CN114428138B - Catecholamine and metabolite liquid chromatography tandem mass spectrometry detection method based on magnetic solid phase extraction - Google Patents
Catecholamine and metabolite liquid chromatography tandem mass spectrometry detection method based on magnetic solid phase extraction Download PDFInfo
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- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/062—Preparation extracting sample from raw material
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- General Physics & Mathematics (AREA)
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Abstract
The invention discloses a liquid chromatography tandem mass spectrometry detection method of catecholamine and metabolites thereof based on magnetic solid phase extraction, which comprises the following steps: 1) Preparing a detection sample: 2) Constructing a standard curve of the target object: 3) Detecting by adopting a liquid chromatography tandem mass spectrometry method, and calculating to obtain the content of catecholamine and metabolites thereof in a detection sample by combining the constructed standard curve; wherein catecholamines include dopamine, norepinephrine and epinephrine, and metabolites of catecholamines include methoxytyramine, phenylephrine and norepinephrine. According to the liquid chromatography tandem mass spectrometry detection method based on catecholamine and metabolites thereof provided by the invention, the catecholamine and metabolites thereof detection sample is subjected to pretreatment by a magnetic solid-phase extraction technology, and the separation and transfer of the to-be-detected substances are rapidly realized by applying external magnetic field energy, so that the automatic pretreatment is conveniently realized, and meanwhile, the advantages of high selectivity, high purification rate and the like of the solid-phase extraction are also maintained.
Description
Technical Field
The invention relates to the field of clinical mass spectrometry in-vitro diagnostic reagents, in particular to a liquid chromatography tandem mass spectrometry detection method of catecholamine and metabolites thereof based on magnetic solid phase extraction.
Background
Catecholamines are synthesized and secreted by adrenal medulla, sympathetic nerves, paraganglionic chromaffins, which are both hormones and neurotransmitters. Catecholamines mainly include Dopamine (DA), norepinephrine (NE) and epinephrine (E), and catecholamine oxymetransferases convert DA, E and NE into methoxytyramine (3-MT), epinephrine (MN) and norepinephrine (NMN), respectively. Catecholamines are endogenous substances with strong physiological effects in living beings and play an important role in brain and nerve signaling. Catecholamine levels in organisms are closely related to a variety of physiological and pathological phenomena. Catecholamine substances have wide regulating effects on physiological activities of the cardiovascular system, nervous system, endocrine system, kidney, respiratory system and the like of human bodies. For example, epinephrine acts on both cardiovascular and catabolic hormones, dopamine being a specific drug for the treatment of parkinson's disease, and can cross the blood brain barrier to decarboxylate neurotransmitters; norepinephrine is a sympathomimetic agent and has the effects of constricting blood vessels and increasing blood pressure. In addition, catecholamines are also associated with the occurrence of several functional disorders such as schizophrenia, depression and organic lesions. By detecting catecholamine and the metabolite level thereof in biological samples, the method has important clinical significance for diagnosis and treatment of diseases such as pheochromocytoma, paraganglioma, neuroblastoma, hypertension, myocardial infarction, adrenal medullary hyperplasia and the like, and is beneficial to diagnosis of diseases such as thyroid dysfunction, congestive heart failure, diabetes, renal insufficiency and the like. In conclusion, the information of catecholamine and the concentration level of the metabolites thereof in organisms is of great significance in neurophysiologic research and diagnosis and prognosis of diseases related to abnormal transmitter metabolism. According to the expert consensus 2020 edition for diagnosis and treatment of pheochromocytoma and paraganglioma, it is proposed to measure the concentration of free plasma or methoxyepinephrine in urine, and to detect the concentration of NE, DA, E and their metabolites in blood or urine simultaneously to help diagnose pheochromocytoma and paraganglioma.
The catecholamine and its metabolite content in plasma is extremely low, reaching pg grade, in the quantitative detection method, the most commonly used is high performance liquid chromatography and electrochemical detection combined method, but the instrument detection sensitivity is low, and the clinical detection requirement cannot be met. In addition, the combined high performance liquid chromatography and electrochemical detection method requires a large amount of plasma sample, and a long time for sample pretreatment, so that chromatographic separation often takes a long time to achieve the required detection sensitivity of the target analyte, and co-eluted interfering components have a great influence on the accuracy of the results. The immune-based detection method can detect the content of phenylephrine in blood plasma, the detection accuracy is low due to cross reaction and nonspecific combination, and the results of different immune tests may not be uniform. Because many analysis methods cannot meet the requirements of quantitative analysis of catecholamine and metabolites thereof in plasma in the aspects of sensitivity, selectivity, analysis speed and the like, the further popularization and development of the analysis methods in clinical detection are limited.
