Malachite green, crystal violet rapid detection pretreatment kit
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Malachite green, crystal violet rapid detection pretreatment kit

Crystal violet (Gentiana violet) and malachite green are triphenylmethane dyes and have strong toxic and side effects. It is prohibited for use as a disinfectant by the State. Because the dye has special effects on the prevention and control of fungal infections in aquatic animals, it is cheap and easy. It is bought and therefore often abused in aquaculture. Studies have shown that most malachite greens quickly convert into the recessive malachite green in muscles in the body of the fish. The rate of discharge is low and mostly remains in the body. Therefore, all countries in the world use this as a technical barrier to prevent the import of aquatic products in China and require a detection limit of 2 ng/g. The domestic media is also very concerned about this. The roasting industry has become even more prominent. The existing inspection methods used by most companies have drawbacks such as cumbersome operation, long time-consuming operation, post-exposure derivation, improper operation, and unstable recovery, resulting in the obstruction of exports.
I. Comparison of the technical parameters of the rapid detection pretreatment kit for crystal violet and malachite green and traditional methods

Technical Parameters

Exercise steps

Do you need to configure the pretreatment reagent?

Pre-processing takes time

Test items

Do you need post-derived

Detection limit

Existing methods

12 steps

need

4 hours

Malachite Green and Leuco Malachite Green

need

0.5ng/g

Kit method

2 steps

Need not

20 minutes

Total amount of malachite green and crystal violet

Need not

0.5ng/g

Second, the technical points:
Malachite green rapid detection pretreatment kit extractant can effectively extract leuco malachite green, malachite green, crystal violet, leuco crystal violet in muscle tissue, and has high extraction efficiency. The test results are malachite green, leucomalachite green, crystal violet, leuco crystal violet.

Third, the principle of the method:
The residue in the sample was extracted with extractant 1 and extractant 2. After centrifugation, the supernatant was taken out and evaporated to dryness. The residue was dissolved in 1 ml of mobile phase, filtered through a microporous membrane, and separated into a liquid phase C18 column. UV or DAD detector detection, external standard method.

Fourth, limit of detection The detection limit of this method is: LC-MS 0.5ug/kg; HPLC 2ug/kg.

V. Recovery rate of 0.5 ug/kg, 2 ug/kg, 10 ug/kg, 20 ug/kg, 50 ug/kg of added malachite green and leucomalachite green recovery rate of 70 to 80%, crystal violet and leucocrystal The purple recovery rate is 80 to 100%.

This method takes only 20 minutes to pre-treat.

Sixth, product packaging methods and prices contain 40 sets of reagents per box, can detect 40 samples; Price: 1000 yuan / box

