Detection method of catecholamine substances and metabolites thereof, freeze-dried powder and application of freeze-dried powder

Abstract

The invention discloses a detection method of catecholamine substances and metabolites thereof, freeze-dried powder and application of the freeze-dried powder. The detection method of catecholamine and metabolites thereof comprises the following step: detecting a sample to be detected by adopting a liquid chromatography-tandem mass spectrometry method. According to the present invention, the sample pretreatment and the detection condition optimization are performed so as to achieve the efficient and accurate detection of noradrenaline, epinephrine, variepinephrine, noradrenaline, dopamine and 3-methoxytyramine, the sensitivity is high, the specificity is good, and the new reliable detection method is provided for the detection of catecholamine and metabolites thereof in the blood sample.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a detection method for catecholamine substances and metabolites thereof, a lyophilized powder and applications thereof. Background Art

[0002] Catecholamine neurotransmitters are closely related to neuroendocrine tumors such as pheochromocytoma, paraganglioma and neuroblastoma. Therefore, if this substance can be detected during the diagnosis and treatment process, it will be of great significance for the prevention, treatment and monitoring of the disease.

[0003] At present, commonly used detection methods include chemiluminescence, electrochemical and fluorescence detection methods. These methods are relatively simple, the detection equipment is small and easy to configure, and the detection cost is low. However, these detection methods have low accuracy in clinical use and are easily interfered by the external environment and matrix components. They cannot completely cover the detection of catecholamine components, and some methods may be harmful to the human body. Therefore, many medical institutions have successively deployed liquid-mass spectrometry equipment for the detection of related projects. This method has high specificity, high throughput, stable and reliable results, and is the first choice of many large medical institutions. However, this method has strict sample processing methods and there is no unified standard.

[0004] Therefore, a simple and complete pretreatment process is also particularly important for the liquid quality detection of catecholamines. Although commonly used pretreatment methods such as protein precipitation and derivatization can meet the detection needs to a certain extent, the detected substances are not perfect, the detection methods are relatively complex and the sensitivity needs to be improved, and they cannot meet the high-throughput needs of medical institutions. The reasons for such defects are, on the one hand, due to the large number of detected substances and poor stability of the substances, and on the other hand, the limitations of the detection method. The method itself has cumbersome steps and cannot be run multiple times at the same time, resulting in insufficient efficiency. To solve the defects of such methods, we can only start from the chemical properties of the detected substances, specifically extract the substances to be tested, and the process cannot be too cumbersome to meet the needs of large samples. Summary of the invention

[0005] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention provides a method for detecting catecholamines and their metabolites.

[0006] The present invention also provides a kit.

[0007] The invention also provides an application of the kit.

[0008] The invention also provides a freeze-dried powder of catecholamine and its metabolites.

[0009] The invention also provides a method for preparing freeze-dried powder of catecholamine and its metabolites.

[0010] The invention also provides an application of freeze-dried powder of catecholamine and its metabolites.

[0011] According to the first aspect of the present invention, the present invention provides a method for detecting catecholamines and their metabolites, the method comprising the following steps: using liquid chromatography-tandem mass spectrometry to detect the sample to be detected; the mobile phase of the liquid chromatography comprises mobile phase A and mobile phase B, the mobile phase A is an aqueous solution containing 0.01-1% formic acid, and the mobile phase B is acetonitrile; the catecholamines and their metabolites include: norepinephrine, epinephrine, metanephrine, nometanephrine, dopamine and 3-methoxytyramine. The detection method is not intended for the diagnosis or treatment of diseases.

[0012] In some embodiments of the present invention, the mobile phase is subjected to gradient elution according to the following gradient:

[0013] 0.00-0.60min, the volume percentage of mobile phase B is 1%-5%, and the balance is mobile phase A;

[0014] 0.60-3.50min, the volume percentage of mobile phase B is 1%-5% to 18%-22%, and the balance is mobile phase A;

[0015] 3.50-3.51min, the volume percentage of mobile phase B is 18%-22% to 92%-98%, and the balance is mobile phase A;

[0016] 3.51-4.20min, the volume percentage of mobile phase B is 92%-98%, and the balance is mobile phase A;

[0017] 4.20-5.00min, the volume percentage of mobile phase B is 92%-98% to 1%-5%, and the balance is mobile phase A.

[0018] In some embodiments of the present invention, the mobile phase is subjected to gradient elution according to the following gradient:

[0019] 0.00-0.60min, the volume percentage of mobile phase B is 2%, and the balance is mobile phase A;

[0020] 0.60-3.50min, the volume percentage of mobile phase B is 2% to 20%, and the balance is mobile phase A;

[0021] 3.50-3.51min, the volume percentage of mobile phase B is 20% to 95%, and the balance is mobile phase A;

[0022] 3.51-4.20min, the volume percentage of mobile phase B is 95%, and the balance is mobile phase A;

[0023] 4.20-5.00min, the volume percentage of mobile phase B is 95% to 2%, and the balance is mobile phase A.

[0024] In some embodiments of the present invention, the flow rate of the mobile phase is 0.1-0.5 mL / min.

[0025] In some embodiments of the present invention, the flow rate of the mobile phase is 0.2-0.4 mL / min.

[0026] In some embodiments of the present invention, the flow rate of the mobile phase is 0.3 mL / min.

[0027] In some embodiments of the present invention, the chromatographic column used for the liquid chromatography separation comprises an ACQUITY UPLCHSS PFP column (2.1×100 mm, 1.8 μm).

