CN107247093B - The detection method for metanephrine substance of dissociating in urine - Google Patents
The detection method for metanephrine substance of dissociating in urine Download PDFInfo
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Abstract
The present invention relates to a kind of detection methods for metanephrine substance of dissociating in urine, include the following steps: that the free metanephrine substance includes metanephrine, methoxyepinephrine and 3-methoxytyramine；Sample pre-treatments: mixing inner mark solution is added in urine sample, obtains eluent by Solid Phase Extraction, dries up, is added and redissolves liquid, obtain sample to be tested；The sample to be tested is detected using Liquid Chromatography-Tandem Mass Spectrometry instrument.The qualitative and quantitative detection of metanephrine substance in urine may be implemented in above-mentioned detection method, has pre-treatment simple, has effectively removed interfering substance, high sensitivity, high specificity are qualitative, quantitative function is powerful, accuracy is good, and the entire detection process time is short, it is high to detect flux.
Description
Technical field
The present invention relates to technical field of medical detection, more particularly to the metanephrine class object that dissociates in a kind of urine
The detection method of matter.
Background technique
Metanephrine substance in blood and urine is in a free form and in conjunction with sulfuric acid or glucuronic acid
Mode exist.Catecholamine matter adjusts basic physiological function in vivo, regulating blood flow amount and blood pressure can be helped, in body
Key player is play under stress situation.On the one hand metanephrine substance derives from catecholamine hormones, a side
Face is directed to pheochromocytoma.
The metabolite of incretion disease based on liquid chromatograph mass spectrography detection is widely used to various at present
It is horizontal can more effective, more acurrate, rapidly to detect pheochromocytoma associated hormone for high-end laboratory.Currently used catechu
The detection method of phenol amine metabolin mainly has radiation zymetology method, chemoluminescence method, fluorescence method and high performance liquid chromatography etc., these
Detection method is primarily present following some problems: first, sample pretreatment process is complicated, and needs initial sample amount larger；
Second, existing detection technique process time is long, and experimental error is larger, and flux is low；Third, since adrenaline and methoxyl group go first
Adrenaline molecular weight is the same, and structure is very similar, can not be distinguished with common detection methods, and specificity is poor.
Main problem is as follows:
1, urine catecholamine substance immunological detection method is at high cost for 24 hours, false positive easily occurs.
2, the detection of associativity metanephrine substance is gone easily to produce in urine due to partially may be from alimentary canal for 24 hours
Raw false positive.
4 kinds of catechols of detection method and detection of patent CN201610935910.8 high throughput Liquid Chromatography-Tandem Mass Spectrometry
In the process patent of amine metabolin, detection process is related to sample preparation, derivative reaction, sample pre-treatments, liquid chromatogram separation
And tandem mass spectrum detection and etc., sample pre-treatments complexity derivatization process is excessively complicated and takes a long time.
Therefore, a kind of those skilled in the art's there is an urgent need to pre-treatments simple, detection quickly, it is method high sensitivity, special
Property the strong urine that is suitable for dissociate the detection method of metanephrine substance.
Summary of the invention
Based on this, the present invention provide a kind of pre-treatment is simple, detection quickly, method high sensitivity, high specificity be applicable in
Dissociate the detection method of metanephrine substance in urine.
Specific technical solution is as follows:
A kind of detection method for metanephrine substance of dissociating in urine, includes the following steps:
The free metanephrine substance includes metanephrine, methoxyepinephrine and 3-
Methoxytyramine；
Sample pre-treatments: mixing inner mark solution is added in urine sample, obtains eluent by Solid Phase Extraction, dries up, adds
Enter to redissolve liquid, obtains sample to be tested；
The mixing inner mark solution be containing metanephrine internal standard compound, methoxyepinephrine internal standard compound and
The aqueous formic acid of 3-methoxytyramine internal standard compound；
The sample to be tested is detected using Liquid Chromatography-Tandem Mass Spectrometry instrument:
Liquid phase chromatogram condition are as follows: use reverse chromatograms column；Mobile phase: A phase is that formic acid content is the water-soluble of 0.1-0.3wt%
Liquid, B phase is the methanol solution that formic acid content is 0.1-0.3wt%, using gradient elution；
Mass Spectrometry Conditions are as follows: positive ion mode, scanning mode are multiple-reaction monitoring ion scan.
