CN102426185B - Application of alkyl phosphonic acid reagent to detection of organic phosphorus pesticide in agricultural product by enzymatic inhibition method - Google Patents
Application of alkyl phosphonic acid reagent to detection of organic phosphorus pesticide in agricultural product by enzymatic inhibition method Download PDFInfo
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- CN102426185B CN102426185B CN201110250656.5A CN201110250656A CN102426185B CN 102426185 B CN102426185 B CN 102426185B CN 201110250656 A CN201110250656 A CN 201110250656A CN 102426185 B CN102426185 B CN 102426185B
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Abstract
The invention relates to application of an alkyl phosphonic acid reagent to detection of an organic phosphorus pesticide in an agricultural product by an enzymatic inhibition method. The invention provides an alkyl phosphonic acid reagent used for increasing inhibition effect of organic phosphorous compounds on acetylcholinesterase and application thereof to agricultural product detection. Activity detection on acetylcholinesterase of the invention employs a method of matrix assisted laser desorption ionization-Fourier transform mass spectrum (MALDI-FTMS). The method comprises the following steps: a) extracting and dissolving a sample; b) the alkyl phosphonic acid reagent acting on acetylcholinesterase; c) using a sample extract as an inhibitor to act on the acetylcholinesterase, and using water of a same volume as a blank controller for the other enzyme reaction; d) adding substrate, carrying out an enzyme hydrolysis reaction, investigating enzyme activity, quenching and mixing with the matrix and carrying out an MALDI-FTMS analysis.
Description
Technical field
The present invention relates to a kind of alkyl phosphoric acid reagent and measure the application in the organophosphorus insecticide in the agricultural product in enzyme inhibition method, binding matrix assisted laser desorption ionization-Fourier Transform Ion cyclotron Resonance mass spectrum (MALDI-FTMS) detects activity of enzyme reaction, introducing by alkyl phosphoric acid reagent, regulate organic phosphorus compound to the inhibiting effect of acetylcholinesterase, the content of high-sensitive reflection organic phosphorus compound, thereby realization is quick to organophosphorus pesticide in the agricultural product, high-throughout examination.
Technical background
Organophosphorus is the highly toxic compound of a class, and activity that can acetylcholine esterase inhibition so that the content of neurotransmitter acetylcholine continues to rise in the biosome, and causes pathology and even the death of life entity.Based on its mechanism of action, in agricultural production, organophosphorus pesticide is widely used as pesticide.In recent years, increasing novel organophosphorus pesticide is generalized in the agricultural production, because the use a large amount of lack of standardization of agricultural chemicals, its residual large hidden danger that endangers public security that also day by day becomes in water body and agricultural product has been subject to people's extensive concern.
Reported for separating of with the method for identifying pesticide, comprise thin-layered chromatography, Capillary Electrophoresis, spectrophotometric method etc. (participates in E.E.Potti, P.M.Kaimal, P.G.Nair, J.Forensic Sci.Soc.1975,15,309-311; J.Wang, M.P.Chatrathi, A.Mulchandani, W.Chen, Anal.Chem.2001,73,1804-1808; S.B.Mathewa, A.K.Pillai, V.K.Gupta, Spectrochimica Acta Part A2007,67,1430-1432).Measure organic phosphorous insecticide content in the agricultural product, existing general method is to use gas chromatography or high performance liquid chromatography and mass spectrum or tandem mass spectrum coupling, identify that by the method for direct compartment analysis the composition of contained organophosphorus pesticide is (referring to N.Amini, M.Shariatgorji, C.Crescenzi, G.Thorsen, Anal.Chem.2010,82,290-296; K.Mastovska, S.J.Lehotay, M.Anastassiades, Anal.Chem.2005,77,8129-8137).But these methods often need long pre-treatment and chromatographic separation process, simultaneously, because the Component comparison of agricultural product matrix is complicated, be easy to instrument is polluted, by the mode of library searching carry out qualitative also can be so that analysis result be subject to composing the limitation of known pesticide variety and quantity in the storehouse.