[0028] In some embodiments of the present invention, the injection volume of the liquid chromatography is 1-20 μL.

[0029] In some embodiments of the present invention, the injection volume of the liquid chromatography is 5-10 μL.

[0030] In some embodiments of the present invention, the column temperature of the liquid chromatography is 30-45°C.

[0031] In some embodiments of the present invention, the mass spectrometer used for the mass spectrometry includes Waters Xevo TQ-SIVD.

[0032] In some embodiments of the present invention, the mass spectrometry detection conditions include: ion source: electrospray ionization source ESI; ion source temperature: 140-160°C; capillary voltage: 0.3-0.7 kV; desolvation gas temperature: 450-600°C; desolvation gas flow rate: 850-1000 L·h -1 ; Cone hole gas flow rate: 140-160L·h -1 .

[0033] In some embodiments of the present invention, the mass spectrometry detection uses an electrospray ion source (ESI) and an MRM detection mode, and the ion pairs for detecting the precursor ions / product ions of norepinephrine, epinephrine, metanephrine, norepinephrine, dopamine and 3-methoxytyramine are as follows: E, m / z 166.1>m / z 107.0 and m / z 166.1>m / z 135.0; NE, m / z 152.0>m / z 107.1 and m / z 152 .0>m / z135.1; NMN, m / z166.1>m / z106.1 and m / z166.1>m / z134.0; MN, m / z180.0>m / z148.1 and m / z180.0>m / z165.0; DA, m / z137.0>m / z91.0 and m / z154.0>m / z91.0; 3-MT, m / z151.0>m / z91.0 and m / z151.0>m / z119.1.

[0034] In some embodiments of the present invention, the precursor ion / product ion pairs for detecting the internal standards of norepinephrine, epinephrine, metanephrine, norepinephrine, dopamine and 3-methoxytyramine are as follows: the detection ion pairs of the internal standard DA_d4 are m / z141.0>m / z95.0 and m / z158.0>m / z95.0; the detection ion pairs of the internal standard 3-MT_d4 are m / z155.0>m / z95.0 and m / z155.0>m / z123.0; the detection ion pairs of the internal standard NE_d6 are The detection ion pairs are m / z158.1>m / z111.0 and m / z158.1>m / z139.1; the detection ion pairs of internal standard NMN_d3 are m / z169.0>m / z109.1 and m / z169.0>m / z137.1; the detection ion pairs of internal standard E_d3 are m / z169.1>m / z107.1 and m / z169.1>m / z135.0; the detection ion pairs of internal standard MN_d3 are m / z183.0>m / z151.1 and m / z183.0>m / z168.1.

[0035] In some embodiments of the present invention, the sample to be tested may be serum, plasma or whole blood.

[0036] In some embodiments of the present invention, a step of pre-treating the sample to be detected is also included, and the step includes:

[0037] (1) adding an internal standard solution to a sample to be tested to obtain a mixed solution of the sample to be tested;

[0038] (2) centrifuging the mixed solution of the sample to be tested and collecting the liquid phase;

[0039] (3) transferring the liquid phase to the SPE solid phase extraction plate, pressing it with positive pressure, adding eluent for cleaning, eluting, and collecting the eluent;

[0040] (4) The eluate is concentrated to dryness, and after adding a reconstitution solution to dissolve it, a sample for machine testing is obtained.

[0041] In some embodiments of the present invention, the internal standard solution is a mixed solution containing E_d3, NE_d6, MN_d3, NMN_d3, DA_d4, and 3-MT_d4.

[0042] In some embodiments of the present invention, in the internal standard solution, the concentration of E_d3 is 300-500 pg / mL, the concentration of NE_d6 is 300-500 pg / mL, the concentration of MN_d3 is 60-100 pg / mL, the concentration of NMN_d3 is 60-100 pg / mL, the concentration of DA_d4 is 300-500 pg / mL, and the concentration of 3-MT_d4 is 60-100 pg / mL.

[0043] In some embodiments of the present invention, in the internal standard solution, the concentration of E_d3 is 350-450 pg / mL, the concentration of NE_d6 is 350-450 pg / mL, the concentration of MN_d3 is 70-90 pg / mL, the concentration of NMN_d3 is 70-90 pg / mL, the concentration of DA_d4 is 350-450 pg / mL, and the concentration of 3-MT_d4 is 70-90 pg / mL.

[0044] In some embodiments of the present invention, in the internal standard solution, the concentration of E_d3 is 380-420 pg / mL, the concentration of NE_d6 is 380-420 pg / mL, the concentration of MN_d3 is 75-85 pg / mL, the concentration of NMN_d3 is 75-85 pg / mL, the concentration of DA_d4 is 380-420 pg / mL, and the concentration of 3-MT_d4 is 75-85 pg / mL.

[0045] In some embodiments of the present invention, the added volume ratio of the sample to be tested to the internal standard solution is 1:(1-3).

[0046] In some embodiments of the present invention, the added volume ratio of the sample to be tested to the internal standard solution is 3:5.

[0047] In some embodiments of the present invention, the centrifugation conditions are 10000-12000 rpm, 2-6°C for 3-6 min.

[0048] In some embodiments of the present invention, the SPE solid phase extraction plate comprises a SPE 96-well plate.

[0049] In some embodiments of the present invention, the liquid phase is transferred to the SPE solid phase extraction plate with a flow rate of 1-2 s / drop under positive pressure.