In wherein some embodiments, the liquid phase chromatogram condition are as follows:
Chromatographic column: reverse phase Agilent Pursuit PFP chromatographic column；
The flow velocity of mobile phase are as follows: 0.2-0.5mL/min；
Column temperature is 28-32 DEG C；
Gradient elution mode are as follows:
0-2.50min, the volume ratio of A phase and B phase is 95:5 in mobile phase；
1.00-2.50min, A phase and the volume ratio of B phase are changed to 40:60 by 95:5 in mobile phase；
2.50-5.50min, the volume ratio of A phase and B phase is 40:60 in mobile phase；
5.51-6.50min, the volume ratio of A phase and B phase is 5:95 in mobile phase；
6.51-8.00min, the volume ratio of A phase and B phase is 95:5 in mobile phase.
In wherein some embodiments, the Mass Spectrometry Conditions are as follows:
Positive ion mode, scanning mode are multiple-reaction monitoring ion scan；
Wherein the parameter of more reaction detection ion scans includes:
Metanephrine quota ion pair is 180.0/148.1；
Metanephrine qualitative ion pair is 180.0/165.1；
Metanephrine internal standard compound ion pair is 183.1/168.0；
Methoxyepinephrine quota ion pair is 166.1/134.1；
Methoxyepinephrine qualitative ion pair is 166.1/149.1；
Methoxyepinephrine internal standard compound ion pair is 169.1/137.1；
3-methoxytyramine quota ion pair is 151.0/91.2；
3-methoxytyramine qualitative ion pair is 151.0/119.2；
3-methoxytyramine internal standard compound ion pair is 155.1/95.0.
In wherein some embodiments, the Mass Spectrometry Conditions further include: carrier gas is nitrogen, and collision gas is argon gas, taper hole gas
Flow velocity is 143-148L/Hr, and ion source temperature is 148-152 DEG C, and desolventizing temperature is 445-455 DEG C, and Desolvention gas velocity is
880-920L/Hr, collision gas flow velocity are 0.15-0.17mL/min.
In wherein some embodiments, the metanephrine internal standard compound is d3- metanephrine, the first
Methoxynoradrenalin element internal standard compound is d3- methoxyepinephrine, and the 3-methoxytyramine internal standard compound is d4-3- methoxy
Tyrasamine.
In wherein some embodiments, the mixing inner mark solution is to contain metanephrine internal standard compound, methoxyl group
The 0.1-0.3wt% aqueous formic acid of norepinephrine internal standard compound and 3-methoxytyramine internal standard compound, wherein methoxyl group adrenal gland
The concentration of plain internal standard compound, methoxyepinephrine internal standard compound and 3-methoxytyramine internal standard compound is respectively as follows: on d3- methoxyl group kidney
Parathyrine: 200-250 μ g/L；D3- methoxyepinephrine: 150-200 μ g/L；D4-3- methoxytyramine: 50-100 μ g/L.
In wherein some embodiments, the volume ratio of the mixing inner mark solution and the urine sample is 0.9-1.1:1；
The volume ratio of the urine sample and the double solvents is 1:0.9-1.1；The double solvents is that 0.1-0.3wt% formic acid is water-soluble
Liquid.
In wherein some embodiments, the filler of the Solid Phase Extraction is weakly positive ion-exchange type.
Elution process in wherein some embodiments, after Solid Phase Extraction are as follows: dense with distilled water, 48-52% volume respectively
The methanol acetonitrile solution of degree and the formic acid acetonitrile solution elution of 1-2% volumetric concentration.
