Kit and detection method for synchronously detecting catecholamine and metabolites thereof

Abstract

The invention provides a kit for synchronously detecting catecholamine and metabolites thereof and a detection method, and relates to the technical field of analysis and detection. The kit comprises a diluent, a solid-phase extraction reagent, an LC-MS / MS detection reagent, a solid-phase extraction device and a solid-phase extraction filler. Wherein the solid-phase extraction filler creatively adopts an amphiphilic ion exchange microsphere containing two functional groups, namely carboxyl and quaternary ammonium at the same time. The kit can be used for synchronously and efficiently separating and detecting eight catecholamines and metabolites thereof in a urine sample; and 6 kinds of catecholamines containing amino groups and metabolites thereof in a blood sample can be separated and detected. When the kit is used for separating and detecting catecholamine and metabolites thereof, the extraction and recovery rate is high, the linear range covers the reference value range of normal detection, the detection time of a urine sample can be remarkably shortened, the detection efficiency of mass spectrum is improved, and popularization and application in clinical detection are facilitated.

Description

Technical Field

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

[0002] Catecholamines CA are a class of neurological substances containing catechol and amine groups. They are important neurotransmitters and hormones in the human body and have strong physiological activity. The concentration of catecholamines and metabolites is mainly determined by measuring epinephrine E, norepinephrine NE and dopamine DA and their intermediate metabolites methylepinephrine MN, methylepinephrine NMN, 3-methoxytyramine 3-MT, and terminal metabolites vanillylmandelic acid VMA and homovanillic acid HVA. Catecholamines and their metabolites have high polarity and similar structures. In addition, they have low content in biological samples, have large interference, and are difficult to detect. Liquid chromatography tandem mass spectrometry LC-MS / MS has become the main method for detecting CA and its metabolites due to its high sensitivity, strong specificity, and the ability to rapidly screen a large number of samples. It is recommended by guidelines and consensus.

[0003] For liquid chromatography-tandem mass spectrometry, a suitable pretreatment method is crucial. Since the concentration of catecholamines and their metabolites in plasma is extremely low, solid phase extraction is usually used for sample concentration during pretreatment. The core of solid phase extraction technology is the filler, and selecting an SPE filler with moderate adsorption to the target is a prerequisite for ensuring accurate detection. Catecholamines and their metabolites are monoamines with amino groups, while the terminal metabolites of catecholamines present in urine, vanillylmandelic acid and homovanillic acid, have strong carboxyl groups. Currently, commercial catecholamine solid phase extraction fillers can only adsorb a class of compounds of the same nature. To fully determine the eight catecholamines and their metabolites on the market, at least two different types of kits are required for pretreatment, which is time-consuming and laborious. There is no suitable kit on the market that can separate and extract the eight catecholamines and their metabolites at one time, which seriously restricts the efficiency of liquid chromatography-tandem mass spectrometry in detecting catecholamines and their metabolites. Therefore, it is of great application value to develop a kit that can simultaneously separate and detect 8 catecholamines and their metabolites in urine and 6 catecholamines and their metabolites in blood at one time. Summary of the invention

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

[0005] In order to solve the above technical problems, the present invention provides a kit for synchronous detection of catecholamines and their metabolites, comprising a diluent, a solid phase extraction reagent, an LC-MS / MS detection reagent, a solid phase extraction device, and a solid phase extraction filler; The solid phase extraction filler is amphoteric ion exchange microspheres; The zwitterionic ion exchange microspheres contain carboxyl functional groups and quaternary ammonium functional groups; Because the amphoteric ion exchange microspheres contain both carboxyl functional groups and quaternary ammonium functional groups, they have the ability to simultaneously separate acidic analytes and alkaline analytes, and can simultaneously separate six alkaline catecholamines and their metabolites and two terminal metabolites of acidic catecholamines contained in urine, which is beneficial to simplify the separation and detection steps and improve the detection efficiency; The solid phase extraction reagent includes an activation solution, a balancing solution, a washing solution, an eluent, and a reconstitution solution; The LC-MS / MS detection reagents include standard products, quality control products, internal standard working solution and mobile phase.

[0006] Furthermore, the carboxyl functional group density of the zwitterionic ion exchange microspheres is 1mmol / g-5mmol / g; the quaternary ammonium functional group density is 0.5mmol / g-2mmol / g; the pore size is 1nm-50nm; the particle size is 10μm-300μm, preferably 30μm-60μm; By selecting zwitterionic ion 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 improved more specifically. Since the concentration range of catecholamines and their metabolites in urine is usually at the μg level, while the concentration range of catecholamines and their metabolites in blood is generally lower, only at the pg level, selecting appropriate zwitterionic ion exchange microspheres as fillers for solid phase extraction is more important for improving the detection sensitivity of blood samples.

[0007] Furthermore, the preparation method of the zwitterionic ion exchange microspheres comprises the following specific steps: Step M1, adding a dispersant to distilled water, stirring thoroughly to obtain a dispersant aqueous solution with a concentration of 0.1%-20%, preferably 3%-10%; The dispersant is hydroxypropyl methylcellulose and / or polyvinyl alcohol; 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; 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); 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.

[0008] The porogen is a mixture of one or more of dichloromethane, n-dodecane, toluene, o-dichlorobenzene, and liquid paraffin; The initiator is azobisisobutyronitrile and / or benzoyl peroxide; 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.

[0009] 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; The acrylic acid ester monomer is selected from methyl acrylate, methyl methacrylate, and ethyl methacrylate; The added mass of the acrylic ester 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, 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; The tertiary amine monomer is selected from N,N-dimethylbutylamine, trimethylamine, and triethylamine; The amount of the tertiary amine monomer added is 1-10 times the mass of the chlorine-containing aromatic compound; Step M5, adding alkaline methanol solution to the second reaction product, stirring at a low speed at 65-75° C., and performing ester hydrolysis reaction for 20-30 hours; after cooling to room temperature, washing with ethanol, sodium bicarbonate solution, and water in sequence, and drying to obtain the zwitterionic ion exchange microsphere adsorption material.

