A method for detecting catecholamines in blood or urine

Abstract

The present invention relates to a method for detecting catecholamines in blood or urine, comprising the following steps: (1) adding 0.1 to 20 mg of a magnetic solid-phase extractant to each milliliter of a sample to be tested, mixing, magnetically separating, and removing the liquid; adding a washing solution, mixing, magnetically separating, and removing the liquid; adding an eluent for elution, mixing, magnetically separating, and collecting the eluate to obtain a test solution; (2) detecting the catecholamine content in the test solution by liquid chromatography and tandem mass spectrometry; the catecholamine is selected from any one of norepinephrine, epinephrine, dopamine, 3-methoxydopamine, metanephrine, and normetanephrine, or a combination thereof; the magnetic solid-phase extractant comprises a magnetic core of ferrosoferric oxide, a middle-layer structure of silica wrapping the magnetic core, and an outer-layer mesoporous structure, wherein the outer-layer mesoporous structure is selected from any one of polystyrene and polystyrene divinylbenzene, or a combination thereof. The present invention solves the problems in the prior art of being difficult to accurately determine the catecholamine content in human serum, and most of the problems are long analysis time, complex sample pretreatment, few types of determination, and inability to simultaneously determine multiple contents. The present invention has the characteristics of good stability, high accuracy and sensitivity, strong specificity, high recovery rate, simple and controllable operation, and fast analysis speed.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to a method for detecting catecholamines in blood or urine. Background Art

[0002] Pheochromocytoma (PA) typically arises in the adrenal medulla and has a certain familial genetic profile. Clinical manifestations include sudden or paroxysmal hypertension, often accompanied by significant cardiovascular problems. Long-term hypertension can lead to left ventricular failure and other serious cardiovascular and cerebrovascular diseases. Numerous studies have found that excessive secretion of catecholamine neurotransmitters and their metabolites caused by pheochromocytoma is directly associated with the aforementioned hypertensive symptoms and is the primary cause of non-essential hypertension caused by pheochromocytoma. Catecholamines primarily include dopamine (DA), epinephrine (E), metanephrine (MN), trimethoxytyramine (3-MT), norepinephrine (NE), and nomethylmetanephrine (NMN). Measuring catecholamines in blood and urine is an important tool for diagnosing and differentiating pheochromocytoma. However, existing research lacks reports on technologies that can simultaneously detect multiple catecholamines. Summary of the Invention

[0003] The object of the present invention is to provide a method for detecting catecholamines in blood or urine, comprising the following steps:

[0004] (1) Add 0.1-20 mg of magnetic solid phase extraction agent per milliliter of the sample to be tested, mix, perform magnetic separation, and remove the liquid; add eluent, mix, perform magnetic separation, and remove the liquid; add eluent for elution, mix, perform magnetic separation, and collect the eluent, which is the sample to be tested;

[0005] (2) detecting the catecholamine content in the test solution by liquid chromatography and tandem mass spectrometry;

[0006] The catecholamine is selected from any one of norepinephrine, epinephrine, dopamine, 3-methoxydopamine, metanephrine, and normetanephrine, or a combination thereof;

[0007] The magnetic solid phase extractant comprises a magnetic core ferrosoferric oxide, a middle layer structure of silicon dioxide wrapping the magnetic core, and an outer layer mesoporous structure, wherein the outer layer mesoporous structure is selected from any one of polystyrene and polystyrene divinylbenzene or a combination thereof.

[0008] In a preferred technical solution of the present invention, the magnetic solid phase extraction material is selected from any one of a water-oil balance magnetic solid phase extractant (HLB), a strong cation exchange magnetic solid phase extractant (MCX), a strong anion exchange magnetic solid phase extractant (MAX), a weak cation exchange magnetic solid phase extractant (WCX), and a weak anion exchange magnetic solid phase extractant (WAX), or a combination thereof.

[0009] According to a preferred technical solution of the present invention, the magnetic extractant further comprises a functional group with selective adsorption properties bonded within the mesoporous structure.

[0010] In a preferred technical solution of the present invention, the functional group is selected from any one of a hydrophilic group, a strong cation exchange group (MCX), a strong anion exchange group (MAX), a weak cation exchange group (WCX), and a weak anion exchange group (WAX).

