CN110987922B - Method for rapidly detecting eugenol anesthetic in aquatic product - Google Patents
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Abstract
The invention discloses a method for rapidly detecting eugenol anesthetic in an aquatic product, which comprises the steps of preparing a separation liquid, preparing a liquid to be detected, detecting an olefin double bond structure, generating a carbamate structure, and detecting and judging the carbamate structure, wherein the method is used for judging whether the eugenol anesthetic is contained in the aquatic product or not by utilizing the color reaction of potassium permanganate, converting the carbamate structure and detecting the carbamate structure. The method can accurately detect whether the eugenol anesthetic is added in the aquatic product or not under the condition of not using large instruments. The method has the advantages of strong specificity, high sensitivity, greatly shortened detection time, simple and convenient operation, low detection cost, high detection accuracy and low requirement on experimental environment, and is suitable for rapid detection of aquatic products.
Description
Technical Field
The invention relates to the field of detection, in particular to a method for rapidly detecting eugenol anesthetic in an aquatic product.
Background
For a long time, Chinese residents prefer to eat fresh and live aquatic products. Due to the limitation of climate and culture conditions, the culture production of Chinese aquatic products has regional differences. In order to meet the market demand, the long-distance transportation condition of fresh and live aquatic products is very common. However, the immunity, survival rate and product value of live fish are reduced due to the influence of factors such as stress, water quality deterioration, injury and infection on the fresh and live aquatic products in the collection, storage and transportation loop, so that the risk is brought to the quality safety of water products, and the development of aquaculture industry is restricted. The eugenol is a fishery anesthetic which is most widely applied to domestic aquatic products due to good drug effect and low price, has good anesthetic effect on fresh and live aquatic products, and improves survival rate and transportation density. Therefore, in order to improve the survival rate of live fish and reduce economic loss, some practitioners gradually begin to use eugenol as an aquatic product anesthetic in a large amount in the links of collection, storage and transportation of fresh and live aquatic products.
Because eugenol has certain toxicity to liver cells, there is a certain controversy about the harm of eugenol to human bodies and the safety of eugenol as a fishing anesthetic. The U.S. food and drug administration also bans eugenol compounds as anesthetics and tranquilizers for live and fresh food aquatic products. At present, no relevant policy and regulation and limit standards are established for the application of eugenol anesthetics in aquatic products in China, but in fact eugenol anesthetics are generally used in the market.
At present, commercial methods for detecting eugenol anesthetics mainly comprise a chromatography method and a ferric chloride color development method. In the patent document of the method for measuring eugenol in aquatic products by using the isotope dilution gas chromatography mass spectrometry with the patent application number of 201610144583.4, a method for detecting eugenol in aquatic products by using the gas chromatography mass spectrometry is disclosed, expensive large-scale instruments and a large amount of auxiliary experimental equipment are required for chromatography, the pretreatment is complex and time-consuming, the cost is high, the treatment steps are complex, the time consumption is long, and the method is not suitable for on-site rapid detection of aquatic products.
Although the ferric trichloride color development method is simple to operate, the ferric trichloride is used as a detection reagent, and the property of a part of target object is reflected only according to the color development condition, so that the detection target object cannot be completely confirmed to be eugenol, the detection work is not facilitated to be carried out in a large range, and the false detection rate is high. The requirement for obtaining the detection result immediately cannot be met, so that the requirement for rapidly detecting aquatic products cannot be met.
Therefore, a method which is simple and rapid to operate, does not need a large-scale analytical instrument, consumes less time, has strong specificity, high sensitivity, high accuracy of test results, low analysis cost and low requirement on experimental environment, and is suitable for rapidly detecting the eugenol anesthetic in the aquatic product needs to be developed.
Disclosure of Invention
The invention aims to provide a method for rapidly detecting eugenol anesthetic in an aquatic product.
