ng / ml;

[0055] The urine internal standard working solution includes epinephrine-d6: 100 ng / ml, norepinephrine-d6: 1000 ng / ml, dopamine-d4: 1500 ng / ml, metanephrine-d3: 100 ng / ml, normetanephrine-d3: 150 ng / ml, 3-methoxytyramine-d4: 150 ng / ml, homovanillic acid-d5: 20000 ng / ml, vanillylmandelic acid-d3: 20000 ng / ml;

[0056] The simulated urine matrix solution is an artificial urine solution containing 0.1% - 0.5% acetic acid.

[0057] On the other hand, the present invention provides a synchronous detection method for catecholamines and their metabolites, and the detection method uses the above-mentioned kit.

[0058] Furthermore, as Figure 1 shown, the specific steps of the detection method are as follows:

[0059] Step 1: Take a solid-phase extraction device filled with zwitterionic exchange microspheres, add an activation solution for activation; then add the equilibration solution and press dry to obtain an activated solid-phase extraction device.

[0060] Step 2: Synchronously pipette 200 μL each of the standard, quality control product, and sample to be tested, and sequentially add 20 μL of the internal standard working solution and 400 μL of the dilution solution, and vortex to mix evenly to obtain the standard working solution, quality control working solution, and sample solution to be tested.

[0061] Step 3: Synchronously transfer the standard working solution, quality control working solution, and sample solution to be tested into the solid-phase extraction device, and press dry; then sequentially add the first eluent and the second eluent, and press dry; then add the elution solution, collect the eluate, and press dry.

[0062] Dry the eluates of each group under nitrogen, add 100 μL of the reconstitution solution for reconstitution to obtain a concentrated detection solution.

[0063] Step 4: Take 10 μL of the concentrated detection solution and perform high-performance liquid chromatography-tandem mass spectrometry analysis.

[0064] Further, the loading amount of the zwitterionic exchange microspheres in Step 1 is 2 mg - 8 mg, preferably 3 mg - 5 mg.

[0065] Further, the density of the quaternary ammonium functional groups of the zwitterionic exchange microspheres is 0.5 mmol / g - 2 mmol / g, and the density of the carboxyl functional groups is 1 mmol / g - 5 mmol / g. The microstructure and adsorption principle are as Figure 2 shown.

[0066] Adopting the above technical solution, the present invention has the following beneficial effects:

[0067] The kit provided by the present invention for the synchronous detection of catecholamines and their metabolites is mainly used for the synchronous detection of catecholamines and their metabolites by high performance liquid chromatography tandem mass spectrometry. During the detection process, an appropriate amount of zwitterionic exchange microspheres are creatively used as the packing material of the solid phase extraction device. Since the zwitterionic exchange microspheres contain both carboxyl functional groups and quaternary ammonium functional groups, they can simultaneously separate 6 kinds of catecholamines and their metabolites with amino functional groups and 2 kinds of terminal metabolites of catecholamines with carboxyl functional groups contained in urine samples; at the same time, they also have high accuracy and sensitivity when separating 6 kinds of catecholamines and their metabolites with amino groups contained in blood samples. The synchronous detection method of catecholamines and their metabolites provided by the present invention can greatly reduce the detection cost of 8 kinds of catecholamines and their metabolites in urine and shorten the detection time on the premise of ensuring the detection accuracy and sensitivity; in addition, the kit is applicable to both blood samples and urine samples, and the detection steps for the two kinds of samples are basically the same, avoiding the problems of numerous kits and complicated detection processes in actual detection work, and having wide practical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for use in the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0069] Figure 1 It is a flow chart for detecting using the kit provided by the present invention;

[0070] Figure 2 It is a schematic diagram of the microscopic structure and adsorption principle of the zwitterionic microspheres;

[0071] Figure 3 It is a chromatogram obtained by detecting 8 kinds of catecholamines and their metabolites in a urine sample using the kit provided in Example 1;

[0072] Figure 4 It is a chromatogram obtained by detecting 6 kinds of catecholamines and their metabolites in a blood sample using the kit provided in Example 5. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0073] The following will clearly and completely describe the technical solutions of the present invention with reference to the drawings. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art without creative efforts based on the embodiments of the present invention belong to the scope of protection of the present invention.

[0074] The present invention will be further explained below in conjunction with specific embodiments.

[0075] The present invention provides a kit for synchronously detecting catecholamines and their metabolites, specifically comprising a diluent, a solid-phase extraction reagent, an LC-MS / MS detection reagent, a solid-phase extraction device, and a solid-phase extraction packing material;

[0076] The solid-phase extraction packing material is zwitterionic exchange microspheres; the zwitterionic exchange microspheres contain carboxyl functional groups and quaternary ammonium functional groups; the density of the carboxyl functional groups is 1 mmol / g - 5 mmol / g; the density of the quaternary ammonium functional groups is 0.5 mmol / g - 2 mmol / g; the pore size is 1 nm - 50 nm; the particle size is 10 μm - 300 μm;

[0077] The solid-phase extraction reagent includes an activation solution, a balance solution, a washing solution, an elution solution, and a reconstitution solution;

[0078] The activation solution is a 0 - 50% methanol solution;

[0079] The balance solution is deionized water;

[0080] The washing solution includes a first washing solution and a second washing solution; the first washing solution is deionized water; the second washing solution is selected from methanol and acetonitrile;

[0081] The elution solution is a methanol solution containing 0.1% - 4% formic acid;

[0082] The reconstitution solution is deionized water;

[0083] The diluent includes a blood detection diluent and / or a urine detection diluent;