Except the method for these direct-detection organic phosphorus compounds, also comprise in addition detection to the pesticide metabolic product (referring to D.Wessels, D.B.Barr, P.Mendola, Environ.Health erspect.2003,111,1939-1946) and the monitoring by the species of organophosphorus phosphorylation (referring to G.D.Liu, J.Wang, R.Barry, C.Petersen, C.Timchalk, P.L.Gassman, Y.H.Lin, Chem.Eur.J.2008,14,9951-9959).But these methods are not suitable for fast detecting pesticide content, and special recognition component and acceptor also is not easy to obtain simultaneously.In existing report, enzyme inhibition method is surveyed pesticide content, and the content from effect toxicity reflection pesticide can directly reflect toxicity of pesticides harm effectively.Particularly to estimating unknown agricultural chemical compound its significant advantage is arranged, solved some novel agrochemicals and can not obtain by the mode of library searching qualitatively problem.Thereby the method for carrying out enzyme activity determination reflection pesticide content of bibliographical information, mainly contain fluorometric assay (referring to A.G.Hadd, S.C.Jacobson, J.M.Ramsey, Anal.Chem.1999,71,5206-5212), and light oxygen element (referring to A.Zitova, F.C.O ' Mahony, I.N.Kurochkin, D.B.Papkovsky1, Anal.Lett.2010,43,746-1755), eucaryote is expressed (S.C.Xu, A.B.Wu, H.D.Chen, Y.Xie, Y.Q.Xu, L.Zhang, J.Li, D.B.Zhang, Biomol.Eng.2007,24,253-261), and the enzyme sensor method is (referring to H.Schulze, R.D.Schmid, T.T.Bachmann, Anal.Bioanal.Chem.2002,372,268-272).By the conversion ratio of unknown sample and dummy relatively, can judge whether the excessive existence of organic phosphorous insecticide and cause inhibition to enzymatic activity.
In recent years, various mass-spectrometric techniques are carried out enzyme kinetics and examination enzyme inhibitor for detection of the activity of enzyme, have caused that equally the extensive concern of scientists is (referring to Y.Z.Chen, W.L.Tang, J.Mou, Z.Li, Angew.Chem.Int.Ed.2010,49,5278-5283).Wherein, resolve ionization massspectrum carry out also bringing into play aspect enzyme biopsy survey and the examination enzyme inhibitor great function (referring to K.P.Nichols, S.Azoz, H.Gardeniers, Anal.Chem.2008,80,8314-8319).Our Development of Laboratories be used for method that the enzyme biopsy surveys (referring to Z.Xu with ground substance assistant laser parsing-Fourier transform mass spectrum (MALDI-FTMS), S.J.Yao, Y.L.Wei, J.Zhou, L.Zhang, C.H.Wang, Y.L.Guo, J.Am.Soc.Mass Spectrom.2008,19,1849-1855; Z.Xu, H.Y.Wang, S.X.Huang, Y.L.Wei, S.j.Yao, Y.L.Guo, Anal.Chem.2010,82,2113-2118).Based on MALDI mass spectrum salt tolerant, efficient, the advantage such as the sample consumption is few has obvious advantage aspect its enzyme inhibitor in analyzing actual sample.
Summary of the invention
The problem to be solved in the present invention: 1) provide a kind of raising organophosphorous pesticides on acetylcholinesteraseand inhibiting adjuvant; 2) provide the applying step of above-mentioned adjuvant; 3) provide the application of this adjuvant on enzymatic assays organophosphorus pesticide content.
This adjuvant that the present invention relates to, alkyl phosphoric acid by name is abbreviated as n-Alkyl-PA, and structural formula is as follows:
R represents alkyl substituent.The alkyl chain of different length has certain difference for the inhibiting effect impact of organophosphorus pesticide.
By improving the action effect of organophosphorus pesticide, under the organophosphorus pesticide effect of same concentrations, can obtain higher to the enzymeinhibition rate, thereby the effect of having amplified the low strength range agricultural chemicals.In actual application, more can show significantly the toxicity of agricultural chemicals, so that detectability further reduces, obtain the test result of more accurate sensitivity.