According to one aspect of the invention, the method for rapidly detecting the eugenol type anesthetic in the aquatic product is provided, and comprises the following steps:
step a, preparation of a separation solution:
taking a certain amount of sample, adding neutral desiccant, absolute ethyl alcohol and acetonitrile, extracting, centrifuging for the first time, and taking supernatant to obtain separation liquid;
step b, preparing a solution to be detected:
adding normal hexane into the separation solution, carrying out second centrifugation, reserving the acetonitrile layer solution, drying to obtain a first object to be detected, adding dichloromethane into the first object to be detected to completely dissolve the first object to be detected, adding dimethyl sulfoxide solution of trimethylsilylimidazole, and adjusting the pH value to obtain a liquid to be detected;
step c, detecting the double bond structure of the olefin:
adding a potassium permanganate solution into the solution to be detected, shaking up to obtain a first detection solution, observing the color change of the first detection solution, judging the olefin double bond structure, and lightening or fading the color after adding the potassium permanganate solution, so as to indicate that the sample contains the olefin double bond structure; the color is unchanged, which indicates that the sample does not contain an olefin double bond structure, namely does not contain eugenol anesthetic, and then the detection is stopped;
step d, generating a carbamate structure:
adding a mixed solution of tetrabutylammonium fluoride solution and hydrogen bromide into the first detection solution, oscillating and mixing uniformly, reacting to obtain a second detection solution, then adding a carbon tetrachloride solution of methylcarbamoyl chloride and triethylamine, stirring at a certain reaction temperature, reacting to obtain a third detection solution, taking the third detection solution, and drying to obtain a second object to be detected;
step e, detecting a carbamate structure:
adding dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer solution into the second object to be detected, dissolving the second object to be detected to obtain fourth detection liquid, detecting a carbamate structure in the fourth detection liquid by adopting a spectrophotometry, and judging whether the carbamate structure is contained;
step f, judging:
if the solution to be detected contains an olefin double bond structure and the fourth detection solution contains a carbamate structure, the eugenol anesthetic can be determined to be contained in the sample; if the solution to be detected contains an olefin double bond structure and the fourth solution to be detected does not contain a carbamate structure, determining that the sample does not contain eugenol anesthetic; and if the liquid to be detected does not contain an olefin double bond structure, judging that the sample does not contain eugenol anesthetic.
The invention has the beneficial effects that: the neutral desiccant and the absolute ethyl alcohol are used in the detection method, so that the dehydration and salting-out effects of the sample are facilitated, and the extraction of the eugenol anesthetic can be promoted. Acetonitrile is used as an extracting solution, so that the extraction efficiency of the eugenol anesthetic can be improved. The n-hexane is used, and the n-hexane layer is discarded, so that the protein and fat in the sample can be removed, and the influence on the subsequent detection result is avoided. The acetonitrile layer solution is dried, and the effect of concentrating and enriching the substance to be detected can be achieved. The addition of dichloromethane can dissolve the eugenol anesthetic in the substance to be detected in dichloromethane, so that the eugenol anesthetic can be conveniently reacted with trimethylsilylimidazole subsequently, phenolic hydroxyl groups are protected, and the phenolic hydroxyl groups are prevented from reacting with potassium permanganate in the next color reaction. The color reaction of the potassium permanganate solution is used for identifying whether the potassium permanganate solution contains an olefin double bond structure. And (3) reacting the product of the color reaction with tetrabutylammonium fluoride to generate a catechol structure, reacting with methyl carbamoyl chloride under the catalytic action of triethylamine to generate a carbamate structure, and determining whether the carbamate structure is contained by a spectrophotometry method so as to determine whether the eugenol anesthetic is contained in the sample.
According to the invention, through concentration enrichment, olefin double bond identification and carbamate structure identification, whether eugenol anesthetic is added in aquatic products can be accurately detected under the condition of not using large-scale instruments. The method has the advantages of strong specificity, high sensitivity, greatly shortened detection time, simple and convenient operation, low detection cost, high detection accuracy and low requirement on experimental environment, and is suitable for rapid detection of aquatic products.
In some embodiments, the preparation of the separation solution comprises putting a certain amount of sample into a centrifuge tube, adding a neutral drying agent and absolute ethyl alcohol according to a certain proportion, uniformly mixing, adding acetonitrile, performing ultrasonic extraction, performing first centrifugation, and taking supernatant to obtain a separation solution; and (3) removing water in the sample by using a neutral drying agent and absolute ethyl alcohol, and performing ultrasonic extraction on the eugenol anesthetic in the acetonitrile pair.
In some embodiments, the eugenol-type anesthetic is selected from at least one of eugenol, isoeugenol, methyl eugenol, or methyl isoeugenol. Eugenol, isoeugenol, methyl eugenol and methyl isoeugenol structurally have branched chain olefin double bond structures, can generate catechol structures through chemical reaction, and can be further converted into carbamate structures, so that the detection can be carried out through the method.
In some embodiments, the pH in step b ranges from 6.8 to 7.2. The pH value is controlled to be 6.8-7.2, so that the reaction system is neutral, and the effect of protecting the reaction product is achieved, because the trimethyl siloxy group of the reaction product has an unstable structure under acidic or alkaline conditions.
