CN116106459A - Method for detecting catecholamine in blood or urine - Google Patents

Method for detecting catecholamine in blood or urine Download PDF

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CN116106459A
CN116106459A CN202211687426.XA CN202211687426A CN116106459A CN 116106459 A CN116106459 A CN 116106459A CN 202211687426 A CN202211687426 A CN 202211687426A CN 116106459 A CN116106459 A CN 116106459A
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朱怀恩
于涛
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Boyi Shanghai Scientific Instrument Equipment Co ltd
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Abstract

The invention relates to a method for detecting catecholamine in blood or urine, which comprises the following steps: (1) Adding 0.1-20 mg of magnetic solid phase extractant into each milliliter of sample to be detected, uniformly mixing, magnetically separating, and removing liquid; adding the eluent, uniformly mixing, magnetically separating, and removing liquid; adding eluent to perform elution, mixing uniformly, performing magnetic separation, and collecting the eluent to obtain the liquid to be detected; (2) Detecting catecholamine content in liquid to be detected by adopting liquid chromatography and tandem mass spectrometry; the catecholamine is selected from any one or combination of norepinephrine, epinephrine, dopamine, 3-methoxy dopamine, epinephrine and norepinephrine; the magnetic solid phase extractant comprises magnetic core ferroferric oxide, middle layer structure silicon dioxide wrapping the magnetic core and outer layer mesoporous structure, wherein the outer layer mesoporous structure is selected from any one or combination of polystyrene and polystyrene divinylbenzene. The invention solves the problems that the catecholamine content in human serum is difficult to accurately measure in the prior art, most of analysis time is long, sample pretreatment is complex, measurement types are few, and various contents cannot be measured simultaneously, and has the characteristics of good stability, high accuracy and sensitivity, strong specificity, high recovery rate, simple and controllable operation, high analysis speed and the like.

