3. FOOD ChEMISTRy & bIOTEChNOLOGy 3.1. Lectures

Chem. Listy, 102, s265–s1311 (2008) Food Chemistry & Biotechnology
P58 NEw APPROACh IN ANALySIS OF FuSARIuM
MyCOTOxINS AND ThEIR “MASKED”
FORMS: IMMuNOAFFINITy COLuMN
CLEAN-uP
ALEXAnDRA MALACHOVá (KRPLOVá), MARTA
KOSTELAnSKá, MILEnA ZACHARIáŠOVá, JAnA
KOHOUTKOVá and JAnA HAJŠLOVá
Institute of Chemical Technology Prague,
Technická 5, 166 28 Prague 6 – Dejvice, Czech Republic,
alexandra.malachova@vscht.cz
Introduction
Mycotoxins are classified as toxic, low molecular weight
secondary metabolites of microscopic filamentous fungi.
Fusarium toxins are the most common contaminants in cereals.
Recently several research study have been focused on
masked deoxynivalenol and other mycotoxins of these group.
The hyphothesis on existence of “masked” mycotoxins was
postulated as soon as in mid-1980s. Mycotoxicosis cases
were observed in farm animals although the laboratory examination
of a feeding stuff did not indicate high levels of
toxins. It was concluded that hydrolysis of precursor toxins in
digestive tracts of animals occurs. Follow up research showed
that mycotoxins can be partly metabolized by living plants
as a result of detoxification process. “Masked” mycotoxins
escape routine analysis for several reasons. These substances
are more polar than the precursor toxins, they are difficult to
extract with the common organic solvents or get lost in the
clean-up procces. In addition, standards of these substances
are not commercially available. Currently deoxynivalenol-3glucoside
(D3G) representing the “masked” form of the most
common mycotoxin deoxynivalenol (DOn) is available.
The most common method in mycotoxin analysis is
liquid chromatography coupled with tandem mass spectrometry
(LC-MS/MS). To reduce interferences due to matrix
coextracts various clean-up procedures are involved in sample
processing step.
This study assessed the afficacy of several immunoaffinity
columns (IACs) designed either for single fusarium toxins
(DOnPREP ® , EASI EXTRACT HT-2�T-2 ® , EASI EXTRACT
ZOn ® ) and DZT Multi Myco IACs for multimycotoxin analysis.
Based on our recent research we investigated occurence
of this phenomenon that documented cross reactivity of D3G
in DOn dedicated ELISA kits in the above IACs.
MycoSep ® 226 AflaZon� columns clean-up based on
adsorbtion chromatography usage were used for crude extract
processing.
Experimental
C h e m i c a l s
Standards of analysed mycotoxins DOn, D3G, HT-2
toxin (HT-2), T-2 toxin (T-2) and zearalenone (ZOn) were
purchased from Biopure (Austria).
Calibration standard solutions were prepared in the LC
mobile phase methanol:water (50 : 50, v/v) by diluting of stock
s700
standard solutions in concentration range 5–1000 ng ml –1 .
While standards in solvent were employed for calibration
in case of purified samples, matrix-matched standards were
used for unpurified extract.
S a m p l e s
naturaly contaminated maize containing several fusarium
toxins (see Table I) was used for our experiments.
Table I
Contents of trichothecene mycotoxins
Trichothecene
Mean s.d.
[µg kg –1 ] [µg kg –1 ]
deoxynivalenol 620 21
deoxynivalenol-3-glucoside 100 26
HT-2 toxin 27 18
T-2 toxin 52 15
zearalenone 462 23
I m m u n o a f f i n i t y C o l u m n s a n d
M y c o S e p
Immunoaffinity columns DOnPREP ® , EASI EXTRACT
HT-2T-2 ® , EASI EXTRACT ZOn ® and DZT Multi Myco
IACs were purchased from R-Biopharm Rhône Ltd (Germany).
DOnPREP ® columns contain a highly specific monoclonal
antibodies purifying and concentrating DOn from a
sample extract. EASI EXTRACT HT-2�T-2 ® columns are
made for determination of HT-2 and T-2 and EASI EXTRACT
ZOn ® are specified for ZOn analysis. DZT Multi Myco
IACs are designed as multi-mycotoxins columns. MycoSep ®
226 AflaZon� columns (Romer Labs ® , Austria) are designed
for complex matrices with more interference such as gluten,
meal feed and processed food.
S a m p l e P r e p a r a t i o n ( E x t r a c t i o n
a n d C l e a n - u p )
For preparation of purified extract column application
instructions recommended by producers were followed.
Extraction processes and all subsequented steps – diluting,
passing through column and eluting are summarized in
Table II.
In addition, the acetonitrile:water (84 : 16, v/v) crude
extract was prepared for LC-MS/MS analyses. This procedure
is accredited to according ČSn En ISO/IEC 17025:2005 for
direct LC-MS/MS analysis and accuracy of generated data is
regularly documented through proficiency tests (FAPAS ® ).
A n a l y s i s o f M y c o t o x i n s U s i n g L C -
M S / M S
High performance liquid chromatograph (HP1100
binary series LC system, Agilent Technologies, USA)
coupled with tandem mass spetrometer (Finnigan LCQ
Deca, USA) were employed. Chromatographic separation of