CN102435474A - Method for preparing sample for urine metabolome nuclear magnetic resonance analysis - Google Patents
Method for preparing sample for urine metabolome nuclear magnetic resonance analysis Download PDFInfo
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Abstract
The invention which discloses a method for preparing a sample for the urine metabolome nuclear magnetic resonance analysis relates to the field of the life science/the analytical chemistry, and concretely relates to the urine metabolome nuclear magnetic resonance analysis. According to the invention, a potassium fluoride solution is added to urine in the sample preparation process to avoid the complexation effect among calcium ions, magnesium ions and metabolites. According to figures shown in the specification, citric acid proton chemical shift differences among samples are substantially reduced, the chemical shift deviation of more metabolite protons is reduced, a lowest field signal of citric acid moves to the high field, the overlap between the lowest field signal of citric acid and a dimethylamine signal is eliminated, and originally covered metabolite signals are acquired in the invention, so the signal consistency is improved and the datum analysis error rate is reduced; and the method has the advantages of simple preparation, convenient operation and low price.
Description
Technical field
The present invention relates to life science/analytical chemistry field, more specifically relate to urine metabolism group nuclear magnetic resonance spectroscopy.
Background technology
Metabolism group is about the integral body of biosome endogenous metabolism material and the science of Changing Pattern thereof; Metabonomic technology based on nuclear magnetic resonance has been applied to many aspects (Tang Huiru etc. such as basic life science, medicament research and development, disease physiology, nutrition and pharmacy,galenic, environmental science; Life science 2007; 19,272-280).Metabolism group research flow process comprises that experimental design, sample collecting and preparation, nmr data acquisition and pre-service, data analysis and metabolin change the explanation of biological significance.Yet; Because the sample of being analyzed is formed complicated; And exist and interact; Make the chemical shift of some metabolin between the different samples have difference in various degree, this can have a negative impact to the explanation of follow-up data analysis and biological significance, and the chemical shift difference of citric acid is exactly a typical example in the urine.This species diversity mainly is (the Cloarec that the difference by the difference of sample room pH value and other environmental factors (phenomenons such as concentration of metal ions, metabolin-combination of proteins and chemical exchange) causes; O. etc.; Analytical Chemistry 2005,77 (2), 517-526).
Existing bibliographical information reduce the method that pH value and ionic strength cause chemical shift difference.These methods adopt wide sectional integration method, spectrum peak align data facture when being included in data analysis, and in the sample preparation, regulate three types of pH values etc. with buffer solution.
Aspect data processing, method commonly used is with the integral breadth of broad the spectrum peak to be carried out integration (Spraul, M. etc.; Journal of Pharmaceutical and Biomedical Analysis 1994,12 (10), 1215-1225); This method can be covered the difference of spectrum peak position largely, but can lose resolution, but also can be incorporated in the same integral breadth by the signal that the variation of different metabolic thing is opposite; The result has covered the variation of metabolin and the contribution of low concentration metabolin, and (Analyst 2009 for Xiao, C. N. etc.; 134 (5), 916-925).Another kind of way is to use automatic spectrum peak correcting algorithm to come difference (Forshed, J. etc., the Analytica Chimica Acta 2003 of calibration spectrum peak position; 487 (2), 189-199), thereby can obtain the data of full resolution; But seriously intersect and bigger Signal Processing still undesirable (Lauridsen, M. etc., Analytical Chemistry 2007 are moved in chemical shift for signal, the chemical shift of new signal, disappearance; 79 (3), 1181-1186).
Therefore, best bet is that (specimen preparation and data acquisition period) eliminated these inconsistencies (Analyst 2009,134 (5), 916-925) for Xiao, C. N. etc. from the source.
People such as Xiao Chaoni adopt a kind of dipotassium hydrogen phosphate/phosphate sodium dihydrogen buffer solution (Xiao in the sample preparation of urine metabolism group nuclear magnetic resonance spectroscopy; C. N. etc.; Analyst 2009,134 (5), 916-925); Normal person's urine pH value is controlled in the 7.1-7.7 scope effectively, and most of chemical shift deviation that has the metabolin of ionogen is controlled in the 0.002ppm; But the proton chemical shifts deviation on citric acid proton and the histidine ring is still bigger, needs to proofread and correct.For the urine of rat, citric acid proton chemical shifts deviation is bigger.Therefore, the high offset issue of citric acid chemical shift of proton needs to be resolved hurrily.
