Introduction

1. General

Aflatoxin is a kind of fungus toxic metabolites. They mainly exist in the peanut, grains, nuts, seeds and some food, animal feed and other related products. Natural pollution of aflatoxin is given priority to aflatoxin B1, which is classified as first-degree carcinogen by WHO is the most toxic . It proved has great harm to human and animal liver, kidneys and other tissues and organs . Aflatoxin contamination can occur in plant growth, harvest and processing, storage, transportation in the process. The timely detection of pollution sources is the best way to prevent aflatoxin contamination.

2. Intended Use

A simple and efficient extraction and purification procedure for Aflatoxins was developed by means of the immunoaffinity column (IAC-SEP^® AFLA) as a cleanup tool. Aflatoxins content in Grain, Food, Feeds, Nuts, Peanuts, Soy sauce, Vinegar, Chili, Pepper, Medicinal herbs and Wine samples are cleaned up by IAC and determined by HPLC or LC-MS/MS. It is a fast, simple, safe and highly accurate method for quantitatively measuring aflatoxins(B₁/B₂/G₁/G₂).

3. Principle

Samples are prepared by mixing with an extraction solution, blending and filtering. The extract is then applied to the Aflatoxins immunoaffinity column bound with specific antibodies to Aflatoxins．At this stage, the Aflatoxins bind to the antibody on the column. The column is then washed with water to remove the impurities. By passing methanol through the column, the Aflatoxins are removed from the antibody. This methanol solution can then be injected into HPLC or LC-MS/MS system.

Preparation of solutions

(1) Extracting solution:

Methanol-water(8+2,V/V): 80mL Methanol+20mL water，mixing blending.

Methanol-water(7+3,V/V): 70mL Methanol+30mL water，mixing blending.

Acetonitrile-water(9+1,V/V): 90mL Methanol+10mL water，mixing blending.

(2) PBS solution(pH=7)：

8.0NaCl+1.44gNa₂HPO₄. 12H₂O+0.24g KH₂PO₄+0.2g KCl, dissolve in approximately 990 mL purified water, adjust the pH to 7.0, adjust 1.0L with purified water.

(3) 0.1%Tween-20: Add 1.0mL Tween-20 into 1L pH=7.0PBS, mixing blending.

(4) 0.5%Tween-20: Add 5.0mL Tween-20 into 1L pH=7.0PBS, mixing blending.

(5) Mobile Phase: Methanol-water: 450mL Methanol+550mLwater,mixing blending.

(6) Standard solution: Dilute aflatoxin B1 stock solution with mobile phase.

(2-8 ℃ storage, valid for 24h)

The column capacity of IAC-AFLA (maximum adsorption amount of aflatoxin) is 400ng, when aflatoxins in sample more than the maximum adsorption amount, please reduced the volume into the detection range, then calculate the accurate content.

HPLC Set up

(1) Column: Cloversil-C18,4.6×150mm（5um）or 4.6*250mm（5um）

(2) Flow rate: 0.8mL/min.

(3) Detector: Fluorescence detector Excitation wavelength: 360nm, Emission wavelength: 440nm

(4) Sample loop: 5-100 mL

(5) Mobile Phase :Methanol-water（45+55,V/V）.

(6) Photochemical derivatization system.

HPLC chromatogram of AFLA standard

Important Notes

(1) Storage: IAC-AFLA should be stored at 2-8 ^oC. Do not freeze.

(2) Shelf Life: IAC-AFLA columns and kits are stable for 18 months from date of manufacture if stored at 2-8^oC.

(3) If Sample recycling test is needed, standard substance should be added to the sample before 2 hours or one night, otherwise, the recovery rate will be low.If standard substance recycling test is needed, make sure methanol concentration <25%,or the adsorption capacity of immunoaffinity column will be influenced.

(4) If you want to modify the operating instructions of the operation steps, please contact with our technology department.