CN103512789A - Reagent for extracting substance to be analyzed in sample and extraction method - Google Patents
Reagent for extracting substance to be analyzed in sample and extraction method Download PDFInfo
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Abstract
The invention provides a reagent for processing a sample before detection. The reagent comprises an extraction assistant, an extraction reagent and a nonpolar modifier, wherein the extraction assistant is used for separating a substance to be analyzed in a sample from the sample, or/and endows the substance to be analyzed with a non-electrified property, or/and enables the substance to be analyzed in a repelled environment; the extraction reagent is used for extracting the substance to be analyzed from an environment after being processed by the extraction assistant; the nonpolar modifier is used for changing the solubility of the substance to be analyzed in a separated extractant and enables the substance to be analyzed to be distributed into an immunoreaction compatibility reagent for subsequent immunological detection.
Description
Technical field
The present invention relates to a kind of reagent and disposal route detecting pre-treatment sample, special, the present invention relates to a kind of in reagent and disposal route with analyte pre-treatment sample in immunization method detection sample.
Background technology
First immunology detection technology grows up from clinical examination field, and it mainly detects sample is blood, urine, the liquid type samples such as saliva.General these liquid type samples can directly be used to immunology detection.And in food safety detection field, generally need to be by analyte, some small-molecule substances for example, from tissue samples, for example liver, fat, kidney etc., middlely first extract, and is used further to follow-up detection.
Aspect analyte extraction, also there are some traditional methods, for example, soxhlet extraction, it is applicable to the project of muting sensitivity detection means, need the detected material in sample to be extracted, the purity after it is extracted also has requirement, to meet as traditional conventional methods such as spectroscopic assay as far as possible.Liquid-liquid extraction method for example again, the method extracts between low pole and intensive polar solvent, but needs a large amount of organic solvents could meet the requirement of detection.
The confirmation method of generally acknowledged agricultural and veterinary chemicals residue detection is instrumental method in the world at present, comprises liquid chromatography, gas chromatography, gas chromatography mass spectrometry and LC-MS.This respect, in order to adapt to the requirement of instrument, normal C18 or the hydrophilic lipophilic multipolymer of HLB(of using) solid-phase extraction column purifies sample, also has in addition molecular engram polymeric material (MIP) as enrichment material (superpolymer in the function Ji Sanwei hole of being combined with target molecule selectivity), it is in fact a kind of bionics principle, and the epi-position with spatial complementary is identified and caught to Artificial Antibodies.In recent years, some great biotech firms, utilize antibody technique to prepare immune affinity chromatographic, for the sample clean before the detection of instrumental method.
Compare with these this traditional method, at present, the extracting method of the analyte in tissue samples, in extraction efficiency, is used in quantity of solvent and has larger improvement, but have the problem of inefficiency.The method that has scholar also to raise the efficiency in exploration, as high flux ，96 hole extraction solid-phase extraction device, can only process 24 samples for one day, and efficiency is lower.
About using instrument to analyze the pretreatment technology using, also constantly making progress at present, as supercritical extract, gel permeation chromatography, accelerated solvent extraction, solid-phase microextraction, matrix solid phase micro-extraction etc.
In a word, for traditional instrument, detect, the pre-treating method of the analyte in complicated tissue samples is a test greatly, and even, some pre-treating method is only applicable to a kind of detection of tissue, and another kind is organized and can not be suitable for.This chief reason is that the detection material means of instrument itself are chromatogram and mass spectral characteristic, and the signal to noise ratio (S/N ratio) of these features is in low-down concentration (10
-9mol/L) under, be difficult to stable maintaining.
At present, although there are some immunoreagents for food safety detection, ELISA kit for example, colloid gold test papers etc., have solved the efficiency and the accuracy problem that detect to a certain extent, still can not accomplish efficiently effective again aspect pre-treatment.About how to extract little molecule in complicated sample, current immunological method is also main with reference to being tending towards ripe instrumental method pre-treating method, and even many methods are direct reference instrument methods.
Take chloromycetin as example, in the Ministry of Agriculture's 1025 bulletin-26-2008 animal-derived foods, residual chloromycetin detects the muscle of enzyme linked immunosorbent assay, the pre-treating method of liver and fishes and shrimps sample, for taking test portion 3g, adds ethyl acetate (organic solvent) 6ml vibration, centrifugal, get upper organic phase, nitrogen dries up, residue n-hexane dissolution, then add buffer solution strongly to vibrate, centrifugal, take off layer (non-normal hexane) layer liquid and carry out immunoassay.The mensuration gas chromatography-mass spectrography of chloramphenicol residue in the method and the bulletin 781-1-2006 of Ministry of Agriculture animal-derived food, the mensuration high performance liquid chromatography-tandem mass method of chloramphenicol residue in No. 781 bulletin-2-2006 animal-derived foods of the Ministry of Agriculture, in No. 1025 bulletin-21-2008 animal-derived foods of the Ministry of Agriculture, the extracting method of residual chloromycetin detection vapor-phase chromatography is basically identical, its core remains uses ethyl acetate that target molecule chloromycetin is separated with impurity, guaranteeing under the prerequisite of effectively extraction, utilize nitrogen to dry up removal ethyl acetate, recycling diverse ways is got rid of the interference to subsequent detection, thereby reach certain accuracy.
The beta-2-agonists class Ractopamine of take is example, in No. 1025 bulletin-6-2008 animal foods of the Ministry of Agriculture, Rct opamine residue detects enzyme linked immunosorbent assay employing acetonitrile and does the little molecule of Ractopamine in extraction tissue, through nitrogen, dry up, the step of redissolving obtains can be used for the sample that euzymelinked immunosorbent assay (ELISA) is measured, substantially similar with the extracting method flow process in beta-receptor activator residue detection Liquid Chromatography-Tandem Mass Spectrometry in the Ministry of Agriculture No. 1025 bulletin-18-2008 animal derived foods, it is organic solvent extraction, remove organic solvent, again residue is dissolved in to examination with computer in the solvent that is suitable for instrument detection.
