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Journal of Chromatography A, 1177 (2008) 5057

Supercritical fluid extraction of macrocyclic lactone mycotoxins in maize

flour samples for rapid amperometric screening and alternative liquid
chromatographic method for confirmation

Mohammed Zougagh, Angel

Ros
Department of Analytical Chemistry and Food Technology, Faculty of Chemistry, University of Castilla-La Mancha,
Av. Camilo Jose Cela s/n, E-13004 Ciudad Real, Spain
Received 10 September 2007; received in revised form 3 November 2007; accepted 7 November 2007
Available online 17 November 2007

Abstract
A rapid and simple method for the direct screening of macrocyclic lactone mycotoxins (zearalenone, ZON; -zearalenol, -ZOL; and zearalenol, -ZOL) in maize flour samples is proposed. The sample screening method comprises supercritical fluid extraction (SFE) and clean-up
on Florisil adsorption cartridge of the selected toxic compounds, followed by continuous flow electrochemical detection. Those samples for which
the total concentration is close to or above the threshold limit established by legislation (0.200 mg kg1 ) are subjected to preconcentration on C18
chromatographic material and liquid chromatographic separation for confirmation purposes. This confirmation method allows the determination
of ZON, -ZOL and -ZOL in the range between 30 and 300 g kg1 , with a average relative standard deviation lower than 5.2 in all cases.
2007 Elsevier B.V. All rights reserved.
Keywords: Micotoxins screening; Chromatographic confirmation; Supercritical fluid extraction; Maize flour samples

1. Introduction
Mycotoxins are secondary metabolites produced by fungal
species, growing on agricultural products during cultivation,
harvest, transport and storage [1]. Their occurrence in food has
been recognized as potential human health hazard either caused
by direct contamination of grains and fruits and their products or by carry over of mycotoxins and their metabolites
in animal tissues [2,3]. Zearalenone (ZON) and its metabolites, namely -zearalenol (-ZOL), -zearalenol (-ZOL) and
zearalanone (ZAN) (Fig. 1) are produced by Fusarium species,
which colonize several grains [4]. High amounts of ZON can
most frequently be found on maize, wheat, oats and barley. It
has relatively low acute toxicity [5] and its carcinogenic properties are controversially estimated [6]. The European Union
has recently set out the maximum levels of Fusarium toxins
to be adopted in July 2006 [7]. Maximum levels of 200 and
100 g kg1 have been fixed for ZON in unprocessed corn and
unprocessed cereals other than corn, respectively.

Corresponding author.
Ros).
E-mail address: angel.rios@uclm.es (A.

0021-9673/$ see front matter 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.chroma.2007.11.021

The main applied techniques for the determination of ZON

and its metabolites in different foods, animal feeds and biological matrices are thin-layer chromatography (TLC) [8], gas
chromatographymass spectrometry (GCMS) [9], liquid chromatography using UV diode array [10], fluorescence [11,12]
or mass spectrometric detection [13,14] and enzyme-linked
immunosorbent assays (ELISAs) [15,16]. An immunosensor
based on a sandwich competitive assay for the analysis of
ZON has also been described [17]. Determination of ZON and
its metabolites in cereals is normally preceded by extensive
and/or selective sample extraction and clean-up procedures to
remove most of the matrix interferents and to pre-concentrate
the analytes in order to reach the required low determination
limits (g kg1 ) [18,19]. Sample pretreatment for ZON and
/ ZOL determination includes liquidliquid partitioning [20],
solid-phase extraction (SPE) [21] and immunoaffinity columns
(IACs) [15,16]. Liquidliquid extraction, which is the basis of
the AOAC Official Method [20], is considered to be inaccurate,
time-consuming and to make use of relatively large volumes of
chlorinated solvents. SPE sorbents show in general a limited
selectivity, since they differentiate chemical species only on the
basis of generic properties, such as hydrophobicity. On the contrary, IACs provide clean extracts from complex matrices such as

 Ros / J. Chromatogr. A 1177 (2008) 5057

M. Zougagh, A.

51

Fig. 1. Chemical structure of ZON and related compounds: namely -ZOL, -ZOL and ZAN.

