CN113203720B - Method for detecting residual amount of dichlorvos in aquatic product by fluorescence analysis method and detection kit - Google Patents
Method for detecting residual amount of dichlorvos in aquatic product by fluorescence analysis method and detection kit Download PDFInfo
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Abstract
The invention relates to a method for detecting the residual amount of dichlorvos in an aquatic product by a fluorescence analysis method and a detection kit. The method comprises the following steps: pretreating an aquatic product sample to obtain an acetonitrile extracting solution, adding a hydrochloric acid solution, sodium chloride and a sodium nonanoate extracting agent into the acetonitrile extracting solution to realize efficient extraction of dichlorvos in the sample, solidifying an organic phase in an ice bath, removing a lower-layer water phase, adding an ammonia water solution into the remaining upper-layer extracting solution, adding a resorcinol solution into the upper-layer extracting solution to react, and detecting a fluorescence value; and preparing standard solutions of the dichlorvos with different concentrations for the detection, drawing a standard curve after measuring a fluorescence value, and obtaining the content of the dichlorvos in the sample according to the standard curve. The method established by the invention has the advantages of environmental protection, high efficiency, good extraction effect, high extraction efficiency and the like, and the used detection instrument is simple to operate, accurate and reliable in experimental result, high in sensitivity and good in reproducibility, and can realize on-site rapid detection.
Description
Technical Field
The invention relates to a method for detecting the residual amount of dichlorvos in aquatic products by a fluorescence analysis method and a detection kit, belonging to the technical field of food detection.
Background
Dichlorvos (DDVP), the scientific name of O, O-dimethyl-O- (2, 2-dichlorovinyl) phosphate, is a high-efficiency organophosphorus insecticide and is widely applied to agricultural production. However, due to unreasonable use and abuse, the amount of the residual dichlorvos in the water body is increased under the influence of rainfall and the like, and the residual dichlorvos is continuously enriched in the aquatic organisms, so that the water environment and the aquatic organisms are seriously influenced. After people eat aquatic products containing dichlorvos, the dichlorvos can be continuously accumulated in the human body, so that the harmful effect is generated on the human body. Dichlorvos combines with cholinesterase in human body, so that cholinesterase loses the function of hydrolyzing acetylcholine, and cholinergic neurotransmitter is accumulated in large quantity, thereby causing serious nerve dysfunction, especially respiratory dysfunction, and further influencing life activities, and death can be caused by pulmonary edema, cerebral edema and respiratory paralysis in serious cases. Sudden delayed death may also occur in severely poisoned patients. Therefore, a rapid and convenient detection method of dichlorvos needs to be established for monitoring the residual quantity in aquatic products.
There are many methods for detecting the residual amount of dichlorvos at home and abroad, such as gas chromatography, thin-layer chromatography, gas chromatography-mass spectrometry, immunosensor, and the like. Although the method has high detection efficiency, good selectivity and wide application range, the method has the defects of expensive instruments, professional operators, complex procedures, fussy sample processing process, unstable synthesized materials and the like, thereby greatly limiting the application of the method in field inspection. Therefore, an efficient and environment-friendly extraction and detection method with accurate detection results and simple instrument operation is needed to be established, and instruments capable of reaching detection conditions in a common laboratory are used for measuring the residual amount of dichlorvos in aquatic products.
Disclosure of Invention
Technical problem to be solved
In order to solve the problems in the prior art, the invention provides a method for detecting the residual amount of dichlorvos in an aquatic product by a fluorescence analysis method and a detection kit, and particularly relates to a rapid, accurate and convenient fluorescence analysis detection method for the residual amount of dichlorvos in the aquatic product.
(II) technical scheme
In order to achieve the purpose, the invention adopts the main technical scheme that:
a method for detecting residual quantity of dichlorvos in aquatic products by a fluorescence analysis method comprises the following steps:
s1, sample pretreatment: crushing an aquatic product sample, accurately weighing the crushed sample in a centrifugal tube, adding an acetonitrile solution, carrying out vortex and standing, filtering a supernatant with a membrane, and placing the filtered supernatant in the centrifugal tube;
s2, sample extraction: adding hydrochloric acid solution, sodium chloride and sodium nonanoate into a new centrifugal tube, centrifuging after sample solution is layered, solidifying in ice bath, pouring out the lower layer solution, and reserving the upper layer extracting solution of a solid phase;
s3, sample detection: taking the upper layer extract after melting, adding ammonia water and resorcinol solution for reaction in water bath, and performing fluorescence detection by using a multifunctional enzyme-linked immunosorbent assay (ELISA) instrument after the color is stable;
s4, making a standard song: preparing standard dichlorvos with different concentrations, directly adding standard dichlorvos solutions with different concentrations into a centrifugal tube, and completely volatilizing an organic solvent after standing; adding acetonitrile, and performing extraction and fluorescence detection according to steps S1, S2 and S3; drawing a standard curve by taking the concentration of the dichlorvos as an abscissa and the fluorescence value as an ordinate;
s5, detection result: and substituting the fluorescence value of the sample measured in the S3 into the standard curve to obtain the residual amount of the dichlorvos in the sample.
