Catecholamine and metabolite detection kit and application thereof

Abstract

The invention discloses a catecholamine and metabolite detection kit and application thereof. The kit comprises a calibration product, a quality control product, an internal standard solution, an eluent and a leacheate, efficient enrichment and purification of a target object in a sample can be realized in cooperation with a magnetic microsphere adsorption-elution pretreatment process, and then liquid chromatography-tandem mass spectrometry (LC-MS / MS) detection is performed. Compared with existing solid-phase extraction (SPE), derivatization or protein precipitation and other methods, the scheme is simplified in operation and high in detection efficiency, can more easily meet clinical high-throughput and automatic detection requirements, and can be widely applied to diagnosis and follow-up monitoring of pheochromocytoma, parangganglioma and other diseases.

Description

Technical Field

[0001] The present invention relates to the technical field of kits, in particular to a kit for detecting catecholamines and metabolites and applications thereof. Background Art

[0002] Catecholamines are an important class of neurotransmitters and hormones, primarily including dopamine (DA), norepinephrine (NE), and epinephrine (E). They are widely present in the central nervous system and peripheral tissues. Catecholamines participate in a variety of physiological and pathological processes in the body, such as blood pressure regulation, metabolic control, and stress response, and are important biomarkers for clinical conditions such as cardiovascular disease and neuroendocrine disorders. Their metabolites, including metanephrine (MN), normetanephrine (NMN), and 3-methoxytyramine (3-MT), can be detected in combination with the parent catecholamines to provide more comprehensive information for disease diagnosis and treatment assessment.

[0003] At present, the main applications of clinical detection of catecholamines and their metabolites in body fluids include screening, diagnosis and follow-up of pheochromocytomas and paragangliomas. Liquid chromatography-tandem mass spectrometry (LC-MS / MS) has become the main means of detecting catecholamines due to its high sensitivity and strong specificity. However, since the concentration of such substances in body fluids is low and they are easily interfered by drugs and endogenous impurities, effective sample pretreatment is required before detection. Currently commonly used pretreatment methods include protein precipitation, solid phase extraction (SPE) and derivatization. Among them, solid phase extraction has good purification ability and can effectively remove interfering substances, but this method has cumbersome steps, long operation time, and high technical requirements for personnel, which is not conducive to the needs of high-throughput and automated detection in clinical laboratories.

[0004] Among the existing disclosed technologies, for example, the Chinese invention patent with publication number CN116223690A discloses the use of SPE combined with nitrogen blow concentration to extract catecholamines and their metabolites from plasma. Although it has a certain separation ability, the overall process is time-consuming, the operating cost is high, and it is not easy to achieve automation. The Chinese invention patent with publication number CN117250288A proposes a detection method based on a derivatization reaction, but this method has high requirements for derivatization reagents and is prone to side reactions, resulting in detection interference. The Chinese invention patent with publication number CN106442837A discloses the extraction of the target by protein precipitation and derivatization, but the derivatization agent dansyl chloride used has high toxicity and can only detect three catecholamine substances, and its scope of application is limited.

[0005] Overall, existing technologies have significant deficiencies in detection efficiency, pre-processing complexity, safety, and automation compatibility. Especially with the growing demand for high-throughput testing, traditional SPE and derivatization methods are no longer able to meet the demands of rapid, efficient, and automated clinical testing. Therefore, there is an urgent need to develop a simple, safe, and automated protocol for the detection of catecholamines and their metabolites. Summary of the Invention

[0006] In view of the shortcomings of the existing technology, the purpose of the present invention is to provide a catecholamine and metabolite detection kit and its application.

