Kit and method for extracting catecholamine and metabolite thereof based on paramagnetic particle method and method for detecting catecholamine and metabolite thereof by LC-MS / MS

Abstract

The invention relates to the technical field of analysis and detection, in particular to a kit and a method for extracting catecholamine and metabolites thereof based on a paramagnetic particle method and a method for detecting catecholamine and metabolites thereof by LC-MS / MS (liquid chromatography-mass spectrometry / mass spectrometry). The kit comprises magnetic beads, an activating solution, an eluent, a washing solution, a leacheate I, a leacheate II and a derivatization reagent, the catecholamine and the metabolite thereof comprise one or more of dopamine hydrochloride, epinephrine, noradrenaline hydrochloride, methoxynoradrenaline hydrochloride, methoxyepinephrine hydrochloride and 3-methoxytyramine hydrochloride. The in-vivo concentration of catecholamine and metabolites thereof in human plasma and urine is detected in a high-flux manner by adopting a magnetic bead and derivation method, and the kit has the characteristics of high sensitivity, good repeatability, high accuracy, good specificity and the like.

Description

Technical Field

[0001] This invention relates to the field of analytical detection technology, and in particular to kits, methods for extracting catecholamines and their metabolites based on magnetic beads, and methods for detecting catecholamines and their metabolites by LC-MS / MS. Background Technology

[0002] Catecholamines (CAs) mainly include epinephrine (E), norepinephrine (NE), and dopamine (DA). CAs are monoamine neurotransmitters synthesized and secreted by the adrenal medulla, adrenal neurons, and extra-adrenal chromaffin bodies. They are endogenous substances with strong physiological activity and play an important role in brain and nerve signal transduction. The levels of CAs and their metabolites in plasma and urine are closely related to various physiological and pathological phenomena in the human body. They not only play a broad regulatory role in the physiological activities of the cardiovascular system, nervous system, endocrine system, kidneys, and smooth muscle tissue, but also affect metabolism. Detecting CA levels in biological samples is of significant clinical importance for the diagnosis and treatment of diseases such as pheochromocytoma, paraganglioma, neuroblastoma, hypertension, myocardial infarction, and adrenal medullary hyperplasia, but also aids in the diagnosis of diseases such as thyroid dysfunction, congestive heart failure, diabetes, and renal insufficiency. At the same time, it is also of great significance for basic medical research such as neuroelectrophysiology.

[0003] The main metabolites of catecholamines include metanphrine (MN), norepinephrine (NMN), 3-methoxytyramine (3-MT), vanillylmandelic acid (VMA), and homovanillic acid (HVA). NMN and MN are intermediate metabolites of norepinephrine (NE) and epinephrine (E), respectively (collectively referred to as MNs). They are metabolized and produced only within adrenal medullary pheochromocytoma or PPGL tumors, and persist at high concentrations. MNs have a longer half-life and are more stable than catecholamines (CA). VMA is the main end metabolite of E and NE. Patients with pheochromocytoma secrete large amounts of epinephrine and norepinephrine, of which approximately 60% is eventually converted into VMA and excreted in the urine. HVA, structurally similar to VMA, is the end metabolite of the monoamine neurotransmitter dopamine.

[0004] Pheochromocytoma (PCC) originates from chromaffin cells in the adrenal medulla and secretes catecholamines. Paraganglioma (PGL) originates from extra-adrenal chromaffin cells and is located in the paravertebral sympathetic chain of the chest, abdomen, and pelvis. It can also originate from the parasympathetic ganglia of the glossopharyngeal and vagus nerves distributed along the neck and skull base; the latter usually do not produce catecholamines (CA). PCC accounts for 80%–85% of cases, and PGL accounts for 15%–20%. The two are collectively referred to as PPGLs (pheochromocytomas or paragangliomas), which are a rare type of secondary hypertension, with a prevalence of 0.2%–0.6% among hypertensive patients in general outpatient clinics. Patients with neuroblastoma can simultaneously secrete excessive amounts of adrenaline, noradrenaline, and dopamine. Their metabolites, VMA and HVA, are excreted through the kidneys and can be elevated in the early stages of the disease. Simultaneous detection of urinary VMA and HVA can not only indirectly reflect the excretion of catecholamines in the body, but also play an important role in the early auxiliary diagnosis and differential diagnosis of central nervous system diseases such as pheochromocytoma and neuroblastoma.