Implementation process of the present invention is that the agricultural product sample solution after extracting is got upper strata 1000 μ L, gets supernatant liquor 500 μ L after centrifugal, and nitrogen dries up, and dissolves with the equal volume ultrapure water; With 10% sodium hydroxide solution alkyl phosphoric acid reagent is neutralized to pH=7.0-8.0, parallel two parts: add alkyl phosphoric acid reagent in the acetylcholinesterase solution, enzyme concentration is 1 with alkyl phosphoric acid reagent concentration ratio: 500-1500, recommend 800-1200,37 ℃ of reaction 5-30min; Another part 2 simultaneously) add ultrapure water with volume in the system as blank; Get the upper strata behind the actual sample solution centrifugal, such as 10 μ L, add in a copy of it enzyme reaction system 37 ℃ of reactions; Add simultaneously the ultrapure water of 10 μ L in another part system as blank; Add afterwards the substrate acetylthiocholine, 37 ℃ of reaction 5-30min, add acetonitrile cancellation enzyme reaction, last reactant liquor adds matrix solution and fully mixes, carry out substance assistant laser desorpted ionization-Fourier Transform Ion cyclotron Resonance mass spectrophotometry, the ratio of enzyme hydrolysis substrate and product is represented by the ratio of peak intensity in the mass spectrum in the sample, thereby enzymatic activity is detected: conversion ratio=product/(product+unreacted substrate), wherein unreacted substrate comprises substrate peak and substrate fragment peak, and the calculating of inhibiting rate is according to formula:
I＝(C
t-C
0)/C
0
C
0Be the enzyme reaction conversion ratio of the blank system that do not add the agricultural product sample, C
tEnzyme reaction system conversion ratio for the participation of agricultural product sample.
The present invention mainly experiences following process:
A) extraction of organophosphorus pesticide in the agricultural product sample;
B) alkyl phosphoric acid reagent acts on acetylcholinesterase, causes the variation of enzymatic structure;
C) with the sample extraction thing a) as inhibitor, act on acetylcholinesterase, simultaneously without inhibitor as the blank group;
D) add substrate, carry out the hydrolysis reaction of enzyme, investigate the activity of enzyme, mix with matrix after the cancellation, carry out substance assistant laser desorpted ionization-Fourier Transform Ion cyclotron Resonance mass spectrophotometry.
Described step is that solvent with extract replaces with ultrapure water a), gets rid of organic solvent to the impact of enzymatic activity; B) alkyl phosphoric acid reagent causes the variation of enzymatic structure; C) sample extracting solution acts on acetylcholinesterase; D) enzyme digestion reaction by substrate, and the activity of MALDI-FTMS Mass Spectrometer Method enzyme, by with the contrast of blank group, investigate the inhibiting effect that sample causes the activity of enzyme.
Above-mentioned target analysis compound is organophosphorus insecticide: Hostathion, acephatemet, parathion, orthene, metrifonate, Rogor etc.
Described acetylcholinesterase and substrate acetylthiocholine solution are all used the trishydroxymethylaminomethane of 50mM-hydrochloric acid Tris-HCl (about pH=8.0) configuration.
The excellent 5mM of the concentration of described substrate acetylthiocholine, the preferred 5unit/mL of the concentration of acetylcholinesterase.
The preferred 0.2mM of described alkyl phosphoric acid reagent concentration.Preferred 37 ℃ of reaction conditions, 30 minutes.
The process of described acetylcholinesterase hydrolysis substrate acetylthiocholine is as follows:
The substrate acetylthiocholine generates acetic acid and thiocholine under the effect of acetylcholinesterase.
Described agricultural product sample pre-treatments is as follows: about agricultural product sample 1g, add 5mL and analyze pure acetone, 0.1gNaCl, 0.4g MgCl
2, vibrated 5 minutes, it is centrifugal to get upper strata 1mL, gets supernatant liquor 500 μ L again, and nitrogen dries up rear with same volume ponding 500 μ L dissolving, and is centrifugal again, and supernatant liquor is as the last enzyme reaction solution that participates in.