The bigger reason of citric acid chemical shift of proton deviation is except that the difference of pH value; The complexing of the calcium in the urine, magnesium ion and citric acid also is a key factor (Potts; B. C. M. etc.; Journal of Pharmaceutical and Biomedical Analysis 2001,26 (3), 463-476).
When collector's urine, add a certain amount of sodium fluoride, can cause on the nmr spectrum chemical shift of citric acid to be moved to High-Field.Metallic ion in the urine (as: calcium, magnesium ion) can and chemical compound lot (particularly citric acid) form complex compound; Make calcium, magnesium ion in the urine form lower calcium fluoride and the magnesium fluoride of solubleness through adding sodium fluoride, thereby eliminated the complexing of calcium, magnesium ion and citric acid, make the Citrated displacement study (Lauridsen that is moved; M. etc.; Analytical Chemistry 2007,79 (3), 1181-1186).
In addition; Hydrofluoride is a growth of microorganism suppressant commonly used in legal medical expert's sample, in urine, add a certain amount of sodium fluoride and can eliminate the fermentation of microorganism effectively, thereby hydrofluoride is the synthetic (Lough that has stoped polysaccharide through the activity that suppresses phosphoglucomutase; P. S. etc.; Journal of Forensic Sciences 1993,38 (2), 266-271).
Summary of the invention
The objective of the invention is: a kind of sample compound method that is used for urine metabolism group nuclear magnetic resonance spectroscopy is provided.This method is through adding potassium fluoride solution in the sample process for preparation; Utilize the combination of fluorine ion and calcium, magnesium ion; Not only reduced the chemical shift of proton difference of sample room metabolin; The chemical shift deviation control of the specific metabolite that will cause because of individual difference in more among a small circle, improved signal consistance, reduced the fault rate of data analysis; And has simple, easy to operate, the cheap advantage of preparation.
For realizing above-mentioned purpose, the present invention has adopted following technical scheme:
The present invention is through adding potassium fluoride solution in urine in the sample process for preparation; Utilize the combination of fluorine ion and calcium, magnesium ion; Form the less salt of solubleness; Form in centrifugal back with deposition is removed, and has avoided the complexing between calcium, magnesium ion and metabolin, thereby has reduced the chemical shift of proton difference of sample room metabolin.
A kind of sample compound method that is used for urine metabolism group nuclear magnetic resonance spectroscopy, this method comprises the following step:
1, at the human of per 100 microlitres or test that to add concentration in the rodentine urine be that 0.1 mol ~ 16 mol, fluorine ion total amount are the micromolar potassium fluoride solutions in 5 micromoles ~ 50, the vortex oscillation mixing left standstill 5 ~ 50 minutes;
2, under 2 ° of C ~ 30 ° C, centrifugal, get supernatant;
3, in the supernatant of per 100 microlitres, adding 10 microlitre concentration is the urine damping fluid of 1.5 mol;
4, under 2 ° of C ~ 30 ° C, centrifugal, get supernatant, this supernatant is the sample that is used for urine metabolism group nuclear magnetic resonance spectroscopy.
Above-mentioned urine damping fluid contains for every liter: 1.2 mole of phosphoric acid hydrogen dipotassiums, and 0.3 mole of phosphoric acid sodium dihydrogen, 1 gram Sodium azide, 0.5 gram 2,2,3,3-d (4)-3-(trimethyl silicon based) propionic acid sodium salt the rest is heavy water.
Conventional sample compound method comprises the following step:
1, in the mankind of per 100 microlitres or rat urine, adding 10 microlitre concentration is the urine damping fluid of 1.5 mol;
2, under 2 ° of C ~ 30 ° C, centrifugal, get supernatant, this supernatant is the sample that is used for urine metabolism group nuclear magnetic resonance spectroscopy.