cereals, due to the specificity of the antibodies; however, multitoxin analysis is not feasible with these columns, since they
are highly specific for just one target mycotoxin and they are
very expensive compared to other less selective SPE sorbents.
Microwave-assisted extraction (MAE) using non-chlorinated
solvents (methanol and acetonitrile) has also been described
as a suitable technique for the extraction of ZON from wheat
and corn samples within short times and with excellent recoveries, although with poorer detection limits with respect to other
approaches [22]. On the other hand, Royer et al. [23] recommended SPE cleaning after pressurised liquid extraction (PLE)
of solid cereals and grains since they observed increased levels
of co-extracted matrix components, being problematic even for
selective MS/MS detection. For other matrices, Huopalahti and
Henion applied supercritical fluid extraction (SFE) without any
further sample clean-up steps and achieved detection limits for
-ZAL of 100 g kg1 in bovine muscle tissue and liver samples
[24], while Lagana et al. proposed matrix solid-phase dispersion
as suitable extraction procedure for ZON, - and -ZOL in fish
tissue samples [25,26].
Recently, Urraca et al. [12] have developed a fast and accurate
method for the determination of ZON and / ZOL in ground
wheat samples using PLE and liquid chromatography (LC) with
fluorescence detection. In the optimised procedure a mixture
of acetonitrilemethanol was selected as the extraction solvent
applying a temperature of 50 C. The application of this procedure allowed automated sample handling and a reduction of the
solvent consumption, providing good recoveries for the target
mycotoxin fortified in ground cereals and swine feed samples
(>96%).
The potential of SFE [27] as an alternative to conventional
extraction procedures has been demonstrated in a wide variety of samples [2831]. One of the greatest advantages of SFE
over other sample preparation techniques is that it can be automated; this makes it highly suitable for rapid, routine analyses.
The efficiency of the different SFE-collection models in online assemblies is very important, as it provides quantitative
transfer of extracted analytes to the analytical instrument and
reduces contamination levels. Four different ways of collecting
supercritical fluid-extracted analytes have been described [32],

namely: (a) solvent collection [33], (b) solid-phase collection

[30] (c) solidliquid phase collection [34], and (d) empty vessel trap collection [35]. Solid-phase collection is widely used
and offers high trapping efficiency for substances with high
vapour pressures, since the trap temperature can be easily be
reduced to 30 C [27]. In These SFE methods, analytes are
retained in a solid trap and eluted with organic solvents. Many
solvents are incompatible with detectors used. This problem can
be overcome by evaporating the solvents with the risk of damaging analytes and losing volatile components. Alternatively,
the solid trap can be replaced with a custom liquid trap. This
allows extracted macrocyclic lactones mycotoxins from maize
samples to be directly collected in a solvent compatible with
electrochemical detection. The use of a liquid trap in SFE was
examined in previous works [3639]. A liquid trap is simpler
and more flexible to use than a solid trap in SFE; however, the
liquid trap is not as efficient as the solid trap for volatile analytes
[39].
The availability of rapid, reliable screening methods is an
important prerequisite when a large number of samples must
be analyzed, in order to meet the urgent need for results. The
screening methods are defined as methods that are used to detect
the presence of an analyte or analyte group at the concentration
level of interest. Screening methods typically feature a high sample throughput and are used to sift large numbers of samples for
potential positives. They avoid or preserve the routine use of
more expensive and sophisticated instrumentation.
In this work, we developed a unique SFE procedure that
uses electrochemical detection for the screening method, and
a RP-C18 preconcentration LC system as the method for confirmation. The screening method is highly responsive, whether
any macrocyclic lactone mycotoxins are present or not. The use
of chromatography, involving RP-C18 preconcentration, identification and quantification of macrocyclic lactone mycotoxins
in maize flour samples can, thus, be dramatically reduced, as
only those samples given positive response in the screening system were further analyzed. Also, the interferences present in
the samples are reduced by the use of a Florisil adsorption cartridge connected to the supercritical fluid extractor behind the
extraction vessel.

