– In our 4th experiment we will show you an aflatoxin soil extraction procedure.
– Spiking is usually done to test the validity of the method through measuring the recovery of the spiked aflatoxin.
– 5 g air-dried soil is weighed in 50 mL labelled centrifuge tubes and spiked with 100 μL spiking standards or pure methanol to obtain three soil concentration levels at 3, 9, and 15 ng g−1 and blank. Various aflatoxin standards are B1, B2, G1, G2 and M1. Here we are spiking aflatoxin B1 standard. We are taking 3 replicates of the soil and blank in order to see the reproducibility.
– It is important to keep in mind that the experiment has to be carried out under the fume hood due to the presence of organic and toxic substances. And also, for safety purposes, we have to wear laboratory coat, goggles and gloves.
– Spiked samples are vortexed for 10 s to obtain a homogeneous sample and left under the fume hood for 30 min to allow the solvent to evaporate.
– Extraction solvent is prepared with acetonitrile and water (84:16 v:v). Acetonitrile has low viscosity, high chemical stability and strong eluting power; therefore, it is a commonly used solvent mixture for multi mycotoxin extraction in different food and feed matrices.
– Samples are extracted using 15 mL extraction solvent.
– The soils are mixed with the extraction solvent for 30 min at 180 rpm in an orbital shaker.
– After shaking, the samples are placed in an ultrasonic bath for 15 min.
– Samples are centrifuged for 15 min at 3500 rpm.
– Aliquots of 4 mL of each extract are evaporated to dryness at 40 °C under a gentle nitrogen stream.
– The samples are reconstituted with 0.8 mL 1:9 methanol: water mixture
– Concentrated extracts are filtered through 0.2 µm PET syringe filters.
– Aliquots of 90 µL of the filtered extracts are mixed with 10 µL isotopically labelled standard mixture into brown glass vials before measurement.
– Now the samples are analysed using Liquid -chromatography mass spectrometry.
– Once the method is validated, we measure the content of aflatoxin from the soil. In that case we have to use 5g of freeze-dried soil samples and the procedure will be the same as mentioned above.