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Supercritical Fluid Extraction of Macrocyclic Lactone Mycotoxins in Maize Flour Samples For Rapid Amperometric Screening and Alternative Liquid Chroma
Supercritical Fluid Extraction of Macrocyclic Lactone Mycotoxins in Maize Flour Samples For Rapid Amperometric Screening and Alternative Liquid Chroma
Supercritical Fluid Extraction of Macrocyclic Lactone Mycotoxins in Maize Flour Samples For Rapid Amperometric Screening and Alternative Liquid Chroma
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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
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],
52
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
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.
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
Fig. 3. Influence of the supercritical-CO2 flow-rates and extraction temperature on macrocyclic lactone mycotoxins recovery.
54
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.
55
Table 1
Figures of merit for the screening system
Mycotoxins
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
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.
Table 2
Figures of merit for the confirmation method
Mycotoxins
Y = aX + b
R2
Sy/x
LOD (g kg1 )
LOQ (g kg1 )
RSD (%)
ZON
-ZOL
-ZOL
38300
38300
30236
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.
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
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.
57
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