[0050] In some embodiments of the invention, the SPE solid phase extraction plate also includes an activation step, specifically: adding 150-250 μL of acetonitrile solution containing 0.8-1.2% formic acid to the sample well, pressing it with positive pressure, and then adding 150-250 μL of pure water to the sample well, and pressing it with positive pressure.

[0051] In some embodiments of the invention, the adding of the eluent for cleaning comprises cleaning with eluent 1 and eluent 2 in sequence and then pressing down with positive pressure;

[0052] The eluent 1 includes pure water, 8-12 mM ammonium formate solution, and 8-12 mM ammonium bicarbonate solution;

[0053] The eluent 2 includes methanol or acetonitrile.

[0054] In some embodiments of the present invention, the flow rate of the cleaning is 1-2 s / drop.

[0055] In some embodiments of the present invention, the elution solution used for elution comprises 80-90% acetonitrile solution of 0.8-1.2% formic acid.

[0056] In some embodiments of the present invention, the flow rate of the elution is 1-2 s / drop.

[0057] In some embodiments of the present invention, the reconstitution solution comprises an aqueous solution containing 0.01%-1% formic acid and water.

[0058] According to a second aspect of the present invention, the present invention provides a detection kit for detecting norepinephrine, epinephrine, metanephrine, nometanephrine, dopamine and 3-methoxytyramine in a sample to be tested, the kit comprising:

[0059] Reagent I: 80-90% acetonitrile in water containing 0.8-1.2% formic acid;

[0060] Reagent II: acetonitrile;

[0061] Reagent III: an aqueous solution containing 0.01%-1% formic acid.

[0062] In some embodiments of the present invention, the kit further comprises: a standard and an internal standard working solution.

[0063] In some embodiments of the present invention, the internal standard working solution includes: E_d3, NE_d6, MN_d3, NMN_d3, DA_d4, and 3-MT_d4.

[0064] In some embodiments of the present invention, in the internal standard working solution, the concentration of E_d3 is 300-500pg / mL, the concentration of NE_d6 is 300-500pg / mL, the concentration of MN_d3 is 60-100pg / mL, the concentration of NMN_d3 is 60-100pg / mL, the concentration of DA_d4 is 300-500pg / mL, and the concentration of 3-MT_d4 is 60-100pg / mL.

[0065] In some embodiments of the present invention, in the internal standard working solution, the concentration of E_d3 is 350-450pg / mL, the concentration of NE_d6 is 350-450pg / mL, the concentration of MN_d3 is 70-90pg / mL, the concentration of NMN_d3 is 70-90pg / mL, the concentration of DA_d4 is 350-450pg / mL, and the concentration of 3-MT_d4 is 70-90pg / mL.

[0066] In some embodiments of the present invention, in the internal standard working solution, the concentration of E_d3 is 380-420 pg / mL, the concentration of NE_d6 is 380-420 pg / mL, the concentration of MN_d3 is 75-85 pg / mL, the concentration of NMN_d3 is 75-85 pg / mL, the concentration of DA_d4 is 380-420 pg / mL, and the concentration of 3-MT_d4 is 75-85 pg / mL.

[0067] In some embodiments of the present invention, the above-mentioned kit can be used with the method for simultaneously detecting catecholamines and their metabolites described in the present invention.

[0068] The detection method of the present invention can be used for the qualitative detection or quantitative detection of norepinephrine, epinephrine, metanephrine, normetanephrine, dopamine and 3-methoxytyramine. When performing quantitative detection, a standard curve is prepared using standard products of different concentrations, with the concentration of the standard solution as the X-axis and the ratio of the peak area of ​​the standard and the internal standard as the Y-axis, and a linear regression analysis is performed, and a regression equation is obtained by weighting with "1 / X". The peak area ratio of the component to be tested in the sample and its internal standard is substituted into the standard curve equation to calculate the concentration of norepinephrine, epinephrine, metanephrine, normetanephrine, dopamine and 3-methoxytyramine in the sample. The ratio of the sample detection signal intensity to the isotope internal standard is proportional to the concentration of the component to be tested in the sample. The present invention finds that the correlation coefficient of the standard curve is high when the detection method of the present invention is used for detection, and the requirements of quantitative detection can be met.

[0069] According to the third aspect of the present invention, the present invention proposes the use of the kit in the detection of norepinephrine, epinephrine, metanephrine, normetanephrine, dopamine and 3-methoxytyramine.

[0070] According to the fourth aspect of the present invention, the present invention also provides a lyophilized powder of catecholamines and their metabolites, wherein the dosage ratio of each component is as follows: catecholamines and their metabolites ≤ 10 mg, 3-8 wt% stabilizer, 1-10 wt% excipient, 0.01-0.1 wt% antioxidant and 0.001-0.1 wt% preservative, based on a preparation volume of 1 L;

[0071] In some embodiments of the invention, the stabilizer comprises BSA.

[0072] In some embodiments of the invention, the excipient comprises mannitol.

[0073] In some embodiments of the present invention, the antioxidant protective agent includes sodium pyrophosphite and ascorbic acid.

[0074] In some embodiments of the present invention, the preservatives include Proclin300 and Krovin300.

[0075] In some embodiments of the present invention, based on a preparation volume of 1 L, the dosage ratio of each component is: catecholamines and their metabolites ≤ 10 mg, 4-6 wt% stabilizer, 2-4 wt% excipient, 0.03-0.07 wt% antioxidant and 0.005-0.02 wt% preservative.