[0084] The blood detection diluent is a 10% - 15% methanol solution; the urine detection diluent is a 0.02 mol / L sodium carbonate - sodium bicarbonate solution;

[0085] The LC-MS / MS detection reagent includes a standard, a quality control product, an internal standard working solution, and a mobile phase; the standard includes a blood standard and / or a urine standard;

[0086] Both the blood standard and the blood quality control product include epinephrine, norepinephrine, dopamine, metanephrine, normetanephrine, and 3-methoxytyramine, and the balance is a blood simulation matrix solution;

[0087] The standard curve concentration ranges of the components in the blood standard are as follows: epinephrine: 23.3 pg / ml - 1800 pg / ml, norepinephrine: 58.31 pg / ml - 4500 pg / ml, dopamine: 17.5 pg / ml - 1350 pg / ml, metanephrine: 11.67 pg / ml - 900 pg / ml, normetanephrine: 23.3 pg / ml - 1800 pg / ml, 3-methoxytyramine: 11.67 pg / ml - 900 pg / ml;

[0088] The target concentrations of the components in the low blood quality control are as follows: epinephrine: 120 pg / ml, norepinephrine: 300 pg / ml, dopamine: 90 pg / ml, metanephrine: 60 pg / ml, normetanephrine: 120 pg / ml, 3-methoxytyramine: 60 pg / ml; The target concentrations of the components in the high blood quality control are as follows: epinephrine: 900 pg / ml, norepinephrine: 2250 pg / ml, dopamine: 675 pg / ml, metanephrine: 450 pg / ml, normetanephrine: 900 pg / ml, 3-methoxytyramine: 450 pg / ml;

[0089] The blood simulation matrix solution includes 4% bovine serum albumin solution and 0.1% - 0.5% ascorbic acid.

[0090] Both the urine standard and the urine quality control include epinephrine, norepinephrine, dopamine, metanephrine, normetanephrine, 3-methoxytyramine, homovanillic acid HVA, and vanillylmandelic acid VMA, with the balance being the simulated urine matrix solution;

[0091] The standard curve concentration ranges of the components in the urine standard are as follows: epinephrine: 0.4 ng / ml - 80 ng / ml, norepinephrine: 6 ng / ml - 1200 ng / ml, dopamine: 10 ng / ml - 2000 ng / ml, metanephrine: 0.5 ng / ml - 100 ng / ml, normetanephrine: 0.8 ng / ml - 160 ng / ml, 3-methoxytyramine: 0.8 ng / ml - 160 ng / ml, homovanillic acid: 125 ng / ml - 25000 ng / ml, vanillylmandelic acid: 125 ng / ml - 25000 ng / ml;

[0092] The target concentrations of the component compounds in the low-quality urine control are as follows: epinephrine: 4 ng / ml, norepinephrine: 60 ng / ml, dopamine: 100 ng / ml, metanephrine: 5 ng / ml, normetanephrine: 8 ng / ml, 3-methoxytyramine: 8 ng / ml, homovanillic acid: 1250 ng / ml, vanillylmandelic acid: 1250 ng / ml;

[0093] The target concentrations of the component compounds in the high-quality urine control are as follows: epinephrine: 32 ng / ml, norepinephrine: 480 ng / ml, dopamine: 800 ng / ml, metanephrine: 40 ng / ml, normetanephrine: 64 ng / ml, 3-methoxytyramine: 64 ng / ml, homovanillic acid: 10000 ng / ml, vanillylmandelic acid: 10000 ng / ml;

[0094] The internal standard working solution includes a blood internal standard working solution and / or a urine internal standard working solution;

[0095] The blood internal standard working solution includes epinephrine-d6: 40 ng / ml, norepinephrine-d6: 100 ng / ml, dopamine-d4: 40 ng / ml, metanephrine-d3: 20 ng / ml, normetanephrine-d3: 40 ng / ml, 3-methoxytyramine-d4: 20 ng / ml;

[0096] The urine internal standard working solution includes epinephrine-d6: 100 ng / ml, norepinephrine-d6: 1000 ng / ml, dopamine-d4: 1500 ng / ml, metanephrine-d3: 100 ng / ml, normetanephrine-d3: 150 ng / ml, 3-methoxytyramine-d4: 150 ng / ml, homovanillic acid-d5: 20000 ng / ml, vanillylmandelic acid-d3: 20000 ng / ml;

[0097] The simulated urine matrix solution is an artificial urine matrix solution containing 0.1% - 0.5% acetic acid.

[0098] The mobile phase includes mobile phase A and mobile phase B; mobile phase A is an aqueous solution of 0.1% formic acid; mobile phase B is a methanol solution.

[0099] The steps for solid-phase extraction and mass spectrometry detection using the above kit are as follows:

[0100] Step 1, take a solid-phase extraction device filled with zwitterionic exchange microspheres, add the activation solution for activation; then add the equilibration solution and press dry to prepare an activated solid-phase extraction device;

[0101] Step 2: Synchronously pipette 200 μL each of the reference standard, quality control product, and sample to be tested, sequentially add 20 μL of the internal standard working solution, add 400 μL of the diluent, and vortex to mix evenly to obtain the reference standard working solution, quality control product working solution, and sample solution to be tested;

[0102] Step 3: Synchronously transfer the reference standard working solution, quality control product working solution, and sample solution to be tested into the solid-phase extraction device, and press dry; then sequentially add the first eluent and the second eluent, and press dry; then add the eluting solution, collect the eluate, and press dry;

[0103] Dry the eluates of each group under nitrogen, add 100 μL of the reconstitution solution for reconstitution to obtain the concentrated test solution;

[0104] Step 4: Take 10 μL of the concentrated test solutions of each group for high performance liquid chromatography-tandem mass spectrometry analysis.