Used matrix solution, preparation process is as follows: 50mg matrix DHB (DHB) dissolves with 500 μ L methyl alcohol, and adds 0.1% (V/V) trifluoroacetic acid, (glycerin/water=60: 40 V/V) is mixed with isopyknic glycerine water solution again.
Mass spectrophotometry afterwards is to resolve the Fourier transform mass spectrum at ground substance assistant laser, carries out under the positive ion mode, quantitatively calculates according to gained spectrogram information again.
Have the following advantages aspect the residual quantity of the present invention's organophosphorus pesticide in measuring agricultural product: at first, alkyl phosphoric acid reagent provided by the invention can be regulated by the different length alkyl inhibiting effect of organophosphorous pesticides on acetylcholinesteraseand.
Secondly, this class alkyl phosphoric acid reagent provided by the present invention is simple and easy to, and goes for improving during conventional enzyme process detects, to obtain lower quantitative detectability.Toxicity according to organophosphorus pesticide is different, and the maximum maximum permission quantity that GB (Ministry of Health P.R.China, GB2763-2005 3) provides is also different.In investigation process of the present invention, high toxicity compound test limit has reached 0.005 μ g/L, and low toxicity compounds has also reached 0.05 μ g/L, and more traditional enzyme suppresses method, and detectability has reduced by 10
2-10
3Doubly.
Again, the present invention uses the activity of MALDI-FTMS Mass Spectrometer Method enzyme, and based on its salt tolerant, high flux does not need the complicated advantages such as chromatographic separation process, has obvious advantage in the organophosphorus pesticide content in detecting actual sample.The present invention adopts the method for mixing point sample, the sample spot good uniformity, and reappearance is high.
At last, the inhibiting method of this raising organophosphorous pesticides on acetylcholinesteraseand provided by the present invention is applied to detecting the content of the organophosphorus pesticide in the cowpea sample, the extracting method normative reference method of sample, the result has verified reliability of the present invention through the contrast of liquid phase tandem mass spectrum.
Description of drawings
Fig. 1 is the analysis synoptic diagram of sample, and MALDI-FTMS is ground substance assistant laser parsing-Fourier transform mass spectrum;
Fig. 2 is the mass spectra peak of acetylcholinesterase hydrolysis substrate, and wherein matrix peak is the peak of matrix;
Fig. 3-6 is the agricultural chemicals orthene, malathion, Rogor, DDVP concentration and its are to the inhibiting relation of acetylcholine esterase active, horizontal ordinate acephate concentration, malathion concentration, rogor concentration, DDVP concentration is the agricultural chemicals orthene, the malathion, Rogor, the concentration of DDVP, unit is every liter of microgram, and ordinate inhibition is the inhibiting rate that acetylcholinesterase is subject to.
Fig. 7 adds n-octyl phosphoric acid and does not add fashionable for contrast, the acetylcholinesterase inhibiting rate of gained relies on the curve of organic phosphorus compound concentration, horizontal ordinate acephate concentration is the concentration of agricultural chemicals orthene, unit is every liter of microgram, and ordinate inhibition is the inhibiting rate that acetylcholinesterase is subject to.
Description of drawings:
Among Fig. 7 ● representative adds the later on inhibiting rate curve of acetylcholinesterase of n-octyl Phosphoric Acid, and ■ is not for adding the inhibiting rate curve of alkyl phosphoric acid adjuvant.
Specific implementation method
Following specific implementation method will help to understand the present invention, but not limit content of the present invention.
Embodiment 1: the n-octyl Phosphoric Acid is applied to the extract of enzymatic assays cowpea sample as adjuvant
One, the investigation acetylcholinesterase is subjected to the relation between inhibiting effect and the organophosphorus pesticide concentration, and checking uses MALDI-FTMS to do the feasibility that enzyme inhibition method is measured pesticide content.