In wherein some embodiments, the urine sample is random urine or (measurement of urine for 24 hours is not necessarily to flesh to urine for 24 hours
Acid anhydride correction, random urine also need to measure the creatinine value of random urine).
The qualitative and quantitative detection of metanephrine substance in urine may be implemented in above-mentioned detection method, before having
Processing is simple, and having effectively removed interfering substance, (a small amount of albumen, the phosphatide etc. in urine sample can be blocked chromatography by pre-treatment
Column generates the removing of matrix effect interfering substance, and the interference of most of chaff interferent is eliminated by Solid Phase Extraction, reduces and enters mass spectrum
In interfering compound, reduce pollution), high sensitivity, high specificity is qualitative, quantitative function is powerful, and accuracy is good, and
The entire detection process time is short, detection flux is high.Also increase detection to 3-methoxytyramine, improves the sensitivity of diagnosis and special
Property, the metabolite content that dissociates in urine for 24 hours is higher than blood plasma, diagnosis accuracy can be improved to the detection of urine.
Above-mentioned detection method uses the scan pattern of mass spectrum multiple-reaction monitoring (MRM), can obtain the total ion current of sample
The selection chromatography of ions figure of chromatogram and each compound, each compound is qualitative by two ion pairs progress of scanning, for examining
Whether the compound during surveying at monitoring appearance time is target compound, in order to avoid there are other interference.In addition, selection monitoring
Signal response is high in ion pair and stable ion pair is as quota ion pair, and internal standard compound is added and is corrected, further
Improve the specificity of accuracy in detection and method.
The reverse-phase chromatographic column that above-mentioned detection method uses is suitable for the separation analysis of catecholamines and its metabolite, most
Low detection limits have reached 5.917ng/L, and detection sensitivity is high.By detection method provided by the invention, single sample detection time
For 8min, compared with existing conventional method, detection time is short, and it is high that instrument detects flux.
Detailed description of the invention
Fig. 1 is the standard curve of metanephrine；
Fig. 2 is the standard curve of methoxyepinephrine；
Fig. 3 is the standard curve of methoxytyramine；
Fig. 4 is the total ion chromatogram of metanephrine substance detection in urine specimen.
Specific embodiment
It to facilitate the understanding of the present invention, below will be to invention is more fully described.But the present invention can be to be permitted
Mostly different form is realized, however it is not limited to embodiment described herein.On the contrary, purpose of providing these embodiments is makes
It is more thorough and comprehensive to the understanding of the disclosure.
Unless otherwise defined, all technical and scientific terms used herein and belong to technical field of the invention
The normally understood meaning of technical staff is identical.Term as used herein in the specification of the present invention is intended merely to description tool
The purpose of the embodiment of body, it is not intended that in the limitation present invention.Term as used herein "and/or" includes one or more phases
Any and all combinations of the listed item of pass.
A kind of detection method for metanephrine substance of dissociating in urine, includes the following steps:
The free metanephrine substance includes metanephrine, methoxyepinephrine and 3-
Methoxytyramine；
1, the pre-treatment of urine sample (2h urine)
1) it takes 200 μ L urine samples (standard curve point and Quality Control synchronization process) in 1.5mL centrifuge tube, 200 μ L is added
Inner mark solution is mixed, concussion is centrifuged, for use；
The mixing inner mark solution be containing metanephrine internal standard compound, methoxyepinephrine internal standard compound and
The 0.1wt% aqueous formic acid of 3-methoxytyramine internal standard compound；The metanephrine internal standard compound is d3- methoxyl group adrenal gland
Element, the methoxyepinephrine internal standard compound are d3- methoxyepinephrine, and the 3-methoxytyramine internal standard compound is
D4-3- methoxytyramine, wherein in metanephrine internal standard compound, methoxyepinephrine internal standard compound and 3-methoxytyramine
The concentration of mark object is respectively as follows: d3- metanephrine: 200 μ g/L；D3- methoxyepinephrine: 150 μ g/L；d4-
3-methoxytyramine: 50 μ g/L；
2) it takes 1mL methanol to activate 96 hole Waters solid-phase extraction plates, drips naturally；
3) 96 hole solid-phase extraction plate of 1mL water balance is taken, is dripped naturally；
4) the sample whole loading for taking step 1) to handle well is dripped naturally；
5) 1mL distilled water is taken, 96 hole solid-phase extraction plates are cleaned, is dripped naturally；
6) it takes the methanol acetonitrile solution of 50% volumetric concentration of 1mL to clean 96 hole solid-phase extraction plates, drips naturally；
7) it takes the formic acid acetonitrile solution of 1.0% volumetric concentration of 1mL in 96 hole solid-phase extraction plates, carries out sample elution, it is natural
Drippage is accepted with the hole 1mL96 bearing plate；
8) eluent that will be gathered is dried with nitrogen at room temperature；
9) in the bearing plate after drying up, 200 μ L is added to redissolve liquid (0.1wt% aqueous formic acid), is obtained after concussion centrifugation to be measured
Solution, upper machine testing.
