CN112505168A - LC-MS/MS detection method and pretreatment kit for catecholamine - Google Patents
LC-MS/MS detection method and pretreatment kit for catecholamine Download PDFInfo
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Abstract
The invention relates to the technical field of catecholamine detection, in particular to an LC-MS/MS (liquid chromatography-mass spectrometry/mass spectrometry) detection method of catecholamine, which mainly comprises the steps of putting a detection sample into a centrifugal tube, and adding an internal standard solution and a diluent for dilution; adding the diluted solution into a WCX solid phase extraction column for extraction, and leaching by using a leacheate after extraction; after washing, vacuum-pumping the WCX solid phase extraction column, extracting a solution, eluting by using a methyl formate solution, collecting an eluent, drying the eluent by using rare gas, and re-dissolving; filtering the re-dissolved solution with superfine filter membrane, and placing in chromatographic column for chromatographic analysis. The kit also comprises a catecholamine LC-MS/MS detection pretreatment kit, wherein the pretreatment kit comprises a WCX solid phase treatment extraction column, diluent and eluent. The invention effectively improves the detection precision of catecholamine in the prior art, and the detection flow improves the detection efficiency.
Description
Technical Field
The invention relates to the technical field of catecholamine detection, in particular to an LC-MS/MS detection method and a pretreatment kit of catecholamine.
Background
Catecholamines are a class of neurological substances containing catechol and amine groups. The catechol and amine groups are combined by enzymatic steps of L-tyrosine at sympathetic, adrenal medulla and chromaffin cell sites. Generally, catecholamines refer to Norepinephrine (NA), epinephrine (Adr), and Dopamine (DA). All three catecholamines are derived from tyrosine precursors. I.e., catechol-containing amines including dopamine, norepinephrine and epinephrine and their derivatives. Norepinephrine and epinephrine are both hormones secreted by the adrenal medulla and the neurotransmitters of the sympathetic and norepinephrine fibers in the central nervous system. Norepinephrine is widely distributed in the central nervous system, and is abundant, while epinephrine is less. Dopamine is mainly concentrated in extrapyramidal sites and is also a neuromediator. They are important classical adrenoreceptor agonists. Catecholamines regulate basic physiological functions in vivo and transmit physiological signals, are important signal media in normal physiological processes, and have corresponding content changes in pathological processes. Can be clinically used for auxiliary diagnosis of endocrine related diseases such as hypertension, hyperthyroidism, pheochromocytoma, neuroblastoma and the like. The chemical structure of catecholamine substances is characterized in that: has a bishydroxybenzene nucleus and a side chain with amino, and has extremely low content of catecholamine substances in a biological sample.
At present, catecholamine detection technology mostly adopts a traditional Mass Spectrometry method for detection, and Mass Spectrometry (MS) is a method for detecting after moving ions (charged atoms, molecules or molecular fragments, ions generated by molecular ions, isotope ions, fragment ions, rearrangement ions, multi-charged ions, metastable ions, negative ions and ions generated by ion-molecule interaction) are separated according to Mass-to-charge ratios thereof by using an electric field and a magnetic field. The accurate mass of the ions is measured, and the compound composition of the ions can be determined. This is because the exact mass of a species is a multi-bit fraction, and never both species will have the same mass, and never will have the mass of one species exactly an integer multiple of the mass of the other species. Analysis of these ions yields information on the molecular weight, chemical structure, cleavage pattern of the compound and some correlation between certain ions formed by the decomposition of single molecules.
The prior catecholamine detection method has the problems of larger measurement result error, insufficient detection precision and the like due to lack of pretreatment on a detection sample or poor treatment effect, which is easy to cause matrix effect.
Disclosure of Invention
The invention aims to provide a catecholamine LC-MS/MS detection method and a catecholamine pretreatment kit, so as to solve the problems in the background technology.
A LC-MS/MS detection method of catecholamine comprises the following steps:
the method comprises the following steps: placing a detection sample in a centrifuge tube, and adding an internal standard solution and a diluent for dilution;
step two: adding the diluted solution into a WCX solid phase extraction column for extraction, and leaching by using a leacheate after extraction;
step three: after washing, vacuum-pumping the WCX solid phase extraction column, extracting a solution, eluting by using a methyl formate solution, collecting an eluent, drying the eluent by using rare gas, and re-dissolving;
step four: filtering the re-dissolved solution with superfine filter membrane, and placing in chromatographic column for chromatographic analysis.
Preferably, the internal standard solution in the step one is an isoproterenol internal standard solution, and the diluted solution is a mixed solution of ascorbic acid and ammonium acetate.
Preferably, the dilution liquid contains 0.1-0.5% ascorbic acid by mass and 30-60 mmol.L ammonium acetate concentration-1And the mixing ratio of the diluent, the internal standard solution and the detection sample is 4: 1.
