*3.5. Extraction of Aflatoxins from Cultures 3.5. Extraction of Aflatoxins from Cultures*

A flow diagram for the extraction of aflatoxins from three different culture conditions is shown in Figure 5. Aflatoxins (B1, B2, G1, and G2) were extracted from the submerged culture by liquid–liquid extraction. Briefly, 1.0 mL aliquot of the fungal culture broth was mixed with 1.5 mL of chloroform in a 15 mL centrifuge tube and vigorously shaken by hand for 10 s followed by vortexing for 30 s. The mixture was then centrifuged for 2.5 m at 5000× *g*. The organic phase in the lower layer was transferred to a new glass vial. The sample residue was re-extracted with another 1.5 mL of chloroform to recover traces of AFs, which might have been present after the first extraction. The two chloroform extracts were combined and evaporated to complete dryness under a gentle stream of air. The dried extract was reconstituted with 1.0 mL of mobile phase. AFs from solid culture were extracted by adding 15.0 mL of 0.1% Tween-80 solution; the conidia were then harvested by gently scraping the top layer and collected into a 50 mL centrifuge tube. Spore suspension was homogenized by vortexing for 30 s. One mL of this suspension was transferred to a new centrifuge tubes (15 mL), and 1.5 mL of chloroform was added. The extractions were performed as described above for the liquid culture. For the liquid slant culture, the cultivated tubes were vortexed for 30 s, and 1.0 mL of the suspension was transferred to a new centrifuge tube (15 mL). Then, 1.5 mL of chloroform was added to the tubes and treated as described above. All samples were filtered into HPLC vials through a PTFE 0.45 µm syringe filter prior to HPLC analysis. A flow diagram for the extraction of aflatoxins from three different culture conditions is shown in Figure 5. Aflatoxins (B1, B2, G1, and G2) were extracted from the submerged culture by liquid– liquid extraction. Briefly, 1.0 mL aliquot of the fungal culture broth was mixed with 1.5 mL of chloroform in a 15 mL centrifuge tube and vigorously shaken by hand for 10 s followed by vortexing for 30 s. The mixture was then centrifuged for 2.5 m at 5000× *g*. The organic phase in the lower layer was transferred to a new glass vial. The sample residue was re-extracted with another 1.5 mL of chloroform to recover traces of AFs, which might have been present after the first extraction. The two chloroform extracts were combined and evaporated to complete dryness under a gentle stream of air. The dried extract was reconstituted with 1.0 mL of mobile phase. AFs from solid culture were extracted by adding 15.0 mL of 0.1% Tween-80 solution; the conidia were then harvested by gently scraping the top layer and collected into a 50 mL centrifuge tube. Spore suspension was homogenized by vortexing for 30 s. One mL of this suspension was transferred to a new centrifuge tubes (15 mL), and 1.5 mL of chloroform was added. The extractions were performed as described above for the liquid culture. For the liquid slant culture, the cultivated tubes were vortexed for 30 s, and 1.0 mL of the suspension was transferred to a new centrifuge tube (15 mL). Then, 1.5 mL of chloroform was added to the tubes and treated as described above. All samples were filtered into HPLC vials through a PTFE 0.45 μm syringe filter prior to HPLC analysis.

**Figure 5.** A process flow diagram for the extraction of aflatoxins from three different culture **Figure 5.** A process flow diagram for the extraction of aflatoxins from three different culture conditions.