Above-mentioned urine damping fluid contains for every liter: 1.2 mole of phosphoric acid hydrogen dipotassiums, and 0.3 mole of phosphoric acid sodium dihydrogen, 1 gram Sodium azide, 0.5 gram 2,2,3,3-d (4)-3-(trimethyl silicon based) propionic acid sodium salt the rest is heavy water.
Advantage of the present invention is: the sample that adopts the inventive method preparation; Citric acid chemical shift of proton significant difference between the sample reduces; The chemical shift of proton deviation of more metabolin reduces, and the minimum field signal of citric acid moves to High-Field, makes the overlapping elimination of minimum field signal of citric acid and dimethylamine signal; Original therebetween concealed metabolite signals is obtained, avoided the adverse effect that to introduce in the follow-up data analytical work.The present invention also has preparation advantage such as simple, easy to operate, cheap.Through the addition of adjustment potassium fluoride solution, the present invention promptly extends to metabolism group nuclear magnetic resonance spectroscopy human and experiment rodent body fluid.
Description of drawings
Fig. 1. rat urine compares synoptic diagram with the final pH value of the inventive method sample of preparing and the sample of preparing with conventional compound method.
Fig. 2. rat urine compares synoptic diagram with the nmr spectrum of the inventive method sample of preparing and the sample of preparing with conventional compound method.
Fig. 3. human urine compares synoptic diagram with the final pH value of the inventive method sample of preparing and the sample of preparing with conventional compound method.
Fig. 4. human urine compares synoptic diagram with the nmr spectrum of the inventive method sample of preparing and the sample of preparing with conventional compound method.
Embodiment
Below in conjunction with accompanying drawing, the present invention is further described.
Embodiment 1:
A kind of sample compound method that is used for urine metabolism group nuclear magnetic resonance spectroscopy, this method comprises the following step:
1, at the human of per 100 microlitres or test that to add 1 microlitre ~ 10 microlitre concentration in the rodentine urine be the potassium fluoride solution of 5 mol, the vortex oscillation mixing left standstill 5 ~ 50 minutes;
2, under 2 ° of C ~ 30 ° C, centrifugal, get supernatant;
3, in the supernatant of per 100 microlitres, adding 10 microlitre concentration is the urine damping fluid of 1.5 mol;
4, under 2 ° of C ~ 30 ° C, centrifugal, get supernatant, this supernatant is the sample that is used for urine metabolism group nuclear magnetic resonance spectroscopy.
Above-mentioned urine damping fluid contains for every liter: 1.2 mole of phosphoric acid hydrogen dipotassiums, and 0.3 mole of phosphoric acid sodium dihydrogen, 1 gram Sodium azide, 0.5 gram 2,2,3,3-d (4)-3-(trimethyl silicon based) propionic acid sodium salt the rest is heavy water.
1 microlitre that in the urine of per 100 microlitres, is added in the above-mentioned steps 1 ~ 10 microlitre concentration are the potassium fluoride solution of 5 mol; Can use concentration is that 0.1 mol ~ 1 mol, fluorine ion total amount are that the micromolar Fluorinse in 5 micromoles ~ 50 substitutes, and can also use concentration is that 0.1 mol ~ 12 mol, fluorine ion total amount are that the micromolar ammonium fluoride solution in 5 micromoles ~ 50 substitutes.
When adopting rat urine in the above-mentioned steps 1, adding 1 microlitre ~ 10 microlitre concentration in the rat urine of per 100 microlitres is the potassium fluoride solution of 5 mol, and preferably adding 3.6 microlitre concentration is the potassium fluoride solution of 5 mol.
When adopting human urine in the above-mentioned steps 1, adding 1 microlitre ~ 10 microlitre concentration in the human urine of per 100 microlitres is the potassium fluoride solution of 5 mol, and preferably adding 2.7 microlitre concentration is the potassium fluoride solution of 5 mol.
Urine damping fluid described in the above-mentioned steps 3 can adopt the urine sample damping fluid of other kind, like sodium dihydrogen phosphate/sodium hydrogen phosphate damping fluid.
When adopting rat urine, rat urine volume: the potassium fluoride solution volume of 5 mol: the urine damping fluid volume=100:1 ~ 10:10 of 1.5 mol, best proportioning is 100:3.6:10.
