CN114965756A - Method for detecting neurotransmitter substances - Google Patents
Method for detecting neurotransmitter substances Download PDFInfo
- Publication number
- CN114965756A CN114965756A CN202210524154.5A CN202210524154A CN114965756A CN 114965756 A CN114965756 A CN 114965756A CN 202210524154 A CN202210524154 A CN 202210524154A CN 114965756 A CN114965756 A CN 114965756A
- Authority
- CN
- China
- Prior art keywords
- mass spectrometry
- liquid chromatography
- performance liquid
- tandem mass
- neurotransmitter
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 239000002858 neurotransmitter agent Substances 0.000 title claims abstract description 106
- 239000000126 substance Substances 0.000 title claims abstract description 94
- 238000000034 method Methods 0.000 title claims abstract description 58
- 238000001514 detection method Methods 0.000 claims abstract description 35
- 210000002700 urine Anatomy 0.000 claims abstract description 29
- 238000004885 tandem mass spectrometry Methods 0.000 claims abstract description 23
- 238000001195 ultra high performance liquid chromatography Methods 0.000 claims abstract description 20
- 210000004369 blood Anatomy 0.000 claims abstract description 18
- 239000008280 blood Substances 0.000 claims abstract description 18
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 claims abstract description 3
- 239000012071 phase Substances 0.000 claims description 79
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 70
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 63
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical group CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 45
- 238000010811 Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry Methods 0.000 claims description 43
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 35
- 235000019253 formic acid Nutrition 0.000 claims description 35
- VYFYYTLLBUKUHU-UHFFFAOYSA-N dopamine Chemical compound NCCC1=CC=C(O)C(O)=C1 VYFYYTLLBUKUHU-UHFFFAOYSA-N 0.000 claims description 32
- 239000011259 mixed solution Substances 0.000 claims description 29
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 25
- 238000002156 mixing Methods 0.000 claims description 20
- QZAYGJVTTNCVMB-UHFFFAOYSA-N serotonin Chemical compound C1=C(O)C=C2C(CCN)=CNC2=C1 QZAYGJVTTNCVMB-UHFFFAOYSA-N 0.000 claims description 18
- 210000002381 plasma Anatomy 0.000 claims description 17
- 229960003638 dopamine Drugs 0.000 claims description 16
- 239000000243 solution Substances 0.000 claims description 16
- HQVFCQRVQFYGRJ-UHFFFAOYSA-N formic acid;hydrate Chemical compound O.OC=O HQVFCQRVQFYGRJ-UHFFFAOYSA-N 0.000 claims description 14
- 239000006228 supernatant Substances 0.000 claims description 13
- SVWLIIFHXFGESG-UHFFFAOYSA-N formic acid;methanol Chemical compound OC.OC=O SVWLIIFHXFGESG-UHFFFAOYSA-N 0.000 claims description 11
- 239000012528 membrane Substances 0.000 claims description 11
- DUUGKQCEGZLZNO-UHFFFAOYSA-N 5-hydroxyindoleacetic acid Chemical compound C1=C(O)C=C2C(CC(=O)O)=CNC2=C1 DUUGKQCEGZLZNO-UHFFFAOYSA-N 0.000 claims description 10
- 239000007864 aqueous solution Substances 0.000 claims description 10
- UCTWMZQNUQWSLP-UHFFFAOYSA-N adrenaline Chemical compound CNCC(O)C1=CC=C(O)C(O)=C1 UCTWMZQNUQWSLP-UHFFFAOYSA-N 0.000 claims description 9
- 238000001914 filtration Methods 0.000 claims description 9
- 239000012086 standard solution Substances 0.000 claims description 9
- QRMZSPFSDQBLIX-UHFFFAOYSA-N homovanillic acid Chemical compound COC1=CC(CC(O)=O)=CC=C1O QRMZSPFSDQBLIX-UHFFFAOYSA-N 0.000 claims description 8
- -1 methoxynoradrenaline Chemical compound 0.000 claims description 8
- 230000001376 precipitating effect Effects 0.000 claims description 8
- 229960002748 norepinephrine Drugs 0.000 claims description 7
- SFLSHLFXELFNJZ-UHFFFAOYSA-N norepinephrine Natural products NCC(O)C1=CC=C(O)C(O)=C1 SFLSHLFXELFNJZ-UHFFFAOYSA-N 0.000 claims description 7
- SFLSHLFXELFNJZ-QMMMGPOBSA-N (-)-norepinephrine Chemical compound NC[C@H](O)C1=CC=C(O)C(O)=C1 SFLSHLFXELFNJZ-QMMMGPOBSA-N 0.000 claims description 6
- DIVQKHQLANKJQO-UHFFFAOYSA-N 3-methoxytyramine Chemical compound COC1=CC(CCN)=CC=C1O DIVQKHQLANKJQO-UHFFFAOYSA-N 0.000 claims description 6
- VJBCNMFKFZIXHC-UHFFFAOYSA-N azanium;2-(4-methyl-5-oxo-4-propan-2-yl-1h-imidazol-2-yl)quinoline-3-carboxylate Chemical compound N.N1C(=O)C(C(C)C)(C)N=C1C1=NC2=CC=CC=C2C=C1C(O)=O VJBCNMFKFZIXHC-UHFFFAOYSA-N 0.000 claims description 6
- QIVBCDIJIAJPQS-XDACSXLQSA-N (2s)-2-(deuterioamino)-3-(1h-indol-3-yl)propanoic acid Chemical compound C1=CC=C2C(C[C@H](N[2H])C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-XDACSXLQSA-N 0.000 claims description 5
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 claims description 5
- 239000005695 Ammonium acetate Substances 0.000 claims description 5
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 claims description 5
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 claims description 5
- QZAYGJVTTNCVMB-BZDFBPEFSA-N OC1=CC=C2NC=C(CC(N([2H])[2H])([2H])[2H])C2=C1 Chemical compound OC1=CC=C2NC=C(CC(N([2H])[2H])([2H])[2H])C2=C1 QZAYGJVTTNCVMB-BZDFBPEFSA-N 0.000 claims description 5
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 claims description 5
- 235000019257 ammonium acetate Nutrition 0.000 claims description 5
- 229940043376 ammonium acetate Drugs 0.000 claims description 5
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 claims description 5
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 claims description 5
- RVWZUOPFHTYIEO-UHFFFAOYSA-N 5-hydroxyindoleacetic acid Natural products C1=C(O)C=C2C(C(=O)O)=CNC2=C1 RVWZUOPFHTYIEO-UHFFFAOYSA-N 0.000 claims description 4
- 239000003310 5-hydroxyindoleacetic acid Substances 0.000 claims description 4
- 201000011243 gastrointestinal stromal tumor Diseases 0.000 claims description 4
- 238000002414 normal-phase solid-phase extraction Methods 0.000 claims description 4
- YDHIAISKMUVSFO-UHFFFAOYSA-N 4-[1-hydroxy-2-(methoxymethylamino)ethyl]benzene-1,2-diol Chemical compound COCNCC(O)C1=CC=C(O)C(O)=C1 YDHIAISKMUVSFO-UHFFFAOYSA-N 0.000 claims description 3
- CGQCWMIAEPEHNQ-UHFFFAOYSA-N Vanillylmandelic acid Chemical compound COC1=CC(C(O)C(O)=O)=CC=C1O CGQCWMIAEPEHNQ-UHFFFAOYSA-N 0.000 claims description 3
- UCTWMZQNUQWSLP-VIFPVBQESA-N (R)-adrenaline Chemical compound CNC[C@H](O)C1=CC=C(O)C(O)=C1 UCTWMZQNUQWSLP-VIFPVBQESA-N 0.000 claims description 2
- 229930182837 (R)-adrenaline Natural products 0.000 claims description 2
- 229960005139 epinephrine Drugs 0.000 claims description 2
- 238000001556 precipitation Methods 0.000 claims description 2
- 230000006920 protein precipitation Effects 0.000 claims description 2
- 238000000108 ultra-filtration Methods 0.000 claims description 2
- 230000037353 metabolic pathway Effects 0.000 abstract description 4
- 238000012545 processing Methods 0.000 abstract description 4
- 230000001575 pathological effect Effects 0.000 abstract description 2
- 238000003908 quality control method Methods 0.000 description 25
- 238000002360 preparation method Methods 0.000 description 22
- 239000000523 sample Substances 0.000 description 22
- 239000012224 working solution Substances 0.000 description 20