[0076] In some embodiments of the present invention, based on a preparation volume of 1 L, the dosage ratio of each component is: catecholamines and their metabolites ≤ 10 mg, 5 wt% stabilizer, 3 wt% excipient, 0.05 wt% antioxidant and 0.005-0.02 wt% preservative.

[0077] In some embodiments of the present invention, the components further include water.

[0078] In some embodiments of the present invention, the catecholamines and their metabolites include at least one of norepinephrine, epinephrine, metanephrine, normetanephrine, dopamine and 3-methoxytyramine.

[0079] According to the fifth aspect of the present invention, the present invention also provides a method for preparing freeze-dried powder of catecholamines and metabolites thereof, comprising the following steps: mixing the components to obtain freeze-dried powder.

[0080] According to the sixth aspect of the present invention, the present invention further proposes a use of the above-mentioned freeze-dried powder of catecholamines and their metabolites in preserving catecholamines and their metabolites.

[0081] In some embodiments of the present invention, the catecholamines and their metabolites include norepinephrine, epinephrine, metanephrine, nometanephrine, dopamine and 3-methoxytyramine.

[0082] According to some embodiments of the present invention, at least the following beneficial effects are achieved: the present invention can achieve efficient and accurate detection of norepinephrine, epinephrine, metanephrine, norepinephrine, dopamine and 3-methoxytyramine by optimizing sample pretreatment and detection conditions. The linear range of epinephrine and norepinephrine is 12-2400 pg / mL, the linear range of metanephrine, norepinephrine and dopamine is 5-1000 pg / mL, the linear range of 3-methoxytyramine is 1-200 pg / mL, the spiked recovery is between 88.42% and 111.19%, and the precision is between 0.77% and 3.88%. The method provided by the present invention realizes the efficient and accurate detection of norepinephrine, epinephrine, metanephrine, normetanephrine, dopamine and 3-methoxytyramine in blood samples, has high sensitivity and good specificity, and provides a new and reliable detection method for the detection of catecholamines and their metabolites in blood samples.

[0083] The present invention also provides a freeze-dried powder of catecholamines and metabolites thereof, which can be stored for a long time by freeze-drying a certain concentration of catecholamine substances and a combination of multiple protective agents. BRIEF DESCRIPTION OF THE DRAWINGS

[0084] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0085] Figure 1 It is a chromatogram of norepinephrine, epinephrine, metanephrine, normetanephrine, dopamine and 3-methoxytyramine in Example 1 of the present invention. DETAILED DESCRIPTION

[0086] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0087] Example 1

[0088] This embodiment provides a method for detecting catecholamines and their metabolites, which specifically comprises the following steps:

[0089] 1. Sample pre-treatment steps

[0090] 1. Preparation of isotope internal standard working solution

[0091] (1) Preparation of mother solution

[0092] Accurately weigh the epinephrine_d3 (E_d3) standard product, and dissolve it in a methanol solution containing 0.1% ascorbic acid to obtain an epinephrine_d3 standard product mother solution, wherein the concentration of epinephrine_d3 is 1 mg / mL;

[0093] Accurately weigh the norepinephrine_d6 (NE_d6) standard, and dissolve it in a methanol solution containing 0.1% ascorbic acid to obtain a norepinephrine_d6 standard stock solution, wherein the concentration of norepinephrine_d6 is 1 mg / mL;

[0094] Accurately weigh the metanephrine_d3 (MN_d3) standard, and dissolve it in a methanol solution containing 0.1% ascorbic acid to obtain a metanephrine_d3 standard mother solution, wherein the concentration of the metanephrine_d3 is 1 mg / mL;

[0095] Accurately weigh the Normetanephrine_d3 (NMN_d3) standard, and dissolve it in a methanol solution containing 0.1% ascorbic acid to obtain a Normetanephrine_d3 standard stock solution, wherein the concentration of Normetanephrine_d3 is 1 mg / mL;

[0096] Accurately weigh the dopamine_d4 (DA_d4) standard, and dissolve it in a methanol solution containing 0.1% ascorbic acid to obtain a dopamine_d4 standard stock solution, wherein the concentration of dopamine_d4 is 1 mg / mL;

[0097] Accurately weigh the 3-methoxytyramine_d4 (3-MT_d4) standard, and dissolve it in a methanol solution containing 0.1% ascorbic acid to obtain a 3-methoxytyramine_d4 standard mother solution, wherein the concentration of 3-methoxytyramine_d4 is 1 mg / mL.

[0098] (2) Preparation of standard working solution

[0099] The isotope internal standard mother solutions of the six compounds prepared above were diluted and mixed with 0.04% formic acid aqueous solution to obtain a mixed internal standard working solution in which the concentrations of epinephrine_d3, norepinephrine_d6, and dopamine_d4 were 400 pg / mL, and the concentrations of metanephrine_d3, normetanephrine_d3, and 3-methoxytyramine_d4 were 80 pg / mL.

[0100] 2. Processing of plasma samples to be tested

[0101] (1) Centrifugation of blood: Take at least 2 mL of the blood to be tested and centrifuge it at 3000 rpm for 15 min to separate the supernatant plasma, which is used as the plasma sample to be tested and stored at 80°C for later use.

[0102] (2) Pretreatment of plasma samples to be tested: 300 μL of the plasma sample to be tested was taken into a 1.5 mL centrifuge tube, and 500 μL of a mixed internal standard working solution containing known concentrations of catecholamines and metabolites stored at low temperature was added. The mixture was vortexed on a vortex mixer for 5 min, and centrifuged at 12,000 rpm at 4°C for 5 min. The supernatant was collected to obtain the pretreated sample.

