CN109725079A - The high performance liquid chromatography tandem mass spectrum detection method of free methoxyepinephrine and metanephrine in human plasma - Google Patents
The high performance liquid chromatography tandem mass spectrum detection method of free methoxyepinephrine and metanephrine in human plasma Download PDFInfo
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Abstract
The invention discloses the high performance liquid chromatography tandem mass spectrum detection methods of dissociate in a kind of blood plasma methoxyepinephrine and metanephrine, include the following steps: (1) sample pretreatment, plasma sample sequentially adds internal standard working solution and ammonium acetate solution, vortex mixed, centrifugation；(2) 96 orifice plate of weak cation exchange Solid Phase Extraction (SPE96 orifice plate) activates；(3) Solid Phase Extraction is handled, and sample loading is to treated 96 orifice plate of SPE after step (1) processing, formic acid acetonitrile solution elution, and eluent is with being dried with nitrogen；(4) it redissolves, high performance liquid chromatography tandem mass spectrometry carries out analysis detection.The method of the present invention has many advantages, such as high accuracy, high reproducibility, high sensitivity, high specificity, high efficiency, high throughput, low cost, easy to operate quick.
Description
Technical field
The present invention relates to technical field of analysis and detection, in particular to a kind of high accuracy, high reproducibility, high sensitivity, height
Specificity, high efficiency, high throughput, low cost, dissociate in quick human plasma easy to operate methoxyepinephrine and first
The adrenergic high performance liquid chromatography tandem mass spectrum detection method of oxygroup.
Background technique
Catecholamine matter is to have the aminated compounds of catechol (i.e. catechol) structure, including dopamine, go first
Adrenaline and adrenaline and their derivative.Norepinephrine and adrenaline are both secreted by adrenal medella
Hormone, and be the neurotransmitter of noradrenergic fiber in sympathetic nerve and central nervous system.Norepinephrine exists
Widely distributed in central nervous system, content is more, and epinephrine contents are then less.Dopamine is concentrated mainly on extrapyramidal system
Position and a kind of neurotransmitter.The above Catecholamine matter is all important typical adrenoceptor agonists.Catechu
Phenol amine substance adjusts basic physiological function in vivo, transmits physiological signal, is signal media important in normal physiological processes,
Its content also will appear apparent variation in pathologic process simultaneously.Therefore they can be used for clinical assistant diagnosis hypertension,
The endocrines related disease such as hyperthyroidism, pheochromocytoma and neuroblastoma, metabolite metanephrine (MN) with
And methoxyepinephrine (NMN) has high susceptibility and accurate to diagnosis pheochromocytoma and neuroblastoma
Degree is the preferred Testing index that division of endocrinology's guide is recommended.
The method of catecholamine metabolite in currently used measurement human plasma mainly has radiation zymetology method, capillary
Electrophoresis, fluorescence method and high performance liquid chromatography etc.；It is primarily present following problems and disadvantages:
1, zymetology method expensive reagents are radiated, are not able to satisfy the demand of clinical mass detection；
2, capillary electrophoresis pre-treatment includes the operation such as dialysis and centrifugation, and need to carry out capillary modification, to prevent
Tea phenol amine substance is had an effect with capillary wall, therefore the cumbersome time-consuming of this method, detection difficulty and higher cost；
3, the fluorescence of catecholamines is weaker, and content is extremely low in biological sample, therefore fluorescence method needs additionally
Derivatization treatment reinforces fluorescence signal, cumbersome time-consuming；
4, liquid chromatography needs relative complex pretreatment process, to separate impurity component in biological sample, time-consuming expense
Power, and sensitivity is lower, analysis time is long, detection flux is low, and matrix interference is serious, poor specificity, and the degree of automation is low.