In some embodiments, the potassium permanganate solution has a potassium permanganate content of 0.5%. The 0.5 percent potassium permanganate solution is a low-concentration potassium permanganate solution, and reacts with an olefin double bond after being added, so that the added potassium permanganate is faded. Meanwhile, the added potassium permanganate solution can not oxidize trimethyl siloxy groups of reactants, so that the aim of protecting phenolic hydroxyl groups is fulfilled.
In some embodiments, the molar ratio of tetrabutylammonium fluoride to hydrogen bromide in the mixed solution of tetrabutylammonium fluoride solution and hydrogen bromide is 2:1, the mass fraction of tetrabutylammonium fluoride in the tetrabutylammonium fluoride solution is 75%, and the mass fraction of hydrobromic acid in the hydrogen bromide is 33%. Tetrabutylammonium fluoride reacts with reactants to generate a catechol structure, so that subsequent detection reaction is facilitated. The reaction is carried out under acidic conditions and the hydrogen bromide provides the acidic environment required for the reaction.
In some embodiments, the reaction temperature in step d is from 20 ℃ to 35 ℃. In this temperature range, the reaction can be smoothly carried out. When the reaction temperature is higher than 35 ℃, more byproducts are generated in the reaction process. The reaction temperature is lower than 20 ℃, which causes lower product yield and influences subsequent detection.
In some embodiments, the concentration of methylcarbamoyl chloride in the carbon tetrachloride solution of methylcarbamoyl chloride is 0.1 mol/L. In the reaction, a methylcarbamoyl structure is provided, replacing the hydrogen of the catechol structure, to form two carbamate groups. Carbon tetrachloride is used to dissolve methylcarbamoyl chloride.
In some embodiments, the first centrifugation is at 4000 rpm and the second centrifugation is at 4000 rpm. The speed of rotation is 4000 rpm, and the first centrifugation can precipitate the neutral desiccant and total insoluble substances of the sample at the bottom, so that the acetonitrile extraction is facilitated. And centrifuging for the second time, and purifying the separated liquid by using n-hexane to remove impurities in the separated liquid.
In some embodiments, the neutral drying agent is anhydrous sodium sulfate, the mass ratio of the anhydrous sodium sulfate to the sample is 1:1, and the volume ratio of the anhydrous ethanol to the acetonitrile is 1: 8. The anhydrous sodium sulfate has strong water absorption and can fully absorb the water in the sample. The anhydrous ethanol has water absorption, is in a liquid state, and can be fully contacted with the sample to absorb the water in the sample so as to make up the incomplete contact between the anhydrous sodium sulfate and the sample.
Detailed Description
The present invention will be described in further detail with reference to examples.
Example 1
In this example, the anhydrous ethanol was selected from the analytically pure anhydrous ethanol provided by the national drug group chemical reagent corporation, the anhydrous sodium sulfate was selected from the analytically pure anhydrous sodium sulfate provided by the national drug group chemical reagent corporation, the acetonitrile was selected from the analytically pure acetonitrile provided by the west longa science corporation, the n-hexane was selected from the analytically pure hexane provided by the national drug group chemical reagent corporation, the dichloromethane was selected from the analytically pure dichloromethane provided by the Nanjing chemical reagent corporation, the carbon tetrachloride was selected from the analytically pure carbon tetrachloride provided by the Nanjing chemical reagent corporation, the hydrogen bromide was selected from the 33% hydrogen bromide provided by the national drug group chemical reagent corporation, the potassium permanganate was selected from the analytically pure potassium permanganate provided by the national drug group chemical reagent corporation, and the tetrabutylammonium fluoride was selected from the tetrabutylammonium fluoride trihydrate provided by the national drug group chemical reagent corporation, the methylamino formyl chloride is selected from methylamino formyl chloride supplied by Beijing coupled technology, Inc., the trimethyl silicane imidazole is selected from trimethyl silicane imidazole supplied by Chijing coupled technology, Inc., the dimethyl sulfoxide is selected from analytically pure dimethyl sulfoxide supplied by Shanghai Aladdin Biotechnology, Inc., the dipotassium hydrogen phosphate is selected from analytically pure dipotassium hydrogen phosphate supplied by national drug group chemical, Inc., the potassium dihydrogen phosphate is selected from analytically pure potassium dihydrogen phosphate supplied by national drug group chemical, Inc., the iodothioacetylcholine is selected from iodothioacetylcholine supplied by national drug group chemical, Inc., the acetylcholinesterase is selected from acetylcholinesterase supplied by national drug group chemical, Inc., the dithiodinitrobenzoic acid (DTNB) is selected from dithiodinitrobenzoic acid supplied by Beijing Solebao technology, Inc., sodium bicarbonate was selected from analytical sodium bicarbonate available from national pharmaceutical group chemical agents, ltd.
