CN108982569A - A kind of two dimensional NMR method of the inside and outside source property glucose of simultaneous quantitative blood plasma - Google Patents
A kind of two dimensional NMR method of the inside and outside source property glucose of simultaneous quantitative blood plasma Download PDFInfo
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Abstract
The present invention provides a kind of two dimensional NMR method of inside and outside source property glucose of simultaneous quantitative blood plasma, utilizes a small amount of D- [U-13C] glucose is as tracer and interior target isotope-dilution analysis, and in conjunction with the nuclear-magnetism list quantum related experiment (ID-HSQC) that adaptability is adapted, the corresponding bimodal and unimodal integral ratio of measurement calculates inside and outside source property concentration of glucose in blood plasma with this.The present invention has many advantages, such as that sample preparation simple (not needing the pre-treatment steps such as removing protein and derivatization), isotope labelling tracer dosage are few, concentration of glucose in inside and outside source in energy while Accurate Determining blood plasma.
Description
Technical field
The invention belongs to analytical chemistry methods fields, and in particular to one kind is based on stable isotope dilution method (isotope
Dilution, ID) combine two dimensional NMR technology for quickly, inside and outside source property concentration of glucose in Accurate Determining blood plasma
Quantitative approach.More specifically to one kind with D- [U-13C]-glucose as marker (tracer), adapts single quantum phase
Nuclear-magnetism experiment (HSQC) is closed to measure the method (ID-HSQC) of inside and outside source property concentration of glucose in blood plasma.
Background technique
Glucose is the important energy source substance of people and animal, and key player is play in energetic supersession.The monitoring of blood glucose
It is of great significance to the diagnosing and treating of a variety of diseases, the measurement of usual blood sugar concentration is the method using enzyme reaction automatic
Test is completed on analyzer.However this method can not distinguish inside and outside source property glucose in blood sample, and inside and outside source property grape
The assay of sugar is clinically to study glucose metabolism, the important parameter in the experiment such as insulin sensitivity.Currently used for
The method main method of these clinical trials has: gas chromatography-mass spectrography method (GC-MS), gas-chromatography-isotopic ratio
Mass spectrometry combination method (GC-IRMS), liquid chromatogram-isotopic ratio mass spectrometry combination method (LC-IRMS) and liquid chromatogram-string
Join mass spectrometry combination method (LC-MS/MS).But these methods need removing protein mostly, the cumbersome pre-treatment step such as derivatization, with
And the disadvantages of needing relatively large amount of isotope tracer.Therefore exploitation sample pre-treatments are simple, isotopic label dosage
The quantitative approach of inside and outside source property glucose will have important practical application value in less and energy Accurate Determining blood sample.
Summary of the invention
The purpose of the present invention is to provide one kind to be tied based on stable isotope dilution method (isotope dilution, ID)
Two dimensional NMR technology is closed for quick, inside and outside source property concentration of glucose in Accurate Determining blood plasma quantitative approach.More specifically
Ground is said, is related to one kind with D- [U-13C] for-glucose as marker (tracer), the single quantum correlation nuclear-magnetism of reorganization tests (HSQC)
To measure the method (ID-HSQC) of inside and outside source property concentration of glucose in blood plasma.The present invention utilizes the D- [U- of low concentration13C]-
Isotope-dilution analysis (Isotope Dilution) of the glucose (~0.035mM) as marker, in conjunction with single quantum of reorganization
Related nuclear-magnetism tests (HSQC), simply, accurately and rapidly measures inside and outside source property concentration of glucose in blood plasma simultaneously.
In order to realize above-mentioned purpose of the invention, the present invention provides the following technical solutions:
A kind of two dimensional NMR method of the inside and outside source property glucose of simultaneous quantitative blood plasma, utilizes a small amount of D- [U-13C] Portugal
Grape sugar is as tracer and interior target isotope-dilution analysis, the nuclear-magnetism list quantum related experiment (ID- adapted in conjunction with adaptability
HSQC), the corresponding bimodal and unimodal integral ratio of measurement, calculates inside and outside source property concentration of glucose in blood plasma with this.
