CN106442837B - A kind of method using Liquid Chromatography-Tandem Mass Spectrometry detection catecholamine levels in plasma - Google Patents
A kind of method using Liquid Chromatography-Tandem Mass Spectrometry detection catecholamine levels in plasma Download PDFInfo
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- 150000003943 catecholamines Chemical class 0.000 title claims abstract description 63
- 238000001514 detection method Methods 0.000 title claims abstract description 53
- 238000000034 method Methods 0.000 title claims abstract description 36
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 title claims abstract description 26
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 54
- 210000002381 plasma Anatomy 0.000 claims abstract description 39
- UCTWMZQNUQWSLP-UHFFFAOYSA-N adrenaline Chemical compound CNCC(O)C1=CC=C(O)C(O)=C1 UCTWMZQNUQWSLP-UHFFFAOYSA-N 0.000 claims abstract description 24
- 125000001295 dansyl group Chemical group [H]C1=C([H])C(N(C([H])([H])[H])C([H])([H])[H])=C2C([H])=C([H])C([H])=C(C2=C1[H])S(*)(=O)=O 0.000 claims abstract description 17
- 238000001212 derivatisation Methods 0.000 claims abstract description 15
- 239000007788 liquid Substances 0.000 claims abstract description 15
- SFLSHLFXELFNJZ-QMMMGPOBSA-N (-)-norepinephrine Chemical compound NC[C@H](O)C1=CC=C(O)C(O)=C1 SFLSHLFXELFNJZ-QMMMGPOBSA-N 0.000 claims abstract description 13
- 229960002748 norepinephrine Drugs 0.000 claims abstract description 13
- SFLSHLFXELFNJZ-UHFFFAOYSA-N norepinephrine Natural products NCC(O)C1=CC=C(O)C(O)=C1 SFLSHLFXELFNJZ-UHFFFAOYSA-N 0.000 claims abstract description 13
- 238000006243 chemical reaction Methods 0.000 claims abstract description 8
- 238000001556 precipitation Methods 0.000 claims abstract description 6
- 238000000926 separation method Methods 0.000 claims abstract description 4
- 239000000243 solution Substances 0.000 claims description 27
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 26
- 238000001819 mass spectrum Methods 0.000 claims description 24
- CGQCWMIAEPEHNQ-UHFFFAOYSA-N Vanillylmandelic acid Chemical class COC1=CC(C(O)C(O)=O)=CC=C1O CGQCWMIAEPEHNQ-UHFFFAOYSA-N 0.000 claims description 22
- 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 13
- -1 catecholamine acetonitrile Chemical compound 0.000 claims description 13
- 235000019253 formic acid Nutrition 0.000 claims description 13
- 239000012071 phase Substances 0.000 claims description 12
- 239000007791 liquid phase Substances 0.000 claims description 11
- 239000006228 supernatant Substances 0.000 claims description 11
- 239000003643 water by type Substances 0.000 claims description 10
- 238000004587 chromatography analysis Methods 0.000 claims description 9
- 238000002552 multiple reaction monitoring Methods 0.000 claims description 9
- 238000005119 centrifugation Methods 0.000 claims description 8
- 239000003153 chemical reaction reagent Substances 0.000 claims description 7
- 238000011068 loading method Methods 0.000 claims description 6
- 239000002904 solvent Substances 0.000 claims description 6
- 238000004458 analytical method Methods 0.000 claims description 5
- 239000007853 buffer solution Substances 0.000 claims description 5
- 238000012937 correction Methods 0.000 claims description 5
- 238000010828 elution Methods 0.000 claims description 5
- 238000002156 mixing Methods 0.000 claims description 5
- UIIMBOGNXHQVGW-UHFFFAOYSA-M sodium bicarbonate Substances [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 5
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims description 5
- 239000011259 mixed solution Substances 0.000 claims description 4
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical class N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 claims description 3
- XBJFCYDKBDVADW-UHFFFAOYSA-N acetonitrile;formic acid Chemical compound CC#N.OC=O XBJFCYDKBDVADW-UHFFFAOYSA-N 0.000 claims description 3
- 235000019257 ammonium acetate Nutrition 0.000 claims description 3
- 230000008859 change Effects 0.000 claims description 3
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- 239000002245 particle Substances 0.000 claims description 3
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- 239000007921 spray Substances 0.000 claims description 3
- 238000004704 ultra performance liquid chromatography Methods 0.000 claims description 3
- 150000001768 cations Chemical class 0.000 claims description 2
- 238000004949 mass spectrometry Methods 0.000 claims description 2
- 238000002360 preparation method Methods 0.000 claims description 2
- LSNNMFCWUKXFEE-UHFFFAOYSA-M Bisulfite Chemical compound OS([O-])=O LSNNMFCWUKXFEE-UHFFFAOYSA-M 0.000 claims 1
- ZZVUWRFHKOJYTH-UHFFFAOYSA-N diphenhydramine Chemical compound C=1C=CC=CC=1C(OCCN(C)C)C1=CC=CC=C1 ZZVUWRFHKOJYTH-UHFFFAOYSA-N 0.000 claims 1
