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

1. Preparation of seeds for fungal challenge

1. Incubate the aflatoxigenic Aspergillus flavus NRRL 3357 on a slant of Potato Dextrose Agar (PDA) in a test tube for 6 days at 30 °C. Harvest fungal spores from the test tube with 10 mL of water with Tween 20 (100 μL of Tween in 1 L of distilled sterile water), filter through glass wool placed in a funnel and count the spores with a hemocytometer referring to the user manual.
2. Dilute the spore suspension with sterile water to 1,000 spores/μL concentration. Confirm the concentration with a hemocytometer.
   NOTE: Prepare the spore suspension no earlier than 2 h before the challenging experiments.
3. Gently crack the peanut pods and remove the hulls. Place the seeds in a sterile beaker so that the volume of seeds does not exceed 1/5 of the beaker volume, add 0.05% hydrogen peroxide solution to approximately 70% of the beaker volume, and allow the seeds to imbibe water for 3 h.
4. Decant the hydrogen peroxide solution from the seeds and add approximately 3x the amount of 80% ethanol-water mixture (v/v) to the beaker to cover the seeds. Allow to stand for 1 min and then rinse the seeds 2x with equal volumes of sterile distilled water.
5. Add 5-fold volume of 3% hydrogen peroxide to the beaker with the ethanol-sterilized seeds and allow to stand for 5 min, then decant the liquid and rinse the seeds twice with equal volumes of sterile water (Figure 2A).

Figure 2: Preparation of peanut seeds for inoculation, incubation, and extraction of aflatoxin and phytoalexin fractions. (A) Sterilizing imbibed seeds with 3% hydrogen peroxide; (B) removing testa (skin) from seeds; (C) cutting off the embryonic axis part; (D,E) drilling cavity in a half cotyledon with a drill bit; (F) placing drilled parts of seeds on agar; (G) applying fungal spores into the drilled cavity. (H) Petri dish with seeds after incubation; (I) placing incubated single seeds (photo of control samples) into reinforced bead tubes; (J) adding measured volume of extracting solvent; (K) pulverizing samples in a bead ruptor; (L) centrifugation of pulverized slurry. Please click here to view a larger version of this figure.

6. Place the seeds on a sterile paper towel, remove the seed testa with forceps, and cut about 1/3 of the seed containing the embryonic axis with a scalpel (Figure 2B,C).
7. Discard this part of the seed or use it to grow a plant in a test tube with growth medium (Figure 1B,E).
8. Split the remaining portion of the seed into two cotyledons, place them in a Petri dish, cover the seed parts with moist sterile filter paper, replace the lid to avoid dehydration and promptly proceed to the inoculation step.
9. Before performing the challenging experiments, prepare a sufficient number of Petri dishes with seed incubation medium by pouring 26 mL of 1.5% sterile agar in water into the 100 x 15 mm dishes and allow them to solidify overnight. 
10. In the middle of the outer side of each remaining cotyledon, make a 1.5-2 mm deep cavity by manually drilling the seed with a 1.6-2.34 mm diameter sterile sharp drill bit. It takes ~2-5 s to make a cavity (Figure 2D,E).
11. Place 4 to 6 "drilled" pieces of the seeds into a Petri dish with agar and apply 2 μL of the 1,000 spores/μL suspension into the drilled cavity of each half-cotyledon piece with a 10 μL pipetter. Instead of the spore suspension, apply sterile water to the control seeds (Figure 2F,G).
12. Cover all Petri dishes with lids and incubate at 30 °C without light for 72 h.

2. Sampling and preparation of the incubated seed pieces for aflatoxin and phytoalexin analysis

1. Collect seed pieces at 72 h by removing them from agar with forceps and placing each of them into labeled 7 mL bead vials with 13 zirconium ceramic beads (12 of 2.8 mm diameter and 1 of 6.5 mm diameter) (Figure 2H,I).
   NOTE: At this point, if not processed immediately, the vials can be placed into a -80 °C freezer and kept for up to 2 months before further processing .
2. Phytoalexins and aflatoxins are determined in the same seed extract. For the extraction, depending on the visual seed size (small, medium, or large), add 2 or 4 mL of precisely measured methanol-water mixture (90:10, v/v) to the bead vials and pulverize at 5.5 m/sec for 45 s. Place the vials with pulverized seeds into a centrifuge and centrifuge at 1,860 × g for 3 min (Figure 2J – L).
   NOTE: The following important^17,22 stilbenoid derivatives are determined (all in trans-configuration): resveratrol, arachidin-1, arachidin-2, arachidin-3, 3′-isopentadienyl-3,5,4′-trihydroxystilbene (IPD), and SB-1.
3. For aflatoxin analyses, before running the challenging experiments, prepare a sufficient number of cleanup columns as follows. Place a matching polyethylene porous (20 μL) frit into a 1.5 mL polypropylene column with the help of a glass rod cut at a 90^o angle. Place 50 mg of the magnesium silica gel (100-200 mesh) into the column with a custom-made scoop and cap the column with an identical frit by pushing it down with a glass rod (Figure 3A – E).
   NOTE: This amount of magnesium silica gel occupies 75 μL volume, and the height of the adsorbent layer is 3 mm.