Take nitrofuran metabolite as example, in DB33T 744-2009 aquatic products, in the Fast Measurement euzymelinked immunosorbent assay (ELISA) of furazolidone, AMOZ, first sample is carried out to derivatization, re-using ethyl acetate extracts furan derivative, remove ethyl acetate, this place is used nitrogen to dry up, recycling normal hexane and biocompatibility buffer solution are dissolved out target substance, for immunoassays.The mensuration high performance liquid chromatography of itrofurans metabolite residue amount in No. 1077 bulletin-2-2008 aquatic products of this kind of method and the Ministry of Agriculture, in No. 783 bulletin-1-2006 aquatic products of the Ministry of Agriculture, the extracting method of the mensuration Liquid Chromatography-Tandem Mass Spectrometry of itrofurans metabolite residue amount is substantially all followed the methods such as extraction and nitrogen or Rotary Evaporators and is removed ethyl acetate, then carries out next step determination step.
Adopt immunology detection, although can realize without equipment at follow-up detection-phase, high-throughout detection, with regard to the whole method of inspection, does not obviously improve the efficiency of check.The main cause that this problem produces is, the main detection of still considering to adapt to instrument of step of the current pre-treating method for immunology detection, as prevented, stop up chromatographic column and take suitable solvent to remove protide or the lipid material in sample, in order to make to detect material to be detected in sample, be easily given instrument and the methods analyst of physicochemical property, usually adopt and purify, and derivatization step.In addition, these little molecules be not be free in sample tissue but with these sample tissues by various chemistry or the organic combination of physical action, allow little molecule and these sample tissues effectively dissociate out, and do not affect follow-up immunology detection, current method can only realize by drying up the method for organic solvent.
Recently, this immunoassay device is adopted by average family or amateur mechanism more and more.Because these detect assessment, to aim at layman designed, so these pick-up units need operating process simple, and can guarantee the accuracy of testing result.Therefore, simple to operate and testing result accurately and reliably pick-up unit be present society in the urgent need to.
Summary of the invention
These defects that exist in order to solve traditional product, a kind of method that the invention provides reagent of before detection, tissue samples being processed and adopt agent treated sample, can simplify processing procedure greatly by the method, for amateur occurrences in human life operation.Especially, when adopting immunoreagent to detect analyte, can improve efficiency and the accuracy of detection.
On the one hand, the invention provides a kind of reagent detecting pre-treatment sample, this reagent comprises: extraction auxiliary agent, allows analyte and this sample separation in sample by extraction auxiliary agent, or/and allow analyte be non-charged character, or/and in the environment of analyte in being ostracised; Extraction agent, allows analyte be extracted in extraction agent from be extracted the environment auxiliary agent is processed by this extraction agent.
In some preferred modes, preferred, the use of extraction auxiliary agent occurs in after sample contacts with extraction agent.Certainly, the two also can contact mixing with sample tissue extraction auxiliary agent and extraction agent simultaneously, or carries out homogenized after mixing.
In some preferred modes, extraction agent is organic extraction reagent.In some preferred modes, the similarity degree of the analyte under the polarity of extraction agent and non-electriferous state is higher than sample and extraction auxiliary agent.Preferred, this extraction agent or part extraction agent and sample and extraction auxiliary agent can not be miscible.In the preferred mode of other, this extraction agent possesses low toxicity or nontoxic, chemical inertness, and without the quick-fried toxicity matter processed of system.These extraction agent can be ester class, nitrile, and the potpourri of ester class and alcohols or ethers.
Reagent of the present invention can further include nonpolar adjusting reagent, and this nonpolar adjusting reagent and extraction agent can the miscible liquid of miscible formation.Preferably, the similarity degree of the target analyte under the polarity of this nonpolar adjusting reagent and non-electriferous state is lower than extraction agent.In some preferred modes, nonpolar adjusting reagent comprises hydrocarbon compound.Preferred, nonpolar adjusting reagent comprises fat hydrocarbon and/or the corresponding derivant of fat hydrocarbon.These nonpolar adjusting reagent include, but are not limited to, pentane, hexane, octane, cyclohexane, cyclohexanone, toluene cyclohexanone, tridecane, dodecane, sherwood oil, decahydronaphthalene, terebinthina, gasoline, kerosene etc.
In the preferred mode of other, reagent of the present invention can further include some immune response intermiscibility reagent.The miscible liquid of preferably, immune response compatibility solution and non-polar solvent and the miscible formation of extraction agent is immiscible.
Another aspect, the present invention also provides a kind of kit of processing sample, and this kit comprises: extraction auxiliary agent, allows analyte and this sample separation in sample by extraction auxiliary agent, or/and allow analyte be non-charged character, or/and in the environment of analyte in being ostracised; Extraction agent, allows analyte be extracted in extraction agent from be extracted the environment auxiliary agent is processed by this extraction agent; Nonpolar adjusting reagent, this nonpolar adjusting reagent and extraction agent can the miscible liquid of miscible formation; With immune response intermiscibility reagent.
In some preferred modes, extraction auxiliary agent, extraction agent, nonpolar adjusting reagent and immune response intermiscibility reagent exist separately respectively and mutually do not mix, and for example above four kinds of reagent are contained in container separately.In other mode, extraction auxiliary agent and extraction agent are contained in a container, for example glass container or plastic containers, and nonpolar adjusting reagent and immune response intermiscibility reagent are carried in another container by splendid attire, for example glass container or plastic containers.
On the other hand, the invention provides a kind of method detecting pre-treatment sample, the method comprises the following steps:
(1), allow extraction auxiliary agent contact with sample, this extracts auxiliary agent by the analyte in sample and sample separation; And/or allow analyte be non-charged character, or/and allow in the environment of analyte in being ostracised;
(2), allow and contacted with extraction agent by sample, allow analyte be extracted in extraction agent;
(3), allow separated extract contact with immune compatibility reagent with nonpolar correctives, allow analyte be assigned in immune compatibility reagent.
In some preferred modes, in sample, can first contact with extraction auxiliary agent, and then and extraction agent; Contact with extraction auxiliary agent; Or, allow sample first contact with extraction agent, and then contact with extraction auxiliary agent; Or extraction auxiliary agent can contact with sample with extraction agent simultaneously.
In some preferred modes, allow the polarity of extraction agent and the similarity degree of the analyte under non-electriferous state higher than sample and extraction auxiliary agent.Preferred, extraction agent or part extraction agent and sample and extraction auxiliary agent can not be mixed.Or allow extraction agent or part extraction agent and sample and extraction auxiliary agent can layering.
In other preferred modes, the method is also included in extraction agent and adds nonpolar adjusting reagent, allows nonpolar adjusting reagent and the extraction agent can the miscible liquid of miscible formation.Preferably, allow the polarity of nonpolar adjusting reagent and the similarity degree of the target organic molecule under non-electriferous state lower than extraction agent.
