CN107478741A - Ultra performance liquid chromatography tandem mass spectrum method that is a kind of while detecting metanephrine and methoxyepinephrine - Google Patents
Ultra performance liquid chromatography tandem mass spectrum method that is a kind of while detecting metanephrine and methoxyepinephrine Download PDFInfo
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- CN107478741A CN107478741A CN201710668915.3A CN201710668915A CN107478741A CN 107478741 A CN107478741 A CN 107478741A CN 201710668915 A CN201710668915 A CN 201710668915A CN 107478741 A CN107478741 A CN 107478741A
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- G01N30/02—Column chromatography
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
It is a kind of while detect the ultra performance liquid chromatography tandem mass spectrum method of metanephrine and methoxyepinephrine in blood plasma/urine, belong to biochemical analysis detection field.The present invention in blood plasma/urine by adding Isotopic Internal Standard solution, 96 orifice plates of excessively activated balance after being well mixed, then scrubbed, elution, finally collects the adrenergic eluent containing methoxy.Sample preprocessing is carried out using offline SPE methods, more online SPE methods save cost, can effectively be enriched with sample, and and can despumation reduces matrix effect.The domestic C18CEX SPE fillers of first Application of the present invention, establish the ultra performance liquid chromatography tandem mass spectrometry of MN and NMN in Simultaneous Determination blood plasma/urine a kind of.The preprocess method is simple to operate and corner on the market is broken in the application of domestic packing, and ultra performance liquid chromatography tandem mass spectrometry high sensitivity, specificity are strong, quantitative accurate, have good potential applicability in clinical practice.
Description
Technical field
The invention belongs to biochemical analysis detection field, is related to a kind of while detects in blood plasma/urine on methoxyl group kidney
The ultra performance liquid chromatography tandem mass spectrum method of parathyrine and methoxyepinephrine.
Background technology
Pheochromocytoma is a kind of rare incretion disease, takes place mostly in the chromaffin cell of medullary substance on kidney, can draw
Play Secretion of Catecholamine increase.Because tumor tissues secrete a large amount of catecholamines, adrenergic receptor, chromaffin cell are acted on
Knurl often using paroxysmal or continuation hypertension and metabolic disorder as main clinical manifestation, other symptoms include headache, it is ictal go out
Sweat, tachycardia, Body weight loss, fatigue, orthostatic hypotension, anxiety are nervous and pale, it is also possible to the lethal heart
Vascular complication.Therefore, carry out early diagnosing and rational therapy is most important.
The diagnosis of pheochromocytoma includes biochemical test, iconography detection and heredity test, and biochemistry detection is the first step
It is a vital step.In the past, the measure of urine and catecholamine levels in plasma is to evaluate the main biochemical of pheochromocytoma
Method, because catecholamine is discontinuity secretion in pheochromocytoma, this diagnostic method is inaccurate.In pheochromocytoma
Middle catecholamine constantly changes into metanephrine（metanephrine, MN）And methoxyepinephrine
（normetanephrine, NMN）Two kinds of metabolites, MN and NMN secretion are independent of catecholamine changeable in vivo.Cause
This, MN and NMN measure in blood plasma or urine, can be used as pheochromocytoma biochemical diagnosis more accurately Testing index[1].For
The adrenergic analytical technology requirement of methoxy has high sensitivity and specificity in biological fluid.Therefore, there is an urgent need to one kind to grasp
Make simple, stable and high sensitivity, the good detection method of specificity.
Radiation enzyme immunoassay, gas-chromatography or the fluoroscopic examination of early stage is connected by the HPLC of high sensitivity and high selectivity
Electrochemistry or fluoroscopic examination are replaced, and the latter is widely accepted in clinical biochemical experiment.Nevertheless, its chromatographic isolation early stage
Purification step it is still extremely important, and can not be ignored.The WCX SPE of more polymerization fillings are applied in the method for recent report mostly,
The present invention is pre-processed by the screening to SPE fillers, the domestic C18CEX SPE of first Application to blood plasma/urine, this method
It is simple to operate, sample can be effectively enriched with, and can despumation reduces matrix effect, meets methoxy adrenaline to pretreatment
The requirement of method, there is good potential applicability in clinical practice.
Ultra performance liquid chromatography tandem mass spectrometry, with its high sensitivity and specificity, adrenergic to methoxy
Suitable advantage is shown in measure.The present invention establishes a kind of detection blood first using ultra performance liquid chromatography tandem mass spectrum technology
The method of methoxy epinephrine contents in slurry/urine, this method high specificity is sensitive, accurately, efficiently, is detected in clinical sample
In embody brilliance advantage.
