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Method for detecting residual quantity of hydroxymetronidazole in food and product

A technology for hydroxy metronidazole and food is applied in the field of detecting hydroxy metronidazole residues in food, and can solve the problems of cumbersome process and high cost

Pending Publication Date: 2021-12-24
北京维德维康生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, most methods for the determination of hydroxymetronidazole content use liquid chromatography or liquid chromatography tandem mass spectrometry, which is cumbersome and costly
Immunochemical analysis makes up for the shortcomings of physical and chemical analysis due to its unique advantages in the qualitative and quantitative aspects of antigens and antibodies, and the advantages of simple and fast operation, low cost, high sensitivity, and large analysis samples. The method research is few, and the present invention has carried out research on this detection method, and immunochemical analysis method has unique advantages in the qualitative and quantitative aspect of antigen antibody, and its simple and fast operation, low cost, high sensitivity, large analysis sample size, make up for The lack of physical and chemical analysis is of great significance for the scientific use of antibacterial drugs, the containment of bacterial resistance, public health safety and food safety

Method used

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  • Method for detecting residual quantity of hydroxymetronidazole in food and product
  • Method for detecting residual quantity of hydroxymetronidazole in food and product
  • Method for detecting residual quantity of hydroxymetronidazole in food and product

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1, the preparation of hydroxymetronidazole hapten

[0040] 1. Preparation of Hydroxymetronidazole Hapten

[0041] Rinse the 50ml round bottom flask, dry it with ethanol, fix it on the stirrer, and add a stirring bar. Weigh 1g of the raw material, dissolve it with 3ml of sodium methoxide, stir, and leave the sample after all the dissolution. At this time, the solution is purple. Weigh 1.38g of terephthalaldehyde diacetal, add it, stir, and when it is found that the reaction is complete by TLC, After treatment, add hydrochloric acid to quench the reaction, add silica gel, perform column chromatography to mix the sample, perform column chromatography after spin-drying, collect product points, and spin-dry to obtain Intermediate 1, a total of 725 mg.

[0042] Weigh 725 mg of intermediate 1, dissolve it with hydrochloric acid solution, stir, monitor the reaction until complete reaction, process, adjust the pH value to 7, spin the solvent to carry out column chrom...

Embodiment 2

[0053] Embodiment 2, the preparation of hydroxymetronidazole artificial antigen

[0054] 1. Preparation of hydroxymetronidazole artificial antigen

[0055] 1. Synthesis of immunogen

[0056] (1) Dissolve 16.1mg of hydroxymetronidazole hapten in 1.5ml of DMF, stir at 200rpm for 10min, add EDC25.75mg, NHS15.46mg to dissolve, stir at room temperature (500rpm) and activate for 2-3h.

[0057] (2) Weigh 50mg of BSA and dissolve it in 3.5ml of 0.1M sodium bicarbonate solution, stir at 200rpm for 10min to fully dissolve, cool down in an ice bath to 0-4°C, stir at 1000rpm, add the reaction solution in step 1 dropwise (1ml / min), stirring at 500rpm for 24h.

[0058] (3) Put the reaction product into distilled water and rinse the dialysis bag (10cm), dialysis with 1L of 0.01M PBS (1×, pH7.2) at 4°C (100rpm) for 3 days, and change the solution 3 times a day (morning, middle and evening) , changing the medium 9 times in total, centrifuge the dialysis product at 5000rpm for 6min, aliquot...

Embodiment 3

[0063] Example 3, Preparation of Monoclonal Antibody by Immunizing Animals with Hydroxymetronidazole Artificial Antigen

[0064] Prepare hydroxymetronidazole monoclonal antibody according to conventional methods, the specific steps are as follows:

[0065] 1. Animal immunity

[0066] Use the immunogen (hydroxymetronidazole-BSA) prepared in Example 2 at 100 μg per mouse, dissolve the immunogen in normal saline and mix it with Freund’s complete adjuvant in an equal volume, and inject subcutaneously on the back of the neck to immunize 6-8-week-old Balb / c female mice, on the 7th, 14th, and 28th day after the initial immunization, the immunogen and Freund's incomplete adjuvant were mixed in equal volumes, and each additional immunization was given once, and the immune complex was 100 μg / rat 3 days before fusion, without Freund's adjuvant. Adjuvant was used for booster immunization once again.

[0067] 2. Cell fusion and cloning

[0068] According to the conventional method, the...

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Abstract

The invention discloses a method for detecting residual quantity of hydroxymetronidazole in food and a product. A product used for detection is a hydroxyl metronidazole hapten compound shown as a formula I. According to the present invention, the small molecule target analyte hapten is designed and synthesized by relying on the basic principles of immunology and immunochemistry and the residue analysis technical means, and is coupled with the carrier protein to prepare the effective artificial antigen, and the animal is immunized to prepare the specific antibody for the small molecule analyte; the micromolecular target analyte in a food sample is qualitatively or quantitatively detected by utilizing the specific immunological reaction of the antigen and the antibody, and the kit has the characteristics of specificity, sensitivity, accuracy, rapidness, convenience, low cost and the like.

Description

technical field [0001] The invention belongs to the field of rapid detection of drug residues, and relates to a method and a product for detecting residual amounts of hydroxymetronidazole in food. Background technique [0002] Hydroxymetronidazole is the metabolite of metronidazole, a nitroimidazole drug. Because metronidazole has antibacterial and antiprotozoal effects, it can kill and prevent anaerobic bacteria and pathogens, so it is often used in agricultural production to prevent and treat Treatment of poultry trichomoniasis and coccidiosis, swine treponema dysentery and various anaerobic infections of animals. However, metronidazole has potential carcinogenic, teratogenic, mutagenic and genotoxic effects, and its residues pose a threat to animal foods. my country's "National Food Safety Standard Maximum Residue Limits of Veterinary Drugs in Food" (GB 31650-2019) stipulates that metronidazole is allowed to be used as a therapeutic veterinary drug, but it must not be de...

Claims

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Application Information

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IPC IPC(8): C07D233/94C07K14/765C07K14/77C07K16/44C07K1/107G01N33/53G01N33/531G01N33/543G01N33/58
CPCC07D233/94C07K14/765C07K14/77C07K16/44G01N33/5308G01N33/531G01N33/581G01N33/543Y02A50/30
Inventor 刘河冰邢维维覃丹凤刘薇金兴刘婷郝建祥
Owner 北京维德维康生物技术有限公司