[0010] Further, 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 detection diluent is a 0.02 mol / L sodium carbonate-sodium bicarbonate solution; preferably, the mass ratio of sodium carbonate:sodium bicarbonate is (1-3):(7-9).

[0011] Further, the activation solution is a 0-50% methanol solution; The balancing solution is deionized water; 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; The eluent is a methanol solution containing 0.1%-4% formic acid; The rehydration solution is deionized water; The standard substance is a blood standard substance and / or a urine standard substance; 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; the mobile phase A is a 0.1% formic acid aqueous solution; and the mobile phase B is methanol.

[0012] Furthermore, the blood standard and blood quality control products both include epinephrine, norepinephrine, dopamine, methylepinephrine, methylnorepinephrine, and 3-methoxytyramine, and the remainder is a blood simulation matrix solution; The standard curve concentration ranges of the components in the blood standard are: epinephrine: 23.3pg / ml-1800pg / ml, norepinephrine: 58.31pg / ml-4500pg / ml, dopamine: 17.5pg / ml-1350pg / ml, methylepinephrine: 11.67pg / ml-900pg / ml, methylnorepinephrine: 23.3pg / ml-1800pg / ml, 3-methoxytyramine: 11.67pg / ml-900pg / ml; The blood quality control products include low-quality blood control products and high-quality blood control products; The target concentrations of the components in the low-quality blood control are: epinephrine: 120 pg / ml, norepinephrine: 300 pg / ml, dopamine: 90 pg / ml, methylepinephrine: 60 pg / ml, methylnorepinephrine: 120 pg / ml, 3-methoxytyramine: 60 pg / ml; The target concentrations of the components in the high-quality blood control product are: epinephrine: 900 pg / ml, norepinephrine: 2250 pg / ml, dopamine: 675 pg / ml, methylepinephrine: 450 pg / ml, methylnorepinephrine: 900 pg / ml, 3-methoxytyramine: 450 pg / ml; The blood internal standard working solution includes epinephrine-d6: 40ng / ml, norepinephrine-d6: 100ng / ml, dopamine-d4: 40ng / ml, methylepinephrine-d3: 20ng / ml, methylnorepinephrine-d3: 40ng / ml, 3-methoxytyramine-d4: 20ng / ml; The blood simulation matrix solution includes 4% bovine serum albumin solution and 0.1%-0.5% ascorbic acid; Preferably, the concentration of ascorbic acid in the blood-simulating matrix solution is 0.3%-0.5%.

[0013] Furthermore, the urine standard and urine quality control products include adrenaline, norepinephrine, dopamine, methylepinephrine, methylnorepinephrine, 3-methoxytyramine, homovanillic acid HVA, and vanillylmandelic acid VMA, and the remainder is a simulated urine matrix solution; The concentration range of the standard curve of each component compound in the urine standard is: epinephrine: 0.4ng / ml-80ng / ml, norepinephrine: 6ng / ml-1200ng / ml, dopamine: 10ng / ml-2000ng / ml, methylepinephrine: 0.5ng / ml-100ng / ml, methylnorepinephrine: 0.8ng / ml-160ng / ml, 3-methoxytyramine: 0.8ng / ml-160ng / ml, homovanillic acid: 125ng / ml-25000ng / ml, vanillylmandelic acid: 125ng / ml-25000ng / ml; The urine quality control products include urine low quality control products and urine high quality control products; The target concentrations of the components in the urine low-quality control product are: epinephrine: 4 ng / ml, norepinephrine: 60 ng / ml, dopamine: 100 ng / ml, methylepinephrine: 5 ng / ml, methylnorepinephrine: 8 ng / ml, 3-methoxytyramine: 8 ng / ml, homovanillic acid: 1250 ng / ml, vanillylmandelic acid: 1250 ng / ml; The target concentrations of the components in the urine high-quality control product are: epinephrine: 32 ng / ml, norepinephrine: 480 ng / ml, dopamine: 800 ng / ml, methylepinephrine: 40 ng / ml, methylnorepinephrine: 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 epinephrine-d6: 100ng / ml, norepinephrine-d6: 1000ng / ml, dopamine-d4: 1500ng / ml, methylepinephrine-d3: 100ng / ml, methylnorepinephrine-d3: 150ng / ml, 3-methoxytyramine-d4: 150ng / ml, homovanillic acid-d5: 20000ng / ml, and vanillylmandelic acid-d3: 20000ng / ml; The simulated urine matrix solution is an artificial urine solution containing 0.1%-0.5% acetic acid.

[0014] In another aspect, the present invention provides a method for the simultaneous detection of catecholamines and their metabolites, wherein the detection method uses the above-mentioned kit.

[0015] Furthermore, if Figure 1 As shown, the specific steps of the detection method are as follows: Step 1, taking a solid phase extraction device filled with zwitterionic ion exchange microspheres, adding an activation solution to activate it; then adding the balancing solution and squeezing it dry to prepare an activated solid phase extraction device; Step 2: Simultaneously pipette 200 µL of each of the standard, quality control, and sample to be tested, add 20 µL of internal standard working solution and 400 µL of diluent, respectively, and vortex to mix to obtain the standard working solution, quality control working solution, and sample solution to be tested; Step 3, synchronously transferring the standard working solution, the quality control working solution, and the sample solution to be tested into the solid phase extraction device and squeezing them dry; then sequentially adding the first eluent and the second eluent, squeezing them dry; then adding the eluent, collecting the eluent, and squeezing them dry; Each group of eluents was blown dry under nitrogen, and 100 μL of reconstitution solution was added for reconstitution to obtain a concentrated detection solution; Step 4, taking 10 μL of the concentrated detection solution and performing high performance liquid chromatography tandem mass spectrometry analysis.