When adopting human urine, human volume of urine: the potassium fluoride solution volume of 5 mol: the urine damping fluid volume=100:1 ~ 10:10 of 1.5 mol, best proportioning is 100:2.7:10.
Embodiment 2:
A kind of sample compound method that is used for rat urine metabolism group nuclear magnetic resonance spectroscopy, this method comprises the following step:
1, in the rat urine of per 100 microlitres, add 1 microlitre ~ 10 microlitres, preferably add 3.6 microlitres, concentration is the potassium fluoride solution of 5 mol, and the vortex oscillation mixing left standstill 5 ~ 50 minutes;
2, under 2 ° of C ~ 30 ° C, centrifugal, get supernatant;
3, in the supernatant of per 100 microlitres, adding 10 microlitre concentration is the urine damping fluid of 1.5 mol;
4, under 2 ° of C ~ 30 ° C, centrifugal, get supernatant, this supernatant is the sample that is used for rat urine metabolism group nuclear magnetic resonance spectroscopy.
Above-mentioned urine damping fluid contains for every liter: 1.2 mole of phosphoric acid hydrogen dipotassiums, and 0.3 mole of phosphoric acid sodium dihydrogen, 1 gram Sodium azide, 0.5 gram 2,2,3,3-d (4)-3-(trimethyl silicon based) propionic acid sodium salt the rest is heavy water.
Can know by Fig. 1, get the urine of 10 portions of rats, numbering R1-R10, the difference of using conventional compound method to prepare between the final pH value of the sample that obtains is 0.35; Difference with between the final pH value of the sample of the inventive method preparation is decreased to 0.15, and the final pH value of most of samples is more near 7.40.Reducing that the pH value difference is different is owing to before adding phosphate buffer, calcium, magnesium ion are removed, and avoided the phosphate in calcium, magnesium ion and the damping fluid to form deposition, thereby guaranteed the surge capability of damping fluid.
Can know by Fig. 2; Get the urine of 10 portions of rats, numbering R1-R10, compare than the nmr spectrum (2.40ppm-2.74ppm) of the sample of preparing with conventional compound method with the sample of the inventive method preparation: the chemical shift difference of citric acid is decreased in the 2.5Hz; The chemical shift deviation of more metabolin reduces; The minimum field signal of citric acid moves about 12Hz to High-Field, eliminated and the dimethylamine signal between overlapping, and original concealed metabolite signals is obtained therebetween; Reduced the adverse effect that to introduce in the follow-up multivariate data analysis work.The chemical shift difference of citric acid reduces, and this is that the form in centrifugal back with deposition is removed, and has eliminated the complexing of calcium, magnesium ion and citric acid because fluorine ion ability and calcium, magnesium ion form the salt more stable, that solubleness is less on the one hand; In addition, owing to before adding phosphate buffer, calcium, magnesium ion are removed, avoided the phosphate in calcium, magnesium ion and the damping fluid to form deposition, thereby guaranteed the surge capability of damping fluid.
To sum up; Adopt 10 parts of rat urine samples of present embodiment preparation, different the reducing of pH value difference between the sample, citric acid chemical shift of proton significant difference reduces; The chemical shift of proton deviation of more metabolin reduces; The minimum field signal of citric acid moves to High-Field, makes the overlapping elimination of minimum field signal of citric acid and dimethylamine signal to be obtained by original concealed metabolite signals therebetween.In addition, the adding of potassium fluoride solution also can be played the effect of certain antisepsis and sterilization, can prolong the holding time of urine to a certain extent.
Embodiment 3:
A kind of sample compound method that is used for human urine metabolism group nuclear magnetic resonance spectroscopy, this method comprises the following step:
1, in the human urine of per 100 microlitres, add 1 microlitre ~ 10 microlitres, preferably add 2.7 microlitres, concentration is the potassium fluoride solution of 5 mol, and the vortex oscillation mixing left standstill 5 ~ 50 minutes;
2, under 2 ° of C ~ 30 ° C, centrifugal, get supernatant;
3, in the supernatant of per 100 microlitres, adding 10 microlitre concentration is the urine damping fluid of 1.5 mol;
4, under 2 ° of C ~ 30 ° C, centrifugal, get supernatant, this supernatant is the sample that is used for human urine metabolism group nuclear magnetic resonance spectroscopy.
