# A Review: Sample Preparation and Chromatographic Technologies for Detection of Aflatoxins in Foods
## 2 Sample Preparation
### Extraction
#### 2.2.3 Immunoaffinity Column Clean-Up
Due to the satisfactory performance of the method, a well-known AOAC Official Method 991.31 [132] was published based on this multilaboratory validated immunoaffinity column clean-up and LC-FLD procedure. Although immunoaffinity column clean-up is often coupled with LC-FLD, [PERSON] et al. [129] used immunoaffinity columns to clean up samples for TLC analysis. Samples were extracted using a mixture of methanol-water (8:2, _v/v_). The extracts were filtered and diluted using PBS buffer prior to loading on the immunoaffinity column. The IAC was washed sequentially using aqueous methanol(11%) containing 0.5% (Tween-20(r)) and water, followed by the elution of aflat using methanol. Sample eluants (in methanol) were evaporated and reconstituted in a hexane-acetone-methanol solution for TLC analysis. This immunoaffinity column clean-up procedure successfully addressed the insufficient elimination of matrix interferences associated with some classical clean-up procedures such as SPE used for TLC [133; 134]. Due to the co-occurrence of aflat and ochratoxin A in some food (e.g., dried fruit, spices and cocoa, nutmeg and botanical roots), it was a logical extension from single class mycotoxin immunoassay to multimycotoxin immunoaffinity columns so that both aflat and ochratoxin A could be purified on the same column, an approach that is more efficient than using two individual columns. [PERSON] et al. [135] used a commercial immunoaffinity column (AflaOchra(tm) HPLC immunoassay immunoaffinity column) to develop a procedure that can simultaneously purify aflat and ochratoxins from maize cereals, whole corn, maize snacks, corn flour, polenta and peanut butter. Average recoveries (within-batch) were 72-94% for ochratoxin A and 73-101% for aflat spiked at 5 ug/kg. RSDr and RSDR of aflat and ochratoxin A in maize cereals were 7.6-10.1% and 10.2-13.8%, respectively. In this work, the estimated method detection limits of aflat and ochratoxin A were around 0.2 ug/kg. [PERSON] et al. [131] compared the method performance using a commercial multimycotoxin column (for both aflat and ochratoxin A) and one single-mycotoxin column (for aflat only). The two types of columns were operated in a very similar manner, and aflat and ochratoxin A were measured using same LC-FLD. Results for aflat (2-16 ng/g) using the single mycotoxin IAC clean-up were comparable (80% recovery) for ginseng and other botanicals such as ginger, liquorice and kava-kava. Using IAC clean-up for both aflat and ochratoxin A, recoveries of aflat in ginseng were about 70% but around 60% in ginger, liquorice and kava-kava. However, recoveries of spiked ochratoxin A (4 ng/g) in these botanicals were about 55%. Based on the results, another single-laboratory study [136] and a multiaboratory collaborative study were conducted to evaluate the performance of the multimycotoxin immunoaffinity column [137]. Similar applications of the multimycotoxin column have been extended to other food matrices such as peanut butter, maize, polenta, maize snacks, corn flour [135], bee pollen [138], olive oil [139] and cocoa product [140]. In recent years, multimycotoxin immunoaffinity columns have been developed and coupled with LC-MS to simultaneously screen for aflat, deoxynivalenol, ochratoxin A, fumonisins, HT-2 and T-2 toxin and zearalenone in one sample clean-up step and instrumental analysis. [PERSON] et al. [141], [PERSON] et al. [142], [PERSON] et al. [143] and [PERSON] et al. [144] demonstrated successful applications of commercial multimycotoxin immunoaffinity columns in various foods. Although the immunoaffinity columns could achieve simultaneous clean-up of multiple mycotoxins, it has been a challenge to achieve satisfactory extraction efficiency of all targeted mycotoxins with different physicochemical properties in a single extraction, where the use of multiple extractions would defeat the purpose of a multimycotoxin analysis. Another practical issue with multimycotoxin immunoaffinity columns is the capacity of mycotoxins purification.