CN101477078B - Detection method for acetylcholine in royal jelly - Google Patents
Detection method for acetylcholine in royal jelly Download PDFInfo
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- CN101477078B CN101477078B CN2009100284549A CN200910028454A CN101477078B CN 101477078 B CN101477078 B CN 101477078B CN 2009100284549 A CN2009100284549 A CN 2009100284549A CN 200910028454 A CN200910028454 A CN 200910028454A CN 101477078 B CN101477078 B CN 101477078B
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Abstract
The invention provides a method for detecting acetylcholine in royal jelly, which is established by combining capillary electrophoresis separation with an electrochemiluminescence detection technique. The method comprises the following: a, a step of pretreating royal jelly; b, a step of performing capillary electrophoresis separation; and c, a step of performing electrochemiluminescence detection, wherein a target component separated out through capillary electrophoresis flows into an electrochemiluminescence detection tank so as to be detected; a selected detection system is a classical tris(bipyridine) ruthenium detection system which contains 2 to 8 mM of ruthenium solution, has the pH approximately between 7.0 and 9.0, adopts a three-electrode system, takes a platinum wire as a counter electrode, takes a silver-silver chloride electrode as a reference electrode, and takes 500-mu m platinum disk electrode as a working electrode; the peaking time of the target component and an interfering component differs by 200 to 250 second; and the quantitative detection of the acetylcholine in a royal jelly sample can be performed by a standard curve method. The method has the advantages of simplicity, economical efficiency and good accuracy.
Description
Technical field
The present invention is the new technology that detects acetylcholine in the royal jelly, belongs to the technical field of phatidylcholine content detection.
Background technology
People's brain tissue has a large amount of acetylcholines, but the content of acetylcholine can descend along with the increase at age, causes various spirit type diseases relevant with memory such as senile dementia etc.Keeping and improve the content of acetylcholine in the brain, is the fundamental way that solves decrease of memory.Acetylcholine exists in the many forms with choline of occurring in nature; After choline must play biochemical reaction with the acetyl coenzyme A in the human body; Could synthesize acetylcholine with physiologically active; Directly exist with the acetylcholine form in the royal jelly, the utilization that can directly be absorbed by the body is so royal jelly has certain effect to prevention senile dementia, decrease of memory.Therefore, the detection of acetylcholine is significant in the royal jelly.
Acetylcholine does not possess electrochemical activity, and does not have to supply the functional group of spectral detection, is difficult to directly detect with galvanochemistry or spectroscopic methodology.Domestic and international research mainly is to be the detection method that the basis is set up with the acetylcholinesterase, and its shortcoming is that the expensive and the application program of used enzyme is complicated.The acetylcholine method research of setting up with liquid chromatography and mass spectrometry recently often, the Mass Spectrometer Method appearance is highly sensitive, selectivity is strong, but used instrument is expensive, the analysis cost height.
Electrochemiluminescdetection detection is the detection technique that a kind of easy, detection sensitivity is high, selectivity is good, and Capillary Electrophoresis is an emerging strong analytical technology of separating power, and they have caused the increasing attention of analytical work person.But as far as we know, these two kinds of analytical technologies all also are not applied to the detection of choline or acetylcholine.
Summary of the invention
Technical matters: the objective of the invention is to utilize capillary electrophoresis separation-electrochemiluminescdetection detection technology to combine and the detection method of acetylcholine in the royal jelly set up, this method is simple, economy, accuracy good.
Technical scheme: the detection method of acetylcholine is specific as follows in the royal jelly of the present invention:
A. the pre-treatment of royal jelly: place 80～100 ℃ baking oven to toast fresh royal jelly, its moisture is volatilized fully, grind then, the powder that takes by weighing 1～2g is contained in the airtight container, places 130～170 ℃ baking oven to toast again 3～5 minutes; In airtight container, inject 2～4mL water, the solution that obtains can be used through centrifugal, filtration;
B. capillary electrophoresis separation: the royal jelly that will pass through pre-treatment is done separating medium with high pressure sample introduction 5～10s with the damping fluid of pH5.0～11.0, under 8～15KV high pressure, separates;
C. electrochemiluminescdetection detection: it is to be detected that the target components of coming out through capillary electrophoresis separation flows into Electrochemiluminescdetection detection cell; The detection architecture of selecting for use is classical tris (bipyridine) ruthenium detection architecture, contains the ruthenium solution of 2～8mM in the system, and pH about 7.0～9.0; Adopt three-electrode system; Platinum filament is that silver-silver chloride electrode is done contrast electrode to electrode, the platinum disk electrode of the 500-μ m electrode of working; Target components and interfering component appearance time differ 200～250s, can be to the acetylcholine detection by quantitative in the royal jelly sample with calibration curve method.