Preferably, the WCX solid phase extraction column before extraction in the second step needs to be activated by a mixed solution of methanol and deionized water in advance, the mass fraction of the methanol in the mixed solution is 30-50%, and the column passing speed of the extraction is 0.5 ml/min.
Preferably, the leacheate in the second step is a mixed solution of 10-30mmol/L of ammonium acetate, acetonitrile and isopropanol in a ratio of 1: 1, and the mixed solution of ammonium acetate, acetonitrile and isopropanol is leached sequentially for 2-3 times during leaching.
Preferably, the vacuum pumping time in the third step is 20-40s, and the mass fraction of the methyl formate is 2-3%.
Preferably, the solvent for redissolution in the third step is 0.2mol/L formic acid solution, and the content of the redissolution solvent is 50-150 ml.
Preferably, the filtration diameter of the superfine filter membrane in the fourth step is 200-500 nm.
Preferably, the chromatographic column in the step four is a pentafluorophenyl reverse phase chromatographic column, and the surface of the pentafluorophenyl reverse phase chromatographic column is bonded with pentafluorophenethyl by using ultrapure silica gel as a matrix.
A LC-MS/MS detection pretreatment kit for catecholamine comprises a WCX solid phase treatment extraction column, a diluent and an eluent.
Compared with the prior art, the invention has the beneficial effects that: compared with the traditional alumina adsorption or liquid-liquid extraction, the extraction of catecholamine in blood plasma by the solid-phase extraction column is simpler and has high recovery rate, meanwhile, the elution by using an eluent (ammonium acetate, mixed liquid of acetonitrile and isopropanol) can effectively remove lipid substances in a sample, reduce ion inhibition and improve benefit, and because the polarity in the catecholamine is larger, a pentafluorophenyl reverse phase chromatographic column is further adopted, and the pentafluorophenyl can generate dipole induction effect, so that the separation capability of the aromatic ring, heterocyclic compound, halide and other easily polarized substances is stronger, and the separation capability is stronger, and because the catecholamine has a plurality of phenolic hydroxyl groups which are easily oxidized, ascorbic acid is added into a diluent in the treatment process, the oxidation of the catecholamine is prevented, and the stability of the catecholamine is effectively ensured.
Detailed Description
The invention discloses a catecholamine LC-MS/MS detection method and a pretreatment kit, and the invention is further detailed by specific embodiments below.
Example 1
The LC-MS/MS detection method of catecholamine mainly comprises the following steps:
the method comprises the following steps: putting blank plasma of a healthy person into a centrifugal tube, and adding an internal standard solution and a diluent for dilution;
step two: adding the diluted solution into a WCX solid phase extraction column for extraction, and leaching by using a leacheate after extraction;
step three: after washing, vacuum-pumping the WCX solid phase extraction column, extracting a solution, eluting by using a methyl formate solution, collecting an eluent, drying the eluent by using rare gas, and re-dissolving;
step four: filtering the re-dissolved solution with superfine filter membrane, and placing in chromatographic column for chromatographic analysis.
The internal standard solution in the step one is specifically isoproterenol internal standard solution, the diluted solution is specifically ascorbic acid and ammonium acetate mixed solution, the mass fraction of ascorbic acid in the diluted solution is 0%, and the concentration of ammonium acetate is 50 mmol.L-1And the mixing ratio of the diluent, the internal standard solution and the detection sample is 4: 1.
And (2) activating the WCX solid-phase extraction small column by using a mixed solution of methanol and deionized water in advance before extraction in the second step, wherein the mass fraction of the methanol in the mixed solution is 50%, the column passing speed of extraction is 0.5ml/min, the leacheate in the second step is a mixed solution of 20mmol/L of ammonium acetate and acetonitrile and isopropanol in a ratio of 1: 1, and the mixed solution of the ammonium acetate and the acetonitrile and the isopropanol is leached sequentially for 2-3 times during leaching.
The vacuum pumping time in the third step is 30s, the mass fraction of the methyl formate is 2 percent, the solvent for redissolution in the third step is 0.2mol/L formic acid solution, and the content of the redissolution solvent is 100 ml.
The diameter of the superfine filter membrane in the step IV is 400nm, the chromatographic column in the step IV is a pentafluorophenyl reverse phase chromatographic column, and the surface of the pentafluorophenyl reverse phase chromatographic column takes ultrapure silica gel as a matrix to be bonded with pentafluorophenethyl.
The kit also comprises a catecholamine LC-MS/MS detection pretreatment kit, wherein the pretreatment kit comprises a WCX solid phase treatment extraction column, diluent and eluent.
