3. FOOD ChEMISTRy & bIOTEChNOLOGy 3.1. Lectures

Chem. Listy, 102, s265–s1311 (2008) Food Chemistry & Biotechnology
L13 ANALySIS OF fusAriuM MyCOTOxINS: A
CRITICAL ASSESSMENT
MILEnA ZACHARIáŠOVá a , JAnA HAJŠLOVá a , JAn
POUSTKA a , MARTA KOSTELAnSKá a , ALEXAnDRA
KRPLOVá a and LUKአVáCLAVíK a
a Institute of Chemical Technology, Prague, Faculty of Food
and Biochemical Technology, Department of Food Chemistry
and Analysis, Technicka 3, 166 28 Prague 6, Czech Republic,
milena.zachariasova@vscht.cz
Introduction
Mycotoxins, secondary metabolites of microscopic filamentary
fungi, are compounds generally considered to be very
toxic. Their occurrence in agricultural commodities represents
a major health concern for humans and animals. One
of the most important fungi genera, that produce mycotoxins,
is Fusarium. Fusarium species are able to produce lots of
structurally different mycotoxins including trichothecenes,
zearalenones and fumonisins. Trichothecenes are a group of
tetracyclic seskviterpene alcohols and can be divided into two
groups: type A and type B (they differ from each others in the
existence of ketone at C8 in trichothecene type B molecule).
The main representatives of type A trichothecenes include
e.g. T-2 toxin, HT-2 toxin, diacetoscirpenol, 15-acetoscirpenol
and neosolaniol. The most important naturally occurring
type B trichothecenes is deoxynivalenol representing the
most abundant trichothecene found in cereals, followed by
15 acetyldeoxynivalenol, 3-acetyldeoxynivalenol, nivalenol
and fusarenone-X. Other mycotoxins belonging to the genus
Fusarium are fumonisins (esters of 20-carbone backbone and
two tricarballylic acids) and zearalenone (substituted lacton
of resorcyclic acid). 1–3 Beside free mycotoxins, the existence
of mycotoxin conjugates, toxins bound to more polar compounds
like glucose, which originate as a result of mycotoxins
metabolism by plants, was proved 4,5 . Deoxynivalenol-3-glucoside
(DOn-3-Glc) was found to be dominating 6 .
Because of relatively high incidence of mycotoxins,
there is an urgent need for effective monitoring of these compounds
in foodstuffs. Over the past 15 years, a great progress
in mycotoxins analysis area has been made. Sampling contributes
to the largest variability in the analysis of mycotoxins ,
thus, obtaining of a representative sample represents a crucial
step. Sampling strategies of agricultural commodities within
mycotoxin analysis are defined by the European legislation
EC/401/2006. 7–9
There are various criteria typically considered when
analytical method is developed. not only the overall cost, but
also other factors such as speed of the analysis, level of technical
skills of an analyst, and the type of results provided by
the method (qualitative or also quantitative) need to be balanced
and the relative importance of each criterion evaluated.
Especially for multidetection methods, when a large number
of mycotoxins possessing a wide range of physicochemical
properties considered, a compromise between the factors
mentioned above is usually needed. 8–10
s571
The first step in multimycotoxin analysis includes the
isolation of particular analytes. The extraction should perform
a compromise between the solvent strength required for
the transfer of analytes from the matrix to the solution and the
compatibility of solvent with further analytical process. An
acetonitrile – water (84 : 16, v/v) mixture is commonly used
providing sufficient recoveries, and simultaneously minimises
number of co-extracted matrix compounds. Additionally,
this solvent mixture represents an azeotropic composition
and thereby its evaporation within further sample preparation
procedure is facilitated11 .
Once the sample extract is obtained, the clean-up step
should be performed to remove impurities potentially interfering
during the determination step. Beside this, the cleanup
procedure helps to concentrate the mycotoxins prior to
their analysis; especially immunoaffinity columns based on
specific immunochemical reaction of analyte and antibody
present in the cartridge are very convenient for this purpose.
The specificity of immunoaffinity clean-up for only one target
mycotoxin or class of mycotoxins can be in some respect
a limiting factor, particularly in the case of multimycotoxin
analysis9,12 . However, multimycotoxin immunoaffinity
clean-up cartridges specific to a wide range of analytes are
already available13,14 . Additionally, common SPE approaches
representing by e.g. MycoSep columns are also widely used.
The main advantage of MycoSep clean-up is the speed of
procedure: no time-consuming rinsing steps as in the case of
immunoaffinity and/or other SPE clean-up are required. 15–17
The final step of the analytical procedure is the determinative
step. nowadays, reference methods for mycotoxins
determination involve almost exclusively liquid chromatography
coupled to tandem mass spectrometry (LC-MS/
MS), which provide both selectivity and sensitivity of the
detection. 7–9 There are no limitations in sample preparation
such as derivatisation step, which is needed within
the gas chromatographic approaches GC-ECD or GC-MS
(Note: derivatisation of hydroxyl groups in order to attain
the volatility is typically performed using heptafluorobutylimidazole
or trimethylsilylether). 18–20 A challenging option
in the field of LC-MS analysis is the use of high-resolution
system LC-TOFMS, which provides a feasible tool for non
target masked mycotoxin screening.
Beside classical confirmatory chromatographic methods
mentioned above, there are also methods based on immunological
assays. Especially ELISA as rapid quantitative tools
for mycotoxin analysis is very suitable. However, possible
overestimation of results caused by cross-reactions with
compounds similar to target analytes could be a limitation of
this approach.
7– 9,17
In the following text, three different analytical approaches
are discussed. Firstly, performance characteristics of
GC-ECD and LC-MS/MS methods are compared. Secondly,
results obtained by means of two commercial ELISA assays
and reference LC-MS/MS method are introduced. A proof of
ELISA capability in terms of its cross-reaction potential is
presented.