A kind of two dimensional NMR method of the inside and outside source property glucose of simultaneous quantitative blood plasma, this method include the following steps:
Sample preparation: taking suitable blood plasma, and a small amount of deuterated reagent is added, is transferred to nuclear magnetic tube after mixing；
Nuclear-magnetism experiment: on the basis of conventional sensitivity enhances the pulse train of single quantum related experiment, by the 180 of carbon channel
It spends non-selective reunion pulse and is changed to No. 1 position of glucose13C1Chemical shift selectivity reunion pulse, to eliminate δ 1, δ 2 and with
Carbon carbon couples in 2 τ times afterwardsEvolution, that is, eliminate carbon carbon couplingTo the shadow of spectrogram phase
It rings；90-G is added in the incipient stage of pulse trainZUnit eliminates -13 longitudinal magnetization vector of carbon；Utilize the pulse train of reorganization
Twodimensional HSQC experiment data are acquired, data must be schemed through Sine-squared window function apodization, zero padding, Fourier transform, phasing
Spectrum, integral obtain the integrated value to induction signal；
Concentration calculation: the integrated value obtained using nuclear-magnetism experiment is calculated inside and outside source glucose by following formula and rubbed
That concentration proportion,
A kind of two dimensional NMR method of the inside and outside source property glucose of simultaneous quantitative blood plasma as mentioned, this method combine
Isotope-dilution analysis and HSQC nuclear-magnetism method have simultaneously carried out adaptability reorganization to it, and sample preparation procedure is simple, label grape
Sugared dosage is few, can the inside and outside source property concentration of glucose of Accurate Determining blood plasma simultaneously.
A kind of two dimensional NMR method of the inside and outside source property glucose of simultaneous quantitative blood plasma as mentioned, this method is in sample
It has used deuterated dimethyl sulfoxide as lock field reagent in the preparation process of product, choosing is utilized in the pulse train of nuclear-magnetism experiment
The reunion pulse of selecting property avoids phase twist.
A kind of two dimensional NMR method of the inside and outside source property glucose of simultaneous quantitative blood plasma as mentioned, sample preparation is only
It needs that a small amount of lock field reagent is added into test plasma, nuclear magnetic tube is transferred to after mixing, it is easy to operate.
A kind of two dimensional NMR method of the inside and outside source property glucose of simultaneous quantitative blood plasma as mentioned, this method use
Deuterated dimethyl sulfoxide avoids active hydrogen from exchanging as lock field reagent.
A kind of two dimensional NMR method of the inside and outside source property glucose of simultaneous quantitative blood plasma as mentioned, this method is to normal
The HSQC pulse train of rule carries out adaptability reorganization, uses No. 1 position of glucose13C chemical shift selectivity reunion pulse is eliminated
Same core13C1-13C2One key couples the influence to map phase, to guarantee the accuracy of map integral and quantitative analysis.
Method of the invention can be described more specifically as:
Sample preparation: taking suitable blood plasma (such as 480uL), and a small amount of deuterated reagent is added, and (nuclear-magnetism experiment lock field is used, such as 20uL
DMSO-d6), nuclear magnetic tube is transferred to after mixing.
Nuclear-magnetism experiment: quantitative detection while in order to realize -13 all mark glucose of natural abundance and carbon, conventional sensitive
On the basis of degree enhances the pulse train of single quantum related experiment, the non-selective reunion pulse of the 180 degree in carbon channel is changed to grape
Sugared No. 1 position13C1Chemical shift selectivity reunion pulse (Fig. 1, the pulse of grey sinusoidal waveform), so that δ 1 is eliminated, δ 2 and subsequent 2 τ
Carbon carbon couples in timeEvolution, that is, eliminate carbon carbon couplingInfluence to spectrogram phase.Together
When in order to eliminate influence of -13 longitudinal magnetization vector of equilibrium state carbon to quantitative result, pulse train incipient stage be added 90-
GZUnit eliminates -13 longitudinal magnetization vector of carbon.Twodimensional HSQC experiment data, data are acquired using the pulse train (Fig. 1) of reorganization
Map, integral acquisition pair are obtained after the processing steps such as Sine-squared window function apodization, zero padding, Fourier transform, phasing
The integrated value of induction signal (Fig. 2).
Concentration calculation: the integrated value obtained using nuclear-magnetism experiment can calculate inside and outside source grape by following formula
Sugared molar concentration ratio.
Compared with the prior art, the advantages of the present invention are as follows: (1) sample preparation simple, it is only necessary to take straight after appropriate blood plasma
Connecing a small amount of deuterated reagent of addition can be detected, and the most of existing method needs removing protein and derivatization etc. cumbersome
Pre-treatment step.(2) there is biggish isotopic dilution ratio (~1% is horizontal), the tracer dosage of isotope labelling is few, thus
Experimental cost can be reduced.It is different from general isotope-dilution analysis, it is general to require in order to obtain accurate quantitative result
The signal strength of the tracer and measured object that detect is suitable, thus the dosage of the tracer of the isotope labelling needed is larger.