[0103] (3) Preparation of plasma samples to be tested

[0104] 1) SPE96 plate balancing: add 200 μL of 85% ACN containing 1% FA to each well of the SPE96 plate, apply positive pressure, and discard; then add 200 μL of purified water to each well of the SPE96 plate, apply positive pressure, and discard, and control the flow rate to 1-2 s / drop;

[0105] 2) Sample loading: add 600 μL of pre-treated sample to each well of the equilibrated SPE96-well plate, apply positive pressure, discard, and control the flow rate to 1-2 s / drop;

[0106] 3) Elution: Add 200 μL of purified water (elution solution 1) to each well, apply positive pressure, and discard; then add 200 μL of ACN (elution solution 2) to each well, apply positive pressure, and discard, and control the flow rate to 1-2 s / drop;

[0107] 4) Elution: Add 50 μL of 85% ACN (elution solution) containing 1% FA to each well for elution, apply positive pressure, collect into 96-well plate, control the flow rate to 1-2 s / drop, and collect the eluate;

[0108] 5) Redissolution: Dry the collected eluate with a nitrogen blower; redissolve with 50 μL 0.1% formic acid water; shake for 5 min at 1200 rpm in a 96-well microplate mixer; centrifuge for 5 min at 4000 rpm and 4° C. to obtain the sample to be tested.

[0109] 3. Treatment of linear solutions to be tested and quality control solutions

[0110] Preparation of analyte working solution: accurately weigh each standard substance and place it in a volumetric flask, dissolve it with a methanol solution containing 1% ascorbic acid, and dilute to the mark to prepare a stock solution with a concentration of 1 mg / mL. Continue to dilute the stock solution with a methanol solution containing 1% ascorbic acid to obtain a mixed intermediate solution, in which the concentration of standard substances E and NE is 6 ng / mL, the concentration of MN, NMN, and DA is 2.5 ng / mL, and the concentration of 3-MT is 0.5 ng / mL.

[0111] The intermediate solution was gradiently diluted with an aqueous solution containing 0.1% ascorbic acid to obtain 8 working solutions with different concentration levels (work 1 to work 6 are linear working solutions, and work 7 to work 8 are quality control working solutions). The concentrations of catecholamines and their metabolites in the working solutions are shown in Table 1 below.

[0112] Table 1 Catecholamine and its metabolite concentrations at each level (unit: ng / mL):

[0113] Serial number E NE MN NMN DA 3-MT work1 0.24 0.24 0.1 0.1 0.1 0.02 work2 0.48 0.48 0.2 0.2 0.2 0.04 work3 1.2 1.2 0.5 0.5 0.5 0.1 work4 4.8 4.8 2 2 2 0.4 work5 19.2 19.2 8 8 8 1.6 work6 48 48 20 20 20 4 work7 6 10 2 3 1 0.4 work8 18 30 6 9 3 1.2

[0114] Preparation of linear solutions and quality control solutions: Accurately pipette 20 μL of the above analyte working solutions of different concentrations and mix them evenly with 380 μL of 5% BSA to obtain linear solutions and quality control solutions of different concentrations. The concentrations are shown in Table 2.

[0115] Table 2 Concentration of each indicator in each linear solution and quality control solution (unit: pg / mL)

[0116]

[0117]

[0118] Treatment of linear solution and quality control solution: Take 300 μL of the solution to be tested (linear solution or quality control solution) into a 1.5 mL centrifuge tube, add 500 μL of the mixed internal standard working solution containing known concentrations of catecholamines and metabolites stored at low temperature, vortex mix on a vortex mixer for 5 minutes, and centrifuge at 12000 rpm and 4°C for 5 minutes to obtain the pretreated sample;

[0119] SPE96 well plate balance: add 200 μL of 85% ACN containing 1% FA to each well of the SPE96 well plate, apply positive pressure, and discard; then add 200 μL of purified water to each well of the SPE96 well plate, apply positive pressure, and discard, and control the flow rate to 1-2 s / drop;

[0120] Sample loading: add 600 μL of pre-treated sample to each well of the equilibrated SPE96-well plate, apply positive pressure, discard, and control the flow rate to 1-2 s / drop;

[0121] Elution: add 200 μL purified water to each well, apply positive pressure, and discard; then add 200 μL ACN to each well, apply positive pressure, and discard, controlling the flow rate to 1-2 s / drop;

[0122] Elution: add 50 μL of 85% ACN containing 1% FA to each well, apply positive pressure, collect into 96-well plate, and control the flow rate to 1-2 s / drop;

[0123] Reconstitution: Dry the collected eluate with a nitrogen blower; reconstitute with 50 μL 0.1% FA water; oscillate at 1200 rpm for 5 min in a 96-well microplate mixer; centrifuge at 4000 rpm and 4°C for 5 min to obtain the linear solution to be tested and the quality control solution.

[0124] 2. Test samples on the machine

[0125] The analytes (plasma samples to be tested, linear solutions to be tested or quality control solutions) are separated by liquid chromatography-mass spectrometry (LC-MS) detection method.

[0126] The liquid phase conditions of high performance liquid chromatography are shown in Table 3, wherein the chromatographic column is ACQUITY UPLC HSS PFP, 2.1*100 mm, 1.8 μm, the flow rate is 0.3 mL / min, and the column temperature is 35° C. The mobile phase A is a 0.1% formic acid aqueous solution, the mobile phase B is an acetonitrile solution, the volume ratio of phase A: phase B is 98%-5%: 2%-95%, and the flow rate is 0.3 mL / min.