[0016] Furthermore, the loading amount of the zwitterionic ion exchange microspheres in step 1 is 2 mg-8 mg, preferably 3 mg-5 mg.

[0017] Furthermore, the quaternary ammonium functional group density of the zwitterionic ion exchange microspheres is 0.5 mmol / g-2mmol / g, the carboxyl functional group density is 1 mmol / g-5mmol / g, and the microstructure and adsorption principle are as follows: Figure 2 shown.

[0018] By adopting the above technical solution, the present invention has the following beneficial effects: The kit for synchronous detection of catecholamines and their metabolites provided by the present invention is mainly used for synchronous detection of catecholamines and their metabolites by high performance liquid chromatography tandem mass spectrometry. In the detection process, an appropriate amount of zwitterionic ion exchange microspheres are creatively used as fillers for a solid phase extraction device. Because the zwitterionic ion exchange microspheres contain both carboxyl functional groups and quaternary ammonium functional groups, the 6 catecholamines with amino functional groups and their metabolites and the terminal metabolites of 2 catecholamines with carboxyl functional groups contained in urine samples can be synchronously separated; at the same time, the 6 catecholamines with amino functional groups and their metabolites contained in blood samples can also be separated with high accuracy and sensitivity. The synchronous detection method of catecholamines and their metabolites provided by the present invention can greatly reduce the detection cost of 8 catecholamines and their metabolites in urine and shorten the detection time under the premise of ensuring detection accuracy and sensitivity; in addition, the kit is suitable for both blood samples and urine samples, and the detection steps for the two samples are basically the same, avoiding the problems of numerous kits and cumbersome detection processes in actual detection work, and has wide practical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings in the following description are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1 A flow chart for performing detection using the kit provided by the present invention; Figure 2 Schematic diagram of the microstructure and adsorption principle of the zwitterionic microspheres; Figure 3 The chromatogram is obtained by detecting 8 catecholamines and their metabolites in a urine sample using the kit provided in Example 1; Figure 4 The chromatogram is obtained by detecting 6 catecholamines and their metabolites in a blood sample using the kit provided in Example 5. DETAILED DESCRIPTION

[0021] The technical solution of the present invention will be described clearly and completely below in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0022] The present invention is further explained below in conjunction with specific implementation modes.

[0023] The present invention provides a kit for synchronously detecting catecholamines and their metabolites, which specifically contains a diluent, a solid phase extraction reagent, an LC-MS / MS detection reagent, a solid phase extraction device, and a solid phase extraction filler; The solid phase extraction filler is a zwitterionic ion exchange microsphere; the zwitterionic ion exchange microsphere contains a carboxyl functional group and a quaternary ammonium functional group; the density of the carboxyl functional group is 1mmol / g-5mmol / g; the density of the quaternary ammonium functional group is 0.5mmol / g-2mmol / g; the pore size is 1nm-50nm; the particle size is 10μm-300μm; Solid phase extraction reagents include activation solution, equilibrium solution, eluent, elution solution, and reconstitution solution; The activation solution is a 0-50% methanol solution; The balancing solution is deionized water; 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; The eluent is a methanol solution containing 0.1%-4% formic acid; The reconstitution solution was deionized water; Diluents include blood test diluents, and / or urine test diluents; The diluent for blood testing is 10%-15% methanol solution; the diluent for urine testing is 0.02 mol / L sodium carbonate-sodium bicarbonate solution; LC-MS / MS detection reagents include standard products, quality control products, internal standard working solution, and mobile phase; standard products include blood standards and / or urine standards; The blood standards and blood quality control products include epinephrine, norepinephrine, dopamine, metanephrine, methylnorepinephrine, 3-methoxytyramine, and the remainder is a blood-simulating matrix solution; The concentration range of the standard curve of each component compound in the blood standard is: epinephrine: 23.3pg / ml-1800pg / ml, norepinephrine: 58.31pg / ml -4500pg / ml, dopamine: 17.5pg / ml-1350pg / ml, methylepinephrine: 11.67pg / ml -900pg / ml, methylnorepinephrine: 23.3pg / ml-1800pg / ml, 3-methoxytyramine: 11.67pg / ml-900pg / ml; The target concentrations of each component compound in the low-quality blood control are: epinephrine: 120pg / ml, norepinephrine: 300pg / ml, dopamine: 90pg / ml, metanephrine: 60pg / ml, methoxynorepinephrine: 120pg / ml, 3-methoxytyramine: 60pg / ml; the target concentrations of each component compound in the high-quality blood control are: epinephrine: 900pg / ml, norepinephrine: 2250pg / ml, dopamine: 675pg / ml, metanephrine: 450pg / ml, methoxynorepinephrine: 900pg / ml, 3-methoxytyramine: 450pg / ml; The blood-simulating matrix solution includes 4% bovine serum albumin solution and 0.1%-0.5% ascorbic acid.