[0007] The above-mentioned object of the present invention is achieved through the following technical solutions:

[0008] Catecholamine and metabolite detection kits, including calibrators, quality controls, internal standards, eluents, and rinses;

[0009] The calibrators and quality control materials are freeze-dried powders containing norepinephrine (NE), epinephrine (E), dopamine (DA), methylnorepinephrine (NMN), methylepinephrine (MN) and 3-methoxytyramine (3-MT);

[0010] The internal standard solution is a methanol aqueous solution including NE-d6, E-d6, DA-d4, NMN-d3, MN-d3 and 3-MT-d4:

[0011] Among them, NE-d6, E-d6, DA-d4, NMN-d3, MN-d3 and 3-MT-d4 are norepinephrine (NE), epinephrine (E), dopamine (DA), methylnorepinephrine (NMN), methylepinephrine (MN) and 3-methoxytyramine (3-MT) with the hydrogen in the original molecule replaced with deuterium;

[0012] "d3" means that there are three deuterium atoms replacing the original three hydrogen atoms;

[0013] "d4" means that there are 4 deuterium atoms replacing the original 4 hydrogen atoms;

[0014] "d6" means that 6 deuterium atoms replace the original 6 hydrogen atoms;

[0015] Eluent: formic acid aqueous solution;

[0016] Eluent: methanol solution.

[0017] Furthermore, the quality control products include three concentrations: low, medium and high;

[0018] The low-concentration quality control product includes the following components:

[0019] Norepinephrine: 300 pg / mL;

[0020] Epinephrine, dopamine, norepinephrine, and metanephrine: 100 pg / mL;

[0021] 3-Methoxytyramine: 40 pg / mL;

[0022] The medium concentration quality control product includes the following components:

[0023] Norepinephrine: 1800 pg / mL;

[0024] Epinephrine, dopamine, norepinephrine, and metanephrine: 600 pg / mL;

[0025] 3-Methoxytyramine: 240 pg / mL;

[0026] The high-concentration quality control product includes the following components:

[0027] Norepinephrine: 4500 pg / mL;

[0028] Epinephrine, dopamine, norepinephrine, and metanephrine: 1500 pg / mL;

[0029] 3-Methoxytyramine: 600 pg / mL.

[0030] Furthermore, the target concentration range of each substance when the calibrator is working is as follows:

[0031] NE: 60 pg / mL to 6000 pg / mL;

[0032] E, DA, MN, NMN: 20.0pg / mL~2000.0pg / mL;

[0033] 3-MT: 10.0pg / mL~1000.0pg / mL.

[0034] Furthermore, the target concentration range of each substance during operation of the internal standard solution is as follows:

[0035] NE-d6: 50 ng / mL;

[0036] E-d6, DA-d4, MN-d3, NMN-d3: 30ng / mL;

[0037] 3-MT-d4: 15ng / mL.

[0038] Furthermore, the concentration of the eluent is 0.05-0.2% (v / v) formic acid aqueous solution.

[0039] A detection method using the above-mentioned catecholamine and metabolite detection kit is characterized by comprising the following steps:

[0040] S1: Reconstitute the calibrator and quality control materials separately with ultrapure water;

[0041] 400 μL of plasma, reconstituted calibrators, and quality controls were mixed with 10 μL of internal standard solution and 290 μL of ultrapure water at 2000 rpm for 2 minutes.

[0042] S2: Add methanol to activate the magnetic microspheres, and then rinse with ultrapure water;

[0043] Equilibration: Rinse with ultrapure water to make the surface environment of the microspheres similar to that of the sample to prevent the target from being adsorbed when the sample is added later.

[0044] S3: Add the sample solution mixed in step S1 and allow it to fully contact and bind with the magnetic microspheres;

[0045] S4: rinse with ultrapure water;

[0046] S5: Remove impurities and interferences with eluent

[0047] S6: Elute the target with an eluent to release it from the microspheres and into the final collection solution.

[0048] S7: Detection of catecholamines and their metabolites by LC-MS / MS.

[0049] Furthermore, the temperature is maintained at 2-8° C. during the entire sample pre-treatment process from step S1 to step S6 .

[0050] Furthermore, after the calibrator, quality control product and plasma sample are redissolved or mixed in step S1, an antioxidant or stabilizer is additionally added, wherein the antioxidant or stabilizer is selected from at least one of ascorbic acid, EDTA, or BHT, and the amount used is 0.01 to 0.1% (v / v) of the total sample volume.