Example 2
The LC-MS/MS detection method of catecholamine mainly comprises the following steps:
the method comprises the following steps: putting blank plasma and a standard substance of a healthy person into a centrifugal tube, and adding an internal standard solution and a diluent for dilution;
step two: adding the diluted solution into a WCX solid phase extraction column for extraction, and leaching by using a leacheate after extraction;
step three: after washing, vacuum-pumping the WCX solid phase extraction column, extracting a solution, eluting by using a methyl formate solution, collecting an eluent, drying the eluent by using rare gas, and re-dissolving;
step four: filtering the re-dissolved solution with superfine filter membrane, and placing in chromatographic column for chromatographic analysis.
The internal standard solution in the step one is specifically isoproterenol internal standard solution, the diluted solution is specifically ascorbic acid and ammonium acetate mixed solution, the mass fraction of ascorbic acid in the diluted solution is 0%, and the concentration of ammonium acetate is 50 mmol.L-1And the mixing ratio of the diluent, the internal standard solution and the detection sample is 4: 1.
And (2) activating the WCX solid-phase extraction small column by using a mixed solution of methanol and deionized water in advance before extraction in the second step, wherein the mass fraction of the methanol in the mixed solution is 50%, the column passing speed of extraction is 0.5ml/min, the leacheate in the second step is a mixed solution of 20mmol/L of ammonium acetate and acetonitrile and isopropanol in a ratio of 1: 1, and the mixed solution of the ammonium acetate and the acetonitrile and the isopropanol is leached sequentially for 2-3 times during leaching.
The vacuum pumping time in the third step is 30s, the mass fraction of the methyl formate is 2 percent, the solvent for redissolution in the third step is 0.2mol/L formic acid solution, and the content of the redissolution solvent is 100 ml.
The diameter of the superfine filter membrane in the step IV is 400nm, the chromatographic column in the step IV is a pentafluorophenyl reverse phase chromatographic column, and the surface of the pentafluorophenyl reverse phase chromatographic column takes ultrapure silica gel as a matrix to be bonded with pentafluorophenethyl.
The kit also comprises a catecholamine LC-MS/MS detection pretreatment kit, wherein the pretreatment kit comprises a WCX solid phase treatment extraction column, diluent and eluent.
Example 3
The LC-MS/MS detection method of catecholamine mainly comprises the following steps:
the method comprises the following steps: placing the plasma of a patient in a centrifugal tube, and adding an internal standard solution and a diluent for dilution;
step two: adding the diluted solution into a WCX solid phase extraction column for extraction, and leaching by using a leacheate after extraction;
step three: after washing, vacuum-pumping the WCX solid phase extraction column, extracting a solution, eluting by using a methyl formate solution, collecting an eluent, drying the eluent by using rare gas, and re-dissolving;
step four: filtering the re-dissolved solution with superfine filter membrane, and placing in chromatographic column for chromatographic analysis.
The internal standard solution in the step one is specifically isoproterenol internal standard solution, the diluted solution is specifically ascorbic acid and ammonium acetate mixed solution, the mass fraction of ascorbic acid in the diluted solution is 0%, and the concentration of ammonium acetate is 50 mmol.L-1And the mixing ratio of the diluent, the internal standard solution and the detection sample is 4: 1.
And (2) activating the WCX solid-phase extraction small column by using a mixed solution of methanol and deionized water in advance before extraction in the second step, wherein the mass fraction of the methanol in the mixed solution is 50%, the column passing speed of extraction is 0.5ml/min, the leacheate in the second step is a mixed solution of 20mmol/L of ammonium acetate and acetonitrile and isopropanol in a ratio of 1: 1, and the mixed solution of the ammonium acetate and the acetonitrile and the isopropanol is leached sequentially for 2-3 times during leaching.
The vacuum pumping time in the third step is 30s, the mass fraction of the formic ester is 2 percent, the solvent for redissolution in the third step is 0.2mol/L formic acid solution, and the content of the redissolution solvent is 100 ml.
The diameter of the superfine filter membrane in the step IV is 400nm, the chromatographic column in the step IV is a pentafluorophenyl reverse phase chromatographic column, and the surface of the pentafluorophenyl reverse phase chromatographic column takes ultrapure silica gel as a matrix to be bonded with pentafluorophenethyl.
The kit also comprises a catecholamine LC-MS/MS detection pretreatment kit, wherein the pretreatment kit comprises a WCX solid phase treatment extraction column, diluent and eluent.
The results of the experimental mass spectrometry are shown in fig. 1, wherein i is the results of the first example, ii, the second example, iii, and the third example.