[0024] Urine standards and urine quality control products include epinephrine, norepinephrine, dopamine, metanephrine, methylnorepinephrine, 3-methoxytyramine, homovanillic acid HVA, vanillylmandelic acid VMA, and the remainder is simulated urine matrix solution; The concentration range of the standard curve of each component compound in the urine standard is: epinephrine: 0.4ng / ml-80ng / ml, norepinephrine: 6ng / ml-1200ng / ml, dopamine: 10ng / ml-2000ng / ml, methylepinephrine: 0.5ng / ml-100ng / ml, methylnorepinephrine: 0.8ng / ml-160ng / ml, 3-methoxytyramine: 0.8ng / ml -160ng / ml, homovanillic acid: 125ng / ml-25000ng / ml, vanillylmandelic acid: 125ng / ml-25000ng / ml; The target concentrations of each component compound in the urine low quality control product are: epinephrine: 4ng / ml, norepinephrine: 60ng / ml, dopamine: 100ng / ml, methylepinephrine: 5ng / ml, methylnorepinephrine: 8ng / ml, 3-methoxytyramine: 8ng / ml, homovanillic acid: 1250ng / ml, vanillylmandelic acid: 1250ng / ml; The target concentrations of each component compound in the urine high-quality control product are: epinephrine: 32ng / ml, norepinephrine: 480ng / ml, dopamine: 800ng / ml, methylepinephrine: 40ng / ml, methylnorepinephrine: 64ng / ml, 3-methoxytyramine: 64ng / ml, homovanillic acid: 10000ng / ml, vanillylmandelic acid: 10000ng / ml; The internal standard working solution includes a blood internal standard working solution and / or a urine internal standard working solution; The blood internal standard working solution includes epinephrine-d6: 40ng / ml, norepinephrine-d6: 100ng / ml, dopamine-d4: 40ng / ml, methylepinephrine-d3: 20ng / ml, methylnorepinephrine-d3: 40ng / ml, 3-methoxytyramine-d4: 20ng / ml; The urine internal standard working solution includes epinephrine-d6: 100ng / ml, norepinephrine-d6: 1000ng / ml, dopamine-d4: 1500ng / ml, methylepinephrine-d3: 100ng / ml, methylnorepinephrine-d3: 150ng / ml, 3-methoxytyramine-d4: 150ng / ml, homovanillic acid-d5: 20000ng / ml, vanillylmandelic acid-d3: 20000ng / ml; The simulated urine matrix solution is an artificial urine matrix solution containing 0.1%-0.5% acetic acid.

[0025] The mobile phase includes mobile phase A and mobile phase B; the mobile phase A is a 0.1% formic acid aqueous solution; and the mobile phase B is a methanol solution.

[0026] The steps of solid phase extraction and mass spectrometry detection using the above kit are as follows: Step 1, taking a solid phase extraction device filled with zwitterionic ion exchange microspheres, adding an activation solution to activate it; then adding the balancing solution and squeezing it dry to prepare an activated solid phase extraction device; Step 2, simultaneously pipette 200 μL of the standard, quality control, and sample to be tested, add 20 μL of internal standard working solution and 400 μL of diluent, respectively, and vortex mix to obtain standard working solution, quality control working solution, and sample solution to be tested; Step 3, synchronously transferring the standard working solution, the quality control working solution, and the sample solution to be tested into the solid phase extraction device and squeezing them dry; then sequentially adding the first eluent and the second eluent, squeezing them dry; then adding the eluent, collecting the eluent, and squeezing them dry; Each group of eluents was blown dry under nitrogen, and 100 μL of reconstitution solution was added for reconstitution to obtain a concentrated detection solution; Step 4, taking 10 μL of the concentrated detection solution of each group, and performing high performance liquid chromatography tandem mass spectrometry analysis.

[0027] The specific conditions of chromatography-mass spectrometry are as follows: Chromatographic column: Phenomenex Kinetex F5 (2.6μm, 3.0×100mm) Column temperature: 35°C Sample chamber temperature: 4°C Injection volume: 10 μL Mobile phase: Mobile phase A: 0.1% formic acid in water; Mobile phase B: methanol solution The mobile phase gradient is shown in Table 1: Table 1 Mobile phase gradient

[0028] The mass spectrometry conditions were as follows: ionization mode: ESI+; capillary voltage: 3.0 kV; desolvation temperature: 600°C; desolvation flow rate: 1100 L / h; source temperature: 150°C; The samples collected during the test are shown in Table 2: Table 2 Ion selection parameters in sample detection

[0029] Example 1

[0030] like Figure 3 As shown, this example verifies that the kit provided by the present invention can be used to perform one-time separation and detection of 8 catecholamines and their metabolites contained in urine.

[0031] A kit for synchronous detection of catecholamines and their metabolites, comprising: (1) Solid phase extraction reagent: 60 ml of 50% methanol activation solution; 60ml deionized water balance solution; 60ml first eluent: deionized water; 60ml second eluent: methanol; 20 ml eluent: methanol solution containing 1% formic acid; 10ml reconstitution solution: deionized water; (2) 50 ml urine test diluent: 0.02 mol / L sodium carbonate-sodium bicarbonate solution; (3) A 96-well solid phase extraction device filled with 5 mg of zwitterionic ion exchange microspheres: the particle size of the zwitterionic ion exchange microspheres is 30 μm, the pore size is 3 nm, the carboxyl group density is 4.8972 mmol / g, and the quaternary ammonium group density is 1.3564 mmol / g; (4) 6 bottles of urine standard of different concentrations: each concentration of urine standard contains 8 catecholamines and their metabolites: epinephrine, norepinephrine, dopamine, methylepinephrine, methylnorepinephrine, 3-methoxytyramine, homovanillic acid, vanillylmandelic acid, and the remainder is simulated urine matrix solution; (5) Urine quality control products: divided into urine high quality control products and urine low quality control products; (6) Urine internal standard working solution: Contains deuterated versions of 8 catecholamines and their metabolites, with concentrations of epinephrine-d6: 100 ng / ml, norepinephrine-d6: 1000 ng / ml, dopamine-d4: 1500 ng / ml, methylepinephrine-d3: 100 ng / ml, methylnorepinephrine-d3: 150 ng / ml, 3-methoxytyramine-d4: 150 ng / ml, homovanillic acid-d5: 20000 ng / ml, and vanillylmandelic acid-d3: 20000 ng / ml.