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Ros / J. Chromatogr. A 1177 (2008) 5057

M. Zougagh, A.

2. Experimental
2.1. Reagents, standards and samples
Zearalenone, -zearalenol, -zearalenol and Florisil adsorbent (1630 mesh) were obtained from SigmaAldrich (St.
Louis, MO, USA). They were used to prepare mixed standard
solutions in acetonitrile. The concentration of the stock standard
solutions was in the range from 2.5 to 3 mg ml1 , depending on
the particular mycotoxin. These standard solutions were stored
at 20 C in the dark. Calibration solutions were made on a daily
basis by appropriate dilution of the stock solutions. Analyticalgrade diatomaceous earth (SigmaAldrich) was used as solid
support. It was washed with hexane and methanol (following
this sequence), dried and stored at room temperature prior to
use.
SFC-grade carbon dioxide (Air Liquide Products, Paris,
France) was used as extraction fluid. Maize flour samples were
obtained locally from local supermarkets. One hundred grams
of maize flour samples was weighed on aluminum foil and a
volume of stock solutions of the mycotoxin analytes was slowly
added dropwise, the solution being spread over the maize and
the sample allowed to stand for 24 h to allow acetonitrile to
evaporate.
A sorbent cartridge was constructed by packing a commercial stainless steel cartridge (2.5 cm 3.9 mm i.d.) with ca. 1 g
of Florisil adsorbent material (1630 mesh particle size) from
SigmaAldrich; stainless steel filters were used to prevent material losses. A solid phase extraction columns (1.0 ml, 200 mg
BondElut-C18) from Varian (Harvor City, CA, USA) were used
for preconcentrating positive samples.
2.2. Instruments, apparatus and chromatographic
conditions
Cyclic voltammetric (CV) studies were conducted using
Metrohm Computrace 757 VA potenciostat equipped with the
electrochemical detection module. The flow system consisted of

a Gilson Minipuls-3 peristaltic pump furnished with poly(vinyl

chloride) pumping tubes, a Rheodyne 5041 injection valve and
PTFE tubing of 0.5 mm i.d. Amperometric detection was accomplished by using a Metrohm 641 VA detector provided with a
Metrohm 65303020 wall-jet flow cell and using a home-made
Labview software. A system of three electrodes has been used:
a Metrohm Model 60727000 Ag/AgCl/3 M KCl reference electrode, a gold auxiliary electrode and glass carbon as a working
electrode.
Supercritical fluid extraction was performed with a Jasco system (Easton, MD, USA) consisting of a PU-1580 CO2 delivery
pump, a BP-1580-81 backpressure regulator and CO-2065 column oven as extraction chamber. The Jasco 2080Plus intelligent
HPLC pump was used for modifier delivery.
The chromatographic system consisted on Hewlett-Packard
HPLC system (model 1090) consisting of an HPLC ternary
pump, an Agilent LC analytical column (model Sorbax SB-C18,
150 mm 4.6 mm i.d., 5 m particle size), a diode array detector
and a Rheodyne injection valve (model 1050i, 20 L). Data were
acquired and the equipment controlled by using Agilent ChemStation software, which was run under Microsoft Windows NT
on an IBM compatible personal computer.
Elution was performed under isocratic conditions, by using
a mobile phase containing a mixture of acetonitrile/water 50/50
(v/v) at a flow-rate of 0.8 ml min1 . The injection volume was
20 l. Detection was performed at 236 nm wavelength.
2.3. Manifold and procedures
The determination of macrocyclic lactone mycotoxins in
maize samples can be approached in two different ways, depending on the type of analytical information required. Thus, the use
of a screening system can be enough to determine the presence or absence of macrocyclic lactone mycotoxins in maize
samples. Preconcentration and separation were carried out in
order to improve both the limits of quantification and selectivity. Moreover, in the present case, a specific macrocyclic lactone
mycotoxin (ZON) has to be identified. Fig. 2 shows the assembly

Fig. 2. Scheme of the screening and confirmation systems for the determination of macrocyclic lactone mycotoxins in maize flour samples. V1 and V2; pressure
selection valves, V3 and V4; pressure injection valve, P1 and P2; high-pressure pump.

