# A Review: Sample Preparation and Chromatographic Technologies for Detection of Aflatoxins in Foods
## 2 Sample Preparation
### Extraction
#### 2.2.3 Immunoaffinity Column Clean-Up  
From a burgeoning research area that focused on the discovery and isolation of monoclonal antibodies that could specifically recognize aflat in the 1980s [114; 115; 116] to a well-accepted sample preparation practice in the 1990s [92; 117; 118], it took less than 10 years for antibody-based immunoaffinity columns (IAC) to become one of the most important and widely used clean-up tools for aflatoxin analysis. Immunoaffinity columns achieve clean-up based on the specificity of binding between antibodies and aflat. The high affinity and specificity of antibodies for aflat have been the cornerstone in the development of the various immunoaffinity columns. Owing to advances in the production of monoclonal antibodies with specificity and affinity, immobilization of antibodies on supporting beads and well-established sample preparation protocols, IACs are applicable to a wide array of food matrices for aflatoxin analysis [117]. Extensive research proves that IACs demonstrate a high degree of specificity in binding aflat even in the presence of complex food and feed matrices [119; 120], providing a methodology for simplifying sample analysis, especially when using non-selective detectors such as FLD [121; 122]. The development of antibody-based immunoaffinity columns comprises the production of the antibodies, immobilization of the antibodies on inert supporting beads such as agarose gel or silica and packing of the beads in cartridges filled with phosphate buffer to maintain physicochemical properties of the antibodies. The evaluation of immunoaffinity columns focus on, but not are limited to, aflatoxin binding capacity and specificity, shelf life, solvent tolerance, cross-reactivity with matrix interferences and reversibility [123; 124; 125]. For sample clean-up, the initial sample extract is loaded on the immunoaffinity column. As the extract passes through the column, aflat are retained on the column by the antibody, followed by a wash step to remove impurities due to nonspecific binding and the elution of aflat using a solvent that disrupts the binding between the antibody and the aflat. [PERSON] and [PERSON] [126; 127] reported the development and evaluation of an early immunoaffinity column for the aflatoxin analysis. After isolating an immunoglobulin M (IgM) monoclonal antibody that recognized aflat B\({}_{1}\), B\({}_{2}\), M\({}_{1}\) and the major aflatoxin-DNA adducts, they developed an immunoassay affinity column by immobilizing the antibody on cyanogen-bromide-activated Sepharose 4B for the analysis of aflat in biological and food samples. Their results clearly demonstrated the potential of monoclonal antibodies as an efficient and simple sample preparation tools for the purification of aflat from food matrices. With commercial immunoaffinity columns, researchers have amassed an enormous amount of knowledge on immunoaffinity chromatography and associated applications for the aflatoxin analysis. Immunoaffinity column clean-up offers the extraction of aflat from various food matrices with simple aqueous mixtures (e.g., methanol/water), making the final extracts compatible with TLC [128; 129; 130], fluorometry, LC-FLD [121; 131] and LC-MS for both single- and multimycotoxin determination without additional sample treatment. In a collaborative study [121], immunoaffinity column clean-up coupled with solution fluorometry with bromine and an FLD was used to determine total and individual aflat in corn, peanuts and peanut butter. Fourteen participating labs extracted samples using methanol-water (7:3, _v/v_) and diluted the resulting extracts to less than 30% methanol with water, followed by an immunoaffinity column clean-up step. At the three spiking concentrations, 10, 20 and 30 ng/g, recoveries of total aflat were 81%, 81% and 83%, respectively with the RSD\({}_{\text{r}}\) ranging from 5.2 to 17%, and the RSD\({}_{\text{R}}\) ranging from 4.7 to 51%.