1. parallel some parts: acetylcholinesterase (5unit/mL, 10 μ L), concentration be the orthene standard solution of 10-100 μ g/L as inhibitor, get 10 μ L and add respectively in the enzyme reaction system, the ultrapure water of 10 μ L is as blank, and 37 ℃ were reacted 30 minutes.
2. the substrate solution acetylthiocholine 10 μ L that add respectively 5mM in each enzyme reaction solution, 37 ℃ were reacted 20 minutes.
3. add respectively 30 μ L acetonitrile cancellation enzyme reactions, add again 65 μ L matrix solutions, get 2 μ L point targets after the mixing and carry out substance assistant laser desorpted ionization-Fourier Transform Ion cyclotron Resonance mass spectrophotometry.
4. according to the MALDI-FTMS analysis result, draw the relation between different orthenes and the corresponding enzyme inhibition rate.
In the MALDI-FTMS spectrogram, we mainly monitor substrate acetylthiocholine (m/z162), product thiocholine (m/z120), and the fragment peak of substrate acetylthiocholine (m/z103), and this three's abundance changes.Because three's structural similarity, through investigate in MALDI-FTMS go out the peak situation and its concentration change has good linear dependence, calculate so refer to concentration with abundance.Conversion ratio=product/(product+unreacted substrate), wherein unreacted substrate comprises substrate peak and substrate fragment peak, and inhibiting rate is calculated as follows:
I＝(C
t-C
0)/C
0
C
0Be the enzyme reaction conversion ratio of the blank system that do not add the agricultural product sample, C
tEnzyme reaction system conversion ratio for the participation of agricultural product sample.
Attached: the configuration of matrix solution is as follows:
50mg matrix DHB (DHB) dissolves with 500 μ L methyl alcohol, and adds 0.1% (V/V) trifluoroacetic acid, and (glycerin/water=60: 40 V/V) is mixed with isopyknic glycerine water solution again.
Two, investigate n-octyl phosphoric acid and acetylcholinesterase is subjected to organic phosphorus compound is inhibiting to be affected.
1. parallel some parts: acetylcholinesterase (5unit/mL, 10 μ L) adds 10 μ L n-octyl Phosphoric Acids (0.2mM is neutralized to pH=7.0-8.0 with NaOH solution), and 37 ℃ were reacted 30 minutes;
Concentration be the orthene standard solution of 10-100 μ g/L as inhibitor, get 10 μ L and add respectively in the enzyme reaction system, the ultrapure water of 10 μ L is as blank, 37 ℃ of reactions 30 minutes.
3. the substrate solution acetylthiocholine 10 μ L that add respectively 5mM in each enzyme reaction solution, 37 ℃ were reacted 20 minutes.
4. add respectively 30 μ L acetonitrile cancellation enzyme reactions, add again 65 μ L matrix solutions, get 2 μ L point targets after the mixing and carry out the MALDI-FTMS analysis.
5. according to the MALDI-FTMS analysis result, calculate substrate conversion efficiency and inhibiting rate, draw the relation between different orthenes and the corresponding enzyme inhibition rate, and relatively map with the enzyme inhibition rate that does not add n-octyl phosphoric acid, as shown in Figure 3.
Three, the n-octyl Phosphoric Acid be applied to enzymatic assays cowpea sample as adjuvant the extract acetylcholinesterase in the presence of n-octyl phosphoric acid, take the cowpea extract as inhibitor, operation steps is as follows:
1. cowpea sample extraction: about cowpea sample 1g, add 5mL and analyze pure acetone, 0.1gNaCl, 0.4g MgCl
2, vibrated 5 minutes, get upper strata 1mL centrifugal (10000rpm, 5 minutes), get again supernatant liquor 500 μ L, nitrogen dries up rear with same volume ponding 500 μ L dissolving, centrifugal (10000rpm, 5 minutes), supernatant liquor is as the last enzyme reaction solution that participates in.