[0051] Furthermore, when liquid chromatography-tandem mass spectrometry (LC-MS / MS) is used for detection in step S7, the chromatographic conditions include:

[0052] (1) Use Phenomenex Kinetex PFP chromatographic column or Shim-pack Velox PFPP chromatographic column;

[0053] (2) Gradient elution was performed using mobile phase A and mobile phase B, wherein:

[0054] Mobile phase A is an aqueous solution containing 0.05-0.1% (v / v) formic acid and 10-50 mM ammonium acetate;

[0055] Mobile phase B was a methanol solution containing 0–0.1% (v / v) formic acid;

[0056] The flow rate was 0.60 mL / min and the injection volume was 5 to 10 μL.

[0057] Furthermore, the gradient elution procedure is as follows:

[0058] 0.00~1.00min: mobile phase A transitions from 100% to 100%, mobile phase B transitions from 0% to 0%;

[0059] 1.00-2.50 min: mobile phase A decreased from 100% to 90%, and mobile phase B increased from 0% to 10%;

[0060] 2.50~4.00min: mobile phase A is reduced from 90% to 20%, and mobile phase B is increased from 10% to 80%;

[0061] 4.00-4.20 min: Mobile phase A is reduced from 20% to 5%, and mobile phase B is increased from 80% to 95%;

[0062] 4.20-4.50 min: mobile phase A is maintained at 5%, mobile phase B is maintained at 95%;

[0063] 4.50-5.00 min: Mobile phase A returns to 100%, mobile phase B returns to 0%, until stop.

[0064] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in the following aspects:

[0065] 1. By using magnetic microspheres to enrich and elute catecholamines and their metabolites in plasma, the tedious operational steps in traditional solid-phase extraction (SPE) or derivatization methods are greatly reduced, the overall detection process is shortened, and the application of automated workstations is facilitated, thereby improving detection throughput.

[0066] 2. There is no need to use highly toxic derivatization reagents, and the pretreatment process is more friendly to the environment and operators; at the same time, the magnetic bead method combined with small volume sample processing can reduce the use of organic solvents and other reagents.

[0067] 3. Based on magnetic bead enrichment, catecholamines and metabolites are quantitatively analyzed by LC-MS / MS, which significantly improves the sensitivity and specificity of the method, reduces the impact of impurities and interferences, lowers the limit of quantification, and expands the scope of application.

[0068] 4. The kit of the present invention can not only detect three common catecholamines, dopamine, norepinephrine, and epinephrine, but also simultaneously detect their metabolites NMN, MN, and 3-MT, greatly improving the comprehensiveness and accuracy of the diagnosis and follow-up of related diseases.

[0069] 5. Through the verification of key methodological indicators such as linearity, accuracy, precision and quantitative limit, the detection kit of the present invention can stably and accurately detect multiple targets, meeting the strict requirements of clinical testing. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 The chromatogram.

[0071] Figure 2 This is the linear correlation graph of norepinephrine (NE).

[0072] Figure 3 This is the linear correlation graph of epinephrine (E).

[0073] Figure 4 This is the linear correlation graph of normetanephrine (NMN).

[0074] Figure 5 This is the linear correlation diagram of dopamine (DA).

[0075] Figure 6 is the linear correlation graph of metanephrine (MN).

[0076] Figure 7 This is the linear correlation diagram of 3-methoxytyramine (3-MT). DETAILED DESCRIPTION

[0077] The present invention is described in detail below with reference to the embodiments and accompanying drawings.

[0078] In the following examples, NE is norepinephrine, E is epinephrine, DA is dopamine, NMN is methylnorepinephrine, MN is methylepinephrine, and 3-MT is 3-methoxytyramine.

[0079] NE-d6, E-d6, DA-d4, NMN-d3, MN-d3 and 3-MT-d4 are norepinephrine (NE), epinephrine (E), dopamine (DA), methylnorepinephrine (NMN), methylepinephrine (MN) and 3-methoxytyramine (3-MT) with hydrogen in the original molecules replaced with deuterium;

[0080] "d3" means that there are three deuterium atoms replacing the original three hydrogen atoms;

[0081] "d4" means that there are 4 deuterium atoms replacing the original 4 hydrogen atoms;

[0082] "D6" means that six deuterium atoms have replaced the original six hydrogen atoms.