In this embodiment, the oscillator is an MIT135C multi-tube vortex oscillator supplied by shangham industries ltd, the ultrasonic extractor is a THC-250B ultrasonic extractor supplied by jingningtianhua ultrasonic electronics ltd, the centrifuge is an H1650 desktop high-speed centrifuge supplied by hunnan xiang instruments laboratory instrument development ltd, the nitrogen blower is a DCY-125 water bath nitrogen blower supplied by shanghai youyi instruments ltd, and the spectrophotometer is a 722S visible spectrophotometer supplied by shanghai essen industries ltd.
Reagents required for formulation in this example:
dimethyl sulfoxide solution of trimethylsilylimidazole: weighing 0.5ml of trimethylsilylimidazole, diluting with dimethyl sulfoxide, and fixing the volume to 10 ml;
0.5% potassium permanganate solution: weighing 5g of potassium permanganate, dissolving with water and fixing the volume to 1L;
75% tetrabutylammonium fluoride solution: weighing 75g of tetrabutylammonium fluoride, dissolving with water and fixing the volume to 1L;
0.1mol/L of methylcarbamoyl chloride in carbon tetrachloride: weighing 0.93g of methylamino formyl chloride, and dissolving in 100ml of carbon tetrachloride;
dipotassium phosphate-potassium dihydrogen phosphate buffer: dissolving 11.9g of anhydrous dipotassium hydrogen phosphate and 3.2g of potassium dihydrogen phosphate with 1000ml of distilled water respectively;
color developing agent: dissolving 160mg of dinitrodithiobenzoic acid (DTNB) and 15.6mg of sodium bicarbonate in 20ml of dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer solution, and storing in a refrigerator at 4 ℃;
substrate: dissolving 25.0mg of iodothioacetylcholine in 3.0ml of distilled water, shaking uniformly, and storing in a refrigerator at 4 ℃ for later use;
acetylcholine esterase solution: dissolving acetylcholinesterase in dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer solution, and measuring absorbance change value delta A for 3min0It should be controlled above 0.3. Shaking, and storing in a refrigerator at 4 deg.C for use with a shelf life of no more than four days;
the reagents, instruments and solutions prepared in example 1 were used in examples 2 to 6.
Example 2
A method for rapidly detecting eugenol anesthetic in aquatic products comprises the following steps:
step a, preparation of a separation solution:
putting 3.5g of fish sample into a 50ml centrifuge tube, adding 3.5g of anhydrous sodium sulfate and 1ml of anhydrous ethanol, carrying out vortex oscillation and uniform mixing for 10s, adding 8ml of acetonitrile, carrying out oscillation and uniform mixing for 2min, carrying out ultrasonic extraction for 4min, carrying out first centrifugation, carrying out centrifugation at a centrifugation speed of 4000r/min for 5min, and taking all supernatant to obtain a separation solution; the anhydrous sodium sulfate and the anhydrous ethanol are matched with each other to absorb the moisture in the sample together, so that the moisture in the sample is removed. And extracting the eugenol anesthetic in the sample by using acetonitrile under the ultrasonic extraction condition.
Step b, preparing a solution to be detected:
adding 3ml of n-hexane into the separation liquid, oscillating and uniformly mixing for 2min, carrying out second centrifugation, wherein the centrifugation speed is 4000r/min, centrifuging for 2min, removing the n-hexane layer, reserving the acetonitrile layer solution, carrying out water bath nitrogen blow-drying on the acetonitrile layer solution at the temperature of 65 ℃ to obtain a first object to be detected, adding 5ml of dichloromethane into the first object to be detected to completely dissolve the first object to be detected, adding 1.0ml of dimethyl sulfoxide solution of trimethylsilylimidazole, adjusting the pH value to be 6.8, and reacting the trimethylsilylimidazole with eugenol anesthetic to obtain a liquid to be detected. In the reaction, the hydrogen in the phenolic hydroxyl group is substituted by the trimethyl silane group and is connected with the oxygen of the phenolic hydroxyl group, so that the phenolic hydroxyl group of the eugenol anesthetic is protected, and the phenolic hydroxyl group is prevented from being oxidized by potassium permanganate in the next reaction to influence the result judgment of the fading of the potassium permanganate.