- 239000000047 product Substances 0.000 claims 1
- 239000013049 sediment Substances 0.000 claims 1
- VYFYYTLLBUKUHU-UHFFFAOYSA-N dopamine Chemical compound NCCC1=CC=C(O)C(O)=C1 VYFYYTLLBUKUHU-UHFFFAOYSA-N 0.000 abstract description 24
- 229960003638 dopamine Drugs 0.000 abstract description 12
- 230000035945 sensitivity Effects 0.000 abstract description 6
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- 150000002500 ions Chemical class 0.000 description 7
- 230000014759 maintenance of location Effects 0.000 description 6
- 239000000126 substance Substances 0.000 description 6
- YCIMNLLNPGFGHC-UHFFFAOYSA-N catechol Chemical compound OC1=CC=CC=C1O YCIMNLLNPGFGHC-UHFFFAOYSA-N 0.000 description 4
- 239000002858 neurotransmitter agent Substances 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- PFTAWBLQPZVEMU-DZGCQCFKSA-N (+)-catechin Chemical compound C1([C@H]2OC3=CC(O)=CC(O)=C3C[C@@H]2O)=CC=C(O)C(O)=C1 PFTAWBLQPZVEMU-DZGCQCFKSA-N 0.000 description 2
- UCTWMZQNUQWSLP-VIFPVBQESA-N (R)-adrenaline Chemical compound CNC[C@H](O)C1=CC=C(O)C(O)=C1 UCTWMZQNUQWSLP-VIFPVBQESA-N 0.000 description 2
- 229930182837 (R)-adrenaline Natural products 0.000 description 2
- 244000080767 Areca catechu Species 0.000 description 2
- 235000006226 Areca catechu Nutrition 0.000 description 2
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- 108060003345 Adrenergic Receptor Proteins 0.000 description 1
- 102000017910 Adrenergic receptor Human genes 0.000 description 1
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- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 1
- 206010029260 Neuroblastoma Diseases 0.000 description 1
- 208000001647 Renal Insufficiency Diseases 0.000 description 1
- 208000026564 adrenal medullary hyperplasia Diseases 0.000 description 1
- 230000001800 adrenalinergic effect Effects 0.000 description 1
- 239000000556 agonist Substances 0.000 description 1
- 125000003368 amide group Chemical group 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 210000003169 central nervous system Anatomy 0.000 description 1
- 210000003737 chromaffin cell Anatomy 0.000 description 1
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- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 239000012224 working solution Substances 0.000 description 1
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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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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- 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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- 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
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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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- 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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- 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
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- G01N2030/065—Preparation using different phases to separate parts of sample
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- G01N30/02—Column chromatography
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- G01N2030/067—Preparation by reaction, e.g. derivatising the sample
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- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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Abstract
The invention discloses a kind of method using Liquid Chromatography-Tandem Mass Spectrometry detection catecholamine levels in plasma, including step：Prepared by standard items, liquid chromatogram separation, Mass Spectrometer Method and standard curve processed, the detection of catecholamine levels in plasma.It carries out the detection of catecholamine the method innovation of the present invention using high-throughput liquid chromatogram-triple tandem quadrupole mass spectrograph, has the advantages that：Albumen precipitation is carried out using acetonitrile, dansyl Cl is performed the derivatization reaction, detected after extraction using liquid chromatogram-triple tandem quadrupole mass spectrograph, and pre-treatment step is simple, can effectively remove plasma matrix interference, and specificity is good；After dansyl Cl derivatization, detection sensitivity can be effectively improved；High-throughput Liquid Chromatography-Tandem Mass Spectrometry instrument is detected, while qualitative and precisely quantify blood plasma and include dopamine, and adrenaline, 3 kinds of catecholamines of norepinephrine, detection time is short, and flux is high, and detection sensitivity is high, specific good, of low cost.