The chromatography-mass spectrometry combined technology which takes chromatography as a separation means and triple quadrupole mass spectrometry as a detection means becomes a powerful means for quantitatively detecting trace target analytes in biological samples, so that the method is used for quantitatively detecting catecholamines and metabolites thereof in plasma. Patent CN109142594a reports a method for detecting catecholamine and phenylephrine in a body fluid sample by using liquid chromatography tandem mass spectrometry, which needs to perform protein precipitation on the sample, remove supernatant, blow-dry the supernatant by using N 2, add PITC to perform derivatization reaction, blow-dry the derivatization product by using N 2, add a complex solvent to obtain a sample to be detected, and perform detection derivatization by using liquid chromatography tandem mass spectrometry, thus having complex pretreatment, long time consumption and low detection efficiency. Patent CN111896643a discloses a method for detecting catecholamine in human plasma by liquid chromatography-mass spectrometry, wherein plasma samples are pretreated by ion exchange solid phase extraction, and the chromatographic column adopts reversed phase pentafluorophenyl (F5) chromatographic column, which is reversed phase action mechanism chromatographic column. According to the method, a large amount of manual participation is needed for sample pretreatment, the experimental efficiency is greatly reduced, meanwhile, because catecholamine and metabolites thereof are strong polar compounds, the retention capacity on the F5 column is very weak, in the experiment, the chromatographic column needs to retain the target object under a high-proportion water phase, the ionization efficiency of mass spectrum is reduced by the high-proportion water phase, and simultaneously, a large amount of endogenous interference components which are weak to retain flow out together, so that matrix interference is caused to the quantification of the target object. Solid phase extraction 96-well plates used in ion exchange solid phase extraction methods commonly used for detecting catecholamines and metabolites thereof are high-cost experimental consumables, swelling phenomenon can be generated after solid phase extraction filler contacts with an organic reagent in experiments, and the flow rate of a solution passing through the solid phase extraction filler can be influenced, so that the problem of poor uniformity among the wells is generated.
Therefore, there is a need to provide a more reliable solution.
Disclosure of Invention
The invention aims to solve the technical problem of providing a liquid chromatography-tandem mass spectrometry detection method of catecholamine and metabolites thereof based on magnetic solid-phase extraction aiming at the defects in the prior art. The invention uses magnetic solid phase extraction technology to pretreat catecholamine and its metabolite in plasma, the mechanism is matrix dispersion type solid phase extraction, in the invention, external magnetic field (such as magnetic solid phase extraction instrument and magnetic bar) can be used to transfer the solid phase extraction filler with magnetism, the whole process is easy to realize automation, and the advantages of high selectivity and high purification rate of solid phase extraction are maintained. The magnetic solid phase extraction filler used in the invention is in mg level, so that the consumable price is low, and the experimental consumable cost can be reduced; the invention also discloses a strategy for detecting the target substance by using the chromatographic column based on the amino functional group, wherein the chromatographic column is a hydrophilic action mechanism, the retention capacity of the chromatographic column to catecholamine and metabolites thereof is stronger under a high proportion of organic phase, the ionization efficiency of the target substance is greatly improved, and meanwhile, the chromatographic column achieves baseline separation of two compounds with common fragments, namely E (epinephrine) and NMN (norepinephrine), which is important for the accurate quantification of the target substance.
