CN103105367B - Method for rapidly detecting malachite green or crystal violet in aquaculture water - Google Patents

Abstract

The invention discloses a method for rapidly detecting malachite green or crystal violet in aquaculture water, that is, a rapid synergistic extraction system composed of extraction aid-extractant-water, so that crystal violet or malachite green is extracted to the organic phase on the upper part of the centrifuge tube In this method, compared with the content of standard malachite green or crystal violet, the detection of malachite green or crystal violet in the sample was carried out, and the detection limit of the method was 0.05 μg/mL; compared with the existing detection methods, the method has mild extraction conditions and rapid detection. The entire analysis and detection process can be completed in 5-10 minutes, the cost is low, no large-scale instruments are required, and the accuracy is high. It is suitable for the detection of the safety of edible aquatic products and has great application potential and broad market prospects.

Description

A method for rapid detection of malachite green or crystal violet in aquaculture water

technical field

The invention relates to a method for quickly detecting malachite green or crystal violet in aquaculture water, which belongs to the detection technology of banned additives for food animals.

Background technique

Both malachite green and crystal violet belong to triphenylmethane dyes, which are bright in color and low in cost, and are widely used in aquaculture, food, medical and textile industries. With the development of science and technology, people have gradually discovered the harm of these two compounds to the human body. Because malachite green and crystal violet have bactericidal and anti-parasitic effects, they can prevent various fish diseases and are widely used in aquaculture. However, both itself and its metabolites have carcinogenic, teratogenic, mutagenic, chromosome breakage and respiratory system toxicity. Malachite green and crystal violet have been banned for use in aquaculture by the EU and US FDA. In order to avoid harm to the health of consumers caused by eating animals, in 2002, the Ministry of Agriculture of my country announced No. 193 "List of Veterinary Drugs and Other Compounds Prohibited for Food Animals" which also listed malachite green as a prohibited drug. However, due to its good antibacterial effect and low price, many farmers still use it illegally. Therefore, the residual monitoring of malachite green and crystal violet is particularly important.

At present, the methods for detecting malachite green and crystal violet mainly include: high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (HPLC-MS), liquid chromatography-tandem mass spectrometry (HPLC-MS-MS), isotope internal standard method. These methods require the use of large instruments such as HPLC and MS, which are costly and time-consuming, and are not suitable for on-site detection.

Rapid collaborative extraction technology is a new type of environmentally friendly solute enrichment and separation method. There is no report on the extraction and separation of malachite green and crystal violet by rapid collaborative extraction. The extraction and enrichment of green and crystal violet requires heating, and the analysis takes tens of minutes to complete. The present invention adopts the rapid cooperative extraction of malachite green and crystal violet in the aquaculture water by using the surfactant auxiliary extraction agent-extractant-water, and has normal temperature operation, and the detection can be completed in 5-10 minutes quickly, the result is accurate, the sensitivity is high, and the cost is low Features.

Contents of the invention

The object of the present invention is to address the deficiencies of the prior art, to provide a method for quickly detecting malachite green or crystal violet in aquaculture water, which can reduce detection cost and shorten detection time while ensuring detection accuracy and sensitivity.

The purpose of the present invention is achieved through the following technical solutions:

(1) Take 5mL of the sample solution containing malachite green or crystal violet and 5mL of the standard solution of malachite green or crystal violet in a 10mL centrifuge tube with a stopper, add the extraction aid surfactant to the standard solution and the sample solution respectively and extractant, vortex, centrifuge;

(2) Compare the colors of the upper organic phases of the two after centrifugation. If the color of the sample solution is darker than that of the standard solution, it means that the content of malachite green or crystal violet in the sample solution is greater than the content of malachite green or crystal violet in the standard solution. On the contrary, it means that the content of malachite green or crystal violet in the sample solution is less than that in the standard solution.

The sample solution containing malachite green or crystal violet described in step (1) of the present invention refers to aquatic product aquaculture water, and the concentration of the malachite green or crystal violet standard solution is 0.05-0.2 μg/mL.