Description
Technical field
The present invention relates to the technical fields of catecholamine detection more particularly to a kind of utilization Liquid Chromatography-Tandem Mass Spectrometry to detect
The method of catecholamine levels in plasma.
Background technology
Catecholamine is a kind of neural class substance containing catechol and amido, including adrenaline (epinephrine,
E), norepinephrine (norepinephrine, NE), dopamine (dopamine, DA) are by adrenal medella and some friendships
Feel a kind of very important neurotransmitter of neuron chromaffin cell secretion and important hormone substance.These three catechols
Amine is all obtained by being converted premised on tyrosine.Norepinephrine and adrenaline are both swashing secreted by adrenal medella
Element, and be the neurotransmitter of noradrenergic fiber in sympathetic nerve and central nervous system.Norepinephrine is in
Widely distributed in pivot nervous system, content is more, and epinephrine contents are then less.Dopamine is concentrated mainly on extrapyramidal system portion
Position and a kind of neurotransmitter, they are important typical adrenoceptor agonists.Catecholamine is in the painstaking effort of human body
Extensive adjustment effect is played in the physiological activity of the organization systems such as guard system, nervous system, incretory, kidney, smooth muscle,
The metabolism of human body is had an effect on simultaneously.Detect blood plasma in catecholamine for pheochromocytoma, neuroblastoma, hypertension,
The clinical diagnosis of the diseases such as heart failure, adrenal medullary hyperplasia is of great significance, and contributes to that hyperthyroidism, first be low, the congested heart
It declines, the diagnosis of the diseases such as diabetes, renal insufficiency, hypoglycemia.
Currently used catecholamine detection method mainly has radiation zymetology method, chemoluminescence method, fluorescence method and efficient liquid
Phase chromatography etc., these detection methods are primarily present following some problems：First, plasma sample pretreatment process is complicated, and
Need initial sample amount larger；Second, existing detection technique process time is long, and experimental error is larger, and flux is low；3rd, due to
For adrenaline as methoxyepinephrine molecular weight, structure is very similar, can not be distinguished with common detection methods, special
The opposite sex is poor.
The content of the invention
In order to solve the above technical problems, the present invention provides catechu in a kind of detection blood plasma using Liquid Chromatography-Tandem Mass Spectrometry
The method of phenol amine, this method sample pre-treatments are simple, can detect 3 kinds of catecholamines simultaneously, and detection specificity is good and whole
A testing process time is short, flux is high.
A kind of method using Liquid Chromatography-Tandem Mass Spectrometry detection catecholamine levels in plasma of the present invention, including walking as follows
Suddenly：
1) prepared by standard items：The catecholamine acetonitrile solution of several various concentrations is taken respectively, is separately added into containing for equivalent
Know target acetonitrile solution in concentration, it is spare；
Under the conditions of being protected from light, using derivatization reagent dansyl Cl to target catecholamine acetonitrile solution in addition, carry out
Derivatization treatment causes catecholamine to chemically react, to improve signal response signal of the catecholamine on mass spectrum；
Aqueous formic acid adjusting is added in after having reacted makes pH=7, and sample supernatant is taken after centrifugation in spare on 96 orifice plates, use
It is analyzed in Liquid Chromatography-Tandem Mass Spectrometry loading, completes the preparation of standard items；
2) liquid chromatogram separates：It is right on reversed C18 analytical columns using ultrahigh pressure liquid phase chromatography and corresponding mobile phase
Catecholamine carries out chromatography eluant separation in standard items, by controlling elution requirement, isolates adrenaline, norepinephrine
And dopamine；
3) Mass Spectrometer Method and standard curve processed：The 3 kinds of catecholamines isolated in liquid chromatogram enter triple level Four bars
Mass spectrum is detected, and 3 kinds of catecholamines are specifically detected using the multiple reaction monitoring mode in triple level Four bar mass spectrums