Description

Method for detecting catecholamine in blood or urine
Technical Field
The invention belongs to the field of biological medicine, and in particular relates to a method for detecting catecholamine in blood or urine.
Background
Pheochromocytoma (PA) generally occurs in the adrenal medulla and has certain familial genetic characteristics. Clinically, hypertension, which is mainly manifested as sudden or paroxysmal, is often accompanied by significant cardiovascular system problems. Long-term hypertension, however, can lead to left ventricular failure and other serious cardiovascular and cerebrovascular diseases. Many studies have found that excessive secretion of catecholamine neurotransmitters and their metabolites by pheochromocytomas has a direct correlation with the above-mentioned symptoms of hypertension, and is also a major cause of non-primary hypertension due to pheochromocytomas. Catecholamines mainly include Dopamine (DA), epinephrine (E), phenylephrine (MN), trimethoxytyramine (3-MT), norepinephrine (NE), norepinephrine (NMN), and the like, and measurement of catecholamines in blood and urine is an important means for diagnosing and identifying pheochromocytomas. However, there is no report on the related art for realizing simultaneous detection of various catecholamines in the existing studies.
Disclosure of Invention
The invention aims to provide a method for detecting catecholamine in blood or urine, which comprises the following steps:
(1) Adding 0.1-20 mg of magnetic solid phase extractant into each milliliter of sample to be detected, uniformly mixing, magnetically separating, and removing liquid; adding the eluent, uniformly mixing, magnetically separating, and removing liquid; adding eluent to perform elution, mixing uniformly, performing magnetic separation, and collecting the eluent to obtain the liquid to be detected;
(2) Detecting catecholamine content in liquid to be detected by adopting liquid chromatography and tandem mass spectrometry;
the catecholamine is selected from any one or combination of norepinephrine, epinephrine, dopamine, 3-methoxy dopamine, epinephrine and norepinephrine;
the magnetic solid phase extractant comprises magnetic core ferroferric oxide, middle layer structure silicon dioxide wrapping the magnetic core and outer layer mesoporous structure, wherein the outer layer mesoporous structure is selected from any one or combination of polystyrene and polystyrene divinylbenzene.
According to the preferred technical scheme, the magnetic solid phase extraction material is selected from any one or combination of water-oil balance magnetic solid phase extractant (HLB), strong cation exchange magnetic solid phase extractant (MCX), strong anion exchange magnetic solid phase extractant (MAX), weak cation exchange magnetic solid phase extractant (WCX) and weak anion exchange magnetic solid phase extractant (WAX).
According to the preferred technical scheme, the magnetic extractant further comprises a functional group with selective adsorption property, wherein the functional group is bonded in the mesoporous structure.
According to a preferred embodiment 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).
According to a preferred embodiment of the present invention, the functional group is selected from any one of an N-vinylpyrrolidone, a sulfonic acid group, a quaternary amine group, a carboxylate group, and a secondary amine group.
According to the preferred technical scheme, the magnetic solid-phase extractant is an activated magnetic solid-phase extractant, the activation step is to dissolve the magnetic solid-phase extractant in an alcohol solution, uniformly mix, add the activator according to a ratio of 1:1-20 (v/v), uniformly mix, magnetically separate and remove liquid, then elute with water, magnetically separate and remove the liquid to obtain the activated magnetic solid-phase extractant, wherein the activator is a 70-95% acetonitrile aqueous solution containing 0.3-5% formic acid, preferably a 85-90% acetonitrile aqueous solution containing 0.2-1% formic acid.
According to a preferred technical scheme, the leaching solution is selected from any one or combination of water and acetonitrile.
According to the preferred technical scheme, the adding amount of the leaching solution is 1-10ml of the leaching solution per mg of the magnetic solid-phase extractant.
According to the preferred technical scheme, the adding method of the eluent comprises the steps of adding water for leaching, uniformly mixing, magnetically separating, removing liquid, adding acetonitrile, uniformly mixing, magnetically separating, and removing liquid.
According to a preferred embodiment of the invention, the eluent is selected from the group consisting of 1-10% aqueous acetonitrile containing 1-5% formic acid, preferably 5% aqueous acetonitrile containing 2% formic acid.
According to the preferred technical scheme, the addition amount of the eluent is 1-10ml of the eluent is added into each mg of the magnetic solid phase extractant.
According to the preferable technical scheme, the mixing mode is ultrasonic mixing, the ultrasonic frequency is 40-60HZ, the ultrasonic power is 60-240w, the ultrasonic temperature is 20-30 ℃, and the ultrasonic mixing time is 30-60 s.
According to the preferred technical scheme, the magnetic separation mode is to adopt a rubidium magnet to adsorb a magnetic solid-phase extraction material for separation.
According to the preferred technical scheme, the sample to be tested is any one of serum obtained by blood coagulation treatment of venous blood of a subject or extracting solution and urine related to the serum.
