# A Review: Sample Preparation and Chromatographic Technologies for Detection of Aflatoxins in Foods
## 2 Sample Preparation
### Extraction
#### 2.2.1 Liquid-Liquid Extraction (Partitioning)  
Aflatoxins are more soluble in polar organic solvents (chloroform, methanol, acetonitrile, etc.) than in non-polar organic solvents such as hexane [62; 63] so liquid-liquid extraction (partitioning) has been widely used to extract and separate aflat from a wide variety of food matrices using immiscible solvent systems containing two or more solvents. In the 1960s and early 1970s, without other extraction and clean-up techniques available, aflatoxin analysis was mainly conducted by liquid-liquid extraction coupled with paper chromatography or thin layer chromatography (TLC). Aflatoxins samples were generally extracted using chloroform, methanol or a mixture of different polar organic solvents, followed by an evaporation step to reduce the volume and concentrate aflat in the extract. The remaining residues were reconstituted and defatted by partitioning between a polar and a non-polar organic solvent (e.g., methanol and petroleum ether). The aflat in the polar phase were processed using TLC. Aflatoxin identification and quantitation was achieved by a comparison of retention factor (Rf) and the intensity of fluorescence of the sample to standards on the TLC plate [64]. For example, in a method developed by [PERSON] and [PERSON] [65], three consecutive liquid-liquid extraction procedures were used to purify aflat from oil samples. In the first extraction, oil samples were extracted three times using methanol/water, partitioning aflat into the methanol phase. In the second extraction, the combined methanol extracts were defatted using hexane (200 mL) to remove coextracted fatty components from the methanol phase. After evaporation of the aqueous methanol solution to 50 mL, the third liquid-liquid partitioning was performed using chloroform. The resulting extracts were analyzed using a TLC procedure. In order to remove lipid and pigment interferences, [PERSON] and [PERSON] [66] used a series of liquid-liquid extractions to extract aflat from cotranseed samples, which were first extracted using acetone and water (70/30, _v/v_). The pigment in the acetone/water phase was precipitated as insoluble lead salt. Aflatoxins were then partitioned into chloroform from the acetone/water phase by repetitive liquid-liquid extractions prior to TLC analysis. Since the late 1960s, liquid-liquid extraction has been gradually coupled with other developed clean-up techniques (e.g., silica gel chromatography) for aflatoxin analysis [67; 68; 69; 70; 71; 72]. For example, to determine aflat in peanut butter, [PERSON] et al. [71] used methanol to extract samples, followed by partitioning fatty interference into hexane. The remaining aflat in the aqueous methanol phase were partitioned into chloroform. Then the chloroform extracts were further purified using an in-house developed silica gel chromatography method. Two laboratories tested the method in eight different matrices, including peanut butter, and achieved consistent results. The mean recoveries of spiked aflat (15-34 ng/g) ranged from 86 to 94% with coefficients of variation less than 7% for spiked samples and 15% for naturally contaminated samples. [PERSON] and [PERSON] [72] used liquid-liquid extractions to prepare samples for a total diet study, focusing on aflatoxin B\({}_{1}\) and M\({}_{1}\), and ochratoxin A. The samples (10 g each) were first extracted with chloroform (100 mL), followed by filtration and evaporation of 50 mL of the extracts. The remaining residue was purified by a liquid-liquid extraction using a mixture of methanol (10 mL), pentane (50 mL) and PBS solution (1 mL). After phase separation, the upper layer (pentane) was re-extracted with 4 mL of PBS solution. Combined PBS and methanol extracts were further mixed with pentane (50 mL) and PBS solution (35 mL) for the second liquid-liquid extraction.