Food Chemistry 135 (2012) 2522–2528

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Food Chemistry
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Analytical Methods

Extraction of ochratoxin A in bread samples by the QuEChERS methodology

Paula Paíga a, Simone Morais a, Teresa Oliva-Teles a, Manuela Correia a, Cristina Delerue-Matos a,
Sofia C. Duarte b, Angelina Pena b, Celeste Matos Lino b,⇑
a
REQUIMTE, Instituto Superior de Engenharia do Porto, Rua Dr. António Bernardino de Almeida 431, 4200-072 Porto, Portugal
b
Group of Health Surveillance, Center of Pharmaceutical Studies, University of Coimbra, Health Sciences Campus, Azinhaga de Santa Comba, 3000-548 Coimbra, Portugal

a r t i c l e i n f o a b s t r a c t

Article history: A QuEChERS method for the extraction of ochratoxin A (OTA) from bread samples was evaluated. A fac-
Received 29 November 2011 torial design (23) was used to find the optimal QuEChERS parameters (extraction time, extraction solvent
Received in revised form 6 June 2012 volume and sample mass). Extracts were analysed by LC with fluorescence detection. The optimal extrac-
Accepted 8 June 2012
tion conditions were: 5 g of sample, 15 mL of acetonitrile and 3 min of agitation. The extraction proce-
Available online 4 July 2012
dure was validated by systematic recovery experiments at three levels. The recoveries obtained ranged
from 94.8% (at 1.0 lg kg1) to 96.6% (at 3.0 lg kg1). The limit of quantification of the method was
Keywords:
0.05 lg kg1. The optimised procedure was applied to 20 samples of different bread types (‘‘Carcaça’’,
Ochratoxin A
Bread
‘‘Broa de Milho’’, and ‘‘Broa de Avintes’’) highly consumed in Portugal. None of the samples exceeded
QuEChERS the established European legal limit of 3 lg kg1.
Factorial design Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction Agency for Research on Cancer has classified OTA in group 2B as

Cereals are consumed nearly worldwide and provide significant
amounts of most nutrients, playing an important position in hu-
man nutrition (Dewettinck et al., 2008). In Portugal, bread is one
of the most consumed cereal derived products (Duarte et al.,
2010). In 2005, 323,194 tons of bread were produced, of which
278,161 tons were wheat bread (INE, 2008). The most recent na-
tional consumption data, from 1994, show an average bread con-
sumption of 32 kg/person/year (IPC, 2005).
Portugal possesses a long standing history of bread-making,
with numerous traditional recipes, different shapes, grains incor-
porated and taste, as the craft of baking is typical for each region.
The different types of bread are therefore part of the national cul-
tural heritage. The traditional maize bread, ‘‘Broa’’, continues to
compete with novel bread types, especially in the Oporto, Coimbra
and Lisbon areas. In Oporto metropolitan area, ‘‘Broa de Avintes’’ is
a variation, with half maize and half rye constituents (Duarte et al.,
2010).
The worldwide contamination of foods and feeds with myco-
toxins poses a significant health problem. Ochratoxin A (OTA),
chemically known as N-{[(3R)-5-chloro-8-hydroxy-3-methyl-
1-oxo-7-isochromanyl]-carbonyl}-3-phenyl-L-alanine, is a myco-
toxin mainly produced by Penicillium verrucosum and some Asper-
gillus species (e.g. A. ochraceus, A. carbonarius and A. niger) (Abarca,
Accensi, Bragulat, Castella, & Cabañes, 2003). The International
⇑ Corresponding author. Tel.: +351 239859994; fax: +351 239827126.
E-mail address: clino@ff.uc.pt (C.M. Lino).
a possible carcinogenic compound to humans (IARC, 1993).
OTA occurs predominantly in cereal grains, cereal products, co-
coa, spices, oilseeds, coffee beans and legumes. However, cereal
products are the major group of food commodities where the toxin
is of greatest impact. The frequency of food contamination with
OTA represents an important source of OTA daily intake (Pena,
Cerejo, Lino, & Silveira, 2005). The maximum levels (MLs) fixed by
European regulations for OTA in cereals and cereal products are 5
and 3 lg kg1 wet weight, respectively (EC, 2006b; FAO, 2002).