2. the cowpea sample solution of gained is got upper strata 10 μ L after centrifugal, adds in a copy of it enzyme reaction system 37 ℃ of reactions 30 minutes; Add simultaneously the ultrapure water of 10 μ L in another part system as blank;
3. parallel two parts: acetylcholinesterase (5unit/mL, 10 μ L) adds 10 μ L n-octyl Phosphoric Acids (0.2mM is neutralized to pH=7.0-8.0 with NaOH solution), and 37 ℃ were reacted 30 minutes;
4. in the solution of two parts of enzyme reactions, add subsequently substrate acetylthiocholine (5mM, 10 μ L), 37 ℃ were reacted 20 minutes, add 30 μ L acetonitrile cancellation enzyme reactions, last reactant liquor adds 65 μ L matrix solutions and fully mixes, get 2 μ L points on the stainless steel target, carry out substance assistant laser desorpted ionization-Fourier Transform Ion cyclotron Resonance mass spectrophotometry.
5.MALDI-FTMS analysis result is as follows: through calculating, the inhibiting rate of three kinds of cowpea samples is respectively common cowpea: 76.8%, 100%; Organic cowpea: 13.3%.
This result shows may contain the organophosphorus pesticide strong to acetylcholinesteraseinhibition inhibition in the common cowpea, second half carries out liquid phase tandem mass spectrum method LC-MS/MS comparison same extract, the result proves and contains respectively organophosphorus pesticide orthene and Hostathion in the common cowpea, and do not detect the component relevant with organophosphorus pesticide in organic cowpea.
Four, the n-octyl Phosphoric Acid is applied to the enzymatic assays green vegetables as adjuvant, and the extract acetylcholinesterase of cucumber and apple sample is in the presence of n-octyl phosphoric acid, and take sample extraction liquid as inhibitor, operation steps is as follows:
1. green vegetables, cucumber, apple sample is extracted: about sample 1g, add 5mL and analyze pure acetone, 0.1gNaCl, 0.4g MgCl
2, vibrated 5 minutes, get upper strata 1mL centrifugal (10000rpm, 5 minutes), get again supernatant liquor 500 μ L, nitrogen dries up rear with same volume ponding 500 μ L dissolving, centrifugal (10000rpm, 5 minutes), supernatant liquor is as the last enzyme reaction solution that participates in.
2. the sample solution of gained is got upper strata 10 μ L after centrifugal, adds in a copy of it enzyme reaction system 37 ℃ of reactions 30 minutes; Add simultaneously the ultrapure water of 10 μ L in another part system as blank;
3. parallel two parts: acetylcholinesterase (5unit/mL, 10 μ L) adds 10 μ L n-octyl Phosphoric Acids (0.2mM is neutralized to pH=7.0-8.0 with NaOH solution), and 37 ℃ were reacted 30 minutes;
4. in the solution of two parts of enzyme reactions, add subsequently substrate acetylthiocholine (5mM, 10 μ L), 37 ℃ were reacted 20 minutes, add 30 μ L acetonitrile cancellation enzyme reactions, last reactant liquor adds 65 μ L matrix solutions and fully mixes, get 2 μ L points on the stainless steel target, carry out substance assistant laser desorpted ionization-Fourier Transform Ion cyclotron Resonance mass spectrophotometry.
5.MALDI-FTMS analysis result is as follows: through calculating, the inhibiting rate of two kinds of samples is respectively common green vegetables: 67.6%; Cucumber: 52.4%; Apple: 100%.
This result shows may contain the organophosphorus pesticide strong to acetylcholinesteraseinhibition inhibition in green vegetables and the apple, utilize the auxiliary reagent enzyme process fast and effeciently to carry out the examination early warning to each agricultural products.