In order to achieve the above purpose, the invention adopts the following technical scheme: a liquid chromatography tandem mass spectrometry detection method of catecholamines and metabolites thereof based on magnetic solid phase extraction, comprising the following steps:
1) Preparing a detection sample:
1-1) taking a sample to be tested, adding an internal standard solution and an aqueous solution into the sample to be tested, and uniformly mixing;
1-2) adding the magnetic solid-phase extraction filler into acetonitrile for filler activation, and then transferring the magnetic solid-phase extraction filler into water for filler balance;
1-3) transferring the magnetic solid phase extraction filler in the product obtained in the step 1-2) into the mixed liquid obtained in the step 1-1), and mixing;
1-4) transferring the magnetic solid-phase extraction filler in the product obtained in the step 1-3) into an aqueous solution, and mixing;
1-5) transferring the magnetic solid phase extraction filler in the product obtained in the step 1-4) into acetonitrile, and mixing;
1-6) transferring the magnetic solid phase extraction filler in the product obtained in the step 1-5) to a mixed solution of acetonitrile and formic acid, and mixing;
1-7) removing the magnetic solid phase extraction filler in the product obtained in the step 1-6), and taking the rest solution as a detection sample;
2) Constructing a standard curve of the target object:
Preparing standard solutions of catecholamine and metabolites thereof with a certain concentration gradient, and detecting by a liquid chromatography-tandem mass spectrometry method to construct and obtain standard curves of catecholamine and metabolites thereof;
3) Detecting a detection sample by adopting a liquid chromatography tandem mass spectrometry method, and calculating the content of catecholamine and metabolites thereof in the detection sample by combining the constructed standard curve;
Wherein catecholamines include dopamine, norepinephrine and epinephrine, and metabolites of catecholamines include methoxytyramine, phenylephrine and norepinephrine.
Preferably, in the step 1-6), the mixed solution of acetonitrile and formic acid further contains 5-20 mug/mL of vitamin C.
Preferably, in the step 1-6), the mixed solution of acetonitrile and formic acid further contains 10 mug/mL of vitamin C.
Preferably, the volume percentage of formic acid in the mixed solution of acetonitrile and formic acid is 0.5-5%.
Preferably, the volume percentage concentration of formic acid in the mixed solution of acetonitrile and formic acid is 1%.
Preferably, the magnetic solid phase extraction filler is transferred by a magnetic solid phase extraction instrument.
Preferably, the step 1) specifically includes:
1-1) taking 250-450 mu L of a sample to be detected, adding 5-20 mu L of an internal standard solution and 200-600 mu L of an aqueous solution into the sample, and uniformly mixing for 1-4min at 500-2000 rpm;
1-2) adding 1-6mg of the magnetic solid phase extraction filler into 100-400 mu L of acetonitrile for filler activation, and then transferring the magnetic solid phase extraction filler into water for filler balance;
1-3) transferring the magnetic solid phase extraction filler in the product obtained in the step 1-2) into the mixed solution obtained in the step 1-1), and mixing for 60-240S;
1-4) transferring the magnetic solid phase extraction filler in the product obtained in the step 1-3) into 200-600 mu L of aqueous solution, and mixing for 30-120S;
1-5) transferring the magnetic solid phase extraction filler in the product obtained in the step 1-4) into 200-600 mu L of acetonitrile, and mixing for 30-120S;
1-6) transferring the magnetic solid phase extraction filler in the product obtained in the step 1-5) into 50-200 mu L of mixed solution of acetonitrile, formic acid and vitamin C, and mixing for 30-120S; the volume percentage concentration of formic acid in the mixed solution is 1 percent;
1-7) transferring the magnetic solid phase extraction filler in the product obtained in the step 1-6) to the residual solution in the product in the step 1), and discarding; taking the residual solution in the products obtained in the steps 1-6) as a detection sample.
Preferably, the step 1) specifically includes:
1-1) taking 390 mu L of a sample to be tested, adding 10 mu L of an internal standard solution and 400 mu L of an aqueous solution into the sample, and uniformly mixing the mixture for 2min at 1000 revolutions per minute;
1-2) adding 3mg of the magnetic solid phase extraction filler into 200 mu L of acetonitrile for filler activation, and then transferring the magnetic solid phase extraction filler into water for filler balance;
1-3) transferring the magnetic solid phase extraction filler in the product obtained in the step 1-2) into the mixed solution obtained in the step 1-1), and mixing for 120S;
1-4) transferring the magnetic solid phase extraction filler in the product obtained in the step 1-3) into 400 mu L of aqueous solution, and mixing for 60S;
1-5) transferring the magnetic solid phase extraction filler in the product obtained in the step 1-4) into 400 mu L of acetonitrile, and mixing for 60S; 1-6) transferring the magnetic solid phase extraction filler in the product obtained in the step 1-5) into 100 mu L of mixed solution of acetonitrile, formic acid and vitamin C, and mixing for 60S; the concentration of formic acid in the mixed solution is 1%;
1-7) transferring the magnetic solid phase extraction filler in the product obtained in the step 1-6) to the residual solution in the product in the step 1), and discarding; taking the residual solution in the products obtained in the steps 1-6) as a detection sample.