[0005] Currently, clinical methods for detecting catecholamines mainly include liquid chromatography-tandem mass spectrometry (LC-MS / MS), high-performance liquid chromatography (HPLC), capillary electrophoresis, and fluorescence spectrometry. The "Expert Consensus on the Diagnosis and Treatment of Pheochromocytoma and Paraganglioma" published in the Chinese Journal of Endocrinology and Metabolism by the Adrenal Group of the Chinese Society of Endocrinology recommends liquid chromatography-mass spectrometry (LC-MS / MS) as the primary method for determining catecholamines and their metabolites.

[0006] Currently, solid-phase extraction is used to pretreat catecholamines in the literature. Although this method can effectively separate analytes from interfering components and improve the response and recovery rate of analytes, solid-phase extraction plates are expensive and the pretreatment operation requires high technical skills from the operators.

[0007] Therefore, how to reduce testing costs, simplify operations, and at the same time ensure testing sensitivity, specificity, and accuracy are urgent problems that need to be solved.

[0008] In view of this, the present invention is proposed. Summary of the Invention

[0009] To address the aforementioned problems in the prior art, this invention proposes a kit, method, and LC-MS / MS method for extracting catecholamines and their metabolites based on magnetic beads. This method meets the requirements of relevant regulations for the linearity, accuracy, precision, residue, and recovery rate of catecholamines and their metabolites, and plays an important role in monitoring catecholamines and their metabolites in patients.

[0010] Based on this, the present invention has the following technical solution: In a first aspect, the present invention provides a kit for extracting catecholamines and their metabolites based on magnetic beads, characterized in that it comprises magnetic beads, an activation solution, a washing solution, an eluent I, an eluent II, an elution solution, and a derivatizing reagent; wherein the catecholamines and their metabolites include one or more of dopamine hydrochloride, adrenaline, norepinephrine hydrochloride, methoxynorepinephrine hydrochloride, methoxyadrenaline hydrochloride, and 3-methoxytyramine hydrochloride.

[0011] Current literature indicates that catecholamines are primarily pretreated using solid-phase extraction (SPE). However, endogenous catecholamines, especially the metabolite 3-MT, are present at extremely low concentrations in vivo. Therefore, the greatest challenge in detecting catecholamines and their metabolites in vivo lies in improving the detection sensitivity of 3-MT. Compared to SPE, magnetic bead extraction minimizes operator error, reducing costs and shortening experimental time. However, it suffers from low sensitivity and high detection limits. Therefore, to balance experimental error, cost, and detection sensitivity, this invention discovers that a derivatization reaction following magnetic bead extraction can significantly improve the detection response value, thereby enhancing detection sensitivity and stability.

[0012] According to the kit for extracting catecholamines and their metabolites based on magnetic beads provided by the present invention, the activation solution contains an aqueous solution of formic acid and acetonitrile; preferably, the volume ratio of formic acid, acetonitrile and water in the activation solution is 1:(260~300):(45~55); more preferably, it is 1:(280~285):(45~55).

[0013] According to the kit for extracting catecholamines and their metabolites based on magnetic beads provided by the present invention, the eluent I is water; and / or the eluent II is acetonitrile.

[0014] The kit for extracting catecholamines and their metabolites based on magnetic beads according to the present invention uses water as the washing solution.

[0015] According to the kit for extracting catecholamines and their metabolites based on magnetic beads provided by the present invention, the eluent is a mixture of formic acid and acetonitrile; preferably, the volume ratio of formic acid to acetonitrile in the eluent is 1:(45~55); more preferably, it is 1:50.

[0016] The kit for extracting catecholamines and their metabolites based on magnetic beads according to the present invention further includes a test sample comprising a matrix, a mixed internal standard, and a buffer solution; the matrix is ​​plasma or urine; the mixed internal standard comprises one or more of dopamine hydrochloride-d4, epinephrine-d6, norepinephrine hydrochloride-d6, methoxynorepinephrine hydrochloride-d3, methoxyepinephrine hydrochloride-d3, and 3-methoxytyramine hydrochloride-d4; and the buffer solution is ammonium acetate solution.