The method as described above, preferably, in step S1, the pulverized sample mass is 1g, and the volume of the acetonitrile is 1 mL; the vortex rotation speed is 2500r/min, the vortex time is 2-5 min, and the standing time is 3-5 min; the filter membrane is a 0.22 μm organic filter membrane.
In the method, preferably, in step S2, the volume of the filtrate is 0.5 to 0.9 mL, the concentration of the hydrochloric acid solution is 0.1 to 0.7mol/L, and the volume is 4.5 mL; the dosage of the sodium chloride is 75-225 mg; the dosage of the sodium nonanoate is 100-200 mg; the centrifugation condition is 3000-8000 r/min, and the centrifugation time is 3-10 min.
In the method, the volume of the filtrate is 0.5mL, and the concentration of the hydrochloric acid solution is 0.5 mol/L; the dosage of the sodium chloride is 150 mg; the dosage of the sodium pelargonate is 150 mg; the centrifugation condition is 5000r/min, and the centrifugation time is 5 min.
In the method as described above, preferably, in step S3, the volume of the upper layer extract is 50 μ L; the mass fraction of the ammonia water is 15-25%, and the volume of the ammonia water is 100 mu L; the concentration of the resorcinol solution is 45-225 mmol/L, absolute ethyl alcohol is used for preparation, and the volume of the resorcinol solution is 200 mu L.
In the method, in step S3, the temperature of the water bath is preferably 25-35 ℃, and the time of the water bath is preferably 5-15 min.
In the method, preferably, in step S3, the ammonia water is 25% by mass, the resorcinol solution has a concentration of 90mmol/L, and is prepared with absolute ethanol, the water bath temperature is 25 ℃, and the water bath time is 10 min.
In the method described above, preferably, in step S3, the excitation wavelength used in the measurement of the fluorescence intensity by the microplate reader is 275nm, and the emission wavelength is 315 nm.
A kit for detecting the residual amount of dichlorvos in aquatic products by a fluorescence analysis method comprises an acetonitrile solution, sodium chloride, a hydrochloric acid solution, ammonia water and a resorcinol solution.
Further, preferably, the concentration of the hydrochloric acid solution is 0.1-0.7 mol/L, the mass fraction of the ammonia water is 15-25%, and the concentration of the resorcinol solution is 45-225 mmol/L.
In the research, the invention discovers that sodium nonanoate is added into an acidic sample solution to generate a nonanoic acid extracting agent in situ, and the efficient extraction of dichlorvos is completed under the assistance of salting-out action in the process of fully contacting with a water phase, namely sodium nonanoate is converted into nonanoic acid under the acidic condition, the nonanoic acid extracts the dichlorvos, the nonanoic acid is layered with the water solution, an organic phase, namely the extracting agent containing the dichlorvos, is positioned at the upper layer, then the organic phase is solidified in ice bath, and the lower water phase is discarded; after the melted extractant is added into alkaline ammonia water solution, the pelargonic acid is converted into pelargonic acid salt which is mixed and dissolved with water to form a uniform phase, thereby facilitating the subsequent detection. In addition, dichlorvos is hydrolyzed into dichloroacetaldehyde under the alkaline condition, and the dichlorovos and resorcinol added subsequently undergo a condensation reaction to generate a fluorescent substance, so that the fluorescent quantitative detection of the dichlorvos is realized. According to the invention, ice bath solidification is adopted for the first time, and multiple experimental researches find that the freezing point of the adopted extractant is higher than that of the sample solution, so that more extractants can be collected by solidification, and the separation from the sample solution is facilitated.
(III) advantageous effects
The invention has the beneficial effects that:
the invention provides a novel method and a detection kit for detecting the residual amount of dichlorvos in aquatic products by a fluorescence analysis method. The sodium nonanoate has the advantages of greenness, no toxicity and small influence on the environment, and meets the requirements of green chemical experiments; and sodium nonanoate has the properties of low density and low melting point, and is solidified at the upper end of a centrifugal tube during ice bath after centrifugation, so that the collection difficulty is reduced, and the extraction efficiency is improved.