Figure 3: Preparation of cleanup columns and filters. (A,B) Placing a porous frit into barrel; (C,D) filling barrel with magnesium silica gel; (E) packing the adsorbent and inserting a top porous frit into barrel. (F) Placing an 11.5 mm dia. glass fiber circle on top of a Pasteur pipette. (G,H) Pushing the center of the circle with a plastic or wooden rod down to the bottom of the Pasteur pipette barrel and firmly compacting the glass fiber. Please click here to view a larger version of this figure.

4. At this point, prepare a matching number of custom-made filters as shown in Figure 3F – H.
5. For aflatoxin analyses, precisely measure up to 0.5 mL aliquot of the supernatant (obtained as described in step 2.2), transfer it into the custom-packed minicolumn, and allow the extract to drain by gravity into a 4-mL glass vial. Transfer 1.0 mL of methanol-water (90:10, v/v) mixture to the column to wash out impurities by gravity into the same 4 mL vial (Figure 4A).

Figure 4: Purification of seed extracts and preparation for the UPLC analyses. (A) Rack with packed columns. (B) Evaporating solvent with N₂ gas from six 4-mL vials with purified extract eluted from cleanup columns. (C) Bringing vials and filters to a subzero temperature in foam containers (1 and 2) with ice before and after addition of the injection solvent (MeOH-H₂O 9:1, v/v) to the 4-mL vials. (D) Filtering cooled extract from a 4-mL vial into a 400 µL autosampler vial. Please click here to view a larger version of this figure.

6. Discard the combined eluates. Elute the aflatoxin fraction by gravity from the column into a clean 4 mL glass vial with 1.2 mL acetone-acetonitrile-water-88% formic acid (65:31:3.5:0.5, v/v) mixture (Figure 4A).
7. Alternatively, elute aflatoxins from the column with 2.0 mL of acetonitrile-water-88% formic acid (96:3.5:0.5, v/v) mixture, and inject an aliquot of the eluate, up to 2 μL, directly into the ultra performance liquid chromatography (UPLC) system (without evaporation of the solvent with N₂).
   NOTE: In this case, expect up to 10-fold lower quantitation limit for aflatoxins; in the majority of the cases such a drop of sensitivity is acceptable since aflatoxin concentrations in challenged seed are often high.
8. Remove the solvent from the vials in a stream of N₂ in a heated block at 45 °C. Cap the vial and place it in a container with crushed ice for 30-45 s to minimize evaporation of the solvent that is added in the following step. Place custom-made filters into disposable test tubes and insert the tubes into ice in another container to minimize evaporation of solvent upon filtration (Figure 4B,C).
9. After evaporation of the solvent, dissolve the dry residue in precisely measured 0.25-1.0 mL of methanol-water mixture (90:10, v/v) and vortex for 1-2 s. Place vials with purified extracts into the UPLC autosampler and inject 0.1 to 3.0 μL into the UPLC system (Figure 5A,B).
   NOTE: If the solution after vortexing is suspected to have some suspended particles, filter the solution through the cooled filter (Figure 4D).

