CN105527136A - Method for pre-treating veterinary drug residual quantity in detection of animal tissue - Google Patents
Method for pre-treating veterinary drug residual quantity in detection of animal tissue Download PDFInfo
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- CN105527136A CN105527136A CN201410572392.9A CN201410572392A CN105527136A CN 105527136 A CN105527136 A CN 105527136A CN 201410572392 A CN201410572392 A CN 201410572392A CN 105527136 A CN105527136 A CN 105527136A
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Abstract
The invention belongs to the field of analytical chemistry, and relates to a method for pre-treating veterinary drug residual quantity in detection of animal tissue. The method comprises the following steps: adding a proper amount of a buffer in the homogenized animal tissue for adjusting the pH value to weak acidic, adding enough amount of an enzymatic hydrolysis solution, then performing constant temperature water-bath or infrared auxiliary microwave processing enzymatic hydrolysis incubation, centrifuging the solution after incubation is carried out, washing by an organic solvent for deposition, centrifuging the materials and collecting the organic solvent for merging with a supernatant, performing rotary evaporation on the solution at room temperature to remove the solvent, using a few organic solvent for re-dissolving the residue to obtain a sample matrix as a functionalization composite material which can effectively enrich the microscale veterinary drug residue in the animal tissue, and conveniently performing subsequent detection. The method has the advantages of simple and easy operation, short time consuming, low cost, and low cost of equipment, and the method is suitable for all animal tissue.
Description
Technical field
The invention belongs to Pharmaceutical Analysis field, relate to a kind of pre-treating method detecting animal tissue's veterinary drug residue amount, this method can be used for the interference also effective gathering trace veterinary drug remnants getting rid of complicated endogenous substance in animal tissue.
Background technology
Along with development that is scientific and technological and medical science, the animal-derived food herbal medicine remaining harm to human body is caused social concern by people's institute's cognition gradually, it is reported, China and European Union have prohibited the illegal interpolation behavior in feed and have put into effect some row relevant regulations.But, complicacy when specifically detecting but because of animal tissue itself, serious matrix effect and the veterinary drug remnants to denier lack effective enrichment method etc., research is made to run into heavy bottleneck, and its extremely low content brings difficulty to follow-up analysis, limits the control of people to food security.
How simple albumen precipitation is only adopted in the disclosed research report of prior art, the method of liquid-liquid extraction or Solid-Phase Extraction etc., along with the continuous increase of residue of veterinary drug kind, multistage chromatography-mass spectrometry (GC-MS/MS, LC-MS/MS) be used as more and more analyzing the means detected, detection material can be analyzed simultaneously, but higher sensitivity requires higher to the pre-treatment of sample, this is stern challenges to current analysis pretreatment technology and means, research is had to attempt realizing extracting completely to accelerate, consider to use the device such as microwave radiation exaraction and infrared accelerated solvent extractor and technology.
Graphene (graphene) is a kind of New Two Dimensional plane carbonaceous Nano-Materials being in recent years found and synthesizing.Graphene shows the character of many excellences, there is electron mobility at a high speed under high specific surface area, good thermal conductivity and room temperature, there is lower production cost (relative to carbon nano-tube), simultaneously containing abundant pi-conjugated system, therefore there is splendid absorption property.Graphene, due to these excellent properties, has become a large study hotspot of field of nanometer material technology.
In view of the situation of prior art, present inventor intends providing a kind of pre-treating method detecting animal tissue's veterinary drug residue amount, makes its more effective interference for getting rid of complicated endogenous substance in animal tissue and effective gathering trace veterinary drug remnants.
Summary of the invention
The object of the invention is to for solve prior art exist animal tissue's veterinary drug residue analytical work in run into matrix effect impact excessive, the problems such as effective enrichment cannot be carried out to micro-veterinary drug, a kind of pre-treating method detecting animal tissue's veterinary drug residue amount is provided.The inventive method effectively can get rid of the interference of protein and other, can carry out enrichment to the veterinary drug composition of denier.
By microwave radiation technology or infraredly to combine with nano material in the inventive method, can settling time long, the problem that process is loaded down with trivial details, nano material can be utilized again both to have had the extra specific surface area of mesopore molecular sieve as the advantage of adsorbent, orderly pore size and unique modification group, enablely to enter in its duct and to be combined by the hydrophobic forces between residue of veterinary drug with alkyl, macromolecular protein repels outside duct simultaneously, thus effective enrichment can be carried out by residual medicine to the agriculture in the biological sample of complexity, thus simplify the analysis difficulty of trace residue of veterinary drug, application for composite nano materials provides new approach.