The method for detecting the residual amount of dichlorvos in the aquatic products by the fluorescence analysis method has the advantages of environmental friendliness, high efficiency, good extraction effect and high extraction efficiency in the detection process, is simple to operate by using an instrument, does not need to build a complex device, does not need to synthesize any substance in the detection process, is less in restriction effect, mild in reaction condition, simple and rapid to operate, high in sensitivity, accurate and reliable in experimental result, good in reproducibility and capable of realizing rapid detection on site.
The detection kit for detecting the residual amount of dichlorvos in the aquatic products by the fluorescence analysis method provided by the invention has the advantages of easily available reagent sources, no toxicity, environmental friendliness and the like.
The method disclosed by the invention is based on switchable medium-chain fatty acids, combines an efficient green microextraction pretreatment technology with a convenient high-throughput fluorescence analysis technology, solves the problem that an organic extractant is not compatible with a water-phase detection system, and establishes a method for efficiently extracting and detecting the residual amount of dichlorvos in aquatic products.
Drawings
FIG. 1 is a photograph of different reaction systems under an ultraviolet lamp;
FIG. 2 is a fluorescence spectrum of different reaction systems;
FIG. 3 is a fluorescence spectrum of the final product at the optimum excitation wavelength (Ex) and emission wavelength (Em);
FIG. 4 is a graph showing the effect of extractant species on fluorescence intensity;
FIG. 5 is a graph showing the effect of extractant dosage on fluorescence intensity;
FIG. 6 is a graph showing the effect of HCl concentration on fluorescence intensity;
FIG. 7 shows the effect of NaCl dosage on fluorescence intensity;
FIG. 8 shows NH 3 ·H 2 The effect of O concentration on fluorescence intensity;
FIG. 9 is a graph showing the effect of resorcinol concentration on fluorescence intensity;
FIG. 10 is a graph showing the effect of heating temperature on fluorescence intensity;
FIG. 11 is a graph showing the effect of heating time on fluorescence intensity;
figure 12 is a linear plot of dichlorvos.
Detailed Description
In the invention, the pretreatment part adopts an organic solvent to complete extraction, and the detection part belongs to a water-phase system. The extraction in the organic phase is realized by the advantage that the extraction agent can switch phases under the acid-base condition, and the reaction with a fluorescent aqueous phase system can be completed in the aqueous phase. The problem that a pretreatment part and a detection part adopt organic phases which are incompatible with each other by adopting water in the prior art is solved.
For the purpose of better explaining the present invention and to facilitate understanding, the present invention will be described in detail by way of specific embodiments with reference to the accompanying drawings. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the reagents used are commercially available from SpectraMax i3x multifunctional microplate reader (Molecular Devices, China).
Example 1 verification of experimental feasibility
The experimental method comprises the following steps: 8 centrifuge tubes of 2mL were prepared, and four different reaction systems a were configured: dichlorvos + NH 3 ·H 2 An O + resorcinol solution; b: dichlorvos + NH 3 ·H 2 O；c：NH 3 ·H 2 O + resorcinol solution; d: dichlorvos + resorcinol solution. Each reaction system is provided with two parallel groups, and the reaction systems are all placed at the same temperature for the same time. The concentrations of the reagents used in the experimental procedure were respectively: the concentration of dichlorvos is 0.1mg/kg (methanol solution, available from Nantong Jiangshan chemical pesticide Co., Ltd.), NH 3 ·H 2 The mass fraction of O is 25 percent, the concentration of the resorcinol solution is 450mmol/L, and ethanol is used as a solventAnd (5) preparing. The addition amount of each reagent is as follows: dichlorvos of 200 mu L, NH 3 ·H 2 The O content is 300 mu L, the resorcinol solution content is 0.5mL, after the water bath reaction is carried out for 10min at the temperature of 25 ℃, whether the fluorescence exists or not is observed under an ultraviolet lamp, and the experimental result is shown in figure 1. The result shows that only the reaction system a has stronger fluorescence under an ultraviolet lamp. The material has strong fluorescence, namely dichlorvos is hydrolyzed under alkaline conditions to generate dichloroacetaldehyde, and the dichloroacetaldehyde and resorcinol undergo condensation reaction to generate a red substance, and the material has strong fluorescence, thereby verifying the feasibility of the method for determining the content of the dichlorvos.