[0083] Example 1

[0084] This embodiment discloses a catecholamine and metabolite detection kit, including a calibrator, a quality control product, an internal standard solution, an eluent, and a rinse solution.

[0085] 1. The target concentration ranges of each substance when working with calibrators are as follows:

[0086] NE: 60 pg / mL to 6000 pg / mL;

[0087] E, DA, MN, NMN: 20.0pg / mL~2000.0pg / mL;

[0088] 3-MT: 10.0pg / mL~1000.0pg / mL.

[0089] 2. Quality control products include low, medium and high concentrations;

[0090] 1) Low-concentration quality control products include the following components:

[0091] NE: 300pg / mL, 4800pg / mL,

[0092] E, DA, MN, NMN: 100pg / mL;

[0093] 3-MT: 40 pg / mL;

[0094] 2) Medium concentration quality control products include the following components:

[0095] NE: 1800 pg / mL;

[0096] E, DA, MN, NMN: 600pg / mL;

[0097] 3-MT: 240 pg / mL;

[0098] 3) High-concentration quality control products include the following components:

[0099] NE-d6: 50 ng / mL;

[0100] E-d6, DA-d4, MN-d3, NMN-d3: 30ng / mL;

[0101] 3-MT-d4: 15 ng / mL;

[0102] 3. The target concentration range of each substance when working with internal standard solution is as follows:

[0103] NE-d6: 50 ng / mL;

[0104] E-d6, DA-d4, MN-d3, NMN-d3: 30ng / mL;

[0105] 3-MT-d4: 15ng / mL.

[0106] 4. Eluent: 0.05-0.2% (v / v) formic acid aqueous solution.

[0107] 5. Eluent: methanol solution.

[0108] A detection method using the above-mentioned catecholamine and metabolite detection kit comprises the following steps:

[0109] S1: After reconstituting the calibrator and quality control with 1 mL of ultrapure water, take out 400 μL of sample and add 10 μL of internal standard solution and 290 μL of ultrapure water into a 2 mL centrifuge tube. Mix at 2000 rpm for 2 minutes.

[0110] S2: Add 400 μL of methanol to 20 mg of magnetic microspheres, vortex for 30 seconds, and discard the supernatant. Then vortex with 400 μL of ultrapure water for 30 seconds and discard the supernatant.

[0111] S3: Add 600 μL of the sample solution mixed in step S1, place it on a sample mixer, mix at 2000 rpm for 2 minutes, adsorb the microspheres on a magnetic stand for 30 seconds, and discard the supernatant.

[0112] S4: Add 400 μL of ultrapure water, mix by inversion for 10 seconds, adsorb on a magnetic stand for 30 seconds, and discard the supernatant.

[0113] S5: Elute with 400 μL of eluent to remove impurities and interferences.

[0114] S6: Elute the target with 200 μL of elution buffer to release it from the microspheres and into the final collection solution.

[0115] S7: Detection of catecholamines and their metabolites by LC-MS / MS.

[0116] The specific LC-MS / MS parameter settings are shown in Tables 1, 2, and 3:

[0117] (1) Use a Phenomenex Kinetex PFP chromatographic column or a Shim-pack Velox PFPP chromatographic column; the former is used in this example.

[0118] (2) Waters TQS liquid chromatography and mass spectrometry parameters:

[0119] Column mobile phase: A is an aqueous solution containing 0.05-0.1% (v / v) formic acid and 10-50 mM ammonium acetate;

[0120] B is a methanol solution containing 0-0.1% (v / v) formic acid;

[0121] The elution gradient was as follows: flow rate: 0.60 mL / min; injection volume: 5-10 μL; the gradient is shown in Table 1 below.

[0122] Table 1 Chromatographic gradient elution program

[0123]

[0124] Table 2 Precursor ions, product ions, and key mass spectrometric parameters of each compound and internal standard in LC-MS / MS detection

[0125]

[0126]

[0127] The mass spectrometry conditions are as shown in Table 3 below.

[0128] Table 3 Mass spectrometry conditions

[0129]

[0130] The chromatogram of this method is as follows Figure 1 shown.

[0131] 1. Linear effect verification:

[0132] (1) Verification plan: According to the test kit detection method, process the calibrators S1 to S6 of the product to be tested and the three concentration quality control products.