Quantitative detection limit and identification detection limit is calculated according to signal noise ratio, according to ratio and known standard items and interior in content
Mark concentration value draws canonical plotting and obtains quantitative correction equation；
4) detection of catecholamine levels in plasma：Plasma sample is taken, adds in and carries out egg containing target acetonitrile solution in known concentration
It is centrifuged for the first time after white precipitation, fully precipitation, pipettes plasma supernatant and be freeze-dried；
Under the conditions of being protected from light, processing is performed the derivatization to plasma supernatant so that catechu using derivatization reagent dansyl Cl
Phenol amine chemically reacts, to improve signal response signal of the catecholamine on mass spectrum；
Aqueous formic acid adjusting is added in after having reacted makes pH=7, and sample supernatant is taken on 96 orifice plates after second of centrifugation
It is spare, it is analyzed for Liquid Chromatography-Tandem Mass Spectrometry loading；
With step 2 and 3), Mass Spectrometer Method obtains catecholamine and interior target ratio for Liquid Chromatography-Tandem Mass Spectrometry analysis, will
Ratio is updated to quantitative correction equation, and the content of catecholamine levels in plasma is calculated.
Further, in the step 1) in be designated as deuterated vanillylmandelic acid (VMA), it is described molten containing target acetonitrile in known concentration
The concentration of liquid is 25nmol/L.
Further, the concentration of the catecholamine acetonitrile solution of several various concentrations controls in the step 1)
Between 1pmol/L~1umol/L.
Further, the step 1) and 4) derivedization reagent dansyl Cl are specially：The dansyl Cl second of 4mg/mL
The Na of nitrile solution and 0.1M pH=11.02CO3–NaHCO3Buffer solution is 3 by volume:1 mixed solution, derivative reaction
Condition is：35 DEG C are protected from light 30min.
Further, the step 1) and 4) in aqueous formic acid be volume fraction 15% aqueous formic acid.
Further, the condition of centrifugation is 4 DEG C, centrifugal force 18000g, time 5min of temperature in the step 1).
Further, the volume ratio of blood plasma and containing the internal standard acetonitrile solution is 1 in the step 4):3, the item centrifuged for the first time
Part is 4 DEG C, centrifugal force 18000g, time 10min of temperature；The condition of second of centrifugation is 4 DEG C, centrifugal force 18000g of temperature, when
Between 5min.
Further, in the step 2), liquid phase chromatogram condition is：Chromatographic column：Waters ACQUITY UPLC BEH
C18Column:pore size particle size 1.7μm,2.1mm×50mm；cat.#186002350IVD；Color
Compose column column temperature：25℃；Sample size：15ul；Flow velocity：0.3ml/min；Flow phase composition：A phases (20mM ammonium acetates and 0.1% formic acid
Aqueous solution, volume fraction), B phases (0.1% formic acid-acetonitrile solution, volume fraction)；
Further, in the step 3), Mass Spectrometry Conditions are：Mass spectrum for Waters Xevo TQD IVD (Waters,
Milford,MA)；Mass Spectrometer Method condition is as follows：Electron spray pin voltage：3.2kV goes solvent stream fast：800L/h removes solvent gas
Temperature：400 DEG C, taper hole gas velocity：50L/h, is detected as cation mode, multiple reaction monitoring, and detection residence time is 25ms.
It applies a kind of method innovation using Liquid Chromatography-Tandem Mass Spectrometry detection catecholamine levels in plasma of the present invention
High-throughput liquid chromatogram-triple tandem quadrupole mass spectrograph carries out the detection of catecholamine, has the advantages that：Using second
Nitrile carries out albumen precipitation, and dansyl Cl performs the derivatization reaction, and liquid chromatogram-triple tandem quadrupole mass spectrograph is used after extraction
Detection, pre-treatment step is simple, can effectively remove plasma matrix interference, and specificity is good；After dansyl Cl derivatization, energy
Effectively improve detection sensitivity；High-throughput Liquid Chromatography-Tandem Mass Spectrometry instrument is detected, at the same it is qualitative and precisely quantify blood plasma in
Including dopamine, adrenaline, 3 kinds of catecholamines of norepinephrine, detection time is short, and flux is high, and detection sensitivity is high,
Specificity is good, of low cost.
