2. ENVIRONMENTAL ChEMISTRy & TEChNOLOGy 2.1. Lectures

Chem. Listy, 102, s265–s1311 (2008) Environmental Chemistry & Technology
P34 MuLTI-RESIDuE METhOD FOR
ThE ANALySIS OF PESTICIDES AND
MyCOTOxINS IN CEREALS by LC-MS/MS
OnDřEJ LACInA, JAnA URBAnOVá, ALEXAnDRA
KRPLOVá and JAnA HAJŠLOVá
Institute of Chemical Technology Prague
Technická 5, 166 28 Prague 6,
ondrej.lacina@vscht.cz
Introduction
In the recent decade, liquid chromatography–tandem
mass spectrometry (LC MS/MS) operated in a selected reaction
monitoring mode, has become the main tool for the analysis
of food and environmental contaminants presenting wide
range of physico-chemical properties. This approach allowed
the introduction of multiresidue methods with up hundreds
target analytes determined in a single run. However, most of
these methods are focused only on one group of food contaminants
such as pesticides, veterinary drugs, mycotoxins,
plant toxins, etc. Considering the fact, that sample preparation/detection
principles are basically similar for most of
these methods, we attempted to determine pesticide residues
and mycotoxins - contaminants representing different sources
of origin in single run. The comprehensive LC-MS-MS
multi-residue method has been developed and validated for
14 Fusarium toxins, 4 aflatoxins, ochratoxin A, 3 Alternaria
toxins and 200 pesticides.
Experimental
C h e m i c a l s a n d R e a g e n t s
Certified pesticide and mycotoxins standards were
purchased from Dr. Ehrenstorfer GmbH (Germany), Riedel
de Haen (Germany) and/or Biopure (Austria). Individual
analyte stock solutions (concentrations in the range
0.3–3mg ml –1 ) were prepared in either methanol or acetonitrile,
depending on the solubility of particular analyte. These
solutions were used for preparation of mixed standard solution
in acetonitrile (10 µg ml) and stored in –18 °C. Deionized
water for preparation of a mobile phase was produced
by Milli–Q apparatus (Millipore, Germany). Ammonium formate
for mass spectrometry was obtained from Fluka (Buchs,
Germany). Acetonitrile (Sigma-Aldrich, Germany) and methanol
(Merck, Germany) were HPLC gradient grade solvents
for pesticide residue analysis.
S a m p l e P r e p a r a t i o n
5 g of sample were weighted into 50 ml PTFE centrifugation
tube. Then, 20 ml of extraction mixture of acetonitrile/
water/formic acid (75 : 24 : 1, v/v/v) were added and the tube
was placed onto laboratory shaker for 90 min. After this time,
the tube was centrifuged (Hettich, Germany) at 11,000 rpm
for 5 min and aliquot of extract was diluted by a water in
a ratio 2 : 1 and filtered through a 0.45 µm PTFE filter Iso-
DiscTM (Supelco, USA), and transferred into a vial.
s404
H P L C - M S - M S A n a l y s i s
The HPLC analyses of selected pesticides and mycotoxins
were performed using an Alliance LC system (Waters,
USA) equipped with an Atlantis T3 column (100 × 2.1 mm
I.D., 3 µm particle size, Waters, USA) maintained at 30 °C.
The mobile phase contained 0.005M ammonium formate
in deionized water (A) and methanol (B), flow rate was
0.3 ml min –1 . The optimized chromatographic method started
at mobile phase composition of 5 % of B and was hold for
0.5 min, then rising linearly to 60 % of B and then 100 %
at 15 min. This composition was held for 8 min to remove
co-extracted matrix from column, 6min re-equilibration to
initial mobile phase composition followed. Sample injection
volume 8µl was used in all experiments.
HPLC system was connected to tandem mass spectrometer
Quattro Premier XE (Waters, USA) operated in positive
electrospray ionization mode. The capillary voltage was set
to 3,500 V, source temperature was maintained at 120 °C and
desolvation temperature was 380 °C. The masses of parent
and daughter ion, cone voltage and collision energy were
tuned previously for each analyte and two MS/MS transitions
were acquired for each of them.
Results
O p t i m i z a t i o n o f S a m p l e
P r e p a r a t i o n
QuEChERS extraction method published and extensively
tested in a recent years 1–3 has become the widely used
method for isolation pesticide residues from various matrices.
QuEChERS method employs acetonitrile (MeCn)
extraction followed by partition induced by added salts. If
necessary dispersive SPE clean-up is performed. The partition
step discriminate a lot of bulk matrix compounds such
as sugars and/or acids, which would interfere with determinative
step, however also recovery of polar target analytes is
reduced. For this reason, the QuEChERS has not become an
extraction method of choice for mycotoxins, especially for
relatively polar B trichothecenes.
It should be noted, that existing multi-mycotoxins
methods are based on the extraction by MeCn i.e. use similar
solvent as QuEChERS. Although the sources of food contamination
by pesticides and mycotoxins are fairly different in
their nature, from analytical point of view there are a large
number of representatives of these two groups, possessing
similar physico-chemical properties. On this account, it is a
conscionable concept to put together analysis of mycotoxins
and pesticide residues in one multi-toxin method.
The bottleneck of such method might be the sample
preparation step, since it is necessary to achieve acceptable
recovery for all analytes and at the same time discriminate
extraction of matrix components, which could cause suppression
of ionization as far as electrospray (ES) is employed
in LC-MS method. In principle, achieving simultaneously
these two requirements is impossible, and, therefore the only
feasible approach is to examine extract directly without any
purification 4–5 .