The content of the invention
It is a kind of to detect metanephrine in blood plasma/urine simultaneously（MN）And methoxyepinephrine（NMN）'s
Ultra performance liquid chromatography tandem mass spectrum method, belongs to biochemical analysis detection field.The present invention in blood plasma/urine by adding
Enter Isotopic Internal Standard solution, 96 orifice plates of excessively activated balance after being well mixed, then scrubbed, elution, finally collect and contain first
The adrenergic eluent of oxygen.Sample preprocessing is carried out using offline SPE methods, more online SPE methods save cost, can be effectively
Sample is enriched with, and can despumation reduces matrix effect.The domestic C18CEX SPE fillers of first Application of the present invention, establish one kind
MN and NMN ultra performance liquid chromatography tandem mass spectrometry in Simultaneous Determination blood plasma/urine.Preprocess method operation letter
Corner on the market is broken in single and domestic packing application, and ultra performance liquid chromatography tandem mass spectrometry high sensitivity, specificity are strong, fixed
Amount is accurate, has good potential applicability in clinical practice.
The technical solution adopted in the present invention is：Ultra performance liquid chromatography string that is a kind of while detecting MN and NMN in blood plasma/urine
Join mass spectrometry method, detection method step is：
A. the preparation of sample：Sample is taken into EP pipes, after adding inner mark solution, is well mixed, 96 holes that overactivation has balanced
Plate, then by 3 elution, finally elute, collect the adrenergic eluent containing methoxy.
B. above-mentioned eluent is analyzed and gathered using HPLC-MS/MS methods.
The concrete operations eluted in step A are：Add 500μL pure water carries out first time elution, and 200 are added after completingμL methanol carries out second and eluted, and is eventually adding 200μL acetonitriles carry out third time elution.
The concrete operations eluted in step A are：Add 200μL acetonitriles, EP pipes collect eluent.
In step in A inner mark solution be deuterated metanephrine (d 3 - MN), deuterated normetanephrine（d 3 -NMN）'s
Mixed solution.
The chromatographic column uses hydrophilic chromatographic post.
The sample is blood plasma/urine.
It is described mixing inner mark solution concentration be：
d 3 - metanephrine： 30 nmol/L
d 3 - normetanephrine： 30 nmol/L
Liquid phase chromatogram condition in step B：Chromatographic column：Waters ACQUITY UPLC@BEH HILIC 2.1 × 100mm, 1.7μ
m；Column temperature：40 DEG C, sample introduction room temperature：4 DEG C, flow velocity：0.4 mL/min, sampling volume：10μL, mobile phase A:0.2% formic acid water
Solution, Mobile phase B:Acetonitrile, gradient elution, elution program are shown in Table 1；
Mass Spectrometry Conditions are：Ion gun：ESI+；Desolvention gas velocity：800 mL/h；Desolventizing temperature：500℃；Capillary voltage：
3KV。
1) standard curve（Quality-control product）Preparation method：Take 30μL standard working solutions（Quality-control product）In 1.5 mL centrifuge tubes,
Add 270μL 4%BSA, vortex 15s, SPE 96 orifice plate are through 500μL methanol activates, and 500μAfter L pure water equilibriums, loading, according to
It is secondary to use 500μL pure water, 200μL methanol, 200μL formic acid acetonitrile elutes, finally with 200μL acetonitriles elute, and collect elution
Liquid, take 150μL is into sample introduction bottle, HPLC-MS/MS methods sample introduction 10μL is analyzed.
2) preparation method of sample：Take 300μL testing sample solutions add 20 in 1.5mL centrifuge tubesμL internal standard solutions,
The orifice plate of vortex 15s, SPE 96 is through 500μL methanol activates, and 500μAfter L pure water equilibriums, loading, successively with 500μL pure water,
200 μL methanol, 200μL acetonitriles elute, finally with 200μL acetonitriles elute, and collect eluent, take 150μL is to sample introduction bottle
In, HPLC-MS/MS methods sample introduction 10μL is analyzed.