According to the preferred technical scheme, 1-15 mg, preferably 5-10mg, of magnetic solid phase extractant is added into each milliliter of sample to be detected.
According to the preferred technical scheme, liquid chromatography and tandem mass spectrometry are adopted for liquid chromatography separation, and triple quadrupole tandem mass spectrometry detection is adopted.
In a preferred embodiment of the present invention, the chromatographic column is selected from the group consisting of a reverse phase C18 chromatographic column, an electrophilic group modified reverse phase C18 chromatographic column, a phenyl modified reverse phase C18 chromatographic column, and a perfluorophenyl modified reverse phase C18 chromatographic column.
In the preferred technical scheme of the invention, the diameter of the chromatographic column is 3-5mm, the length of the chromatographic column is 20-300mm, and the particle size of the filler in the chromatographic column is 2-5 mu m.
According to the preferred technical scheme, the chromatographic column of the liquid chromatograph is Phenomenex Kinetex C, 2.6um and 2.1x50mm.
According to the preferred technical scheme, the mobile phase of the liquid chromatograph is a mobile phase A and a mobile phase B, wherein the mobile phase A is an aqueous solution containing 0.1-1% formic acid; mobile phase B was methanol.
According to the preferred technical scheme, the flow rate of the liquid chromatography is 0.1-1mL/min, preferably 0.4-0.8mL/min.
According to the preferred technical scheme of the invention, the column temperature of the liquid chromatograph is 30-40 ℃, preferably 35-37 ℃.
According to the preferred technical scheme of the invention, the sample injection volume of the liquid chromatograph is 1-10 mu L, preferably 2-5 mu L.
According to the preferred technical scheme, the mobile phase elution program comprises the following steps:
time (min) A％ B％
0 95 5
2 95 5
2.5 10 90
3.5 10 90
4 95 5
5 95 5
According to the preferred technical scheme, the tandem mass spectrum conditions are as follows: ion source portion: atomizing gas at 50psi; heating the assist gas 55psi; the air curtain gas is 25psi; the ionization positive voltage is 3000-5500V, and the ionization negative voltage is-2000-4500V.
Compared with the prior art, the invention has the following beneficial effects:
1. according to the method, catecholamine and metabolites thereof in serum are enriched through a dispersion solid-phase extraction technology, and trace catecholamine in serum is analyzed by combining a liquid chromatography-tandem mass spectrometry (LC-MS/MS), so that the problems that in the prior art, the content of catecholamine in human serum is difficult to accurately determine, most of analysis time is long, sample pretreatment is complex, the determination types are few, and various contents cannot be determined simultaneously are solved.
2. The method has the characteristics of good stability, high accuracy and sensitivity, strong specificity, high recovery rate, simple and controllable operation, high analysis speed and the like, and can be used for simultaneous quantitative detection of various androgens in clinic.
Drawings
FIG. 1 is an MRM chromatogram of catecholamines of example 1.
Detailed Description
The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only. All techniques implemented based on the above description of the invention are within the scope of the invention.
Ultrasonic conditions: ultrasonic frequency 45HZ, ultrasonic power 100w and ultrasonic temperature 25 ℃.
Magnetic separation: a rubidium magnet is arranged on the outer side of the bottom of the test tube to carry out magnetic separation.
EXAMPLE 1 preparation of catecholamine test sample in serum
1. Preparation of internal standard working solution
The I S original stock solutions were serially diluted to I S single standard stock solutions I, I I, ii by adding 900 μl of methanol per 100 μl of stock solution in table 1.
TABLE 1
Figure BDA0004019980400000061
Taking 100 mu L of NE, DA, E-I S single standard stock solution I I and 20 mu L of 3-MT-I S, MN-I S and NMN-I S single standard stock solution I, and adding 640 mu L of 50% methanol solution to obtain I S mixed standard working solution I.
100 mu L of I S mixed standard working solution I is taken, 500 mu L of 1 mol/L ammonium formate aqueous solution (6.3 g ammonium formate is weighed and dissolved in 100mL deionized water, and dissolved and mixed uniformly) is added, 9.4mL of water is added, and I S mixed standard working solution I I is obtained after mixing uniformly. Wherein, the concentration of NE-I S, DA-I S and E-I S is 1ng/mL, and the concentration of 3-MT-I S, MN-I S and NMN-I S is 0.2ng/mL.
2. Standard sample preparation
The original stock solutions were serially diluted to single standard stock solutions I, I I, ii by adding 900 μl of methanol per 100 μl of stock solution in table 2.
TABLE 2
Figure BDA0004019980400000071
100 mu L of NE, DA, E, MN, NMN single-standard stock solution I I and 100 mu L of 3-MT single-standard stock solution II are taken, 400 mu L of 50% methanol solution is added, and the mixture is uniformly mixed to obtain mixed standard working solution I, wherein the concentration is NE, DA, E, MN, NMN 1.0.0 mu g/mL, and the concentration is 3-MT 0.10 mu g/mL.
100 mu L of mixed standard working solution I is taken, 900 mu L of 50% methanol solution is added, and the mixed standard working solution I I with the concentration of NE, DA, E, MN, NMN ng/mL and 3-MT 10ng/mL is obtained after uniform mixing. Standard working curves were prepared according to the method of table 3. In actual use, 360. Mu.L of water was added to each 40. Mu.L of working solution, simulating 400. Mu.L of serum solution.