Preferably, in the step 3), the liquid chromatography detection conditions are:
Chromatographic column: waters XBridge BEH Amide;
Mobile phase: phase A is an aqueous solution containing 5% acetonitrile (v/v) and containing ammonium acetate at a concentration of 10mM, and phase B comprises: an acetonitrile solution containing 5% water (v/v), 0.1% (v/v) formic acid;
gradient elution procedure: the starting ratio was 90% b phase, 1.5min down to 70% b phase, 70% b hold to 3min,3.1min up to 90% b phase, and hold to 6.6min;
Flow rate: 0.6mL/min; sample injection amount: 20. Mu.L; column temperature: 35 ℃.
Preferably, in the step 3), the mass spectrometry conditions are:
ion source: an electrospray ion source;
the detection mode is as follows: monitoring multiple reactions;
The air curtain gas is 20psi, the sprayer is 55psi, the auxiliary heating gas is 45psi, the temperature is 450 ℃, the ionization voltage is 3500V, and the collision gas is 8psi.
The beneficial effects of the invention are as follows:
according to the liquid chromatography tandem mass spectrometry detection method based on catecholamine and metabolites thereof, which is provided by the invention, the catecholamine and metabolites thereof detection sample is subjected to pretreatment by a magnetic solid-phase extraction technology, and the separation and transfer of the to-be-detected substances are rapidly realized by applying external magnetic field energy, so that the automatic pretreatment is conveniently realized, and meanwhile, the advantages of high selectivity, high purification rate and the like of solid-phase extraction are also maintained;
according to the invention, a chromatographic column based on a hydrophilic action mechanism of an amino functional group is used for detecting a target object, so that the ionization efficiency of mass spectrum on the target object is greatly improved, the quantitative limit of the target object is reduced, and meanwhile, the chromatographic column can separate E (epinephrine) and NMN (norepinephrine) compounds with common fragments from each other to achieve a baseline, so that the quantitative accuracy of the target object is improved;
The invention adopts a high proportion of organic phase as an eluent, which is beneficial to reducing the co-outflow of endogenous interference components and reducing the matrix effect;
The invention increases the contact area through the dispersion adsorption of the magnetic solid phase extraction filler, can improve the adsorption rate and the enrichment effect, and shortens the balance time; the invention can avoid the errors of SPE flow speed difference, filler swelling and the like, and reduce the variability of mass spectrum detection;
The sample treatment step can remove the interference of endogenous phospholipid, protein and other substances, and reduce the matrix effect and instrument pollution risk.
Drawings
FIG. 1 is a graph of a target standard obtained in an embodiment of the present invention;
FIG. 2 is a graph of a target object obtained in an embodiment of the present invention;
FIG. 3 is a schematic diagram of the principle of magnetic solid phase extraction packing transfer;
Detailed Description
The present invention is described in further detail below with reference to examples to enable those skilled in the art to practice the same by referring to the description.
It will be understood that terms, such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.
Description of the sources of the reagents
(1) Epinephrine (E, shanghai Selfie Biotechnology Co., ltd.);
(2) Epinephrine-d 6 (E-d 6, shanghai Seifeng Biotechnology Co., ltd.);
(3) DL-norepinephrine HCl (NE, shanghai Seifeng Biotechnology Co., ltd.);
(4) DL-norepinephrine-d 6 hydrochloride (NE-d 6, shanghai Seifen Biotechnology Co., ltd.);
(5) Dopamine hydrochloride (DA, shanghai spectral Fender Biotechnology Co., ltd.);
(6) Dopamine-d 4 hydrochloride (DA-d 4, shanghai spectral Fender Biotechnology Co., ltd.);
(7) 3-methoprenamide hydrochloride (3-MT, shanghai Seifeng Biotechnology Co., ltd.);
(8) 3-methoxytyramine-D4 hydrochloride (3-MT-D4, shanghai Seifen Biotechnology Co., ltd.);
(9) DL-norepinephrine hydrochloride (NMN, shanghai Seifeng Biotechnology Co., ltd.);
(10) DL-norepinephrine hydrochloride-d 3 (NMN-d 3, shanghai Seifeng Biotechnology Co., ltd.);
(11) Phenylephrine hydrochloride (MN, shanghai spectral Fender Biotechnology Co., ltd.);
(12) DL-phenylephrine hydrochloride-d 3 (MN-d 3, shanghai Seifeng Biotechnology Co., ltd.);
(13) Acetonitrile (conkede technologies);
(14) Ammonium acetate (Thermo Fisher);
(15) Formic acid (Thermo Fisher);
(16) Vitamin C (sigma);
The high-purity water is primary water conforming to International GB/T6682-2008.