In addition, compounds similar in structure to resorcinol have also been tested, such as: 1,2,3, 5-benzenetetraol, 2, 5-dichlorophenol, m-cresol, phenol, p-cresol, nitrosophenol; however, these substances do not undergo a condensation reaction with dichloroacetaldehyde and no fluorescence is produced.
Example 2 determination of optimal excitation wavelength (Ex) and emission wavelength (Em) of the final product and fluorescence spectra of different reaction systems
The system a after the reaction in the embodiment 1 is taken to be 200 mu L and placed in a 96-well plate, a multifunctional microplate reader is adopted, the excitation wavelength (Ex) is adjusted to be the lowest within the available range, on the basis, the emission spectrum is swept, the highest point on the spectrum is the emission wavelength, then the emission wavelength (Em) is unchanged, the excitation spectrum is swept, the highest point on the spectrum is the excitation wavelength, and the like, when the excitation spectrum and the emission spectrum are in an axisymmetric pattern, namely, the optimal emission wavelength in the emission spectrum obtained by fixing the scanning of a certain excitation wavelength is consistent with the optimal excitation wavelength in the excitation spectrum obtained by fixing the scanning of a certain emission wavelength, the optimal excitation wavelength (Ex) and the emission wavelength (Em) of the substance are determined. The emission spectra of different reaction systems scanned at the optimal excitation wavelength are shown in fig. 2 and 3.
The results show that the optimal excitation wavelength (Ex) of the final product of the reaction system a is 275nm, and the optimal emission wavelength (Em) is 315 nm. Only the reaction system a in the four reaction systems generates strong fluorescence intensity at 315nm, which shows that dichlorvos are hydrolyzed to generate dichloroacetaldehyde only under alkaline conditions, and the dichloroacetaldehyde and resorcinol undergo condensation reaction to generate fluorescent substances, so that the method is feasible for detecting the dichlorvos content in the aquatic products.
Example 3 optimization of reaction conditions
A. Kinds of the extracting agent
(1) The experimental method comprises the following steps: weighing 1g of pulverized sample (sea cucumber, grass carp, or abalone), adding 100 μ L of dichlorvos (0.1mg/kg, methanol solution), standing for 20min, adding 1mL of acetonitrile solution, performing vortex extraction at 2500r/min for 3min, and filtering the extractive solution with organic filter membrane (0.22 μm). A500. mu.L sample of the filtrate was taken and added to 4.5mL of a 0.5mol/L aqueous hydrochloric acid solution in a 10mL centrifuge tube. Then 150mg of sodium chloride was added, and 150mg of sodium salt of medium-chain fatty acid (sodium valerate, sodium caproate, sodium caprylate, sodium nonanoate and sodium decanoate) was added after the sodium chloride was completely dissolved. The sodium salt of the medium-chain fatty acid originally generates the medium-chain fatty acid under the acidic condition and is uniformly dispersed into the sample solution as an extracting agent (valeric acid, caproic acid, caprylic acid, pelargonic acid and capric acid), so that the high-efficiency extraction of the object to be detected is realized. Centrifuging for 5min under the condition of 5000r/min, wherein the medium-chain fatty acid is not dissolved in the water solution, layering occurs, the dichlorvos is dissolved in the medium-chain fatty acid layer, the organic phase is positioned on the upper layer of the centrifugal tube, and the lower water phase is discarded after ice bath solidification. Putting 50 mu L of the extract into a 2mL centrifuge tube, and then adding 100 mu L of 25% ammonia water by mass fraction. Finally 200. mu.L resorcinol (prepared with absolute ethanol (AR)) was added at a concentration of 90 mmol/L. After reacting in water bath at 25 ℃ for 10min, 200. mu.L of the mixture was pipetted and placed in a 96-well plate, and the fluorescence intensity was measured at an optimum excitation wavelength (Ex) of 275nm and an optimum emission wavelength (Em) of 315 nm.
(2) The experimental results are as follows: as shown in fig. 4.
(3) And (4) analyzing results: the extractant plays a decisive role in the extraction process. Under the same extraction conditions, the influence of five extracting agents, namely sodium valerate, sodium caproate, sodium caprylate, sodium nonanoate and sodium caprate, on the extraction effect is examined. The results in FIG. 4 show that sodium nonanoate has the highest fluorescence intensity, indicating that it is most effective in extraction. The sodium caprate has a high freezing point and is easily influenced by the change of room temperature, so that the recovery volume of the extractant is unstable. Sodium nonanoate was therefore chosen as extractant.