The reaction formula is as follows:
step c, detecting the double bond structure of the olefin:
adding 100 mu l of 0.5% potassium permanganate solution into the solution to be detected, shaking up to obtain a first detection solution, observing the color change of the first detection solution, judging the olefin double bond structure, and after the potassium permanganate solution is dropped, lightening or fading the color, which indicates that the sample contains the olefin double bond structure; the color is unchanged, which indicates that the sample does not contain an olefin double bond structure, namely does not contain eugenol anesthetic, and then the detection is stopped; and performing oxidation reaction on an olefin double-bond group connected to a benzene ring in the liquid to be detected and potassium permanganate, oxidizing the olefin double-bond group into an aldehyde group, reducing the potassium permanganate into manganese oxide in a neutral environment, and fading the purple color of the first detection liquid.
The reaction formula is as follows:
step d, generating a carbamate structure:
adding 0.3ml of mixed solution of tetrabutylammonium fluoride solution and hydrogen bromide into the first detection solution, wherein the molar ratio of tetrabutylammonium fluoride to hydrogen bromide is 0.002mol and the molar ratio of hydrogen bromide to tetrabutylammonium fluoride to hydrogen bromide is 2:1, oscillating and mixing uniformly, heating to 35 ℃ for reacting for 30min to obtain a second detection solution, then adding 20ml of carbon tetrachloride solution of methylamino formyl chloride and 0.2g of triethylamine into the second detection solution, stirring for 30min at 20 ℃ to obtain a third detection solution, taking the third detection solution, and drying to obtain a second object to be detected; the stability of the F-Si bond is higher than that of the O-Si bond, and by utilizing the difference of bond energy, the reactant in the first detection liquid reacts with tetrabutylammonium fluoride, the O-Si bond of the reactant in the first detection liquid is broken, the trimethylsilyl group is lost, and the catechol structure is generated. The generated product with the catechol structure and methyl carbamyl chloride are subjected to esterification reaction to generate a carbamate structure.
The reaction formula is as follows:
step e, detecting a carbamate structure:
and adding 7.5ml of dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer solution into the second substance to be detected, and dissolving the second substance to be detected to obtain a fourth detection solution.
And (3) measuring the carbamate structure in the fourth detection solution by a spectrophotometry method:
1. blank control solution determination:
adding 2.5ml of dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer solution into a blank test tube, adding 0.1ml of acetylcholine esterase solution and 0.1ml of color developing agent, shaking uniformly, standing at 37 ℃ for 20min, adding 0.1ml of substrate, shaking uniformly, immediately placing into a colorimetric pool of a spectrophotometer, and measuring the absorbance change value delta A of 3min0Is 0.570.
2. And (3) measuring a fourth detection solution:
adding 2.5ml of fourth detection solution into a blank test tube, adding 0.1ml of acetylcholine esterase solution and 0.1ml of color developing agent, shaking uniformly, standing at 37 ℃ for 20min, adding 0.1ml of substrate, shaking uniformly, immediately placing into a colorimetric pool of a spectrophotometer to obtain the 3min absorbance change value delta A of the fourth detection solutiontIs 0.177.
3. Judging the inhibition rate:
and calculating the inhibition rate according to a formula, and judging that the fourth detection solution contains a carbamate structure when the inhibition rate is more than or equal to 50%. The inhibition rate of the fourth test solution is calculated according to an inhibition rate formula and is 68.9% and more than 50%, and the fourth test solution can be judged to contain a carbamate structure. The fourth detection solution is composed of a dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer solution and a second object to be detected, and can judge that the second object to be detected contains a carbamate structure.
The inhibition ratio formula is as follows:
step f, judging:
the liquid to be detected prepared by the sample contains an olefin double bond structure, the fourth detection liquid contains a carbamate structure, namely the second object to be detected contains the carbamate structure, and the eugenol anesthetic can be judged to be contained in the sample.
Example 3
A method for rapidly detecting eugenol anesthetic in aquatic products comprises the following steps:
step a, preparation of a separation solution:
putting 2.0g of shrimp sample into a 50ml centrifuge tube, adding 2g of anhydrous sodium sulfate and 1ml of anhydrous ethanol, carrying out vortex oscillation and uniform mixing for 10s, adding 8ml of acetonitrile, carrying out oscillation and uniform mixing for 2min, carrying out ultrasonic extraction for 5min, carrying out first centrifugation at a centrifugation speed of 4000r/min for 5min, and taking all supernatant to obtain a separation solution.