Example 1
The present embodiment provides a liquid chromatography tandem mass spectrometry detection method of catecholamines and metabolites thereof based on magnetic solid phase extraction, wherein the catecholamines include Dopamine (DA), norepinephrine (NE) and epinephrine (E), and the metabolites of catecholamines include methoxytyramine (3-MT), epinephrine (MN) and norepinephrine (NMN).
The method comprises the following steps:
1) Preparing a detection sample:
1-1) taking 390 mu L of a sample to be tested, adding 10 mu L of an internal standard solution and 400 mu L of an aqueous solution into the sample, and uniformly mixing the mixture for 2min at 1000 revolutions per minute;
1-2) adding 3mg of magnetic solid phase extraction filler into a clean sample tube, using 200 mu L of acetonitrile to activate the filler, transferring the filler into 400 mu L of water by a magnetic solid phase extraction instrument to perform filler balance;
1-3) transferring the magnetic solid phase extraction filler in the product obtained in the step 1-2) into the mixed solution obtained in the step 1-1), and mixing for 120S;
1-34) transferring the magnetic solid phase extraction filler in the product obtained in the step 1-3) into 400 mu L of aqueous solution, and mixing for 60S;
1-5) transferring the magnetic solid phase extraction filler in the product obtained in the step 1-4) into 400 mu L of acetonitrile, and mixing for 60S;
1-6) transferring the magnetic solid phase extraction filler in the product obtained in the step 1-5) into 100 mu L of acetonitrile solution containing 1% of formic acid by volume percentage (and containing 10 mu g/mL of vitamin C), and mixing for 60S;
1-7) transferring the magnetic solid phase extraction filler in the product obtained in the step 1-6) to the residual solution in the product in the step 1), and discarding; taking the residual solution in the products obtained in the steps 1-6) as a detection sample.
2) Constructing a standard curve of the target object:
Preparing standard liquid of catecholamine and its metabolite with a certain concentration gradient, detecting by liquid chromatography tandem mass spectrometry, and constructing to obtain standard curve of catecholamine and its metabolite.
In this embodiment, the detection range is determined according to the clinical detection requirements of catecholamines and metabolites, standard solutions of catecholamines and metabolites with serial concentration gradients are set in the detection range requirements, and are used for liquid chromatography tandem mass spectrometry detection, and the ratio of the target analyte to the internal standard peak area is fitted with the gradient concentration to obtain a working curve.
In this example, stock solutions of the target substances were prepared using methanol/water (volume ratio: 1/1 and containing 10. Mu.g/mL of vitamin C), and then diluted to obtain standard working solutions, wherein the concentrations of E, NE and NMN standard working solutions were 20ng/mL, 50ng/mL and 1000ng/mL, the concentrations of DA standard working solutions were 20ng/mL, 100ng/mL and 1000ng/mL, and the concentrations of 3-MT and MN standard working solutions were 2ng/mL, 50ng/mL and 1000ng/mL. And mixing the target standard working solution with a diluent to form a series of standard solutions with concentration gradients, and detecting by using a liquid chromatography tandem mass spectrometry to obtain a standard working curve, wherein the concentration of the standard solution is shown in a table 1, and the concentration of the internal standard working solution is 20ng/mL.
TABLE 1 Standard solution concentration
In this embodiment, the obtained target standard curves are shown in fig. 1 (a) - (f), wherein (a) is the standard curve of DA; (b) a standard curve of E; (c) a standard curve of NE; (d) a standard curve of MN; (e) a standard curve of NMN; (f) standard curve of 3-MT. The linear relation of the fitting is good, r values are above 0.995, and the linear range of the target is shown in Table 2.
TABLE 2 target linear range and linear relationship
3) Detecting a detection sample by adopting a liquid chromatography tandem mass spectrometry method, and calculating the content of catecholamine and metabolites thereof in the detection sample by combining the constructed standard curve;
The liquid chromatography detection conditions are as follows:
1. Chromatographic column: waters XBridge BEH Amide (2.1 x 100mm,2.5 μm);
2. Mobile phase: phase A is an aqueous solution containing 5% acetonitrile (v/v) and containing ammonium acetate at a concentration of 10mM, and phase B comprises: an acetonitrile solution containing 5% water (v/v), 0.1% (v/v) formic acid;
3. gradient elution procedure: the starting ratio was 90% b phase, 1.5min down to 70% b phase, 70% b hold to 3min,3.1min up to 90% b phase, and hold to 6.6min;