B. The amount of the extractant
(1) The experimental method comprises the following steps: the same experimental procedure as in A in example 3, except that different amounts of sodium nonanoate, 50mg, 100mg, 150mg, 200mg, 300mg, respectively, were added. Other reaction conditions are as follows: the concentration of dichlorvos is 0.1mg/kg, the concentration of hydrochloric acid is 0.5mol/L, the concentration of sodium chloride is 150mg, the mass fraction of ammonia water is 25%, and the concentration of resorcinol is 90 mmol/L.
(2) The experimental results are as follows: as shown in fig. 5.
(3) And (4) analyzing results: when the dosage of the sodium nonanoate is within the range of 50-150 mg, the fluorescence value is gradually increased along with the increase of the content of the sodium nonanoate, and when the dosage is within the range of 150-300 mg, the fluorescence value is gradually reduced along with the increase of the content of the sodium nonanoate. The content of sodium nonanoate reaches the maximum when the content is 150mg, because the substance to be detected cannot be completely recovered when the dosage of the extracting agent is less, the fluorescence intensity gradually increases as the dosage of the extracting agent increases and decreases when the dosage of the extracting agent further increases. Therefore, 100-200 mg of sodium nonanoate is preferred for extraction, and 150mg of sodium nonanoate is most preferred for extraction.
Concentration of C.HCl
(1) The experimental conditions are as follows: the same experimental method as A in example 3, except that hydrochloric acid solutions with different concentrations of 0.1mol/L, 0.5mol/L and 1mol/L are added respectively, and other reaction conditions: the concentration of dichlorvos is 0.1mg/kg, the concentration of sodium chloride is 150mg, the concentration of sodium nonanoate is 150mg, the mass fraction of ammonia water is 25%, and the concentration of resorcinol is 90 mmol/L.
(2) The experimental results are as follows: as shown in fig. 6.
(3) And (4) analyzing results: when the concentration of the hydrochloric acid is within the range of 0.1-0.5 mol/L, the fluorescence value gradually increases and remarkably increases along with the decrease of the concentration of the hydrochloric acid, and when the concentration of the hydrochloric acid is within the range of 0.5-1 mol/L, the fluorescence value remarkably decreases along with the increase of the concentration of the hydrochloric acid. This is because sodium nonanoate cannot be completely converted into nonanoic acid extractant due to too low a hydrochloric acid concentration, which leads to a decrease in extraction efficiency; when the concentration of hydrochloric acid is too high, excessive ammonia water is consumed to waste reagents when sufficient alkaline environment is created for the dichlorvos. The subsequent operation of the experiment is affected by too high or too low concentration of hydrochloric acid, so the concentration of HCl needs to be strictly controlled, preferably 0.1-0.7 mol/L, and most preferably 0.5mol/L hydrochloric acid solution.
Amount of NaCl used
(1) The reaction conditions are as follows: the same experimental procedure as in A in example 3, except that different contents of sodium chloride were added, 50mg, 75mg, 100mg, 125mg, 150mg, 175mg, 200mg, 250mg, 275mg, and 300mg, respectively. Other reaction conditions were: the concentration of dichlorvos is 0.1mg/kg, the concentration of hydrochloric acid is 0.5mol/L, the concentration of sodium nonanoate is 150mg, the mass fraction of ammonia water is 25%, and the concentration of resorcinol solution is 90 mmol/L.
(2) The experimental results are as follows: as shown in fig. 7.
(3) And (4) analyzing results: when the dosage of the sodium chloride is within the range of 50-150 mg, the fluorescence value is gradually increased along with the increase of the dosage of the sodium chloride, and when the dosage of the sodium chloride is within the range of 150-300 mg, the fluorescence value is gradually decreased along with the increase of the dosage of the sodium chloride. When the using amount of sodium chloride is insufficient, organic components (namely dichlorvos and pelargonic acid) in the solution cannot be completely separated out from the mixed solution, and the insufficient salting-out can reduce the recovery rate of the dichlorvos and influence the fluorescence of the final product; the excessive use amount of sodium chloride can increase the ionic viscosity of the solution and reduce the recovery rate of the dichlorvos to a certain extent. Therefore, the amount of NaCl to be used is preferably 75 to 225mg, and the most preferable amount of NaCl to be used is 150 mg.
E.NH 3 ·H 2 Concentration of O
(1) The experimental method comprises the following steps: the method is the same as the experimental method A in the example 3, except that ammonia water with different mass fractions is respectively added, and specifically comprises the following steps: 25%, 20%, 15%, 12.5%, 10%, 7.5%, 5%, 2.5%, 1.25%, other reaction conditions: the concentration of dichlorvos is 0.1mg/kg, the concentration of hydrochloric acid is 0.5mol/L, the concentration of sodium chloride is 150mg, the concentration of sodium nonanoate is 150mg, and the concentration of resorcinol solution is 90 mmol/L.