- 239000007789 gas Substances 0.000 description 15
- 239000013062 quality control Sample Substances 0.000 description 10
- 239000011550 stock solution Substances 0.000 description 9
- 238000009210 therapy by ultrasound Methods 0.000 description 7
- 238000006243 chemical reaction Methods 0.000 description 5
- 238000010438 heat treatment Methods 0.000 description 5
- 239000007791 liquid phase Substances 0.000 description 5
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 210000004027 cell Anatomy 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 3
- 230000009471 action Effects 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 235000013922 glutamic acid Nutrition 0.000 description 3
- 239000004220 glutamic acid Substances 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 241001251200 Agelas Species 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
- 238000004949 mass spectrometry Methods 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 238000002203 pretreatment Methods 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- CGQCWMIAEPEHNQ-QMMMGPOBSA-N (2s)-2-hydroxy-2-(4-hydroxy-3-methoxyphenyl)acetic acid Chemical compound COC1=CC([C@H](O)C(O)=O)=CC=C1O CGQCWMIAEPEHNQ-QMMMGPOBSA-N 0.000 description 1
- QTBSBXVTEAMEQO-DICFDUPASA-N 2,2-dideuterioacetic acid Chemical compound C(C([2H])[2H])(=O)O QTBSBXVTEAMEQO-DICFDUPASA-N 0.000 description 1
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 1
- KCWQSNFJXVMEQC-DYCDLGHISA-N [2H]OC(CC1=CC2=CC(O)=CC=C2N1)=O Chemical compound [2H]OC(CC1=CC2=CC(O)=CC=C2N1)=O KCWQSNFJXVMEQC-DYCDLGHISA-N 0.000 description 1
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000000861 blow drying Methods 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 150000003943 catecholamines Chemical class 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000001212 derivatisation Methods 0.000 description 1
- 239000003651 drinking water Substances 0.000 description 1
- 235000020188 drinking water Nutrition 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000010926 purge Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
Classifications
-
- 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
-
- 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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- 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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
-
- 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
-
- 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
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
-
- 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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N2030/042—Standards
- G01N2030/045—Standards internal
-
- 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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/062—Preparation extracting sample from raw material
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The application provides a method for detecting neurotransmitter substances, which takes blood and urine as detection objects and adopts an ultra-high performance liquid chromatography tandem mass spectrometry method to carry out ultra-high performance liquid chromatography tandem mass spectrometry on the neurotransmitter substances. The method for detecting the neurotransmitter substances is provided for comprehensively detecting the metabolic pathways of the patient, and clinical comprehensive and effective interpretation of pathological causes of the patient is facilitated; the method for detecting the neurotransmitter substances through the ultra-high performance liquid chromatography-tandem mass spectrometry is simple, has low requirements on detection instruments, is simple in pretreatment operation, consumes short time, and is suitable for processing of large-batch samples.
Description
Technical Field
The application relates to the technical field of neurotransmitter detection, in particular to a method for detecting neurotransmitter.
Background
The main disadvantages of the prior art processes for the detection of neurotransmitters are: firstly, the content of the substances in blood plasma is low, and the requirements on a detection method and an instrument are high; secondly, the substances are pretreated by a common derivatization method, the operation is complex, the time consumption is long, and the substances are not suitable for processing a large batch of samples; thirdly, most of the existing methods only detect catecholamine substances, and the basic metabolic pathway is not detected comprehensively, so that the clinical interpretation is not facilitated; and fourthly, the neurotransmitter exists in plasma and urine, and 2 persons can detect the neurotransmitter to better reflect the level of the neurotransmitter in the human body.
Disclosure of Invention
The embodiment of the application provides a method for detecting neurotransmitter substances, and aims to solve the technical problems that the existing method for detecting neurotransmitter substances has high requirements on methods and instruments, complex pretreatment operation, long time consumption, unsuitability for processing of large-batch samples, incomplete detection of basic metabolic pathways and inconvenience for clinical interpretation.
The invention provides a method for detecting neurotransmitter substances, which takes blood and urine as detection objects and adopts ultra-high performance liquid chromatography tandem mass spectrometry to detect the neurotransmitter substances.
In some embodiments, in order to effectively detect the neurotransmitter substances in the blood and the urine, the two groups of neurotransmitter substances are respectively subjected to ultra performance liquid chromatography tandem mass spectrometry detection, and in order to effectively separate and detect various neurotransmitter substances in the blood, the first, second and third groups of neurotransmitter substances in the blood are detected by adopting first, second and third different ultra performance liquid chromatography tandem mass spectrometry methods; in order to effectively separate and detect various neurotransmitter substances in the urine, a fourth and a fifth groups of neurotransmitter substances in the urine are detected by adopting a fourth and a fifth different ultra performance liquid chromatography tandem mass spectrometry;
wherein the first group of neurotransmitter substances comprises dopamine, adrenaline, noradrenaline, methoxyadrenaline, methoxynoradrenaline, 3-methoxytyramine, dopamine d4, noradrenaline-d 6 and methoxynoradrenaline-d 3 A second group of neurotransmitter substances including tyrosine, tryptophan and tryptophan-d 5, a third group of neurotransmitter substances including 5-hydroxytryptamine and 5-hydroxytryptamine-d 4, and a fourth group of neurotransmitter substances including dopamine, epinephrine, norepinephrine, methoxyepinephrine, methoxynorepinephrine, 3-methoxytyramine, dopamine d4, norepinephrine-d 6 and methoxynorepinephrine-d 3 A fifth group of neurotransmitter substances includes homovanillic acidVanilmandelic acid, 5-hydroxyindoleacetic acid and 5-hydroxyindoleacetic acid-d 2.