[0133] (2) Acceptance criteria: The concentration deviation of each point is within ±15%, and the linear correlation coefficient r is ≥0.990

[0134] (3) The experimental results are shown in Tables 4, 5 and Figure 2 、 3 , 4, 5, 6, and 7.

[0135] Table 4

[0136]

[0137]

[0138] Table 5

[0139]

[0140] 2. Precision effect verification:

[0141] (1) Validation method: Prepared samples at low, medium, and high concentrations were used and processed according to the pretreatment protocol of the kit. Each concentration was measured three times and the coefficient of variation (CV) was calculated.

[0142] (2) Acceptance criteria: Sample concentration deviation is within ±15% and CV≤15%.

[0143] (3) The experimental results are shown in Table 6 below.

[0144] Table 6

[0145]

[0146]

[0147] 3. Verification of accuracy effect:

[0148] (1) Verification method: Three standard solutions of known concentrations, high, medium, and low, were added to human samples (the volume of the added standard solution was 2% of the total volume) to prepare spiked samples of three concentrations. Each concentration sample was divided into five portions, and each sample was tested three times. The recovery rate was calculated according to formula (1).

[0149]

[0150] Where:

[0151] R——recovery rate;

[0152] V——the volume of standard solution added;

[0153] V0 – volume of matrix;

[0154] c——the mean test concentration after the matrix is added to the standard solution;

[0155] c0——mean test concentration of matrix;

[0156] c s ——Concentration of standard solution.

[0157] (2) Acceptance criteria: The sample concentration deviation is within ±15%.

[0158] (3) The experimental results are shown in Tables 7 and 8 below.

[0159] Table 7 Kit accuracy verification results

[0160]

[0161] Table 8 Calculation results of kit accuracy verification data

[0162]

[0163] 4. Verification of the quantitative lower limit effect:

[0164] (1) Verification method: Use the prepared quantitative limit sample and process the sample according to the pre-treatment method of the kit.

[0165] (1) Acceptance criteria: The sample concentration deviation is within ±15%.

[0166] (2) The experimental results are shown in Table 9 below.

[0167] Table 9

[0168]

[0169]

[0170]

[0171] In other embodiments, the temperature may be maintained at 4° C. during the entire sample pre-treatment process from step S1 to step S6 .

[0172] In other embodiments, after the calibrator, quality control material and plasma sample are re-dissolved or mixed in step S1, an antioxidant or stabilizer may be additionally added, wherein the antioxidant or stabilizer is selected from at least one of ascorbic acid, EDTA, or BHT, and the amount used is 0.01-0.1% (v / v) of the total volume of the sample.

[0173] After operating under low temperature conditions and adding appropriate antioxidants or stabilizers, the results obtained were basically consistent with the scheme described in the examples. Key indicators such as repeatability (CV), linear correlation coefficient (r), recovery rate and quantification limit all met the above acceptance criteria, indicating that the pretreatment process under these conditions can still obtain accurate and stable test results.

[0174] The above description is merely a preferred embodiment of the present invention. The scope of protection of the present invention is not limited to the above embodiment. All technical solutions based on the concept of the present invention are within the scope of protection of the present invention. It should be noted that for those skilled in the art, various improvements and modifications that do not depart from the principles of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. A kit for detecting catecholamines and metabolites, characterized in that: Including calibrators, quality control products, internal standard solutions, eluents and rinses; The calibrators and quality control materials are freeze-dried powders containing norepinephrine (NE), epinephrine (E), dopamine (DA), methylnorepinephrine (NMN), methylepinephrine (MN) and 3-methoxytyramine (3-MT); The internal standard solution is a methanol aqueous solution including NE-d6, E-d6, DA-d4, NMN-d3, MN-d3 and 3-MT-d4: Among them, NE-d6, E-d6, DA-d4, NMN-d3, MN-d3 and 3-MT-d4 are norepinephrine (NE), epinephrine (E), dopamine (DA), methylnorepinephrine (NMN), methylepinephrine (MN) and 3-methoxytyramine (3-MT) with the hydrogen in the original molecule replaced with deuterium; "d3" means that three deuterium atoms replace the original three hydrogen atoms; "d4" means that there are 4 deuterium atoms replacing the original 4 hydrogen atoms; "d6" means that 6 deuterium atoms replace the original 6 hydrogen atoms; Eluent: formic acid aqueous solution; Eluent: methanol solution.