Beneficial effect: this method can be used for the detection of acetylcholine in the samples such as royal jelly.
Mark-on reclaims experiment, and the recovery is 92% to 107%; In a few days and in the daytime standard deviation is respectively less than 4.9% and 6.8%.Prove that this detection technique is accurately, reliably.
Description of drawings
Fig. 1 is the capillary electrophoresis separation-electrochemiluminescdetection detection collection of illustrative plates of acetylcholine in the royal jelly.
Embodiment
1 reagent and instrument
The acetylcholine standard items; Royal jelly; Baking oven; HPCE; The electrogenerated chemiluminescence appearance.
2 methods
1) pre-treatment of royal jelly:
Acetylcholine is a quaternary amine alkali, and itself can not strengthen or weaken the electrogenerated chemiluminescence signal of ruthenium dipyridine.Present technique places 100 ℃ baking oven to toast 10 hours royal jelly, and its moisture is volatilized fully, grinds then, takes by weighing and is contained in (this container is depressurized earlier) in the airtight container about the about 1g of powder, places 150 ℃ baking oven to toast again 3 minutes.In this process, decomposition reaction can take place and generate trimethylamine soluble in water in acetylcholine.In airtight container, inject 3mL water with syringe immediately and absorb trimethylamine, the solution that obtains can be employed through centrifugal, filtration.
2) capillary electrophoresis separation technology
In the solution that the pre-treatment of royal jelly process obtains many chaff interferences are arranged, necessary process is separated could be by accurately detection.Electrokinetic injection 10s under the high pressure of 10KV does separating medium with the phosphate buffer of 35mM pH8.0, under the high pressure of 15KV, separates, and target components went out the peak in the time of 4 minutes, and interfering component goes out the peak about 6.5 minutes, and both are separated fully.
3) electrochemiluminescdetection detection condition
The detection architecture of selecting for use is classical tris (bipyridine) ruthenium detection architecture, contains the ruthenium solution of 5mM in the system, pH=8.0; Adopt three-electrode system; Platinum filament is to electrode, and silver-silver chloride electrode (3.0M NaCl) is done contrast electrode, the platinum disk electrode of the 500-μ m electrode of working.The photomultiplier high pressure is that 600V. detection current potential is the detection that 1.15V. comes acetylcholine in the health board royal jelly:
One, the pre-treatment of royal jelly:
Get that 40.44g is fresh to come health board royal jelly (Nanjing Pearl Spring honeybee product factory) in 100 ℃ baking oven, to heat 12 hours, behind the royal jelly bone dry, be weighed as 22.68g, it is ground to form fine powder in mortar.Claim that the 0.343g fine powder places vial (drying 5 minutes at 135 ℃ baking oven in advance), seals rapidly with rubber stopper.Place 160 ℃ baking oven heating to take out in 3 minutes again.In this air-tight bottle, inject 5mL water rapidly with syringe, shake up, filter, collect subsequent filtrate.
Two, handle the separation and the detection of back sample
With 10 times of electrokinetic injection 10s under the high pressure of 10KV of subsequent filtrate dilution, do separating medium with the phosphate buffer of 35mM pH8.0, under the high pressure of 15KV, separate.Be the ruthenium solution of 5mM in the detection architecture, pH=8.0 adopts three-electrode system, and platinum filament is to electrode, and silver-silver chloride electrode (3.0MNaCl) is done contrast electrode, the platinum disk electrode of the 500-μ m electrode of working.It is that the 1.15V. collection of illustrative plates shows that other composition in trimethylamine and the royal jelly is separately complete that the photomultiplier high pressure is made as 600V. detection current potential.Triplicate, the average honeybee height that records the effective constituent trimethylamine is 135.
Three, the drafting of typical curve.
The acetylcholine of getting the 0.1075g standard is placed in the vial of drying in 135 ℃ the baking oven, seals rapidly with rubber stopper, places 160 ℃ baking oven heating to take out in 1 minute.In this air-tight bottle, inject 10mL water rapidly with syringe, shake up.This 10mL solution is transferred to constant volume in the 100mL volumetric flask.Compound concentration is from 7.5 * 10
-7To 4.3 * 10
-5The series of standards solution of g/mL, the drawing standard curve, try to achieve regression equation and be: y=6.2+126.3x (r=0.9996, n=6).
Four, calculate.