[0017] According to the kit for extracting catecholamines and their metabolites based on magnetic beads provided by the present invention, the derivatizing reagent is a mixture of propionic anhydride and acetonitrile. Preferably, the volume ratio of propionic anhydride to acetonitrile in the derivatizing reagent is 1:(2~6); more preferably, it is 1:(3~5).

[0018] In this invention, the test samples obtained by magnetic bead extraction are derivatized using the aforementioned derivatization reagents, which can reduce the interference of other endogenous substances on the analytes. The high-throughput detection of catecholamines and their metabolites in human plasma and urine by magnetic bead combined with derivatization can simultaneously obtain the in vivo concentrations of dopamine (DA), adrenaline (E), norepinephrine (NE), methoxynorepinephrine (NMN), methoxyadrenaline (MN), and 3-methoxytyramine (3-MT) in a single detection, which has the characteristics of high sensitivity, good repeatability, high accuracy, and good specificity.

[0019] The kit for extracting catecholamines and their metabolites based on magnetic beads according to the present invention further includes a pH adjuster; the pH adjuster is an alkaline solution containing at least one of an ammonium compound, a hydroxide compound, and a carbonate compound; preferably, the ammonium compound includes at least one of ammonia and triethylamine; the hydroxide compound includes at least one of potassium hydroxide and sodium hydroxide; and the carbonate compound includes at least one of sodium carbonate, sodium bicarbonate, potassium carbonate, and potassium bicarbonate.

[0020] Preferably, the alkaline solution is a sodium bicarbonate solution with a concentration of 15 g / L to 25 g / L, more preferably 18 g / L to 22 g / L.

[0021] Secondly, the present invention provides a method for extracting catecholamines and their metabolites based on magnetic beads, comprising: setting an operating program, starting an automated magnetic bead extractor, causing the magnetic beads to be activated sequentially by an activation solution, washed and balanced by a washing solution, grabbing the sample to be tested, washing away interfering substances with a rinsing solution, and eluting the target substance with an elution solution; after the elution solution containing the target substance is dried by nitrogen blowing, it is mixed with a pH adjuster and a derivatizing reagent for derivatization.

[0022] Preferably, the activating solution, washing solution, rinsing solution, and elution solution are the reagents in the above-mentioned kit.

[0023] Preferably, the pH adjuster and the derivatizing reagent are the reagents in the above-described kit.

[0024] Thirdly, the present invention provides a method for detecting catecholamines and their metabolites by LC-MS / MS, comprising: pretreatment using the above-described method for extracting catecholamines and their metabolites based on magnetic beads, followed by detection on the instrument; The mobile phase for liquid chromatography detection includes mobile phase A and mobile phase B. Mobile phase A is a 0.05%~0.15% (v / v) aqueous solution of formic acid, and mobile phase B is a 0.05%~0.15% (v / v) methanol solution of formic acid.

[0025] In this invention, catecholamines and their metabolites are first extracted from the sample using magnetic beads, then processed using a derivatization method, and finally detected by instrument. Sample processing is performed in a magnetic bead analyzer; after processing, derivatization reagent is added to the sample, and pretreatment is completed in half an hour, followed by instrument detection.

[0026] The sample to be tested includes a matrix, a mixed internal standard, and a buffer solution; the matrix is ​​plasma or urine; the mixed internal standard includes one or more of dopamine hydrochloride-d4, epinephrine-d6, norepinephrine hydrochloride-d6, methoxynorepinephrine hydrochloride-d3, methoxyepinephrine hydrochloride-d3, and 3-methoxytyramine hydrochloride-d4; the buffer solution is ammonium acetate solution.

[0027] According to the present invention, a method for detecting catecholamines and their metabolites by LC-MS / MS is provided, wherein the gradient elution conditions of the mobile phase include:

[0028] "%" represents volume percentage, and the sum of the volume percentages of mobile phase A and mobile phase B is 1.

[0029] According to the present invention, a method for detecting catecholamines and their metabolites by LC-MS / MS is provided, wherein the flow rate of the mobile phase is 0.5 mL / min.

[0030] According to the present invention, a method for detecting catecholamines and their metabolites by LC-MS / MS is provided, wherein the chromatographic column for liquid phase detection includes C18.

[0031] Preferably, the chromatographic column is a C18 column with dimensions of 3.0*100mm and 3.0μm.

