CN109633032B - Method for simultaneously measuring content of malachite green and crystal violet - Google Patents
Method for simultaneously measuring content of malachite green and crystal violet Download PDFInfo
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- CN109633032B CN109633032B CN201910082937.0A CN201910082937A CN109633032B CN 109633032 B CN109633032 B CN 109633032B CN 201910082937 A CN201910082937 A CN 201910082937A CN 109633032 B CN109633032 B CN 109633032B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/062—Preparation extracting sample from raw material
Abstract
The invention discloses a method for simultaneously measuring the contents of malachite green and crystal violet. Mixing and heating choline chloride and phenol to be used as an extracting agent, filtering a sample solution by using a water-phase microporous filter membrane, adding the extracting agent and an organic silicon emulsifier, performing vortex to form uniform emulsion, standing, performing centrifugal phase separation, and taking an upper-layer extraction phase to perform high performance liquid chromatography determination. According to the method, the eutectic solvent prepared from choline chloride and phenol is used as an extracting agent, the organic silicon emulsifying agent is used as an emulsion increasing agent for the first time, an extraction target object in the formed emulsion is fully contacted with the extracting agent and is completely extracted, and the low-content malachite green and crystal violet can be simultaneously and accurately detected by combining a high performance liquid chromatography method. The method has the characteristics of fast and uniform emulsion formation, easy phase separation operation, high efficiency, low toxicity, simple operation, high sensitivity and good reproducibility. Is particularly suitable for the quantitative analysis of malachite green and crystal violet residue in bodies of aquatic animals such as fish, shrimps, soft-shelled turtles and the like and in water bodies.
Description
Technical Field
The invention belongs to the technical field of harmful substance residue detection, and particularly relates to a method capable of simultaneously and accurately measuring contents of malachite green and crystal violet in bodies and water bodies of aquatic animals.
Background
Malachite green and crystal violet belong to alkaline triphenylmethane dyes, have similar structures, and are often used as bactericides and antiparasitic drugs for preventing and treating various fish diseases in the aquaculture process. The products generated by the metabolism in organisms have high toxicity, high residue and teratogenic carcinogenic effect. Malachite green has been listed as a forbidden drug for aquaculture in many countries in the world, and China also announces that malachite green and crystal violet are strictly forbidden to be used in aquaculture and stipulates that malachite green cannot be detected. Malachite green and crystal violet are important organic pollutants monitored in the sanitary safety of edible aquatic products in recent years.
The dispersion liquid micro-extraction is a sample pretreatment technology integrating sampling, extraction and concentration. The trace extractant forms countless tiny droplets in the sample solution under the action of the dispersing solvent, and because the dosage of the extractant is small and the contact area with the water phase is large, the mass transfer balance can be quickly achieved, the extraction is hardly influenced by the extraction time, and the method has the advantages of simple and quick operation, low cost, environmental friendliness and the like, thereby being concerned in the pretreatment and analysis of food samples. However, the extractant for liquid-liquid microextraction with the eutectic solvent is mainly hydrophobic, and because of the extremely small amount of the extractant, the contact between the extractant and target molecules is quite limited, and a good extraction effect is difficult to achieve.
Disclosure of Invention
The invention aims to provide a method which has high sensitivity and good reproducibility and is particularly suitable for accurately detecting the residual trace malachite green and crystal violet in the bodies of aquatic animals and in water aiming at the defects of the prior art.
The purpose of the invention is realized by the following technical scheme.
All percentages used herein are by weight unless otherwise indicated.
A method for simultaneously measuring the contents of malachite green and crystal violet is characterized in that: taking 50-100 mL of sample solution, filtering the sample solution by using a water-phase microporous filter membrane, adding 0.5-2 mL of extracting agent and 0.5-1 mL of organic silicon emulsifier, swirling for 1-2 min to form a uniform emulsion, standing for 5-10 min, performing centrifugal phase separation, and taking an upper-layer extract phase to perform determination by using a high performance liquid chromatography; the preparation method of the extracting agent comprises the following steps: mixing choline chloride and phenol according to a molar ratio of 1: 2-4, and heating to 70-80 ℃ to form a uniform and transparent eutectic solvent; the organic silicon emulsifier is one of TMN-6 and TMN-10.
The centrifugal speed is 3000-5000 rpm, and the centrifugal time is 5-10 min.
The phenol is one of phenol, nitrophenol and chlorophenol.
The sample detected by the invention can be edible parts of aquatic animals or culture water bodies.
Compared with the prior art, the invention has the following advantages:
1. according to the method, the eutectic solvent prepared from choline chloride and phenol is used as an extracting agent, the organic silicon emulsifier is used as an emulsion increasing agent for the first time, an extraction target object in the formed emulsion is fully contacted with the extracting agent, the extraction is complete, the phase separation is easy, and the low-content malachite green and crystal violet can be simultaneously and accurately detected by combining a high performance liquid chromatography method.
2. The organosilicon emulsifier has no absorption under ultraviolet spectrum, so that the liquid chromatogram detection baseline is stable and the interference is small.