[0105] The specific conditions of chromatography and mass spectrometry are as follows:

[0106] Chromatographic column: Phenomenex Kinetex F5 (2.6 μm, 3.0×100 mm)

[0107] Column temperature: 35 °C

[0108] Sample chamber temperature: 4 °C

[0109] Injection volume: 10 μL

[0110] Mobile phase: Mobile phase A: 0.1% formic acid aqueous solution; Mobile phase B: methanol solution

[0111] The mobile phase gradient is shown in Table 1:

[0112] Table 1 Mobile phase gradient

[0113]

[0114] The mass spectrometry conditions are ionization mode: ESI+; capillary voltage: 3.0 kV; desolvation gas temperature: 600 °C; desolvation gas flow rate: 1100 L / h; source temperature: 150 °C;

[0115] The collection in sample detection is shown in Table 2:

[0116] Table 2 Ion selection parameter table in sample detection

[0117]

[0118] Example 1

[0119] As Figure 3As shown, this embodiment verifies that the kit provided by the present invention can be used to simultaneously separate and detect 8 kinds of catecholamines and their metabolites contained in urine.

[0120] A kit for simultaneously detecting catecholamines and their metabolites, comprising the following:

[0121] (1) Solid-phase extraction reagents: 60 ml of activation solution of 50% methanol;

[0122] 60 ml of deionized water balance solution;

[0123] 60 ml of the first eluent: deionized water;

[0124] 60 ml of the second eluent: methanol;

[0125] 20 ml of eluent: methanol solution containing 1% formic acid;

[0126] 10 ml of reconstitution solution: deionized water;

[0127] (2) 50 ml of urine detection diluent: 0.02 mol / L sodium carbonate-sodium bicarbonate solution;

[0128] (3) One 96-well solid-phase extraction device filled with 5 mg of zwitterionic exchange microspheres: the particle size of the zwitterionic exchange microspheres is 30 μm, the pore size is 3 nm, the carboxyl density is 4.8972 mmol / g, and the quaternary ammonium group density is 1.3564 mmol / g;

[0129] (4) 6 bottles of urine standard products with different concentrations: each concentration of urine standard product contains 8 kinds of catecholamines and their metabolites: epinephrine, norepinephrine, dopamine, metanephrine, normetanephrine, 3-methoxytyramine, homovanillic acid, vanillylmandelic acid, and the balance is a simulated urine matrix solution;

[0130] (5) Urine quality control products: divided into high urine quality control products and low urine quality control products;

[0131] (6) Urine internal standard working solution: containing deuterated products of 8 kinds of catecholamines and their metabolites, with concentrations of epinephrine-d6: 100 ng / ml, norepinephrine-d6: 1000 ng / ml, dopamine-d4: 1500 ng / ml, metanephrine-d3: 100 ng / ml, normetanephrine-d3: 150 ng / ml, 3-methoxytyramine-d4: 150 ng / ml, homovanillic acid-d5: 20000 ng / ml, vanillylmandelic acid-d3: 20000 ng / ml.

[0132] The specific detection steps are as follows:

[0133] Step 1: Add 600 μL of methanol to a 96-well solid-phase extraction plate filled with zwitterionic exchange microspheres for activation, and then use a positive pressure instrument to control the flow rate to press dry; then add 600 μL of deionized water for equilibration, and use a positive pressure instrument to control the flow rate to press dry to prepare an activated solid-phase extraction plate;

[0134] Step 2: Synchronously pipette 200 μL each of urine standard, urine quality control, and urine sample spiked with the analyte, and sequentially add 20 μL of urine internal standard working solution and 400 μL of urine detection diluent, and vortex to mix evenly to obtain urine standard working solution, urine quality control working solution, and urine sample spiked with the analyte solution;

[0135] Step 3: Take 600 μL of urine standard working solution, urine quality control working solution, and urine sample spiked with the analyte solution, and synchronously transfer them into the activated solid-phase extraction column prepared in Step 1, and use a positive pressure instrument to control the flow rate to press dry; then sequentially add 600 μL of deionized water and 600 μL of methanol for two elutions, and use a positive pressure instrument to control the flow rate to press dry; then add 200 μL of 1% formic acid-methanol solution for elution, collect the eluate, and use a positive pressure instrument to control the flow rate to press dry;

[0136] Dry the collected eluates of each group under nitrogen at 30 °C, add 100 μL of deionized water for reconstitution to obtain concentrated detection solutions for standby;

[0137] Step 4: Take 10 μL of the concentrated detection solutions of each group for high performance liquid chromatography-tandem mass spectrometry analysis. The results are as Figure 3 shown. It can be clearly seen in the chromatogram that through the solid-phase extraction device using zwitterionic exchange microspheres as the packing material, the simultaneous separation and mass spectrometry detection of 8 catecholamines and their metabolites in urine can be achieved.

[0138] Table 3 Recovery test results of urine samples spiked with the analyte using the detection kit provided in this example

[0139]

[0140] It can be seen from Table 3 that in this example, by using the kit provided by the present invention, not only the simultaneous separation and detection of 8 catecholamines and their metabolites are achieved, but also the recoveries of the 8 target analytes are between 93.8% and 105.3%, indicating that the solid-phase extraction method using the kit provided by the present invention has good extraction effects.

[0141] Example 2

[0142] This example verifies the influence of using different zwitterionic exchange microspheres on urine test results.