In some embodiments, in the first ultra high performance liquid chromatography tandem mass spectrometry, the mobile phase a is a mixed solution of formic acid and water, and the phase B is a mixed solution of formic acid and methanol, in the second ultra high performance liquid chromatography tandem mass spectrometry, the mobile phase a is a mixed solution of formic acid and water, and the phase B is acetonitrile, in the third ultra high performance liquid chromatography tandem mass spectrometry, the mobile phase a is a mixed solution of formic acid and water, and the phase B is acetonitrile, in the fourth ultra high performance liquid chromatography tandem mass spectrometry, the mobile phase a is a mixed solution of formic acid and water, and the phase B is a mixed solution of formic acid and methanol, in the fifth ultra high performance liquid chromatography tandem mass spectrometry, the mobile phase a is a mixed solution of formic acid and water, and the phase B is acetonitrile.
In some embodiments, in the first hplc tandem mass spectrometry, the mobile phase a is 0.1% formic acid in water, and the mobile phase B is 0.1% formic acid-methanol mixed solution;
in the second ultra performance liquid chromatography-tandem mass spectrometry, the mobile phase A is 0.1% formic acid aqueous solution;
in the third ultra performance liquid chromatography-tandem mass spectrometry, the mobile phase A is 0.1% formic acid aqueous solution;
in the fourth ultra-high performance liquid chromatography-tandem mass spectrometry, a mobile phase A is 0.1% formic acid aqueous solution, and a mobile phase B is 0.1% formic acid-methanol mixed solution;
in the fifth ultra performance liquid chromatography-tandem mass spectrometry, the mobile phase A is 0.1% formic acid aqueous solution. In some embodiments, the flow rate of the mobile phase in the first, second, third, fourth and fifth types of ultra high performance liquid chromatography tandem mass spectrometry is 0.2ml/min to 0.5 ml/min.
In some embodiments, the first ultra performance liquid chromatography-tandem mass spectrometry has a mobile phase flow rate of 0.4 mL/min;
in the second ultra performance liquid chromatography-tandem mass spectrometry, the flow rate of a mobile phase is 0.3 mL/min;
in the third ultra performance liquid chromatography-tandem mass spectrometry, the flow rate of a mobile phase is 0.3 mL/min;
in the fourth ultra performance liquid chromatography-tandem mass spectrometry, the flow rate of a mobile phase is 0.4 mL/min;
in the fifth ultra performance liquid chromatography-tandem mass spectrometry, the flow rate of the mobile phase is 0.4 mL/min.
In some embodiments, in the first hplc tandem mass spectrometry, the column is a Shim-pack Velox PFPP column;
in the second ultra performance liquid chromatography-tandem mass spectrometry, the chromatographic column is a Shim-pack Scepter Phenyl-120 chromatographic column;
in the third ultra performance liquid chromatography-tandem mass spectrometry, the chromatographic column is a Shim-pack Scepter Phenyl-120 chromatographic column;
in the fourth ultra performance liquid chromatography-tandem mass spectrometry, the chromatographic column is a Shim-pack Scepter Phenyl-120 chromatographic column;
in the fifth ultra performance liquid chromatography-tandem mass spectrometry, a chromatographic column is a Shim-pack GIST C18 chromatographic column.
In some embodiments, in the first, second, third, fourth and fifth types of hplc tandem mass spectrometry, the column temperature is 40 ℃.
In some embodiments, urine and blood are pretreated by protein precipitation or solid phase extraction, respectively, and then subjected to ultra performance liquid chromatography tandem mass spectrometry.
In some embodiments, in order to realize effective separation and detection of various neurotransmitter substances in blood and urine, the blood is treated by different pretreatment methods, and the urine is treated by different pretreatment methods.
In some embodiments, in order to realize effective separation and detection of various neurotransmitter substances in blood and urine, blood is pretreated by three different methods, and then the ultrahigh performance liquid chromatography tandem mass spectrometry detection is respectively carried out on the first group of neurotransmitter substances, the second group of neurotransmitter substances and the third group of neurotransmitter substances in extracting solution; pretreating urine by two different methods, and respectively performing ultra high performance liquid chromatography tandem mass spectrometry detection on a fourth group of neurotransmitter substances and a fifth group of neurotransmitter substances in the extracting solution.
In one embodiment, the plasma to be tested comprises 5x10 -8 g/mL dopamine D4, 5X10 -8 g/mL norepinephrine-d 6, 5x10 -9 g/mL methoxy norepinephrine-d 3 The mixed internal standard solution and 50mmol/L ammonium acetate solution are subjected to WCX solid phase extraction and then a first group of neurotransmitter substances are detected;
in one embodiment, the plasma to be tested comprises 1 × 10 -5 g/mL Tryptophan-d 5 Performing methanol precipitation on the internal standard solution, centrifuging to obtain 20 mu L of supernatant, adding 180 mu L of mobile phase containing 0.1% formic acid water and 0.1% formic acid-methanol, centrifuging to obtain 30 mu L of supernatant, and detecting a second group of neurotransmitter substances in the supernatant;
in one embodiment, the plasma to be tested comprises 5x10 -7 g/mL 5-hydroxytryptamine-d 4 Precipitating the internal standard solution by methanol, and centrifuging to detect a third group of neurotransmitter substances;
in one embodiment, the urine to be tested, containing internal standard 1x10 -6 g/mL dopamine D4, 1X10 -6 g/mL norepinephrine-d 6, 1x10 -6 g/mL Methoxymethylepinephrine-d 3 Mixing, precipitating with methanol, centrifuging, filtering with ultrafiltration membrane, bottling, and detecting the fourth group of neurotransmitter;
in one embodiment, the urine to be tested comprises 5x10 -7 And mixing g/mL 5-hydroxyindoleacetic acid-d 2 internal standard solution, precipitating with methanol, mixing uniformly, centrifuging, taking supernatant, passing through a membrane, and detecting a fifth group of neurotransmitter substances.
The beneficial effect that technical scheme that this application provided brought includes:
the embodiment of the application provides a method for detecting neurotransmitter substances, which is characterized in that blood and urine are used as neurotransmitter substance detection objects, so that metabolic pathways of a patient are comprehensively detected, and clinical comprehensive and effective interpretation of pathological causes of the patient is facilitated;
the method for detecting the neurotransmitter substances through the ultra-high performance liquid chromatography tandem mass spectrometry has the advantages of simple detection method, low requirement on a detection instrument, simple pretreatment operation and short consumed time, and is suitable for processing a large number of samples.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below in conjunction with the embodiments of the present application, and it is obvious that the described embodiments are some embodiments of the present application, but not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.