[0011] In a preferred technical solution of the present invention, the functional group is selected from any one of N-vinyl pyrrolidone, sulfonic acid group, quaternary amine group, carboxylate group and secondary amine group.

[0012] According to a preferred technical solution of the present invention, the magnetic solid-phase extractant is an activated magnetic solid-phase extractant. The activation step comprises dissolving the magnetic solid-phase extractant in an alcohol solution, mixing, adding an activator at a ratio of 1:1-20 (v / v), mixing, magnetic separation, removing the liquid, eluting with water, magnetic separation, and removing the liquid to obtain the activated magnetic solid-phase extractant. The activator is a 70-95% acetonitrile aqueous solution containing 0.3-5% formic acid, preferably an 85-90% acetonitrile aqueous solution containing 0.2-1% formic acid.

[0013] According to a preferred technical solution of the present invention, the eluent is selected from any one of water and acetonitrile or a combination thereof.

[0014] According to a preferred technical solution of the present invention, the amount of the eluent added is 1-10 ml of eluent per mg of magnetic solid phase extraction agent.

[0015] According to a preferred technical solution of the present invention, the eluent is added by first adding water for elution, mixing, magnetic separation, removing the liquid, and then adding acetonitrile, mixing, magnetic separation, and removing the liquid.

[0016] According to a preferred technical solution of the present invention, the eluent is selected from a 1-10% acetonitrile aqueous solution containing 1-5% formic acid, preferably a 5% acetonitrile aqueous solution containing 2% formic acid.

[0017] According to a preferred technical solution of the present invention, the amount of the eluent added is 1-10 ml of the eluent per mg of the magnetic solid phase extractant.

[0018] According to a preferred technical solution of the present invention, the mixing method is ultrasonic mixing, with an ultrasonic frequency of 40-60 Hz, an ultrasonic power of 60-240 W, an ultrasonic temperature of 20-30 degrees, and an ultrasonic mixing time of 30 to 60 seconds.

[0019] In a preferred technical solution of the present invention, the magnetic separation method is to use a neodymium magnet to adsorb a magnetic solid phase extraction material for separation.

[0020] According to a preferred technical solution of the present invention, the sample to be tested is any one of serum, extracts related thereto, and urine obtained after coagulation treatment of the subject's venous blood.

[0021] According to a preferred technical solution of the present invention, 1 to 15 mg, preferably 5 to 10 mg, of magnetic solid phase extraction agent is added to each milliliter of the sample to be tested.

[0022] In a preferred technical solution of the present invention, the liquid chromatography and tandem mass spectrometry method adopts liquid chromatography separation and triple quadrupole tandem mass spectrometry detection.

[0023] In the preferred technical solution of the present invention, the chromatographic column is selected from a reversed-phase C18 chromatographic column, an electrophilic group-modified reversed-phase C18 chromatographic column, a phenyl-modified reversed-phase C18 chromatographic column, and a perfluorophenyl-modified reversed-phase C18 chromatographic column.

[0024] In a preferred technical solution of the present invention, the diameter of the chromatographic column is 3-5 mm, the length of the chromatographic column is 20-300 mm, and the particle size of the filler in the chromatographic column is 2-5 μm.

[0025] According to a preferred technical solution of the present invention, the liquid chromatography column is Phenomenex Kinetex C18, 2.6um, 2.1x50mm.

[0026] According to a preferred technical solution of the present invention, the mobile phases of the liquid chromatography are mobile phase A and mobile phase B, wherein mobile phase A is an aqueous solution containing 0.1-1% formic acid; and mobile phase B is methanol.

[0027] According to a preferred technical solution of the present invention, the flow rate of the liquid chromatography is 0.1-1 mL / min, preferably 0.4-0.8 mL / min.

[0028] According to a preferred technical solution of the present invention, the column temperature of the liquid chromatography is 30-40°C, preferably 35-37°C.

[0029] According to a preferred technical solution of the present invention, the injection volume of the liquid chromatography is 1-10 μL, preferably 2-5 μL.