Figure 5: The process and results of UPLC analyses of purified extracts. (A) Placing a rack with purified sample extracts into UPLC autosampler. (B) Performing the instrumental analyses, obtaining data, and interpreting results. (C) UPLC of four major aflatoxins; the major peaks of interest, aflatoxins B₁ and B₂ represent 0.04 and 0.004 ng, respectively. (D) UPLC of aflatoxins B₁ and B₂; peak 2 represents aflatoxin B₁ at a 2 pg level (with an 8 µL flow cell). (E) UPLC of purified extract of a seed harvested from a local peanut field with a wild Arachis species. (F) UPLC of the extract of a challenged peanut seed purified with a basic aluminum oxide minicolumn. (G) UPLC of the extract of challenged peanut seed purified with a Florisil column; blue chromatogram presents impurities that are washed from the column with 1 mL of methanol-water (90:10 v/v) mixture; black chromatogram shows peaks of aflatoxins M₁ and B₁. The level of aflatoxin B₁ is 48 ng/g. (H) A typical chromatogram of peanut stilbenoids from a challenged seed; abbreviation: IPD = 3′-isopentadienyl-3,5,4′-trihydroxystilbene. Note: All stilbenoids in seed extracts are in trans-configuration. Please click here to view a larger version of this figure. 

10. For phytoalexin analyses, from each bead vial, transfer 200 μL of supernatant into the above Pasteur pipette filter. Use N₂ gas from a compressed nitrogen tank to expedite filtration into a 400 μL UPLC autosampler vial and cap the vial with a matching cap with a PTFE septum (Figure 4D).

3. Aflatoxin analyses

1. For separations of aflatoxins in seed extracts, use a UPLC system equipped with an autosampler, a quaternary pump, a fluorescent detector, a UPLC BEH C18 3.0 mm x 100 mm, 1.7 µm column with a matching precolumn, and a postcolumn photochemical reactor (PHRED) with a standard knitted PTFE coil 0.25 internal diameter cut to 25% of its original length.
   NOTE: UPLC instrument and associated equipment used by the authors are listed in the Table of Materials.
2. Use water (A), methanol (B), and acetonitrile (C) in the following gradient: initial conditions, 62.7% A, 24% B, 13.3% C, changed linearly to 30% A, 45% B, 25% C in 3.75 min, changed to 0% A, 64.4% B, 35.6% C in 3.751 min, held isocratic for 3.249 min, then changed to initial conditions in 0.01 min and held isocratic for 2.499 min before next injection. Set the flow rate to 0.45 mL/min and run time to 9.5 min. Maintain the column at 40 °C in the system column heater. Use 362 nm and 440 nm as the excitation and emission wavelengths, respectively, for aflatoxins B₁, B₂, G₁, G₂, and M₁ quantification. The structures of major aflatoxins are shown in Figure 6A.
   NOTE: Under the experimental conditions, only aflatoxins B₁ and B₂ are expected to be produced by A. flavus NRRL 3357; if an A. parasiticus strain is used for seed challenging, the production of aflatoxins B₁, B₂, G₁, and G₂ is expected.
3. Determine concentrations of aflatoxins by reference to peak areas of corresponding authentic standards (calibration curve) as described in the software user manual.
   NOTE: Since the UPLC procedure exhibits baseline noise at the high sensitivity settings for the fluorescent detector, Detection Limit (LOD) and Quantitation Limit (LOQ) for aflatoxins are evaluated using the widely accepted Signal-to-Noise ratio of 3:1 for LOD and 10:1 for LOQ. In the routine experimental setup for peanut seed analysis, described in sections 2 and 3, the quantitation limits for aflatoxins G₁ and B₁ are expected to be less or equal to 0.8 and 0.08 ng/g for aflatoxins G₂ and B₂, respectively.

4. Stilbenoid phytoalexin analyses

1. For separation of stilbenoids, use water (A), methanol (B), and 88% formic acid (C) in the following gradient: initial conditions, 53% A, 42% B, 5% C, changed linearly to 35% A, 60% B, 5% C in 2.0 min, held isocratic for 4 min, changed to 0% A, 95% B, 5% C in 0.5 min, held isocratic for 2.5 min, then changed to initial conditions in 0.1 min and held isocratic for 2.9 min before next injection. Set flow rate to 0.5 mL/min and run time to 12.0 min.
2. Determine concentrations of stilbenoids by reference to peak areas of corresponding authentic standards (calibration curve) and/or to published coefficients of molar extinctions as described in the software user manual. The structures of major stilbene-derived phytoalexins are shown in Figure 6B.
   NOTE: Separation of phytoalexins is achieved using the same system but without a fluorescent detector and a PHRED reactor. Instead, a Diode Array Detector is used for the detection and quantification of peanut stilbenoids. Quantitation Limit (LOQ) for the major stilbenoid phytoalexins does not have a practical meaning as stilbenoid concentrations exceed concentrations of aflatoxins in the same extract by some orders of magnitude; after 3 days of incubation, stilbenoids are reliably quantitated.