[0127] Electrospray ionization (ESI) and MRM were used for mass spectrometry detection. The mass spectrometer was Waters Xevo TQ-S IVD. The mass spectrometry conditions are shown in Table 4, and the detection results are shown in Table 5.

[0128] Table 3

[0129]

[0130] Table 4 Liquid quality parameters

[0131] Mass spectrometry conditions ESI+ Capillary voltage(kV) 0.5 Ion source temperature (℃) 150 Desolvation temperature (℃) 500 Desolvation gas flow rate (L / Hr) 900 Cone gas flow rate (L / hr) 150

[0132] Table 5 Ion pair information

[0133]

[0134]

[0135] Table 6 Retention time

[0136]

[0137] Typical spectra of the detected catecholamines and their metabolites norepinephrine, epinephrine, metanephrine, norepinephrine, dopamine and 3-methoxytyramine are shown in Figure 1 As shown, an electrospray ion source (ESI) and MRM detection mode are used to detect the precursor ion / product ion ion pairs of norepinephrine, epinephrine, metanephrine, normetanephrine, dopamine and 3-methoxytyramine as shown in Table 5, and the retention time is shown in Table 6. It can be seen from the chart that the peak shape detected by each substance is well symmetrical, the peak shape is narrow, there is no interfering peak around the peak of the analyte, and the analysis time is short.

[0138] 3. Construction of standard curve

[0139] Construction method: Standard curves were prepared using standards of different concentrations, with the concentration of the standard solution as the X-axis and the ratio of the standard and internal standard peak areas as the Y-axis. Linear regression analysis was performed and the regression equation was obtained by weighting with "1 / X". The peak area ratio of the component to be tested in the sample and its internal standard was substituted into the standard curve equation to calculate the concentrations of norepinephrine, epinephrine, metanephrine, normetanephrine, dopamine and 3-methoxytyramine in the sample.

[0140] Table 7 Linearity, limit of quantitation and limit of detection results

[0141]

[0142]

[0143] The results are shown in Table 7. It can be seen from the table that E is within the linear range of 12-2400 pg / mL; NE is within the linear range of 12-2400 pg / mL; MN is within the linear range of 5-1000 pg / mL; NMN is within the linear range of 5-1000 pg / mL; DA is within the linear range of 5-1000 pg / mL; 3-MT is within the linear range of 1-200 pg / mL; the linear correlation is good, and the correlation coefficient r 2 They are 0.9994, 0.9991, 0.9994, 0.9994, 0.9964 and 0.9971 respectively.

[0144] Example 2 Methodology Verification

[0145] This example tests the recovery and precision of the detection method in Example 1.

[0146] Detection method: The standard working solution (work7, work8) provided in Example 1 was prepared into two concentrations, high and low, for spike recovery experiment and precision experiment. The determination was carried out according to the method provided in Example 1. The analysis was repeated for 3 batches. The recovery and precision results are shown in the table below.

[0147] Table 8 Recovery and precision results

[0148]

[0149]

[0150] The results are shown in Table 8. It can be seen from the table that the recovery rate of this method is 88.42%-111.19%, and the precision is within 4%.

[0151] Example 3

[0152] In this example, the components of the eluent 1, eluent 2, eluent and reconstitution solution used in part 2 of the method for detecting catecholamines and their metabolites in Example 1, i.e., the processing part of the plasma sample to be tested, are screened.

[0153] 1. Optimization of eluent 1:

[0154] The experimental method is the same as that of Example 1, except that the eluent 1 is purified water, 10 mM ammonium formate solution, and 10 mM ammonium bicarbonate solution, respectively.

[0155] The results are shown in Table 9. It can be seen from the table that when purified water is used as eluent 1, the responses of epinephrine, norepinephrine, dopamine, and 3-methoxytyramine are the highest, while for metanephrine and norepinephrine, the responses are better when 10 mmol / L ammonium bicarbonate solution is used as eluent 1. However, compared with pure water, the advantage is not obvious, and purified water is simple and easy to obtain, and no additional preparation steps are required. Therefore, purified water is used as eluent 1.

[0156] Table 9

[0157] Eluent 1 Name E NE MN NMN DA 3-MT Purified water 11746.0 6796.1 6658.2 3763.8 3864.5 5510.9 10 mM ammonium formate 8976.0 3922.9 6325.4 3627.9 3693.8 2766.4 10 mM ammonium bicarbonate 9913.0 4876.3 6831.2 3857.1 3471.1 3039.8

[0158] 2. Optimization of eluent 2:

[0159] The experimental method is the same as that of Example 1, except that the eluent 2 is methanol or acetonitrile.

[0160] The results are shown in Table 10. It can be seen from the table that when acetonitrile is used as eluent 2, the responses of the six indicators are all stronger, and acetonitrile is preferably used as eluent 2.

[0161] Table 10

[0162] Eluent 2 Name E NE MN NMN DA 3-MT Methanol 42501.6 21508.8 42571.6 25988.1 21165.2 24001.8 Acetonitrile 45923.6 21845.7 43819.8 27233.7 21973.0 25208.5

[0163] 3. Elution buffer optimization:

[0164] The experimental method is the same as that of Example 1, except that the eluents are a methanol solution containing 1% formic acid and an acetonitrile aqueous solution containing 1% formic acid.