Step b, preparing a solution to be detected:
adding 3ml of n-hexane into the separation liquid, oscillating and uniformly mixing for 2min, carrying out second centrifugation, centrifuging at the centrifugal rotation speed of 4000r/min for 2min, discarding the n-hexane layer, reserving the acetonitrile layer solution, carrying out water bath and nitrogen blow-drying on the acetonitrile layer solution at the temperature of 65 ℃ to obtain a first object to be detected, adding 5ml of dichloromethane into the first object to be detected to completely dissolve the first object to be detected, adding 1ml of dimethyl sulfoxide solution of trimethylsilylimidazole, adjusting the pH value to be 7.0, and reacting the trimethylsilylimidazole with eugenol anesthetic to obtain a liquid to be detected. In the reaction, the hydrogen in the phenolic hydroxyl group is substituted by the trimethyl silane group and is connected with the oxygen of the phenolic hydroxyl group, so that the phenolic hydroxyl group of the eugenol anesthetic is protected, and the phenolic hydroxyl group is prevented from being oxidized by potassium permanganate in the next reaction to influence the result judgment of the fading of the potassium permanganate.
Step c, detecting the double bond structure of the olefin:
and adding 100 mu l of 0.5% potassium permanganate solution into the solution to be detected, shaking up to obtain a first detection solution, observing the color change of the first detection solution, judging the olefin double bond structure, and stopping detection if the color does not change after the potassium permanganate solution is dropped into the solution, indicating that the sample does not contain the olefin double bond structure, namely does not contain eugenol anesthetic.
Step d, judging:
the liquid to be detected does not contain an olefin double bond structure, so that the sample is judged to contain no eugenol anesthetic.
Example 4
A method for rapidly detecting eugenol anesthetic in aquatic products comprises the following steps:
step a, preparation of a separation solution:
putting 2.0g of fish sample into a 50ml centrifuge tube, adding 2g of anhydrous sodium sulfate and 1ml of anhydrous ethanol, carrying out vortex oscillation and uniform mixing for 10s, adding 8ml of acetonitrile, carrying out oscillation and uniform mixing for 2min, carrying out ultrasonic extraction for 5min, carrying out first centrifugation, carrying out centrifugation at a centrifugation speed of 4000r/min for 5min, and taking all supernatant to obtain a separation solution.
Step b, preparing a solution to be detected:
adding 3ml of n-hexane into the separation liquid, oscillating and uniformly mixing for 2min, carrying out secondary centrifugation, centrifuging at the centrifugal rotation speed of 4000r/min for 2min, discarding the n-hexane layer, reserving the acetonitrile layer solution, carrying out water bath nitrogen blow-drying on the acetonitrile layer solution at the temperature of 65 ℃ to obtain an object to be detected, adding 1ml of dichloromethane into the first object to be detected to completely dissolve the first object to be detected, adding 1ml of dimethyl sulfoxide solution of trimethylsilylimidazole, adjusting the pH value to be 7.2, and reacting the trimethylsilylimidazole with eugenol anesthetic to obtain a liquid to be detected. In the reaction, the hydrogen in the phenolic hydroxyl group is substituted by the trimethyl silane group and is connected with the oxygen of the phenolic hydroxyl group, so that the phenolic hydroxyl group of the eugenol anesthetic is protected, and the phenolic hydroxyl group is prevented from being oxidized by potassium permanganate in the next reaction to influence the result judgment of the fading of the potassium permanganate.
Step c, detecting the double bond structure of the olefin:
and adding 100 mu l of 0.5% potassium permanganate solution into the solution to be detected, shaking up to obtain a first detection solution, observing the color change of the first detection solution, judging the olefin double bond structure, and after the potassium permanganate solution is dropped, lightening or fading the color, which indicates that the sample contains the olefin double bond structure. And carrying out oxidation reaction on an olefin double-bond group connected to a benzene ring in the liquid to be detected and potassium permanganate, oxidizing the olefin double-bond group into an aldehyde group, reducing the potassium permanganate into manganese oxide, and lightening or fading the color of the first detection liquid.