By above-mentioned pre-treatment, a small amount of albumen, the phosphatide etc. in urine sample are blocked into chromatographic column, generate matrix effect
Interfering substance removes, and the interference of most of chaff interferent is eliminated by Solid Phase Extraction, reduces the interfering compound entered in mass spectrum,
Reduce pollution.
2, the detection for metanephrine substance of dissociating in sample to be tested
Sample to be tested is detected using liquid chromatograph-mass spectrometer.
Liquid phase chromatogram condition includes: chromatographic column using Agilent Pursuit PFP (Agilent, Pursuit
3PFP150*2.0mm) chromatographic column, separation elution is gradient elution, and liquid phase gradient is as shown in 1 liquid phase separation condition of table:
1 liquid phase separation condition of table
Mass Spectrometer Method uses electrospray ionisation (ESI) mode, is scanned using the multiple-reaction monitoring (MRM) under positive ion mode
Mode.Mass Spectrometry Conditions include: that carrier gas is nitrogen, and collision gas is argon gas, taper hole gas velocity 145L/Hr, ion source temperature 150
DEG C, desolventizing temperature is 450 DEG C, Desolvention gas velocity 900L/Hr, and collision gas flow velocity is 0.16mL/min.Multiple-reaction monitoring
(MRM) scan pattern parameter is as shown in table 2.
2 multiple-reaction monitoring pattern parameter of table
The qualitative and quantitative analysis of metanephrine substance in urine: with metanephrine class each in sample
Two ion pairs of substance are compared as qualitative foundation, each metanephrine class with the Compound Retention time of standard items
Substance retention time is as shown in table 3.The ion ratio of qualitative and quantitative ion selected in sample chromatogram figure and standard solution from
The deviation of sub- ratio is no more than preset rules range, then may determine that there are corresponding target substances in sample.It is bent with internal standard standard
Collimation method is quantitative, and the ratio of the peak area of each metanephrine substance and corresponding internal standard compound area is ordinate, respectively
The concentration of metanephrine substance and the concentration proportion of internal standard compound are abscissa, and weighting scheme is weight 1/x, draw mark
Directrix curve, check curve low spot and low middle high three horizontal Quality Control situation, quantitative calculating, knot can be carried out to actual sample
Fruit is as shown in Figure 1-Figure 3, calculates the content of each metanephrine substance in human urine.
The retention time of 3 metanephrine substance of table
3, the detection of actual sample
Using the above method, the metanephrine substance in 1 urine specimen is detected, has obtained 3
The kind respective content of metanephrine substance, total ion chromatogram are as shown in Figure 4.From fig. 4, it can be seen that each methoxy
Base adrenaline substance peak type is symmetrical, and response is suitable, and separating effect is fine.
Above-mentioned detection method minimum detectability has reached 5.917ng/L, obtains in normal human urine 3 kinds of methoxyl groups in sample
Adrenaline substance is in the concentration range of measurement, and linear good, related coefficient is 0.999 or more.
