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Method for extracting salinomycin compound from animal sample and special immune affinity sorbent thereof

A technology of salinomycin and compounds, which is applied in the field of extracting salinomycin compounds, can solve the problems of excessive waste of organic solvents, cumbersome treatment process, and long time required to achieve improved analysis quality, simple method operation, and pretreatment process simplified effect

Inactive Publication Date: 2009-05-20
CHINA AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The methods for detecting the residual amount of salinomycin and methyl salinomycin mainly include high-performance liquid chromatography (HPLC), liquid-mass spectrometry (LC-MS), liquid-mass tandem mass spectrometry (LC-MS / MS), etc.; The pretreatment of these methods utilizes liquid-liquid distribution, conventional SPE column purification and separation, all of which have shortcomings such as cumbersome treatment process, poor purification effect, waste of organic solvents, and long time required to varying degrees.

Method used

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  • Method for extracting salinomycin compound from animal sample and special immune affinity sorbent thereof
  • Method for extracting salinomycin compound from animal sample and special immune affinity sorbent thereof
  • Method for extracting salinomycin compound from animal sample and special immune affinity sorbent thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0040] Embodiment 1, the preparation of the immunochromatographic column of purifying salinomycin and methyl salinomycin

[0041] Salinomycin was purchased from Sigma, USA, and its product catalog number is 53003-10-4.

[0042] 1. Preparation of salinomycin monoclonal antibody

[0043] (1) Synthesis of salinomycin hapten:

[0044] Salinomycin and thionyl chloride are subjected to condensation reaction to obtain salinomycin acyl chloride, and then salinomycin acyl chloride is reacted with aminoacetic acid to obtain a salinomycin hapten.

[0045] The specific steps of hapten synthesis are:

[0046] (1) Stir and mix the salinomycin and thionyl chloride at a molar ratio of 1:1, and react at room temperature (25° C.) for 4 hours to obtain salinomycin acid chloride;

[0047] (2) Salinomycin acid chloride and aminoacetic acid were mixed uniformly at a volume ratio of 1:1, pyridine was added for catalysis, and the reaction was stirred under ice bath conditions to obtain the salinom...

Embodiment 2

[0096] Example 2, the preparation of the kit containing the immunochromatographic column in Example 1 and its purification effect on salinomycin and methyl salinomycin

[0097] 1. Preparation of kits for purifying salinomycin and methyl salinomycin

[0098] The kit is composed of immunochromatographic column (IAC column), salinomycin, methyl salinomycin standard solution, washing solution, eluent, preservation solution, sponge bracket, and the sponge bracket is provided with holes and grooves. groove. There are reagent bottles containing salinomycin, methyl salinomycin standard solution, washing solution, eluent and preservation solution in the groove of the sponge bracket, and IAC columns are housed in the holes of the sponge bracket. The immunochromatographic column is the immunochromatographic column prepared in Example 1 coupled with the monoclonal antibody secreted by the salinomycin monoclonal antibody hybridoma cell line C-3-3 CGMCC No.2587.

[0099] The washing solut...

Embodiment 3

[0114] Embodiment 3, use the kit described in embodiment 2 to detect salinomycin and methyl salinomycin in the sample

[0115] 1. Sample pretreatment:

[0116] 1) Take chicken muscle and chicken liver tissues respectively and put them in a homogenizer, and homogenize at 10000rpm for 3 minutes. Store at -20°C for later use.

[0117] 2) Weigh 5.0 grams of homogenized chicken muscle and chicken liver tissue samples, respectively, and place them in 100ml polypropylene centrifuge tubes, then add 10g of anhydrous sodium sulfate to the tubes, mash them with a glass rod, and let stand for 5 minutes Then add 20ml of acetonitrile (muscle) or methanol (liver), vortex to mix, oscillate for 20 minutes, centrifuge at 3500g for 10 minutes, take the supernatant, repeat the extraction once, combine the supernatant, and put it into a chicken heart bottle. Rotate evaporated at 50°C to 2-3ml, transfer it to a 10ml centrifuge tube, wash the heart bottle with 2ml methanol and incorporate it, cent...

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Abstract

The invention discloses a method for extracting salinomycin compounds from an animal sample and a special immunoaffinity absorbent thereof. The immunoaffinity absorbent for extracting the salinomycin compounds from the animal sample consists of a solid phase carrier and a salinomycin monoclonal antibody which is coupled with the solid phase carrier; the salinomycin monoclonal antibody is an antibody obtained by taking a conjugate of salinomycin semiantigen and carrier protein as immunogen; and the salinomycin compounds are salinomycin or methylsalinomycin. The immunoaffinity absorbent and a chromatographic column use the high specific salinomycin monoclonal antibody, have high selectivity, ensure the reliability of detection effect, greatly simplify the pretreatment processes of samples at the same time, are particularly applicable to the pretreatment of a trace amount of salinomycin and methylsalinomycin in muscle and liver, and improve the analysis quality. A detection method of the invention can efficiently detect the content of the salinomycin and the methylsalinomycin, and remedy the disadvantages that the direct assay of the samples by a single immunoassay technology has less information amount, poor quantification accuracy, or low selectivity of physical and chemical methods and so on, so the immunoaffinity absorbent, the chromatographic column, a reagent kit and the methods for extracting and detecting the salinomycin compounds in the animal sample are suitable to be promoted and applied.

Description

technical field [0001] The invention relates to a method for extracting salinomycin compounds from animal samples and a special immunoaffinity adsorbent thereof. Background technique [0002] With the development of life sciences, people have become more and more interested in the substances and their changes in living organisms, and the analysis of biological samples has become a necessary means to explore and discover the mysteries of life. Due to the complex components of biological samples, the concentration of analytes is low, and most of the sample volumes are small, which puts forward higher requirements for the selectivity and sensitivity of the analytical method. Immunoaffinity chromatography (IAC, immunoaffinity chromatography) is an analytical method that combines an immune response with a chromatographic analysis method. Its high selectivity and high affinity undoubtedly simplify the analysis process. In the analysis of veterinary drug residues, the simplest an...

Claims

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Application Information

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IPC IPC(8): B01J20/286C07D493/20
Inventor 沈建忠张素霞史为民万宇平曹兴元江海洋冯才伟李建成王战辉
Owner CHINA AGRI UNIV
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