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Strong carcinogenic substance aflatoxin M1 detection program
Aflatoxins are mainly found in foods such as grains, nuts, peanuts, cottonseed meal, corn, dried paprika, and some products related to human blood and animal feed. The growth of mold does not necessarily indicate the presence of aflatoxin, because aflatoxin is produced only when it is suitable for humidity, temperature, and ventilation. Aflatoxin B has blue fluorescence and Class G has green fluorescence. In addition to the common (B1, B2, G1, G2) in vegetables, there is also aflatoxin M in the milk of cows fed toxic feed, where the most toxic metabolite of M is the 4-hydroxylated product Bs. Aflatoxin M1 is a hydroxylated metabolite of aflatoxin B1 and is also a potent carcinogen. The harm is huge, and human consumption of food such as cow's milk and its products is one of the foods susceptible to aflatoxin M1 contamination. The harmfulness of aflatoxin is that it has a destructive effect on the liver tissue of humans and animals. In severe cases, liver cancer may even cause death. Toxicity is 100 times that of arsenic. In fact, it mainly refers to aflatoxin than arsenic.
At present, the detection methods of Aflatoxin M1 include high performance liquid chromatography (HPLC) and enzyme-linked immunosorbent assay. Liquid chromatography can accurately detect the small amount of aflatoxin in dairy products. The aflatoxin M1 ELISA kit allows rapid and accurate analysis of aflatoxin M1 residues in the sample.
As a major domestic supplier of mycotoxin detection technology and product services, in 2010, Tellabs Technology invested heavily in the establishment of the "Inspection Application Laboratory", focusing on food safety incidents, especially on the detection of mycotoxin safety. Propose a solution quickly with expectations. In the near future, the issue of aflatoxin M1 in milk should be exceeded. Tellabs Technology provides two detection schemes to help companies easily cope with the limited detection of aflatoxin M1 in milk.
High performance liquid chromatography:
Determination of aflatoxin M1 in milk and dairy products according to national standard GB 5413.37-2010
Pre-treatment of the experiment:
Experimental instruments and consumables include:
Constant temperature water bath solvent filter (with vacuum pump) 1000ml high speed homogenizer aflatoxin immunity M1 affinity column glass fiber filter paper glass syringe no fluorescence characteristics glass test tube air pressure pump ultrasonic cleaning machine quantitative filter paper cylinder sample bottle volumetric pipette or Pipette
Experimental reagent: chromatographic acetonitrile chromatography methanol sodium hydroxide petroleum ether ultrapure water or deionized water, etc.
HPLC conditions:
High performance liquid chromatography
Column
C18 column, 150*4.6mm, 5um
Mobile phase
Acetonitrile-water (25:75)
Fluorescence detector:
The excitation wavelength is 365 nm, and the emission wavelength is 435 nm;
Column temperature
Room temperature
Flow rate
1.0ml/min
Injection volume
20ul
Peak time
10 minutes or so
Sample Sampling: The largest source of error in mycotoxin analysis is the collection and sampling of samples, so sample collection and sampling must be representative.
Sample preparation: different samples
milk The water bath is heated to 40 degrees, and the supernatant is centrifuged to purify the column;
Milk powder and powdered infant formula : Weigh a proper amount of milk powder sample, use it in distilled water, mix it evenly to make it fully dissolved, and centrifuge it to purify the column with the supernatant;
Fermented milk ( including solid, semi-solid and fleshy ) : Weigh a proper amount of milk powder sample, use it in distilled water, adjust the pH to slightly alkaline, homogenize with high-speed homogenizer, centrifuge after heating in water bath, take supernatant The liquid is uniformly mixed to make it fully dissolved, and the supernatant is centrifuged and purified by the column;
Cheese: Weigh the appropriate amount of shredded or pulverized, pass through the sieve and dissolve with pure water, homogenize with a high-speed homogenizer, collect the supernatant after ultrasonic extraction, then extract twice with petroleum ether, and then extract with a suitable organic solution. Take a number of supernatants to purify the column
The milk powder sample is used for dissolving distilled water, adjusting the pH value to slightly alkaline, and the mixture is heated and centrifuged, and the supernatant is mixed uniformly to be fully dissolved, and the supernatant is centrifuged and purified by the column;
Cream: Weigh the appropriate amount and add methanol water to dissolve. After shaking, the separating funnel takes the lower layer solution, and after evaporation, it is dissolved in water for column purification.