Claims (8)
1. the application of class alkyl phosphoric acid reagent organophosphorus pesticide in enzyme inhibition method mensuration agricultural product is characterized in that described alkyl phosphoric acid reagent has following structural formula:
2. application as claimed in claim 1 is characterized in that being undertaken by following steps:
1) sample solution after extracting agricultural product is got upper strata 1000 μ L, gets supernatant liquor 500 μ L after centrifugal, and nitrogen dries up, and with the water-soluble solution of equal volume;
2) with 10% sodium hydroxide solution alkyl phosphoric acid reagent is neutralized to pH=7.0-8.0, parallel two parts: acetylcholinesterase adds equal-volume alkyl phosphoric acid reagent, and enzyme concentration is 1:500-1500 with alkyl phosphoric acid reagent concentration ratio;
3) step 1) sample solution of gained is got the upper strata after centrifugal, adds a copy of it step 2) the enzyme reaction system in react 5-30min; Another part 2 simultaneously) add ultrapure water with volume in the system as blank;
4) in the solution of step 3), add and the isopyknic substrate acetylthiocholine of inhibitor, 37 ℃ of reaction 5-30min, add acetonitrile cancellation enzyme reaction, add at last and fully mix with the isopyknic matrix solution of end reaction liquid, carry out substance assistant laser desorpted ionization-Fourier Transform Ion cyclotron Resonance mass spectrophotometry, the ratio of enzyme hydrolysis substrate and product is represented by the ratio of peak intensity in the mass spectrum in the sample, enzymatic activity is detected: conversion ratio=product/(product+unreacted substrate), wherein unreacted substrate comprises substrate peak and substrate fragment peak, inhibiting rate I=(C
0-C
t)/C
0, C
0Be the enzyme reaction conversion ratio of the blank system that do not add the agricultural product sample, C
tEnzyme reaction system conversion ratio for the participation of agricultural product sample.
3. application as claimed in claim 2 is characterized in that step 1) in the solvent of extract is replaced with ultrapure water, get rid of organic solvent to the impact of enzymatic activity; Step 2) alkyl phosphoric acid reagent acts on acetylcholinesterase in, causes the variation of enzymatic structure; 3) in the sample extraction thing as inhibitor, act on acetylcholinesterase, simultaneously without inhibitor as the blank group; 4) add substrate in, carry out the hydrolysis reaction of enzyme, investigate the activity of enzyme, and cancellation mixes with matrix, do substance assistant laser desorpted ionization-Fourier Transform Ion cyclotron Resonance mass spectrophotometry.
4. application as claimed in claim 1 is characterized in that described organophosphorus pesticide is Hostathion, acephatemet, parathion, orthene, metrifonate or Rogor.
5. application as claimed in claim 2 is characterized in that described acetylcholinesterase and substrate acetylthiocholine solution all uses the buffer preparation of trishydroxymethylaminomethane-hydrochloric acid pH=8.0.
6. application as claimed in claim 2, it is characterized in that described sample solution after extracting agricultural product: agricultural product sample 1g, add 5mL and analyze pure acetone, 0.1g sodium chloride, 0.4g magnesium chloride, vibrated 5 minutes, it is centrifugal to get upper strata 1mL, gets supernatant liquor again, and nitrogen dries up rear with the water-soluble solution of equal volume, centrifugal again, supernatant liquor is as the last enzyme reaction solution that participates in.
7. application as claimed in claim 2 is characterized in that the process of described acetylcholinesterase hydrolysis substrate acetylthiocholine is as follows:
8. application as claimed in claim 2 is characterized in that the preparation process of described matrix solution is as follows: 50mg matrix DHB dissolves with 500 μ L methyl alcohol, and adding 0.1%V/V trifluoroacetic acid, mix again glycerin/water=60:40, V/V with isopyknic glycerine water solution.
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| EP1460135A1 (en) * | 2002-12-09 | 2004-09-22 | Quality Engineering s.r.l. | Acetylcholinesterase method to detect organophosphorate and carbamate insecticides in water, foods and beverages |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1460135A1 (en) * | 2002-12-09 | 2004-09-22 | Quality Engineering s.r.l. | Acetylcholinesterase method to detect organophosphorate and carbamate insecticides in water, foods and beverages |
| CN1632538A (en) * | 2003-12-25 | 2005-06-29 | 中国科学院大连化学物理研究所 | Fast detection method with high sensitivity for pesticide residue |
| CN101706431A (en) * | 2009-06-09 | 2010-05-12 | 华中农业大学 | Chemiluminescence method for detecting organophosphorus pesticide |
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