[0032] According to the present invention, a method for detecting catecholamines and their metabolites by LC-MS / MS is provided, wherein the mass spectrometry parameters include:

[0033] Based on this, the technical solution of the present invention has the following beneficial effects: The kits, methods, and LC-MS / MS methods for the extraction of catecholamines and their metabolites based on magnetic beads provided by this invention enable high-throughput detection of catecholamines and their metabolites in human plasma and urine using a magnetic bead-derivative method. A single detection can simultaneously obtain the in vivo concentrations of dopamine (DA), epinephrine (E), norepinephrine (NE), methoxynorepinephrine (NMN), methoxyepinephrine (MN), and 3-methoxytyramine (3-MT), exhibiting high sensitivity, good repeatability, high accuracy, and good specificity. In this method, the linear range of DA, E, NMN and MN is 10~10000 pg / mL, the linear range of NE is 20~20000 pg / mL, and the linear range of 3-MT is 4~4000 pg / mL, with correlation coefficients r≥0.990 for all. The coefficient of variation (CV) of the precision of the lower limit of quantitation, low value, medium value and high value quality control samples is ≤15%, the relative deviation (B) of the accuracy is ≤±15%, the spiked recovery rate is 85%~115%, and the residue meets the requirements. Detailed Implementation

[0034] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0035] Unless otherwise specified, all raw materials used in the examples and comparative examples are commercially available conventional raw materials, and the technical means used are conventional means well known to those skilled in the art.

[0036] Example 1 This embodiment provides a kit for extracting catecholamines and their metabolites based on magnetic beads, comprising: 1. Main compounds and reagents: Standards: Dopamine hydrochloride, epinephrine, norepinephrine hydrochloride, methoxynorepinephrine hydrochloride, methoxytyramine hydrochloride.

[0037] Isotope markers: dopamine hydrochloride-d4, adrenaline-d6, norepinephrine hydrochloride-d6, methoxynorepinephrine hydrochloride-d3, methoxyadrenaline hydrochloride-d3, 3-methoxytyramine hydrochloride-d4.

[0038] Main reagents: methanol, formic acid, isoascorbic acid, sodium bicarbonate, propionic anhydride, BSA, PBS, purified water.

[0039] 2. Reagent preparation: Reagent 1: Weigh 100 mg of isoascorbic acid into 100 mL of purified water, then add 0.5 mL of formic acid and mix well.

[0040] Reagent 2: Weigh 10g of BSA into 1000mL of PBS and mix well.

[0041] Reagent 3: Weigh 2g of sodium bicarbonate into 100mL of purified water and mix well.

[0042] Reagent 4: Transfer 10 mL of propionic anhydride and 40 mL of acetonitrile and mix well.

[0043] Reagent 5: Transfer 30 μL of formic acid, 8.5 mL of acetonitrile and 1.5 mL of purified water and mix well.

[0044] Reagent 6: Transfer 0.2 mL of formic acid and 10 mL of acetonitrile and mix well.

[0045] Reagent 7: Weigh 200 mg of magnetic beads (WCX magnetic beads), add 2 mL of 50% methanol, and mix well.

[0046] 3. Preparation of stock solution, secondary stock solution and working solution: Stock solutions: Using a 0.0001 g electronic balance, weigh appropriate amounts of dopamine hydrochloride, epinephrine, norepinephrine hydrochloride, methoxynorepinephrine hydrochloride, methoxynorepinephrine hydrochloride, 3-methoxytyramine hydrochloride, dopamine-d4 hydrochloride, epinephrine-d6, norepinephrine-d6 hydrochloride, methoxynorepinephrine-d3 hydrochloride, methoxynorepinephrine-d3 hydrochloride, and 3-methoxytyramine-d4 hydrochloride, and place them in 2 mL volumetric flasks respectively. Dissolve them with reagent 1 and dilute to the mark to prepare a stock solution with a concentration of 1 mg / mL.

[0047] Secondary stock solutions: Transfer 20 μL of dopamine stock solution, 20 μL of adrenaline stock solution, 100 μL of norepinephrine stock solution, 20 μL of methoxynorepinephrine stock solution, 20 μL of methoxynorepinephrine stock solution, and 10 μL of 3-methoxytyramine stock solution. Transfer 3810 μL of reagent 1 and mix thoroughly. This is the secondary stock solution of the compound, with the following concentrations: dopamine: 5000 ng / mL, adrenaline: 5000 ng / mL, norepinephrine: 25000 ng / mL, methoxynorepinephrine: 5000 ng / mL, methoxynorepinephrine: 5000 ng / mL, and 3-methoxytyramine: 2500 ng / mL.