Beneficial effects of the present invention：The present invention adds Isotopic Internal Standard solution using by blood plasma/urine, is well mixed, mistake
96 orifice plates of activated balance, then scrubbed, elution, finally collect the adrenergic eluent containing methoxy.Entered using SPE methods
Row sample preprocessing, method flux is high, is effectively enriched with sample, and and can despumation reduces matrix effect.The present invention first should
With the preprocess method based on C18CEX SPE fillers, a kind of the super of MN and NMN in Simultaneous Determination blood plasma/urine is established
High performance liquid chromatography tandem mass spectrum method, this method high sensitivity, specificity are strong, quantitative accurate, before having good clinical practice
Scape.
Brief description of the drawings
Fig. 1 is the MRM chromatograms containing MN and NMN hybrid standards product and each determinand inner mark solution described in the embodiment of the present invention
Figure.
Fig. 2 is the plasma sample and inner mark solution MRM chromatograms described in the embodiment of the present invention.
Embodiment
Below using the best orifice plates of C18CEX 96 of concentration effect as instantiation, the present invention is expanded on further.The application is real
Example is only illustrative of the invention and is not intended to limit the scope of the invention.
1. instrument and reagent
Liquid chromatograph-mass spectrometer：UHPLC-Xevo Waters
High speed freezing centrifuge：TECHCOMP model Cs T18RT
Constant temperature DL instrument：THERMO-SHAKER models AS20130565138
Ultrasonic cleaner：New sesame model SB-25-12D
Formic acid（Chromatographic grade）, acetonitrile（Chromatographic grade）, water（Wahaha Pure Water）
2. the preparation of standard liquid
Take MN, NMN standard items appropriate respectively, it is accurately weighed, it is placed in EP pipes, acetonitrile is dissolved, and is made containing MN, NMN
Hybrid standard product solution, it is standby.With the above-mentioned hybrid standard product solution of dilution in acetonitrile, be made following concentration series standard solution and
QC solution, is shown in Table 2.
Table 2 MN, NMN series standard solution and QC solution
3. chromatographic condition：
Chromatographic column：Waters ACQUITY UPLC@BEH HILIC 2.1 × 100mm, 1.7μm；
Mobile phase：0.2% aqueous formic acid（A）, acetonitrile（B）；Gradient elution, elution program are shown in Table 1；
Flow velocity：0.4 mL/min；
Column temperature：40℃；
Sample size：10μL
4. Mass Spectrometry Conditions：
Ion gun：ESI+
Source temperature：150℃
Precipitation is vented one's spleen temperature：500 ℃；
Desolvention gas velocity：800 L/h；
Capillary voltage：3 KV；
Determinand MRM sweep parameters are shown in Table 3.
Table 3 MN, NMN and interior target MRM sweep parameters
5. pre-treating method：
5.1 standard working solutions/quality-control product pre-treating method：
Take 30μL standard working solutions（Quality-control product）In 1.5 mL centrifuge tubes, 270 are addedμL 4%BSA, vortex 15s, SPE 96
Orifice plate is through 500μL methanol activates, and 500μAfter L pure water equilibriums, loading, successively with 500μL pure water, 200μL methanol, 200μL acetonitriles elute, finally with 200μL acetonitriles elute, and collect eluent, take 150μL is into sample introduction bottle, HPLC-MS/MS side
Method sample introduction 10μL is analyzed.
5.2 plasma sample pre-treating methods：
Take 300μL sample solutions add 20 in 1.5 mL centrifuge tubesμL internal standard solutions, the orifice plate of vortex 15s, SPE 96 are through 500μL methanol activates, and 500μAfter L pure water equilibriums, loading, successively with 500μL pure water, 200μL methanol, 200μL acetonitriles drench
Wash, finally with 200μL acetonitriles elute, and collect eluent, take 150μL is into sample introduction bottle, HPLC-MS/MS methods sample introduction 10μL is analyzed.
6. linear equation
Sample is prepared according to " standard working solution pre-treating method ", carries out HPLC-MS/MS analyses.Respectively with MN, NMN concentration
For abscissa, using MN, NMN and interior target peak area ratio as ordinate, with weighting（W=1/C2）Least square method carries out recurrence meter
Calculate, least square method carries out regressing calculation, and the linear regression equation tried to achieve is standard curve.Typical regression equation and linear
Scope is shown in Table 4.
Table 4 MN, NMN retention time, linear equation and linearly dependent coefficient
7. precision test
Sample, each concentration are prepared according to " Quality Control pre-treating method "（QC is low, and in QC, QC is high）6 samples are prepared respectively, continuously
Measure 3 days, and the concentration of quality-control sample is calculated with the standard curve on the same day, tries to achieve the essence of method with measure is criticized with standard curve
Density（RSD）, the results are shown in Table 5.