 Ros / J. Chromatogr. A 1177 (2008) 5057

M. Zougagh, A.

used to combine a flow manifold for screening and a chromatograph for identification.
In the extraction step, CO2 is aspirated from a dip-tube cylinder at a constant flow-rate of 1 ml min1 (liquid) by means of a
peltier pump and passed through the extraction vessel. A homogenized mixture of 2 g of sample and 0.5 g of diatomaceous earth
is manually placed into a 10 ml stainless steel extraction vessel
accommodated in the extraction chamber. The intimate contact with the extracting supercritical fluid is allowed through
the combination of static and dynamic extraction steps. Thus,
once the target pressure (25 MPa) and temperature (80 C) are
reached, the sample is extracted in the static mode for 10 min.
In this step, the modified CO2 (9.1%, v/v methanol), bypasses
the extraction cell and Florisil adsorption cartridge. Then,
dynamic extraction is performed for 30 min, after which CO2
is passed through the sample and Florisil adsorption cartridge
connected behind the extraction vessel, in order to clean-up
the samples. The extracted macrocyclic lactone mycotoxins are
collected in the flask containing 2 mL of borax (20 mM, pH
9.2) inserted behind the programmable back-pressure regulator
maintained at 40 C. The screening of the extract is monitored
in the flow FI system coupled to the electrochemical detector. The manifold used for the flow measurements consisted
of two channels. In the first channel the sample was carried
to the injection valve and the second stream was for the borax
buffer solution at pH 9.2, which acted as a carrier. The sample
was introduced into the system by the buffer stream, reaching the cell that was connected to the equipment, which also
captured the signal from the flow cell. Once the sample had
passed the detector, it was driven to waste. The sample volume injected was 60 l and a flow-rate of 1.5 ml min1 was
employed. For confirmation, the 1.5 ml of extracts for positive
samples are preconcentrated in RP-C18, eluted with 0.5 ml of
methanol and therefore injected into the LC system for separation and quantification of individual macrocyclic lactone
mycotoxins.

53

3. Results and discussion

3.1. Extraction system
3.1.1. Optimization of the extraction procedure
The extractability of macrocyclic lactone mycotoxins from
test samples was studied by using SFE technique in order to
maximize recovery. Macrocyclic lactone mycotoxins extracts in
borax (20 mM, pH 9.2) were preconcentrated in RP-C18 eluted
with methanol and therefore quantified by using a HPLC system. For this purpose, the mixture of 2 g of spiked sample and
0.5 g of diatomaceous earth was homogenised and inserted in
the extraction cell. Individual macrocyclic lactone mycotoxins are recovered at variable CO2 flow-rate values by using
four different extraction temperatures (50, 60, 70 and 80 C)
and 10 min of static extraction followed by 30 min of dynamic
extraction. Four isotherms were thus constructed at CO2 flowrate values from 0.4 to 1.5 ml min1 . Fig. 3 shows the effect
of the supercritical-CO2 flow-rates and extraction temperature
on macrocyclic lactone mycotoxins recovery. As can be seen,
the best recoveries were achieved by using 1 ml min1 at 80 C.
Increasing the extraction time up to 45 min and using on equilibration time in the range 1030 min resulted in no improved
extraction. The addition of a modifier in the CO2 stream was
then tested. Methanol was used for this purpose, as macrocyclic
lactone mycotoxins are highly soluble in this solvent. Modifying
the polarity of the medium, a substantial effect on macrocyclic
lactone mycotoxins recovery was obtained. Thus, a flow-rate
of 0.05 ml min1 modifier increased the recovery from 8488%
to 9092%, whereas a flow-rate of 0.1 ml min1 modifier was
enough for achieving quantitative extraction (97100%) of each
mycotoxin. Once the optimum conditions for the extraction
of macrocyclic lactone mycotoxins (viz. 1 ml min1 at 80 C,
10 min static extraction and 30 min dynamic extraction, 9.1%
methanol as CO2 modifier) were established, the macrocyclic
lactone mycotoxins extracted from maize flour samples and col-

Fig. 3. Influence of the supercritical-CO2 flow-rates and extraction temperature on macrocyclic lactone mycotoxins recovery.

54

Ros / J. Chromatogr. A 1177 (2008) 5057

M. Zougagh, A.

lected in the flask containing 2 mL of Na2 B4 O7 (20 mM, pH 9.2),

were screened by the electrochemical detector.
3.1.2. SFE plus Florisil absorption cartridge for the
determination of macrocyclic lactone mycotoxins in maize
matrix
The use of SFE in conjunction with Florisil absorption
cartridge and liquid trap was tested on real sample matrices
(viz. maize flours). No macrocyclic lactone mycotoxins were
detected in the commercial product, so 2 g of the previously
prepared maize flour samples spiked with a mixture of the
three macrocyclic lactone mycotoxins (ZON, -ZOL and ZOL) were transferred to an extraction vessel. Triplicate samples
were extracted by conventional SFE, using a liquid-trap under
the optimum conditions for each sample set without using the
Florisil adsorption cartridge. Similarly, an identical preparation
ofmacrocyclic lactone mycotoxins spiked maize flours was
also extracted using a Florisil adsorption cartridge connected
to the supercritical fluid extractor behind the extraction vessel.
The extracts obtained by the SFE Florisil absorption cartridge
were transparent, while those provided by conventional SFE
without use of a Florisil adsorption cartridge were cloudy due
to the interferences present in the extracts. When injected in
the electrochemical detector, off-range signals were obtained
in the electrochemical detector. SFE recoveries of macrocyclic
lactone mycotoxins obtained by using the Florisil absorption
cartridge and liquid-trap under the previously established optimum extraction conditions revealed that the three macrocyclic
lactone mycotoxins (ZON, -ZOL and -ZOL) were extracted
highly efficiently (recoveries ranged from 96% to 104%), and
compatible with the electrochemical detector.