3. The method has the biggest advantages of fast and uniform emulsion formation, easy phase separation operation, high efficiency, low toxicity, simple operation, high sensitivity, good reproducibility and the like. Is particularly suitable for the quantitative analysis of malachite green and crystal violet residue in bodies of aquatic animals such as fish, shrimps, soft-shelled turtles and the like and in water bodies.
Drawings
FIG. 1 is a chromatogram comparison of Malachite green and crystal violet added with 5. mu.g/kg for example 1. a is subjected to the micro-extraction treatment of the invention, and b is not subjected to the micro-extraction treatment of the invention.
Detailed Description
The invention is described in further detail with reference to the following drawings and examples, which are not intended to limit the technical scope of the invention, and all changes and equivalents that can be made based on the teachings of the invention shall fall within the protective scope of the invention.
Example 1: detection of content of malachite green and crystal violet in fish pond culture water
1. Preparation of eutectic solvent: mixing choline chloride and phenol according to a molar ratio of 1: 2, mixing, and heating to 70 ℃ to obtain a uniform and transparent solution, namely the required eutectic solvent.
2. Manufacturing a working curve: preparing mixed standard solutions of malachite green and crystal violet of 0.01, 0.05, 0.2, 1, 2, 5 and 10 mu g/mL respectively, and carrying out liquid chromatography under the conditions as follows: the chromatographic column is C18Column (250 mm. times.4.6 mm,5 μm), column temperature: 35 ℃, mobile phase: acetonitrile +50m mol/L ammonium acetate buffer solution (pH 4.50) ═ 70+30, flow rate 1.0mL/min, sample volume 20. mu.L, malachite green detection wavelength of 618nm, crystal violet detection wavelength of 588 nm. The linear range, regression equation and correlation coefficient are shown in table 1, and the recovery rate by adding standard is shown in table 2.
TABLE 1 Linear Range of Malachite Green and Crystal Violet, regression equation, correlation coefficient
Table 2 peacock stone green and crystal violet normalized recovery (%) n ═ 6
3. Determining the contents of malachite green and crystal violet in the fish pond culture water body: filtering 50mL of fish farming water through a 0.45-micron water-phase microporous membrane, adding 0.5mL of the eutectic solvent prepared in the step 1 as an extracting agent and 0.5mL of TMN-6 as an emulsifier, carrying out vortex mixing for 1min to form a uniform emulsion, standing for 5min, centrifuging at 3000rpm for 10min, carrying out phase separation, taking 20 mu L of upper-layer extract phase, carrying out high performance liquid chromatography analysis, substituting into a regression equation to calculate that the content of malachite green is 0.5 mu g/L, and not detecting crystal violet.
4. The measurement of the residual quantity of the malachite green and the crystal violet in the GB/T20361-2006 water product commonly used for water body detection-a high performance liquid chromatography fluorescence detection method is taken as a control, and the result is shown in figure 1. As can be seen from FIG. 1, the detection sensitivity of the method of the present invention is significantly improved.
Example 2: cytosine green and Crystal Violet detection in shrimp
1. Preparation of eutectic solvent: choline chloride and nitrophenol are mixed according to a molar ratio of 1: 3, mixing and heating to 75 ℃ to form a eutectic solvent.
2. Manufacturing a working curve: the same as in example 1.
3. Detecting the contents of malachite green and crystal violet in the shrimps:
(1) sample preparation: removing head, shell and intestinal gland of shrimp, collecting muscle part, and homogenizing with high speed tissue triturator, and collecting small blocks of 0.5cm x 0.5cm x 0.5 cm.
(2) Sample extraction: weighing (0.01 g of 5.00 g of soil) sample into a 50mL centrifuge tube, adding 5g of acidic alumina, adding 20mL of acetonitrile, carrying out vortex oscillation for 2min, adding 1mL0.5mol/L of potassium borohydride, carrying out ultrasonic treatment for 5min, standing for 10min, centrifuging for 10min at 4000r/min, and transferring the supernatant into a multi-sample concentration bottle; and adding 10mL of acetonitrile into the centrifuge tube, carrying out vortex oscillation for 2min, centrifuging for 10min, combining the supernatants, carrying out vacuum concentration at 45 ℃ to be nearly dry, dissolving the residue with 5.0mL of acetonitrile, and fixing the volume to 50m L with high-purity water to prepare a sample solution to be detected for later use. And simultaneously, reagent blank experiments are carried out.
(3) And (3) sample determination: and (2) filtering 50mL of the sample solution to be detected by a 0.45-micron water-phase microporous membrane, adding 1mL of the eutectic solvent prepared in the step (1) as an extracting agent and 1mL of TMN-10 as an emulsifier, carrying out vortex mixing for 2min to form a uniform emulsion, standing for 10min, centrifuging at 5000rpm for 5min, carrying out phase separation, carrying out high performance liquid chromatography analysis on 20-micron upper-layer extract phase, substituting into a regression equation for calculation, wherein malachite green is not detected, and the content of crystal violet is 0.1-micron g/kg.