In the following examples, the test compound standards and manufacturer information used are shown in tables 1-5:
TABLE 1 group I neurotransmitters
TABLE 2 second group of neurotransmitter substances
TABLE 3 third group of neurotransmitter substances
TABLE 4 fourth group of neurotransmitter substances
TABLE 5 group V neurotransmitters
| Name of Chinese | English name | Molecular formula | Manufacturer of the product | Molecular weight g/mol |
| Homovanillic acid | homovanillic acid | C 9 H 10 O 4 | SHANGHAI YUANYE BIOTECHNOLOGY Co.,Ltd. | 182.17 |
| Vanillymandelic acid | vanillylmandelic acid | C 9 H 10 O 5 | SHANGHAI YUANYE BIOTECHNOLOGY Co.,Ltd. | 198.17 |
| 5-Hydroxyindoleacetic acid | 5-HIAA | C 10 H 9 NO 3 | SHANGHAI YUANYE BIOTECHNOLOGY Co.,Ltd. | 191.19 |
| 5-Hydroxyindoleacetic acid-d 2 | 5-HIAA-d 2 | C 10 H 7 NO 3 -d 2 | ISOREAG | 193.19 |
The following examples employ the following materials and equipment:
1. ultra-high performance liquid chromatography tandem mass spectrometry equipment
Shimadzu 8050LCMSM (Jingdu Shimadzu, Japan)
Analytical column Shim-pack Velock PFPP column (2.7 μm, 2.1X 100mm)
Analytical column Shim Phenyl 120 Shim-pack column (1.9 μm, 2.1X 100mm)
Analytical column Shim-pack GIST C18 column (2 μm, 2.1X 100mm) 2, sample pretreatment apparatus
Vortex mixer (German IKA company)
Desk type high speed micro freezing centrifuge 24 x 1.5ml (Sallocke SCILOGEX CF1524R)
PWN224ZH/E electronic analytical balance (Aohaus instruments (Changzhou) Inc.)
JXDC-400 nitrogen purging instrument (Shanghai Jing Xin Shi Kogyo Co., Ltd.)
A liquid transfer device: dragon
Cleanert PWCX 98Well Microplates(Agela)
Cleanert V96 nitrogen blowing device (Bonaaijier science and technology limited)
Cleanert PWCX 96Well Microplates(2mg/1mL/Well 2/pkg)
96-hole receiving plate (Shimadzu)
3. Chemicals and reagents
Methanol (chromatographically pure methanol, Sigma-Aldrich, Germany)
Acetonitrile (chromatographically pure acetonitrile, Sigma-Aldrich, Germany)
Formic acid 88% (national drug group chemical reagent company, Inc. 20171107)
Formic acid 98% (national drug group chemical reagent company, Inc. 20180525)
Drech drinking water (105 ℃ special distilled boiled water preparation method)
Ammonium acetate (Shanghai test 20181115)
Concentrated hydrochloric acid 12mmoL (national drug group chemical reagent Co., Ltd.)
Example 1
1.1 sample preparation
1.1.1 preparation of Standard Curve and quality control sample
Preparing a mixed solution from dopamine, adrenaline, noradrenaline, methoxyadrenaline, methoxynoradrenaline and 3-methoxytyramine standard substances, using the mixed solution as a stock solution of standard working solution and quality control working solution, mixing the stock solution with blank human plasma respectively, and preparing a standard curve and a quality control sample.
The 6 neurotransmitters had 9 series of concentrations in the standard (S1-S9), as shown in table 6:
TABLE 6 9 series concentrations of 6 neurotransmitters in the Standard (S1-S9)
The 6 neurotransmitters had three series of concentrations in the quality control, low (L), medium (M), and high (H), as shown in table 7:
TABLE 7, 6 three series of concentrations of neurotransmitter in quality control
1.1.2 preparation of internal standard substance working solution
Preparing mixed internal standard working solution with the concentrations of dopamine d4, norepinephrine-d 6 and methoxy norepinephrine-d 3 of 5x10 -8 、5x10 -8 、5x10 -9 g/mL。
1.2 sample pretreatment
The sample comprises human plasma to be detected, a standard substance and a quality control substance, and is processed by the following method:
(1) adding 290 mu L of standard substance/quality control substance/human plasma to be detected into a 1.5ml ep tube;
(2) adding 10 mu L of internal standard mixed solution, and uniformly mixing;
(3) add 300. mu.L of 50mmol/L ammonium acetate solution, mix well and wait for spe column elution.
(4) Rinse SPE cartridges (Cleanert PWCX 98Well Microplates (Agela)):
adding 200 μ L of methanol, and draining
Adding 50mmol/L ammonium acetate 200 μ L, draining
Loading: adding 250 μ L of sample for 2 times, loading 500ul, draining
And (3) elution: adding acetonitrile 500 μ L, draining
Adding 500 μ L of water, draining
Collecting: adding 2% formic acid water-acetonitrile 100 μ L for collection
(5)N 2 Blow-drying, adding 60 μ L of 0.1% formic acid water for redissolving, centrifuging, collecting 40 μ L, and bottling.
1.3 ultra performance liquid chromatography tandem mass spectrometry detection
10 μ L of sample was injected and tested under the following conditions:
1.3.1 Mobile phase
The mobile phase A is 0.1% formic acid water solution, the mobile phase B is 0.1% formic acid-methanol, and the total flow rate is 0.4000 mL/min;
the 1.3.1.1A phase is 0.1% formic acid water solution, and its preparation method comprises adding 1000 μ L of 88% formic acid into 1L volumetric flask, adding Drech water to reach volume of 1L scale, performing ultrasonic treatment for 2min, filtering with membrane (water system), and storing at-4 deg.C (longest period of 1 week).
1.3.1.2 the B phase is 0.1% formic acid-methanol, and the preparation method comprises the following steps: adding 1000 μ L88% formic acid into a 1L volumetric flask, diluting to a constant volume of 1L with methanol, performing ultrasonic treatment for 2min, adding a membrane (water system), filtering, and storing at-4 deg.C (1 week at most);
1.3.2 chromatography column: shim-pack Velock PFPP column (2.7 μm, 2.1X 100 mm);
1.3.3 the temperature of the column oven is 40 ℃;
TABLE 8 first set of neurotransmitter detection liquid phase parameters
| Time | Unit cell | Command | Set value |
| 0.00 | Pump and method of operating the same | B.Conc | 1％ |
| 0.50 | Pump and method of operating the same | B.Conc | 1％ |
| 6.50 | Pump and method of operating the same | B.Conc | 65％ |
| 6.60 | Pump and method of operating the same | B.Conc | 99％ |
| 8.50 | Pump | B.Conc | 99％ |
| 8.60 | Pump and method of operating the same | B.Conc | 1％ |
| 12.00 | Controller | Stop |
TABLE 9 first set of neurotransmitter parameters
| Mass spectrometer parameters | Set value |
| Flow rate of atomized gas | 1.8L/min |
| Dry gas flow | 3.0L/min |
| Flow of heated air | 17.0L/min |
| Interface voltage | 4.0kV |
| Interface current | 7.4uA |
| Interface temperature | 250℃ |
| DL temperature | 250℃ |
| Temperature of heating block | 400℃ |
| Conversion of dynode voltage | 10.0kV |
| Voltage of detector | 1.75kV |
| IG vacuum degree | 2.0e-003Pa |
| PG vacuum degree | 1.1e+002Pa |
| CID gas | 270kpa |
Example 2
2.1 sample preparation
2.1.1 preparation of Standard Curve and quality control sample
Preparing a mixed solution from tryptophan, glutamic acid and tyrosine standard substances as stock solutions of standard working solution and quality control working solution, mixing with blank human plasma respectively, and preparing a standard curve and a quality control sample.
