Agriculture Reference
In-Depth Information
Samples
Samples of soybean from the 2004−2007 harvests were collected from different locations
in the Republic of Serbia. Samples of the 2004 harvest were taken from barns, i.e., storages,
where they had been kept for one year, while those of the 2005−2007 harvests were taken
directly off fields immediately after the harvest. Soybean meal samples, originated from the
same harvests, were taken from several oil factories after deoling by extraction. After
sampling, 1000 g of each sample were ground in a laboratory mill in such a way that > 93%
passed through a sieve with pore diameter of 0.8 mm. After that, the sample was
homogenized by mixing. Samples thus prepared were packed in plastic bags and stored in a
freezer at −20 °C until the analysis. Prior to each analysis, the samples were allowed to attain
room temperature.
Extraction and Clean-up
Activated charcoal-alumina-Celite-cation exchange resin column was used for
purification; 25.0 g of the sample were extracted with 100 ml of acetonitrile−water (84:16,
v/v) and shaken on a magnetic stirrer for 60 minutes. After filtration through Advantec filter
paper, 6.0 ml of the extract were applied to the prepared column. The column was then
washed with 5 ml of the solvent mixture comprising of acetonitrile−water (84:16, v/v) at
about 0.6 ml/min. The cleaned-up extract was evaporated to dryness, dissolved in 3.0 ml of
ethyl acetate and quantitatively transferred to an evaporation vessel by triple washing with 1.5
ml ethyl acetate. The eluate was evaporated just to dryness.
Activated Charcoal-alumina-Celite-cation Exchange Resin (CACC) Column
The column was prepared in the following way (32): a plug of glass wool was inserted
into the tapered end of a glass tube (9 cm x 1.5 cm i.d.), then 0.1 g of Celite, 1.5 g of activated
charcoal, alumina, and Celite mixture (7:5:3) were added, loosely packed, and tapped to level.
Two grams of prewashed cation exchange resin were added and lightly compacted above
activated charcoal-alumina-Celite by pushing down a second glass wool plug.
Liquid Chromatographic Analysis
The equipment consisted of an HP 1090 Liquid Chromatograph (Hewlett Packard, Palo
Alto, CA, USA) with a DAD detector (Hewlett Packard, Palo Alto, CA, USA) and a Hypersil
ODS column (100 x 4.6 mm i.d., particle size 5 μm, Agilent Technologies, USA). DON
analysis was performed after evaporation, the residue was redissolved in 300 μl of methanol,
and a 15 μl aliquot of the solution was injected into the LC system. The mobile phase
consisting of a mixture of acetonitrile−water (16:84, v/v) was used at a flow rate of 0.6
ml/min. UV detection was performed at 220 nm. The mobile phase was filtered through a
0.45 μm membrane (Aura industries, TFM, Hewlett Packard).
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