3.2. Screening system

3.2.1. Optimisation of variables
The glass carbon electrode (GCE) was tested as an amperometric detector in flowing streams. For this purpose, some
standard solutions were injected in the carrier stream in order to
obtain their responses. Prior to use, electrode treatment was necessary. This treatment consisted of polishing with emery paper
and immersing the glass carbon surface in 0.3 g ml1 ZON
solution during 5 min with stirring.
The choice of the potential to be applied to the composite
electrode for its use as an amperometric detector was established by plotting the peak current values measured at different
pH by applying different potentials within the range 0.11.5 V.
Fig. 4a shows the data obtained for 0.3 g ml1 for ZON, -ZOL
and -ZOL in 0.1 M Na2 B4 O7 obtained under flow injection
conditions and using a wall jet flow cell. A volume of 60 l
of macrocyclic lactone mycotoxins solution (ZON, -ZOL or
-ZOL) was injected into the 0.1 M Na2 B4 O7 carrier solution at flow-rate of 1.5 ml min1 . Peak-shaped profiles, with
the maximum current at 0.9, 0.7 and 0.8 V for ZON, -ZOL
or -ZOL, respectively, were observed, but an intermediate
potential of 0.8 V could be suitable for the determination of
the three macrocyclic lactone mycotoxins. Under these experimental conditions, no cleaning or pretreatment of the electrode
after each injection was required. It was enough to polish the
electrode and immerse them in 0.3 g ml1 ZON solution during 5 min at the beginning of the experiments, in order to obtain
a fresh electrode surface, and no appreciable fouling signals
were observed after successive scans. This is one of the most
advantageous characteristic from a practical point of view, which

Fig. 4. Effect of the most influential variables of the screening system on the analytical signal: (a) applied potential electrode, (b) supporting electrolyte concentration,
(c) flow-rate and (d) sample volume injected.

 Ros / J. Chromatogr. A 1177 (2008) 5057

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55

Table 1
Figures of merit for the screening system
Mycotoxins

Linear range (mg kg1 )

ZON
-ZOL
-ZOL

5.4 102

R2

Y = aX + b
Y = (29.53 0.37)X + (3.65 0.16)
Y = (30.06 0.35)X + (1.04 0.14)
Y = (34.44 0.32)X + (1.62 0.12)

to 1.0
4.6 102 to 1.0
3.4 102 to 0.8

0.9966
0.9985
0.9978

Sy/x

LOD (mg kg1 )

LOQ (mg kg1 )

RSD (%)

0.58
0.54
0.44

1.6 102

5.4 102

6.7
6.3
5.2

1.4 102
1.0 102

4.6 102
3.4 102

A: slope; b: intercept; R: regression coefficient; Sy/x : standard deviation of residuals; LOD: limit of detection; LOQ: limit of quantification, RSD: relative standard
deviation.