The 3 neurotransmitters had 9 series of concentrations in the standard (S1-S9), as shown in table 10:
TABLE 10 9 series concentrations of 3 neurotransmitters in the Standard (S1-S9)
| (*10 4 nmol/L) | Tryptophan | Glutamic acid | Tyrosine |
| S1 | 0.5 | 0.5 | 0.5 |
| S2 | 1 | 1 | 1 |
| S3 | 2 | 2 | 2 |
| S4 | 4 | 4 | 4 |
| S5 | 8 | 8 | 8 |
| S6 | 10 | 10 | 10 |
| S7 | 20 | 20 | 20 |
| S8 | 50 | 50 | 50 |
| S9 | 100 | 100 | 100 |
The 3 neurotransmitters had three series of concentrations in the quality control, low (L), medium (M), and high (H), as shown in table 4:
TABLE 11, 3 three series of concentrations of neurotransmitter in quality control
| (*10 4 nmol/L) | Tryptophan | Glutamic acid | Tyrosine |
| L | 1.5 | 1.5 | 1.5 |
| M | 5 | 5 | 5 |
| H | 12 | 12 | 12 |
2.1.2 preparation of internal standard substance working solution
The concentration of tryptophan-d 5 in the internal standard working solution is 1x10-5 g/mL.
2.2 sample pretreatment
The sample comprises human plasma to be detected, a standard substance and a quality control substance, and is processed by the following method:
(1) adding 40 mu L of standard substance/quality control substance/human plasma to be detected into a 1.5ml ep tube;
(2) adding 10 mu L of internal standard solution and mixing uniformly;
(3) precipitating with 400 μ L methanol, mixing, and centrifuging at 10000rpm for 5 min;
the supernatant was taken 20. mu.L, and 180. mu.L of 0.1% formic acid water was added: 0.1% formic acid-methanol 1:1 solution, centrifuged at 10000rpm for 5min, 30 μ L of supernatant was taken and bottled.
2.3 ultra high performance liquid chromatography tandem mass spectrometry detection
5 μ L of sample was injected and tested under the following conditions:
2.3.1 Mobile phase
The mobile phase A is 0.1% formic acid water solution, the mobile phase B is acetonitrile, the flow rate is 0.3000 mL/min;
2.3.1.1 the mobile phase A is 0.1% formic acid water solution, and its preparation method comprises adding 1000 μ L88% formic acid into 1L volumetric flask, adding 1L of water to desired volume, performing ultrasonic treatment for 2min, and filtering with membrane (water system) at-4 deg.C (maximum 1 week).
2.3.1.2 the mobile phase B is acetonitrile
2.3.2 columns Shim scanner Phenyl 120 columns (1.9 μm, 2.1X 100 mm);
2.3.3 the temperature of the column incubator is 40 ℃;
TABLE 12 second set of neurotransmitter detection liquid phase parameters
| Time (min) | Unit cell | Command | Set value |
| 0.00 | Pump and method of operating the same | B.Conc | 15％ |
| 3.50 | Pump and method of operating the same | B.Conc | 50％ |
| 3.51 | Pump and method of operating the same | B.Conc | 15％ |
| 6.00 | Controller | Stop |
TABLE 13 second set of neurotransmitter parameters
| Mass spectrometer parameters | Set value |
| Flow rate of atomized gas | 1.8L/min |
| Dry gas flow | 3.0L/min |
| Flow of heated air | 17.0L/min |
| Interface voltage | 4.0kV |
| Interface current | 7.4uA |
| Interface temperature | 250℃ |
| DL temperature | 250℃ |
| Temperature of heating block | 400℃ |
| Conversion of dynode voltage | 10.0kV |
| Voltage of detector | 1.75kV |
| IG vacuum degree | 2.0e-003Pa |
| PG vacuum degree | 1.1e+002Pa |
| CID gas | 270kpa |
Example 3
3.1 sample preparation
3.1.1 preparation of Standard Curve and quality control sample
Preparing a 5-hydroxytryptamine standard substance into a solution serving as a stock solution of a standard working solution and a quality control working solution, mixing the stock solution with blank human plasma respectively, and preparing a standard curve and a quality control sample.
5-hydroxytryptamine has 9 series of concentrations (S1-S9) in the standard, as shown in Table 14:
TABLE 14 9 series concentrations of 5-hydroxytryptamine in Standard substance (S1-S9)
| (μmol/L) | 5-hydroxytryptamine |
| S1 | 0.05 |
| S2 | 0.1 |
| S3 | 0.2 |
| S4 | 0.5 |
| S5 | 1 |
| S6 | 2 |
| S7 | 5 |
| S8 | 10 |
| S9 | 20 |
The 5-hydroxytryptamine has three series of concentrations of low (L), medium (M) and high (H) in the quality control product, as shown in the following table 15:
TABLE 15 three series of concentrations of 5-hydroxytryptamine in quality control products
| (μmol/L) | 5-hydroxytryptamine |
| L | 0.25 |
| M | 1.25 |
| H | 4 |
3.1.2 preparation of internal standard substance working solution
The concentration of the prepared internal standard working solution 5-hydroxytryptamine-d 4 is 5x10-7 g/mL.
3.2 sample pretreatment
The sample comprises human plasma to be detected, a standard substance and a quality control substance, and is processed by the following method:
(1) adding 40 mu L of standard substance/quality control substance/human plasma to be detected into a 1.5ml ep tube;
(2) adding 10 mu L of internal standard mixed solution, and uniformly mixing;
(3) precipitating with 400 μ L methanol, mixing, centrifuging at 10000rpm for 5min, collecting supernatant 50ul, and bottling.
3.3 ultra high performance liquid chromatography tandem mass spectrometry detection
5ul of sample was injected and the detection was performed under the following conditions:
3.3.1 Mobile phase
A mobile phase A phase of 0.1 percent formic acid water solution and a mobile phase B phase of acetonitrile, wherein the total flow rate is 0.3000 mL/min;
3.3.1.1 preparation method of mobile phase A as 0.1% formic acid-water (0.1% formic acid water): adding 1000 μ L of 88% formic acid into a 1L volumetric flask, diluting to 1L scale with Drech water, performing ultrasonic treatment for 2min, adding membrane (water system), filtering, and storing at-4 deg.C (1 week at most).
3.3.1.2 the mobile phase B is acetonitrile
3.3.2 chromatography column: column Shim Phenyl 120 column Shim Phenyl (1.9 μm, 2.1X 100 mm);
3.3.3 column temperature 40 ℃;
TABLE 16 measurement of liquid phase parameters for a third group of neurotransmitter substances
| Time (min) | Unit cell | Command | Value (%) |
| 0.00 | Pump | B.Conc | 10 |
| 3.50 | Pump and method of operating the same | B.Conc | 40 |
| 3.51 | Pump and method of operating the same | B.Conc | 10 |
| 6.00 | Controller | Stop | - |
TABLE 17 measurement of Mass Spectrometry parameters for a third group of neurotransmitter species
| Parameters of the instrument | Set value |
| Flow rate of atomized gas | 3L/min |
| Dry gas flow | 10L/min |
| Flow of heated air | 10L/min |
| Interface voltage | 4.0kV |
| Interface current | 7.4uA |
| Interface temperature | 300℃ |
| DL temperature | 250℃ |
| Temperature of heating block | 400℃ |
| Conversion of dynode voltage | 10.0kV |
| Voltage of detector | 1.75kV |
| IG vacuum degree | 2.0e-003Pa |
| PG vacuum degree | 1.1e+002Pa |
| CID gas | 270kpa |
TABLE 18 Mass spectrometric detection parameters of neurotransmitters in blood
TABLE 19 Mass spectrometric detection of neurotransmitters in blood Linear equation, Range and r-value Table
From tables 18 and 19, it can be seen that by the method for detecting neurotransmitter substances provided by the application, effective separation detection can be realized for various neurotransmitters in blood.