[0048] Internal standard secondary stock solutions: Mix 10 μL of dopamine-d4 with 990 μL of reagent 1, 10 μL of epinephrine-d6 with 990 μL of reagent 1, 10 μL of norepinephrine-d6 with 990 μL of reagent 1, 10 μL of methoxynorepinephrine-d3 with 990 μL of reagent 1, 10 μL of methoxynorepinephrine-d3 with 990 μL of reagent 1, and 10 μL of 3-methoxytyramine-d4 with 990 μL of reagent 1. The concentration of each of the six isotope markers in the secondary stock solutions is 10 μg / mL.

[0049] Preparation of working solution: Transfer 800 μL of secondary stock solution and 3200 μL of reagent 1, and mix thoroughly.

[0050] Preparation of internal standard working solution: Transfer 40 μL of dopamine-d4 secondary stock solution, 40 μL of adrenaline-d6 secondary stock solution, 80 μL of norepinephrine-d6 secondary stock solution, 40 μL of methoxynorepinephrine-d3 secondary stock solution, 40 μL of methoxynorepinephrine-d3 secondary stock solution, and 20 μL of 3-methoxytyramine-d4 secondary stock solution, and transfer 3740 μL of methanol to prepare 4 mL of internal standard secondary stock solution 2. Transfer 200 μL of internal standard secondary stock solution 2, add 3800 μL of methanol, and mix well to obtain the internal standard working solution.

[0051] 4. Preparation of calibrators and quality control samples: Transfer 150 μL of working solution and 14.85 mL of reagent 2 (H0), mix well, and the result is calibrator C6.

[0052] Prepare calibrators C1~C6, and quality control samples LQC, MQC, and HQC according to the preparation methods in the table below.

[0053] Table 1 This embodiment further provides a method for extracting catecholamines and their metabolites based on magnetic beads, including: 1. Sample pretreatment 1) Add reagents and samples to a 2 mL 96-well plate: Add reagent 5 and reagent 7 to the first and / or seventh columns, and add 20 μL of reagent 7 (magnetic bead solution) and 280 μL of reagent 5 (activation solution) to each well. Add purified water (washing solution) to the second and / or eighth columns, 300 μL to each well; Add the test sample (plasma / urine) to the third and / or ninth column, 400 μL of sample to each well. Plasma: Transfer 200 μL of plasma, add 50 μL of internal standard, and then add 150 μL of 20 mM ammonium acetate solution; Urine: Take 20 μL of urine sample, dilute with purified water to 2 mL, mix well, take 200 μL of diluted urine, add 50 μL of internal standard, and then add 150 μL of 20 mM ammonium acetate solution (buffer solution); Add purified water (eluent I) to the fourth and / or tenth columns, 300 μL per well; Add acetonitrile (eluent II) to the fifth and / or eleventh columns, 300 μL per well; Add reagent 6 (elution buffer) to the sixth and / or twelfth column, 200 μL to each well; 2) Set the operating program, start the automated magnetic bead extractor, and after the program is completed, transfer the reagents in the sixth and / or twelfth columns to the sample plate and blow them dry with nitrogen. 3) Transfer 100 μL of reagent 3 (pH adjuster) and 50 μL of reagent 4 (derivative) into the injection plate and shake at 1000 rpm for 10 min; 4) On-machine testing and analysis.

[0054] This embodiment further provides a method for detecting catecholamines and their metabolites by LC-MS / MS, including: 1. Instrument parameters 1) Mass spectrometry parameters Table 2 2) Liquid phase parameters Table 3 Experimental Example 1 1. Precision Prepare samples at three concentrations: limit of quantitation (LOQ) and quality control. Process the samples according to the pretreatment method, processing five parallel samples and three batches consecutively. Calculate precision and accuracy. Calculate the coefficient of variation (CV) and relative deviation (B) of the samples, requiring a CV ≤ 15% and a B ≤ ±15%.