Concrete, a kind of pre-treating method detecting animal tissue's veterinary drug residue amount provided by the invention, comprises the following steps:
1) solution preparation: preparation sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution, GRD beta-glucuronidase solution; Add appropriate amount of buffer solution after animal tissue being pulverized homogenization process and be adjusted to faintly acid;
2) enzymolysis is hatched: in tissue homogenate, add enough GRD beta-glucuronidase solution or Luo Man snail (Helixpomatia) tissue fluid, water bath with thermostatic control or infrared assisted microwave synthesis process;
3) subsequent treatment: get supernatant after rear gained solution centrifugal will be hatched, with ethyl acetate washing precipitation and collected by centrifugation ethyl acetate and supernatant merge.Gained solution room temperature backspin turns evaporation of solvent, and a small amount of ethanol of residue redissolves, then by the sample substrate that obtains with the process of a kind of functionalization compound substance, adopts the Graphene mesoporous silicon compound substance of alkyl modified in embodiments of the invention.
In the method for the invention,
1) sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution concentration is 0.01-0.1M.;
2) tissue homogenate is adjusted to about pH=1-5 with damping fluid;
3) in enzymolysis incubation step, water bath with thermostatic control or infrared, microwave temperature control should ensure that temperature is constant about 40-60 DEG C maintenance; Microwave power is in the scope of 200-600W;
4) incubation time of enzymolysis incubation step is in the scope of 0.1-640 minute;
5) in centrifugal treating step, centrifugation rate is 6000-10000rpm, and centrifugation time is 5-20 minute;
6) with in the step of organic solvent washing precipitation, the volume ratio of organic solvent and precipitation is 5:1-1:1; Wash 2 times altogether;
7) organic solvent volume used that redissolves is unsuitable many, ensures to redissolve rear solution clear; Can ultrasonic process it be made fully to dissolve if desired.
Invention has been the pre-treatment of glucocorticoid medicine method for detecting residue in animal's liver, result shows, and this method can micro-veterinary drug remnants effectively in enriched animal tissue, conveniently carry out follow-up detecting step; Method of the present invention is simple and easy to operate, and consuming time short, cost is low, and equipment is cheap, and the inventive method goes for all animal tissues.
Method of the present invention contrasts existing animal tissue pretreatment technology and has the following advantages:
1) traditional simple liquid-liquid extraction is not limited to, but carry out enzymolysis thoroughly after fully homogenate is pulverized to tissue, the thorough digestion not only making animal tissue reach mechanical crushing cannot to complete, also the medicine making part be combined with biomacromolecule is free out, is conducive to the accuracy improving subsequent detection;
2) optimize the top condition of enzymolytic tissue, comprise temperature, pH and incubation time, the control proposed for incubation temperature can adopt the modes such as water bath with thermostatic control, microwave treatment or infrared temperature control;
3) adopt organic solvent cyclic washing precipitation, ensure that the left drug in animal tissue is fully extracted;
4) rotary evaporation redissolves with a small amount of organic solvent except after desolventizing, concentrates drug concentration, is conducive to follow-up mass spectrophotometry.
Accompanying drawing explanation
Fig. 1 is the inventive method operational flowchart.
Fig. 2 is the techniqueflow chart of the pre-treating method that glucocorticoid remains in pork liver in embodiment.
Fig. 3 is microwave radiation technology enzymolysis in embodiment, and nano composite material is the chromatogram of dexamethasone, betamethasone and prednisolone in adsorbent enrichment pork liver.
Embodiment
The following examples further illustrate of the present invention, instead of limit the scope of the invention.
The pre-treating method of glucocorticoid medicine method for detecting residue in embodiment 1 one kinds of animal's livers
The Graphene mesoporous silicon compound substance of alkyl modified is distributed in the methyl alcohol of 50%, and the dispersion liquid being made into 10mg/mL is stand-by;
5g fresh liver is ground into tissue homogenate, add 10mL sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution and regulate pH to 5.2, add the GRD beta-glucuronidase solution of 100 μ L160KU/mL and the hydrocortisone solution of 50 μ L0.2ng/mL, be incubated 4 hours or microwave (600W) assistance enzymolysis 0.5min or infrared assistance enzymolysis (400W) 0.5h in 40 DEG C of waters bath with thermostatic control;
Hatch rear gained solution with 8000rpm centrifugal 15 minutes, get supernatant liquid, with the washing precipitation of 10mL ethyl acetate, after washing twice, the ethyl acetate of collected by centrifugation and supernatant are merged; Gained solution room temperature backspin turns evaporation of solvent, and residue ethanol redissolves and makes into 2mL solution.Again the sample substrate obtained is analyzed with sample introduction HPLC-MS/MS after the Graphene mesoporous silicon compound substance process of C8 alkyl modified (high-efficient liquid be in series mass spectrum) and detect, result shows, this method can micro-veterinary drug effectively in enriched animal tissue remaining, conveniently carry out follow-up detecting step; Method of the present invention is simple and easy to operate, and consuming time short, cost is low, and equipment is cheap.
