Food Control 60 (2016) 196e204

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Food Control
journal homepage: www.elsevier.com/locate/foodcont

Occurrence of Alternaria toxins in food products in The Netherlands

 pez a, *, Dini Venema a, Theo de Rijk a, Andre
Patricia Lo  de Kok b, Jos M. Scholten b,
a a
Hans G.J. Mol , Monique de Nijs
a
RIKILT e Wageningen UR, Akkermaalsbos 2, 6708 WB, Wageningen, The Netherlands
b
NVWA e Netherlands Food and Consumer Product Safety Authority, Chemistry Laboratory, Akkermaalsbos 4, 6708 WB, Wageningen, The Netherlands

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

Article history: Alternaria toxins are mycotoxins that can contaminate cereals, oilseeds and various fruits and vegetables
Received 2 March 2015 such as apples, tomatoes, citrus fruits and olives. The fungi can grow at low temperatures, thus causing
Received in revised form spoilage even during transport and storage. Currently, there are relatively little occurrence data on
20 July 2015
Alternaria toxins in food products and in the Netherlands most data are limited to the presence of
Accepted 25 July 2015
Available online 29 July 2015
alternariol (AOH) and alternariol methyl ether (AME) in cereals. Tenuazonic acid (TeA), tentoxin (TEN)
and altenuene (ALT) have been recently identified by the Standing Committee on the food Chain and
animal health as Alternaria toxins of concern. A survey (95 samples) was conducted in the Netherlands in
Keywords:
Liquid chromatography tandem mass-
2013. The aim was to screen the levels of the five Alternaria toxins in wine (n ¼ 5), fresh apples (n ¼ 11),
spectrometry apple juices (n ¼ 7), fresh tomatoes (n ¼ 19), tomato sauces (n ¼ 8), fresh citrus (n ¼ 11), dried figs
Mycotoxins (n ¼ 5), olives (n ¼ 10), sunflower seeds (n ¼ 5) and cereals (n ¼ 14). Multi-mycotoxin methods for the
Alternaria toxins analysis of mycotoxins were adapted for this purpose. Their performance characteristics were assessed
Occurrence and were as follows: recoveries and precision ranged from 85 to 112% and from 4 to 20%, respectively.
Processed food LOQs were between 1.5 and 5.0 mg kg1. In the subsequent survey, AOH, AME, TeA, and TEN were
detected in one or more food commodities, while ALT was not detected in any of the samples. TeA was
found in 27% of the samples and at relatively high concentrations in sunflower seeds, tomato sauces and
dried figs (up to 2345 mg kg1). Alternaria toxins occurred regularly in cereals, tomato sauces, figs, wine
and sunflower seeds. Only incidental occurrence of the Alternaria toxins was observed in fresh apples,
fresh citrus fruits, fresh tomatoes and olives.
© 2015 Elsevier Ltd. All rights reserved.

1. Introduction
Alternaria species are pathogenic and saprophytic fungi widely
distributed in soil. They are widespread in both humid and semi-
arid regions and can infect growing plants in the field. They are
the principal contaminating fungi in wheat, sorghum and barley. In
addition to cereal crops, Alternaria species have been reported to
occur in oilseeds such as sunflower and rapeseed, tomato, apples,
citrus fruits, olives and several other fruits and vegetables. Alter-
naria species grow at low temperature; hence they are generally
associated with extensive spoilage during refrigerated transport
and storage (Ostry, 2008).
Alternaria species can produce around 70 toxic secondary me-
tabolites. The German Federal Institute of Risk Assessment (BfR)
* Corresponding author.
 pez).
E-mail address: patricia.lopezsanchez@wur.nl (P. Lo
already pointed out in 2013 that there was an urgent need for more
information about the toxicity of Alternaria toxins, because data
published until then did not allow assessment of the health risks for
the consumer (BfR, 2003). A scientific opinion in 2007 of the Czech
Scientific Committee on Food on Alternaria mycotoxins led to the
same conclusion (SCF, 2008). The EFSA CONTAM panel published
their opinion on the risks for animal and public health related to the
presence of Alternaria toxins in feed and food in 2011 (EFSA, 2011).
The EFSA risk assessment was inconclusive due to limited repre-
sentative occurrence and toxicity data. In reply to this, the Standing
Committee on the food chain and animal health identified the
following Alternaria toxins to be of relevance: alternariol (AOH),
alternariol monomethyl ether (AME), tenuazonic acid (TeA), ten-
toxin (TEN) and altenuene (ALT). EFSA also recommended their
monitoring in national surveys. Other Alternaria toxins such as
altertoxins (ATX) and Alternaria alternate f sp lycopersici toxins (AAL
toxin) can also be analysed but were considered of less relevance by
EFSA (EFSA, 2011).