2. The catecholamine and metabolite detection kit according to claim 1, characterized in that: The quality control products include three concentrations: low, medium and high; The low-concentration quality control product includes the following components: Norepinephrine: 300 pg / mL; Epinephrine, dopamine, norepinephrine, and metanephrine: 100 pg / mL; 3-Methoxytyramine: 40 pg / mL; The medium concentration quality control product includes the following components: Norepinephrine: 1800 pg / mL; Epinephrine, dopamine, norepinephrine, and metanephrine: 600 pg / mL; 3-Methoxytyramine: 240 pg / mL; The high-concentration quality control product includes the following components: Norepinephrine: 4500 pg / mL; Epinephrine, dopamine, norepinephrine, and metanephrine: 1500 pg / mL; 3-Methoxytyramine: 600 pg / mL.

3. The catecholamine and metabolite detection kit according to claim 1, characterized in that: The target concentration ranges of the various substances when the calibrators are working are as follows: NE: 60 pg / mL to 6000 pg / mL; E, DA, MN, NMN: 20.0pg / mL~2000.0pg / mL; 3-MT: 10.0pg / mL~1000.0pg / mL.

4. The catecholamine and metabolite detection kit according to claim 1, characterized in that: The target concentration range of each substance when the internal standard solution is working is as follows: NE-d6: 50 ng / mL; E-d6, DA-d4, MN-d3, NMN-d3: 30ng / mL; 3-MT-d4: 15ng / mL.

5. The catecholamine and metabolite detection kit according to claim 1, characterized in that: The concentration of the eluent is 0.05-0.2% (v / v) formic acid aqueous solution.

6. A detection method using the catecholamine and metabolite detection kit according to any one of claims 1 to 5, characterized in that: The following steps are involved: S1: Reconstitute the calibrator and quality control materials separately with ultrapure water; 400 μL of plasma, reconstituted calibrators, and quality controls were mixed with 10 μL of internal standard solution and 290 μL of ultrapure water at 2000 rpm for 2 minutes. S2: Add methanol to activate the magnetic microspheres, and then rinse with ultrapure water; Equilibration: Rinse with ultrapure water to make the surface environment of the microspheres similar to that of the sample to prevent the target from being adsorbed when the sample is added later. S3: Add the sample solution mixed in step S1 and allow it to fully contact and bind with the magnetic microspheres; S4: rinse with ultrapure water; S5: Remove impurities and interferences with eluent S6: Elute the target with an eluent to release it from the microspheres and into the final collection solution. S7: Detection of catecholamines and their metabolites by LC-MS / MS.

7. The detection method of the catecholamine and metabolite detection kit according to claim 6, characterized in that: During the entire sample pre-treatment process from step S1 to step S6, the temperature is maintained at 2-8°C.

8. The detection method of the catecholamine and metabolite detection kit according to claim 6, characterized in that: After the calibrator, quality control material and plasma sample are redissolved or mixed in step S1, an antioxidant or stabilizer is additionally added, wherein the antioxidant or stabilizer is selected from at least one of ascorbic acid, EDTA, or BHT, and the amount used is 0.01-0.1% (v / v) of the total volume of the sample.

9. The detection method of the catecholamine and metabolite detection kit according to claim 8, characterized in that: When liquid chromatography-tandem mass spectrometry (LC-MS / MS) is used for detection in step S7, the chromatographic conditions include: (1) Use Phenomenex Kinetex PFP chromatographic column or Shim-pack Velox PFPP chromatographic column; (2) Gradient elution was performed using mobile phase A and mobile phase B, wherein: Mobile phase A is an aqueous solution containing 0.05-0.1% (v / v) formic acid and 10-50 mM ammonium acetate; Mobile phase B was a methanol solution containing 0–0.1% (v / v) formic acid; The flow rate was 0.60 mL / min and the injection volume was 5 to 10 μL.

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