Description of the drawings
Fig. 1 is the method and step schematic diagram of the present invention；
Fig. 2 is the chromatogram of liquid chromatography qualitative catecholamine and the deuterated vanillylmandelic acid (VMA) of internal standard in the present invention；
Fig. 3 is the adrenergic mass spectrum canonical plotting of catecholamine in the present invention；
Fig. 4 is the mass spectrum canonical plotting of catecholamine dopamine in the present invention；
Fig. 5 is the mass spectrum canonical plotting of catecholamine norepinephrine in the present invention.
Specific embodiment
The technical solution in the embodiment of the present invention is clearly and completely described below, it is clear that described embodiment
Only part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiment of the present invention, ordinary skill
Personnel's all other embodiments obtained without making creative work, belong to the scope of protection of the invention.
3 kinds of catecholamine standard items (adrenaline, norepinephrine, dopamine) and corresponding isotope marks it is interior
Mark (deuterated vanillylmandelic acid (VMA), D3-VMA) is purchased from Sigma Aldriches.Standard items and internal standard are carried out first with acetonitrile
Then dissolving is stored in -80 DEG C, it is necessary to when carrying out actual sample detection, recycles acetonitrile that these internal standards are diluted to about 25nM
Concentration is as working solution.
As shown in Figure 1, a kind of method using Liquid Chromatography-Tandem Mass Spectrometry detection catecholamine levels in plasma of the present embodiment,
Include the following steps：
1) prepared by standard items：The catecholamine acetonitrile solution of 0.3ml various concentrations is taken respectively into 1.5ml EP pipes, then is divided
Not Jia Ru 0.9ml Cord bloods the deuterated vanillylmandelic acid (VMA) acetonitrile solution of containing the internal standard, after being sufficiently mixed with vortex mixer, use
The catecholamine acetonitrile solution of the liquid-transfering gun transfer deuterated vanillylmandelic acid (VMA) of 1mL containing the internal standards of 1mL is managed to another 1.5ml EP,
Then freezed with vacuum freeze dryer；
After lyophilized under the conditions of being protected from light, the dansyl Cl acetonitrile solution of 150uL 4mg/mL is added in above-mentioned 1.5ml EP pipes
With the Na of 50uL 0.1M pH=11.02CO3–NaHCO3Buffer solution, vortex mixing 30s, is then protected from light at 35 DEG C
30min；
Mixed solution pH is transferred to 7 or so by adding in the aqueous formic acid of 5uL volume fractions 15% after having reacted, mixing
Component centrifuges 5min using refrigerated centrifuge at 4 DEG C of 18000g centrifugal force, and supernatant 100uL is taken to be used for liquid phase color in 96 orifice plates
Compose the analysis of tandem mass spectrum loading；
2) liquid chromatogram separates：It is right on reversed C18 analytical columns using ultrahigh pressure liquid phase chromatography and corresponding mobile phase
Catecholamine carries out chromatography eluant separation in sample supernatant, by controlling elution requirement, isolates adrenaline, goes on first kidney
Parathyrine and dopamine；
Liquid phase chromatogram condition is：Chromatographic column：Waters ACQUITY UPLC BEH C18Column:pore sizeparticle size 1.7μm,2.1mm×50mm；cat.#186002350IVD；Chromatographic column column temperature：25℃；Sample introduction
Amount：15ul；Flow velocity：0.3ml/min；Flow phase composition：A phases (20mM ammonium acetates and 0.1% aqueous formic acid, volume fraction), B
Phase (0.1% formic acid-acetonitrile solution, volume fraction)；
Gradient is as shown in the following table 1 or table 2：
Table 1
| Time (min) | Flow velocity (ml/min) | A% | B% | Curve values | |
| 1 | - | 0.3 | 75 | 25 | - |
| 2 | 3 | 0.3 | 75 | 25 | 1 |
| 3 | 4 | 0.3 | 50 | 50 | 6 |
| 4 | 10 | 0.3 | 10 | 90 | 6 |
| 5 | 13.5 | 0.3 | 10 | 90 | 6 |
| 6 | 15 | 0.3 | 75 | 25 | 11 |
Table 2
| Time (min) | Flow velocity (ml/min) | A% | B% | Curve values | |
| 1 | - | 0.3 | 75 | 25 | - |
| 2 | 1.5 | 0.3 | 75 | 25 | 1 |
| 3 | 3 | 0.3 | 50 | 50 | 6 |
| 4 | 3.5 | 0.3 | 10 | 90 | 6 |
| 5 | 4 | 0.3 | 10 | 90 | 6 |
| 6 | 5 | 0.3 | 75 | 25 | 11 |
3) Mass Spectrometer Method and standard curve processed：The 3 kinds of catecholamines isolated in liquid chromatogram enter triple level Four bars
Mass spectrum is detected, and 3 kinds of catecholamines are specifically detected using the multiple reaction monitoring mode in triple level Four bar mass spectrums