(2) The experimental results are as follows: as shown in fig. 8.
(3) And (4) analyzing results: the effect of ammonia in this experiment was two: firstly, creating alkaline conditions for dichlorovos to be hydrolyzed to generate dichloroacetaldehyde, and carrying out condensation reaction on dichloroacetaldehyde and resorcinol to generate red fluorescent substances; the second is to react with pelargonic acid in the organic phase to generate water-soluble substances, which is convenient for detecting results. The image shows that the fluorescence value is sequentially reduced along with the reduction of the mass fraction of the ammonia water, because when the concentration of the ammonia water is reduced, the alkaline environment is weaker and weaker, so that the DDVP is incompletely hydrolyzed, and the fluorescent products of the final reaction are reduced, the ammonia water solution is preferably 15-25%, and the ammonia water solution with the mass fraction of 25% is optimally selected.
F. Concentration of Resorcinol solution
(1) Reaction conditions are as follows: the same experimental method as that of the experiment A in the example 3 is adopted, except that resorcinol solutions with different concentrations are respectively added, specifically: 4.5mmol/L, 15mmol/L, 45 mmol/L, 90mmol/L, 225mmol/L, 450mmol/L, other reaction conditions: the concentration of dichlorvos is 0.1mg/kg, the concentration of hydrochloric acid is 0.5mol/L, the concentration of sodium chloride is 150mg, the concentration of sodium nonanoate is 150mg, and the mass fraction of ammonia water is 25%.
(2) The results of the experiment are shown in FIG. 9.
(3) And (4) analyzing results: dichlorvos is hydrolyzed only under alkaline conditions to generate dichloroacetaldehyde, and the hydrolysis product dichloroacetaldehyde and resorcinol solution are subjected to condensation reaction to generate red fluorescent substances. When the concentration of the resorcinol is 15-90 mmol/L, the fluorescence value increases with the increase of the concentration of the resorcinol, and when the concentration is 90-450 mmol/L, the fluorescence value gradually decreases with the increase of the concentration of the resorcinol. Therefore, the concentration of the resorcinol solution is preferably 45 to 225mmol/L, and most preferably 90 mmol/L.
G. Temperature of heating
(1) The experimental method comprises the following steps: the same experimental procedure as in A in example 3 was followed, except that the reaction temperature was changed to 25 deg.C, 35 deg.C, 45 deg.C, and 55 deg.C, respectively, for 20min in water bath. Other reaction conditions were as follows: the concentration of dichlorvos is 0.1mg/kg, the concentration of hydrochloric acid is 0.5mol/L, the concentration of sodium chloride is 150mg, the concentration of sodium nonanoate is 150mg, the mass fraction of ammonia water is 25%, and the concentration of resorcinol is 90 mmol/L.
(2) The experimental results are as follows: as shown in fig. 10.
(3) And (4) analyzing results: according to the verification experiment, the effect of the experiment under the heating condition is more remarkable, so that the temperature can influence the generation speed of the product, and substances in the reaction system can be decomposed to cause loss when the temperature is too high; when the temperature is too low, the reaction speed is slow. Therefore, the heating temperature needs to be strictly controlled, the constant temperature is preferably 25-35 ℃, the heating is most preferably carried out at 25 ℃, and the effect is best.
H. Time of heating
(1) The experimental method comprises the following steps: the same experimental method as A in example 3, except that the reaction time in water bath at 25 ℃ is 5min, 10min, 15min and 20 min; other reaction conditions are as follows: the concentration of dichlorvos is 0.1mg/kg, the concentration of hydrochloric acid is 0.5mol/L, the concentration of sodium chloride is 150mg, the concentration of sodium nonanoate is 150mg, the mass fraction of ammonia water is 25%, and the concentration of resorcinol is 90 mmol/L.
(2) The experimental results are as follows: as shown in fig. 11.
(3) And (4) analyzing results: the temperature can influence the synthesis of the product, and the result shows that the heating effect is best when the temperature is kept at 25 ℃ for 10min, and when the heating time is too long, substances in a reaction system can be decomposed to cause loss; the reaction speed is slow due to the low temperature. Therefore, the heating time is preferably 5 to 15min, and most preferably 10 min.
