# A Review: Sample Preparation and Chromatographic Technologies for Detection of Aflatoxins in Foods
## 2 Sample Preparation
### Extraction
#### 2.2.1 Liquid-Liquid Extraction (Partitioning)
With clear phase separation, the lower layer was filtered and an aliquot (40 mL) of the filtrate (equivalent to 4 g test portion) was mixed with 4 mL of 0.1 M citric acid and 36 mL of water and processed using immunoaffinity columns prior to the LC-FLD analysis. [PERSON] et al. [73] developed a two-phase clean-up system to analyze aflat in barley and milk based on partitioning of aflat between toluene and an in-house aqueous solvent, PBS-methanol (1:1, _v/v_). Briefly, the aflat were extracted from samples with methanol or chloroform. The resulting extracts were evaporated and reconstituted in toluene, a hydrophobic solvent, followed by the partitioning of aflat to the PBS/methanol phase, while interferences remained in the organic phase. The partitioned aflat were directly determined by FLD. However, such an approach required exhaustive optimization of partitioning conditions (e.g., combinations of different solvents at various ratios, extraction time) for different matrices, a common drawback of liquid-liquid extraction [74]. To minimize solvent consumption of conventional liquid-liquid extraction and increase concentration factors, liquid-liquid microextraction or dispersive liquid-liquid microextraction (DLLME) [75] has been increasingly used for the determination of aflat in dairy and oil products [76; 77; 78; 79]. As a miniaturization of liquid-liquid extraction, DLLME allows extracting and preconcentrating target aflat into a very small amount (microvolume) of the extraction phase. For example, [PERSON] [80] used a DLLME procedure for aflatoxin M\({}_{1}\) determination in whole, skimmed and powdered milk with LC-MS. Aflatoxin M\({}_{1}\) was extracted and salted out by the addition of sodium chloride and acetonitrile. After centrifugation, the acetonitrile supernatant was used as the dispersive solvent and extractant (chloroform, 1.5 mL) and water (5 mL) were added sequentially to perform DLLME. Hand shaking and centrifugation were used to facilitate extraction and achieve phase separation. The developed method using DLLME for aflatoxin detection in milk gave a method LOQ of 0.6 ng/kg, which is sufficient to measure aflatoxin M\({}_{1}\) below the EU regulations (25-50 ng/kg). To improve extraction efficiency and accurately control the volume of extraction solution, [PERSON] et al. [81] used a drop-on-demand inkjet device to inject extraction solvent (10 \(\upmu\)L, chloroform) as ultrafine droplets (20 \(\upmu\)m diameter) at high frequency into wheat extracts. The extraction could be finished within 1 min and recoveries of the aflat (spiked at 0.05-0.5 \(\upmu\)g/L) ranged from 83% to 93% with RSDs \(<\) 5%. However, evaporation and solvent exchange had to be performed to remove chloroform prior to the LC-MS analysis, which is a disadvantage when coupling DLLME with LC-MS.  
Another interesting derivative of the liquid-liquid extraction is centrifugal partition chromatography, in which both stationary and mobile phases are liquids and a strong centrifugal force is used to immobilize a solvent on a stacked and interconnected rotating-discs as the stationary phase. Just like conventional liquid chromatography, whereby solvent (the mobile phase) and sample partition into a stationary phase, different analytes are eluted according to their partition coefficients in the two solvents. In a recent study, [PERSON] et al. [82] reported the separation and purification of aflat using centrifugal partition chromatography. After testing different biphasic systems, a toluene-acetic acid-water (30:20:50, _v/v/v_/%) solvent system was selected to provide optimized purification of aflatoxin B\({}_{1}\), B\({}_{2}\), G\({}_{1}\) and G\({}_{2}\) from an _Aspergillus parasitic_s fermented material in a 250 mL centrifugal partition chromatography column. Mass spectrometry was then used to confirm the identity of the purified aflat and their purity was determined to be greater than 96% using LC-UV.