Content, and draw canonical plotting processed；
3 kinds of catecholamines after being separated in chromatography enter Waters Xevo TQD mass spectrums and are detected, and utilize triple four
Multiple reaction monitoring mode in grade bar mass spectrum specifically detects 3 kinds of catecholamines, according to different chromatographic elution times, if
Put different detection windows and parameter；
Dopamine retention time is 9.89min, and adrenaline retention time is 7.53min, when norepinephrine retains
Between for 9.28min, the retention time of internal standard vanillylmandelic acid (VMA)-D3 is 4.88min, as shown in Fig. 2, sample passes through ultrahigh pressure liquid phase
After chromatographic isolation, different catecholamine substances is examined in different elution time appearances, and by mass spectrum selection reaction monitoring pattern
It measures, the substance detected from top to bottom is respectively：Adrenaline, norepinephrine, dopamine, deuterated vanillylmandelic acid (VMA).
Mass spectrum is Waters Xevo TQD IVD (Waters, Milford, MA)；Mass Spectrometer Method condition is as follows：Electron spray
Pin voltage：3.2kV goes solvent stream fast：800L/h removes solvent temperature degree：400 DEG C, taper hole gas velocity：50L/h is detected as sun
Ion mode, multiple reaction monitoring, detection residence time are 25ms, the specific reactive ion of each determinand to, orifice potential,
Collision energy etc., as shown in table 3 below (different type instrument, collision energy, orifice potential value are different, it is necessary to independent excellent
Change)：Mass spectrum multiple reaction monitoring parameter is as follows：
Table 3
Multiple reaction monitoring by screening twice, i.e., first level Four bar carries out specific parent ion screening, second level Four
Bar carries out parent ion fragmentation and generates daughter ion, and the 3rd level Four bar carries out specific daughter ion screening, special with extraordinary detection
The opposite sex.It can be by selecting the ion stream and corresponding retention time that reaction monitoring detects, to determine catecholamine substance
Detection, recycle addition known quantity vanillylmandelic acid (VMA) internal standard quantified.
Canonical plotting is shown in Fig. 3, Fig. 4, Fig. 5, and detection limit is defined as signal-to-noise ratio>3, quantitative limit is defined as signal-to-noise ratio>10, it protects
The absolute retention time that the time is chromatography eluant appearance is stayed, the coefficient R 2 of all detection substances is equal>0.99, each catechol
The retention time of amine, detection limit, quantitative limit and the range of linearity as shown in FIG., were tested respectively by the precision of three days, bag
Basic, normal, high three concentration is included, in a few days day to day precision RSD is respectively less than 15%, shows that testing result is accurate, repeats.
4) detection of catecholamine levels in plasma：0.3ml plasma samples is taken to add 0.9ml low temperature into 1.5ml EP pipes
The deuterated vanillylmandelic acid (VMA) acetonitrile solution of containing the internal standard of preservation after being sufficiently mixed with vortex mixer, is shifted with the liquid-transfering gun of 1mL
The catecholamine acetonitrile solution of the deuterated vanillylmandelic acid (VMA) of 1mL containing the internal standards is managed to another 1.5ml EP, then uses vacuum refrigeration
Drier freezes；
After lyophilized under the conditions of being protected from light, the dansyl Cl acetonitrile solution of 150uL 4mg/mL is added in above-mentioned 1.5ml EP pipes
With the Na of 50uL 0.1M pH=11.02CO3–NaHCO3Buffer solution, vortex mixing 30s, is then protected from light at 35 DEG C
30min；
Mixed solution pH is transferred to 7 or so by adding in the aqueous formic acid of 5uL volume fractions 15% after having reacted, mixing
Component centrifuges 5min using refrigerated centrifuge at 4 DEG C of 18000g centrifugal force, and supernatant 100uL is taken to be used for liquid phase color in 96 orifice plates
Compose the analysis of tandem mass spectrum loading；
With step 2 and 3), Mass Spectrometer Method obtains catecholamine and interior target ratio for Liquid Chromatography-Tandem Mass Spectrometry analysis, will
Ratio is updated to quantitative correction equation, and the content of catecholamine levels in plasma is calculated.