[0165] The results are shown in Table 11. It can be seen from the table that when 85% acetonitrile solution containing 1% formic acid is used as the eluent, the responses of the six indicators are all stronger, indicating that this solution has a stronger elution effect, and 85% acetonitrile solution containing 1% formic acid is preferably used as the eluent.

[0166] Table 11

[0167] Eluent Name E NE MN NMN DA 3-MT Methanol containing 1% formic acid 40906.9 17616.0 44250.3 28524.2 21435.2 24200.1 Contains 1% formic acid 85% acetonitrile 53649.6 24041.8 47951.7 28997.2 24324.2 25036.1

[0168] 4. Reconstitution solution optimization:

[0169] The experimental method is the same as that of Example 1, except that the reconstitution solutions are pure water and 0.1% formic acid aqueous solution, respectively.

[0170] The results are shown in Table 12. It can be seen from the table that when the 0.1% formic acid aqueous solution is used as the re-solution, the responses of the six indicators are all stronger, and after adding formic acid, the compound is easier to ionize. The 0.1% formic acid aqueous solution is preferably used as the re-solution.

[0171] Table 12

[0172] Reconstitution solution name E NE MN NMN DA 3-MT Purified water 45577.6 18982.3 45370.5 28122.6 21449.4 24454.5 0.1% formic acid aqueous solution 47838.1 20176.9 45923.0 28768.2 22532.1 24853.8

[0173] Example 4

[0174] In this embodiment, a lyophilized powder of catecholamines and their metabolites is prepared, which is obtained by freeze-drying a lyophilized powder solution of catecholamines and their metabolites. The composition range of the components contained in the lyophilized powder solution of catecholamines and their metabolites per liter of aqueous solution is as follows: 5wt% BSA, 3wt% mannitol, 0.05wt% ascorbic acid (VC), 0.01wt% Krovin300, and catecholamines and their metabolites ≤10mg.

[0175] Catecholamines and their metabolites are tested by preparing freeze-dried powder solutions of catecholamines and their metabolites using quality control products 1 and 2 as examples. The components of quality control products 1 and 2 are shown in Table 13. Freeze-dried powder solutions of catecholamines and their metabolites can also be prepared using other calibrators 1-6.

[0176] Quality control product 1 Preparation method of freeze-dried powder solution of catecholamines and their metabolites: freeze-drying the freeze-dried powder solution containing catecholamines and their metabolites; wherein, in the freeze-dried powder solution containing catecholamines and their metabolites, the final concentration of epinephrine (E) is 300pg / mL, the final concentration of norepinephrine (NE) is 500pg / mL, the final concentration of metanephrine (MN) is 100pg / mL, the final concentration of normetanephrine (NMN) is 150pg / mL, the final concentration of dopamine (DA) is 50pg / mL, the final concentration of 3-methoxytyramine (3-MT) is 20pg / mL, the mass fraction of BSA is 5%, the mass fraction of mannitol is 3%, the mass fraction of ascorbic acid is 0.05%, the mass fraction of Krovin300 is 0.01%, and the balance is water.

[0177] Preparation method of quality control product 2 freeze-dried powder solution of catecholamines and their metabolites: freeze-dry the freeze-dried powder solution containing catecholamines and their metabolites; wherein, in the freeze-dried powder solution containing catecholamines and their metabolites, the final concentration of epinephrine (E) is 900pg / mL, the final concentration of norepinephrine (NE) is 1500pg / mL, the final concentration of metanephrine (MN) is 300pg / mL, the final concentration of normetanephrine (NMN) is 450pg / mL, the final concentration of dopamine (DA) is 150pg / mL, the final concentration of 3-methoxytyramine (3-MT) is 60pg / mL, the mass fraction of BSA is 5%, the mass fraction of mannitol is 3%, the mass fraction of ascorbic acid is 0.05%, the mass fraction of Krovin300 is 0.01%, and the balance is water.

[0178] Table 13 The concentrations of catecholamines and their metabolites in the lyophilized products are as follows (unit: pg / mL)

[0179]

[0180]

[0181] After preparing the lyophilized powder solution of catecholamine and its metabolites according to the above formula, lyophilization was performed to obtain the corresponding lyophilized powder. The lyophilized powder was placed in a 60°C constant temperature and humidity chamber for 10 days and 19 days, respectively, and compared with the detection value at -80°C, and the test results obtained are shown in Table 14 below.

[0182] Table 14

[0183]

[0184]

[0185] As can be seen from Table 14, the relative deviations of the test results at 60°C for 10 days and 19 days compared with the test results at -20°C are within the range of ±15.0. Calculation based on the Arrhenius formula shows that the catecholamine and metabolite solutions prepared according to the formulation of the present invention can be stably stored at 2-8°C for 2.5 years after freeze-drying.

[0186] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A method for detecting catecholamines and their metabolites, characterized in that: The method comprises the following steps: using liquid chromatography-tandem mass spectrometry to detect the sample to be detected; The mobile phase of the liquid chromatography comprises mobile phase A and mobile phase B, wherein the mobile phase A is an aqueous solution containing 0.01-1% formic acid, and the mobile phase B is acetonitrile; The catecholamines and their metabolites include norepinephrine, epinephrine, metanephrine, nometanephrine, dopamine and 3-methoxytyramine.

2. The detection method according to claim 1, characterized in that: The mobile phase is gradient eluted according to the following gradient: 0.00-0.60min, the volume percentage of mobile phase B is 1%-5%, and the balance is mobile phase A; 0.60-3.50min, the volume percentage of mobile phase B is 1%-5% to 18%-22%, and the balance is mobile phase A; 3.50-3.51min, the volume percentage of mobile phase B is 18%-22% to 92%-98%, and the balance is mobile phase A; 3.51-4.20min, the volume percentage of mobile phase B is 92%-98%, and the balance is mobile phase A; 4.20-5.00min, the volume percentage of mobile phase B is 92%-98% to 1%-5%, and the balance is mobile phase A.