Compared with traditional detection method, efficient liquid phase tandem mass spectrometry has the advantages such as high specific and high sensitivity, but
It is existing there are two the Liquid Chromatography-Tandem Mass Spectrometry detection method of disclosed catecholamines, there are defects below not
Foot:
1. disclosed on State Intellectual Property Office website " with Liquid Chromatography-Tandem Mass Spectrometry technology detection catecholamine and metabolism
The method of product " comprising Solid Phase Extraction processing and the scanning of mass spectrum positive ion mode can detect 6 kinds of catecholamines objects simultaneously
Matter, this method have the disadvantage that (1) does not provide using the consistent stable isotope labeling object internal standard of structure.With reference to liquid phase matter
Spectral method detects clinical guidance principle, and internal standard should select the consistent stable isotope labeling object of determinand structure, to correct mass spectrum
The deviation that analytic process mesostroma effect and sample extraction, chromatographic isolation and ionization process generate, other kinds of chemical combination
Object internal standard may influence the accuracy of testing result；(2) it is quantified using cation scan pattern, and guideline is not used
The multiple-reaction monitoring pattern (MRM) of defined parent ion and daughter ion screening.Compared with MRM mode, cation scan pattern
The interference of complex matrices vulnerable to biological sample, the specificity and specificity of method are relatively poor.
2. disclosed " the detection method and detection of high-throughput Liquid Chromatography-Tandem Mass Spectrometry on State Intellectual Property Office website
The method of 4 kinds of catecholamine metabolism objects ", method include S1 sample preparation, S2 derivative reaction, S3 sample pre-treatments, S4 liquid phase
Chromatographic isolation and the detection of S5 tandem mass spectrum and etc., this method has the disadvantage that (1) pretreatment process complexity, including egg
The processes such as white precipitating, vacuum freeze drying processing and derivative reaction, cumbersome time-consuming, instrument and reagent cost are higher, are not suitable for
Clinical high-volume sample analysis；(2) for the detection of NMN, the consistent isotopic label internal standard of structure is not used, may influence
The accuracy and precision of actual clinical pattern detection；(3) the instrument analysis time of sample is longer, and the analysis of single sample needs
15min, efficiency is lower, is not suitable for the detection of clinic high-volume sample.
Summary of the invention
Present invention aim to address subproblem of the existing technology, the methoxyl group that dissociates in a kind of human plasma is provided and is gone
The high performance liquid chromatography tandem mass spectrum detection method of methylepinephrine and metanephrine, this method have high efficiency, height
The low advantage of flux, high sensitivity, easy to operate, high specificity, accuracy height, high sensitivity, testing cost.
The purpose of the present invention is what is be achieved through the following technical solutions: in human plasma dissociate methoxyepinephrine with
The high performance liquid chromatography tandem mass spectrum detection method of metanephrine, includes the following steps:
(1) sample preprocessing: taking 100~500 μ L human plasmas, sequentially adds 10~50 μ L internal standard working solutions and 0.1~1mL
50mM pH=8 ammonium acetate solution, vortex oscillation 30 seconds, 4000rpm was centrifuged 5 minutes；
(2) 96 orifice plate of weak cation exchange Solid Phase Extraction is taken, 200 μ L methanol is sequentially added and water elutes, flowed naturally to it
It is dry；
(3) solid phase extraction procedure: by step (1) treated sample is transferred to step (2) treated SPE96 orifice plate
On, it is pressed dry with positive pressure of nitrogen device, sequentially adds 50~500 μ L 50mM ammonium acetate solutions (pH=8) and 50~500 μ L acetonitriles
It is eluted；The elution of 200 μ L, 5% formic acid acetonitrile solution is added, eluent is collected with 96 hole receiver boards, leads to 25~60 DEG C of nitrogen
Gas is until dry；
(4) residue for obtaining step (3) further redissolves solution with 50~200 μ L and redissolves, and vortex makes sufficiently to dissolve,
Upper 5~50 μ L analysis detection of high performance liquid chromatography tandem mass spectrum system sample introduction.
Preferably, the configuration method of internal standard working solution is as follows in the step (1): being matched with 0.5% sodium metabisulfite solution
Set concentration be 1mg/mL deuterated methoxyepinephrine (NMN-d3) and deuterated metanephrine (MN-d3) it is interior
Stock solution is marked, then further dilutes that be mixed to get containing NMN-d3 concentration be 10ng/ml and MN-d3 with 0.1% formic acid solution
Concentration be 1ng/mL internal standard working solution.