The Precision test result of table 5
8. recovery test
Precision measures the 4%BSA aqueous solution 270μL, add 30μSeries standard solution of the L containing each determinand, prepares MN's and NMN
The QC samples of basic, normal, high three concentration（Per the sample analysis of concentration five）, the retinue standard curve on the gained peak area substitution same day,
Concentration C is obtained, with the concentration measured compared with adding concentration, calculates average recovery.It the results are shown in Table 6.
The recovery test result of table 6
9. matrix effect
Precision measures the 4%BSA aqueous solution 270μL, add 30μSeries standard solution of the L containing each determinand, prepares MN's and NMN
The QC samples of basic, normal, high three concentration（Per the sample analysis of concentration five）, the peak area measured is designated as A1；Another precision measures pure water
270 μL, add 30μL, by " plasma sample pre-treatment " item, similarly hereinafter method is tested, and is carried out LC-MS/MS analyses, is obtained corresponding peak
Area（The average value of five measure）, A2 is designated as, with the peak area ratio A of each two kinds of processing methods of concentration1/A2× 100% meter
Calculate matrix effect.It the results are shown in Table 7.
The matrix effect result of the test of table 7
10. the calculating of analysis result
The peak area of testing sample is read, the MN and NMN standard curve established according to step 6, the MN of sample is calculated
Content with NMN is respectively that 0.4 μm of ol/L and 0.7 μm of ol/L appearance time MN as shown in Figure 1 is 1.57min, and NMN is
1.59min。
Above-mentioned although the embodiment of the present invention is described with reference to accompanying drawing, not to invention protection domain
Limitation, on the basis of technical scheme, those skilled in the art need not pay creative work and can make
Various modifications or deformation it is still within the scope of the present invention.
Claims (4)
- It is 1. a kind of while detect the ultra high efficiency liquid phase color of metanephrine and methoxyepinephrine in blood plasma/urine Compose tandem mass spectrum method, it is characterised in that comprise the following steps：（1）The preparation of sampleTesting sample is taken into EP pipes, after adding Isotopic Internal Standard solution, 96 orifice plates are activated, loading after balance, then by 3 times Elution, finally elutes, collects the eluent containing metanephrine and methoxyepinephrine；The Isotopic Internal Standard Solution be containingd 3 - metanephrine,d 3 - normetanephrine, the leacheate of 3 times are followed successively by：Pure water, methanol, acetonitrile, are washed De- liquid is：Acetonitrile（2）The preparation of standard curveUsing the concentration of metanephrine as abscissa, using metanephrine and interior target peak area ratio as ordinate, Recurrence calculating is carried out with weighted least-squares method, the linear regression equation tried to achieve is standard curve；Metanephrine Standard curve is Y=0.728X ﹣ 0.052；Using the concentration of methoxyepinephrine as abscissa, gone with methoxyl group on first kidney Parathyrine and interior target peak area ratio are ordinate, carry out recurrence calculating with weighted least-squares method, the linear regression equation tried to achieve As standard curve；The standard curve of methoxyepinephrine is Y=0.488X+0.001；（3）Above-mentioned eluent is analyzed and gathered using HPLC-MS/MSTake 300μL testing samples add 20 in EP pipesμL internal standard solutions, mix stand-by, and 96 orifice plates are with 500μL methanol activates, 500 μL pure water equilibriums, loading 300μL, successively with pure water, methanol, acetonitrile elution, finally with 200μL acetonitriles elute, and obtain Eluent containing metanephrine and methoxyepinephrine, draw 150μThe above-mentioned eluents of L carry out series connection matter Spectrum analysis；Liquid phase chromatogram condition is chromatographic column：Waters ACQUITY UPLC@BEH HILIC 2.1 × 100mm, 1.7μm；Post Temperature：40 DEG C, sample introduction room temperature：4 DEG C, flow velocity：0.4mL/min, sampling volume：10μL, mobile phase A:0.2% aqueous formic acid, Mobile phase B:Acetonitrile, gradient elution, elution program are shown in Table 1；Mass Spectrometry Conditions are：Ion gun：ESI+；Desolvention gas velocity：800 mL/h；Desolventizing temperature：500℃；Capillary voltage： 3KV；The gradient elution program of table 1
Time（min） Mobile phase A（v%） Mobile phase B（v%） 0.0 5 95 1.0 30 70 2.0 30 70 3.0 40 60 5.0 5 95 Metanephrine and NMN and interior target peak area ratio, bring metanephrine into and methoxyl group remove first kidney respectively The standard curve of upper parathyrine, obtain the concentration of metanephrine and methoxyepinephrine. - 2. detect metanephrine and methoxyl group in blood plasma/urine removes first kidney to one kind according to claim 1 simultaneously The ultra performance liquid chromatography tandem mass spectrum method of upper parathyrine, it is characterised in that：96 described orifice plate fillers are PSA, C18SAX, One or more in 18CEX.