permits working several days without any deterioration of the

signals.
Different salts were tested as supporting electrolytes at
different concentrations by applying different potentials, and
Na2 B4 O7 was finally selected, as a compromise between electrochemical detection and retention of macrocyclic lactone
micotoxyns in the preconcentration step. Different concentrations between 5 and 50 mM were studied and 20 mM was
selected for sensitivity reasons. As can be seen in Fig. 4b, maximum signals were obtained for the analytes in 20 mM Na2 B4 O7 .
As expected, the peak height depended on the flow-rate of the
carrier solution. An increase in the signals and a decrease in
peak width were observed when the flow-rate increased. Different flow-rate values, from 1 to 2.5 ml min1 were tested, and
a value of 1.5 ml min1 was finally chosen for (giving the best
signal (Fig. 4c). The influence of sample volume on peak height
was also studied. Different sample volumes (from 20 to 100 l)
were tested, and a volume of 60 l was chosen (for giving the
best signal (Fig. 4d).
3.2.2. Calibration equations and reliability of the proposed
screening method
The screening system was designed to provide a rapid
response to the presence or not of macrocyclic lactone mycotoxins at a threshold limit concentration established by legislation.
This system is based on the amperometric detection of extracted
macrocyclic lactone mycotoxins from supercritical fluid extractor. The responses of the macrocyclic lactone mycotoxins
extracts were obtained by using flow injection experiments
at 0.8 V for ZON, -ZOL and -ZOL. All compounds were
injected at the same concentration (0.2 mg kg1 ) into a 20 mM
carrier solution (flow-rate 1.5 ml min1 ). Reproducible flow
injection responses were observed at this characteristic potential.
Under these conditions, the sample were analysed by triplicate
injections. For the calibration, amperometric measurements in
the flow-cell were carried out. Linear calibration graphs for
ZON, -ZOL and -ZOL were obtained within the ranges
0.0541.0, 0.0461.0, and 0.0340.8 mg kg1 , respectively. The

peak height increased linearly with analyte concentration. The

analytical characteristics of the proposed method are summarised in Table 1. The precision of the method, expressed as
RSD, for the determination of 0.2 mg kg1 of each mycotoxin,
was in all cases <7% (n = 11). The limits of detection (LODs)
were 0.016, 0.014 and 0.010 mg kg1 for ZON, -ZOL and ZOL, respectively, calculated as the intercept plus three times
its standard deviation (SD).
The crucial requirements for measuring the total macrocyclic lactone mycotoxins content of maize flour samples with
electrochemical measurements are that all macrocyclic lactone
mycotoxins components presented the same electrochemical
properties and, thus, the current peak containing the overall
response for the total amount of macrocyclic lactone mycotoxins in the sample can be obtained. However, there are some
differences in the response factor (i.e. slope of the calibration
curves). Table 2 shows that -ZOL was the analyte that gave
the lower analytical limit and ZON was the analyte that give
the highest response factor. To quantify total mycotoxin content
the bestworst-case scenario approach can be used, by alternatively assuming that either the whole current signal comes
from the compound with highest response (ZON) or from the
compound with the lowest response (-ZOL). To avoid false
negatives, the -ZOL calibration curve was chosen. The cut-off
concentration was defined as the concentration corresponding to
0.200 mg kg1 (reference value established for ZON by directive 2005/38/EC). In this work, we establish a worst scenario
for sample screening by selecting the least sensitive macrocyclic
lactone mycotoxins as a criterion to classify samples as negative
if the current signal is <7.0 nA or positive otherwise if the current signal is >7.0 nA. The final decision, however, can be made
by subjecting them to the LC confirmatory method.
3.3. Confirmation chromatographic system
Combined HPLC with SPE seemed to be an excellent way to
determine macrocyclic lactone mycotoxins in maize flour samples, which would provide the advantages of HPLC, as well as

Table 2
Figures of merit for the confirmation method
Mycotoxins

Linear range (g kg1 )

Y = aX + b

R2

Sy/x

LOD (g kg1 )

LOQ (g kg1 )

RSD (%)

ZON
-ZOL
-ZOL

38300
38300
30236

Y = (0.0251 0.0004)X + (0.2774 0.0669)

Y = (0.0265 0.0003)X + (0.4479 0.0581)
Y = (0.0491 0.0018)X + (0.6241 0.2647)

0.9994
0.9996
0.9985

0.0779
0.0676
0.3082

9
8
19

31
26
63

4.5
3.9
5.2

A: slope; b: intercept; R: regression coefficient; Sy/x : standard deviation of residuals; LOD: limit of detection; LOQ: limit of quantification, RSD: relative standard
deviation.