Recently, Anastassiades, Lehotay, Stajnbaher, and Schenck
(2003) developed an original analytical methodology combining
the extraction/isolation of pesticides from food matrices and ex-
tract cleanup. This technique involves micro-scale extraction with
acetonitrile coupled to a clean-up based on a dispersive solid-
phase extraction (d-SPE). Since the development of the ‘‘Quick,
Easy, Cheap, Effective, Rugged and Safe’’ (QuEChERS) method, it
has been gaining significant popularity. It is the method of choice
for food analysis because it combines several steps and extends
the range of pesticides recovered over older and more tedious
extraction techniques. Recent studies showed that QuEChERS can
be successfully used for extraction of other analytes besides pesti-
cides. Such studies include analysis of polycyclic aromatic hydro-
carbons (PAHs) in fish (Ramalhosa, Paíga, Morais, Delerue-Matos,
& Oliveira, 2009), acrylamide in food (Mastovska & Lehotay,
2006), veterinary drugs in animal tissue (Stubbings & Bigwood,
2009), and in milk (Keegan et al., 2009), as well as in other matrices
besides foods (Wilkowska & Biziuk, 2011).
Analysis of mycotoxins using QuEChERS extraction is starting to
appear in the literature. Extraction of Fusarium toxins in cereals

0308-8146/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodchem.2012.06.045
 P. Paíga et al. / Food Chemistry 135 (2012) 2522–2528
and cereal based products (Zachariasova et al., 2010), zearalenone
in barley (Wu, Zhao, Chen, & Yang, 2011), aflatoxins and OTA in
dried figs. (Broszat, Welle, Wojnowski, Ernst, & Spangenberg,
2010), 27 mycotoxins and other secondary metabolites in maize si-
lage (Rasmussen, Storm, Rasmussen, Smedsgaard, & Nielsen, 2010),
deoxynivalenol, nivalenol, zearalenone, actyldeoxynivalenol, deep-
oxy-deoxynivalenol, fusarenon-X, altenuene, alternariol, alternari-
olmethylether, diacetoxyscirpenol, sterigmatocystin in wheat and
maize (Vaclavik, Zachariasova, Hrbek, & Hajslova, 2010) and type
A- and B-trichothecenes in wheat flour samples (Sospedra, Blesa,
Soriano, & Mañes, 2010) has already been described.
The state-of-the-art regarding the analysis of ochratoxin A in
food is presented by Duarte, Silva, Lino, and Pena (2011), who con-
clude that the majority of the reported methods generally involve
extraction with mixtures of polar solvents followed by a clean-up
procedure that usually employs solid-phase extraction (SPE), such
as immunoaffinity columns (IACs), that present high specificity.
Detection and quantification are traditionally performed by liquid
chromatography with fluorescence (FLD) or mass spectrometry
(MS) detection (Duarte et al., 2011).
In the current study, the possibility to apply QuEChERS extrac-
tion for OTA determination in bread samples was investigated. This
is a very convenient extraction technique that due to its simplicity
and rapidity is being applied in the analysis of complex matrices
and replacing traditional extraction methods. A factorial design
was used to examine the effect on extraction efficiency of the QuE-
ChERS conditions (x1 – extraction time, x2 – extraction solvent vol-
ume and x3 – sample mass). The method was applied to 20 bread
samples commercially available in Oporto metropolitan area (NW
Portugal).
2. Experimental
2.1. Reagents, solvents and materials
Acetonitrile, methanol and n-hexane were acquired from Merck
(Darmstadt, Germany), acetone (purity P99.8%) and glacial acetic
acid (purity P99.7%) from Carlo Erba (Rodano, Italy). OTA (P98%
purity) was purchased from Sigma Chemical Co. (St. Louis, MO,
USA).
The water used was previously purified (15.0 MX cm) using an
Elix apparatus (Millipore, Molsheim, France) and subsequently a
Simplicity 185 system (18.2 MX cm) (Millipore, Molsheim,
France). All chromatographic solvents were filtered through a
0.20 lm Supelco membrane filter (Ø 47 mm, Bellefonte, PA, USA)
under vacuum and degassed for 15 min in an ultrasonic bath (Ray-
paÒ Trade, Terrassa, Spain).
All solutions and samples extracts were filtered through an
OlimPeak syringe filter, PTFE, 0.20 lm, from Teknokroma (Ø
13 mm, Barcelona, Spain). For homogenisation a vortex mixer Nah-
ita 681/5 (Navarra, Spain) was used.
A standard stock solution was prepared by diluting OTA with
95% acetonitrile and 5% water solution pH 2.60 (adjusted with ace-
tic acid) at 250 lg mL1, and stored at 20 °C. For calibration
curves, six standard solutions (0.5–10.0 lg L1) were also prepared
in 95% acetonitrile and 5% water solution pH 2.60. Amber glass-
ware was used to prevent light deterioration of the mycotoxin.