It answers a kind of method innovation using Liquid Chromatography-Tandem Mass Spectrometry detection catecholamine levels in plasma of the present embodiment
The detection of catecholamine is carried out with high-throughput liquid chromatogram-triple tandem quadrupole mass spectrograph, is had the advantages that：Using
Acetonitrile carries out albumen precipitation, and dansyl Cl performs the derivatization reaction, and liquid chromatogram-triple tandem quadrupole mass spectrum is used after extraction
Instrument detects, and pre-treatment step is simple, can effectively remove plasma matrix interference, and specificity is good；After dansyl Cl derivatization,
Detection sensitivity can be effectively improved；High-throughput Liquid Chromatography-Tandem Mass Spectrometry instrument is detected, at the same it is qualitative and precisely quantify blood plasma
Include dopamine, adrenaline, 3 kinds of catecholamines of norepinephrine, detection time is short, and flux is high, detection sensitivity
Height, specificity is good, of low cost.
A kind of high-throughput liquid chromatography tandem mass spectrometry detection catecholamine provided above the embodiment of the present invention
Method is described in detail, and specific case used herein carries out the principle and embodiment of the embodiment of the present invention
It illustrates, the explanation of above example is only intended to the method and its core concept that help understands the embodiment of the present invention；Meanwhile for
Those of ordinary skill in the art according to the thought of the embodiment of the present invention, can in specific embodiment and application range
There is change part, in conclusion this specification content should not be construed as limiting the invention.
Claims (7)
- A kind of 1. method using Liquid Chromatography-Tandem Mass Spectrometry detection catecholamine levels in plasma, which is characterized in that including walking as follows Suddenly：1) prepared by standard items：The catecholamine acetonitrile solution of several various concentrations is taken respectively, and it is known dense to be separately added into containing for equivalent Target acetonitrile solution in degree, it is spare；Under the conditions of being protected from light, using derivatization reagent dansyl Cl to target catecholamine acetonitrile solution in addition, derived Change processing so that catecholamine chemically reacts, to improve signal response of the catecholamine on mass spectrum；Derivatization reagent is red Sulfonic acid chloride is specially：The dansyl Cl acetonitrile solution of 4mg/mL and the Na of 0.1M pH=11.02CO3–NaHCO3Buffer solution is pressed Volume ratio is 3:1 mixed solution, derivative reaction condition are：35 DEG C are protected from light 30min；Aqueous formic acid adjusting is added in after having reacted makes pH=7, sample supernatant is taken after centrifugation in spare on 96 orifice plates, for liquid Phase chromatographic tandem mass spectrum loading is analyzed, and completes the preparation of standard items；2) liquid chromatogram separates：Using ultrahigh pressure liquid phase chromatography and corresponding mobile phase, to standard on reversed C18 analytical columns Catecholamine carries out chromatography eluant separation in product, by controlling elution requirement, isolates adrenaline, norepinephrine and more Bar amine；Liquid phase chromatogram condition is：Chromatographic column：Waters ACQUITY UPLC BEH C18Column:pore size particle size 1.7μm,2.1mm×50mm；cat.#186002350IVD；Chromatographic column column temperature：25℃；Sample size：15μ l；Flow velocity：0.3ml/min；Flow phase composition：A phases：20mM ammonium acetates and 0.1% aqueous formic acid, volume fraction, B phases： 0.1% formic acid-acetonitrile solution, volume fraction；3) Mass Spectrometer Method and standard curve processed：The 3 kinds of catecholamines isolated in liquid chromatogram enter triple level Four bar mass spectrums It is detected, the content of 3 kinds of catecholamines is specifically detected using the multiple reaction monitoring mode in triple level Four bar mass spectrums, Quantitative detection limit and identification detection limit are calculated