[0055] The experimental results are shown in the table below: Table 4 Table 5 Table 6 Inter-batch precision is shown in the table below: Table 7 Conclusion: The coefficient of variation (CV) for the lower limit of quantitation, low-value quality control, medium-value quality control and high-value quality control of each compound is ≤15%, and the relative deviation (B) of the accuracy is ≤ ±15%.

[0056] 2. Linear Configure calibrators and quality control samples, process the samples according to the pretreatment method, and repeat the test three times for each calibrator. Calculate the mean test result (Y). Calculate a linear regression equation with the calibrator target concentration (Xi) as the independent variable and the mean test result as the dependent variable, requiring a correlation coefficient r ≥ 0.990.

[0057] Table 8 Conclusion: The linear range of DA, E, NMN and MN is 10~10000 pg / mL, the linear range of NE is 20~20000 pg / mL, and the linear range of 3-MT is 4~4000 pg / mL. All compounds show good linearity within the linear range.

[0058] 3. Accuracy Prepare low- and high-value spiked samples. Add standard at a 5% spiking rate, i.e., transfer 10 μL of standard to 190 μL of plasma / diluted urine to prepare two spiked recovery samples. Process the samples according to the pretreatment method. The required spiked recovery rate is 85%–115%.

[0059] Table 9 Conclusion: The recoveries of spiked samples with low and high values ​​were both 85%–115%.

[0060] 4. Residue Prepare upper limit of quantitation (UPQ) and lower limit of quantitation (LLQ) samples, perform pretreatment, and evaluate for residues by first testing the UQ (C6) sample, followed by the LQ sample, repeating this process 6 times. The accuracy of the LQ should be ≤15%, and the CV should be ≤15%.

[0061] Table 10 Conclusion: The accuracy of the lower limit of quantitation in the experimental results is ≤15%, and the CV is ≤15%.

[0062] Comparative Example 1 This comparative example provides a kit, method, and LC-MS / MS method for the extraction of catecholamines and their metabolites based on magnetic beads. The only difference between this example and Example 1 is that the pretreatment only uses derivatization reaction and does not involve magnetic bead extraction.

[0063] The specific steps are as follows: Sample pretreatment: (1) Accurately pipette 200 μL of calibrator / quality control / sample to be tested into different 1.5 mL centrifuge tubes, add 50 μL of internal standard to each tube, and vortex mix for 30 s; (2) Add 400 μL of acetonitrile solution sequentially, vortex at 2500 rpm for 5 min, and centrifuge at 13000 rpm for 5 min at 4℃; (3) Take 200 μL of the supernatant after centrifugation into a 96-well plate and place the 96-well plate on a nitrogen blower to blow nitrogen for 20 min; (4) Transfer 50 μL of reagent 3 (pH adjuster) into a 96-well plate after nitrogen blowing, and shake at 1000 rpm for 2 min; (5) Transfer 20 μL of reagent 4 (derivative reagent) into the above 96-well plate, seal the film, place it in a 96-well plate constant temperature shaker, shake at 40℃ and 1000 rpm for 15 min; (6) On-machine testing and analysis.

[0064] Comparative Example 2 This comparative example provides a kit, method, and LC-MS / MS method for extracting catecholamines and their metabolites based on magnetic beads. The only difference between this example and Example 1 is that the pretreatment method uses solid phase extraction (SPE).

[0065] The specific steps are performed according to the "sample preparation" method in Example 1 of CN115327008A "Detection Method of Catecholamines and Their Metabolites in Plasma and Urine".

[0066] Experimental Example 2 The detection performance of Comparative Examples 1 and 2 was tested according to the method of Experimental Example 1. At the same time, the same sample was treated as a control according to the detection method of Example 1. The sample concentrations are shown in Table 11 below.

[0067] Table 11 The above samples were tested according to the detection methods of Example 1, Comparative Example 1, and Comparative Example 2, respectively.

[0068] The test results are shown in Table 12 below.

[0069] Table 12 The results for Example 1 are obtained using magnetic beads and derivatization, while those for Comparative Example 1 are obtained using derivatization. SPE is the result of the test in Comparative Example 2. Based on the comparison of different methods, lower limits of quantitation (LOQs), and corresponding peak areas, it can be concluded that Example 1 is superior to Comparative Examples 1 and 2.