- 3. detect metanephrine and methoxyl group in blood plasma/urine removes first kidney to one kind according to claim 1 simultaneously The ultra performance liquid chromatography tandem mass spectrum method of upper parathyrine, it is characterised in that：The leacheate is pure water, methanol or acetonitrile, is washed De- liquid is acetonitrile；The chromatographic column uses hydrophilic chromatographic post.
- 4. detect metanephrine and methoxyl group in blood plasma/urine removes first kidney to one kind according to claim 1 simultaneously The ultra performance liquid chromatography tandem mass spectrum method of upper parathyrine, it is characterised in that：The Isotopic Internal Standard solution concentration be containingd 3 - become 30 nmol/L of element, contain on kidneyd 3 The nmol/L of-normetanephrine 30.
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Cited By (7)
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| CN109060983A (en) * | 2018-08-20 | 2018-12-21 | 广州金域医学检验中心有限公司 | A kind of method of liquid chromatography-tandem mass spectrometry detection metanephrine substance |
| CN109633181A (en) * | 2018-12-20 | 2019-04-16 | 天津国科医工科技发展有限公司 | The detection kit of metanephrine and normetanephrine in a kind of blood plasma |
| CN109725079A (en) * | 2018-12-30 | 2019-05-07 | 杭州凯莱谱精准医疗检测技术有限公司 | The high performance liquid chromatography tandem mass spectrum detection method of free methoxyepinephrine and metanephrine in human plasma |
| CN110161140A (en) * | 2019-06-06 | 2019-08-23 | 中国医学科学院北京协和医院 | The Liquid Chromatography-Tandem Mass Spectrometry method of urine citric acid and oxalic acid content is detected simultaneously |
| CN110274974A (en) * | 2019-07-16 | 2019-09-24 | 大连润生康泰医学检验实验室有限公司 | The detection method and its enrichment material of adrenaline substance in a kind of serum |
| CN112782328A (en) * | 2019-11-11 | 2021-05-11 | 深圳华大临床检验中心 | Method and kit for detecting catecholamine and metabolites thereof in urine and application of kit |
| CN114674963A (en) * | 2022-04-26 | 2022-06-28 | 中国医学科学院北京协和医院 | Method for simultaneously detecting four oxidation-related markers and detection kit thereof |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109060983A (en) * | 2018-08-20 | 2018-12-21 | 广州金域医学检验中心有限公司 | A kind of method of liquid chromatography-tandem mass spectrometry detection metanephrine substance |
| CN109633181A (en) * | 2018-12-20 | 2019-04-16 | 天津国科医工科技发展有限公司 | The detection kit of metanephrine and normetanephrine in a kind of blood plasma |
| CN109725079A (en) * | 2018-12-30 | 2019-05-07 | 杭州凯莱谱精准医疗检测技术有限公司 | The high performance liquid chromatography tandem mass spectrum detection method of free methoxyepinephrine and metanephrine in human plasma |
| CN110161140A (en) * | 2019-06-06 | 2019-08-23 | 中国医学科学院北京协和医院 | The Liquid Chromatography-Tandem Mass Spectrometry method of urine citric acid and oxalic acid content is detected simultaneously |
| CN110274974A (en) * | 2019-07-16 | 2019-09-24 | 大连润生康泰医学检验实验室有限公司 | The detection method and its enrichment material of adrenaline substance in a kind of serum |
| CN112782328A (en) * | 2019-11-11 | 2021-05-11 | 深圳华大临床检验中心 | Method and kit for detecting catecholamine and metabolites thereof in urine and application of kit |
| CN114674963A (en) * | 2022-04-26 | 2022-06-28 | 中国医学科学院北京协和医院 | Method for simultaneously detecting four oxidation-related markers and detection kit thereof |
| CN114674963B (en) * | 2022-04-26 | 2024-04-12 | 中国医学科学院北京协和医院 | Method for simultaneously detecting four oxidation related markers and detection kit thereof |
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Application publication date: 20171215 |