Step d, generating a carbamate structure:
adding 0.3ml of mixed solution of tetrabutylammonium fluoride solution and hydrogen bromide into the first detection solution, wherein the molar ratio of tetrabutylammonium fluoride to hydrogen bromide is 0.002mol, and the molar ratio of hydrogen bromide to tetrabutylammonium fluoride to hydrogen bromide is 2:1, oscillating and mixing uniformly, heating to 35 ℃ for reacting for 30min to obtain a second detection solution, then adding 20ml of carbon tetrachloride solution of methylamino formyl chloride and 0.2g of triethylamine into the second detection solution, stirring for 30min at 35 ℃ to obtain a third detection solution, taking the third detection solution, and drying to obtain a second object to be detected. The stability of the F-Si bond is higher than that of the O-Si bond, and by utilizing the difference of bond energy, the reactant in the first detection liquid reacts with tetrabutylammonium fluoride, the O-Si bond of the reactant in the first detection liquid is broken, the trimethylsilyl group is lost, and the catechol structure is generated. The generated product with the catechol structure and methyl carbamyl chloride are subjected to esterification reaction to generate a carbamate structure.
Step e, detecting a carbamate structure:
adding 7.5ml of dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer solution into the second object to be detected, and dissolving the second object to be detected to obtain fourth detection solution;
and (3) measuring the carbamate structure in the fourth detection solution by a spectrophotometry method:
1. blank control solution determination:
adding 2.5ml of dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer solution into a blank test tube, adding 0.1ml of acetylcholine esterase solution and 0.1ml of color developing agent, shaking uniformly, standing at 37 ℃ for 20min, adding 0.1ml of substrate, shaking uniformly, immediately placing into a colorimetric pool of a spectrophotometer, and measuring the absorbance change value delta A of 3min0Is 0.570.
2. And (3) measuring a fourth detection solution:
adding 2.5ml of the fourth detection solution into a blank test tube, adding 0.1ml of acetylcholine esterase solution and 0.1ml of color developing agent, shaking, standing at 37 deg.C for 20min, adding 0.1ml of substrate, shaking, and immediately adding into spectrophotometer for color comparisonIn the pool, obtaining the 3min absorbance change value delta A of the fourth detection liquidtIs 0.522.
3. Judging the inhibition rate:
and calculating the inhibition rate according to a formula, and judging that the fourth detection solution contains a carbamate structure when the inhibition rate is more than or equal to 50%. The inhibition rate of the fourth detection solution calculated according to the inhibition rate formula is 8.5% and less than 50%, and it can be determined that the fourth detection solution does not contain a carbamate structure. The fourth to-be-detected solution consists of a dipotassium hydrogen phosphate-monopotassium phosphate buffer solution and a second to-be-detected object, so that the second to-be-detected object does not contain a carbamate structure;
the inhibition ratio formula is as follows:
step f, judging:
the liquid to be detected prepared by the sample contains an olefin double bond structure, the fourth detection liquid does not contain a carbamate structure, namely the second object to be detected does not contain a carbamate structure, and the sample can be judged to contain no eugenol anesthetic.
Example 5
The method (refer to the method of example 2) and the high performance liquid chromatography (refer to high performance liquid chromatography in Chenghui research on the detection method of eugenol type anesthetic residues in aquatic products for detecting five eugenol type anesthetics in aquatic products, pages 9-14) are adopted to detect eugenol type anesthetics in the same aquatic product samples, and the following table is obtained:
| categories | The method of the invention | High performance liquid chromatography |
| Extraction, purification and enrichment time/h | 0.50 | 1.22 |
| Preparation time of standard solution/h | 0.25 | 2.00 |
| Measurement time/h | 1.25 | 1.50 |
| Total time/h | 2.00 | 4.72 |
| Detection time saving rate/%) | 57.6 | — |
In the embodiment, the total time of using the method to detect the eugenol type anesthetic in the aquatic product is only 2.00 hours, while the time of using the high performance liquid chromatography method needs 4.72 hours, the total time of using the method is reduced by 2.72 hours, and the detection time is shortened by 57.6%. In addition, the detection method provided by the invention does not need to prepare a standard solution, reduces the detection steps, improves the detection efficiency, and is suitable for rapid detection of a large amount of aquatic products.
In conclusion, the detection method provided by the invention can qualitatively detect whether the eugenol anesthetic is added in the aquatic product or not by concentrating and enriching the eugenol anesthetic remaining in the aquatic product sample, identifying the double bond of the olefin, identifying the carbamate group and the like without using a large-scale instrument.
The method has the advantages of strong specificity, greatly shortened detection time, simple operation, low detection cost, high sensitivity, high detection accuracy and low requirement on experimental environment, and is suitable for rapid detection of aquatic products.
What has been described above are merely some embodiments of the present invention.
It will be apparent to those skilled in the art that various changes and modifications can be made without departing from the inventive concept thereof, and these changes and modifications can be made without departing from the spirit and scope of the invention.