Five different types of QuEChERS and one clean-up column were
tested. Their corresponding content in the 50 mL Teflon centrifuge
tubes were: QuEChERS A (6 g of magnesium sulphate and 1.5 g of
anhydrous sodium acetate); QuEChERS B (4 g of magnesium sul-
phate and 1 g of sodium chloride); QuEChERS C (6 g of magnesium
sulphate, 1.5 g of sodium chloride, 1.5 g of sodium citrate dihy-
drate and 0.750 g of sodium citrate sesquihydrate), all supplied
by Unit Chemical Technologies, Inc. (Bristol, PA, USA); the clean-
2523
up column D contained 1.5 g of magnesium sulphate and 0.5 g of
CEC18 and was supplied by Unit Chemical Technologies, Inc. (Bris-
tol, PA, USA); QuEChERS E (4 g of magnesium sulphate, 1 g of so-
dium chloride, 1 g of trisodium citrate dihydrate and 0.5 g of
disodium hydrogencitrate sesquihydrate), supplied by Restek RES-
PREP (Bellefonte, PA, USA) and QuEChERS F (4 g of magnesium sul-
phate, 1 g sodium chloride, 1 g of sodium citrate and 0.5 g of
sodium hydrogencitrate sesquihydrate), supplied by Agilent Tech-
nologies (Santa Clara, PA, USA).
2.2. Bread samples collection
Fresh bread samples of wheat bread ‘‘Carcaça’’, maize bread
‘‘Broa de Milho’’, and half maize and half rye bread ‘‘Broa de Avin-
tes’’, were purchased from different local markets and supermar-
kets in Oporto metropolitan region. Each bread sample was
mechanically homogenised with a blender (Brio 400 WMAX, Ufesa,
Spain). The samples were divided accordingly to Commission
Directive (EC) No. 401/2006/EC (EC, 2006a) and a subsample of
200 g was collected in a plastic bag and kept at 20 °C until anal-
ysis. Moisture was determined in the fresh bread samples using a
MLS moisture analyzer from Kern (Balingen, Germany).
2.3. Liquid chromatography
OTA analysis was carried out by LC using a Shimadzu system
(Shimadzu Corporation, Kyoto, Japan) equipped with a LC 20AB
prominence pump, a DGU-20A5 prominence degasser, a SIL 20A
prominence autosampler and a RF-10AXL fluorescence detector.
An Atlantis column (dC18, 5 lm, 2.1  150 mm, Waters, Milford,
MA, USA) was used and maintained at room temperature. The fluo-
rescence intensity was measured at the following optimised exci-
tation/emission wavelength pair: 333/460 nm. LCsolution
software version 2.1 (Shimadzu Corporation, Kyoto, Japan) was
used for control and data processing. The injection volume was
15 lL. The mobile phase consisted of 40% of acetonitrile and 60%
water solution pH 2.60, adjusted with acetic acid, at a flow rate
of 0.5 mL min1. Using the described experimental conditions,
OTA retention time was 11.53 min (OTA standard solution,
RSD = 0.18%, n = 10).
2.4. Extraction procedure
An aliquot (5.0 g) of homogenised bread sample was placed into
a 50 mL Teflon tube (QuEChERS). For recovery studies, samples
were fortified at three levels, 3.0 (I), 2.0 (II), and 1.0 (III) lg kg1
wet weight. The spiking volume was 300 lL in all cases. The
concentrations of the OTA standard solutions used in the spiking
procedure were 50.0, 33.3 and 16.7 lg L1, respectively. For non-
spiked samples 300 lL of the solvent mixture (95% acetonitrile
and 5% water solution pH 2.60) were added. Fortified and non-
spiked samples were allowed to stand for 30 min before extraction,
protected from light. Then, the tested extraction solvent (acetoni-
trile, acetonitrile:methanol (1:1, v/v), methanol, methanol:water
(95:5, v/v), n-hexane:acetone (1:1, v/v)) and volume (13.5–
16.5 mL) were added to the selected QuEChERS (A; B; C; E; F).
The QuEChERS tubes were shaken vigorously during a tested time
(2–4 min). After centrifugation in a 2.16 Sartorius centrifuge (Sig-
ma, Goettingen, Germany), for 10 min at 2575g, the solvent layer
was filtered through a 0.20 lm syringe filter. A 2.0 mL aliquot
was transferred to an amber vial, evaporated with a gentle stream
of nitrogen to dryness and redissolved with 200 lL 95% acetoni-
trile–5% water solution (pH 2.60). The extract was placed in an
autosampler amber vial for LC–FLD analysis.