according to signal noise ratio, according to ratio and known standard items and internal standard concentration Value draws canonical plotting and obtains quantitative correction equation；4) detection of catecholamine levels in plasma：Plasma sample is taken, adds in and sinks containing target acetonitrile solution progress albumen in known concentration It forms sediment, is fully centrifuged for the first time after precipitation, pipette plasma supernatant and be freeze-dried；Under the conditions of being protected from light, processing is performed the derivatization to plasma supernatant so that catecholamine using derivatization reagent dansyl Cl It chemically reacts, is responded with improving signal of the catecholamine on mass spectrum；Derivatization reagent dansyl Cl is specially：4mg/mL Dansyl Cl acetonitrile solution and 0.1M pH=11.0 Na2CO3–NaHCO3Buffer solution is 3 by volume:1 mixing is molten Liquid, derivative reaction condition are：35 DEG C are protected from light 30min；Aqueous formic acid adjusting is added in after having reacted makes pH=7, and sample supernatant is taken after centrifuging for second in spare on 96 orifice plates, It is analyzed for Liquid Chromatography-Tandem Mass Spectrometry loading；Liquid Chromatography-Tandem Mass Spectrometry analysis is with step 2 and 3), and Mass Spectrometer Method obtains catecholamine and interior target ratio, by ratio Quantitative correction equation is updated to, the content of catecholamine levels in plasma is calculated.
- 2. a kind of method using Liquid Chromatography-Tandem Mass Spectrometry detection catecholamine levels in plasma according to claim 1, It is characterized in that, it is interior in the step 1) to be designated as deuterated vanillylmandelic acid (VMA), the concentration containing target acetonitrile solution in known concentration For 25nmol/L.
- 3. a kind of method using Liquid Chromatography-Tandem Mass Spectrometry detection catecholamine levels in plasma according to claim 1, It is characterized in that, the concentration of the catecholamine acetonitrile solution of several various concentrations is controlled in the μ of 1pmol/L~1 in the step 1) Between mol/L.
- 4. a kind of method using Liquid Chromatography-Tandem Mass Spectrometry detection catecholamine levels in plasma according to claim 1, Be characterized in that, the step 1) and 4) in aqueous formic acid be volume fraction 15% aqueous formic acid.
- 5. a kind of method using Liquid Chromatography-Tandem Mass Spectrometry detection catecholamine levels in plasma according to claim 1, It is characterized in that, the condition of centrifugation is 4 DEG C, centrifugal force 18000g, time 5min of temperature in the step 1).
- 6. a kind of method using Liquid Chromatography-Tandem Mass Spectrometry detection catecholamine levels in plasma according to claim 1, It is characterized in that, the volume ratio of blood plasma and containing the internal standard acetonitrile solution is 1 in the step 4):3, the condition of centrifugation is temperature for the first time 4 DEG C, centrifugal force 18000g, time 10min；The condition of second of centrifugation is 4 DEG C, centrifugal force 18000g, time 5min of temperature.
- 7. a kind of method using Liquid Chromatography-Tandem Mass Spectrometry detection catecholamine levels in plasma according to claim 1, It is characterized in that, in the step 3), Mass Spectrometry Conditions are：Mass spectrum is Waters Xevo TQD IVD：Waters,Milford,MA； Mass Spectrometer Method condition is as follows：Electron spray pin voltage：3.2kV goes solvent stream fast：800L/h removes solvent temperature degree：400 DEG C, cone Gas flow hole speed：50L/h, is detected as cation mode, multiple reaction monitoring, and detection residence time is 25ms.
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| CN111721853A (en) * | 2020-05-09 | 2020-09-29 | 浙江湖州三体生物科技有限公司 | Method for detecting catecholamine substances in blood plasma by using liquid chromatography tandem mass spectrometry |
| CN111896643A (en) * | 2020-07-20 | 2020-11-06 | 上海药明奥测医疗科技有限公司 | Liquid chromatography tandem mass spectrometry detection method for catecholamine in human plasma |
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