 Ros / J. Chromatogr. A 1177 (2008) 5057

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56

Table 3
Determination of three mycotoxins in maize flavour samples using the LC method
Sample

Analyte
ZON (g kg1 )

Sample 1
Sample 2
Sample 3
Sample 4
Sample 5
Sample 6
Sample 7
Sample 8

-ZOL (g kg1 )

-ZOL (g kg1 )

Added

Found

Error (%)

Added

Found

Error (%)

Added

Found

Error (%)

75.0
50.0
100.0
200.0
150.0
125.0
225.0
250.0

73.3
44.7
109.3
208.1
148.3
120.4
225.4
262.2

2.3
11.8
8.5
3.9
1.2
3.8
0.2
4.8

100.0
75.0
225.0
50.0
125.0
150.0
50.0
200.0

92.5
81.1
216.9
53.1
117.6
154.1
51.0
189.3

8.1
7.6
3.7
6.6
6.3
2.6
1.9
5.7

50.0
200.0
75.0
50.0
100.0
125.0
150.0
75.0

53.1
207.8
80.3
52.4
94.5
126.4
159.6
72.8

5.9
3.8
6.5
4.6
5.8
1.1
6.0
3.0

3.4. Analytical applications

Fig. 5. Chromatograms for the extract from a maize flour sample spiked with
three macrocyclic lactone mycotoxins. The peaks labelled with numbers correspond to the following macrocyclic lactone mycotoxins: (1) -ZOL, (2) -ZOL
and (3) ZON.

The proposed method was used to analyze various maize flour

samples that were found to contain none of the three abovementioned macrocyclic lactone mycotoxins. The samples were
then spiked with ZON, ZOL and -ZOL at variable concentrations, in order to study the presence of potential matrix effects.
Table 3 shows the results obtained in the determination of the
three macrocyclic lactone mycotoxins. In all these cases, a positive response has been obtained in the screening method. As can
be seen, the differences between the concentrations added and
those found were very small. The static texp values were 0.88,
1.00 and 1.55 for ZON, -ZOL and -ZOL, respectively. These
values are smaller than the corresponding tabulated tcrit values
(2.36 for 7 degrees of freedom at the 95% confidence level).
4. Conclusions

the high sensitivity achieved after SPE treatment. Analytes were

isolated from maize flour samples using the supercritical fluid
extraction followed by preconcentration in RP-C18. Macrocyclic lactone mycotoxins were retained in C18 material, eluted
with methanol and therefore injected into the chromatographic
system for separation and quantification of individual macrocyclic lactone mycotoxins. A polar endcapped C18 column was
applied for the separation of ZON, -ZOL and -ZOL. A secondary mobile phase consisting of acetonitrile/water 50/50 (v/v)
at a flow-rate of 0.8 ml min1 was selected for the separation of
the three analytes within 10 min with excellent resolution, as
is it shown in the chromatogram depicted in Fig. 5. The mean
retention times (min SD; n = 10), under such conditions were:
3.7 0.2 (ZON), 5.1 0.3 (-ZOL), 8.1 0.2 (-ZOL). These
well separated values allowed to achieve resolutions higher than
2.5 between consecutive macrocyclic lactone mycotoxins.
Linear calibration graphs for ZON, -ZOL and -ZOL were
obtained within the concentration ranges 38300, 38300 and
30236 g kg1 , respectively. The limits of detection and quantification were calculated according to the IUPAC criterion.
The corresponding regression equation and other characteristic parameters analytical for the determination of these three
macrocyclic lactone mycotoxins are shown in Table 2. The precision of the method, expressed as RSD, for the determination of
200 g kg1 of each mycotoxin, was in all cases <6% (n = 11).

A rapid SFE method for the isolation and clean-up of

macrocyclic lactone mycotoxins from maize flour samples was
developed. The use of a Florisil adsorption cartridge connected
to the supercritical fluid extractor allows interfering substances
to be retained and their presence in the extract reduced. The joint
use of a screening system (electrochemical) and the partially
confirmatory (identification) technique using preconcentration
on RP-C18 and LC-DAD (in accordance with the European
Union Commission decision 2002/657/EC additional specific
requirements should be met), to control macrocyclic lactone
mycotoxins in maize flour samples present a number of practical advantages. Thus, when large numbers of samples are to
be processed, the screening system allows them to be classified them as positive or negative. Only positive samples needed
to be confirmed, afterwards, using the LC method previous
the preconcentration step. This screeningconfirmation system
provides an interesting approach to raising productivity while
reducing analysis times and costs in routine laboratories.
Acknowledgements
The Spanish Ministerio de Educacion y Ciencia and JJCC
Castilla-La Mancha are gratefully acknowledged for funding
this work with Grants CTQ2004-02362 and PAC05-001-1. The

 Ros / J. Chromatogr. A 1177 (2008) 5057

M. Zougagh, A.

support given through a Juan de la Cierva research contract

for M.Z. is also acknowledged.
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