Bring the average peak height value of sample subsequent filtrate the regression equation of typical curve into, through converting to such an extent that this comes the content of acetylcholine in the health board royal jelly to be: 825 ± 64 μ g/g.Do mark-on and reclaim experiment, the recovery is between 87-111%.In the daytime be respectively 5.2% and 9.8% with withinday precision.
Claims (1)
1. the detection method of acetylcholine in the royal jelly is characterized in that this method is:
A. the pre-treatment of royal jelly: place 80～100 ℃ baking oven to toast fresh royal jelly, its moisture is volatilized fully, grind then, the powder that takes by weighing 1～2g is contained in the airtight container, places 130～170 ℃ baking oven to toast again 3～5 minutes; In airtight container, inject 2～4mL water, the solution that obtains can be used through centrifugal, filtration;
B. capillary electrophoresis separation: the royal jelly that will pass through pre-treatment is with high pressure sample introduction 5～10s, and the phosphate buffer of 35mM pH=8.0 is done separating medium, under 8～15KV high pressure, separates;
C. electrochemiluminescdetection detection: it is to be detected that the target components of coming out through capillary electrophoresis separation flows into Electrochemiluminescdetection detection cell; The detection architecture of selecting for use is classical tris (bipyridine) ruthenium detection architecture, contains the ruthenium solution of 2～8mM in the system, and pH is 7.0～9.0, adopts three-electrode system, and platinum filament is that silver-silver chloride electrode is done contrast electrode to electrode, the platinum disk electrode of the 500 μ m electrode of working; Target components and interfering component appearance time differ 200～250s, can be to the acetylcholine detection by quantitative in the royal jelly sample with calibration curve method.
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| CN101949885A (en) * | 2010-08-12 | 2011-01-19 | 东南大学 | Capillary electrophoresis-electrochemiluminescence detection method for spectinomycin |
| CN103149345B (en) * | 2013-02-25 | 2015-07-15 | 浙江省电力公司电力科学研究院 | Detecting method and detecting device for intelligent dissolved gas-in-oil component |
| CN113358528B (en) * | 2021-06-08 | 2022-06-17 | 山东大学 | Method for detecting acetylcholinesterase and inhibitor thereof based on pendant drop method |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1161478C (en) * | 2002-01-09 | 2004-08-11 | 南京医科大学 | Optical fibre bio-sensor of acetylcholinesterase and its preparing process |
| JP2007135474A (en) * | 2005-11-18 | 2007-06-07 | Chiba Univ | Method for determining quantity of acetylcholine |
| CN101236169A (en) * | 2007-11-21 | 2008-08-06 | 上海理工大学 | Acetylcholine esterase electrode preparation method |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1161478C (en) * | 2002-01-09 | 2004-08-11 | 南京医科大学 | Optical fibre bio-sensor of acetylcholinesterase and its preparing process |
| JP2007135474A (en) * | 2005-11-18 | 2007-06-07 | Chiba Univ | Method for determining quantity of acetylcholine |
| JP4677558B2 (en) * | 2005-11-18 | 2011-04-27 | 国立大学法人 千葉大学 | Determination of acetylcholine |
| CN101236169A (en) * | 2007-11-21 | 2008-08-06 | 上海理工大学 | Acetylcholine esterase electrode preparation method |
Non-Patent Citations (4)
| Title |
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| Emteborg M等.Peroxyoxalate chemiluminescence in aqueous solutions: Coupling of immobilized enzyme reactors and 1,1 "-oxalyldiimidazole chemiluminescence reaction to flow-injection analysis and liquid chromatographic systems.《ANALYTICA CHIMICA ACTA》.1997,第357卷(第1-2期),111-118. * |
| EmteborgM等.Peroxyoxalatechemiluminescenceinaqueoussolutions:Couplingofimmobilizedenzymereactorsand1 1 "-oxalyldiimidazole chemiluminescence reaction to flow-injection analysis and liquid chromatographic systems.《ANALYTICA CHIMICA ACTA》.1997 |
| Jianguo Li等.Electrogenerated Chemiluminescence Detection of Amino Acids Based on Precolumn Derivatization Coupled with Capillary Electrophoresis Separation.《Analytical Chemistry》.2006,第78卷(第8期),2694-2699. * |
| Yuan Jipei等.Characterization of procaine metabolism as probe for the butyrylcholinesterase enzyme investigation by simultaneous determination of procaine and its metabolite using capillary electrophoresis with electrochemiluminescence detection.《 JOURNAL OF CHROMATOGRAPHY A》.2007,第1154卷(第1-2期),368-372. * |
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