Example 4
4.1 sample preparation
4.1.1 preparation of Standard Curve and quality control sample
Preparing a mixed solution from dopamine, adrenaline, noradrenaline, methoxy adrenaline, methoxy noradrenaline and 3-methoxytyramine standard substances, using the mixed solution as a stock solution of a standard working solution and a quality control working solution, mixing the stock solution with blank human urine respectively, and preparing a standard curve and a quality control sample.
The 6 neurotransmitters had 9 series of concentrations in the standard (S1-S9), as shown in table 20:
TABLE 20, 9 series concentrations of 6 neurotransmitters in Standard substance (S1-S9)
The 6 neurotransmitters had three series of concentrations in the quality control, low (L), medium (M), and high (H), as shown in table 21:
TABLE 21, 8 three series of concentrations of neurotransmitter in quality control
4.1.2 preparation of internal standard substance working solution
And preparing a mixed internal standard working solution, wherein the concentrations of dopamine d4, noradrenaline-d 6 and methoxy noradrenaline-d 3 are 1x10-6, 1x10-6 and 1x10-6g/mL respectively.
4.2 pretreatment of samples
The sample comprises human urine to be detected, a standard substance and a quality control substance, and is processed by the following method:
(1) adding 90 mu L of standard substance/quality control substance/human urine to be detected into a 1.5ml ep tube;
(2) adding 10 mu L of internal standard mixed solution, and uniformly mixing;
(3) adding 300 μ L methanol, mixing, centrifuging at 10000rpm for 5min, collecting supernatant 50ul, and bottling.
4.3 ultra high performance liquid chromatography tandem mass spectrometry detection
5ul of sample was injected and the detection was performed under the following conditions:
4.3.1 Mobile phase
The mobile phase A is 0.1% formic acid water solution, the mobile phase B is 0.1% formic acid-methanol, and the total flow rate of the mobile phases is 0.4000 mL/min;
4.3.1.1 the phase A is 0.1% formic acid aqueous solution, and the preparation method comprises adding 1000 μ L88% formic acid into 1L volumetric flask, adding Drech water to reach volume of 1L scale mark, performing ultrasonic treatment for 2min, adding membrane (water system), filtering, and storing at-4 deg.C (1 week at most).
4.3.1.2 the B phase is 0.1% formic acid-methanol, and is prepared by adding 1000 μ L88% formic acid into 1L volumetric flask, adding methanol to reach volume of 1L scale mark, performing ultrasonic treatment for 2min, filtering with membrane (water system), and storing at-4 deg.C (1 week at most).
4.3.2 columns Shim-pack Velock phenyl column (2.7 μm, 2.1X 100 mm);
4.3.3 temperature of column oven 40 deg.C
TABLE 22 fourth group of neurotransmitter detection liquid phase parameters
| Time | Unit cell | Command | Set value |
| 0 | Pump and method of operating the same | B.Conc | 3％ |
| 2 | Pump and method of operating the same | B.Conc | 3％ |
| 2.1 | Pump and method of operating the same | B.Conc | 45％ |
| 4 | Pump and method of operating the same | B.Conc | 55％ |
| 7 | Pump and method of operating the same | B.Conc | 95％ |
| 8 | Pump and method of operating the same | B.Conc | 95％ |
| 8.1 | Pump and method of operating the same | B.Conc | 3％ |
| 12.00 | Controller | Stop |
TABLE 23 fourth set of neurotransmitter parameters
| Parameters of the instrument | Set value |
| Flow rate of atomized gas | 1.8L/min |
| Dry gas flow | 3.0L/min |
| Flow of heated air | 17.0L/min |
| Interface voltage | 4.0kV |
| Interface current | 7.4uA |
| Interface temperature | 250℃ |
| DL temperature | 250℃ |
| Temperature of heating block | 400℃ |
| Conversion of dynode voltage | 10.0kV |
| Voltage of detector | 1.75kV |
| IG vacuum degree | 2.0e-003Pa |
| PG vacuum degree | 1.1e+002Pa |
| CID gas | 270kpa |
Example 5
5.1 sample preparation
5.1.1 preparation of Standard Curve and quality control sample
Preparing a mixed solution of the standard substance of the vanillylmandelic acid, the homovanillic acid and the 5-hydroxyindoleacetic acid, taking the mixed solution as stock solutions of a standard working solution and a quality control working solution, mixing the stock solutions with blank human urine respectively, and preparing a standard curve and a quality control sample.
The 3 neurotransmitters had 9 series of concentrations in the standard (S1-S9), as shown in table 24:
TABLE 24, 9 series concentrations of 3 neurotransmitters in the Standard (S1-S9)
The 3 neurotransmitters had three series of concentrations in the quality control, low (L), medium (M), and high (H), as shown in table 25:
TABLE 25, 3 three series of concentrations of neurotransmitter in quality control
5.1.2 preparation of internal standard substance working solution
The concentration of the prepared internal standard working solution 5-oxindole acetic acid-d 2 is 5x10-7 g/mL.
5.2 sample pretreatment
The sample comprises human urine to be detected, a standard substance and a quality control substance, and is processed by the following method:
(1) adding 90 mu L of standard substance/quality control substance/human urine to be detected into a 1.5ml ep tube;
(2) adding 10 mu L of internal standard mixed solution, and uniformly mixing;
(3) adding 200 μ L methanol, mixing well, centrifuging at 10000rpm for 5min, collecting supernatant 50ul, and bottling. 5ul of sample was injected.
5.3 ultra high performance liquid chromatography tandem mass spectrometry detection
5ul of sample was injected and the detection was performed under the following conditions:
5.3.1 Mobile phase
The phase A of the mobile phase is 0.1 percent formic acid-water, the phase B of the mobile phase is acetonitrile, and the total flow rate of the mobile phase is 0.4000 mL/min;
5.3.1.1 the mobile phase A is 0.1% formic acid water solution, and its preparation method comprises adding 1000 μ L88% formic acid into 1L volumetric flask, adding Drech water to reach volume of 1L scale, performing ultrasonic treatment for 2min, filtering with membrane (water system), and storing at-4 deg.C (1 week at most).
5.3.1.2 mobile phase B: and (3) acetonitrile.
