Non-destructive SPE-UPLC-based Quantification of Aflatoxins and Stilbenoid Phytoalexins in Single Peanut (Arachis spp.) Seeds

The scope of the research was to develop a method of quantification of aflatoxins and defensive phytoalexin in thousands of peanut seeds for the exploration of wild Arachis germplasm to identify species resistant to Aspergillus, and to determine and characterize novel sources of genetic resistance to this fungal pathogen. Our research group has found that the most promising approach to identify peanut germplasm that does not accumulate aflatoxin is to perform a holistic analysis of each individual seed, including its transcriptomics, genomics, phytoalexin, and mycotoxin profiles. We have developed protocols for wild and cultivated peanut seed analysis, which means using a quarter of a single seed for chemical profiling.

Developing high throughput methods for effective seed challenging, extraction, and accurate quantification of phytoalexin in peanut seed samples. We have discovered numerous peanut phytoalexin and developed efficient and inexpensive methods for aflatoxin analysis, as well as for screening peanut germplasm with reduced or no aflatoxin accumulation. The methods include genotyping and determination of seed viability.

After growing aflatoxigenic Aspergillus flavus NRRL 3357 on a potato dextrose agar slant for six days at 30 degrees Celsius elute the fungal spores from the test tube with 10 milliliters of water containing Tween 20. Vortex the test tube for 20 seconds. Filter the fungal spore suspension through glass wool placed in a funnel, and use the vortexed filtrate for dilution.

Dilute a 100 microliter aliquot of the filtered suspension with 9.9 milliliters of water. After vortexing, count the spores with a hemocytometer. Using the onscreen formula, calculate the volume of water needed to dilute one volume of the original suspension from the slant to 1, 000 spores per microliter.

Add one volume of the original suspension to the calculated amount of water. Next, gently crack the peanut pods, and remove the hulls. Place the seeds in a sterile beaker.

Add 0.05%hydrogen peroxide solution until the beaker is 70%full, allowing seeds to imbibe water for three hours. Decant the hydrogen peroxide solution from the seeds and add approximately three times the amount of 80%ethanol water mixture to cover the seeds. Incubate for one minute, then rinse the seeds twice with equal volumes of sterile distilled water.

Add the fivefold volume of 3%hydrogen peroxide solution to the beaker, and allow it to stand for five minutes. After decanting the hydrogen peroxide solution, rinse the seeds twice with equal volumes of sterile water. Place a 90 millimeter diameter filter paper into the lid and moisten the paper with one milliliter of sterile water.

Place the lid on the dish. Place the seeds on a sterile paper towel. Using forceps, remove the seeds skin.

Place the deskinned seeds promptly in the Petri dish to avoid dehydration. Cut about one third of the seed containing the embryonic axis. Split the seed into cotyledons, and using a sharp drill bit drill approximately 1.5 to 2 millimeter deep into the middle of the outer side of each cotyledon.

Place four to six drilled pieces of the seeds on a 1.5%agar dish. Using a 10 microliter pipette, add two microliters of the spore suspension into the drilled cavity of each half cotyledon piece. After covering the dish with a lid, incubate at 30 degrees Celsius without light for 72 hours.

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 phytoalexin 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 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, acetonitrile water, 88%formic acid mixture into the column to elute the aflatoxin fraction into a clean glass vial. Evaporate 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 ice 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 measured 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 auto sampler vial. Place the vial in the UPLC auto sampler 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 acetonitrile with specific percentage compositions and run times. Set the flow rate to 0.45 milliliters per minute and run time 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 still 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 run time 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 Arachis 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 phytoalexin.