In the preferred mode of other, method of the present invention can further include in the miscible liquid of nonpolar adjusting reagent and extraction agent formation and adds some immune response compatibilizing agents.Preferably, the liquid that allows immune response compatibility solution and non-polar solvent and extraction agent be mixed to form is immiscible.
At immune response compatibility solution, extraction agent, and in the mixed process of non-polar solvent, analyte, will be ionized, the effect of hydrogen bond etc., redistributes back in immune response compatibility solution, finally realizes the detection to analyte.
In above aforesaid all embodiments, extraction auxiliary agent can comprise some inorganic salts.These inorganic salts comprise, but are not limited to ammonium sulfate, sodium sulphate, magnesium sulphate etc.Preferred, these inorganic salts are high concentration, for example the inorganic salts of inorganic salts for being greater than 20% the solubleness of 10-50 ℃ with water, comprise sodium, potassium, caesium, magnesium, calcium, copper, silver, iron, the hydrochloric acid of ammonium, nitric acid, sulfate, and the alkaloids that to meet dissolubility be water is greater than 20% the solubleness of 10-50 ℃.
In above aforesaid all embodiments, sample can be 0.2-4 with the volume ratio of extraction auxiliary agent.
In above aforesaid all embodiments, the potpourri of sample/extraction auxiliary agent and the volume ratio of extraction agent can be 0.2-10.
In above aforesaid all embodiments, separated extract and the volume ratio of nonpolar correctives are 0.2-10.
In above aforesaid all embodiments, sample can be the tissue of animal body, for example musculature, adipose tissue, internal organs tissue, hair tissue; Or comprise blood, blood plasma, serum, sweat, saliva, the body fluid such as urine.
In above aforesaid all embodiments, analyte can comprise any small-molecule substance as medicine, for example some small molecule organic compounds.Small molecule organic compound can be hormone medicine, microbiotic, and industrial chemical.Small molecule organic compound can be, but be not limited to beta-2-agonists class medicine, chloromycetin, sulfa drugs, tetracycline, nitrofurans metabolin, fluoroquinolones, beta-lactam antibiotic, malachite green, concealed malachite green, crystal violet, melamine, olaquindox, 19-nortestosterone, Trenbolone, norethindrone, diethylstilbestrol, Florfenicol, medroxyprogesterone acetate, estradiol, or one or more in stable.
In above aforesaid all embodiments, immune response compatibilizing agent or solution include, but are not limited to, phosphate buffered solution, citric acid solution, carbonate buffer solution, MES, TRIS, HEPES, MOPES, physiological saline.
In above aforesaid all embodiments, detect the detection that comprises any means, for example comprise the instrumental methods such as liquid chromatography, gas chromatography, gas chromatography mass spectrometry and LC-MS.Detection method also comprises the immunology detection based on immunology principle, for example enzyme-linked immunoassay method based on antibody and antigen combination principle and cross flow reagent strip method, and the instrument read method being used in conjunction with these immunization methods.
Beneficial effect
Utilize reagent of the present invention effectively from sample, to extract analyte, make follow-up detection consistent with real level, improved the accuracy detecting; Because it has reduced traditional concentrated and evaporation, utilize reagent of the present invention and disposal route not to need complicated step and complicated utility appliance, the people generally without professional standards can operate; In addition, the analyte that uses reagent of the present invention and method to extract can directly utilize immunological detection method to detect, and can well coordinate with some current commercially available immunologic detection methods, is beneficial to popularization.
Of the present invention elaborating
The invention provides the using method of a group reagent and this group reagent.By the use of this group reagent, can be by 10
-5mol/kg below sample, especially, 10
-7mol/kg sample 10
-10the small molecule organic compound of mol/kg sample, mainly comprises various agricultural chemicals, veterinary drug, and hormone, environmental contaminants are residual to be extracted from biological specimen, for immunology detection.
So-called sample, mainly comprises animal tissue, dairy products, birds, beasts and eggs, and various body fluid, and hair.
So-called extraction agent refers to middle polarity, and chemical inertness is insoluble or be slightly soluble in water, or with water can not be with the miscible solvent of arbitrary volume ratio.
So-called nonpolar adjusting reagent, itself and extraction agent have low pole or the nonpolarity solvent of good compatibility.
So-called extraction auxiliary agent, refers to energy and water immiscible liquid, includes but not limited to strong acid, example hydrochloric acid; Or highly basic, as NaOH; Or oxygenant, as sodium nitrite, or protein denaturant, as urea; Or acidity or alkaline buffer solution, as glycine buffer, Tris damping fluid; Or Lewis acid, as aluminium choride; Or Lewis alkali, as ammoniacal liquor; Proteinase, as pepsin; Or in water the high inorganic salts of solubleness under normal temperature and pressure environment; And one or more mixed solutions as above.
So-called layering, refers to that out of phase outward appearance distinguishes obviously, and method comprises standing, centrifugal, but be not limited to standing, or centrifugal method.
Analyte can be organic molecule medicine, so-called organic molecule medicine: molecular weight is lower than 1000 daltonian organic compounds, and its physiological activity tool to living organism has a certain impact.
So-called residual: in edible animal derived food, residual medicine, or the metabolin of medicine, content is lower than 10-7 mol/kg below sample.
So-called immune response intermiscibility solution refers to conventional immunological response buffer solution, and with the potpourri of particular matter, this not appreciable impact of solution immunological response and follow-up detection.
Characteristics of principle one of the present invention is: extraction auxiliary agent, and its function includes but not limited at strong acid, highly basic, oxidation/reduction, or under the effect of enzyme, the covalent bond small molecule organic compound in tissue to be checked is dissociated out; Include but not limited under the effect of denaturant, by the structural failure of physisorption Medicine small molecule in tissue to be checked; Include but not limited to change little molecule in the band point feature of self, make it be tending towards being assigned in suitable extraction agent.
Characteristics of principle two of the present invention is: suitable extraction agent, fully mix with sample and/or extraction auxiliary agent, utilize external force, include but not limited to centrifugal, or stratification, by target organic molecule be extracted to extraction agent mutually in.