TABLE 3 Table 3
Figure BDA0004019980400000081
3. Sample preparation
400 mu L of plasma is taken, 400 mu LNE-I S, DA-I S and I S mixed labeling working solution I I with E-I S concentration of 1ng/mL,3MT-I S, MN-I S and NMN-I S concentration of 0.2ng/mL are respectively added, and the mixture is uniformly vortex mixed to obtain a sample solution to be detected.
4. Activation of magnetic extractant
0.5g WCX magnetic extractant is weighed, 5mL ethanol is added, and the mixture is uniformly oscillated until all solids are suspended in the ethanol, and the bottom of the bottle is not settled, thus obtaining magnetic extractant suspension.
40. Mu.L of the magnetic extractant suspension was placed in a 1.5mL polypropylene tube, 400. Mu.L of 85% acetonitrile aqueous solution containing 2% formic acid was added, and the mixture was sonicated and homogenized for 30 seconds.
600 μl of water was added, sonicated for 30 seconds, magnetically separated for 30 seconds, and the supernatant removed completely and discarded. Thus obtaining the activated magnetic extractant.
6. The activated magnetic extractant is used for treating samples
Adding 800 mu L of sample solution to be purified into the test tube filled with the activated magnetic extractant in the step (5), uniformly mixing by ultrasonic for 60 seconds, magnetically separating for 30 seconds, and completely removing and discarding the upper layer liquid.
800 μl of water was added, sonicated for 30 seconds, magnetically separated for 30 seconds, and the supernatant removed completely and discarded.
600 μl of acetonitrile was added, sonicated for 30 seconds, magnetically separated for 30 seconds, and the supernatant removed completely and discarded.
100 mu L of 5% acetonitrile aqueous solution containing 2% formic acid is added, the mixture is uniformly mixed for 60 seconds by ultrasonic, and magnetic separation is carried out for 30 seconds, so that eluent is obtained, and the sample to be detected is obtained.
Example 2 detection of catecholamine test sample in serum
1. Conditions of liquid chromatography
Chromatographic column: phenomenex Ki netex PFP 2.6.6 um, 2.1X105 mm
Flow rate: 0.4 mL/min
Column temperature: 35 DEG C
Sample injection volume: 5 mu L
Mobile phase: a, aqueous solution containing 0.1% formic acid; b, methanol
Chromatographic gradient
Figure BDA0004019980400000091
Figure BDA0004019980400000101
2. Tandem quadrupole mass spectrometry parameters
The following applies to serial quadrupole mass spectra of SCI EX API 4000, API 4500, AP I5000, API 5500. For other manufacturers and their models, tandem quadrupole mass spectrometry was optimized to the best conditions, respectively.
Ion source portion: the atomization gas is 50ps i; heating the auxiliary gas 55 pi; a gas curtain gas 25 pi; ionization voltage 5500V.
Mass analyzer section:
Figure BDA0004019980400000102
Figure BDA0004019980400000111
3. analysis of detection results:
after each sample of the standard working curve acquires LC-MS/MS data, a ratio of the peak area of the target compound in each standard sample data to the corresponding internal standard peak area is taken as an ordinate, a ratio of the target compound concentration of each standard sample to the corresponding internal standard concentration is taken as an abscissa, and a least square method is used to obtain the standard working curve and a regression equation. Substituting the ratio of the peak area of the target compound to the peak area of the internal standard in LC-MS/MS data acquired by the sample to be detected into a regression equation of a standard working curve to obtain the concentrations of the above six catecholamines in the sample to be detected. The detection results are shown in FIG. 1.
Example 2 methodological validation results:
1. linearity and range
3-MT and MN are in the range of 5-2000 pg/mL, DA, E and NMN are in the range of 10-2000 pg/mL, NE is in the range of 20-2000 pg/mL, the linear relation is good, and the correlation coefficients are all larger than 0.9968.
2. Quantitative limit
The quantitative limit of 3-MT and MN was 5pg/mL, the quantitative limit of DA, E and NMN was 10pg/mL, and the quantitative limit of NE was 20pg/mL.
3. Matrix effect
The actual response values a and the standard response values B of the six substances were measured, respectively. The matrix influencing factors of the six substances, namely the response value ratio A/B, are all between 84.4 and 118 percent, and meet the verification requirement.
4. Accuracy of
The method of the invention is repeated for seven times, the recovery rates of six substances are all between 98.8 and 111 percent, and RSD (n=7) is less than or equal to 7.2 percent, thereby meeting the verification requirement.
5. Precision of
Taking blank plasma, adding a proper amount of six catecholamine standard substances, and preparing precision test samples with low, medium and high concentrations (50, 200 and 500 pg/mL). The above samples were measured in four batches, five times per batch, using the method of the invention. The relative standard deviation of the measurement results of the six substances in the batch and the batch is less than or equal to 11 percent, and the recovery rate is between 88.6 and 111 percent, thereby meeting the methodological requirements.
The above description of the embodiments of the present invention is not intended to limit the present invention, and those skilled in the art can make various changes or modifications according to the present invention without departing from the spirit of the present invention, and shall fall within the scope of the claims of the present invention.