Above-mentioned urine damping fluid contains for every liter: 1.2 mole of phosphoric acid hydrogen dipotassiums, and 0.3 mole of phosphoric acid sodium dihydrogen, 1 gram Sodium azide, 0.5 gram 2,2,3,3-d (4)-3-(trimethyl silicon based) propionic acid sodium salt the rest is heavy water.
Can know by Fig. 3, get 9 parts of human urines, numbering H1-H9, the difference of using conventional compound method to prepare between the final pH value of the sample that obtains is 0.33; Difference with between the final pH value of the sample of the inventive method preparation is decreased to 0.30.
Can know by Fig. 4; Get 9 parts of human urines; Numbering H1-H9, the nmr spectrum (2.50ppm-2.74ppm) of the sample of preparing with the sample of the inventive method preparation with conventional compound method is compared: the chemical shift difference of citric acid reduces, and the chemical shift deviation of more metabolin reduces; The minimum field signal of citric acid moves to High-Field; Eliminated and the dimethylamine signal between overlapping, and original concealed metabolite signals is obtained therebetween, reduced the adverse effect that possibly introduce in the follow-up multivariate data analysis work.
To sum up; Adopt 9 parts of human urine samples of present embodiment preparation, different the reducing of pH value difference between the sample, citric acid chemical shift of proton significant difference reduces; The chemical shift of proton deviation of more metabolin reduces; The minimum field signal of citric acid moves to High-Field, makes the overlapping elimination of minimum field signal of citric acid and dimethylamine signal to be obtained by original concealed metabolite signals therebetween.In addition, the adding of potassium fluoride solution also can be played the effect of certain antisepsis and sterilization, can prolong the holding time of urine to a certain extent.
Claims (13)
1. sample compound method that is used for urine metabolism group nuclear magnetic resonance spectroscopy is characterized in that this method comprises the following step:
A, at the human of per 100 microlitres or test that to add concentration in the rodentine urine be that 0.1 mol ~ 16 mol, fluorine ion total amount are the micromolar potassium fluoride solutions in 5 micromoles ~ 50, the vortex oscillation mixing left standstill 5 ~ 50 minutes;
B, under 2 ° of C ~ 30 ° C, centrifugal, get supernatant;
C, in the supernatant of per 100 microlitres, to add 10 microlitre concentration be the urine damping fluid of 1.5 mol;
D, under 2 ° of C ~ 30 ° C, centrifugal, get supernatant, this supernatant is the sample that is used for urine metabolism group nuclear magnetic resonance spectroscopy;
Urine damping fluid among the step c contains for every liter: 1.2 mole of phosphoric acid hydrogen dipotassiums, and 0.3 mole of phosphoric acid sodium dihydrogen, 1 gram Sodium azide, 0.5 gram 2,2,3,3-d (4)-3-(trimethyl silicon based) propionic acid sodium salt the rest is heavy water.
2. a kind of sample compound method that is used for urine metabolism group nuclear magnetic resonance spectroscopy according to claim 1 is characterized in that, in the urine of per 100 microlitres, adding 1 microlitre ~ 10 microlitre concentration among the step a is the potassium fluoride solution of 5 mol.
3. a kind of sample compound method that is used for urine metabolism group nuclear magnetic resonance spectroscopy according to claim 1; It is characterized in that it is that 0.1 mol ~ 1 mol, fluorine ion total amount are the micromolar Fluorinses in 5 micromoles ~ 50 that potassium fluoride solution described in the step a adopts concentration.
4. a kind of sample compound method that is used for urine metabolism group nuclear magnetic resonance spectroscopy according to claim 1; It is characterized in that it is that 0.1 mol ~ 12 mol, fluorine ion total amount are the micromolar ammonium fluoride solutions in 5 micromoles ~ 50 that potassium fluoride solution described in the step a adopts concentration.