4. Flow rate: 0.6mL/min; sample injection amount: 20. Mu.L; column temperature: 35 ℃.
The mass spectrum conditions are as follows:
1. ion source: electrospray ion source (ESI);
2. The detection mode is as follows: multiple Reaction Monitoring (MRM);
3. Air Curtain (CUR) 20psi, atomizer (GS 1) 55psi, auxiliary heating gas (GS 2) 45psi, temperature (TEM) 450 ℃, ionization voltage (IS) 3500V, collision gas (CAD) 8psi;
4. The mass spectral parameters of parent ion, residence time, cone-hole voltage, collision energy, etc. for each compound are shown in table 3 below.
Table 3 mass spectrum acquisition parameters
| Q1 | Q3 | Dwell time | ID | DP | EP | CE |
| 184.2 | 166 | 15 | E-1 | 36 | 7 | 12 |
| 190.2 | 172.1 | 15 | E-d6 | 38 | 6 | 13 |
| 170.3 | 152.1 | 15 | NE-1 | 36 | 7 | 11 |
| 176.1 | 158.1 | 15 | NE-d6 | 55 | 7 | 9 |
| 198.1 | 180.1 | 15 | MN-1 | 35 | 7 | 11 |
| 183.2 | 151.2 | 15 | MN-d3 | 80 | 8 | 24 |
| 184.1 | 166.1 | 15 | NMN-1 | 35 | 6 | 12 |
| 187.1 | 169.1 | 15 | NMN-d3 | 35 | 6 | 12 |
| 168.1 | 91 | 15 | 3-MT-1 | 44 | 7 | 32 |
| 172 | 155.1 | 15 | 3-MT-d4 | 43 | 6 | 14 |
| 154.1 | 91.1 | 15 | DA-1 | 52 | 7 | 33 |
| 158.1 | 141 | 15 | DA-d4 | 20 | 10 | 30 |
Referring to fig. 2, there is a target chromatogram, wherein (a) a chromatogram of DA; (b) a chromatogram of E; (c) a chromatogram of NE; (d) chromatogram of MN; (e) chromatogram of NMN; (f) chromatogram of 3-MT. The 2b graph and the 2E graph are respectively E and NMN chromatograms, but two peaks appear, because E and NMN have cracking phenomena in the detection process to cause common fragments of the E and NMN, and the chromatographic column and the established gradient condition adopted by the invention can separate the E and NMN from each other to achieve a base line without interfering with the accurate quantification of the E and NMN. Wherein E has a retention time of 2.87min and NMN has a retention time of 2.74min.
In the step 1), transferring a magnetic solid-phase extraction instrument through an external magnetic field; in a preferred embodiment, a magnetic solid phase extraction instrument is used to transfer the magnetic solid phase extraction packing. Referring to fig. 3, a schematic diagram of magnetic solid phase extraction packing transfer is shown, wherein a magnetic solid phase extraction instrument is a magnetic rod, a stirring sleeve is sleeved outside the magnetic rod, and magnetic beads (magnetic solid phase extraction packing) are collected through magnetism of the magnetic rod. The magnetic solid phase extraction filler can be weak cation exchange type magnetic solid phase extraction filler such as magnetic microspheres or magnetic beads.
Example 2 experimental evaluation of the detection method of the present invention
1. Evaluation of matrix Effect
In the experiment, matrix effect is evaluated by a sample treatment post-addition method, namely mf=response of the extracted blank matrix to the analyte/response of the analyte in the pure solvent. Internal standard normalization MF = test substance MF/internal standard MF, when the internal standard normalization MF ranges from 0.8 to 1.2 acceptable. Three concentrations were selected for each analyte, and 5 samples of each concentration were assayed. The results are shown in tables 4-9, indicating that the MF of each compound is within the accepted range.
Internal standard normalization of Table 4E
Internal standard normalization of Table 5 NE
Internal standard normalization of Table 6 DA
Internal standard normalization of Table 7 MN
Internal standard normalization of Table 8 NMN
Internal standard normalization of Table 9 3-MT
2. Lower limit of quantification
According to clinical detection requirements and sensitivity of instruments, the method for quantifying the target is defined as follows: the measurement was continued 10 times with E of 12.5pg/mL, NE of 40pg/mL, DA of 12.5pg/mL, 3-MT of 5pg/mL, MN of 5pg/mL, NMN of 12.5pg/mL, and the deviation between the measured value and the theoretical value and the coefficient of variation CV value of the measurement result were calculated. The test results are shown in Table 10. The result shows that the deviation of the test value and the theoretical value is within 15%, the variation coefficient of the tested parallel samples is within 20%, and the clinical detection method meets the requirements.