5.3.2 column Shim-pack GIST C18 column (2 μm, 100X 2.1 mm);
5.3.3 the temperature of the chromatographic column is 40 ℃;
TABLE 26 fifth group neurotransmitter detection liquid phase parameters
TABLE 27 fifth set of neurotransmitter species detection mass spectrometry parameters
| Parameters of the instrument | Set value |
| Flow rate of atomized gas | 1.8L/min |
| Dry gas flow | 3.0L/min |
| Flow of heated air | 17.0L/min |
| Interface voltage | 4.0kV |
| Interface current | 7.4uA |
| Interface temperature | 250℃ |
| DL temperature | 250℃ |
| Temperature of heating block | 400℃ |
| Conversion of dynode voltage | 10.0kV |
| Voltage of detector | 1.75kV |
| IG vacuum degree | 2.0e-003Pa |
| PG vacuum degree | 1.1e+002Pa |
| CID gas | 270kpa |
TABLE 28 Mass spectrometric parameters of neurotransmitter substances in urine
TABLE 29 Mass spectrometric detection of neurotransmitter in urine Linear equation, Range and r-value Table
It can be seen from tables 28 and 29 that the neurotransmitter detection method provided by the present application can effectively separate and detect various neurotransmitters in urine.
It is noted that, in the present application, relational terms such as "first" and "second", and the like, are used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising an … …" does not exclude the presence of other identical elements in a process, method, article, or apparatus that comprises the element.
The previous description is only an example of the present application, and is provided to enable any person skilled in the art to understand or implement the present application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the application. Thus, the present application is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (10)
1. A method for detecting a neurotransmitter is provided,
blood and urine are used as detection objects, and the ultra-high performance liquid chromatography tandem mass spectrometry is adopted to carry out ultra-high performance liquid chromatography tandem mass spectrometry detection on neurotransmitter substances in the blood and urine.
2. The method for detecting a neurotransmitter according to claim 1,
detecting a first group of neurotransmitter substances, a second group of neurotransmitter substances and a third group of neurotransmitter substances in blood by adopting a first ultra performance liquid chromatography-tandem mass spectrometry, a second ultra performance liquid chromatography-tandem mass spectrometry and a third ultra performance liquid chromatography-tandem mass spectrometry;
detecting a fourth group of neurotransmitter substances and a fifth group of neurotransmitter substances in the urine by adopting a fourth ultra performance liquid chromatography-tandem mass spectrometry and a fifth ultra performance liquid chromatography-tandem mass spectrometry;
wherein the first group of neurotransmitter substances comprises dopamine, adrenaline, noradrenaline, methoxyadrenaline, methoxynoradrenaline, 3-methoxytyramine, dopamine d4, noradrenaline-d 6 and methoxynoradrenaline-d 3 The second group of neurotransmitter substances includes tyrosine, tryptophan and tryptophan-d 5 The third group of neurotransmitter substances includes 5-hydroxytryptamine and 5-hydroxytryptamine-d 4 The fourth group of neurotransmitter substances includes dopamine, epinephrine, norepinephrine, methoxyepinephrine, methoxynorepinephrine, 3-methoxytyramine, dopamine d4, norepinephrine-d 6, and methoxynorepinephrine-d 3 The fifth group of neurotransmitter substances includes homovanillic acid, vanillylmandelic acid, 5-hydroxyindoleacetic acid and 5-hydroxyindoleacetic acid-d 2 。
3. The method for detecting a neurotransmitter according to claim 2,
in the first ultra performance liquid chromatography-tandem mass spectrometry, a mobile phase A is a mixed solution of formic acid and water, and a mobile phase B is a mixed solution of formic acid and methanol;
in the second ultra performance liquid chromatography-tandem mass spectrometry, a mobile phase A is a mixed solution of formic acid and water, and a phase B is acetonitrile;
in the third ultra performance liquid chromatography-tandem mass spectrometry, a mobile phase A is a mixed solution of formic acid and water, and a phase B is acetonitrile;
in the fourth ultra performance liquid chromatography-tandem mass spectrometry, a mobile phase A is a mixed solution of formic acid and water, and a mobile phase B is a mixed solution of formic acid and methanol;
in the fifth ultra performance liquid chromatography-tandem mass spectrometry, a mobile phase A is a mixed solution of formic acid and water, and a mobile phase B is acetonitrile.
4. The method for detecting a neurotransmitter according to claim 3,
in the first ultra performance liquid chromatography-tandem mass spectrometry, a mobile phase A is 0.1% formic acid aqueous solution, and a mobile phase B is 0.1% formic acid-methanol mixed solution;
in the second ultra performance liquid chromatography-tandem mass spectrometry, the mobile phase A is 0.1% formic acid aqueous solution;
in the third ultra performance liquid chromatography-tandem mass spectrometry, the mobile phase A is 0.1% formic acid aqueous solution;
in the fourth ultra performance liquid chromatography-tandem mass spectrometry, a mobile phase A is a 0.1% formic acid aqueous solution, and a mobile phase B is a 0.1% formic acid-methanol mixed solution;
in the fifth ultra performance liquid chromatography-tandem mass spectrometry, the mobile phase A is 0.1% formic acid aqueous solution.
5. The method for detecting a neurotransmitter according to claim 3,
in the first, second, third, fourth and fifth ultra performance liquid chromatography tandem mass spectrometry, the flow rate of the mobile phase is 0.2ml/min-0.5 ml/min.
6. The method for detecting a neurotransmitter according to claim 5,
in the first ultra performance liquid chromatography-tandem mass spectrometry, the flow rate of a mobile phase is 0.4 mL/min;
in the second ultra performance liquid chromatography-tandem mass spectrometry, the flow rate of a mobile phase is 0.3 mL/min;
in the third ultra performance liquid chromatography-tandem mass spectrometry, the flow rate of a mobile phase is 0.3 mL/min;
in the fourth ultra high performance liquid chromatography-tandem mass spectrometry, the flow rate of a mobile phase is 0.4 mL/min;
in the fifth ultra performance liquid chromatography-tandem mass spectrometry, the flow rate of the mobile phase is 0.4 mL/min.
7. The method for detecting a neurotransmitter according to claim 2,
in the first ultra performance liquid chromatography-tandem mass spectrometry, a chromatographic column is a Shim-pack Velock PFPP chromatographic column;
in the second ultra performance liquid chromatography-tandem mass spectrometry, the chromatographic column is a Shim-pack Scepter Phenyl-120 chromatographic column;
in the third ultra performance liquid chromatography tandem mass spectrometry, a chromatographic column is a Shim-pack Scepter Phenyl-120 chromatographic column;
in the fourth ultra performance liquid chromatography-tandem mass spectrometry, the chromatographic column is a Shim-pack Scepter Phenyl-120 chromatographic column;
in the fifth ultra performance liquid chromatography-tandem mass spectrometry, a chromatographic column is a Shim-pack GIST C18 chromatographic column.
8. The method for detecting a neurotransmitter according to claim 4, wherein the first, second, third, fourth and fifth types of ultra high performance liquid chromatography tandem mass spectrometry have a column temperature of 40 ℃.
9. The method for detecting a neurotransmitter according to claim 2, wherein the urine and the blood are pretreated by protein precipitation or solid phase extraction, respectively, and then are subjected to ultra performance liquid chromatography tandem mass spectrometry.