[0030] The preferred technical solution of the present invention is that the mobile phase elution procedure is:

[0031] Time (min) A％ B％ 0 95 5 2 95 5 2.5 10 90 3.5 10 90 4 95 5 5 95 5

[0032] According to a preferred technical solution of the present invention, the tandem mass spectrometry conditions are as follows: ion source part: nebulizing gas 50 psi; heating auxiliary gas 55 psi; curtain gas 25 psi; ionization positive voltage 3000-5500 V, ionization negative voltage -2000 to -4500 V.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] 1. The method of the present invention enriches catecholamines and their metabolites in serum through dispersed solid-phase extraction technology, and combines it with liquid chromatography-tandem mass spectrometry (LC-MS / MS) to analyze trace catecholamines in serum. This solves the problems of the existing technology in accurately determining the content of catecholamines in human serum, and most of them have long analysis time, complex sample pretreatment, limited number of assay types, and inability to simultaneously determine multiple contents.

[0035] 2. The method of the present invention has the characteristics of good stability, high accuracy and sensitivity, strong specificity, high recovery rate, simple and controllable operation, and fast analysis speed. It can be used for the simultaneous quantitative detection of multiple androgens in clinical practice. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is the MRM chromatogram of catecholamines in Example 1. DETAILED DESCRIPTION

[0037] The following further describes the above content of the present invention in detail through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention.

[0038] Ultrasonic conditions: ultrasonic frequency 45 Hz, ultrasonic power 100 w, ultrasonic temperature 25 °C.

[0039] Magnetic separation: Place a rubidium magnet on the outside of the test tube near the bottom for magnetic separation.

[0040] Example 1 Preparation of Catecholamine Test Samples in Serum

[0041] 1. Preparation of internal standard working solution

[0042] According to Table 1, the IS original stock solution was diluted stepwise to IS single-label stock solutions I, II, and III by adding 100 μL of each IS stock solution to 900 μL of methanol.

[0043] Table 1

[0044]

[0045] Take 100 μL of NE, DA, and EIS single-label stock solution III and 20 μL of 3-MT-IS, MN-IS, and NMN-IS single-label stock solution III, add 640 μL of 50% methanol solution to obtain IS mixed standard working solution I.

[0046] Take 100 μL of IS mixed standard working solution I, add 500 μL of a 1 mol / L aqueous solution of ammonium formate (dissolve 6.3 g of ammonium formate in 100 mL of deionized water, and mix thoroughly). Then add 9.4 mL of water and mix thoroughly to obtain IS mixed standard working solution II. The concentrations of NE-IS, DA-IS, and EIS are 1 ng / mL, respectively, and the concentrations of 3-MT-IS, MN-IS, and NMN-IS are 0.2 ng / mL, respectively.

[0047] 2. Preparation of Standard Samples

[0048] According to Table 2, the original stock solution was diluted stepwise to single-label stock solutions I, II, and III by adding 900 μL of methanol at a time of 100 μL.

[0049] Table 2

[0050]

[0051] Take 100 μL each of NE, DA, E, MN, and NMN single-label stock solution II and 100 μL of 3-MT single-label stock solution III, add 400 μL of 50% methanol solution, and mix well to obtain mixed standard working solution I with a concentration of 1.0 μg / mL for NE, DA, E, MN, and NMN, and 0.10 μg / mL for 3-MT.

[0052] Take 100 μL of mixed standard working solution I and add 900 μL of 50% methanol solution. Mix thoroughly to obtain mixed standard working solution II. The concentrations are 100 ng / mL for NE, DA, E, MN, and NMN, and 10 ng / mL for 3-MT. Prepare a standard working curve according to Table 3. In practice, add 360 μL of water to every 40 μL of working solution to simulate 400 μL of serum solution.

[0053] Table 3

[0054]

[0055] 3. Sample Preparation

[0056] Take 400 μL of plasma and add 400 μL of IS mixed standard working solution II with a concentration of 1 ng / mL for NE-IS, DA-IS, and EIS, and a concentration of 0.2 ng / mL for 3MT-IS, MN-IS, and NMN-IS, respectively. Vortex and mix thoroughly to obtain the sample solution to be tested.

[0057] 4. Activation of magnetic extractant

[0058] Weigh 0.5 g of WCX magnetic extractant, add 5 mL of ethanol, and shake evenly until all the solids are suspended in the ethanol and there is no sedimentation at the bottom of the bottle to obtain a magnetic extractant suspension.