Table 10 quantitative lower limit sample test results
3. Precision of
And selecting the precision of the low, medium and high concentration evaluation methods, wherein each concentration sample of each batch is processed for at least 5 times, 3 batches are continuously measured, the total number of samples is at least 45, and the intra-batch precision, the inter-batch precision and the total precision of each concentration sample are respectively evaluated. The results are shown in tables 11 to 16.
Table 11E method precision
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Table 12 NE method precision
Table 13 DA method precision
Table 14 MN method precision
Table 15 NMN method precision
TABLE 16 precision of method for 3-MT
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As shown in the data results, the precision in batch, the precision between batches and the total precision CV of the low, medium and high concentrations are all within 15 percent, which meets the requirements of methodological verification.
4. Accuracy of
And (3) truly mixing human serum+ samples, testing each sample three times, and calculating the standard adding recovery rate and the variation coefficient of the detection result. The results are shown in tables 17 to 22. From the data, the deviation between the detection value and the theoretical value of the marked sample is within + -15%.
Table 17E method recovery
Table 18 NE method recovery
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Table 19 DA Process recovery
Table 20 MN method recovery
Table 21 NMN Process recovery
TABLE 22 method recovery of 3-MT
Although embodiments of the present invention have been disclosed above, it is not limited to the use of the description and embodiments, it is well suited to various fields of use for the invention, and further modifications may be readily apparent to those skilled in the art, and accordingly, the invention is not limited to the particular details without departing from the general concepts defined in the claims and the equivalents thereof.
Claims (7)
1. A liquid chromatography tandem mass spectrometry detection method of catecholamines and metabolites thereof based on magnetic solid phase extraction, which is characterized by comprising the following steps:
1) Preparing a detection sample:
1-1) taking a sample to be tested, adding an internal standard solution and an aqueous solution into the sample to be tested, and uniformly mixing;
1-2) adding the magnetic solid-phase extraction filler into acetonitrile for filler activation, and then transferring the magnetic solid-phase extraction filler into water for filler balance;
1-3) transferring the magnetic solid phase extraction filler in the product obtained in the step 1-2) into the mixed liquid obtained in the step 1-1), and mixing;
1-4) transferring the magnetic solid-phase extraction filler in the product obtained in the step 1-3) into an aqueous solution, and mixing;
1-5) transferring the magnetic solid phase extraction filler in the product obtained in the step 1-4) into acetonitrile, and mixing;
1-6) transferring the magnetic solid phase extraction filler in the product obtained in the step 1-5) to a mixed solution of acetonitrile and formic acid, and mixing;
1-7) removing the magnetic solid phase extraction filler in the product obtained in the step 1-6), and taking the rest solution as a detection sample;
2) Constructing a standard curve of the target object:
Preparing standard solutions of catecholamine and metabolites thereof with a certain concentration gradient, and detecting by a liquid chromatography-tandem mass spectrometry method to construct and obtain standard curves of catecholamine and metabolites thereof;
3) Detecting a detection sample by adopting a liquid chromatography tandem mass spectrometry method, and calculating the content of catecholamine and metabolites thereof in the detection sample by combining the constructed standard curve;
Wherein catecholamines include dopamine, norepinephrine and epinephrine, and metabolites of catecholamines include methoxytyramine, phenylephrine and norepinephrine;
The step 1) specifically comprises the following steps:
1-1) taking 250-450 mu L of a sample to be detected, adding 5-20 mu L of an internal standard solution and 200-600 mu L of an aqueous solution into the sample, and uniformly mixing for 1-4min at 500-2000 rpm;
1-2) adding 1-6mg of the magnetic solid phase extraction filler into 100-400 mu L of acetonitrile for filler activation, and then transferring the magnetic solid phase extraction filler into water for filler balance;
1-3) transferring the magnetic solid phase extraction filler in the product obtained in the step 1-2) into the mixed solution obtained in the step 1-1), and mixing for 60-240S; 1-4) transferring the magnetic solid phase extraction filler in the product obtained in the step 1-3) into 200-600 mu L of aqueous solution, and mixing for 30-120S;