4, methodology validation
Repeated experiment: each 12 parts of urine (basic, normal, high) sample of three separate sources is taken, is carried out on the same day identical
Pre-treatment and detection, as a result the RSD of 3 kinds of metanephrine substance contents shows this between 0.4%~3.5%
Method it is in a few days reproducible.
Take each 20 parts of urine (basic, normal, high) sample of three separate sources, each daily two groups of measurements of concentration, continuous 10
It carries out identical pre-treatment and detection, and as a result the RSD of 3 kinds of metanephrine substance contents is 1.5%~5.0%
Between, show the reproducible in the daytime of this method.
Through the accuracy rate of recovery of recovery of standard addition experimental verification method between 85%-115%, method is quasi-
Really；The range of linearity and Reportable range covering are extensively, as shown in table 4 in detail:
The 4 method range of linearity of table and detection limit
Each technical characteristic of embodiment described above can be combined arbitrarily, for simplicity of description, not to above-mentioned reality
It applies all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited
In contradiction, all should be considered as described in this specification.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
It cannot therefore be construed as limiting the scope of the patent.It should be pointed out that coming for those of ordinary skill in the art
It says, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to protection of the invention
Range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
Claims (10)
1. a kind of detection method for metanephrine substance of dissociating in urine, which comprises the steps of:
The free metanephrine substance includes metanephrine, methoxyepinephrine and 3- methoxy
Tyrasamine；
Sample pre-treatments: mixing inner mark solution being added in urine sample, obtains eluent by Solid Phase Extraction, dries up, and is added multiple
Solution obtains sample to be tested；
The mixing inner mark solution is to contain metanephrine internal standard compound, methoxyepinephrine internal standard compound and 3- first
The aqueous formic acid of oxygen tyrasamine internal standard compound；
The sample to be tested is detected using Liquid Chromatography-Tandem Mass Spectrometry instrument:
Liquid phase chromatogram condition are as follows: use reverse phase Agilent Pursuit PFP chromatographic column；Mobile phase: A phase is for formic acid content
The aqueous solution of 0.1-0.3wt%, B phase is the methanol solution that formic acid content is 0.1-0.3wt%, using gradient elution, the gradient
The mode of elution are as follows:
0-1.00min, the volume ratio of A phase and B phase is 95:5 in mobile phase；
1.00-2.50min, A phase and the volume ratio of B phase are changed to 40:60 by 95:5 in mobile phase；
2.50-5.50min, the volume ratio of A phase and B phase is 40:60 in mobile phase；
5.51-6.50min, the volume ratio of A phase and B phase is 5:95 in mobile phase；
6.51-8.00min, the volume ratio of A phase and B phase is 95:5 in mobile phase；
The flow velocity of the mobile phase are as follows: 0.2-0.5mL/min；
Column temperature is 28-32 DEG C；
Mass Spectrometry Conditions are as follows:
Positive ion mode, scanning mode are multiple-reaction monitoring ion scan；
Wherein the parameter of more reaction detection ion scans includes:
Metanephrine quota ion pair is 180.0/148.1；
Metanephrine qualitative ion pair is 180.0/165.1；
Metanephrine internal standard compound ion pair is 183.1/168.0；
Methoxyepinephrine quota ion pair is 166.1/134.1；
Methoxyepinephrine qualitative ion pair is 166.1/149.1；
Methoxyepinephrine internal standard compound ion pair is 169.1/137.1；
3-methoxytyramine quota ion pair is 151.0/91.2；
3-methoxytyramine qualitative ion pair is 151.0/119.2；
3-methoxytyramine internal standard compound ion pair is 155.1/95.0；
Carrier gas is nitrogen, and collision gas is argon gas, and taper hole gas velocity 143-148L/Hr, ion source temperature is 148-152 DEG C, is taken off
Solvent temperature is 445-455 DEG C, Desolvention gas velocity 880-920L/Hr, and collision gas flow velocity is 0.15-0.17 mL/min.