(3) present invention tests (HSQC) using single quantum correlation nuclear-magnetism of reorganization, has used No. 1 position of glucose13C1Chemical shift selection
Property reunion pulse (refocusing pulses), eliminate one key of same core coupling (13C1-13C2) influence to map phase, with
Improve the accuracy of map integral and quantitative analysis.
Detailed description of the invention
Sensitivity of the Fig. 1 for the reorganization of inside and outside source property glucose quantitation enhances HSQC pulse sequence diagram.
Fig. 2 blood plasma ID-HSQC map (No. 1 group signal area partial enlarged view of α-D- glucopyranose).A) three
Dimension figure；B) contour map, unimodal centre is α-D- glucopyranose1H-13C1(-12C2) group signal, the bimodal of both sides be
D- glucopyranose1H-13C1(-13C2) group signal.
Fig. 3 standard curve.Abscissa is the theoretical molar concentration proportion of standard specimen, and ordinate is by testing mole measured
Concentration proportion.
Two dimensional NMR method (ID-HSQC) Technology Roadmap of the inside and outside source property glucose of Fig. 4 simultaneous quantitative blood plasma.
Specific embodiment
With reference to the accompanying drawing, essentiality content of the invention is further illustrated with the embodiment of the present invention, but not with
This limits the present invention.
The data acquisition and processing (DAP) of the embodiment of the present invention carries out by the following method:
All nuclear-magnetism experiments are in the Brooker AVANCE III 800 of configuration QCI-P cryogenic probe involved in the invention
It is carried out on nmr spectrometer.Pulse train such as Fig. 1 manipulates software TopSpin3.2.Data processing is carried out using TopSpin 3.5.
Measurement result is verified using the LC-MS/MS method developed before this study group.LC-MS data processing uses
DataAnalysis 4.1(Bruker)。
Embodiment 1:
In order to confirm that the present invention is first using the accuracy of the method for the present invention measurement inside and outside source property concentration of glucose of blood plasma
Prepare the D-Glucose and D- [U- of a series of various concentration ratios13C] glucose mixture standard sample, investigate the inventive method
Quantitative dynamic range, accuracy and accuracy.
90.6mg natural glucose and 95.4mg D- [U- are weighed respectively13C] glucose in 5mL volumetric flask, adds water fixed
Hold, obtaining mother liquid concentration is respectively 100.4mM and 99.5mM.Take the D- [U- of 20uL99.5mM13C] glucose mother liquid, add water dilute
It releases to 2mL, obtains the D- [U- that concentration is 1mM13C] glucose solution.
20 μ L, 40 μ L, 70 μ L, 100 μ L, 140 μ L, 170 μ L, 200 μ L99.9mM natural glucoses are taken with liquid-transfering gun respectively
Mother liquor is managed in the EP of 2mL, and 35 μ L1mM D- [U- of equivalent is added in each EP pipe13C] glucose solution, add suitable water and 500 μ
L DMSO-d6Be diluted to 1mL, be vortexed mix, abundant balance, be prepared natural glucose (concentration is respectively 2,4,7,10,
14,17,20mM) and D- [U-13C] glucose (0.035mM) various concentration ratio mixed standard solution.Successively 480uL is taken to mark
Quasi- solution is managed in the EP of 1.5mL, and 20 μ L DMSO-d are added6, it is vortexed and mixes, the solution of mixing is transferred to the nuclear magnetic tube of 5mm,
Retest is three times in different times for every sample.
Experimental data is handled using 3.5 software of TopSpin, is integrated, using the integrated value of acquisition according to concentration
Natural glucose and D- [U- is calculated in calculation formula (1)13C] glucose molar concentration ratio, and with theoretical mole it is dense
Degree ratio is compared (table 1).All sample test relative standard deviation (relative standard deviation,
RSD it) is respectively less than 9%, relative error (relative error, RE) is respectively less than 3%, and the method for the present invention has good as the result is shown
Accuracy and accuracy (Fig. 2).
The quantitative performance of 1 ID-HSQC method of table measurement standard sample
aAverage value ± standard deviation, n=3
Embodiment 2:
Accuracy: accuracy and accuracy in order to investigate glucose in measurement blood plasma take monkey plasma sample, are added dropwise
[the U- of 0.035mM13C] glucose, 20 μ L DMSO-d are added6, it is vortexed and mixes, the sample after mixing is transferred to the nuclear-magnetism of 5mm
Pipe.It is tested 8 times in three days, relative standard deviation 4.02%.