Immune column purification step: fully in line with GB/T 18980-2003
The immunoaffinity chromatography column was taken out, returned to room temperature, and the upper plug was taken out obliquely and cut, and then inserted back into the affinity column.
连接 Attach the immunoaffinity column to the glass syringe. Accurately remove the clear liquid into the glass syringe;
连接 Connect the air pressure pump to the glass syringe, adjust the pressure so that the solution slowly passes through the immunoaffinity column at a flow rate of about 2-3 mL/min until 2~3 mL of air passes through the column;
â”…â”… Replace the glass syringe with the affinity column, add 10 ml of water to the syringe, adjust the pressure so that the water cleans the affinity column at a flow rate of about 6 mL/min.
â”…â”… Accurately add 4 mL of chromatographic grade acetonitrile to elute at a flow rate of 1 mL/min to 2 mL/min. Collect all eluates in glass tubes for testing.
Note : The eluent in the collection tube is the purified sample, which can be directly detected by ELISA, fluorescence and high performance liquid phase.
HPLC testing requires instruments and consumables
product name
Description
specification
Aflatoxin M1 solid standard
Reference control
100ug 1mg
Aflatoxin M1 liquid standard
Concentration 0.5ug/ml, soluble in acetonitrile
1ml
PriboFast Aflatoxin M1 Immunoaffinity Column
For sample pretreatment column analysis of aflatoxin
25/50T
PriboFast pump flow operator (8-bit)
For immunoaffinity column purification sample processing, convenient control of flow rate
Special glass fiber filter paper for aflatoxin detection
It is used to remove some peaks during HPLC detection and effectively improve sensitivity. 110, 1.5um
100P
Stainless steel homogenizer
High-speed homogenization of samples to effectively improve extraction efficiency
Rotary evaporator - ultrasonic generator
Enzyme-linked immunosorbent assay: in line with national standards
The enzyme-linked immunosorbent assay uses a competition ELISA to pre-coat aflatoxin B1 antigen on a microplate, add a sample (or aflatoxin M1 standard solution) and horseradish peroxidase-labeled aflatoxin M1 antibody. The aflatoxin M1 in the sample or standard solution competes with the aflatoxin M1 antigen pre-coated on the plate well for binding to the horseradish peroxidase-labeled aflatoxin M1 antibody. Unbound enzyme-labeled antibodies are removed upon washing. Add TMB coloring solution and read the absorbance. The absorbance of the sample is inversely related to the content of the aflatoxin M1 antigen contained in the residue. The content of the corresponding residue aflatoxin M1 can be obtained by comparing the standard curve.
Sample processing
Directly centrifuge the milk to take the supernatant for testing
Milk powder and other solid dairy products: need to be dissolved in deionized water, centrifuged to take the supernatant for testing
Kit detection time: 30 minutes + 30 minutes
Cross-reaction rate:
Aflatoxin M1
100%
Aflatoxin B1
8%
Aflatoxin B2
10%
Aflatoxin G1
7%
Aflatoxin G2
5%
ELISA method detection needs:
Description
PriboFast aflatoxin M1 enzyme-linked immunosorbent assay kit
Detection range (0.1 ppb -8.1ppb)
96 holes
Leibo semi-sterilized pipette (color)
Multiple ranges: 0.5-10; 20-200; 5-50; 1000-5000ul
support
Leibo eight-and-a-half sterilization pipette
Range: 5-50ul; 50-300ul
support
Microplate reader
Imported, domestically produced instruments,
station
Analyst requirements:
Chemical analysis instruments and related disciplines or those with experience in high performance liquid chromatography.
Note: As one of the leading providers of solutions for mycotoxin detection technology worldwide, PriboLab provides professional solutions for the global agricultural production, food processing and food, feed industry, etc. with a strong research and development team and research on mycotoxin detection technology. Mycotoxin detection technology products and technical services . As the only partner of PriboLab in China, Beijing Tellabs actively provides high-quality mycotoxin detection technology and product services to domestic customers, providing domestic customers with suitable mycotoxin detection solutions to help domestic customers establish efficient and high-quality mycotoxin project testing. platform.