[0032] The specific detection steps are as follows: Step 1, adding 600 μL of methanol to a 96-well solid phase extraction plate filled with zwitterionic ion exchange microspheres for activation, and then controlling the flow rate with a positive pressure instrument to press dry; then adding 600 μL of deionized water for equilibration, and controlling the flow rate with a positive pressure instrument to press dry, to prepare an activated solid phase extraction plate; Step 2, simultaneously pipette 200 μL of urine standard, urine quality control, and urine spiked sample to be tested, add 20 μL of urine internal standard working solution and 400 μL of urine test diluent, respectively, and vortex mix to obtain urine standard working solution, urine quality control working solution, and urine spiked sample solution to be tested; Step 3, take 600 μL of urine standard working solution, urine quality control working solution, and urine spiked sample solution to be tested, and transfer them to the activated solid phase extraction column prepared in step 1 simultaneously, and press dry with a positive pressure instrument to control the flow rate; then add 600 μL of deionized water and 600 μL of methanol in turn for two rinses, and press dry with a positive pressure instrument to control the flow rate; then add 200 μL of 1% formic acid-methanol solution for elution, collect the eluate, and press dry with a positive pressure instrument to control the flow rate; The collected eluates from each group were blown dry under nitrogen at 30°C, and 100 μL of deionized water was added for re-dissolution to obtain a concentrated detection solution for later use; Step 4, take 10 μL of each concentrated test solution and perform high performance liquid chromatography tandem mass spectrometry analysis. Figure 3 As shown in the chromatogram, it can be clearly seen that the solid phase extraction device with zwitterionic ion exchange microspheres as filler can achieve one-time separation and mass spectrometry detection of 8 catecholamines and their metabolites in urine.

[0033] Table 3 Recovery test results of urine spiked samples using the test kit provided in this example

[0034] As can be seen from Table 3, this example not only achieves the one-time separation and detection of 8 catecholamines and their metabolites by using the kit provided by the present invention, but also the recovery rates 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 a good extraction effect.

[0035] Example 2

[0036] This example verifies the effect of using different zwitterionic ion exchange microspheres on urine test results.

[0037] The kit and detection method used in this example are basically the same as those in Example 1, except that this example uses multiple groups of different zwitterionic ion exchange microspheres in Table 4 as fillers for the solid phase extraction device, and performs recovery rate detection of 8 catecholamines and their metabolites on the same spiked urine sample. The results are shown in Table 5: Table 4 Differences of the zwitterionic ion exchange microspheres used in each condition of this example

[0038] Table 5: The recovery rates of 8 catecholamines and their metabolites determined by using 10 kits provided in this example

[0039] When the density of the carboxyl functional group of the zwitterionic ion exchange microspheres is higher, the recovery rate of the kit for the six alkaline catecholamines and their metabolites is higher; when the density of the quaternary ammonium functional group is higher, the recovery rate of the kit for the two acidic catecholamines is higher. It can be seen from the test results in Table 5 that when zwitterionic ion exchange microspheres with a carboxyl density of 3-5mmol / L and a quaternary ammonium density of 1-2mmol / L are used as fillers for solid phase extraction columns, the recovery rates of the eight catecholamines and their metabolites can reach a high level. In addition, under the premise that the functional group density remains unchanged, the smaller the pore size of the zwitterionic ion exchange microspheres, the more significant the adsorption effect, because the smaller the pore size, the larger the surface area of ​​the microspheres, the more active sites that can be grafted with the functional groups, and the better the adsorption effect; and the pore size is too large, which may cause interference from impurities such as macromolecular proteins and reduce the adsorption specificity of the microspheres. Therefore, the pore size of the zwitterionic ion exchange microspheres used in the kit provided by the present invention is preferably 1nm-50nm.

[0040] In terms of the preferred particle size of the zwitterionic ion exchange microspheres, it can be seen from Table 5 that the smaller the particle size, the larger the specific surface area of ​​the zwitterionic ion exchange microspheres, the more active sites there are, which is beneficial to increasing the adsorption capacity; but when the particle size is too small, the mass transfer efficiency of the analyte will be reduced, the solid phase extraction time will be increased, and the extraction efficiency will be reduced. Therefore, the particle size of the zwitterionic ion exchange microspheres in the kit provided by the present invention is preferably 30 μm-60 μm.

[0041] This example verifies through multiple groups of experiments that the carboxyl functional group density of the zwitterionic ion exchange microspheres in the kit provided by the present invention is preferably 3mmol / L-5mmol / L; the quaternary ammonium functional group density is preferably 1mmol / L-2mmol / L; the pore size is preferably 1nm-50nm; and the particle size is preferably 30μm-60μm.

[0042] Example 3

[0043] This example verifies the effect of the amount of zwitterionic ion exchange microspheres in the kit provided by the present invention on the recovery rate of catecholamines and their metabolites.

[0044] The kit and detection method used in this example are basically the same as those in Example 1, except that the dosage of the zwitterionic ion exchange microspheres is 2 mg, 3 mg, 5 mg, and 8 mg, respectively. The recovery rate detection data of the four groups obtained are shown in Table 6 below: Table 6 Effect of different dosages of zwitterionic ion exchange microspheres on analyte detection recovery

[0045] As can be seen from Table 6, the greater the amount of zwitterionic ion exchange microspheres used, the higher the recovery rate of the kit provided by the present invention for the eight catecholamines and their metabolites; when the amount of microspheres used 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 amount of zwitterionic ion exchange microspheres used in the kit provided by the present invention is 2 mg-8 mg, preferably 3 mg-5 mg.

[0046] Example 4

[0047] This example verifies the effects of different volumes of eluent used in the kit and formic acid content on the recovery rates of eight catecholamines and their metabolites.