The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, and the above embodiments and descriptions are only preferred examples of the present invention and are not intended to limit the present invention, and that various changes and modifications may be made without departing from the spirit and scope of the present invention, which fall within the scope of the claimed invention. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (10)
1. A LC-MS/MS detection method of catecholamine is characterized in that: comprises the following steps:
the method comprises the following steps: placing a detection sample in a centrifuge tube, and adding an internal standard solution and a diluent for dilution;
step two: adding the diluted solution into a WCX solid phase extraction column for extraction, and leaching by using a leacheate after extraction;
step three: after washing, vacuum-pumping the WCX solid phase extraction column, extracting a solution, eluting by using a methyl formate solution, collecting an eluent, drying the eluent by using rare gas, and re-dissolving;
step four: filtering the re-dissolved solution with superfine filter membrane, and placing in chromatographic column for chromatographic analysis.
2. The LC-MS/MS detection method of catecholamines according to claim 1, wherein: the internal standard solution in the step one is specifically isoproterenol internal standard solution, and the diluted solution is specifically ascorbic acid and ammonium acetate mixed solution.
3. The LC-MS/MS detection method of catecholamines according to claim 2, wherein: the dilution contains ascorbic acid 0.1-0.5 wt%, and ammonium acetate 30-60 mmol.L-1And the mixing ratio of the diluent, the internal standard solution and the detection sample is 4: 1.
4. The LC-MS/MS detection method of catecholamines according to claim 1, wherein: and the WCX solid phase extraction small column is activated by using a mixed solution of methanol and deionized water in advance before extraction in the second step, wherein the mass fraction of the methanol in the mixed solution is 30-50%, and the column passing speed of the extraction is 0.5 ml/min.
5. The LC-MS/MS detection method of catecholamines according to claim 1, wherein: and in the second step, the leacheate is a mixed solution of 10-30mmol/L of ammonium acetate, acetonitrile and isopropanol in a ratio of 1: 1, and the mixed solution of the ammonium acetate, the acetonitrile and the isopropanol is leached sequentially for 2-3 times during leaching.
6. The LC-MS/MS detection method of catecholamines according to claim 1, wherein: in the third step, the vacuum pumping time is 20-40s, and the mass fraction of the methyl formate is 2-3%.
7. The LC-MS/MS detection method of catecholamines according to claim 1, wherein: in the third step, the solvent for redissolution is 0.2mol/L formic acid solution, and the content of the redissolution solvent is 50-150 ml.
8. The LC-MS/MS detection method of catecholamines according to claim 1, wherein: the filtering diameter of the superfine filter membrane in the fourth step is 200-500 nm.
9. The LC-MS/MS detection method of catecholamines according to claim 1, wherein: the chromatographic column in the fourth step is a pentafluorophenyl reverse phase chromatographic column, and the surface of the pentafluorophenyl reverse phase chromatographic column is bonded with pentafluorophenethyl by taking ultrapure silica gel as a matrix.
10. A LC-MS/MS detection pretreatment kit for catecholamine is characterized in that: the pretreatment kit comprises a WCX solid phase treatment extraction column, diluent and eluent.
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Cited By (3)
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| CN114034805A (en) * | 2021-11-12 | 2022-02-11 | 大连润生康泰医学检验实验室有限公司 | Metabolic kinetic analysis method of pentafluorouracil drugs |
| CN114428138A (en) * | 2022-03-08 | 2022-05-03 | 天津国科医工科技发展有限公司 | Liquid chromatography tandem mass spectrometry detection method of catecholamine and metabolites thereof based on magnetic solid phase extraction |
| CN115389682A (en) * | 2022-09-13 | 2022-11-25 | 北京豪思生物科技股份有限公司 | Kit for detecting catecholamine and metabolites thereof in blood plasma and urine and application thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114034805A (en) * | 2021-11-12 | 2022-02-11 | 大连润生康泰医学检验实验室有限公司 | Metabolic kinetic analysis method of pentafluorouracil drugs |
| CN114428138A (en) * | 2022-03-08 | 2022-05-03 | 天津国科医工科技发展有限公司 | Liquid chromatography tandem mass spectrometry detection method of catecholamine and metabolites thereof based on magnetic solid phase extraction |
| CN114428138B (en) * | 2022-03-08 | 2024-05-10 | 天津国科医疗科技发展有限公司 | Catecholamine and metabolite liquid chromatography tandem mass spectrometry detection method based on magnetic solid phase extraction |
| CN115389682A (en) * | 2022-09-13 | 2022-11-25 | 北京豪思生物科技股份有限公司 | Kit for detecting catecholamine and metabolites thereof in blood plasma and urine and application thereof |
| CN115389682B (en) * | 2022-09-13 | 2023-08-04 | 北京豪思生物科技股份有限公司 | Kit for detecting catecholamines and metabolites thereof in blood plasma and urine and application of kit |
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Application publication date: 20210316 |