3. The detection method according to claim 1, characterized in that: The flow rate of the mobile phase is 0.1-0.5 mL / min; And / or, the chromatographic column used in the liquid chromatography comprises an ACQUITY UPLC HSS PFP column; And / or, the injection volume of the liquid chromatography is 1-20 μL; And / or, the column temperature of the liquid chromatography is 30-45°C.

4. The detection method according to claim 1, characterized in that: The mass spectrometer used in the mass spectrometry includes WatersXevo TQ-S IVD; And / or, the mass spectrometry detection conditions include: ion source: electrospray ionization source ESI; ion source temperature: 140-160°C; capillary voltage: 0.3-0.7 kV; desolvation gas temperature: 450-600°C; desolvation gas flow rate: 850-1000 L·h -1 ; Cone hole gas flow rate: 140-160L·h -1 .

5. The detection method according to claim 1, characterized in that: The mass spectrometry detection adopts an electrospray ion source and an MRM detection mode, and the ion pairs for detecting the precursor ions / product ions of norepinephrine, epinephrine, metanephrine, norepinephrine, dopamine and 3-methoxytyramine are as follows: E, m / z 166.1>m / z 107.0 and m / z 166.1>m / z 135.0; NE, m / z 152.0>m / z 107.1 and m / z 152.0>m / z 135 .1; NMN, m / z166.1>m / z106.1 and m / z166.1>m / z134.0; MN, m / z180.0>m / z148.1 and m / z180.0>m / z165.0; DA, m / z137.0>m / z91.0 and m / z154.0>m / z91.0; 3-MT, m / z151.0>m / z91.0 and m / z151.0>m / z119.1; and / or, the precursor ion / product ion pairs for detecting the internal standards of norepinephrine, epinephrine, metanephrine, norepinephrine, dopamine, and 3-methoxytyramine are as follows: the detection ion pairs of the internal standard DA_d4 are m / z 141.0>m / z 95.0 and m / z 158.0>m / z 95.0; the detection ion pairs of the internal standard 3-MT_d4 are m / z 155.0>m / z 95.0 and m / z 155.0>m / z 123.0; the detection ion pairs of the internal standard NE_d6 are m / z 158.1>m / z111.0 and m / z158.1>m / z139.1; the detection ion pairs of internal standard NMN_d3 are m / z169.0>m / z109.1 and m / z169.0>m / z137.1; the detection ion pairs of internal standard E_d3 are m / z169.1>m / z107.1 and m / z169.1>m / z135.0; the detection ion pairs of internal standard MN_d3 are m / z183.0>m / z151.1 and m / z183.0>m / z168.

1.

6. The detection method according to claim 1, characterized in that: The method also includes the steps of pre-treating the sample to be detected, the steps comprising: (1) adding an internal standard solution to a sample to be tested to obtain a mixed solution of the sample to be tested; (2) centrifuging the mixed solution of the sample to be tested and collecting the liquid phase; (3) transferring the liquid phase to the SPE solid phase extraction plate, pressing it with positive pressure, adding eluent for cleaning, eluting, and collecting the eluate; (4) Concentrating the eluate to dryness, adding a reconstitution solution to dissolve the eluate, and obtaining a sample for machine testing; Preferably, the internal standard solution is a mixed solution containing E_d3, NE_d6, MN_d3, NMN_d3, DA_d4, and 3-MT_d4; Preferably, the added volume ratio of the sample to be tested to the internal standard solution is 1:(1-3); Preferably, the adding of the eluent for cleaning comprises cleaning with eluent 1 and eluent 2 in sequence and then pressing down with positive pressure; The eluent 1 includes pure water, 8-12 mM ammonium formate solution, and 8-12 mM ammonium bicarbonate solution; The eluent 2 includes methanol or acetonitrile.

7. A detection kit for detecting norepinephrine, epinephrine, metanephrine, normetanephrine, dopamine and 3-methoxytyramine in a sample to be tested, characterized in that: The kit contains: Reagent I: 80-90% acetonitrile in water containing 0.8-1.2% formic acid; Reagent II: acetonitrile; Reagent III: an aqueous solution containing 0.01%-1% formic acid.

8. Use of the kit according to claim 7 in the detection of norepinephrine, epinephrine, metanephrine, normetanephrine, dopamine and 3-methoxytyramine.

9. A lyophilized powder of catecholamines and their metabolites, characterized in that: Based on a preparation volume of 1 L, the dosage ratio of each component is: catecholamine and its metabolites ≤ 10 mg, 3-8 wt% stabilizer, 1-10 wt% excipient, 0.01-0.1 wt% antioxidant and 0.001-0.1 wt% preservative; Preferably, the stabilizer comprises BSA; Preferably, the excipient comprises mannitol; Preferably, the antioxidant protective agent includes sodium pyrophosphite and ascorbic acid; Preferably, the preservatives include Proclin300 and Krovin300.

10. Use of the lyophilized powder of catecholamines and their metabolites according to claim 9 in preserving catecholamines and their metabolites; Preferably, the catecholamines and their metabolites include at least one of norepinephrine, epinephrine, metanephrine, nometanephrine, dopamine and 3-methoxytyramine.

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