[0143] The kit and detection method used in this example are basically the same as those in Example 1. The difference is that in this example, multiple groups of different zwitterionic exchange microspheres in Table 4 are used as the packing material of the solid-phase extraction device, and the recovery rates of 8 catecholamines and their metabolites in the same spiked urine sample are detected respectively. The results are shown in Table 5:

[0144] Table 4 Differences in zwitterionic exchange microspheres used under various conditions in this example

[0145]

[0146] Table 5 Recovery rates of 8 catecholamines and their metabolites measured using 10 groups of kits provided in this example

[0147]

[0148] When the density of carboxyl functional groups in the zwitterionic exchange microspheres is higher, the recovery rates of the 6 basic catecholamines and their metabolites by the kit are higher; when the density of quaternary ammonium functional groups is higher, the recovery rates of the 2 acidic catecholamines by the kit are higher. It can be seen from the detection results in Table 5 that when zwitterionic exchange microspheres with a carboxyl density of 3 - 5 mmol / L and a quaternary ammonium density of 1 - 2 mmol / L are used as the packing material of the solid-phase extraction column, the recovery rates of 8 catecholamines and their metabolites can all reach a relatively high level. In addition, on the premise that the density of functional groups remains unchanged, the smaller the pore size of the zwitterionic exchange microspheres, the more significant the adsorption effect. This is because the smaller the pore size, the larger the surface area of the microspheres, and the more active sites available for grafting with functional groups, resulting in a better adsorption effect; moreover, if the pore size is too large, it may cause interference from impurities such as macromolecular proteins, reducing the adsorption specificity of the microspheres. Therefore, the pore size of the zwitterionic exchange microspheres used in the kit provided by the present invention is preferably 1 nm - 50 nm.

[0149] Regarding the preferred particle size of the zwitterionic exchange microspheres, it can be seen from Table 5 that the smaller the particle size, the larger the specific surface area of the zwitterionic exchange microspheres and the more active sites, which is beneficial to increasing the adsorption capacity; however, when the particle size is too small, it will reduce the mass transfer efficiency of the analyte, increase the solid-phase extraction time, and reduce the extraction efficiency. Therefore, the particle size of the zwitterionic exchange microspheres in the kit provided by the present invention is preferably 30 μm - 60 μm.

[0150] This example verified through multiple groups of experiments that the density of carboxyl functional groups in the zwitterionic exchange microspheres in the kit provided by the present invention is preferably 3 mmol / L - 5 mmol / L; the density of quaternary ammonium functional groups is preferably 1 mmol / L - 2 mmol / L; the pore size is preferably 1 nm - 50 nm; and the particle size is preferably 30 μm - 60 μm.

[0151] Example 3

[0152] This example verified the influence of the dosage of zwitterionic exchange microspheres in the kit provided by the present invention on the recovery rates of catecholamines and their metabolites.

[0153] The kit and detection method used in this example were basically the same as those in Example 1, except that the dosages of zwitterionic exchange microspheres were 2 mg, 3 mg, 5 mg, and 8 mg respectively. The 4 groups of recovery rate detection data obtained are shown in Table 6 below:

[0154] Table 6 Influence of the dosage of different zwitterionic exchange microspheres on the detection recovery rate of analytes

[0155]

[0156] It can be seen from Table 6 that the greater the dosage of zwitterionic exchange microspheres, the higher the recovery rates of the 8 catecholamines and their metabolites by the kit provided by the present invention; when the dosage of microspheres reaches 5 mg, the recovery rate reaches the maximum value and no longer increases significantly, proving that almost all analytes in the sample have been separated at this time. Therefore, the dosage of zwitterionic exchange microspheres in the kit provided by the present invention is 2 mg - 8 mg, preferably 3 mg - 5 mg.

[0157] Example 4

[0158] This example verified the influence of different usage volumes of the eluent in the kit and the formic acid content on the recovery rates of detecting 8 catecholamines and their metabolites.

[0159] The kit and detection process used in this example were basically the same as those in Example 1, except that:

[0160] A. On the premise of constant volume, the formic acid concentrations of the eluent used in the kit were 0.1%, 0.5%, 1%, 2%, 3%, and 4% respectively;

[0161] B. On the premise of a certain formic acid concentration in the eluent, 60 μL and 300 μL of the eluent were respectively used to elute the solid-phase extraction column. The recovery rates of the 8 catecholamines and their metabolites obtained under each condition in this example are shown in Table 7:

[0162] Table 7 Influence of the formic acid content and its usage volume in the eluent on the recovery rate

[0163]

[0164] As can be seen from Table 7, when the formic acid concentration is 1%, the recovery rates of 8 kinds of catecholamines and their metabolites all reach the maximum value; when the formic acid concentration continues to increase, the recovery rates of each component show varying degrees of decline, proving that under the premise of a certain added volume, when using an eluent with a formic acid concentration of 1%, the recovery rates of the 8 kinds of catecholamines and their metabolites obtained are the highest. When the formic acid content in the eluent used is certain, the recovery rates of the 8 kinds of catecholamines and their metabolites obtained increase with the increase of the eluent volume. When the eluent volume reaches 200 μL, the recovery rates of each substance no longer increase significantly, indicating that the substances retained by the solid-phase extraction column have been basically eluted. The eluent provided by the present invention can achieve one-step elution of basic and acidic catecholamine analytes. Therefore, in the detection method provided by the present invention, the preferred formic acid concentration of the eluent is 1%, and the preferred use volume is 200 μL.

[0165] Example 5

[0166] As Figure 4 shown, this example verifies that the kit provided by the present invention can be used for one-time separation and detection of 6 kinds of catecholamines and their metabolites in blood.