Characteristics of principle three of the present invention is: the nonpolar correctives of certain proper proportion and extraction agent and immune response compatibility solution fully mix, utilize external force, include but not limited to centrifugal, or the standing target organic molecule that makes is dissolved in immune response compatibility solution, for follow-up immunological test.
analyte
Analyte can be organic molecule, the agricultural chemicals that comprises as the organic molecule of medicine, veterinary drug, hormone and environmental contaminants etc., and the compound of some carbon elements (beyond the oxide of de-carbon, carbonic acid, carbonate, cyanogen, prussiate, oxygen cyanogen, cyanate, sulphur cyanogen, metal carbide etc.) is referred to as organism.It is generally acknowledged that molecular weight is lower than 1000 daltonian molecules, is little molecule.Take veterinary drug as example mainly refers to the medicine as promoting that growth of animal or treatment Animal diseases are artificially added in breeding process, be mainly microbiotic, sulfa drugs, nitrofurans, expelling parasite chemicals, hormone etc.
Drug effect essence as the organic molecule of medicine
From macroscopic scale, observe, with reacting that body interacts and produces, i.e. medicament contact or enter after body, promote the biochemical functions of body surface and internal environment to change, or the pathogen of inhibition invasion, assist body to improve resistance against diseases, reach the effect of disease preventing and treating.As the explanation of microscopic scale, be known as at present after the ad hoc structure effect of mutually combining of medicine and body or pathogen, body is brought into play corresponding curative effect in conjunction with self-regulation function.The core of the combination of medicine and body or pathogen is drug molecule and effective combination of target position accordingly.Source, acting force You Liang aspect between drug molecule and body or pathogen target position, is the complementarity of space three-dimensional structure on the one hand, derives from the other hand the interaction in the corresponding site of complementary structure, and in conjunction with the stability of rear ligand.
The essence of organic molecule and body tissue sample (can be called part again) combination.
Combination between little molecule and tissue samples is that the biomacromolecule surface of body or pathogen exists and the target position structure of medicine complementation, and often the essence of this structure is the protein of body or pathogen self amino acid chain naturally folding and the result of the specificity conformation that produces under physiological condition, simultaneously, on the amino acid residue of protein in conjunction with lipid and glucide, under physiological condition, also can form specific conformation, under certain drug concentration, with medicine, non-covalent property occur and be combined.Utilize these principles, can allow small-molecule substance with part under extreme condition, as strong acid, highly basic, high salt under the effect of scaling agent, by destroying the conformation of the part of being combined with medicine, dissociates out little molecule from sample.
The essence that small molecule organic compound dissolves
Depend mainly on the character of small-molecule substance itself, under extreme case, the protein of tissue will thoroughly be destroyed, thereby lost the character of being combined with little molecular specificity, in different phase-splittings, micromolecular distribution, depends on the originally distribution character between difference phase of little molecule.According to the similar principle that mixes, little molecule in the abundant mixed distribution of homophase not, little molecule be tending towards polarity similar mutually in.This will be detailed in the following description book elaboration.
Concentration 10 when analyte
-7mol/kg sample 10
-10during mol/kg sample, the organic molecule medicament residue in tissue due to normal physiological state under the non-covalent combination of target position, or metabolin and the covalent bond of reactive group under the catalysis of biology enzyme, present 10
-7mol/kg sample-10
-10mol/kg concentration of specimens and high dispersion, generally can not exist with crystalline state.Therefore micromolecular extraction essence is to select suitable extraction agent in this case, and destroys body or the pathogen part complementary with it.
When low concentration organic molecule, as 10
-7mol/kg sample-10
-10mol/kg or after following small-molecule substance is extracted, by adding a certain amount of nonpolar adjusting reagent, with immune compatibility solution, can change the solvation of extraction agent to organic molecule, in suitable formula and ratio situation, the organic molecule of low concentration will be redistributed back in the compatible solution of immune response, at this moment can be for the follow-up high sensitivity testing method based on immunological response.
sample
Any tissue that may contain analyte can be as sample, the body tissue that for example contains organic molecule medicine also can be used as sample, these body tissues can be the tissues of animal body, for example musculature, adipose tissue, internal organs tissue, hair tissue; Also can be to comprise blood, blood plasma, serum, sweat, saliva, the body fluid sample of urine etc.Animal tissue can be any animal, for example, as various cultivated animals or the wild animal of food, and for example pig, sheep, chicken, duck, the animals such as ox.
extraction auxiliary agent
In the present invention, at sample to be measured, for example, in tissue samples, add after extraction auxiliary agent, carry out physical mechanical vibration.In this process, extract auxiliary agent will be to sample in the complementary ligand structure of organic molecule produce irreversible destruction.Owing to extracting the strong acid of the high concentration (final concentration is in 0.5M left and right conventionally) of auxiliary agent, example hydrochloric acid, or highly basic, as NaOH, or oxygenant, as sodium nitrite, will be hydrolyzed the protein of bearing in sample with the effect of little complementary element ligand structure support, protein chain is ruptured.Allow the analyte be non-charged character, or/and allow in the environment of analyte in being ostracised.Or protein denaturant, as urea, under existence, make protein that sex change further occur.Under this extreme case, there is irreversible variation in the ligand structure of being originally combined with little molecule, and the product after variation will pass through various weak forces at this kind of extreme environment again, as Van der Waals force, hydrogen bond, sat linkage, electrostatic interaction and water delivery effect, mutually combine again.This mutually combining, has not possessed physiological characteristic, due to the flexibility of chain structure, makes this interaction very strong on the one hand, has got rid of on the other hand the small-molecule substance possibility of combination with it.Under this kind of environment, organic molecule presents non-charged character, and the electrostatic interaction of the material in little molecule self and environment is further weakened simultaneously.The so little molecule part complementary with it thoroughly separates, thereby dissociates out.
Meanwhile, extraction auxiliary agent comprises the inorganic salts that contain high concentration, as ammonium sulfate.Extraction auxiliary agent can comprise some inorganic salts.These inorganic salts comprise, but are not limited to ammonium sulfate, sodium sulphate, magnesium sulphate etc.Preferred, these inorganic salts are high concentration, for example the inorganic salts of inorganic salts for being greater than 20% the solubleness of 10-50 ℃ with water, comprise sodium, potassium, caesium, magnesium, calcium, copper, silver, iron, the hydrochloric acid of ammonium, nitric acid, sulfate, and the alkaloids that to meet dissolubility be water is greater than 20% the solubleness of 10-50 ℃.Inorganic salts contribute to the separated of sample and extraction agent, and the extraction efficiency that regulates organic molecule.