2524 P. Paíga et al. / Food Chemistry 135 (2012) 2522–2528
2.5. Strategy for optimization of QuEChERS extraction
To find the optimum conditions for OTA extraction in bread
samples, the experimental design as a function of the selected
main factors had to be determined. The assays were organised in
a 23 factorial design (Box & Hunter, 1957). The levels chosen for
each process variable were based on preliminary studies (see Sec-
tions 2.4 and 3.1.2). Three variables with two levels (high (+) and
low ()) were studied: extraction time (x1: 1, +1 (min)), extrac-
tion solvent volume (x2: 1, +1 (mL)) and sample mass (x3: 1,
+1 (g)).
The OTA recovery after QuEChERS extraction from bread spiked
samples at 3.0 lg kg1 wet weight was taken as the dependent var-
iable or response of the design experiments. There are seven de-
grees of freedom between the eight treatment combinations in
the 23 design. Three degrees of freedom are associated with the
main effects x1, x2 and x3. Four degrees of freedom are associated
with interactions, being x1x2, x1x3, x2x3 and x1x2x3 (Montgomery,
2001).
The analysis of variance (ANOVA) was used for determination of
the significant factors affecting OTA recovery using STATISTICA
program version 6.0 (Statsoft, USA) which equation was deter-
mined by multiple regression analysis (Montgomery, 2001).
In most response surface methodology (RSM) problems, the
form of the relationship between the response (y) and the indepen-
dent variables (xi) is unknown. Thus, the first step is to find a suit-
able approximation for the true functional relationship between
the response and the set of independent variables, usually applying
a second-order model, where bo represents the global mean, bi the
other regression coefficients and e the error of the model
(Montgomery, 2001):
X
k X
k XX
y ¼ b0 þ bi xi þ bii x2i þ    þ bij xi xj þ e
i¼1 i¼1 ii <j j was tested in order to improve the highest results of OTA recovery
Pareto chart, normal probability and response surface plots
were constructed from the OTA recovery results after application
of 23 factorial design.
The quality of the polynomial model equations was judged sta-
tistically by the lack of fit, coefficient of determination, R2, and its
statistical significance was determined by the F-test, within a level
of confidence of 95% and p values <0.05. Pure Error of the model
was determined by central point duplications.
After optimization of QuEChERS extraction using the
3.0 lg kg1 wet weight level and RSM, the overall QuEChERS-LC
procedure for analysis of OTA in bread samples was validated by
systematic recovery experiments at two other fortification levels
(2.0 and 1.0 lg kg1 wet weight).
3. Results and discussion
3.1. QuEChERS extraction
3.1.1. Preliminary tests
OTA recovery can be affected by several factors, including QuE-
ChERS composition, extraction solvent, volume and time, sample
mass, contamination level and bread type. The preliminary tests
were carried out with ‘‘Carcaça’’ samples (wheat bread), the most
consumed type of bread in Portugal (INE, 2008).
The original QuEChERS procedure consists of extracting a cer-
tain amount of homogenised sample by hand-shake or vortex dur-
ing 1 min with the same amount of solvent (acetonitrile; 1 mL final
extract/g sample). A preliminary study was performed testing dif-
ferent ratios of mass of sample per volume of solvent, ranging from
1 to 0.33. In order to maximise sensitivity and to obtain a suitable
mixture/dispersion and OTA extraction, 15 mL of solvent were se-
lected as a preliminary volume to treat 5 g of sample. In a previous
study, concerning the QuEChERS extraction of PAHs (Ramalhosa
et al., 2009), it was concluded that vortex allowed an efficient mix-
ture of the QuEChERS content with the sample and solvent, with an
optimum extraction time of 3 min. For this reason, shaking was
performed by vortex, and an extraction time of 3 min was chosen
for the preliminary studies.