Preferably, the mixture that solution is formic acid and acetonitrile of redissolving in the step (4), volume ratio 1:1000.
Preferably, high performance liquid chromatography tandem mass spectrum described in the step (4) is triple level four bars mass spectrographs.
Preferably, the step (4) measures sample solution after pretreatment by high performance liquid chromatography tandem mass spectrometry,
The separation condition for using gradient elution normal phase chromatography to establish determinand is as follows: using water-acetonitrile-ammonium formate-formic acid as mobile phase
System, chromatographic column are Silica column, and flow velocity is 0.4~1mL/min, and column temperature is 30~45 DEG C, scan mould using multiple-reaction monitoring
Formula (MRM) analysis is quantitative.
Preferably, the high performance liquid chromatography tandem mass spectrometry measures the mobile phase that sample solution after pretreatment uses
It is made of following mobile phase A and Mobile phase B；Wherein mobile phase A is 0.1% formic acid -30mM ammonium formate solution of volume fraction, flowing
Phase B is 0.1% formic acid acetonitrile solution of volume fraction, and the volume ratio of mobile phase A and Mobile phase B is 5~22:95~78%.
Preferably, the filler of the chromatographic column be Silica column, packing material size be 1.7~5 μm, internal diameter be 2.1~
4.6mm, column length are 25~150mm.
Preferably, in the step (4), Mass Spectrometer Method uses electrospray ionisation source (ESI) and MRM mode, for detecting
Methoxyepinephrine (NMN) and parent ion/daughter ion detection ion pair of metanephrine (MN) are as follows:
166.1 > m/z of NMN, m/z 134.1 and 166.1 > m/z of m/z 121.1；180.0 > m/z of MN, m/z 149.1 and m/z
180.0>m/z 120.9；The detection ion pair of internal standard NMN-d3 is 169.1 > m/z of m/z 137.1；The detection of internal standard MN-d3 from
Son is to for 183.1 > m/z of m/z 151.1.
It based on above-mentioned experiment condition, is verified by test of many times, precision RSD % < 15% of the invention.
Compared with prior art, beneficial effects of the present invention are as follows:
Compared with traditional detection, efficient liquid phase tandem mass spectrometry has the advantage that (1) high specific: when detecting may be used
The interference and influence of complex component in bio-matrix is effectively reduced, thus reduced sample pretreatment process；(2) highly sensitive, it is fixed
Amount lower limit can achieve pg/mL rank；(3) automation and high throughput: LC-MS instrument is furnished with automatic sample handling system, sample measurement
It can be with uninterrupted progress in 24 hours, it can be achieved that automating, high-throughput, to meet hospital laboratory and third party's medical test list
The actually detected demand of position；(4) pretreatment process is relatively easy: not needing to be kept completely separate with interfering component in detection process
Accurate quantitative analysis is completed, complicated pre-treatment extraction and derivatization operating process can be simplified.
Compared with existing Mass Spectrometry detection method, dissociate methoxynoradrenalin in a kind of human plasma of the present invention
The high performance liquid chromatography tandem mass spectrum detection method of element (NMN) and metanephrine (MN) have following significant
Advantage (1) uses stable isotope labeling object as internal standard, and the more acurrate reproducibility of testing result is more preferable: this method using MN and
The consistent isotopic label internal standard of NMN structure, the influence for the matrix effect that can effectively correct actual clinical sample are (such as high in fat
Matter, the matrix and haemolysis of hepatic and renal function impaired subjects), it is ensured that the accuracy of testing result；(2) this method is faced with reference to liquid chromatography mass spectrometric
Bed detection guideline is detected using the multiple-reaction monitoring pattern (MRM) that parent ion and daughter ion screen, by feature
The screening of parent ion and daughter ion, so that the interference of complex component in biological sample be effectively reduced, the specificity of improvement method and
Sensitivity.In addition, making NMN sensitivity be increased to 10pg/mL from 20pg/mL by drying concentration；(3) this method detects
The instrument analysis time of single sample is only 5 minutes, and more traditional high performance liquid chromatography greatly shortens.Pre-treatment uses 96 holes
Plate greatly improves operating efficiency；Furthermore the automatic sample handling system of LC-MS instrument is used in combination, it can be achieved that automation and high throughput
Detection；(4) Sample pretreatment process is simple, sample is handled using weak cation exchange solid phase extraction method, to catecholamine
The substance rate of recovery with higher, while the interference of complex matrices in sample is effectively reduced, obtain better sensitivity；This
Outside, the reagent consumptive material used is routinely easily obtained and at low cost, does not need complicated derivatization operation.