Claims (10)

1. A method for detecting catecholamines in blood or urine, comprising the steps of:
(1) Adding 0.1-20 mg of magnetic solid phase extractant into each milliliter of sample to be measured, uniformly mixing,
magnetically separating, removing the liquid; adding the eluent, uniformly mixing, magnetically separating, and removing liquid;
adding eluent to perform elution, mixing uniformly, performing magnetic separation, and collecting the eluent to obtain the liquid to be detected;
(2) Detecting catecholamine content in liquid to be detected by adopting liquid chromatography and tandem mass spectrometry;
the catecholamine is selected from any one or combination of norepinephrine, epinephrine, dopamine, 3-methoxy dopamine, epinephrine and norepinephrine;
the magnetic solid phase extractant comprises magnetic core ferroferric oxide, middle layer structure silicon dioxide wrapping the magnetic core and outer layer mesoporous structure, wherein the outer layer mesoporous structure is selected from any one or combination of polystyrene and polystyrene divinylbenzene.
2. The method of claim 1, wherein the magnetic solid phase extraction material is selected from any one of water-oil balanced magnetic solid phase extractant (HLB), strong cation exchange magnetic solid phase extractant (MCX), strong anion exchange magnetic solid phase extractant (MAX), weak cation exchange magnetic solid phase extractant (WCX), weak anion exchange magnetic solid phase extractant (WAX), or a combination thereof.
3. The method according to claim 1, wherein the magnetic extractant further comprises a selectively adsorptive functional group bonded within the mesoporous structure, preferably the functional group is selected from any one of hydrophilic group, strong cation exchange group (MCX), strong anion exchange group (MAX), weak cation exchange group (WCX), weak anion exchange group (WAX), more preferably the functional group is selected from any one of N-vinylpyrrolidone, sulfonic acid group, quaternary amine group, carboxylate group, secondary amine group.
4. A method according to any one of claims 1 to 3, wherein the magnetic solid phase extractant is an activated magnetic solid phase extractant, and the activation step comprises dissolving the magnetic solid phase extractant in an alcohol solution, mixing, adding an activator at a ratio of 1:1 to 20 (v/v), mixing, magnetically separating, removing the liquid, eluting with water, magnetically separating, and removing the liquid to obtain an activated magnetic solid phase extractant, wherein the activator is a 70-95% acetonitrile aqueous solution containing 0.3-5% formic acid, preferably a 85-90% acetonitrile aqueous solution containing 0.2-1% formic acid.
5. The method according to any one of claims 1 to 4, wherein the eluent is selected from any one of water and acetonitrile or a combination thereof, preferably the eluent is added in an amount of 1 to 10ml of eluent per mg of magnetic solid phase extractant, more preferably the eluent is added by adding water first, mixing, magnetic separation, removing liquid, then acetonitrile, mixing, magnetic separation, removing liquid.
6. The method according to any one of claims 1 to 5, wherein the eluent is selected from 1 to 10% acetonitrile in water containing 1 to 5% formic acid, preferably 5% acetonitrile in water containing 2% formic acid, preferably the eluent is added in an amount of 1 to 10ml eluent per mg of magnetic solid phase extractant.
7. The method of any one of claims 1-6, wherein the mixing mode is ultrasonic mixing, ultrasonic frequency is 40-60HZ, ultrasonic power is 60-240w, ultrasonic temperature is 20-30 degrees, and ultrasonic mixing time is 30-60 s.
8. The method of any one of claims 1-7, wherein the liquid chromatography and tandem mass spectrometry is performed using liquid chromatography separation, triple quadrupole tandem mass spectrometry, and wherein the chromatography column is selected from the group consisting of reversed-phase C18 chromatography column, electrophilic group modified reversed-phase C18 chromatography column, phenyl modified reversed-phase C18 chromatography column, and perfluorinated phenyl modified reversed-phase C18 chromatography column, more preferably wherein the liquid chromatography column is Phenomenex Kinetex C, 2.6um,2.1x50mm.
9. The method of any one of claims 1-8, wherein the mobile phase of the liquid chromatograph is mobile phase a and mobile phase B, wherein mobile phase a is an aqueous solution comprising 0.1-1% formic acid; mobile phase B is methanol, preferably the mobile phase elution procedure is:
time (min) A％ B％ 0 95 5 2 95 5 2.5 10 90 3.5 10 90 4 95 5 5 95 5
10. The method of any one of claims 1-9, wherein the tandem mass spectrometry conditions are: ion source portion: atomizing gas at 50psi; heating the assist gas 55psi; the air curtain gas is 25psi; the ionization positive voltage is 3000-5500V, and the ionization negative voltage is-2000-4500V.
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CN116448923A (en) * 2023-06-20 2023-07-18 中国医学科学院北京协和医院 Methods and kits for extracting and detecting steroid hormones and catecholamine metabolites

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116448923A (en) * 2023-06-20 2023-07-18 中国医学科学院北京协和医院 Methods and kits for extracting and detecting steroid hormones and catecholamine metabolites

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