[0059] Take 40 μL of magnetic extractant suspension, place it in a 1.5 mL polypropylene test tube, add 400 μL of 85% acetonitrile aqueous solution containing 2% formic acid, and mix by ultrasonication for 30 seconds.

[0060] Add 600 μL of water, mix by ultrasonication for 30 seconds, and perform magnetic separation for 30 seconds. Remove the upper layer of liquid completely and discard it. This is the activated magnetic extractant.

[0061] 6. The activated magnetic extractant is used to process the sample

[0062] 800 μL of the sample solution to be purified was added to the test tube containing the activated magnetic extractant in step (5), ultrasonically mixed for 60 seconds, magnetically separated for 30 seconds, and the upper liquid was completely removed and discarded.

[0063] Add 800 μL of water, mix by ultrasonication for 30 seconds, and perform magnetic separation for 30 seconds. Remove the upper layer of liquid completely and discard it.

[0064] Then add 600 μL of acetonitrile, mix by ultrasonication for 30 seconds, and perform magnetic separation for 30 seconds. The upper layer of liquid is completely removed and discarded.

[0065] Add 100 μL of 5% acetonitrile aqueous solution containing 2% formic acid, mix by ultrasonication for 60 seconds, and perform magnetic separation for 30 seconds to obtain the eluate, which is the sample to be tested.

[0066] Example 2 Detection of Catecholamines in Serum

[0067] 1. Liquid chromatography conditions

[0068] Column: Phenomenex Kinetex PFP 2.6um, 2.1x50mm

[0069] Flow rate: 0.4 mL / min

[0070] Column temperature: 35°C

[0071] Injection volume: 5 μL

[0072] Mobile phase: A, aqueous solution containing 0.1% formic acid; B, methanol

[0073] Chromatographic gradient

[0074]

[0075]

[0076] 2. Tandem quadrupole mass spectrometry parameters

[0077] The following conditions apply to SCI EX API 4000, API 4500, API 5000, and API 5500 series tandem quadrupole mass spectrometers. For tandem quadrupole mass spectrometers from other manufacturers and models, the optimal conditions for each manufacturer shall prevail.

[0078] Ion source part: nebulizer gas 50 pSi; heating auxiliary gas 55 pSi; curtain gas 25 pSi; ionization voltage 5500V.

[0079] Mass analyzer part:

[0080]

[0081]

[0082] 3. Analysis of test results:

[0083] After collecting LC-MS / MS data for each sample of the standard working curve, the ratio of the peak area of ​​the target compound in each standard sample data to the corresponding internal standard peak area is used as the vertical coordinate, and the ratio of the target compound concentration in each standard sample to the corresponding internal standard concentration is used as the horizontal coordinate. The standard working curve and regression equation are obtained using the least squares method. The ratio of the peak area of ​​the target compound and the peak area of ​​the internal standard in the LC-MS / MS data collected for the sample to be tested is substituted into the regression equation of the standard working curve to obtain the concentration of the above six catecholamines in the sample to be tested. The test results are shown in Figure 1 .

[0084] Example 2 Methodology Verification Results:

[0085] 1. Linearity and range

[0086] The linear relationships were good for 3-MT and MN in the range of 5-2000 pg / mL, for DA, E, and NMN in the range of 10-2000 pg / mL, and for NE in the range of 20-2000 pg / mL, with correlation coefficients greater than 0.9968.

[0087] 2. Limit of Quantitation

[0088] The limits of quantification for 3-MT and MN were 5 pg / mL, for DA, E, and NMN were 10 pg / mL, and for NE was 20 pg / mL.

[0089] 3. Matrix Effect

[0090] The actual response values ​​A and standard response values ​​B of the six substances were measured respectively. The matrix influence factors of the six substances, that is, the response value ratios A / B, were all between 84.4% and 118%, meeting the verification requirements.

[0091] 4. Accuracy

[0092] The method of the present invention was used to repeat the test seven times, and the recoveries of the six substances were all between 98.8% and 111%, and the RSD (n=7) was ≤7.2%, meeting the verification requirements.