Acetonitrile is the extraction solvent of preference in the QuE-
ChERS methodology because extracts contain fewer interfering
substances than the corresponding ethyl acetate and acetone ex-
tracts, and acetonitrile can be separated fairly easily from water
by the salting out effect (Wilkowska & Biziuk, 2011). Moreover,
acetonitrile, methanol, methanol–acetonitrile mixtures and aque-
ous solutions of acetonitrile or methanol were tested as extraction
solvents for other mycotoxins (type A- and B-trichothecenes) from
wheat flour using QuEChERS (Sospedra et al., 2010). Additionally,
the mixture n-hexane:acetone (1:1, v/v) has proven to be an effi-
cient solvent system for the extraction of different pollutants from
environmental samples (U.S. Environmental Protection Agency,
2000). In this study, for the selection of the adequate extraction
solvent and QuEChERS composition, extraction efficiency was
firstly evaluated testing five solvents (acetonitrile, acetoni-
trile:methanol (1:1, v/v), methanol, methanol:water (95:5, v/v),
and n-hexane:acetone (1:1, v/v)) and three different QuEChERS
types using spiked bread samples. The results are presented in
Fig. 1. The highest recoveries were obtained, by descending order,
with QuEChERS C, B and A. Concerning QuEChERS A, quantitative
extraction was not attained with any of the extraction solvents
and they were not further considered. Using QuEChERS B, the best
recoveries were reached using methanol mixtures (86.0 ± 2.5% and
67.6 ± 3.2%, for acetonitrile:methanol (1:1, v/v) and metha-
nol:water (95:5, v/v), respectively) and methanol (75.9 ± 2.5%).
ð1Þ Clean-up suggested by the supplier (Unit Chemical Technologies)
in QuEChERS B. No improvement was observed in the recovery val-
ues (81.1%, 62.5% and 66.9%, for acetonitrile–methanol (1:1, v/v),
methanol and methanol:water (95:5, v/v), respectively). Thus, the
approach of using a clean-up step after extraction was discarded.
Acetonitrile (96.7 ± 1.3%), acetonitrile:methanol (1:1, v/v)
(88.1 ± 3.3%) and n-hexane:acetone (1:1, v/v) (87.2 ± 2.0%) pro-
vided the overall highest average recoveries with QuEChERS C.
Excellent quantitative extraction efficiencies were clearly attained
with QuEChERS C and acetonitrile. These results were obtained
without the inclusion of a clean-up step, thus minimising sample
preparation. Taking all these observations in consideration, aceto-
nitrile was chosen as the extraction solvent to be used with QuE-
ChERS C.
In order to evaluate different suppliers, two types of QuEChERS
with a composition similar to QuEChERS C were tested, namely,
QuEChERS E (Restek) and F (Agilent) (Section 2.1). No significant
differences were observed in the chromatograms or in the recovery
values obtained with acetonitrile as the extraction solvent
(96.4 ± 2.0% and 96.3 ± 1.4%, for QuEChERS E and F, respectively).
Therefore, the tested commercial QuEChERS composed of magne-
sium sulphate, sodium chloride, sodium citrate dihydrate and so-
dium citrate sesquihydrate (4:1:1:0.5, w/w/w/w) are adequate
for OTA extraction from bread samples, according to the minimum
performance criteria established by EC (2006a). QuEChERS C were
used in the subsequent essays.
3.1.2. Optimization studies
Experiments were organised in a typical 23 factorial design,
with three process variables (x1 – extraction time, x2 – extraction
solvent volume and x3 – sample mass) evaluated at two levels
(high and low) for a total of eight independent experiments
(Table 1).
 P. Paíga et al. / Food Chemistry 135 (2012) 2522–2528 2525

Fig. 1. Recoveries of OTA (n P 3) from ‘‘Carcaça’’ spiked bread sample (5 g fortified at 3.0 lg kg1 wet weight) using QuEChERS extraction with different QuEChERS
compositions (A, B and C defined in Section 2) and 15 mL of extraction solvent (ACN – acetonitrile; MeOH – methanol; Hex – n-hexane; Ace – acetone).

Table 1
Real values and coded levels for the experimental design 23 (x1 – extraction time (min), x2 – extraction solvent volume (mL) and x3 – sample mass (g)), and results for OTA
experimental (y1 observed, %) and model predicted (y1 predicted, %) recovery from ‘‘Carcaça’’ spiked bread sample at 3.0 lg kg1 wet weight.

Experiment x1 (min) x2 (mL) x3 (g) y1 (%) Observed y1 (%) Predicted

1 2 () 13.5 () 4.5 () 95.89 95.90
2 2 () 13.5 () 5.5 (+) 94.06 94.57
3 2 () 16.5 (+) 4.5 () 94.77 95.29
4 2 () 16.5 (+) 5.5 (+) 94.32 94.28
5 4 (+) 13.5 () 4.5 () 94.74 95.27
6 4 (+) 13.5 () 5.5 (+) 93.52 93.50
7 4 (+) 16.5 (+) 4.5 () 95.68 95.68
8 4 (+) 16.5 (+) 5.5 (+) 93.75 94.23
9 (CP) 3 (0) 15.0 (0) 5.0 (0) 96.34 94.84
10 (CP) 3 (0) 15.0 (0) 5.0 (0) 96.59 94.84

CP, central point.