In short, this method quickly can timely detect methoxyepinephrine in human plasma (NMN) and methoxyl group kidney
The concentration of upper parathyrine (MN) provides accurate inspection result for the diagnosis of clinical disease.Compared with the conventional method compared with this method
Have many advantages, such as high accuracy, high reproducibility, high sensitivity, high efficiency, easy to operate, is suitable for clinical expansion, has extensive
Clinical value.
Detailed description of the invention:
Fig. 1 a and Fig. 1 b are the chromatogram for detecting the lower limit of quantitation of 20pg/mL NMN and 10pg/mL MN respectively；
Fig. 2 a and Fig. 2 b are the standard curve of NMN and MN respectively.
Specific embodiment
In order to enable those skilled in the art to better understand the present invention with reference to specific embodiments this hair is further described
The bright specific implementation process for metanephrine (MN) in human plasma and methoxyepinephrine (NMN) detection.
It should be understood that examples are only for illustrating the present invention and not for limiting the scope of the present invention, unmentioned tool in the following example
Body experimental method is usually carried out according to routine experiment method.
Embodiment: dissociate in a kind of human plasma methoxyepinephrine (NMN) and metanephrine (MN)
High performance liquid chromatography tandem mass spectrum detection method.
One, solution allocation
Standard working solution configuration: precision weighs NMN and MN standard items, and 0.5% sodium metabisulfite solution is added and dissolves to obtain
Concentration be 10mg/mL level-one stock solution, then with 0.1% formic acid solution be further diluted to concentration be 10 μ g/mL MN with
The secondary reserves liquid of NMN；Take suitable 0.1% formic acid solution to be placed in the centrifuge tube of 2mL, sequentially add suitable NMN and
The secondary reserves liquid of MN is into the centrifuge tube, vortex mixed 10s, and obtaining NMN concentration is 50ng/mL, and MN concentration is 25ng/mL's
Three-level stock solution is mixed, three-level stock solution 4%BSA solution is diluted into configuration and obtains series standard working solution, specific concentration is such as
The following table 1.
1 standard curve concentration of table
The configuration of internal standard working solution: precision weighs 2 kinds of internal standard standard items, and 0.5% sodium metabisulfite solution dissolved dilution is added
At the deuterated methoxyepinephrine (NMN-d3) of 1mg/mL and the internal standard deposit of deuterated metanephrine (MN-d3)
Liquid, then further diluting that be mixed to get NMN-d3 concentration be 10ng/mL and MN-d3 concentration with 0.1% formic acid solution is 1ng/
The internal standard working solution of mL.