5. a kind of sample compound method that is used for urine metabolism group nuclear magnetic resonance spectroscopy according to claim 1; It is characterized in that; When adopting rat urine among the step a, adding 1 microlitre ~ 10 microlitre concentration in the rat urine of per 100 microlitres is the potassium fluoride solution of 5 mol.
6. a kind of sample compound method that is used for urine metabolism group nuclear magnetic resonance spectroscopy according to claim 1 is characterized in that, when adopting rat urine among the step a, adding 3.6 microlitre concentration in the rat urine of per 100 microlitres is the potassium fluoride solution of 5 mol.
7. a kind of sample compound method that is used for urine metabolism group nuclear magnetic resonance spectroscopy according to claim 1; It is characterized in that; When adopting human urine among the step a, adding 1 microlitre ~ 10 microlitre concentration in the human urine of per 100 microlitres is the potassium fluoride solution of 5 mol.
8. a kind of sample compound method that is used for urine metabolism group nuclear magnetic resonance spectroscopy according to claim 1 is characterized in that, when adopting human urine among the step a, adding 2.7 microlitre concentration in the human urine of per 100 microlitres is the potassium fluoride solution of 5 mol.
9. a kind of sample compound method that is used for urine metabolism group nuclear magnetic resonance spectroscopy according to claim 1 is characterized in that, the urine damping fluid described in the step c adopts sodium dihydrogen phosphate/sodium hydrogen phosphate damping fluid.
10. a kind of sample compound method that is used for urine metabolism group nuclear magnetic resonance spectroscopy according to claim 1; It is characterized in that; When adopting rat urine, rat urine volume: the potassium fluoride solution volume of 5 mol: the urine damping fluid volume=100:1 ~ 10:10 of 1.5 mol.
11. a kind of sample compound method that is used for urine metabolism group nuclear magnetic resonance spectroscopy according to claim 1; It is characterized in that; When adopting rat urine, rat urine volume: the potassium fluoride solution volume of 5 mol: the urine damping fluid volume=100:3.6:10 of 1.5 mol.
12. a kind of sample compound method that is used for urine metabolism group nuclear magnetic resonance spectroscopy according to claim 1; It is characterized in that; When adopting human urine, human volume of urine: the potassium fluoride solution volume of 5 mol: the urine damping fluid volume=100:1 ~ 10:10 of 1.5 mol.
13. a kind of sample compound method that is used for urine metabolism group nuclear magnetic resonance spectroscopy according to claim 1; It is characterized in that; When adopting human urine, human volume of urine: the potassium fluoride solution volume of 5 mol: the urine damping fluid volume=100:2.7:10 of 1.5 mol.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107505346A (en) * | 2016-06-14 | 2017-12-22 | 布鲁克碧奥斯平有限公司 | Predict the method that the chemical displacement value of NMR spin systems in biological fluid sample is particularly in class of fluids sample |
| CN107607572A (en) * | 2017-09-15 | 2018-01-19 | 山东大学齐鲁医院 | A kind of two dimensional NMR detection method for isolated cells water-soluble metabolic thing |
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| CN101206203A (en) * | 2007-12-14 | 2008-06-25 | 上海交通大学 | Method for detecting urine metabolite based on deriving method |
Non-Patent Citations (2)
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| MICHAEL LAURIDSEN ET AL: "Human Urine as Test Material in 1H NMR-Based Metabonomics: Recommendations for Sample Preparation and Storage", 《ANALYTICAL CHEMISTRY》 * |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107505346A (en) * | 2016-06-14 | 2017-12-22 | 布鲁克碧奥斯平有限公司 | Predict the method that the chemical displacement value of NMR spin systems in biological fluid sample is particularly in class of fluids sample |
| US10401312B2 (en) | 2016-06-14 | 2019-09-03 | Bruker Biospin Gmbh | Method for predicting chemical shift values of NMR spin systems in a sample of a fluid class, in particular in a sample of a biofluid |
| CN107607572A (en) * | 2017-09-15 | 2018-01-19 | 山东大学齐鲁医院 | A kind of two dimensional NMR detection method for isolated cells water-soluble metabolic thing |
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Application publication date: 20120502 |