[0167] A kit for simultaneously detecting catecholamines and their metabolites, comprising the following:

[0168] (1) Solid-phase extraction reagents: 60 ml of activation solution of 50% methanol;

[0169] 60 ml of deionized water balance solution;

[0170] 60 ml of the first eluent: deionized water;

[0171] 60 ml of the second eluent: methanol;

[0172] 20 ml of eluent: methanol solution containing 1% formic acid;

[0173] 10 ml of reconstitution solution: deionized water;

[0174] (2) 50 ml of blood detection diluent: methanol solution of 10 - 15%;

[0175] (3) One 96-well solid-phase extraction device filled with 5 mg of zwitterionic exchange microspheres: the particle size of the zwitterionic exchange microspheres is 30 μm, the pore size is 3 nm, the carboxyl density is 4.8972 mmol / g, and the quaternary ammonium group density is 1.3564 mmol / g;

[0176] (4)6 vials of blood standard products with different concentrations: Each concentration of blood standard product contains 6 catecholamines and their metabolites: epinephrine, norepinephrine, dopamine, metanephrine, normetanephrine, 3-methoxytyramine, and the balance is blood simulation matrix solution;

[0177] (5)Blood quality control products: Divided into high blood quality control products and low blood quality control products;

[0178] (6)Blood internal standard working solution: Contains deuterated forms of 6 catecholamines and their metabolites, with concentrations of epinephrine-d6: 40 ng / ml, norepinephrine-d6: 100 ng / ml, dopamine-d4: 40 ng / ml, metanephrine-d3: 20 ng / ml, normetanephrine-d3: 40 ng / ml, 3-methoxytyramine-d4: 20 ng / ml.

[0179] The specific detection steps are as follows:

[0180] Step 1, Add 600 μL of methanol to a 96-well solid-phase extraction plate filled with zwitterionic exchange microspheres for activation, and then dry it under positive pressure control of the flow rate; then add 600 μL of deionized water for equilibration, and dry it under positive pressure control of the flow rate to prepare an activated solid-phase extraction plate;

[0181] Step 2, Synchronously pipette 200 μL each of blood standard products, blood quality control products, and blood samples to be tested with added standards, and successively add 20 μL of blood internal standard working solution and 400 μL of blood detection diluent, and vortex and mix well to obtain blood standard working solution, blood quality control working solution, and blood samples to be tested with added standards solution;

[0182] Step 3, Take 600 μL of blood standard working solution, blood quality control working solution, and blood samples to be tested with added standards solution, and synchronously transfer them into the activated solid-phase extraction column prepared in Step 1, and dry it under positive pressure control of the flow rate; then successively add 600 μL of deionized water and 600 μL of methanol for two elutions, and dry it under positive pressure control of the flow rate; then add 100 μL of 1% formic acid-methanol solution for elution, collect the eluate, and dry it under positive pressure control of the flow rate; Dry the collected eluates of each group under nitrogen at 30 °C, add 100 μL of deionized water for reconstitution to obtain a concentrated detection solution for standby;

[0183] Step 4, Take 10 μL of each group of concentrated detection solutions for high-performance liquid chromatography-tandem mass spectrometry analysis. The results are as Figure 4 shown. It can be clearly seen in the chromatogram that the solid-phase extraction device filled with zwitterionic exchange microspheres can also separate and perform mass spectrometry detection on 6 catecholamines and their metabolites in blood samples, and the recovery rate is relatively high.

[0184] Example 6

[0185] This example verified the influence of adding ascorbic acid to the blood-simulating matrix solution of the kit on the stability of the LC-MS / MS blood detection reagent.

[0186] Four blood standards in the kit provided in this example were selected, and the influence of the added amount of ascorbic acid on their stability was tested at three storage temperatures: room temperature, 4°C, and -20°C. The specific experimental conditions and results are shown in Table 8.

[0187] Table 8 Influence of antioxidants and storage temperature on the stability of LC-MS / MS blood detection reagent

[0188]

[0189] As can be seen from Table 8, even in the storage environment of -20°C, dopamine (DA), epinephrine (E), and norepinephrine (NE) could not remain stable in the blood standard for a long time without adding ascorbic acid; when 0.3% ascorbic acid was added to the blood standard, all six catecholamines and their metabolites could maintain long-term relative stability in the storage environment of -20°C. Therefore, a blood-simulating matrix solution containing 0.1% - 0.5% ascorbic acid was added to both the blood standard solution and each group of blood quality control products in the kit provided by the present invention.

[0190] Example 7

[0191] The application of the kit provided in this example in urine sample detection and blood sample detection.

[0192] A kit that can be used to detect catecholamines and their metabolites in urine or blood samples, including the following:

[0193] 1. Solid-phase extraction reagents: 60 ml of activation solution of 50% methanol;

[0194] 60 ml of deionized water balance solution;

[0195] 60 ml of the first eluent: deionized water;

[0196] 60 ml of the second eluent: methanol;

[0197] 20 ml of elution solution: methanol solution containing 1% formic acid;

[0198] 10 ml of reconstitution solution: deionized water;

[0199] 2. 50 ml of urine detection diluent: 0.02 mol / L sodium carbonate - sodium bicarbonate solution;

[0200] 3.50 ml blood test diluent: 10%-15% methanol solution;

[0201] 4.1 96-well solid-phase extraction device filled with 5 mg zwitterionic exchange microspheres: the particle size of the zwitterionic exchange microspheres is 30 μm, the pore size is 3 nm, the carboxyl density is 4.8972 mmol / g, and the quaternary ammonium density is 1.3564 mmol / g;

[0202] 5.6 bottles of urine standards with different concentrations: each urine standard contains 8 catecholamines and their metabolites: epinephrine, norepinephrine, dopamine, metanephrine, normetanephrine, 3-methoxytyramine, homovanillic acid, vanillylmandelic acid, and the balance is a simulated urine matrix solution;