The preliminary tests allowed defining the values of the vari- extraction was performed (Daniel, 1959) and an unreplicated fac-
ables at the central point (5 g of sample, 15 mL of acetonitrile torial design was used. The experimental error of the method
and 3 min of agitation). Subsequently, the increments in the vari- was derived from duplicate experiments carried out at the central
ables (x2 – extraction solvent volume and x3 – sample mass) were point of the selected variables (3.0 min of extraction time, 15.0 mL
studied in attempt to achieve the maximum mass and solvent vol- of extraction solvent volume and 5.0 g of ‘‘Carcaça’’ bread sample).
ume inside the Teflon QuEChERS tube. Firstly, increments of Results of OTA recovery from spiked bread sample are shown in
2.5 mL, in solvent volume, and 1 g, in sample mass, were assessed. Table 1.
For these increments the solvent volume ranged from 10 to 20 mL Regression coefficients for the applied 23 design are shown in
(1.682 and +1.682 levels) and the mass ranged from 3 to 7 g Table 2. Substituting the coefficients in Eq. (1):
(1.682 and +1.682 levels), respectively. A solvent volume of
Y ¼ 114:60  1:62x1  1:04x2  2:38x3 þ 0:17x1 x2
10 mL was not sufficient to promote the adequate mixture be-
tween the sample and solvent with the QuEChERS content. Simi-  0:22x1 x3 þ 0:11x2 x3 ð2Þ
larly, the use of a sample mass of 7 g inside the QuEChERS tube
did not allow enough space to make the correct mixture with the
solvent and the QuEChERS content. Table 2
Increments of 1.5 mL and 0.5 g were then studied. The solvent Analysis of variance (ANOVA) of the regression model for OTA recovery (y1, %) from
‘‘Carcaça’’ spiked bread sample at 3.0 lg kg1 wet weight.
volume ranged from 12 to 18 mL (1.682 and +1.682 levels) and
the mass ranged from 4 to 6 g (1.682 and +1.682 levels), respec- Response Source SS DF MS F-value p
tively. An adequate mixture was observed between the sample and OTA, y1 (%) Model 9.54 4 2.39 9.60 0.014483
solvent with the QuEChERS content for all the sample mass/solvent Residual 1.24 5 0.25
volume combinations and these were used to investigate the ef- Lack of fit 1.21 4 0.30 9.69 0.235835
Pure error 0.031 1 0.031 76.34 0.085604
fects of the variables in the OTA QuEChERS extraction.
Total 10.78 9
The results obtained in preliminary tests showed that QuE- R2 0.8847
ChERS extraction of OTA was highly reproducible. Therefore, to
SS, Sum of squares; DF, Degree of freedom; MS, Mean square; R2, quadratic corre-
simplify the optimization study only one experiment for each
lation coefficient.
2526 P. Paíga et al. / Food Chemistry 135 (2012) 2522–2528
The model reached statistical significance (p < 0.05) and the lack
of fit was not significant (p > 0.05) as desired. The model presented
a quadratic correlation coefficient of 0.8847, fitting the statistical
model quite well.
An attempt of applying a face-centred central composite design
was made. Six additional experiments were carried out at the low-
est (1.682) and the highest (+1.682) levels. However, no differ-
ence in OTA recovery was observed and the obtained values
ranged from 95.2 (x1 = 1.0 min, x2 = 15.0 mL and x3 = 5.0 g) to
96.8% (x1 = 3.0 min, x2 = 15.0 mL and x3 = 6.0 g). Thus, the model
quadratic correlation coefficient decreased significantly
(R2 = 0.1103) and so, standard design (Box & Hunter, 1957) without
model expansion was chosen.
For determination of the significant factors affecting OTA recov-
ery an analysis of variance (ANOVA) was performed. Main (x1, x2
and x3) and interaction effects (x1x2, x1x3 and x2x3), were not statis-
tically significant for p < 0.05 or p < 0.1. The values of these esti-
mated effects on OTA recovery (for p value < 0.05 for a 95%
confidence level) were: 0.33215 for x1 (extraction time, min),
0.07627 for x2 (solvent volume, mL), 1.33600 for x3 (sample mass,
g), 0.49946 for x1x2, 0.21405 for x1x3 and 0.16485 for x2x3.
According to the analysis of variance and the Pareto charts (not
shown) all effects were discarded, because they did not exhibit
any statistical significance.
The distribution of the residual values, defined as the difference
between the predicted (model) and the observed (experimental)
ones were examined. Normal probability plot (not shown) indi-
cated a close relation between the values predicted by the model
and the experimental ones (Table 1).