10. The method for detecting a neurotransmitter-like substance according to claim 8,
the blood plasma to be tested contains 5x10 -8 g/mL dopamine D4, 5X10 -8 g/mL norepinephrine-d 6, 5x10 -9 g/mL methoxy norepinephrine-d 3 Uniformly mixing the mixed internal standard solution and 50mmol/L ammonium acetate solution, and detecting a first group of neurotransmitter substances after wcx solid-phase extraction;
the blood plasma to be tested contains 1x10 -5 g/mL Tryptophan-d 5 Performing methanol precipitation on the internal standard solution, centrifuging to obtain 20 mu L of supernatant, adding 180 mu L of mobile phase containing 0.1% formic acid water and 0.1% formic acid-methanol, centrifuging to obtain 30 mu L of supernatant, and detecting a second group of neurotransmitter substances in the supernatant;
the blood plasma to be tested contains 5x10 -7 g/mL 5-hydroxytryptamine-d 4 Precipitating the internal standard solution by methanol, and centrifuging to detect a third group of neurotransmitter substances;
the urine to be tested, containing internal standard 1x10 -6 g/mL dopamine D4, 1X10 -6 g/mL norepinephrine-d 6, 1x10 -6 g/mL methoxy norepinephrine-d 3 Mixing, precipitating with methanol, centrifuging, filtering with ultrafiltration membrane, bottling, and detecting the fourth group of neurotransmitter;
the urine to be tested contains 5x10 -7 And mixing g/mL 5-hydroxyindoleacetic acid-d 2 internal standard solution, precipitating with methanol, centrifuging, taking supernatant, passing through a membrane, and detecting a fifth group of neurotransmitter substances.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210524154.5A CN114965756A (en) | 2022-05-13 | 2022-05-13 | Method for detecting neurotransmitter substances |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210524154.5A CN114965756A (en) | 2022-05-13 | 2022-05-13 | Method for detecting neurotransmitter substances |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN114965756A true CN114965756A (en) | 2022-08-30 |
Family
ID=82983197
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210524154.5A Pending CN114965756A (en) | 2022-05-13 | 2022-05-13 | Method for detecting neurotransmitter substances |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114965756A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115327008A (en) * | 2022-09-13 | 2022-11-11 | 北京豪思生物科技股份有限公司 | Method for detecting catecholamine and metabolite thereof in blood plasma and urine |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108693294A (en) * | 2018-05-22 | 2018-10-23 | 复旦大学 | A variety of amino metabolins based on N- ethylization methods synchronize quantitative analysis method |
| CN111024874A (en) * | 2019-12-30 | 2020-04-17 | 上海百趣生物医学科技有限公司 | Method for quantitatively detecting catecholamine and metabolites thereof by liquid chromatography-mass spectrometry |
| CN112630311A (en) * | 2020-07-09 | 2021-04-09 | 深圳云合医药科技合伙企业(有限合伙) | Metabolic markers and kits for detecting affective disorders and methods of use |
| CN113917007A (en) * | 2021-09-08 | 2022-01-11 | 上海睿质科技有限公司 | Kit for detecting human urine catecholamine and intermediate metabolites thereof and test method |
| CN114441665A (en) * | 2020-11-04 | 2022-05-06 | 中国科学院大连化学物理研究所 | Detection method of catecholamine and metabolite thereof |
| CN114460188A (en) * | 2022-01-14 | 2022-05-10 | 上海睿质科技有限公司 | Kit for detecting human plasma catecholamine intermediate metabolite and testing method |
-
2022
- 2022-05-13 CN CN202210524154.5A patent/CN114965756A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108693294A (en) * | 2018-05-22 | 2018-10-23 | 复旦大学 | A variety of amino metabolins based on N- ethylization methods synchronize quantitative analysis method |
| CN111024874A (en) * | 2019-12-30 | 2020-04-17 | 上海百趣生物医学科技有限公司 | Method for quantitatively detecting catecholamine and metabolites thereof by liquid chromatography-mass spectrometry |
| CN112630311A (en) * | 2020-07-09 | 2021-04-09 | 深圳云合医药科技合伙企业(有限合伙) | Metabolic markers and kits for detecting affective disorders and methods of use |
| CN114441665A (en) * | 2020-11-04 | 2022-05-06 | 中国科学院大连化学物理研究所 | Detection method of catecholamine and metabolite thereof |
| CN113917007A (en) * | 2021-09-08 | 2022-01-11 | 上海睿质科技有限公司 | Kit for detecting human urine catecholamine and intermediate metabolites thereof and test method |
| CN114460188A (en) * | 2022-01-14 | 2022-05-10 | 上海睿质科技有限公司 | Kit for detecting human plasma catecholamine intermediate metabolite and testing method |
Non-Patent Citations (4)
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115327008A (en) * | 2022-09-13 | 2022-11-11 | 北京豪思生物科技股份有限公司 | Method for detecting catecholamine and metabolite thereof in blood plasma and urine |
| CN115327008B (en) * | 2022-09-13 | 2023-05-26 | 北京豪思生物科技股份有限公司 | Method for detecting catecholamine and metabolite thereof in blood plasma and urine |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107621399B (en) | Method for detecting oligosaccharide in breast milk | |
| CN111307993B (en) | Method for detecting bile acid content in blood | |
| CN114965756A (en) | Method for detecting neurotransmitter substances | |
| CN102565251B (en) | Method for detecting contents of acylcarnitines in serum or blood scrip | |
| CN108845063B (en) | Detection reagent combination and detection method of aquatic product additive | |
| CN112684068A (en) | Method for measuring content of hydroxy-alpha-sanshool | |
| CN112526021A (en) | Method for detecting 2' -fucosyllactose in milk | |
| CN112005110B (en) | Analysis method and application of dalteparin sodium nitrite degradation product | |
| CN109100463B (en) | Method for extracting, purifying and inspecting astragaloside in Astragalus membranaceus mushroom | |
| CN115963205A (en) | Method for determining content of diazepam, chlorpromazine and ribavirin in animal derived food by accelerating solvent extraction-liquid chromatography-mass spectrometry | |
| CN111198235B (en) | Method for detecting content of isosinensetin in plasma | |
| Hou et al. | Liquid chromatography-tandem mass spectrometry analysis for identification and quantification of antimicrobial compounds in distillery wastewater | |
| CN114113361A (en) | Extraction paper, preparation method thereof and application of extraction paper in detection of acidic drugs | |
| CN114113394A (en) | Magnetic microsphere for extracting and purifying paraquat metabolite, preparation method, kit and extraction method | |
| CN115963169B (en) | Detection method of carnitine and detection kit | |
| CN113237982A (en) | Derivatization method of hydroxyl metabolites and efficient non-targeted metabonomics analysis method | |
| CN111239277B (en) | Method and kit for determining N-dimethyl nitrosamine in water and application | |
| CN113671090A (en) | Method for measuring sialic acid content in serum | |
| CN108226350B (en) | Method for determining ginsenoside in rat plasma | |
| CN113156027A (en) | Derivatization method of carboxyl metabolites and efficient non-targeted metabonomics analysis method | |
| CN110988210A (en) | Method for reducing nonspecific adsorption of fat-soluble vitamin 96-well plate | |
| CN112129856A (en) | Method for identifying ellagic acid from gallnut | |
| CN112547031A (en) | Solid-phase microextraction probe fiber and preparation method and application thereof | |
| CN114814012B (en) | Determination method of lincolamine antibiotics in feed | |
| CN110161169A (en) | The rapid detection method of a variety of pharmaceutically active substances in a kind of water environment |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |