*4.1. Mycotoxin Extraction Methods*

Food and feed sample pretreatment mainly requires the selective isolation and enrichment of target analytes from the complex matrix. After the size reduction of solid samples, extraction of mycotoxins can be performed. Most of the mycotoxins are soluble in organic solvents—including methanol, acetone, acetonitrile, ethyl acetate, and dichloromethane but are hardly soluble in water except fumonisins and patulin [92]. Solvent extraction is the most common method for the extraction and purification of mycotoxins in colorimetric assays. In a representative extraction procedure, liquid samples such as milk, juice, and wine are directly subjected to liquid-liquid extraction for the initial isolation of mycotoxins. However, solid-liquid extraction is used for the extraction of mycotoxins from solid samples such as grains and cereals. Generally, a mixture of organic solvent and acidic buffer or water is extensively used to extract mycotoxins. In this mixture, the water improves the organic solvent's penetration in the food/feed matrix, while the acidic solvent can decompose the strong bonds between the analyte and other food components (e.g., protein and sugars), resulting in increasing the extraction efficiency [93].

Because of the destructive effect of organic solvents on enzyme or antibody bioreceptors (e.g., denaturing properties), many efforts have been made to minimize these effects or replace new extraction methods. In the first case, diluting the sample after solvent extraction can reduce the solvent's damaging effect. In the latter case, supercritical CO<sup>2</sup> extraction and microwave-assisted extraction can be promising methods for replacement with conventional solvent extraction.

After mycotoxin extraction, filtration and centrifugation are used to remove any suspended particles. In most cases, no further purification steps are required, and the sample is used for detection. However, in biosensors/bioassays with high detection limits, clean-up procedures such as solid-phase extraction (SPE), dispersive solid-phase extraction (DSPE), solid-phase micro-extraction (SPME), and immunoaffinity column (IAC) can be performed in order to improve the detection sensitivity and specificity [9]. These advanced extraction methods were recently reviewed by Agriopoulou et al. [94].