Example 4
The manufacturing experiment method of the standard curve of the concentration of the dichlorvos comprises the following steps: 100 mu L of dichlorvos with different concentrations are added into a 10mL centrifuge tube, the concentration is respectively 0.04mg/kg, 0.08mg/kg, 0.12mg/kg, 0.16mg/kg, 0.20mg/kg, 0.24mg/kg and 0.28mg/kg, and the organic solvent is completely volatilized after standing for 20 min. Then adding 1mL acetonitrile solution, vortex extracting for 3min at 2500r/min, and filtering the extractive solution with organic filter (0.22 μm). mu.L of the filtrate was taken and added with 4.5mL of aqueous hydrochloric acid solution of 0.5mol/L concentration to a 10mL centrifuge tube. Then 150mg of sodium chloride was added, and 150mg of sodium nonanoate was added after the sodium chloride was completely dissolved. The sodium nonanoate is originally generated into the nonanoic acid extractant which is uniformly dispersed into the sample solution under the acidic condition, so that the high-efficiency extraction of the object to be detected is realized. After the sample solution is layered, centrifuging for 5min under the condition of 5000r/min, placing the organic phase on the upper layer of a centrifuge tube, solidifying in ice bath (solidifying the extracted centrifuge tube on the frozen layer of a refrigerator, or placing ice blocks in a heat-insulating container, and placing the centrifuge tube into a centrifuge tube for solidification), and then removing the lower-layer water phase. Putting 50 mu L of the extract into a 2mL centrifuge tube, adding 100 mu L of 25% ammonia water, converting hydrophobic medium-chain fatty acid into hydrophilic fatty acid salt under alkaline conditions, and hydrolyzing dichlorvos into dichloroacetaldehyde which can be subjected to condensation reaction with resorcinol. Finally, 200. mu.L of resorcinol (prepared with absolute ethanol (AR)) at a concentration of 90mmol/L was added. After 10min of water bath reaction at 25 ℃, 200. mu.L of the solution is absorbed and placed in a 96-well plate, and the fluorescence intensity is measured under the conditions that the optimal excitation wavelength (Ex) is 275nm and the optimal emission wavelength (Em) is 315 nm. The standard deviation of each substrate was calculated for 10 blanks according to the formula for limit of quantitation (LOQ): LOQ is 10 σ/K and limit of detection (LOD) calculation formula: the LOD is 3 sigma/K (sigma is the standard deviation of blank sample; the slope of K working curve) to calculate the limit of quantification and detection under the optimized condition.
The standard curve was plotted with the concentration of dichlorvos as abscissa and the fluorescence value as ordinate, and the result is shown in fig. 12. The results show that the concentration of the dichlorvos presents good linearity in the range of 0.04mg/kg to 0.28 mg/kg: 12.1339x +8.7186, R 2 0.9904. The limit of quantitation (LOQ) is 8.1 mug/kg, and the limit of detection (LOD) is 2.4 mug/kg, which indicates that the method has higher sensitivity.
Example 5 simulated sample detection
(1) Simulating sample treatment: crushing aquatic product samples, accurately weighing 1g of crushed actual samples (sea cucumber, grass carp and abalone) in a 10mL centrifuge tube, and carrying out actual sample detection and labeling recovery experiments. When the actual sample is detected, directly carrying out the subsequent steps; when standard recovery experiments are carried out, 100 mu L of dichlorvos (40 mu g/kg, 160 mu g/kg and 280 mu g/kg) with different concentrations are added into samples respectively and placed for 20min, and then the subsequent steps are carried out. The subsequent steps are as follows: 1mL of acetonitrile solution was added to the prepared sample, vortex extraction was performed at 2500r/min for 3min, and the extract was filtered through an organic filter (0.22 μm) and placed in a 2mL centrifuge tube. A500. mu.L sample of the filtrate was taken and added to 4.5mL of a 0.5mol/L aqueous hydrochloric acid solution in a 10mL centrifuge tube. Then 150mg of sodium chloride was added, and 150mg of sodium nonanoate was added after the sodium chloride was completely dissolved. And after the sample solution is layered, centrifuging for 5min under the condition of 5000r/min, placing the organic phase on the upper layer of a centrifugal tube, solidifying in an ice bath, and removing the lower-layer water phase. Putting 50 μ L of the above extractive solution into a 2mL centrifuge tube, adding 100 μ L of 25% ammonia water, and finally adding 200 μ L of 90mmol/L resorcinol solution (prepared with anhydrous ethanol (AR)). After the reaction was carried out in a water bath at 25 ℃ for 10min, 200. mu.L of the resulting solution was pipetted and placed in a 96-well plate, and the fluorescence intensity was measured at an optimum excitation wavelength (Ex) of 275nm and an optimum emission wavelength (Em) of 315 nm. The concentration of dichlorvos in the actual sample and the recovery of spiked standard were calculated from the standard concentration curve prepared in example 4. The recovery was calculated according to the following formula:
C found : the method comprises the steps of (1) indicating the total concentration of dichlorvos measured after adding known concentrations of dichlorvos into actual aquatic product samples; c real : representing the initial concentration of dichlorvos in the actual aquatic product sample; c added : indicating the concentration of actual added dichlorvos.