http://dx.doi.org/10.1016/j.foodcont.2015.07.032
0956-7135/© 2015 Elsevier Ltd. All rights reserved.
 pez et al. / Food Control 60 (2016) 196e204
P. Lo
The CONTAM panel applied the threshold of toxicological
concern (TTC) approach on Alternaria toxins, indicating that the
Alternaria toxins are suspected mutagenic-carcinogens. TeA has
been proven to be toxic to several animal species, e.g. mice, chicken,
dogs (Ostry, 2008). The acute toxicity of AOH, AME, ALT and TEN is
low, although there are several reports on the mutagenic and
genotoxic activities of AOH and AME: AOH and AME are teratogenic
and fetotoxic; altertoxin I is cytotoxic and mutagenic; TeA and AME
cause precancerous changes in the esophageal mucosa of mice;
AOH and AME also seem to be mutagenic and act as antagonists to
topoisomerase; furthermore, AME provokes DNA strand breaks
(EFSA, 2011). AOH can be very rapidly absorbed by the human in-
testinal lumen (Ostry, 2008). Recently, altertoxin II has been
demonstrated to be much a stronger mutagen and DNA strand
breaking mycotoxin than AOH and AME in cultured mammalian
cells (Fleck, Burkhardt, Pfeiffer, & Metzler, 2012).
The methods for the determination of Alternaria toxins in food
and feed have been reviewed by Scott (Scott, 2001) and more
recently by Ostry (Ostry, 2008), although most of the reported
literature only refers to AOH and AME. Organic solvents as such or
in mixtures are commonly used to extract Alternaria toxins from
food commodities, while an acidic extraction solvent is preferable
for TeA. Purification and concentration procedures, when appli-
cable, usually include solvent partitioning, solid phase extraction
(SPE) or solid phase microextraction (SPME) (Ostry, 2008). In 2004
Solfrizzo et al. developed a method to determine Alternaria toxins
in carrots based on an acidified extraction with water-
emethanoleacetonitrile. They split the filtered extract into two for
further purification by solid-phase extraction on a C18 column for
the analysis of altertoxin-I (ATX-I), AOH and AME, and on a poly-
meric Oasis® HLB column for the analysis of TeA. The analysis was
performed by HPLC-photodiode array (PDA) (Solfrizzo, De
Girolamo, Vitti, Visconti, & van den Bulk, 2004).
Due to its sensitivity, selectivity and specificity, liquid
chromatography-tandem mass spectrometry (LC-MS/MS) is
currently the most applied method of analysis for mycotoxins. The
new generation of analytical instruments allows the use of the
“dilute-and-shoot” approach to analyse multiple mycotoxins in one
run without prior clean-up and thus reducing analysis time. AOH
and AME were the first Alternaria toxins to be included in multi-
mycotoxin methods. More recently other Alternaria toxins are be-
ing integrated in these methods (Varga et al., 2013). Dedicated
methods for the analysis of the relevant Alternaria toxins have been
recently developed and applied to tomato products (Noser,
Schneider, Rother, & Schmutz, 2011) and to cereals (Walravens
et al., 2014). Due to its poor chromatographic behaviour, the limit
of detection of TeA is higher than that for the other Alternaria
toxins. To improve the chromatographic behaviour of TeA, its
derivatization with 2,4-dinitrophenylhydrazine (Siegel, Rasenko,
Koch, & Nehis, 2009) was performed and shown to achieve
20e30 times lower limits of detection. The use of atmospheric
pressure chemical ionization (APCI) enhanced the signal of TeA at
least a factor of three compared to electrospray ionization (ESI)
(Prelle, Spadaro, Garibaldi, & Gullino, 2013).
Most of the occurrence studies on Alternaria toxins (EFSA, 2011)
reported high concentrations of toxins on mouldy foods, which are
not appropriate for human consumption and hence these data were
not suitable for risk assessment. Due to the lack of statistically
robust figures for risk assessment, EFSA launched a call in October
2010 to compile occurrence data on Alternaria toxins in food and
feed. The vast majority of reported data were on grains products,
very limited on fruits and vegetables and none on wines or feed.
Moreover, only scarce information about the fate of Alternaria
toxins during processing was available. Hence, EFSA concluded that
more occurrence data in food, feed and processed food/feed are
197
required to refine the exposure assessment (EFSA, 2011).
This work aims to provide more occurrence data of the selected
Alternaria toxins in the food commodities recommended by EFSA.
Two different in-house multi-mycotoxin methods devoted for the
determination of mycotoxins in feed/food by LC-ESI-MS/MS were
slightly modified for the simultaneous detection of the AOH, AME,
TEN, ALT and TeA. The performance characteristics of both methods
were assessed and they were applied for a market survey of ten
food commodities in the Netherlands, which also included pro-
cessed food.
2. Material and methods
2.1. Chemicals and reagents
Acetonitrile and methanol, both UPLC gradient grade, were
purchased from Actu-all Chemicals (Oss, the Netherlands), formic
acid (98e100%) from Merck (Amsterdam, the Netherlands), and
ammonium formate (99%) from Across Organics (Geel, Belgium).
MilliQ water (>18 MU) was used. AOH and AME were purchased
from Biosolve (Greyhound Chromatography and Allied Chemicals,
Merseyside, UK). TeA, TEN and ALT were supplied by Sigma-
eAldrich (Zwijndrecht, the Netherlands). All standards had a purity
higher than 99%.
Individual stock solutions were prepared as follows: ALT at
1000 mg mL1 and TeA at 100 mg mL1 in methanol, AOH, AME and
TEN at 100 mg mL1 in acetonitrile. An intermediate solution
mixture containing the Alternaria mycotoxins at 1 mg mL1
(5 mg mL1 for TeA) was prepared in the extraction solvent
(acetonitrile/water/formic acid, 84/16/1, v/v/v). The working solu-
tions to obtain calibration curves, to determine matrix effect (ion
suppression/enhancement), recoveries and limits of quantification
for each mycotoxin were prepared in extraction solvent and in
blank matrix extract.
2.2. Samples
A total of 95 samples from retail stores in the Netherlands
classified in 10 different food categories were analysed: 14 cereal
products, 27 tomato products (19 fresh tomatoes and 8 processed
tomatoes), 5 sunflower seeds, 36 fruits (11 citrus, 11 apples, 10 ol-
ives and 5 figs), 7 apple juices and 5 red wines. Cereals, citrus, fresh
apples and tomatoes and wines were collected in the frame of the
regular Dutch 2013 survey programme, while the other 35 samples
were purchased from retails shops in the Netherlands and stored at
the recommended temperature until processing.
Wine samples were homogenised and stored at 20  C. Olives,
apples, citrus and tomatoes were cryogenically milled, homoge-
nized and stored at 20  C. Figs, sunflower seeds and cereals were
homogenized, milled with water to form a slurry and stored
at 20  C. The slurry for figs, for sunflower seeds and for cereal
grains contained water in a ratio of 1/1.5, 1/1.5 and 1/3, respectively.
2.3. Method 1: cereal samples
Cereal grain samples were processed as follows: 12.5 g of the
slurry were mixed with 10 mL of acetonitrile acidified at 1% with
acetic acid and shaken manually for 1 min. Then, 4.5 g of magne-
sium sulphate were added to induce phase separation. The mixture
was shaken for 1 min and centrifuged at 3000 rpm for 3 min.
Finally, 0.5 mL of the supernatant were diluted with 0.5 mL of
methanol in an autosampler vial. The extracts were analysed by
injection of 2 mL of extract into an Acquity UPLC coupled with a
Xevo-TQ-S mass spectrometer from Waters (Waters, Hertfordshire,
UK).
198 pez et al. / Food Control 60 (2016) 196e204
P. Lo

