After incubation of peanut seeds, following Aspergillus flavus spore inoculation, use forceps to remove the seed pieces from the agar. Place the seed pieces into labeled seven milliliter bead vials containing 13 zirconium ceramic beads. For the extraction of phytoalexins and aflatoxins, depending on the seed size, add two or four milliliters of a methanol water mixture to the bead vial and pulverize at 5.5 meters per second for 45 seconds.
Then centrifuge at 1, 860 x g for three minutes, and collect the supernatant in a fresh vial. For aflatoxins analysis, using a glass rod, cut at a 90 degree angle, insert a polyethylene porous frit into a 1.5 milliliter polypropylene column. With a custom made scoop, add 50 milligrams of magnesium silica gel into the column.
Cap the column with an identical frit and push it down with a glass rod. Add 0.5 milliliters of supernatant into the custom packed mini column and allow the extract to drain by gravity into a four milliliter glass vial. Add one milliliter of methanol water mixture into the column to wash out impurities by gravity into the same vial.
After discarding the combined eluates from the vial, add 1.2 milliliters of acetone acetyl nitrile water, 88%formic acid mixture into the column to elute the aflatoxin fraction into a clean glass vial. Evaporates solvent from the vial in a stream of nitrogen in a heating block at 45 degrees Celsius. After evaporation, cap the vial and cool it in crushed dice for one minute.
Place the custom made Pasteur pipette filters in disposable test tubes and put them on ice to minimize evaporation upon filtration. Dissolve the dry residue in precisely measure 0.25 to 1.0 milliliter of methanol water mixture. After vortexing, transfer an aliquot of 0.2 milliliters into the filter.
Use nitrogen gas to expedite the filtration into a 400 microliter autosampler vial. Place the vial in the UPLC autosampler and set the injection volume to 0.1 to 3.0 microliters. For phytoalexin, transfer 200 microliters of supernatant from the seven milliliter centrifuge vial into a Pasteur pipette filter.
After filtration, place a matching cap with polytetrafluoroethylene septum on the vial. For separations of aflatoxins, implement a gradient using water, methanol, and acetyl nitrile with specific percentage compositions and runtimes. Set the flow rate to 0.45 milliliters per minute and runtime to 9.5 minutes.
In the system column heater, set the column temperature to 40 degrees Celsius. To quantify aflatoxins, set excitation and emission wavelengths to 362 and 440 nanometers, respectively. Then using the calibration curve method and UPCL software manual, perform the analysis to determine aflatoxin concentrations in the test samples.
For the separation of stilbenoids, use a gradient consisting of water, methanol, and 88%formic acid with specific percentage conditions and times. Set the flow rate to 0.5 milliliters per minute and runtime to 12 minutes. Using the calibration curve method, determine the concentrations of stilbenoids and compare the peak areas of the test sample with pure, authentic standards.
The UPLC based aflatoxin quantification method achieve sensitive quantification of aflatoxin B1 with a limit of two picograms per injection. The purified extract of seeds harvested from a wild Areca species showed unambiguous quantification of aflatoxins. The magnesium silica gel column approach effectively removes impurities from the aflatoxin fraction and demonstrates high efficiency of quantification in contrast to previous methods.
In addition, this method also aids in the quantitation of peanut phytoalexins.