(2) The experimental results are as follows: as shown in table 1.
TABLE 1 determination of the concentration of dichlorvos in a sample of an actual aquatic product
The result shows that the content of the dichlorvos in the actual sample (the adding concentration is 0) is not detected, and an adding recovery experiment is carried out to verify the feasibility of the method (carried out according to the final concentrations of the dichlorvos of 40 mu g/kg, 160 mu g/kg and 280 mu g/kg), and the result shows that the adding recovery rate is 97.23-107.25 percent and the relative standard deviation (RSD is less than or equal to 4.06 percent). The result shows that the method has accurate and reliable detection result and high precision, and can be widely used for quickly detecting the content of the dichlorvos in the aquatic product samples.
Claims (8)
1. A method for detecting the residual quantity of dichlorvos in aquatic products by a fluorescence analysis method is characterized by comprising the following steps:
s1, sample pretreatment: crushing an aquatic product sample, accurately weighing the crushed sample in a centrifugal tube, adding an acetonitrile solution, carrying out vortex and standing, filtering a supernatant with a membrane, and placing the filtered supernatant in the centrifugal tube;
s2, sample extraction: adding hydrochloric acid solution, sodium chloride and sodium nonanoate into a new centrifugal tube, centrifuging after sample solution is layered, solidifying in ice bath, pouring out the lower layer solution, and reserving the upper layer extracting solution;
s3, sample detection: adding ammonia water and resorcinol solution into the melted upper layer extracting solution, reacting in water bath, and performing fluorescence detection by using a multifunctional enzyme-linked immunosorbent assay (ELISA) instrument after the color is stable;
s4, making a standard song: preparing standard dichlorvos with different concentrations, directly adding standard dichlorvos solutions with different concentrations into a centrifugal tube, and completely volatilizing an organic solvent after standing; adding acetonitrile, and performing extraction and fluorescence detection according to steps S1, S2 and S3; drawing a standard curve by taking the concentration of the dichlorvos as an abscissa and the fluorescence value as an ordinate;
s5, detection result: and substituting the fluorescence value of the sample measured in the step S3 into the standard curve to obtain the residual amount of the dichlorvos in the sample.
2. The method of claim 1, wherein in step S1, the pulverized sample mass is 1g, and the volume of the acetonitrile is 1 mL; the vortex rotation speed is 2500r/min, the vortex time is 2-5 min, and the standing time is 3-5 min; the filter membrane is an organic filter membrane of 0.22 mu m.
3. The method according to claim 1, wherein in step S2, the filtrate has a volume of 0.5 to 0.9 mL, the hydrochloric acid solution has a concentration of 0.1 to 0.7mol/L and a volume of 4.5 mL; the dosage of the sodium chloride is 75-225 mg; the dosage of the sodium nonanoate is 100-200 mg; the centrifugation condition is 3000-8000 r/min, and the centrifugation time is 3-10 min.
4. The method of claim 1, wherein the filtrate volume is 0.5mL, the hydrochloric acid solution concentration is 0.5 mol/L; the dosage of the sodium chloride is 150 mg; the dosage of sodium nonanoate is 150 mg; the centrifugation condition is 5000r/min, and the centrifugation time is 5 min.
5. The method of claim 1, wherein in step S3, the volume of the upper extract is 50 μ L; the mass fraction of the ammonia water is 15-25%, and the volume of the ammonia water is 100 mu L; the concentration of the resorcinol solution is 45-225 mmol/L, absolute ethyl alcohol is used for preparation, and the volume of the resorcinol solution is 200 mu L.
6. The method of claim 1, wherein in step S3, the temperature of the water bath is 25-35 ℃ and the time of the water bath is 5-15 min.
7. The method of claim 5, wherein the ammonia water is 25% by mass, the resorcinol solution has a concentration of 90mmol/L, the water bath temperature is 25 ℃, and the water bath time is 10 min.
8. The method of claim 1, wherein in step S3, when the fluorescence intensity is measured by a multifunctional plate reader, the excitation wavelength is 275nm and the emission wavelength is 315 nm.
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