[0203] 6.6 bottles of blood standards with different concentrations: each blood standard contains 6 catecholamines and their metabolites: epinephrine, norepinephrine, dopamine, metanephrine, normetanephrine, 3-methoxytyramine, and the balance is a blood-simulated matrix solution;

[0204] 7. Urine quality control products: divided into high urine quality control products and low urine quality control products;

[0205] 8. Blood quality control products: divided into high blood quality control products and low blood quality control products;

[0206] 9. Urine internal standard working solution: contains deuterated products of 8 catecholamines and their metabolites, with concentrations of epinephrine-d6: 100 ng / ml, norepinephrine-d6: 1000 ng / ml, dopamine-d4: 1500 ng / ml, metanephrine-d3: 100 ng / ml, normetanephrine-d3: 150 ng / ml, 3-methoxytyramine-d4: 150 ng / ml, homovanillic acid-d5: 20000 ng / ml, vanillylmandelic acid-d3: 20000 ng / ml;

[0207] 10. Blood internal standard working solution: contains deuterated products of 6 catecholamines and their metabolites, with concentrations of epinephrine-d6: 40 ng / ml, norepinephrine-d6: 100 ng / ml, dopamine-d4: 40 ng / ml, metanephrine-d3: 20 ng / ml, normetanephrine-d3: 40 ng / ml, 3-methoxytyramine-d4: 20 ng / ml.

[0208] Using the kit provided in this example to detect 8 randomly selected urine samples, the specific steps are basically the same as those in Example 1, and the detection results are shown in Table 9:

[0209] Table 9 Comparison of measured values of 8 groups of urine samples

[0210]

[0211] As can be seen from Table 9, the kit provided by the present invention can accurately detect the contents of 8 kinds of catecholamines and their metabolites in urine at one time.

[0212] The kit provided by this embodiment was used to detect 5 randomly selected groups of serum samples. The specific steps were basically the same as those in Example 5, and the detection results are shown in Table 10:

[0213] Table 10 Comparison of measured values of 5 groups of serum samples

[0214]

[0215] As shown in Table 10, the kit provided by the present invention can be well used for the detection of 6 kinds of catecholamines and their metabolites in serum samples.

[0216] This embodiment verifies that the detection kit provided by the present invention can not only accurately detect serum samples, but also accurately separate and detect 8 kinds of catecholamines and their metabolites in urine samples at one time, greatly shortening the detection cost and detection time.

[0217] Experimental Example 1

[0218] This experimental example provides the linear range, detection limit and quantification limit of the detection kit provided by the present invention for detecting 8 kinds of catecholamines and their metabolites.

[0219] The kit and detection steps used in this experimental example were basically the same as those in Example 1. The difference was that a series of standard products were processed in parallel for 3 groups. When the recovery rate was within the range of 100% ± 20% and the total coefficient of variation did not exceed 20%, the measurement range of the analytical method was determined. Taking the concentrations of 8 target analytes as the abscissa and the peak area ratio of the analyte to its isotope internal standard as the ordinate, a linear regression operation was performed with a weighting factor of 1 / x to obtain a linear regression equation, which was the working curve.

[0220] The signal-to-noise ratio method was used to evaluate the detection limit and quantification limit. A series of dilutions of the standard product with the lowest concentration were performed and detected. The concentration corresponding to the signal-to-noise ratio equal to 3 was defined as the detection limit LOD; the concentration corresponding to the signal-to-noise ratio greater than or equal to 10 was determined as the quantification limit LOQ.

[0221] Table 11 Linear range, detection limit and quantification limit of 8 kinds of catecholamines and their metabolites

[0222]

[0223] As can be seen from Table 11, the detection limit and quantification limit of the detection kit provided by the present invention for urine samples; among which the quantification limit is significantly lower than that of the commercially available kit, which indicates that using the kit provided by the present invention can simplify the detection steps, reduce the detection cost, and ensure the accuracy of the detection results.

[0224] Experimental Example 2

[0225] In this example, the detection limit and quantification limit of detecting 6 catecholamines and their metabolites in blood samples using the kit provided by the present invention were determined.

[0226] The kit and detection steps used in this example were basically the same as those in Experimental Example 5. The difference was that a series of standard products were processed in parallel in 3 groups. When ensuring that the recovery rate was within the range of 100% ± 20% and the total coefficient of variation did not exceed 20%, the measurement range of the analytical method was determined. Taking the concentrations of 6 target analytes as the abscissa and the peak area ratio of the analyte to its isotope internal standard as the ordinate, a linear regression operation was performed with a weighting factor of 1 / x to obtain a linear regression equation, which was the working curve.

[0227] The signal-to-noise ratio method was used to evaluate the detection limit and quantification limit. A series of dilutions of the standard product with the lowest concentration were carried out for detection. The concentration corresponding to the signal-to-noise ratio equal to 3 was defined as the detection limit LOD; the concentration corresponding to the signal-to-noise ratio greater than or equal to 10 was determined as the quantification limit LOQ.

[0228] Table 12 Linear range, detection limit and quantification limit of 6 catecholamines and their metabolites in blood

[0229]

[0230] As can be seen from Table 12, when using the kit and detection steps provided by the present invention to detect blood samples, the obtained detection limit and quantification limit. Among them, the quantification limit of the detection kit provided by the present invention for blood samples is significantly lower than that of the commercially available kit, and it can completely replace the existing kit in the field of detecting blood samples of catecholamines and their metabolites.