In the present invention, the extractant is one of octanol, isooctyl alcohol, renyl alcohol and isononyl alcohol, and the consumption is 0.2-0.5 mL.

The extraction aid in the present invention is surfactant polyethylene glycol octyl phenyl ether (Triton-100 or Triton-114), nonylphenol polyoxyethylene ether (Np-7 or Np-9), branched secondary alcohol One of the polyoxyethylene ethers (TMN-6), the volume percentage concentration of the extraction aid is 5%-10% (v/v), and the dosage is 10-50μl.

In the present invention, the vortex time is 5-30s, the centrifugation time is 3-8min, and the centrifugation rate is 2500-5000r/min.

Compared with the prior art, the present invention has the following significant advantages:

1. The present invention uses extraction aid-extractant-water rapid synergistic extraction as a pretreatment method for sample enrichment to enrich malachite green or crystal violet in a very small extractant, and the enrichment factor reaches 50 times. Before enrichment, the solution is colorless. After enrichment, a blue-green or purple ring appears on the upper layer of the centrifuge tube. The color is obvious and easy to observe. The detection limit can reach 0.05 μg/mL;

2. There is no need to purchase expensive testing equipment such as HPLC and MS. The instruments required for the experiment are simple, the conditions are mild, the operation is simple, and the accuracy is high. Non-professionals can also complete the entire analysis and testing process in 5-10 minutes. A fast, simple, intuitive and economical method for detecting malachite green or crystal violet in aquaculture water is provided, which is suitable for the detection of the safety of edible aquatic products. This method has great application potential and broad market application prospects.

Detailed ways

The present invention will be further described below in conjunction with the examples, but the protection scope of the present invention is not limited thereto.

Embodiment 1: the method for rapid detection of malachite green in aquaculture water, concrete operations are as follows:

① Put 5 mL of eel breeding water and 5 mL of malachite green standard series solutions with concentrations of 0.05, 0.1, 0.15, and 0.2 μg/mL respectively into 10 mL centrifuge tubes with stoppers, and add 300 μL isooctyl alcohol and 10 μL polymer to each centrifuge tube. Ethylene glycol octyl phenyl ether Triton -100 (volume percent concentration: 10%); vortex for 5s to form a uniform emulsified system; centrifuge at 3500 r/min for 3 min, demulsification, phase separation, extractant A layer of ring is formed on the upper part of the centrifuge tube, and malachite green is extracted into this layer of ring; ②Under the same conditions, a layer of blue-green ring formed by the eel breeding water on the upper part of the centrifuge tube and a layer of color formed by the standard series solution Compared with the ring color scale, the color ring formed by eel culture water is darker than that formed by 0.2 μg/mL standard malachite green, and the content of malachite green in eel culture water is higher than 0.2 μg/mL.

Embodiment 2: the method for rapid detection of crystal violet in aquaculture water, the specific operations are as follows:

①Put 5mL of carp breeding water and 5mL of crystal violet standard series solutions with concentrations of 0.05, 0.1, and 0.15μg/mL respectively into 10ml stoppered centrifuge tubes, and add 200μl octanol and 50μl nonylphenol polyoxygen to each centrifuge tube Vinyl ether Np-7 (volume percent concentration: 5%), vortex for 20s to form a uniform emulsified system; centrifuge at 4000 r/min for 5min, demulsify, separate phases, and the extractant forms a ring on the upper part of the centrifuge tube , crystal violet is extracted into this ring; ②Under the same conditions, compare the purple ring formed by the carp culture water on the upper part of the centrifuge tube with the color ring formed by the standard series of solutions, the color of the color ring formed by the carp culture water It is lighter than the color formed by 0.05μg/mL standard crystal violet, and the content of crystal violet in carp breeding water is lower than 0.05μg/mL.