Example 3: detection of content of malachite green and crystal violet in fish
1. Preparation of eutectic solvent: choline chloride and chlorophenol are mixed according to a molar ratio of 1: 4, mixing and heating to 80 ℃ to obtain the eutectic solvent.
2. Manufacturing a working curve: the same as in example 1.
3. Detecting the contents of malachite green and crystal violet in the fish:
(1) sample preparation: removing scales and skin of fish, taking muscle part along dorsal spine, cutting into small pieces no greater than 0.5cm x 0.5cm x 0.5cm, and homogenizing with high speed tissue mashing machine.
(2) Sample extraction: same as example 2
(3) And (3) sample determination: and (3) filtering 50mL of the sample solution to be detected obtained in the step (2) through a 0.45-micrometer water-phase microporous filter membrane, adding 2mL of the eutectic solvent prepared in the step (1) as an extracting agent and 1mL of TMN-6 as an emulsifier, carrying out vortex mixing for 2min to form a uniform emulsion, standing for 10min, centrifuging at 5000rpm for 6min, carrying out phase separation, taking 20 mu L of an upper-layer extraction phase, carrying out high performance liquid chromatography analysis, substituting into a regression equation to calculate, wherein the content of malachite green is 0.3 mu g/kg, and crystal violet is not detected.
Example 4: detection of malachite green and crystal violet in Chinese softshell turtle
1. Preparation of eutectic solvent: mixing choline chloride and phenol according to a molar ratio of 1: 3, mixing and heating to 80 ℃ to obtain the eutectic solvent.
2. Manufacturing a working curve: the same as in example 1.
3. Detecting the content of malachite green and crystal violet in the Chinese softshell turtle:
(1) sample preparation: taking the edible part of muscle of the leg of the Chinese softshell turtle, cutting into small pieces with the size of not more than 0.5cm x 0.5cm x 0.5cm, and fully homogenizing by a high-speed tissue triturator.
(2) Sample extraction: same as example 2
(3) And (3) sample determination: and (3) filtering 50mL of the sample solution to be detected obtained in the step (2) by using a 0.45-micron water-phase microporous membrane, adding 1.5mL of the eutectic solvent prepared in the step (1) as an extracting agent and 0.5mL of TMN-10 as an emulsifier, carrying out vortex mixing for 1min to form a uniform emulsion, standing for 10min, centrifuging at 4000rpm for 8min, carrying out phase separation, taking 20-mu L of the upper-layer extract phase, carrying out high performance liquid chromatography analysis, substituting into a regression equation to calculate that the content of malachite green is 0.2 mu g/kg and the content of crystal violet is 0.1 mu g/kg.
As can be seen from the above examples, the determination method of the invention has high detection sensitivity, and the detection limit of the method is higher than the detection limit of the high performance liquid chromatography fluorescence detection method for determining the residual quantity of the malachite green and the crystal violet in the GB/T20361-2006 aquatic product by 0.5 mu g/kg.
Claims (3)
1. A method for simultaneously measuring the contents of malachite green and crystal violet is characterized in that: taking 50-100 mL of sample solution, filtering the sample solution by using a water-phase microporous filter membrane, adding 0.5-2 mL of extracting agent and 0.5-1 mL of organic silicon emulsifier, swirling for 1-2 min to form a uniform emulsion, standing for 5-10 min, performing centrifugal phase separation, and taking an upper-layer extract phase to perform determination by using a high performance liquid chromatography; the preparation method of the extracting agent comprises the following steps: mixing choline chloride and phenol according to a molar ratio of 1: 2-4, and heating to 70-80 ℃ to form a uniform and transparent eutectic solvent; the organic silicon emulsifier is one of TMN-6 and TMN-10.
2. The method for simultaneously determining the content of malachite green and crystal violet as claimed in claim 1, wherein: the centrifugal speed is 3000-5000 rpm, and the centrifugal time is 5-10 min.
3. The method for simultaneously measuring the content of malachite green and crystal violet as claimed in claim 1, wherein the phenol is one of phenol, nitrophenol and chlorophenol.
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| CN103105367A (en) * | 2013-01-24 | 2013-05-15 | 昆明理工大学 | Method for rapidly detecting malachite green or crystal violet in aquaculture water |
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| CN103105367A (en) * | 2013-01-24 | 2013-05-15 | 昆明理工大学 | Method for rapidly detecting malachite green or crystal violet in aquaculture water |
| WO2016145242A1 (en) * | 2015-03-11 | 2016-09-15 | Agenus Inc. | Methods and compositions for high throughput screening of biomolecules using gel microdrops |
| CN108473484A (en) * | 2015-10-01 | 2018-08-31 | 赛诺米克斯公司 | It can be used as the compound of TRPM8 conditioning agents |
| CN108593799A (en) * | 2018-04-27 | 2018-09-28 | 甘肃省食品检验研究院 | A kind of method of the preparation and analysis water-like Malachite Green and crystal violet and metabolite of magnetism eutectic solvent |
| CN109115898A (en) * | 2018-06-14 | 2019-01-01 | 浙江工业大学 | Based on the remaining method of pesticide in eutectic solvent-inorganic salts double-aqueous phase system analysis water-like |
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