In addition, in the environment of extraction auxiliary agent, organic molecule is in the environment of repulsion, and when the extraction agent with below extracts, free organic molecule more easily enters in extraction agent from these tissue samples, thereby reaches separated completely.For example more than at least 60% organic molecule of separating from tissue enters in extraction agent, and this ratio can be more than at least 70%, 80% or 90%, 95%.
extraction agent
In actual extraction process, can first to sample, extract auxiliary agent and process, and then add extraction agent; Also extraction auxiliary agent and extraction agent can be joined in sample simultaneously, two kinds of effects are carried out simultaneously.
Composition according to material, is generally divided into extraction agent, and 1. arene, as (poisonous) such as benzene,toluene,xylenes; 2. fat hydrocarbon: pentane, hexane, octane etc. (low toxicity); 3. alicyclic hydrocarbon type, as (low toxicities) such as cyclohexane, cyclohexanone, toluene cyclohexanone; 4. halogenated hydrocarbons, as (poisonous) such as chlorobenzene, dichloro-benzenes, methylene chloride; 5. alcohols, as (low toxicities) such as methyl alcohol, ethanol, isopropyl alcohols; 6. ethers: as (poisonous) such as ether, epoxypropane; 7. ester class: as (low toxicities) such as methyl acetate, ethyl acetate, propyl acetate, butyl acetates; 8. ketone: as acetone, espeleton, methylisobutylketone etc. (easily make quick-fried, be subject to control); 9. diol, derivatives: as (poisonous) such as glycol monoethyl ether, ethylene glycol monoethyl ether, ethylene glycol monobutyl ethers;
other: are as (poisonous) such as acetonitrile, pyridine, phenol.These extraction agent can be used as extraction agent above.
Generally, when selecting suitable extraction agent, allow the extraction agent of selecting preferably possess such characteristic: the similarity degree of the target organic molecule under the polarity of this extraction agent and non-electriferous state is higher than sample and extraction auxiliary agent.Certainly, as a preferred mode, this extraction agent or part extraction agent and sample and extraction auxiliary agent can not mix, to guarantee layering (or phase-splitting), and like this can be better separated.As a preferred mode, this extraction agent possesses low toxicity or nontoxic, chemical inertness, and without the quick-fried toxicity matter processed of system, can under any circumstance can operate like this, and safer and easy.In addition, as preferred, supplement, at normal temperatures and pressures, extraction agent is liquid, and low-viscosity.Meeting in the present invention above-mentioned characteristic extraction reagent, can be ester class, nitrile, and the potpourri of ester class and alcohols or ethers or nitrile.
In the present invention, extraction agent preferably can be separated with sample and extraction auxiliary agent, and for example naturally layering or by conventional mechanical effect is for example centrifugally carried out separation.For example, due to the low pole feature of extraction agent, and the cohesion of biomacromolecule and the multidigit point effect between large molecule in sample, in standing long enough time situation, extraction agent and extraction auxiliary agent and sample will natural separation, show as layering in appearance, and layering interfaces are obvious.Yet in actual applications,, in order better to extract to improve the efficiency of extraction, also can adopt centrifugal method.The in the situation that of certain centrifugal force, make immiscible each separation fast mutually.General separating effect is determined by the product of centrifugal force and centrifugation time.In low centrifugal force situation, by extending centrifugation time, can realize out of phase separation, in high centrifugal force situation, can complete separation at short notice.For the present invention, centrifugal force can be for more than 1000g, and centrifugation time was set in below 20 minutes.
nonpolar adjusting reagent and immune response compatibilizing agent
In the present invention, when analyte is low concentration, as 10
-7mol/kg sample 10
-10mol/kg sample or following.Under this kind of concentration, the solubleness of analyte is decided by polarity or the ionization characteristics of analyte itself, and the environment of this kind of feature and its existence has relativity to a certain degree.In this kind of situation, in isolated extraction agent, add can be miscible with extraction agent nonpolar adjusting reagent, or add immune response compatibility solution simultaneously, can change like this solubleness of analyte in extraction agent.In this kind of situation, analyte will be in extraction/non-polar solvent blended liquid phase, and redistributes in immune response compatibility solution.
A kind of situation is not for changing the band electrical feature of analyte itself.Extraction/nonpolar adjusting reagent mixed liquor shows as low pole, strong polarity during immune response compatibility solution shows as, now, the little molecule of low concentration will be redistributed according to a certain percentage in two, by regulating the extraction of different proportion and the ratio of nonpolar adjusting reagent, and select containing the buffer solution of suitable organic solvent and can realize this kind of process.By the method for centrifuging, finally get rid of extraction/non-polar solvent.
Another kind of situation is the band electrical feature of analyte itself for a change, and as for carboxylic analyte, immune response compatibility solution is meta-alkali solution, and little molecule, with negative ion state, is re-assigned in immune response compatibility solution; Otherwise for containing amino analyte, immune response compatibility solution is slant acidity solution, little molecule is re-assigned in immune response compatibility solution with kation state.
In the present invention, nonpolar correctives mainly refers to non-polar solvent.Selecting this kind of solvent should at least possess one or more in following characteristics: the similarity degree of the target organic molecule under the polarity of this non-polar solvent and non-electriferous state is lower than extraction agent.In some preferred modes, this non-polar solvent and extraction agent are miscible, and miscible liquid and immune response compatible solvent do not dissolve each other, can natural layering.In other preferred modes, this is nonpolar possesses low toxicity or nontoxic, chemical inertness, and without the quick-fried toxicity matter processed of system.In more preferred mode, at normal temperatures and pressures, this kind of material is in a liquid state nonpolar correctives, and the feature such as low viscosity.Nonpolar correctives can comprise hydro carbons, and corresponding derivant; Or fat hydrocarbon, and corresponding derivant.Nonpolar correctives can include but not limited to pentane, hexane, octane cyclohexane, cyclohexanone, toluene cyclohexanone, tridecane, dodecane, sherwood oil, decahydronaphthalene, terebinthina, gasoline, kerosene etc.
Nonpolar correctives can comprise vegetable oil, the oil that squeezes or extract from vegetable seeds or fruit is all usingd as nonpolar correctives, for example soya-bean oil, sesame oil, tung oil, castor oil, coconut oil, safflower oil, sunflower oil, camellia seed oil, maize germ, canola, olive oil and blending stock etc.
immune response intermiscibility reagent
In the present invention, immune response compatibilizing agent is an experimental concept, as long as meet the not obvious immune response that affects, in this kind of solution, can there is specific recognition in antigen and antibody, in conjunction with, and do not have influence on involved biological fixation effect, any solution weakening of the tracer signal relating to through this solution.Reagent can be to include, but are not limited to like this, phosphate buffered solution, citric acid solution, carbonate buffer solution, MES, TRIS, HEPES, MOPES, physiological saline.
immunology detection
So-called immunology detection is the high sensitivity based on antigen-antibody, the feature of high selectivity association reaction, and develop for detection of antibody or antibody, can identify the method for antigen or similar structures compound.