The rate of recovery:
Sample preparation: four parts of identical monkey plasma samples, wherein three parts of samples are separately added into 2mM, 4mM, 6mM (are to add
Concentration after entering) D-Glucose, another be not added D-Glucose as control (matrix).Then 20 μ L are added in every part of sample
DMSO-d6, it is vortexed and mixes, the sample after mixing is transferred to the nuclear magnetic tube of 5mm.Every ocean NMR test is three times.By formula (2)
It calculates the rate of recovery (table 2).
C in formulaN,m(addition), CN,m(matrix) is the concentration of glucose measured being added dropwise after D-Glucose and before, CN
(dropwise addition) is the concentration of glucose being added.
2 sample recovery rate experimental result of table
aAverage value ± standard deviation, n=3
Embodiment 3:
Healthy monkey and type-2 diabetes mellitus model monkey plasma sample are measured (with D- [U-13C] glucose is as exogenous glucose
Tracer clamp experiment plasma sample) in inside and outside source property glucose concentration:
Nuclear-magnetism sample preparation steps:
It takes 480uL blood plasma to manage in the EP of 1.5mL, 20 μ L DMSO-d is added6, it is vortexed and mixes, the sample after mixing is shifted
Nuclear magnetic tube to 5mm is tested for nuclear-magnetism.
LC-MS confirmatory experiment sample treatment and preparation process comprising the following three steps:
(1) plasma sample protein precipitation is handled: take 100 μ L blood plasma, be added 400 μ L acetonitriles, vortex 30s, at 4 DEG C with
12000 × g is centrifuged 10min, takes supernatant liquor, is rotarily dried using traditional vacuum concentrating instrument, then is again molten with 100 μ L ultrapure waters
Solution.
(2) it derivative reaction: takes 20 μ L or more to pass through the blood sample of protein precipitation processing, adds 100 μ L mixing derivatization reagents
(configuration process: weighing 0.385g Butesin, 0.625g sodium cyanoborohydride is in 4mL methanol), in 80 DEG C of water-baths
1h is reacted in pot, takes out, is cooled to room temperature.
(3) purification process: purify the mixing sample that derivative reaction obtains with the solid phase extraction column of C18 filler, solid phase
After extracting pillar activation, balance, sample is transferred to solid phase extraction column, is washed twice with 1ml acetonitrile-water (5:95, V/V), then
It is eluted with 1ml acetonitrile-water (30:70, V/V), eluent is collected in sample introduction bottle, carries out LC-MS/MS experiment.
Test result (table 3) shows that ID-HSQC method is consistent with LC-MS/MS methods and results, therefore ID-HSQC method can
With the inside and outside source glucose assays for plasma sample.
Quantitative healthy monkey (1~6) and type-2 diabetes mellitus model monkey (7~12) the blood plasma sample of table 3 ID-HSQC and LC-MS/MS
Interior exogenous glucose (C in productendo/Cexo) result comparison
aIn blood plasma the concentration range of glucose be 3~5.5mM (b) or 10~15mM (c)
Embodiment 4:
In order to further evaluate ID-HSQC in the performance (accuracy and precision) of measurement plasma sample.Three parts identical
Suffer from diabetes monkey plasma sample (no D- [U-13C] glucose tracer): wherein 0.5mM, 10mM are added dropwise respectively (after dropwise addition for two parts
Final concentration in blood sample selects the two concentration to be to make to be respectively adopted two kinds of detection methods of nuclear magnetic resonance and LC-MS
The signal strength of the natural glucose and labelled glucose that detect is suitable, to improve the accuracy of measurement result) D- [U-13C] glucose is as reference sample.D- [the U- of 0.035mM is added dropwise in another plasma sample13C] glucose is (in blood after dropwise addition
Final concentration in sample) be used as test sample, to investigate the method for the present invention ID-HSQC test plasma sample quantitative accuracy and
Accuracy.The step of with embodiment 2, reference sample is detected with ID-HSQC and LC-MS/MS method respectively, two kinds of detection methods obtain
The average value for obtaining result regards " theoretical concentration " of blood glucose as.It takes 480uL test sample to be placed in the EP pipe of 1.5mL, 20 μ L is added
DMSO-d6, it is vortexed and mixes, the effective the method for the present invention ID-HSQC of nuclear-magnetism that the solution of mixing is transferred to 5mm is tested into (weight
Repetition measurement tries three times).Table 4 shows that the relative error (RE) of ID-HSQC measurement is 2.1%, and relative standard deviation (RSD) is 5.2%.