[0070] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A kit for extracting catecholamines and their metabolites based on magnetic beads, characterized in that, Includes magnetic beads, activation solution, washing solution, eluent I and eluent II, elution solution, and derivatizing reagent; The catecholamines and their metabolites include one or more of dopamine hydrochloride, epinephrine, norepinephrine hydrochloride, methoxynorepinephrine hydrochloride, and 3-methoxytyramine hydrochloride.

2. The kit for extracting catecholamines and their metabolites based on magnetic beads according to claim 1, characterized in that, The activation solution contains an aqueous solution of formic acid and acetonitrile; Preferably, the volume ratio of formic acid, acetonitrile and water in the activation solution is 1:(260~300):(45~55).

3. The kit for extracting catecholamines and their metabolites based on magnetic beads according to claim 1 or 2, characterized in that, The rinsing solution I is water; And / or, the eluent II is acetonitrile.

4. The kit for extracting catecholamines and their metabolites based on magnetic beads according to any one of claims 1 to 3, characterized in that, The washing solution is water; And / or, the eluent is a mixture of formic acid and acetonitrile; preferably, the volume ratio of formic acid to acetonitrile in the eluent is 1:(45~55).

5. The kit for extracting catecholamines and their metabolites based on magnetic beads according to any one of claims 1 to 4, characterized in that, The kit also contains a test sample, which includes a matrix, a mixed internal standard, and a buffer solution; The matrix is ​​plasma or urine; The mixed internal standard includes one or more of dopamine hydrochloride-d4, adrenaline-d6, norepinephrine hydrochloride-d6, methoxynorepinephrine hydrochloride-d3, methoxyadrenaline hydrochloride-d3, and 3-methoxytyramine hydrochloride-d4. The buffer solution is an ammonium acetate solution.

6. The kit for extracting catecholamines and their metabolites based on magnetic beads according to any one of claims 1 to 5, characterized in that, The derivatizing reagent is a mixture of propionic anhydride and acetonitrile. Preferably, the volume ratio of propionic anhydride to acetonitrile in the derivatizing reagent is 1:(2~6). And / or, the kit further contains a pH adjuster; preferably, the pH adjuster is an alkaline solution; preferably, the alkaline solution contains at least one of an ammonium compound, a hydroxide compound, and a carbonate compound; preferably, the ammonium compound includes at least one of ammonia and triethylamine; the hydroxide compound includes at least one of potassium hydroxide and sodium hydroxide; the carbonate compound includes at least one of sodium carbonate, sodium bicarbonate, potassium carbonate, and potassium bicarbonate. Preferably, the alkaline solution is a sodium bicarbonate solution with a concentration of 15 g / L to 25 g / L.

7. A method for extracting catecholamines and their metabolites based on magnetic beads, characterized in that, include: The operating procedure is set, and the automated magnetic bead extractor is started. The magnetic beads are then activated by an activation solution, washed and balanced by a washing solution, the sample to be tested is picked up, interfering substances are washed away by a rinsing solution, and the target substance is eluted by an elution solution. The elution solution containing the target substance is dried by nitrogen blowing and then mixed with a pH adjuster and a derivatizing reagent for derivatization.

8. The method for extracting catecholamines and their metabolites based on magnetic beads according to claim 7, characterized in that, The activation solution, washing solution, rinsing solution, and elution solution are the reagents in the kit described in any one of claims 1 to 6; And / or, the pH adjuster and the derivatizing reagent are the reagents in the kit described in any one of claims 1 to 6.

9. A method for detecting catecholamines and their metabolites by LC-MS / MS, characterized in that, include: After pretreatment using the method for extracting catecholamines and their metabolites based on magnetic beads as described in claim 7 or 8, the samples are then subjected to instrumental detection. The mobile phase for liquid chromatography detection includes mobile phase A and mobile phase B, wherein mobile phase A is a 0.05%~0.15% (v / v) aqueous solution of formic acid, and mobile phase B is a 0.05%~0.15% (v / v) methanol solution of formic acid. Preferably, the gradient elution conditions of the mobile phase include: "%" represents volume percentage, and the sum of the volume percentages of mobile phase A and mobile phase B is 1; Preferably, the flow rate of the mobile phase is 0.5 mL / min; Preferably, the chromatographic column for liquid phase detection includes C18.

10. The method for detecting catecholamines and their metabolites by LC-MS / MS according to claim 9, characterized in that, Mass spectrometry parameters include: 。

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