Antibody (antibody): body under antigenic substance stimulates, the thick liquid cell being become by B Cell Differentiation produces, the immunoglobulin (Ig) that can react with corresponding antigens generation specific binding.All antibody is all immunoglobulin (Ig).Utilize the repetitious stimulation of antigen to animal, collect serum, or prepare monoclonal antibody by monoclonal technigue, can obtain the antibody of specific recognition antigen,, under suitable solution environmental, finite concentration antigen can be by antibody recognition, combination occurs, and this kind of concentration is low especially, generally all 10
-12under mol/L level, simultaneously this identification possesses height selectivity, for example, identify the antibody that faces the benzene ring structure of a nitro on epitope, can not or exceed the benzene ring structure of identifying interband position nitro under the condition of thousands of times of concentration at same isoconcentration.Utilize this kind of feature, the immunology detection technology based on antigen-antibody reaction starts to grow up from eighties of last century the fifties, and in medical science, microbiological Test aspect has progressively started commercial applications.
The essence of antibody is protein, applies the character of its amino acid residue and protein itself, antibody can be combined with various probe materials or be fixed on certain polymer surface, for detection of antigenic substance.These probe materials comprise collaurum, enzyme, and radiomaterial, fluorescent dye, fixedly macromolecular material comprises nitrocellulose filter, polystyrene, Polyvinylchloride, tygon, nylon etc.
In concrete application, for various probe materials, developed respectively the detection method with different performance, as colloidal gold technique.Gold immunological technique is a kind of with the serve as a mark immunolabelling technique of thing of collaurum, collaurum refers to that golden fine particle (1-100nm) is dispersed in formed system in another kind of material, be often referred to golden fine particle and be dispersed in formed aurosol in solution, with this aurosol labelled protein (antigen, antibody or SPA, SPG), colloid gold particle has the characteristic of high electron density, therefore in the antigen-antibody junction of gold mark albumen, visible pitchy particle under microscope; When these labels are when corresponding mark is assembled in a large number, can on carrier macromolecular material, present naked eyes red color visible or pink spot, thereby for the location of antigen or antibody materials or qualitative.The device that utilization contains immunoreagent bar detects and in sample, whether contains analyte and having and disclosing or describe in a lot of prior aries.These reagent strips generally comprise reagent areas and surveyed area, and wherein reagent areas can comprise sample region of acceptance and marked region.On surveyed area, comprise the region that shows testing result and the testing result control zone that is positioned at surveyed area downstream.Conventionally, the sample region of acceptance of these reagent strips comprises sample blank film, marked region comprises marker slip, Zhe Liangge region forms reagent areas jointly, in reagent areas, comprised the necessary reagent of reaction, surveyed area comprises detection lug, on detection lug, comprise the region showing test results and the testing result control control zone that is positioned at this downstream, region, general testing result region is linear, on testing result region Show Color change to represent sample in analyte whether exist; Suction zone comprises water suction sheet, and these parts join end to end and allow liquid along the direction of arrow indication, flow to the other end from one end of reagent strip to complete test.General conventional reagent strip is nitrocellulose filter reagent strip, and surveyed area comprises nitrocellulose filter, and on nitrocellulose filter, fixedly specific bond molecule shows the result of detection; Can also be cellulose acetate membrane or nylon membrane etc.The reagent strip that for example the following patent is described or the device that contains reagent strip: US 4857453; US 5073484; US 5119831; US 5185127; US5275785; US 5416000; US 5504013; US 5602040; US 5622871; US 5654162; US 5656503; US 5686315; US 5766961; US 5770460; US 5916815; US 5976895; US 6248598; US 6140136; US 6187269; US 6187598; US 6228660; US 6235241; US 6306642; US 6352862; US 6372515; US 6379620; And US6403383.
For Enzyme-multiplied immune technique, by antigen or antibody labeling enzyme, corresponding with it antibody and antigen or antigen and antibody are fixed on macromolecular material, under liquid environment, make antigen or antibody free diffusing in liquid, while containing detected material in liquid, this material will have influence on reacting of the antigen of mark or antibody and fixing antigen or antibody, by washing, in connection with separating of the antigen that is labeled enzyme of state or antibody and free state, by enzyme, touch reaction, amplifying signal, the power of comparison signal, reflect in detected liquid and whether contain target antigen.This kind of method be with respect to the highly sensitive several times of colloidal gold method, and by the typical curve of Offered target antigen or antibody, can accomplish quantitative measurement target substance.As above two kinds of methods all do not need expensive experimental facilities to complete, and with respect to mass spectrum, the instrumental methods such as chromatogram have incomparable cost and can operational advantage.
Organic molecule medicine generally can not independently become the antigen that causes organism immune response, claims that this class material is haptens in specialty.When with carrier covalent bond after, can be used as holoantigen, in order to Dispersal risk.Due to the high sensitivity of immunological response, and height selectivity, and in the widespread use of field of medical examination, correlation technique is comparatively ripe.And various technology based on same principle, progressively to the mensuration infiltration of small-molecule drug, especially, in food safety detection field, application is carried out gradually recently.
Immunology detection can be to comprise enzyme linked immunosorbent assay, collaurum emulsion process, immune agglutination method, radioimmunoassay method, immune affine series of strata technology, immunofluorescence technique, and the method such as immunochemiluminescence.
Embodiment
For further the present invention will be described in detail, now describe for example, these explanations are just carried out limited enumerating under marrow of the present invention, can not make any restriction to the scope of claim of the present invention.
Embodiment 1: by principle involved in the present invention, utilize enzyme linked immunological kit to measure pork, and pork liver, beef, in the analog sample of beef liver tissue, Clenbuterol molecule is residual.
The preparation of sample: at beef, pork, beef liver, adds forbidden drugs Clenbuterol molecule (being commonly called as " clenbuterol hydrochloride ") in pork liver sample, to its content be 0.5ug/Kg, for Reality simulation sample, the sample that adds clenbuterol hydrochloride is standing 48 hours at 4-8 ℃.