Based on the high statistical quadratic correlation coefficient
(R2 = 0.8847; Table 2), the mathematical model was used to gener-
ate the response surface plots (Fig. 2) and to calculate the condi-
tions for maximum OTA recovery from bread samples. According
to the model, these conditions are likely to provide the highest re-
sponse. The optimum conditions were: x1 = 3.0 min, x2 = 15.0 mL
and x3 = 5.0 g (central point). Five experiments were carried out
using these conditions and the average response (y1, OTA recovery)
was 96.6 ± 1.0%. Hence, there was a considerable agreement be-
tween the experimental and the predicted values and this con-
firmed that the 23 factorial design was suitable to predict the
course of QuEChERS extraction of OTA and high reproducibility
was attained. The results show that the method is quite robust
as changes in the extraction time, solvent volume and sample mass
have little effect on OTA recovery.
The accuracy of the method was also evaluated by analysing
OTA in ‘‘Carcaça’’ bread sample spiked at two additional fortifica-
Fig. 2. Three-dimensional response surface showing OTA recovery from ‘‘Carcaça’’ spiked bread sample at 3.0 lg kg1 wet weight as a function of: (a) extraction time (x1) and
solvent volume (x2) (sample mass (x3) = 5.0 g), (b) extraction time (x1) and sample mass (x3) (solvent volume (x2) = 15.0 mL) and (c) solvent volume (x2) and sample mass (x3)
(extraction time (x1) = 3 min).
tion levels, level II (2.0 lg kg1 wet weight) and level III
(1.0 lg kg1 wet weight) using the optimum extraction conditions.
Level I corresponds to the maximum level fixed by European regu-
lation for OTA in cereal products (EC, 2006b) and levels II and III
were chosen to be below that level. The extraction efficiency was
nearly constant for all fortifications levels with average recoveries
for fortification levels II (96.1 ± 2.0%) and III (94.8 ± 1.6%) similar to
the one obtained for level I (96.6 ± 1.0%).
In order to assess the adequacy of the optimised methodol-
ogy, samples of different bread types fortified at 3.0 lg kg1
wet weight were studied. The types chosen are the most com-
monly consumed in Portugal, namely, ‘‘Broa de Milho’’ (maize
bread) and ‘‘Broa de Avintes’’ (half maize and half rye bread).
OTA recoveries were 96.0 ± 1.2% and 94.3 ± 2.0% for ‘‘Broa de
Milho’’ and ‘‘Broa de Avintes’’, respectively. No significant differ-
ences were observed in OTA recovery for the three types of
bread and therefore QuEChERS extraction may be considered a
suitable technique for OTA extraction from the different types
of bread that were studied.
Representative chromatograms of naturally contaminated sam-
ples are presented in Fig. 3. When analysing the chromatograms of
the last two types of bread (maize and maize/rye bread), these
showed, in some cases, more matrix interference than from wheat
bread. Therefore quantification by standard addition was used for
all the types of bread. Nevertheless, the matrix effect for wheat
bread was almost neglectable as the results obtained by external
standard and standard addition were very similar (slope values:
40,896 (external standard in solvent) and 40,930 (standard addi-
tion to a wheat sample)).
The detection (LOD) and quantification (LOQ) limits were 0.02
and 0.05 lg kg1 wet weight, respectively, in the bread samples.
They were calculated according to Miller and Miller (1989) from
the standard addition calibration curve of an unfortified wheat
bread sample. Considering the EC maximum level of 3 lg kg1
wet weight for OTA, the LOQ attained permits the use of the de-
scribed method for monitoring purposes.
The reported results are better than the ones presented in pre-
vious studies concerning OTA extraction from bread samples using
immunoaffinity columns, which in spite of providing very high
recoveries (80.4–102.0%) (Juan et al., 2007), are considered a more
expensive technique. As already reported for pesticides, the use of
QuEChERS extraction for OTA is also clearly advantageous over
other extraction techniques involving intensive treatments since
accurate results can be achieved with minimal sample preparation
in a short time. This method is suitable for laboratories engaged in
routine analysis of a large number of samples.
 P. Paíga et al. / Food Chemistry 135 (2012) 2522–2528
Fig. 3. Overlay chromatograms of OTA standard solution (10 lg L1) and naturally contaminated bread sample extracts: (a) ‘‘Broa de Avintes’’ (2.24 ± 0.03 lg kg1), (b) ’’Broa
de Milho’’ (1.32 ± 0.05 lg kg1) and (c) ‘‘Carcaça’’ (0.06 ± 0.006 lg kg1).
Table 3
Moisture contents and OTA contamination of the analysed bread samples.
