# A Review: Sample Preparation and Chromatographic Technologies for Detection of Aflatoxins in Foods
## 2 Sample Preparation
### Extraction
#### 2.2.2 Solid Phase Extraction  
After SPE columns became commercially available, SPE has been widely used as a replacement for conventional liquid-liquid extraction [91; 92]. SPE achieves clean-up via selective retention of analytes while limiting interferences in extracts due to the careful selection of an adsorbent that would have strong analyte affinity. Compared to liquid-liquid extraction, SPE is more sample- and solvent-efficient, selective and flexible due to a wide range of adsorbents available for selection. For aflatoxin analysis, the most commonly used SPE cartridges contain silica gel, Florisil, C18 and other phases. For the aflatoxin analysis, the early SPE applications were mainly focused on silica gel- and Florisil-based clean-up. Without commercially available SPE cartridges, researchers had to prepare in-house glass columns using activated silica gel [93], silicic acid [94], silica gel [95] or Florisil [96; 97] as the adsorbent and different combinations of alcohols (methanol or ethanol) and chloroform as elution solutions. SPE conditions had to be determined by trial and error, varying by SPE columns, adsorbents (type and amount) and matrices. These early SPE procedures offered unsatisfactory separation between aflat and matrix components and consumed a large amount of organic solvents due to inconsistent quality of adsorbents and packing. Very often, two SPE columns had to be used consecutively to purify aflat. [PERSON] et al. [95] reported a stepwise procedure for the purification of aflat from inoculated wheat. After the initial extraction and partitioning, the resulting crude mixtures of aflat in chloroform (10 mL) was loaded on a glass column packed with silica gel (70 g, particle size, 0.05-0.20 mm) and capped with 25 g of anhydrous sodium sulfate. The column was washed with ethyl ether (500-600 mL) to remove pigments and other impurities, followed by eluting aflat using 3% methanol in chloroform (600 mL). The collection of fractions was guided by intermittent inspection of the glass column with an ultraviolet lamp. The combined fractions were evaporated to dryness, but the resulting aflat were further purified using a second column due to low purity (60%). In the second column, a slurry of silica gel and 20% methanol in benzene was used as an adsorbent permitting selective partitioning of aflat on the silica gel. Aflatoxins were eluted in 1.75% methanol in benzene. The four aflat (B\({}_{1}\), B\({}_{2}\), G\({}_{1}\) and G\({}_{2}\)) were collected in different fractions and checked with TLC. In total, more than 100 fractions (20 mL each) were collected and 2 L of the eluting solvent was used. To address insufficient sensitivity and selectivity issues of early SPE applications [91; 98], [PERSON] and [PERSON] [99] developed an SPE clean-up procedure using basic aluminum oxide as the adsorbent. Samples were extracted with methanol-water (80:20, _v/v_). An aliquot of the extract was mixed with acetonitrile (1:1, _v/v_) and pipetted onto an Alltech Extract-Clean cartridge packed with basic aluminum oxide where aflat were eluted by gravity. The purified extract was injected onto an LC system with fluorescence detection. Recoveries of aflat B1, B2, G1 and G2 from peanuts spiked at 5.0-2.5 mg/kg were between 80 and 87% with RSDs \(<\) 5%. The basic aluminum oxide sorbent retained compounds of medium to high polarity in polar solvents like methanol, acetonitrile and water, while significantly removing low polarity and nonpolar (both neutral and acidic) interferences from the extract. Since food matrices that are prone to aflatoxin contamination contain similar components such as water, carbohydrates, lipids and proteins, a careful selection of SPE sorbent chemistry is required to promote the purification of aflat from co-extracted food matrix components prior to downstream analysis. [PERSON] and [PERSON] [100] developed the Mycosep multifunctional clean-up column to retain co-extracted compounds that could create interferences in aflatoxin analysis but allow targeted aflat to pass through.