2. detection method according to claim 1, which is characterized in that in the Mass Spectrometry Conditions:
Carrier gas is nitrogen, and collision gas is argon gas, and taper hole gas velocity 145L/Hr, ion source temperature is 150 °C, desolventizing temperature
It is 450 °C, Desolvention gas velocity 900L/Hr, collision gas flow velocity is 0.16mL/min.
3. detection method according to claim 1 or 2, which is characterized in that the metanephrine internal standard compound is d3-
Metanephrine, the methoxyepinephrine internal standard compound are d3- methoxyepinephrine, the 3- methoxy
Tyrasamine internal standard compound is d4-3- methoxytyramine.
4. detection method according to claim 1 or 2, which is characterized in that the mixing inner mark solution is to contain methoxyl group
The 0.1-0.3wt% formic acid of adrenaline internal standard compound, methoxyepinephrine internal standard compound and 3-methoxytyramine internal standard compound is water-soluble
Liquid, the wherein concentration of metanephrine internal standard compound, methoxyepinephrine internal standard compound and 3-methoxytyramine internal standard compound
Being respectively as follows: d3- metanephrine concentration is 200-250 μ g/L；D3- methoxyepinephrine concentration is 150-
200 μg/L；D4-3- methoxytyramine concentration is 50-100 μ g/L.
5. detection method according to claim 4, which is characterized in that the mixing inner mark solution is containing on methoxyl group kidney
The 0.1wt% aqueous formic acid of parathyrine internal standard compound, methoxyepinephrine internal standard compound and 3-methoxytyramine internal standard compound, wherein
The concentration of metanephrine internal standard compound, methoxyepinephrine internal standard compound and 3-methoxytyramine internal standard compound is respectively as follows:
D3- metanephrine concentration is 200 μ g/L；D3- methoxyepinephrine concentration is 150 μ g/L；D4-3- methoxy
Tyrasamine concentration is 50 μ g/L.
6. detection method according to claim 1 or 2, which is characterized in that the mixing inner mark solution and the urine sample
The volume ratio of product is 0.9-1.1:1；The urine sample and the volume ratio for redissolving liquid are 1:0.9-1.1；The redissolution liquid
For 0.1-0.3wt% aqueous formic acid.
7. detection method according to claim 6, which is characterized in that the mixing inner mark solution and the urine sample
Volume ratio is 1:1；The urine sample and the volume ratio for redissolving liquid are 1:1；The redissolution liquid is that 0.1wt% formic acid is water-soluble
Liquid.
8. detection method according to claim 1 or 2, which is characterized in that the filler of the Solid Phase Extraction be weakly positive from
Sub- crossover.
9. detection method according to claim 8, which is characterized in that the elution process after the Solid Phase Extraction are as follows: respectively
It is eluted with the formic acid acetonitrile solution of water, the methanol acetonitrile solution of 48-52% volumetric concentration and 1-2% volumetric concentration.
10. detection method according to claim 1 or 2, which is characterized in that the urine sample is random urine or urinates for 24 hours
Liquid.
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| CN110568093A (en) * | 2019-08-28 | 2019-12-13 | 重庆同怡生物技术研究院有限公司 | Liquid chromatography tandem mass spectrometry method for quantitatively detecting catecholamine metabolites |
| CN111398448A (en) * | 2020-03-12 | 2020-07-10 | 南京品生医学检验实验室有限公司 | Method for detecting 8 catecholamines and metabolites thereof in urine by ultra-high performance liquid chromatography tandem mass spectrometry technology |
| CN113030330A (en) * | 2021-03-15 | 2021-06-25 | 北京医院 | Method for measuring plasma methoxy adrenaline substances |
| CN114994211B (en) * | 2022-06-15 | 2023-10-27 | 北京豪思生物科技股份有限公司 | Kit for detecting catecholamine metabolite content in human urine and application thereof |
| CN116448923A (en) * | 2023-06-20 | 2023-07-18 | 中国医学科学院北京协和医院 | Methods and kits for extracting and detecting steroid hormones and catecholamine metabolites |
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