The solvent that pre-treatment is used comprises:
Extraction auxiliary agent: the sodium hydrate aqueous solution that concentration is 0.1-10M;
Extraction agent: ethyl acetate;
Nonpolar correctives: heptane;
Immune response compatibility solution: the phosphate buffer solution that pH is 6.0-7.0.
The concrete implementation and operation of pre-treatment:
Accurately take 1g tissue samples, add NaOH (extraction auxiliary agent) 1mL, add ethyl acetate (extraction agent) 1mL, quick oscillation is after 10 minutes, under the condition that is 4000g at centrifugal force centrifugal 5 minutes;
The phosphate buffer solution that is 6.0-7.0 with pH by supernatant liquid (immune response compatibility solution) and heptane (nonpolar correctives) be take the ratio that volume ratio is 2:1:4 and are mixed, standing or centrifugal after layering, take off layer liquid and carry out immunoassays.The immune detection of carrying out is as follows:
(1), with commercially available Clenbuterol enzyme linked immunological kit (r-biopharm,
clenbuterol Fast, Lot:12280), the sensitivity of its kit is 0.1ppb.
Result:
Embodiment 2: according to commercially available Clenbuterol enzyme linked immunological kit (r-biopharm,
clenbuterol Fast, Lot:
12280) instructions carrying on carries out method of operating and carries out Sample pretreatment and carry out Clenbuterol molecule, and concrete method is specific as follows:
Take sample 5g sample in embodiment 1 in centrifuge tube, add the Tris damping fluid of 25ml 50mM, homogenate 30 minutes; Add 15mL normal hexane, and vibrate 5 minutes;
At 10-15 ℃, take centrifugal force as 4000g centrifugal 5 minutes
Re-use normal hexane grease removal once
Add 0.5mL 5-6M hydrochloric acid vibration 1 hour, or spend the night;
Take 6g(and be equivalent to 1g sample) with centrifuge tube in;
At 10-15 ℃, take centrifugal force as 4000g centrifugal 5 minutes;
Supernatant is moved in another centrifuge tube, add 300 μ L NaOH, and mix 15 minutes.
Add 4mL 500mM potassium phosphate buffer PH3.0, at 4 ℃, mix 1.5 hours
At 10-15 ℃, take centrifugal force as 4000g centrifugal 5 minutes
Supernatant is used
c18 crosses post.
First use the drip washing of 3mL methyl alcohol
Use again 50mM potassium phosphate buffer PH3.0 drip washing
Add sample
Add again 50mM potassium phosphate buffer PH3.0 drip washing
Use vacuum filtration method to drain
Use again methanol-eluted fractions
Collect eluent, at 50-60 ℃ of nitrogen, blow
With 0.4mL pure water, dissolve, to be measured.
In order to do horizontal contrast with the present invention, 1.6mL pure water will be added again, to reach and identical extension rate in sample.Result is as follows:
By as above implementing 1.2 contrast, the method simple and fast that the present invention uses, under the same conditions, to carry methods and results suitable with the traditional kit that needs complex steps pre-treatment.Complete whole mensuration, the present invention only need can complete about 1 hour, does not relate to Nitrogen evaporator, vacuum pump, the experiment equipments such as decontaminating column; And commercial reagent cassette method not only needs experiments supporting equipment at present, and step is complicated, consuming time at least wanting more than 6 hours.
Embodiment 3: (nine ancient cooking vessels are biological, SJJ1) to sample being processed by mode of the present invention and being carried the comparison to the testing result of sample processing method of recording in instructions for the horizontal immunoreagent bar of commercially available Clenbuterol detection kit.
reagent of the present invention is processed
The preparation of sample: in pork sample, add forbidden drugs Clenbuterol molecule (being commonly called as " clenbuterol hydrochloride "), to its content be 3ug/Kg, for Reality simulation sample, the sample that adds clenbuterol hydrochloride is standing 48 hours at 4-8 ℃.
Disposal route is shown in the method in embodiment 1, is specially:
Accurately take 1g tissue samples, add NaOH (extraction auxiliary agent) 1mL, add ethyl acetate (extraction agent) 1mL, quick oscillation is after 10 minutes, under the condition that is 4000g at centrifugal force centrifugal 5 minutes;
The phosphate buffer solution that is 6.0-7.0 with pH by upper strata (immune response compatibility solution) and heptane (nonpolar correctives) be take the ratio that volume ratio is 1:1:4 and are mixed, standing or centrifugal after layering, take off layer liquid and carry out immunoassays.
Concrete outcome is as follows:
| Sample | Negative number | Positive number | Invalid |
| 20 parts, blank pork sample | 20 | 0 | 0 |
| 20 parts, 3ug/Kg pork sample | 0 | 20 | 0 |
Inefficiency: 0/40 * 100%=0%
False negative rate: 0/20 * 100%=0%
False positive rate: 0/20 * 100%=0%.
according in the horizontal immunoreagent bar of commercially available Clenbuterol detection kit, (nine ancient cooking vessels are biological, and instructions SJJ1) carrying enters sample row is processed, specific as follows:
By 20 grams, same pork sample, pack in the centrifuge tube of 50ml, tighten pipe lid (not allowing water enter in pipe).The centrifuge tube that sample is housed, at more than 90 ℃ heating water bath 10-15 minute, is taken out and is cooled to room temperature, get transudate and measure.
The concrete outcome of measuring after application specifications operation is as follows:
| Sample | Negative number | Positive number | Invalid |
| 20 parts, blank pork sample | 17 | 2 | 1 |
| 20 parts, 3ug/Kg pork sample | 4 | 14 | 2 |
Inefficiency: 3/40 * 100%=7.5%
False negative rate: 4/18 * 100%=22%
False positive rate: 2/19 * 100%=10.5%
By as above contrasting, method provided by the invention can effectively be extracted material to be checked, eliminates matrix effect simultaneously.Although the method that test strips instructions carries looks simply, add gentleness and rise again consuming time also quite longly, can not effectively remove matrix effect simultaneously, it is a certain proportion of invalid to make to occur, false negative and false positive results.
Embodiment 4: by principle involved in the present invention, utilize enzyme linked immunological kit to measure pork, and pork liver, beef, in the analog sample of beef liver tissue, Ractopamine molecule is residual.
The preparation of sample: at beef, pork, beef liver, adds forbidden drugs Ractopamine molecule in pork liver sample, to its content be 0.5ug/Kg, for Reality simulation sample, the sample that adds clenbuterol hydrochloride is standing 48 hours at 4-8 ℃.