1-5) transferring the magnetic solid phase extraction filler in the product obtained in the step 1-4) into 200-600 mu L of acetonitrile, and mixing for 30-120S;
1-6) transferring the magnetic solid phase extraction filler in the product obtained in the step 1-5) into 50-200 mu L of mixed solution of acetonitrile, formic acid and vitamin C, and mixing for 30-120S; the volume percentage of formic acid in the mixed solution is 1 percent;
1-7) transferring the magnetic solid phase extraction filler in the product obtained in the step 1-6) to the residual solution in the product in the step 1), and discarding; taking the residual solution in the product obtained in the step 1-6) as a detection sample;
in the step 3), the liquid chromatography detection conditions are as follows:
chromatographic column: waters XBridge BEH Amide;
Mobile phase: phase a is an aqueous solution containing 5% acetonitrile by volume and contains ammonium acetate at a concentration of 10mM, and phase B comprises: an acetonitrile solution containing 5% by volume of water and 0.1% by volume of formic acid;
gradient elution procedure: the starting ratio was 90% b phase, 1.5min down to 70% b phase, 70% b hold to 3min,3.1min up to 90% b phase, and hold to 6.6min;
Flow rate: 0.6mL/min; sample injection amount: 20 [ mu ] L; column temperature: 35 ℃;
In step 3), the mass spectrometry conditions are:
ion source: an electrospray ion source;
the detection mode is as follows: monitoring multiple reactions;
The air curtain gas is 20psi, the sprayer is 55psi, the auxiliary heating gas is 45psi, the temperature is 450 ℃, the ionization voltage is 3500V, and the collision gas is 8psi.
2. The method for detecting the catecholamine and the metabolites thereof by liquid chromatography tandem mass spectrometry based on the magnetic solid phase extraction according to claim 1, wherein in the step 1-6), the mixed solution of acetonitrile and formic acid further contains vitamin C of 5-20 μg/mL.
3. The method for detecting the catecholamine and the metabolites thereof by liquid chromatography tandem mass spectrometry based on the magnetic solid phase extraction according to claim 2, wherein in the step 1-6), the mixed solution of acetonitrile and formic acid further contains 10 μg/mL of vitamin C.
4. The method for detecting catecholamine and its metabolites by liquid chromatography tandem mass spectrometry based on magnetic solid phase extraction according to claim 3, wherein the volume percentage of formic acid in the mixed solution of acetonitrile and formic acid is 0.5-5%.
5. The method for detecting catecholamine and its metabolites by liquid chromatography tandem mass spectrometry based on magnetic solid phase extraction according to claim 4, wherein the volume percentage of formic acid in the mixed solution of acetonitrile and formic acid is 1%.
6. The method for detecting the catecholamine and the metabolites thereof by liquid chromatography tandem mass spectrometry based on the magnetic solid phase extraction according to claim 5, wherein the magnetic solid phase extraction filler is transferred by a magnetic solid phase extraction instrument.
7. The method for detecting the catecholamine and the metabolites thereof by liquid chromatography tandem mass spectrometry based on the magnetic solid phase extraction according to claim 1, wherein the step 1) specifically comprises:
1-1) taking 390 mu L of a sample to be tested, adding 10 mu L of an internal standard solution and 400 mu L of an aqueous solution into the sample, and uniformly mixing the mixture for 2min at 1000 revolutions per minute;
1-2) adding 3mg of the magnetic solid phase extraction filler into 200 mu L of acetonitrile for filler activation, and then transferring the magnetic solid phase extraction filler into water for filler balance;
1-3) transferring the magnetic solid phase extraction filler in the product obtained in the step 1-2) into the mixed solution obtained in the step 1-1), and mixing for 120S;
1-4) transferring the magnetic solid phase extraction filler in the product obtained in the step 1-3) into 400 mu L of aqueous solution, and mixing for 60S;
1-5) transferring the magnetic solid phase extraction filler in the product obtained in the step 1-4) into 400 mu L of acetonitrile, and mixing for 60S;
1-6) transferring the magnetic solid phase extraction filler in the product obtained in the step 1-5) into 100 mu L of mixed solution of acetonitrile, formic acid and vitamin C, and mixing for 60S; the volume percentage of formic acid in the mixed solution is 1 percent;
1-7) transferring the magnetic solid phase extraction filler in the product obtained in the step 1-6) to the residual solution in the product in the step 1), and discarding; taking the residual solution in the products obtained in the steps 1-6) as a detection sample.
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