[0093] 5. Precision

[0094] Blank plasma was spiked with appropriate amounts of six catecholamine standards to prepare precision test samples at low, medium, and high concentrations (50, 200, and 500 pg / mL). Using the method described in this invention, these samples were assayed in four batches, with five replicates per batch. The relative standard deviations (RSDs) for the six standards, both within and between batches, were ≤11%, and the recoveries ranged from 88.6% to 111%, meeting the methodological requirements.

[0095] The above description of the specific embodiments of the present invention does not limit the present invention. Those skilled in the art can make various changes or modifications based on the present invention. As long as they do not depart from the spirit of the present invention, they should fall within the scope of protection of the claims of the present invention.

Claims

1. A method for detecting catecholamines in blood or urine, comprising the following steps: (1) Add 1-15 mg of activated magnetic solid phase extractant to each milliliter of the sample to be tested, mix, magnetically separate, and remove the liquid; first add water to rinse, magnetically separate, and remove the liquid; then add acetonitrile to rinse, magnetically separate, and remove the liquid; then add 1-10% acetonitrile solution containing 1-5% formic acid to elute, magnetically separate, and collect the eluate as the test liquid, wherein the sample to be tested is selected from the plasma or urine of the subject, and the magnetic solid phase extractant is a weak cation exchange magnetic solid phase extractant, which comprises a magnetic core ferroferric oxide, a middle layer structure of dioxygen wrapped around the magnetic core, and a magnetic solid phase extractant. Silicon and an outer mesoporous structure and a weak cation exchange group with selective adsorption performance bonded to the mesoporous structure, wherein the outer mesoporous structure is selected from any one of polystyrene and polystyrene divinylbenzene or a combination thereof, and the activation of the magnetic solid phase extractant comprises the following steps: dissolving the magnetic solid phase extractant in an alcohol solution, mixing, adding a 70-95% acetonitrile solution containing 0.3-5% formic acid in a volume ratio of 1:1-20, mixing, magnetically separating, removing the liquid, eluting with water, magnetically separating, and removing the liquid to obtain an activated magnetic solid phase extractant; (2) using liquid chromatography to separate catecholamines in the test solution, and then detecting the catecholamines in the test solution by triple quadrupole tandem mass spectrometry, wherein the catecholamines include separation and detection of norepinephrine, epinephrine, dopamine, 3-methoxydopamine, metanephrine, and normetanephrine; The chromatographic column of the liquid chromatography was Phenomenex Kinetex C18, 2.6 μm, 2.1×50 mm, with a flow rate of The mobile phase consists of mobile phase A and mobile phase B, wherein mobile phase A is an aqueous solution containing 0.1-1% formic acid; mobile phase B is methanol, the flow rate is 0.1-1 mL / min, the column temperature is 30-40°C, the injection volume is 1-10 μL, and the gradient elution conditions are: 。 2. The method according to claim 1, wherein the activation of the magnetic solid phase extractant comprises the steps of dissolving the magnetic solid phase extractant in an alcohol solution, mixing, adding 0.2-1% formic acid in 85-90% acetonitrile solution, mixing, magnetic separation, removing the liquid, eluting with water, magnetic separation, and removing the liquid to obtain the product.

3. The method according to claim 1, wherein the amount of eluent added is 1 mg of magnetic solid phase extraction agent added to 1-10 ml of eluent.

4. The method of claim 1 , wherein the eluent is selected from a 5% acetonitrile aqueous solution containing 2% formic acid.

5. The method according to claim 1, wherein the volume mass ratio of the eluent to the magnetic solid phase extractant is 1-10 ml:1 mg.

6. The method according to claim 1, wherein the mixing is ultrasonic mixing, and the conditions are ultrasonic frequency 40-60 Hz, ultrasonic power 60-240 w, ultrasonic temperature 20-30 ° C, and ultrasonic mixing time 30-60 s.

7. The method according to claim 1, wherein the magnetic separation method is to use a neodymium magnet to adsorb a magnetic solid phase extraction material for separation.

8. The method of claim 1, wherein 5-10 mg of activated magnetic solid phase extraction agent is added to each milliliter of the sample to be tested.

9. The method of claim 1, wherein the flow rate of the liquid chromatography is 0.4-0.8 mL / min.

10. The method according to claim 1, wherein the column temperature of the liquid chromatography is 35-37°C. The method according to claim 1 , wherein the injection volume of the liquid chromatography is 2-5 μL.

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