Claims (10)
1. A method for rapidly detecting eugenol anesthetic in aquatic products is characterized by comprising the following steps:
step a, preparation of a separation solution:
taking a certain amount of sample, adding neutral desiccant, absolute ethyl alcohol and acetonitrile, extracting, centrifuging for the first time, and taking supernatant to obtain separation liquid;
step b, preparing a solution to be detected:
adding normal hexane into the separation solution, carrying out second centrifugation, reserving the acetonitrile layer solution, drying to obtain a first object to be detected, adding dichloromethane into the first object to be detected to completely dissolve the first object to be detected, adding dimethyl sulfoxide solution of trimethylsilylimidazole, and adjusting the pH value to obtain a liquid to be detected;
step c, detecting the double bond structure of the olefin:
adding a potassium permanganate solution into the solution to be detected, shaking up to obtain a first detection solution, observing the color change of the first detection solution, judging the olefin double bond structure, and lightening or fading the color after adding the potassium permanganate solution, so as to indicate that the sample contains the olefin double bond structure; the color is unchanged, which indicates that the sample does not contain an olefin double bond structure, namely does not contain eugenol anesthetic, and then the detection is stopped;
step d, generating a carbamate structure:
adding a mixed solution of tetrabutylammonium fluoride solution and hydrobromic acid into the first detection solution, oscillating and mixing uniformly, reacting to obtain a second detection solution, then adding a carbon tetrachloride solution of methylcarbamoyl chloride and triethylamine, stirring at a certain reaction temperature, reacting to obtain a third detection solution, taking the third detection solution, and drying to obtain a second object to be detected;
step e, detecting a carbamate structure:
adding dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer solution into the second object to be detected, dissolving the second object to be detected to obtain fourth detection liquid, detecting a carbamate structure in the fourth detection liquid by adopting a spectrophotometry, and judging whether the carbamate structure is contained;
step f, judging:
if the solution to be detected contains an olefin double bond structure and the fourth detection solution contains a carbamate structure, the eugenol anesthetic can be determined to be contained in the sample; if the to-be-detected liquid contains an olefin double bond structure and the fourth detection liquid does not contain a carbamate structure, determining that the sample does not contain eugenol anesthetic; and if the liquid to be detected does not contain an olefin double bond structure, judging that the sample does not contain eugenol anesthetic.
2. The method for rapidly detecting eugenol type anesthetic in aquatic products according to claim 1,
step a, preparing the separation liquid comprising
Putting a certain amount of sample into a centrifuge tube, adding neutral desiccant and absolute ethanol according to a certain proportion, mixing uniformly, adding acetonitrile, performing ultrasonic extraction, performing first centrifugation, and taking supernatant to obtain a separation solution.
3. The method for rapidly detecting eugenol type anesthetic in aquatic products according to claim 1, wherein the eugenol type anesthetic is selected from at least one of eugenol, isoeugenol, methyl eugenol or methyl isoeugenol.
4. The method for rapidly detecting eugenol type anesthetic in aquatic products according to claim 1, wherein the pH in the step b is in the range of 6.8-7.2.
5. The method for rapidly detecting eugenol type anesthetic in aquatic products according to claim 1, wherein the content of potassium permanganate in the potassium permanganate solution is 0.5%.
6. The method of claim 1, wherein the molar ratio of tetrabutylammonium fluoride to hydrogen bromide in the mixed solution of tetrabutylammonium fluoride solution and hydrobromic acid is 2:1, the mass fraction of tetrabutylammonium fluoride in the tetrabutylammonium fluoride solution is 75%, and the mass fraction of hydrogen bromide in the hydrobromic acid is 33%.
7. The method for rapidly detecting eugenol type anesthetic in aquatic products according to claim 1, wherein the reaction temperature in the step d is 20-35 ℃.
8. The method for rapidly detecting eugenol type anesthetic in aquatic products according to claim 1, wherein the concentration of the methylcarbamoyl chloride in the carbon tetrachloride solution of the methylcarbamoyl chloride is 0.1 mol/L.
9. The method for rapidly detecting eugenol type anesthetic in aquatic products according to claim 1, wherein the rotation speed of the first centrifugation is 4000 revolutions per minute, and the rotation speed of the second centrifugation is 4000 revolutions per minute.
10. The method for rapidly detecting eugenol type anesthetic in aquatic product according to claim 1, wherein the neutral drying agent is anhydrous sodium sulfate, the mass ratio of the anhydrous sodium sulfate to the sample is 1:1, and the volume ratio of the anhydrous ethanol to the acetonitrile is 1: 8.
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