[0048] The kit and detection process used in this example are basically the same as those in Example 1, except that: A. Under the premise of unchanged volume, the formic acid concentrations of the eluents used in the kit are 0.1%, 0.5%, 1%, 2%, 3%, and 4% respectively; B. Under the premise of a certain formic acid concentration in the eluent, 60 μL and 300 μL of eluent were used to elute the solid phase extraction column, respectively. The recovery rates of 8 catecholamines and their metabolites obtained under various conditions in this embodiment are shown in Table 7: Table 7 Effect of formic acid content in eluent and its volume on recovery

[0049] It can be seen from Table 7 that when the formic acid concentration is 1%, the recovery rates of the eight catecholamines and their metabolites all reach the maximum value; when the formic acid concentration continues to increase, the recovery rates of each component decrease to varying degrees, proving that under the premise of a certain added volume, when an eluent with a formic acid concentration of 1% is used, the recovery rates of the eight catecholamines and their metabolites are the highest. When the formic acid content of the eluent used is constant, the recovery rates of the eight catecholamines and their metabolites obtained increase with the increase in the volume of the eluent used. When the volume of the eluent used reaches 200 μL, the recovery rates of each substance no longer increase significantly, indicating that each group of substances retained by the solid phase extraction column has been basically eluted. The eluent provided by the present invention can achieve one-step elution of alkaline and acidic catecholamine analytes. Therefore, the formic acid concentration of the eluent in the detection method provided by the present invention is preferably 1%, and the volume used is preferably 200 μL.

[0050] Example 5

[0051] like Figure 4 As shown, this example verifies that the kit provided by the present invention can be used to separate and detect six catecholamines and their metabolites in the blood at one time.

[0052] A kit for synchronous detection of catecholamines and their metabolites, comprising: (1) Solid phase extraction reagent: 60 ml of 50% methanol activation solution; 60ml deionized water balance solution; 60ml first eluent: deionized water; 60ml second eluent: methanol; 20 ml eluent: methanol solution containing 1% formic acid; 10ml reconstitution solution: deionized water; (2) 50 ml blood test diluent: 10-15% methanol solution; (3) A 96-well solid phase extraction device filled with 5 mg of zwitterionic ion exchange microspheres: the particle size of the zwitterionic ion exchange microspheres is 30 μm, the pore size is 3 nm, the carboxyl group density is 4.8972 mmol / g, and the quaternary ammonium group density is 1.3564 mmol / g; (4) 6 bottles of blood standards of different concentrations: each concentration of blood standard contains 6 catecholamines and their metabolites: epinephrine, norepinephrine, dopamine, methylepinephrine, methylnorepinephrine, 3-methoxytyramine, and the remainder is blood-simulating matrix solution; (5) Blood quality control products: divided into high-quality blood control products and low-quality blood control products; (6) Blood internal standard working solution: contains deuterated versions of six catecholamines and their metabolites, with concentrations of epinephrine-d6: 40 ng / ml, norepinephrine-d6: 100 ng / ml, dopamine-d4: 40 ng / ml, methylepinephrine-d3: 20 ng / ml, methylnorepinephrine-d3: 40 ng / ml, and 3-methoxytyramine-d4: 20 ng / ml.

[0053] The specific detection steps are as follows: Step 1, adding 600 μL of methanol to a 96-well solid phase extraction plate filled with zwitterionic ion exchange microspheres for activation, and then controlling the flow rate with a positive pressure instrument to press dry; then adding 600 μL of deionized water for equilibration, and controlling the flow rate with a positive pressure instrument to press dry, to prepare an activated solid phase extraction plate; Step 2, simultaneously pipette 200 μL of each of the blood standard, blood quality control, and blood spiked sample to be tested, add 20 μL of blood internal standard working solution and 400 μL of blood test diluent, respectively, and vortex mix to obtain blood standard working solution, blood quality control working solution, and blood spiked sample solution to be tested; Step 3, take 600 μL of blood standard working solution, blood quality control working solution, and blood spiked sample solution to be tested, and transfer them to the activated solid phase extraction column prepared in step 1 simultaneously, and press dry by controlling the flow rate with a positive pressure instrument; then add 600 μL of deionized water and 600 μL of methanol in turn for two rinses, and press dry by controlling the flow rate with a positive pressure instrument; then add 100 μL of 1% formic acid-methanol solution for elution, collect the eluate, and press dry by controlling the flow rate with a positive pressure instrument; blow dry the collected eluate groups under nitrogen at 30°C, add 100 μL of deionized water for re-dissolution, and obtain a concentrated detection solution for standby use; Step 4, take 10 μL of each concentrated test solution and perform high performance liquid chromatography tandem mass spectrometry analysis. Figure 4 As shown in the chromatogram, it can be clearly seen that the solid phase extraction device with zwitterionic ion exchange microspheres as filler can also separate and mass spectrometrically detect the six catecholamines and their metabolites in blood samples with a high recovery rate.

[0054] Example 6

[0055] This example verifies the effect of adding ascorbic acid to the blood simulation matrix solution of the kit on the stability of the LC-MS / MS blood detection reagent.

[0056] Four blood standards in the kit provided in this example were selected and the effect of the amount of ascorbic acid added 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. Table 8 Effects of antioxidants and storage temperature on the stability of LC-MS / MS blood test reagents

[0057] As can be seen from Table 8, dopamine DA, epinephrine E, and norepinephrine NE cannot remain stable in the blood standard even in a storage environment of -20°C without the addition of ascorbic acid; when 0.3% ascorbic acid is added to the blood standard, the six catecholamines and their metabolites can remain relatively stable for a long time in a storage environment of -20°C. Therefore, in the kit provided by the present invention, a blood simulation matrix solution containing 0.1%-0.5% ascorbic acid is added to the blood standard solution and each group of blood quality control products.

[0058] Example 7

[0059] The kit provided in this embodiment is used in urine sample detection and blood sample detection.