Map

Rapid Determination Method of Residues of Malachite Green and Crystal Violet in Aquatic Products 1 Scope This standard specifies the malachite green and its metabolites in the aquatic products and their products. The leucomalachite green, crystal violet and its metabolites are leuco crystal violet. Residues High Performance Liquid Chromatography-Tandem Mass Spectrometry and Rapid Determination of High Performance Liquid Chromatography. This standard applies to the determination of residual amounts of malachite green and its metabolites, malachite green, crystal violet, and their metabolites, leuco crystal violet in aquatic products and their products. 2 Principle The residue in the sample was treated with Malachite Green and Crystal Violet Rapid Pretreatment Kit, centrifuged, and the supernatant was rotated and evaporated to dryness. The mobile phase dissolved the residue. After high-speed centrifugation, the microporous membrane was filtered and used efficiently. Liquid chromatography-tandem mass spectrometry or high performance liquid chromatography. 3 Reagents and materials Unless otherwise specified, all reagents were of analytical grade and water was redistilled. 3.1 Acetonitrile: Liquid chromatography. 3.2 Methanol: Pure liquid chromatography. 3.2 Anhydrous ammonium acetate. 3.3 glacial acetic acid: pure liquid chromatography. 3.4 5mol/l ammonium acetate buffer solution: Dissolve 38.5g of anhydrous ammonium acetate in 90ml of water, adjust the pH to 7.0 with glacial acetic acid, and finally dilute to 100ml with water. 3.5 0.1mol/l ammonium acetate buffer solution: Dissolve 7.71g of anhydrous ammonium acetate in 1000ml of water and adjust the pH to 4.5 with glacial acetic acid. 3.7 5mmol/l ammonium acetate buffer solution: Dissolve 0.385g of anhydrous ammonium acetate in 1000ml of water. Glacial acetic acid adjusts the pH to 4.5 over a 0.2um filter. 3.8 Acetate buffer: Dissolve 4.95g of anhydrous sodium acetate and 0.95g of p-toluenesulfonic acid in 950ml of water, adjust the solution to pH 4.5 with glacial acetic acid, and finally dilute to 1L with water. 3.9 Standards: The purity of malachite green (MG), leucomalachite green (LMG), crystal violet (CV), and leuco crystal violet (LCV) is greater than 98%. 3.10 Standard solution: Accurately weighed malachite green, leucomalachite green, crystal violet, leuco crystal violet, prepared into a standard stock solution of 100 μg/mL with acetonitrile, and diluted to 1 μg/mL with acetonitrile. Solution. -18°C protected from light. 3.11 mixed standard working solution: Mix standard solution of malachite green, leucomalachite green, crystal violet, and leuco crystal violet 20ng/ml diluted with mobile phase diluted standard solution (3.2.5). 0 ~ 4 °C protected from light. 3.12 Malachite green and crystal violet rapid detection pretreatment kit (China National Academy of Inspection and Quarantine, developed by Beijing Luqiao Commercial Inspection New Technology Company). 4 instruments and equipment 4.1 high performance liquid chromatography - tandem mass spectrometer: equipped with electrospray (ESI) ion source. 4.2 HPLC: equipped with UV-visible detector. 4.3 Homogenizer. 4.4 Rotary evaporator. 4.5 Centrifuge: 4000r/min, 8000r/min 4.6 oscillator. 4.7 PTFE centrifuge tube: 2.5 ml, 50 ml, 100 ml, with stopper. 4.8 Microporous filter: 0.45um. 4.9 Ultrasonic water bath. 5 Sample Preparation Weigh the sample about 5.0g into a 50ml centrifuge tube. Add malachite green and crystal violet to quickly detect the extractant in the pretreatment kit. Homogenize at a speed of 8000r/min for 30s. Add the extraction. The agent 2 was shaken for 1 min, centrifuged at 4000r/min for 5 min, and 10 ml of the supernatant was taken in a heart bottle and 10 ml of isopropanol was added to a 50-55° C. water bath and evaporated to dryness. The residue was dissolved by mobile phase (HPLC, 1 ml, mass spectrometry, 2.5 ml), the solution was placed in a 2.5 ml centrifuge tube, centrifuged at 8000 r/min for 5 min, filtered through a microporous membrane, and the filtrate was measured by the instrument. 6 Determination 6.1 High Performance Liquid Chromatography - Tandem Mass Spectrometry Conditions Refer to National Standard GB/T19857-20056.2 Liquid Chromatography Determination 6.2.1 Liquid Chromatography Conditions a) Column: C18 Column, 250mm4.6mm (id), Particle Size 5um; b) Flow Phase: acetonitrile + acetate buffer (80+20, V/V); c) flow rate: 1.0 mL/min; d) column temperature: room temperature; e) detection wavelength: 0-5.0 min, 618 nm; 12 min, 588 nm; 12-18 min, 267 nm; Note: Malachite green 618 nm, crystal violet 588 nm, leuco malachite green, leuco crystal violet 267 nm. f) Sample volume: 50uL According to the measured malachite green, leucomalachite green, crystal violet, and leuco crystal violet content in the sample solution, the standard working solution with similar peak height was selected. The response values ​​of malachite green, leucomalachite green, crystal violet, and leuco crystal violet in the standard working solution and sample solution should be within the linear range of the instrument detection. The standard working solution and the sample volume were measured by inserting the sample. Under the above chromatographic conditions, the retention times of malachite green, leucomalachite green, crystal violet, leuco crystal violet were approximately 3.9 min, 5.8 min, 13.5 min, and 14.7 min. 7 The blank test is carried out according to the above determination procedure except that no sample is added. 8 Calculation and expression of results Calculate the residual amounts of malachite green, leucomalachite green, crystal violet and leuco crystal violet in the test sample by the following formula. The calculation result must be deducted from the blank value.

20VACSX= NMAS


Residue of test components in X-test tissue, ug/kg; V-final volume, mL, malachite green, leucomalachite green, crystal violet, or leuco crystal violet in A-solution Peak area of ​​the peak. M - Sample area weight of sample, peak area of ​​gAS-malachite green, leucomalachite green, crystal violet or leuco crystal violet standard working solution. 20-Malachite green and crystal violet were used to quickly detect 1 volume of extractant in the pretreatment kit. N - Take the supernatant volume, mL. The concentration of CS-malachite green, leucomalachite green, crystal violet or leuco crystal violet standard working solution, ug/L. 9 Method detection limit Malachite green, leucomalachite green, crystal violet, leuco crystal violet detection limit: high-performance liquid chromatography - mass spectrometry are 0.5ug/kg; high performance liquid chromatography is 2ug/kg. 10 Recovery rate This method is malachite green, leucomalachite green, crystal violet, leuco crystal violet. The recovery rate is 85% to 105% for both high performance liquid chromatography and mass spectrometry, and 85 for high performance liquid chromatography. % to 105%. 11 Precision The RSD of this method for malachite green, leucomalachite green, crystal violet, leuco crystal violet is: the high performance liquid chromatography-mass spectrometry method is less than 10%; the high performance liquid chromatography method is less than 10%. Appendix A (Informative Appendix) Figure 1. High-Performance Liquid Chromatography Malachite Green, Leuco Malachite Green, Crystal Violet, Leuco Crystal Violet Spectra Note: Malachite Green 4.0 min, Malachite Green 13.6 Min, crystal violet 5.4 min, leuco crystal violet 14.7 min.

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