Bread type Moisture (%)
Range Mean ± SD
‘‘Broa de Avintes’’ (Half maize half 32.6–41.1 36.1 ± 3.1
rye bread)
‘‘Broa de Milho’’ (maize bread) 27.6–43.1 34.6 ± 6.4
‘‘Carcaça’’ (wheat bread) 21.8–31.6 27.3 ± 3.2
*
Contaminated samples.
3.2. Application to bread samples
The proposed QuEChERS method for OTA extraction followed by
LC–FLD was applied to 20 bread samples (‘‘Carcaça’’, ‘‘Broa de Mil-
ho’’, and ‘‘Broa de Avintes’’). Representative chromatograms of an
OTA standard solution (10 lg L1) and bread samples of ‘‘Carcaça’’,
‘‘Broa de Milho’’ and ‘‘Broa de Avintes’’ are presented in Fig. 3. Ta-
ble 3 shows the results of samples’ moisture contents and OTA con-
centrations. Differences concerning moisture contents were
detected between bread types that contain maize (‘‘Broa de Avin-
tes’’ and ‘‘Broa de Milho’’) and those having wheat in their compo-
sition (‘‘Carcaça’’). Average moisture contents ranged from 21.8%
(‘‘Carcaça’’) to 43.1% (‘‘Broa de Milho’’).
OTA was detected in 13 of the 20 samples in levels below the
established European legal limit for cereal based products
(3 lg kg1 wet weight; EC, 2006a). In the contaminated bread sam-
ples, OTA concentration ranged from 0.05 to 1.93 lg kg1 wet
weight. The repeatability expressed as the relative standard devia-
tion (n = 3) ranged between 1.02% and 10.1%, with a mean value of
5.1% for all the 20 samples analysed.
As regards the different types of bread considered in this study,
‘‘Broa de Avintes’’ presents a higher percentage of contamination
(100% of the samples showed values P the LOQ, 0.05 lg kg1) with
OTA levels in the range 0.05–1.93 lg kg1 wet weight and an aver-
age contamination of 0.89 lg kg1 wet weight. Samples of ‘‘Broa de
milho’’ showed OTA levels between n.d. – 1.01 lg kg1 wet weight
and contamination of 75% of the samples. The OTA mean value for
the contaminated samples was 0.42 lg kg1 wet weight. Finally,
the least contaminated bread samples were ‘‘Carcaça’’ (wheat
bread) (25% of contaminated samples). OTA concentrations ranged
2527
OTA
Frequency of contamination (%) Range (lg kg1 wet weight) Mean ± SD (lg kg1 wet weight)*
8/8 (100%) 0.05–1.93 0.89 ± 0.74
3=
4 (75%) n.d. – 1.01 0.42 ± 0.51
2/8 (25%) n.d. – 0.12 0.12 ± 0.00
between n.d. – 0.12 lg kg1 wet weight, with an OTA mean value,
for the contaminated samples, of 0.12 lg kg1 wet weight. These
results are in line with the ones presented in previous studies.
Duarte et al. (2010) concluded that a widespread low level of
OTA contamination was observed in all Portuguese regions and
types of bread products analysed, especially in the Oporto and
Coimbra regions, and in the maize and whole-grain or fibre-
enriched bread.
4. Conclusions
This paper describes a simple, rapid, inexpensive and effective
procedure for quantification of OTA in bread samples by QuEChERS
and LC–FLD. Good accuracy and precision were obtained using an
extraction time of 3.0 min, 15.0 mL of acetonitrile, 5.0 g of bread
sample and QuEChERS containing magnesium sulphate, sodium
chloride, sodium citrate dihydrate and sodium citrate sesquihy-
drate (4:1:1:0.5, w/w/w/w).
The method was applied to different types of bread very con-
sumed in Portugal and none of the samples exceeded the legal
European limit of 3 lg kg1 wet weight for cereal based products.
However, maize-based breads seem to present higher OTA levels.
The results of this study show that QuEChERS methodology is
clearly useful beyond pesticide analysis, particularly in food
matrices.
Acknowledgements
The authors are grateful to FCT-Fundação para a Ciência e
Tecnologia, for supporting this work through the Project
2528 P. Paíga et al. / Food Chemistry 135 (2012) 2522–2528
[PTDC/AGR-ALI/65528/2006] and to Unit Chemical Technologies,
Inc., Resprep and Agilent Technologies for the QuEChERS supplied.
The authors thank Ana M.M. Sousa (REQUIMTE, Faculty of Engi-
neering, University of Porto) for her precious help in the data sta-
tistical analysis.
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