[0060] A kit for detecting catecholamines and their metabolites in urine or blood samples, comprising: 1. Solid phase extraction reagent: 60 ml of 50% methanol activation solution; 60ml deionized water balance solution; 60ml first eluent: deionized water; 60ml second eluent: methanol; 20 ml eluent: methanol solution containing 1% formic acid; 10ml reconstitution solution: deionized water; 2.50ml urine test diluent: 0.02mol / L sodium carbonate-sodium bicarbonate solution; 3.50ml blood test diluent: 10%-15% methanol solution; 4.1 96-well solid phase extraction device filled with 5 mg of zwitterionic ion exchange microspheres: the particle size of the zwitterionic ion 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; 5.6 bottles of urine standards of different concentrations: each concentration of urine standard contains 8 catecholamine machine metabolites: epinephrine, norepinephrine, dopamine, metanephrine, methylnorepinephrine, 3-methoxytyramine, homovanillic acid, vanillylmandelic acid, and the rest is simulated urine matrix solution; 6.6 bottles of blood standards of different concentrations: each concentration of blood standard contains 6 catecholamines and their metabolites: epinephrine, norepinephrine, dopamine, metanephrine, methylnephrine, 3-methoxytyramine, and the balance is blood simulation matrix solution; 7. Urine quality control products: divided into urine high quality control products and urine low quality control products; 8. Blood quality control products: divided into high-quality blood control products and low-quality blood control products; 9. Urine internal standard working solution: contains 8 kinds of deuterated catecholamines and their metabolites, with concentrations of epinephrine-d6: 100ng / ml, norepinephrine-d6: 1000ng / ml, dopamine-d4: 1500ng / ml, methylepinephrine-d3: 100ng / ml, methylnorepinephrine-d3: 150ng / ml, 3-methoxytyramine-d4: 150ng / ml, homovanillic acid-d5: 20000ng / ml, vanillylmandelic acid-d3: 20000ng / ml; 10. Blood internal standard working solution: contains deuterated versions of 6 catecholamines and their metabolites, with concentrations of epinephrine-d6: 40 ng / ml, norepinephrine-d6: 100 ng / ml, dopamine-d4: 40 ng / ml, methylepinephrine-d3: 20 ng / ml, methylnorepinephrine-d3: 40 ng / ml, and 3-methoxytyramine-d4: 20 ng / ml.

[0061] The kit provided in this embodiment was used to test 8 groups of urine samples randomly selected. The specific steps were basically the same as those in Example 1. The test results are shown in Table 9: Table 9 Comparison of measured values ​​of 8 groups of urine samples

[0062] As can be seen from Table 9, the kit provided by the present invention can accurately detect the contents of 8 catecholamines and their metabolites in urine at one time.

[0063] The kit provided in this example was used to test 5 groups of serum samples randomly selected. The specific steps were basically the same as those in Example 5. The test results are shown in Table 10: Table 10 Comparison of measured values ​​of 5 groups of serum samples

[0064] As shown in Table 10, the kit provided by the present invention can be well used for detecting 6 catecholamines and their metabolites in serum samples.

[0065] This example verifies that the detection kit provided by the present invention can not only accurately detect serum samples, but also accurately separate and detect 8 catecholamines and their metabolites in urine samples at one time, greatly shortening the detection cost and detection time.

[0066] Experimental Example 1 This experimental example provides the linear range, detection limit and quantitation limit of the detection kit provided by the present invention when used to detect 8 catecholamines and their metabolites.

[0067] The kit and detection steps used in this experimental example are basically the same as those in Example 1, except that a series of standard products are used to process three groups in parallel, and the measurement range of the analytical method is determined when the recovery rate is within the range of 100% ± 20% and the total coefficient of variation does not exceed 20%. The concentration of the eight target analytes is used as the abscissa, and the peak area ratio of the analyte and its isotope internal standard is used as the ordinate. A linear regression operation is performed with a weight factor of 1 / x to obtain a linear regression equation, which is the working curve.

[0068] The detection limit and quantification limit were evaluated by the signal-to-noise ratio method. The lowest concentration of the standard was diluted in series and tested. The concentration corresponding to the signal-to-noise ratio of 3 was determined as the detection limit (LOD); the concentration corresponding to the signal-to-noise ratio of 10 or more was determined as the quantification limit (LOQ).

[0069] Table 11 Linear range, detection limit and quantification limit of 8 catecholamines and their metabolites

[0070] It can be seen from Table 11 that the detection limit and quantitative limit of the detection kit provided by the present invention for urine samples; the quantitative limit is significantly lower than that of commercially available kits, which shows that the use of the kit provided by the present invention can simplify the detection steps, reduce the detection cost, and ensure the accuracy of the detection results.

[0071] Experimental Example 2 This example determines the detection limit and quantification limit of 6 catecholamines and their metabolites in blood samples using the kit provided by the present invention.

[0072] The kit and detection steps used in this example are basically the same as those in Experimental Example 5, except that a series of standard products are used to process three groups in parallel, and the measurement range of the analytical method is determined when the recovery rate is within the range of 100% ± 20% and the total coefficient of variation does not exceed 20%. The concentration of the six target analytes is used as the abscissa, and the peak area ratio of the analyte and its isotope internal standard is used as the ordinate. A linear regression operation is performed with a weight factor of 1 / x to obtain a linear regression equation, which is the working curve.

[0073] The detection limit and quantification limit were evaluated by the signal-to-noise ratio method. The lowest concentration of the standard was diluted in series and tested. The concentration corresponding to the signal-to-noise ratio of 3 was determined as the detection limit (LOD); the concentration corresponding to the signal-to-noise ratio of 10 or more was determined as the quantification limit (LOQ).

[0074] Table 12 Linear range, detection limit and quantification limit of 6 catecholamines and their metabolites in blood

[0075] As can be seen from Table 12, the detection limit and quantification limit of blood samples were detected using the kit and detection steps provided by the present invention. The quantification limit of blood samples for the detection kit provided by the present invention is significantly lower than that of commercially available kits, and the existing kits can be completely replaced in the field of blood sample detection of catecholamines and their metabolites.