[0231] In summary, the present invention provides a kit for synchronously detecting catecholamines and their metabolites, and zwitterionic exchange microspheres are used as the packing material for solid-phase extraction in the kit. By using zwitterionic exchange microspheres containing both carboxyl groups and quaternary ammonium groups as the packing material of the solid-phase extraction column, six catecholamines and their metabolites containing amino functional groups and two terminal metabolites of catecholamines containing carboxyl functional groups can be synchronously and efficiently separated, which is beneficial to the detection of catecholamines and their metabolites by high performance liquid chromatography-tandem mass spectrometry; by optimizing the pore size, particle size and the density of carboxyl and quaternary ammonium functional groups of the zwitterionic exchange microspheres, adjusting the dosage of the eluent and the concentration of formic acid after solid-phase extraction, the recovery rate of the solid-phase extraction column for eight catecholamines and their metabolites is improved; while simplifying the detection steps, the accuracy of the detection of eight catecholamines and their metabolites in urine is ensured. In addition, the kit provided by the present invention can also be used for the detection of catecholamines and their metabolites in blood samples. The kit and its application proposed by the present invention have far-reaching significance for optimizing the mass spectrometry detection process and improving the application value of catecholamines and their metabolites.

[0232] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A kit for synchronous detection of catecholamines and their metabolites, characterized in that, The kit contains a diluent, a solid-phase extraction reagent, an LC-MS / MS detection reagent, a solid-phase extraction device, and a solid-phase extraction packing material; The solid-phase extraction packing material is zwitterionic exchange microspheres; the zwitterionic exchange microspheres contain carboxyl functional groups and quaternary ammonium functional groups; The solid-phase extraction reagent includes an activation solution, a balance solution, a washing solution, an elution solution, and a reconstitution solution; The washing solution includes a first washing solution and a second washing solution; The LC-MS / MS detection reagent includes a standard product, a quality control product, an internal standard working solution, and a mobile phase.

2. The kit according to claim 1, characterized in that, The carboxyl functional group density of the zwitterionic exchange microspheres is 1 mmol / g - 5 mmol / g; the quaternary ammonium functional group density is 0.5 mmol / g - 2 mmol / g.

3. The kit according to claim 1 or 2, characterized in that, The preparation method of the zwitterionic exchange microspheres is as follows: Step M1, adding a dispersant to distilled water and stirring well to obtain a dispersant aqueous solution with a concentration of 0.1% - 20%; The dispersant is hydroxypropyl methylcellulose and / or polyvinyl alcohol; Step M2, weighing 50 - 90 parts of a polymer monomer mixture, 1 - 90 parts of a pore-forming agent, and 0.003 - 1 part of an initiator according to mass parts, and stirring well to obtain 100 parts of an oil-phase mixture; The polymer monomer mixture includes divinylbenzene and a chlorine-containing aromatic compound; the mass ratio of divinylbenzene to the chlorine-containing aromatic compound is 1:(1 - 3); The chlorine-containing aromatic compound is selected from p-chloromethylstyrene and 4-chlorostyrene; The pore-forming agent is one or a mixture of dichloromethane, n-dodecane, toluene, o-dichlorobenzene, and liquid paraffin; The initiator is azobisisobutyronitrile and / or benzoyl peroxide; Step M3, fully mixing an acrylate monomer with the oil-phase mixture and the dispersant aqueous solution, and carrying out a condensation reflux reaction at 50 - 80 °C for 4 - 20 h; after the reaction is completed, cooling to room temperature, washing successively with water and ethanol, and drying at 60 °C for 10 - 12 h to obtain a first reaction product; The acrylate monomer is selected from methyl acrylate, methyl methacrylate, and ethyl methacrylate; The added mass of the acrylate monomer is 1% - 20% of the polymer monomer mixture; The mass ratio of the oil-phase mixture to the dispersant aqueous solution is (30 - 60):100; Step M4, placing the first reaction product in a toluene solution and swelling for 0.5 - 1.5 h to obtain a swollen mixture; adding a tertiary amine monomer to the swollen mixture and continuously stirring well at 80 - 90 °C for 4 - 20 h, and after the reaction is completed, obtaining a second reaction product; The tertiary amine monomer is selected from N,N-dimethylbutylamine, trimethylamine, and triethylamine; The added amount of the tertiary amine monomer is 1 - 10 times the mass of the chlorine-containing aromatic compound; Step M5, adding an alkaline methanol solution to the second reaction product, stirring at 65 - 75 °C, and carrying out an ester hydrolysis reaction for 20 - 30 h; after cooling to room temperature, washing successively with ethanol, a sodium bicarbonate solution, and water, and drying to obtain the zwitterionic exchange microsphere adsorbent material.

4. The kit according to claim 1, wherein The diluent is a blood test diluent and / or a urine test diluent; The blood test diluent is a 10%-15% methanol solution; The urine test diluent is a 0.02 mol / L sodium carbonate-sodium bicarbonate solution.

5. The kit according to claim 1, characterized in that The activation solution is a 0-50% methanol solution; The equilibration solution is deionized water; The first eluent is deionized water; the second eluent is selected from methanol and acetonitrile; The elution solution is a methanol solution containing 0.1%-4% formic acid; The reconstitution solution is deionized water; The standard is a blood standard and / or a urine standard; The quality control product is a blood quality control product and / or a urine quality control product; The internal standard working solution is a blood internal standard working solution and / or a urine internal standard working solution; The mobile phase includes mobile phase A and mobile phase B; mobile phase A is a 0.1% aqueous formic acid solution; mobile phase B is methanol.