The solvent using comprises:
Extraction auxiliary agent: the aqueous solution of the NaOH that concentration is 0.1-10M and 1-20% sodium sulphate;
Extraction agent: ethylacetate/acetonitrile mixed solution (ethyl acetate: the ratio of acetonitrile is from 4:1 to 9:1);
Nonpolar correctives: sherwood oil;
Immune response compatibility solution: the citric acid solution that pH is 5.5-6.5.
Concrete disposal route is as follows:
Accurately take 1g tissue samples, add the NaOH of 0.1-10M and (extraction auxiliary agent) 1mL of 1-20% sodium sulphate, add ethylacetate/acetonitrile mixed solution (extraction agent) 1mL, quick oscillation is after 5 minutes, under the condition that is 4000g at centrifugal force centrifugal 5 minutes;
The citric acid solution that is 5.5-6.5 with pH by upper solution (immune response compatibility solution) and sherwood oil (nonpolar correctives) be take the ratio that volume ratio is 1:1:3 and are mixed, standing or centrifugal after layering, take off a layer liquid assay.
That use is commercially available Ractopamine enzyme linked immunological kit (Beijing Qin Bang Bioisystech Co., Ltd, RACT-CA055, lot:B-110413-B10), and the sensitivity of its kit is 0.1ppb.
Result:
(2), same sample, the instructions method carrying by commercially available Ractopamine enzyme linked immunological kit (Beijing Qin Bang Bioisystech Co., Ltd, RACT-CA055, lot:B-110413-B10) is processed sample, specific as follows:
Get 2g sample and join in 50mL centrifuge tube, add 8mL to extract working fluid, vibrate 10 minutes, centrifugal 10 minutes of 3000g, gets supernatant and measures.
The result of measuring is:
As above data show, the pre-treating method of by specification operation can not be distinguished significantly dummy and contain the difference between 0.5 μ g/Kg Rct opamine residue sample.Adopt method of the present invention obviously to increase difference, in fact needn't be by instrument, naked eyes can be distinguished.In order to reduce operator in practical operation, can coordinate instrument reading out data, make result have more objectivity.
Claims (17)
1. a reagent that extracts analyte in sample, this reagent comprises: extraction auxiliary agent, by this extraction auxiliary agent, allow analyte and this sample separation in sample, or/and allow analyte be non-charged character, or/and allow in the environment of analyte in being ostracised; Extraction agent, allows analyte be extracted in extraction agent from be extracted the environment auxiliary agent is processed by this extraction agent; Wherein, described reagent also comprises nonpolar adjusting reagent, and this nonpolar adjusting reagent and extraction agent can the miscible liquid of miscible formation.
2. according to the reagent of claim 1, wherein, extraction agent is organic extraction reagent, and the similarity degree of the analyte under the polarity of extraction agent and non-electriferous state is higher than sample and extraction auxiliary agent.
3. according to the reagent of claim 1, wherein, this extraction agent or part extraction agent and sample and extraction auxiliary agent are not miscible.
4. according to the reagent of claim 1, wherein, the similarity degree of the analyte under the polarity of this nonpolar adjusting reagent and non-electriferous state is lower than extraction agent.
5. according to the reagent of claim 1, the volume ratio of extraction agent and nonpolar correctives is 0.2-10.
6. according to the reagent of claim 1, wherein, described reagent also comprises immune response intermiscibility reagent, and the miscible liquid of this immune response compatibility solution and nonpolar reagent and the miscible formation of extraction agent is immiscible.
7. according to the reagent of claim 6, the volume ratio of extraction agent and immune response intermiscibility reagent is 0.2-2.
8. according to the reagent one of claim 1-7 Suo Shu, wherein, extraction auxiliary agent comprises the inorganic salts that is greater than 20% the solubleness of 10-50 ℃ with water.
9. according to the reagent one of claim 1-7 Suo Shu, wherein, extraction agent is ester class, nitrile, alcohols, ethers, and the potpourri of ester class and nitrile, alcohols or ethers.
10. according to the reagent one of claim 1-7 Suo Shu, wherein, nonpolar correctives is varsol or vegetable oil.
11. according to the reagent of claim 6, and wherein, immune response intermiscibility reagent comprises phosphate buffered solution, citric acid solution, carbonate buffer solution, MES, TRIS, HEPES, one or more in MOPES or physiological saline.
12. according to the reagent of claim 1, and wherein, analyte comprises beta-2-agonists class medicine, chloromycetin, sulfa drugs, tetracycline, nitrofurans metabolin, fluoroquinolones, beta-lactam antibiotic, malachite green, concealed malachite green, crystal violet, melamine, olaquindox, 19-nortestosterone, Trenbolone, norethindrone, stilbestrol, Florfenicol, medroxyprogesterone acetate, estradiol or stable in one or more.
13. 1 kinds of methods of extracting analyte from sample, the method comprises the following steps: (1), allow extraction auxiliary agent contact with sample, this extracts auxiliary agent by the analyte in sample and sample separation; And/or allow analyte be non-charged character, or/and allow in the environment of analyte in being ostracised; (2), allow sample contact with extraction agent, allow analyte be extracted in extraction agent; Wherein, the method is also included in extraction agent and adds nonpolar adjusting reagent, allows nonpolar adjusting reagent and the extraction agent can the miscible liquid of miscible formation.
14. methods according to claim 13, allow sample first contact with extraction auxiliary agent, and then allow sample contact with extraction agent; Or, allow extraction auxiliary agent contact with sample with extraction agent simultaneously; Or, allow sample first contact with extraction agent, and then allow the potpourri of sample and extraction agent contact with extraction auxiliary agent.
15. methods according to claim 13, allow the polarity of extraction agent and the similarity degree of the analyte under non-electriferous state higher than sample and extraction auxiliary agent.
16. methods according to claim 13, the method also comprises allows described nonpolar adjusting reagent mix mutually with immune response intermiscibility reagent with the miscible liquid that extraction agent forms.
17. 1 kinds of methods of extracting analyte from sample, the method comprises the following steps: (1), allow extraction compounding agent solution contact with sample, this extracts auxiliary agent by the analyte in sample and sample separation; And/or allow analyte be non-charged character, or/and allow in the environment of analyte in being ostracised; (2), allow sample contact with extraction agent solution, allow analyte be extracted in extraction agent; Wherein, at step (2) relief, contain the mix reagent that extracts auxiliary agent, extraction agent and analyte and carry out layering, and then allow the upper solution of layering mix with nonpolar adjusting reagent and immunity compatibility solution.
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