Two, concrete operation step:
(1) 500 μ L of blood plasma is taken, 15 μ L internal standard working solutions and 500 μ L 50mM ammonium acetate solutions (pH=8), whirlpool are sequentially added
Rotation mixes, and 4000rpm is centrifuged 5min；
(2) 96 orifice plate of weak cation exchange Solid Phase Extraction (SPE96 orifice plate) sequentially adds 200 μ L methanol and water in hole, to
It drains off naturally；
(3) step (1) is handled resulting sample to be added in the activated SPE96 orifice plate of step (2), uses positive pressure of nitrogen
Device slowly press dry, and sequentially adds 200 μ L 50mM ammonium acetate solutions (pH=8) and acetonitrile is eluted, positive pressure of nitrogen device pressure
It is dry, 200 μ L, 5% formic acid acetonitrile solution is then added and is eluted, collects eluent with 96 hole receiver boards, is placed under nitrogen evaporator
Logical 40 DEG C of nitrogen are until dry；
(4) residue that step (3) obtains is added 80 μ L and redissolves solution redissolution, and vortex makes sufficiently to dissolve, upper high-efficient liquid phase color
Compose 35 μ L of tandem mass spectrum combined system sample introduction analysis；
The mixture that solution is formic acid and acetonitrile of redissolving in step (4), volume ratio 1:1000；
Mass spectrum described in step (4) is triple level four bars mass spectrographs；
Step (4) measures sample solution after pretreatment by high performance liquid chromatography tandem mass spectrometry, is washed using gradient
The separation condition that de- normal phase chromatography establishes determinand is as follows: chromatographic column be Venusil XBP Silica (A) (3 μm, 2.1 ×
100mm, 150A), flow velocity 0.6mL/min, column temperature is 30 DEG C；
Mobile phase A is containing 0.1% formic acid -30mM ammonium acetate solution of volume fraction, and Mobile phase B is 0.1% first of volume fraction
Sour acetonitrile solution；A phase: B phase volume ratio is 5~22%:95~78%, and flow velocity 0.6mL/min is eluted by 2 Gradient program of table；
Table 2: gradient elution program
The retention time of NMN and MN is 2.75min.
Mass Spectrometry Conditions are as shown in table 3；
3 Mass Spectrometry Conditions of table
After the separation of the above liquid-phase condition, Mass Spectrometer Method is carried out using electrospray ionisation source (ESI) and MRM mode, wherein
The parent ion of metanephrine (MN) and methoxyepinephrine (NMN)/daughter ion detection ion pair is as follows:
180.0 > m/z of MN, m/z 149.1 and 180.0 > m/z of m/z 120.9；166.1 > m/z of NMN, m/z 134.1 and m/z
166.1>m/z 121.1；The detection ion pair of internal standard NMN-d3 and MN-d3 are as follows: 169.1 > m/z of NMN-d3, m/z 137.1；
183.1 > m/z of MN-d3, m/z 151.1.The MRM mass spectrometry parameters of the above detection ion pair are as shown in table 4；
Table 4 detects ion pair mass spectrum MRM parameter
Fig. 1 a and Fig. 1 b are the chromatogram for detecting the lower limit of quantitation of 20pg/mL NMN and 10pg/mL MN respectively；
Standard curve is established using internal standard method.Such as Fig. 2 a, Fig. 2 b and table 5 it is found that NMN is in the linear of 20~1000pg/mL
In range, MN is good in 10~500pg/mL linear dependence, and correlation coefficient r is respectively 0.9992 and 0.9995, accuracy model
Enclose respectively 91.1%-111% and 93.7%-111%.
Table 5: standard curve result
The present invention has been used for the inspection measurement of clinical batches sample, the results showed that this method range of linearity can satisfy clinic
The needs of inspection.
Above-mentioned, although the foregoing specific embodiments of the present invention is described with reference to the accompanying drawings, not protects model to the present invention
The limitation enclosed, based on the technical solutions of the present invention, those skilled in the art are not needed to make the creative labor and can be done
Various modifications or changes out are still within protection scope of the present invention.
Claims (8)
1. the high performance liquid chromatography tandem mass spectrum inspection of dissociate in human plasma methoxyepinephrine and metanephrine
Survey method, which comprises the following steps:
(1) sample preprocessing: taking 100~500 μ L human plasmas, sequentially adds 10~50 μ L internal standard working solutions and 0.1~1mL
50mM pH=8 ammonium acetate solution, vortex oscillation 30 seconds, 4000rpm was centrifuged 5 minutes；
(2) 96 orifice plate of weak cation exchange Solid Phase Extraction is taken, 200 μ L methanol is sequentially added and water elutes, drain off naturally to it；
(3) it solid phase extraction procedure: by step (1) treated sample is transferred to step (2) treated SPE96 orifice plate, uses
Positive pressure of nitrogen device press dry, and sequentially adds 50~500 μ L 50mM ammonium acetate solutions (pH=8) and 50~500 μ L acetonitriles are drenched
It washes；Add the elution of 200 μ L, 5% formic acid acetonitrile solution, collect eluent with 96 hole receiver boards, lead to 25~60 DEG C of nitrogen until
It is dry；
(4) residue for obtaining step (3) further redissolves solution with 50~200 μ L and redissolves, and vortex makes sufficiently to dissolve, upper height
5~50 μ L analysis detection of effect liquid phase chromatogram tandem mass spectrum system sample introduction.