Embodiment 3: the method for rapid detection of malachite green in aquaculture water, concrete operations are as follows:

① Put 5 mL of shrimp culture water and 5 mL of malachite green standard series solutions with concentrations of 0.05, 0.1, 0.15, and 0.2 μg/mL respectively into centrifuge tubes with stoppers, and add 500 μl of Ren alcohol and 20 μl of branched chain paraffin to each centrifuge tube. Alcohol polyoxyethylene ether TMN-6 (volume percent concentration: 10%); vortex for 15s to form a uniform emulsified system; centrifuge at 2500 r/min for 6min to break the emulsion and separate phases, and the extractant is on the upper part of the centrifuge tube A ring is formed, and malachite green is extracted into this layer of ring; ②Under the same conditions, compare the color scale of a layer of blue-green ring formed by the shrimp culture water on the upper part of the centrifuge tube and a layer of color ring formed by the standard series solution , the color of the ring formed by the shrimp breeding water is between the color ring formed by 0.05-0.1 μg/mL standard malachite green, and the content of malachite green in the shrimp breeding water is between 0.05-0.1 μg/mL.

The comparison results of the above examples and high performance liquid chromatography are shown in Table 1.

Table 1: Example results and high performance liquid chromatography comparative results

As can be seen from table 1 result: the content result of the content result of malachite green determined by the high performance liquid chromatography that national standard adopts in the aquaculture water that measures with the present invention and the malachite green compares, and illustrates that method has certain reliability. The method for detecting malachite green in the invention is convenient and fast, and the accuracy of the result is difficult to achieve by other methods.

Embodiment 4: the method for rapid detection of crystal violet in aquaculture water, the specific operations are as follows:

①Put 5mL of crucian carp breeding water and 5mL of crystal violet standard series solutions with concentrations of 0.05, 0.1, 0.15, and 0.2μg/mL respectively into 10ml centrifuge tubes with stoppers, and add 400μl isononyl alcohol and 30μl nonyl to each centrifuge tube Phenol polyoxyethylene ether Np-9 (8% concentration by volume), vortex for 30s to form a uniform emulsified system; centrifuge at 5000 r/min for 8min, demulsify, separate phases, and the extractant forms on the upper part of the centrifuge tube One layer of ring, crystal violet is extracted into this layer of ring; ② Under the same conditions, compare the layer of purple ring formed by the carp culture water on the upper part of the centrifuge tube with the color ring formed by the standard series of solutions, the color formed by the carp culture water The color of the ring is between the colors formed by standard crystal violet at 0.1-0.15 μg/mL, and the content of crystal violet in carp breeding water is between 0.1-0.15 μg/mL.

Claims (3)

1. a method for fast detecting aquaculture water Malachite Green or crystal violet, comprises the following steps:

(1) get 5mL and be placed in tool plug centrifuge tube containing malachite green or the sample solution of crystal violet and the standard solution of 5mL malachite green or crystal violet, in standard solution and sample solution, add respectively and help extraction agent and extractant, vortex, centrifugal;

(2) color of more centrifugal rear both upper organic phase, if the color of sample solution is deeper than standard solution, interpret sample solution Malachite Green or crystal violet content are greater than malachite green in standard solution or the content of crystal violet, otherwise interpret sample solution Malachite Green or crystal violet content are less than the content in standard solution;

Described extractant is the one in octanol, isooctyl alcohol, nonyl alcohol, isononyl alcohol, and consumption is 0.2-0.5mL;

Described help extraction agent be the one in surfactant polyethylene octyl phenyl ether Triton-100, Triton X-100 Triton-114, NPE Np-7, NPE Np-9, branched secondary alcohol polyoxyethylene ether TMN-6, helping extraction agent concentration of volume percent is 5-10%, and consumption is 10-50 μ l.

2. the method for fast detecting aquaculture water Malachite Green according to claim 1 or crystal violet, it is characterized in that: described in step (1) is aquaculture water containing malachite green or crystal violet sample solution, and malachite green or crystal violet concentration of standard solution are 0.05-0.2 μ g/mL.

3. the method for fast detecting aquaculture water Malachite Green according to claim 1 or crystal violet, is characterized in that: vortex time is 5-30s, centrifugation time is 3-8min, and centrifugation rate is 2500-5000r/min.