Claims (9)
1. detect a pre-treating method for animal tissue's veterinary drug residue amount, it is characterized in that, it comprises step:
1) solution preparation: preparation sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution, GRD beta-glucuronidase solution; Add appropriate amount of buffer solution after animal tissue being pulverized homogenization process and be adjusted to faintly acid;
2) enzymolysis is hatched: in tissue homogenate, add enough GRD beta-glucuronidase solution or Luo Man snail (Helixpomatia) tissue fluid, water bath with thermostatic control or infrared assisted microwave synthesis process;
3) subsequent treatment: get supernatant after rear gained solution centrifugal will be hatched, with ethyl acetate washing precipitation and collected by centrifugation ethyl acetate and supernatant merge.Gained solution room temperature backspin turns evaporation of solvent, and a small amount of ethanol of residue redissolves, then by the sample substrate that obtains with the process of a kind of functionalization compound substance.
2. the pre-treating method of detection animal tissue according to claim 1 veterinary drug residue amount, is characterized in that: described sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution concentration is 0.01-0.1M.
3. the pre-treating method of detection animal tissue according to claim 1 veterinary drug residue amount, is characterized in that: the homogenate of knitting described in group is adjusted to pH=5-9 with damping fluid.
4. the pre-treating method of detection animal tissue veterinary drug residue amount according to claim 1, is characterized in that: in described enzymolysis incubation step, water bath with thermostatic control or infrared, microwave temperature control should ensure that temperature is at 40 DEG C-60 DEG C; Microwave power is in the scope of 200-600W.
5. the pre-treating method of detection animal tissue according to claim 1 veterinary drug residue amount, is characterized in that: described water bath with thermostatic control or incubation time that is infrared, microwave temperature control enzymolysis incubation step are in the scope of 0.1-640 minute.
6. the pre-treating method of detection animal tissue according to claim 1 veterinary drug residue amount, it is characterized in that: in described centrifugal treating step, centrifugation rate is 4000-8000rpm, and centrifugation time is 10-20 minute.
7. the pre-treating method of detection animal tissue according to claim 1 veterinary drug residue amount, is characterized in that: in the step of described organic solvent washing precipitation, the volume ratio of organic solvent and precipitation is 5:1-1:1; Wash 2-6 time.
8. the pre-treating method of detection animal tissue veterinary drug residue amount according to claim 1, is characterized in that: described redissolution organic solvent volume used is as the criterion to redissolve rear solution clear.
9. the pre-treating method of detection animal tissue according to claim 1 veterinary drug residue amount, is characterized in that: described a kind of functionalization compound substance is selected from the Graphene mesoporous silicon compound substance of alkyl modified.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106769282A (en) * | 2016-11-28 | 2017-05-31 | 无锡艾科瑞思产品设计与研究有限公司 | Left-handed 18 methylnorethindron detection method and kit in a kind of animal tissue |
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| US20050196702A1 (en) * | 2004-02-24 | 2005-09-08 | Bryant Stephanie J. | Methods for photopatterning hydrogels |
| US20060035889A1 (en) * | 2004-08-13 | 2006-02-16 | Tedford Clark E | Treatment for methamphetamine addiction and reduction of methamphetamine use using serotonin antagonists |
| CN102288464A (en) * | 2011-07-05 | 2011-12-21 | 时文春 | Preparation method for standard sample of chloramphenicol residual lyophiled powder in muscle of carp |
| CN103760269A (en) * | 2014-01-23 | 2014-04-30 | 姜艳彬 | Detection method for veterinary drug residues |
| CN104280437A (en) * | 2013-12-17 | 2015-01-14 | 南京师范大学 | Immunosensor and method for detecting various beta-adrenergic receptor stimulant residues |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050196702A1 (en) * | 2004-02-24 | 2005-09-08 | Bryant Stephanie J. | Methods for photopatterning hydrogels |
| US20060035889A1 (en) * | 2004-08-13 | 2006-02-16 | Tedford Clark E | Treatment for methamphetamine addiction and reduction of methamphetamine use using serotonin antagonists |
| CN102288464A (en) * | 2011-07-05 | 2011-12-21 | 时文春 | Preparation method for standard sample of chloramphenicol residual lyophiled powder in muscle of carp |
| CN104280437A (en) * | 2013-12-17 | 2015-01-14 | 南京师范大学 | Immunosensor and method for detecting various beta-adrenergic receptor stimulant residues |
| CN103760269A (en) * | 2014-01-23 | 2014-04-30 | 姜艳彬 | Detection method for veterinary drug residues |
Non-Patent Citations (1)
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| 王凤美 等: "超高效液相色谱-串联质谱测定动物源性食品和尿液中4中β-受体激动剂残留", 《分析化学研究报告》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106769282A (en) * | 2016-11-28 | 2017-05-31 | 无锡艾科瑞思产品设计与研究有限公司 | Left-handed 18 methylnorethindron detection method and kit in a kind of animal tissue |
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