[0076] In summary, the present invention provides a kit for synchronous detection of catecholamines and their metabolites, in which zwitterionic ion exchange microspheres are used as fillers for solid phase extraction. By using zwitterionic ion exchange microspheres containing both carboxyl and quaternary ammonium groups as fillers for solid phase extraction columns, 6 catecholamines containing amino functional groups and their metabolites and 2 terminal metabolites of catecholamines containing carboxyl functional groups can be synchronously and efficiently separated, which is conducive to the detection of catecholamines and their metabolites by high performance liquid chromatography tandem mass spectrometry; the present invention improves the recovery rate of 8 catecholamines and their metabolites by solid phase extraction columns by optimizing the pore size, particle size and density of carboxyl and quaternary ammonium functional groups of zwitterionic ion exchange microspheres, adjusting the amount of eluent after solid phase extraction and the concentration of formic acid; achieving the accuracy of detecting 8 catecholamines and their metabolites in urine while simplifying the detection steps. 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 application thereof proposed in the present invention have far-reaching significance for optimizing the mass spectrometry detection process and improving the application value of catecholamines and their metabolites.

[0077] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, 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 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 filler; The solid phase extraction filler is a zwitterionic ion exchange microsphere; the zwitterionic ion exchange microsphere contains a carboxyl functional group and a quaternary ammonium functional group; The solid phase extraction reagent includes an activation solution, a balancing solution, a washing solution, an eluent, and a reconstitution solution; The LC-MS / MS detection reagents include standard products, quality control products, internal standard working solution and mobile phase.

2. The kit according to claim 1, characterized in that The carboxyl functional group density of the zwitterionic ion 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 ion exchange microspheres comprises the following specific steps: Step M1, adding a dispersant to distilled water, and stirring thoroughly 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 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; 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); The chlorine-containing aromatic compound is selected from p-chloromethylstyrene and 4-chlorostyrene; The porogen is a mixture of one or more of dichloromethane, n-dodecane, toluene, o-dichlorobenzene, and liquid paraffin; The initiator is azobisisobutyronitrile and / or benzoyl peroxide; 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; The acrylic acid ester monomer is selected from methyl acrylate, methyl methacrylate, and ethyl methacrylate; The added mass of the acrylic ester 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, 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; The tertiary amine monomer is selected from N,N-dimethylbutylamine, trimethylamine, and triethylamine; The amount of the tertiary amine monomer added is 1-10 times the mass of the chlorine-containing aromatic compound; Step M5, adding alkaline methanol solution to the second reaction product, stirring at 65-75° C., and performing ester hydrolysis reaction for 20-30 h; after cooling to room temperature, washing with ethanol, sodium bicarbonate solution, and water in sequence, and drying to obtain the zwitterionic ion exchange microsphere adsorption material.

4. The kit according to claim 1, characterized in that 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 balancing solution is deionized water; 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; The eluent is a methanol solution containing 0.1%-4% formic acid; The rehydration solution is deionized water; The standard substance is a blood standard substance and / or a urine standard substance; 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; the mobile phase A is a 0.1% formic acid aqueous solution; and the mobile phase B is methanol.

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

7. The kit according to claim 5, characterized in that The urine standard and urine quality control products include the compounds epinephrine, norepinephrine, dopamine, methylepinephrine, methylnorepinephrine, 3-methoxytyramine, homovanillic acid HVA, and vanillylmandelic acid VMA, and the remainder is a simulated urine matrix solution; The concentration range of the standard curve of each component compound in the urine standard is: epinephrine: 0.4ng / ml-80ng / ml, norepinephrine: 6ng / ml-1200ng / ml, dopamine: 10ng / ml-2000ng / ml, methylepinephrine: 0.5ng / ml-100ng / ml, methyloxynorepinephrine: 0.8ng / ml-160ng / ml, 3-methoxytyramine: 0.8ng / ml-160ng / ml, homovanillic acid: 125ng / ml-25000ng / ml, vanillylmandelic acid: 125ng / ml-25000ng / ml; The urine quality control products include urine low quality control products and urine high quality control products; The target concentrations of the components in the urine low-quality control product are: epinephrine: 4 ng / ml, norepinephrine: 60 ng / ml, dopamine: 100 ng / ml, methylepinephrine: 5 ng / ml, methylnorepinephrine: 8 ng / ml, 3-methoxytyramine: 8 ng / ml, homovanillic acid: 1250 ng / ml, vanillylmandelic acid: 1250 ng / ml; The target concentrations of the components in the urine high-quality control product are: epinephrine: 32 ng / ml, norepinephrine: 480 ng / ml, dopamine: 800 ng / ml, methylepinephrine: 40 ng / ml, methylnorepinephrine: 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 epinephrine-d6: 100ng / ml, norepinephrine-d6: 1000ng / ml, dopamine-d4: 1500ng / ml, methylepinephrine-d3: 100ng / ml, methylnorepinephrine-d3: 150ng / ml, 3-methoxytyramine-d4: 150ng / ml, homovanillic acid-d5: 20000ng / ml, and vanillylmandelic acid-d3: 20000ng / ml; The simulated urine matrix solution is an artificial urine solution containing 0.1%-0.5% acetic acid.

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

9. The detection method according to claim 8, characterized in that: The specific steps of the detection method are as follows: Step 1, taking a solid phase extraction device filled with zwitterionic ion exchange microspheres, adding an activation solution to activate it; then adding the balancing solution and squeezing it dry to prepare an activated solid phase extraction device; Step 2, simultaneously pipette 200 μL of each of the standard, quality control, and sample to be tested, add 20 μL of internal standard working solution and 400 μL of diluent, respectively, and mix to obtain standard working solution, quality control working solution, and sample solution to be tested; Step 3, synchronously transferring the standard working solution, the quality control working solution, and the sample solution to be tested into the solid phase extraction device and pressing them dry; Then, the first eluent and the second eluent are added in sequence and squeezed dry; then, the eluent is added, the eluent is collected, and squeezed dry; Each group of eluents was blown dry under nitrogen, and 100 μL of reconstitution solution was added for reconstitution to obtain a concentrated detection solution; Step 4, taking 10 μL of the concentrated detection solution of each group, and performing high performance liquid chromatography tandem mass spectrometry analysis.

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

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