6. The kit according to claim 5, characterized in that, Both the blood standard and the blood quality control product include the compounds epinephrine, norepinephrine, dopamine, metanephrine, normetanephrine, and 3-methoxytyramine, and the balance is a blood mimic matrix solution; The standard curve concentration ranges of the component compounds in the blood standard are: epinephrine: 23.3 pg / ml - 1800 pg / ml, norepinephrine: 58.31 pg / ml - 4500 pg / ml, dopamine: 17.5 pg / ml - 1350 pg / ml, metanephrine: 11.67 pg / ml - 900 pg / ml, normetanephrine: 23.3 pg / ml - 1800 pg / ml, 3-methoxytyramine: 11.67 pg / ml - 900 pg / ml; The blood quality control product includes a low blood quality control product and a high blood quality control product; The target concentrations of the component compounds in the low blood quality control product are: epinephrine: 120 pg / ml, norepinephrine: 300 pg / ml, dopamine: 90 pg / ml, metanephrine: 60 pg / ml, normetanephrine: 120 pg / ml, 3-methoxytyramine: 60 pg / ml; The target concentrations of the component compounds in the high blood quality control product are: epinephrine: 900 pg / ml, norepinephrine: 2250 pg / ml, dopamine: 675 pg / ml, metanephrine: 450 pg / ml, normetanephrine: 900 pg / ml, 3-methoxytyramine: 450 pg / ml; The blood internal standard working solution includes epinephrine-d6: 40 ng / ml, norepinephrine-d6: 100 ng / ml, dopamine-d4: 40 ng / ml, metanephrine-d3: 20 ng / ml, normetanephrine-d3: 40 ng / ml, 3-methoxytyramine-d4: 20 ng / ml; The blood mimic matrix solution includes a 4% bovine serum albumin solution and 0.1%-0.5% ascorbic acid.

7. The kit according to claim 5, characterized in that, Both the urine standard and the urine quality control product contain compounds adrenaline, noradrenaline, dopamine, metanephrine, normetanephrine, 3-methoxytyramine, homovanillic acid (HVA), and vanillylmandelic acid (VMA), with the balance being a simulated urine matrix solution; The standard curve concentration ranges of the component compounds in the urine standard are as follows: adrenaline: 0.4 ng / ml - 80 ng / ml, noradrenaline: 6 ng / ml - 1200 ng / ml, dopamine: 10 ng / ml - 2000 ng / ml, metanephrine: 0.5 ng / ml - 100 ng / ml, normetanephrine: 0.8 ng / ml - 160 ng / ml, 3-methoxytyramine: 0.8 ng / ml - 160 ng / ml, homovanillic acid: 125 ng / ml - 25000 ng / ml, vanillylmandelic acid: 125 ng / ml - 25000 ng / ml; The urine quality control product includes a low urine quality control product and a high urine quality control product; The target concentrations of the component compounds in the low urine quality control product are as follows: adrenaline: 4 ng / ml, noradrenaline: 60 ng / ml, dopamine: 100 ng / ml, metanephrine: 5 ng / ml, normetanephrine: 8 ng / ml, 3-methoxytyramine: 8 ng / ml, homovanillic acid: 1250 ng / ml, vanillylmandelic acid: 1250 ng / ml; The target concentrations of the component compounds in the high urine quality control product are as follows: adrenaline: 32 ng / ml, noradrenaline: 480 ng / ml, dopamine: 800 ng / ml, metanephrine: 40 ng / ml, normetanephrine: 64 ng / ml, 3-methoxytyramine: 64 ng / ml, homovanillic acid: 10000 ng / ml, vanillylmandelic acid: 10000 ng / ml; The urine internal standard working solution includes adrenaline-d6: 100 ng / ml, noradrenaline-d6: 1000 ng / ml, dopamine-d4: 1500 ng / ml, metanephrine-d3: 100 ng / ml, normetanephrine-d3: 150 ng / ml, 3-methoxytyramine-d4: 150 ng / ml, homovanillic acid-d5: 20000 ng / ml, vanillylmandelic acid-d3: 20000 ng / ml; The simulated urine matrix solution is an artificial urine solution containing 0.1% - 0.5% acetic acid.

8. A synchronous detection method for catecholamine and its metabolites, characterized in that, The detection method uses the kit according to any one of claims 1 - 7.

9. The detection method according to claim 8, wherein The specific steps of the detection method are as follows: Step 1, take a solid-phase extraction device filled with zwitterionic exchange microspheres, add an activation solution for activation; then add the equilibration solution and press it dry to prepare an activated solid-phase extraction device; Step 2: Synchronously pipette 200 μL each of the reference standard, quality control sample, and sample to be tested, sequentially add 20 μL of the internal standard working solution to each, and then add 400 μL of diluent, and mix to obtain the reference standard working solution, quality control working solution, and sample solution to be tested respectively; Step 3: Synchronously transfer the reference standard working solution, quality control working solution, and sample solution to be tested into the solid-phase extraction device, and press dry; Then sequentially add the first eluent and the second eluent, and press dry; then add the elution solution, collect the eluate, and press dry; Dry the eluates of each group under nitrogen, add 100 μL of the reconstitution solution for reconstitution to obtain the concentrated detection solution; Step 4: Take 10 μL of the concentrated detection solutions of each group for high performance liquid chromatography-tandem mass spectrometry analysis.

10. The detection method according to claim 9, characterized in that, The packing amount of the zwitterionic exchange microspheres in Step 1 is 2 mg - 8 mg; The density of quaternary ammonium functional groups of the zwitterionic exchange microspheres is 0.5 mmol / g - 2 mmol / g, and the density of carboxyl functional groups is 1 mmol / g - 5 mmol / g.

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