2. dissociate the efficient of methoxyepinephrine and metanephrine in human plasma according to claim 1
Liquid Chromatography-Tandem Mass Spectrometry detection method, which is characterized in that the configuration method of internal standard working solution is as follows in the step (1): essence
It is close to weigh 2 kinds of internal standard standard items, it dissolves to obtain the deuterated methoxyl group that concentration is 1mg/mL with 0.5% sodium metabisulfite solution and goes first
The internal standard stock solution of adrenaline (NMN-d3) and deuterated metanephrine (MN-d3), then with 0.1% formic acid solution into
The internal standard working solution that it is 10ng/ml containing NMN-d3 concentration that the dilution of one step, which is mixed to get, and the concentration of MN-d3 is 1ng/mL.
3. dissociate the efficient of methoxyepinephrine and metanephrine in human plasma according to claim 1
Liquid Chromatography-Tandem Mass Spectrometry detection method, which is characterized in that the mixing that solution is formic acid and acetonitrile of redissolving in the step (4)
Object, volume ratio 1:1000.
4. dissociate the efficient of methoxyepinephrine and metanephrine in human plasma according to claim 1
Liquid Chromatography-Tandem Mass Spectrometry detection method, which is characterized in that the high performance liquid chromatography tandem mass spectrum of the step (4) is triple four
Grade bar mass spectrograph.
5. dissociate the efficient of methoxyepinephrine and metanephrine in human plasma according to claim 1
Liquid Chromatography-Tandem Mass Spectrometry detection method, which is characterized in that the step (4) is measured by high performance liquid chromatography tandem mass spectrometry
Sample solution after pretreatment, the separation condition for using gradient elution normal phase chromatography to establish determinand are as follows: with water-second
Nitrile-ammonium formate-formic acid is flow visualizing, and chromatographic column is Silica column, and flow velocity is 0.4~1.0mL/min, and column temperature is 30~45
DEG C, using multiple-reaction monitoring scan pattern (MRM).
6. dissociate the efficient of methoxyepinephrine and metanephrine in human plasma according to claim 5
Liquid Chromatography-Tandem Mass Spectrometry detection method, which is characterized in that the high performance liquid chromatography tandem mass spectrometry measurement is after pretreatment
The mobile phase that uses of sample solution be made of mobile phase A and Mobile phase B；Wherein mobile phase A is 0.1% formic acid of volume fraction-
30mM ammonium formate solution, Mobile phase B are 0.1% formic acid acetonitrile solution of volume fraction, and the volume ratio of mobile phase A and Mobile phase B is 5
~22%:95~78%.
7. dissociate the efficient of methoxyepinephrine and metanephrine in human plasma according to claim 5
Liquid Chromatography-Tandem Mass Spectrometry detection method, which is characterized in that the chromatographic column is Silica column, and packing material size is 1.7~5 μ
M, internal diameter are 2.1~4.6mm, and column length is 25~150mm.
8. dissociate in human plasma methoxyepinephrine and metanephrine according to claim 1 or 5
High performance liquid chromatography tandem mass spectrum detection method, which is characterized in that step (4) Mass Spectrometer Method uses electric spray ion source
(ESI) and MRM mode, parent ion/daughter ion detection ion pair of NMN and MN for detection are as follows: NMN, m/z
166.1 > m/z 134.1 and 166.1 > m/z of m/z 121.1；180.0 > m/z of 180.0 > m/z of MN, m/z 149.1 and m/z
120.9；The detection ion pair of internal standard NMN-d3 is 169.1 > m/z of m/z 137.1；The detection ion pair of internal standard MN-d3 is m/z
183.1>m/z 151.1。
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