4 ID-HSQC method of table is directed to the glucose quantitation performance of true blood sample
Claims (7)
1. a kind of two dimensional NMR method of the inside and outside source property glucose of simultaneous quantitative blood plasma, utilizes a small amount of D- [U-13C] grape
Sugar is used as tracer and interior target isotope-dilution analysis, in conjunction with the nuclear-magnetism list quantum related experiment ID-HSQC that adaptability is adapted, surveys
Fixed corresponding bimodal and unimodal integral ratio, calculates inside and outside source property concentration of glucose in blood plasma with this.
2. a kind of two dimensional NMR method of the inside and outside source property glucose of simultaneous quantitative blood plasma, this method include the following steps:
Sample preparation: taking suitable blood plasma, and a small amount of deuterated reagent is added, is transferred to nuclear magnetic tube after mixing；
Nuclear-magnetism experiment: on the basis of conventional sensitivity enhances the pulse train of single quantum related experiment, the 180 degree in carbon channel is non-
Selective reunion pulse is changed to No. 1 position of glucose13C1Chemical shift selectivity reunion pulse, so that δ 1 is eliminated, δ 2 and subsequent 2 τ
Carbon carbon couples in timeEvolution, that is, eliminate carbon carbon couplingInfluence to spectrogram phase；?
90-G is added in the incipient stage of pulse trainZUnit eliminates -13 longitudinal magnetization vector of carbon；Utilize the pulse sequence acquisition two of reorganization
HSQC experimental data is tieed up, data obtain map through Sine-squared window function apodization, zero padding, Fourier transform, phasing, integrate
Obtain the integrated value to induction signal；
Concentration calculation: it is dense to calculate inside and outside source glucose mole by following formula for the integrated value obtained using nuclear-magnetism experiment
Ratio is spent,
3. a kind of two dimensional NMR method of the inside and outside source property glucose of simultaneous quantitative blood plasma as claimed in claim 1 or 2,
It is characterized in that this method combines isotope-dilution analysis and HSQC nuclear-magnetism method and has carried out adaptability reorganization, sample system to it
Standby process is simple, labelled glucose dosage is few, can the inside and outside source property concentration of glucose of Accurate Determining blood plasma simultaneously.
4. a kind of two dimensional NMR method of the inside and outside source property glucose of simultaneous quantitative blood plasma as claimed in claim 1 or 2,
It is characterized in that this method has used deuterated dimethyl sulfoxide as lock field reagent in the preparation process of sample, tested in nuclear-magnetism
Pulse train in selective reunion pulse be utilized avoid phase twist.
5. a kind of two dimensional NMR method of the inside and outside source property glucose of simultaneous quantitative blood plasma as claimed in claim 1 or 2,
It is characterized in that a small amount of lock field reagent need to be only added in sample preparation into test plasma, it is transferred to nuclear magnetic tube after mixing, operates
Simply.
6. a kind of two dimensional NMR method of the inside and outside source property glucose of simultaneous quantitative blood plasma as claimed in claim 1 or 2,
It is characterized in that the method use deuterated dimethyl sulfoxides to be used as lock field reagent that active hydrogen is avoided to exchange.
7. a kind of two dimensional NMR method of the inside and outside source property glucose of simultaneous quantitative blood plasma as claimed in claim 1 or 2,
It is characterized in that this method carries out adaptability reorganization to conventional HSQC pulse train, No. 1 position of glucose is used13C chemical potential
Selective reunion pulse is moved, same core is eliminated13C1-13C2One key couples influence to map phase, thus guarantee map integral and
The accuracy of quantitative analysis.
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| WO2014161960A1 (en) * | 2013-04-03 | 2014-10-09 | Asociación Centro De Investigación Cooperativa En Biomateriales | Synthesis and use of isotopically-labelled glycans |
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| JPH1171431A (en) * | 1996-08-16 | 1999-03-16 | Idemitsu Petrochem Co Ltd | Polypropylene-based resin and polypropylene-based resin composition |
| WO2002004945A1 (en) * | 1999-03-26 | 2002-01-17 | Neo Gen Screening, Inc. | Clinical method for the genetic screening of newborns using tandem mass spectrometry |
| CN101730842A (en) * | 2007-07-23 | 2010-06-09 | F·霍夫曼-拉·罗奇股份有限公司 | Method by the magnetic resonance detection target substance |
| CN101910841A (en) * | 2007-10-29 | 2010-12-08 | 佐治亚大学研究基金会 | The interior isotope labeling method of body that is used for quantitatively sugar group |
| CN103105444A (en) * | 2013-01-25 | 2013-05-15 | 贵州同济堂制药有限公司 | Method for detecting quality of Xianing Gubao capsules |
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Application publication date: 20181211 |