3. FOOD ChEMISTRy & bIOTEChNOLOGy 3.1. Lectures

Chem. Listy, 102, s265–s1311 (2008) Food Chemistry & Biotechnology
Experimental
M a t e r i a l s a n d M e t h o d s
Standards of mycotoxins nivalenol (nIV), deoxynivalenol
(DOn), deoxynivalenole-3-glucoside (DOn-3-Glc),
3-acetyldeoxynivalenol (3-ADOn), 15-acetyldeoxynivalenol
(15-ADOn), fusarenone-X (fus-X), T-2 toxin (T-2),
HT-2 toxin (HT-2) and zearalenone (ZEA) were purchased
from Biopure (Tulln, Austria). A Synergi Hydro RP column
(Phenomenex, Torrance, CA, USA), a liquid chromatograph
HP1100 Binary Series LC system (Agilent Technologies,
Palo Alto, CA, USA) coupled to an ion trap mass analyser
LCQ Deca (Finnigan, San Jose, CA, USA) were employed
for LC separation and MS/MS detection. For GC–ECD
method, a gas chromatograf HP 5890 Series II with an
electron capture detector ( 63 ni) and a capillary column HP-
35 (30 m × 0,25 mm I.D. × 0,25 μm phase) with (35%-fenyl)methylpolysiloxan
stationary phase (Agilent Technologies,
Palo Alto, CA, USA) were used. For ELISA analysis, two
commercial available DOn kits, i.e. Ridascreen ® DOn (R-
Biopharm, Darmstadt, Germany) and AgraQuant ® DOn
Assay 0.25/5.0 Test Kit (Romer Labs, Tulln, Austria), were
purchased.
gC-eCD method: An amount of 10 g of homogenised
ground sample was extracted by shaking with 100 mL of an
acetonitrile–water mixture (84 : 16, v/v) for one hour and
extract was filtered (Filtrak no. 390, VEB Freiberger, Berlin,
Germany). The crude extract was passed through MycoSep
#225 and four millilitres of cleaned extract was evaporated.
Residue after evaporation was redissolved in methanol,
transferred into the derivatisation vial, and after methanol
evaporation using a gentle stream of nitrogen, derivatisation
for 20 min at 60 °C using 100 μl trifluoracetanhydride with
addition of 10 mg naHCO 3 was performed. Further, 500 μl
of isooctane, 1 ml of deionised water and 0.5 g of anhydrous
sodium sulphate was added followed by removing of 300 µl
of the organic layer and addition of 200 μl of isooctane to the
vial for GC–ECD analysis.
lC-Ms/Ms method: An amount of 12.5 g of homogenised
ground sample was extracted by shaking with 50 mL
of an acetonitrile–water mixture (84 : 16, v/v) for one hour
and the crude extract was filtered (Filtrak no. 390, VEB Freiberger,
Berlin, Germany). Eight millilitres of filtered extract
were passed through the MycoSep #226 column and four
millilitres of filtered extract were evaporated to dryness and
dissolved in 1 mL of a water–methanol mixture (50 : 50, v/v).
Finally, the sample was passed through a 0.2 µm microfilter
(Alltech, Deerfield, IL, USA) prior to analysis.
elisA: In case of immunochemical assays, both sample
preparation and the ELISA analysis itself were carried
out strictly according to manufacturer recommendations. An
amount of 20 g of ground sample was extracted with 100 ml
of deionised water and vigorously shaken for 3 minutes. Further,
the extract was filtered and an appropriate aliquot was
placed into the microwell.
s572
Fig. 1. Correlation of the results of wheat samples obtained by
LC–MS/MS and GC–ECD methods for a) NIV b) DON.
Fig. 2. DON content in reference materials measured by LC–
MS/MS and two types of commercial ELISA kits.
G C - E C D v s . L C - M S / M S
Using the previously used GC–ECD method, nIV,
DOn, FUS-X, 3-ADOn, 15-ADOn, HT-2 and T-2 were
analysed within a single run. Recoveries 68–97% and relative
standard deviations (RSDs) ≤ 16 % were obtained for
all of analytes analysed. As regards LC–MS/MS method,
the scope of target analytes has been extended for ZEA and
“masked” mycotoxin DOn-3-Glc. Recoveries of LC-MS/MS