4.1.4. Sample Extraction (Quaternary Sampling)

Before blending, dry-run of equipment was carried out to locate particulate contaminants and then cleaned with 70% ethanol. Two (2) 25 g test portions were homogenized by a wet milling step through 2.5-fold dilution proposed earlier for maize samples [59], followed by high speed slurring in water. Briefly, a 25 g test portion was transferred to a kitchen blender cup, 37.5 mL water added (matrix/water ratio of 1:1.5, *w*/*v*) and blended at high speed (Moulinex®, Model: Type LM240, Écully, France) for 5 min using a two-step milling protocol (3 min running; 1.5 min pulse; 2 min running) and 1.3 g slurry (0.52 g feed: 0.78 g water) immediately weighed out for extraction and quantification for AFB1. Regular tapping of the blending cup during blending was critical for successful sample homogenization. This reduced splashing of contents on sides of container away from the

macerating rotor. Further, the slurry aliquot was weighed out immediately after slurring, directly to the bottom avoiding the neck of the extraction bottle. In each slurry sample, 86.5 mL of acetonitrile (HPLC Grade):water (Milli Q) (80:20, *v*/*v*) was added, contents agitated at 300 rpm for 15 min in orbital shaker-incubator (MRC®, Model: Tou-50, Holon, Israel), allowed to settle for 40 min at ambient temperature, supernatant extract decanted and stored at 4 ◦C.