The separation was carried out using a Waters Acquity HSS-T3, into a Waters Atlantis HSS-T3, (3 mm), 3.0  100 mm column. The
(1.8 mm), 2.1  100 mm column, set at 40  C. Mobile phase A con- chromatographic conditions were as follows: column temperature
sisted of water, and mobile phase B of methanol, both with 5 mM 35  C and mobile phases consisting of water as eluent A and 4% (v/
ammonium formate and 0.1% formic acid. The flow rate was set at v) water in methanol as eluent B. Both mobile phases contained
0.4 mL min1. The gradient elution was applied as follows: after an 1 mM of ammonium formate and 1% of formic acid. The flow rate
initial hold time of 1 min at 90% eluent A, 90% eluent B was reached was set at 0.4 mL min1. A gradient elution was applied as follows:
within 6.5 min and kept for 2 min. After 0.01 min the gradient was after an initial hold time of 1 min at 100% eluent A, 50% eluent B was
changed to the initial conditions: 90% eluent A 10% eluent B. This reached within 2 min and 100% eluent B within the next 2.5 min.
composition was maintained for 0.5 min for column re- This composition was kept for 6 min, after which 100% eluent A was
equilibration. reached within the next 0.5 min and kept for 3.5 min for column re-
Table 1a shows the optimum settings for the transitions used to equilibration.
determine the analytes under study. Other settings for the MS were The precursor and product ion selection and the optimization of
as follows: source and desolvation temperature were 150  C and the fragmentation voltages and the collision energies were con-
500  C, respectively; cone gas flow and desolvation gas flow were ducted with infusion of single-analyte solutions (Table 1b). Opti-
150 and 1000 L/h, respectively; capillary voltage in ESI þ was set at mization was conducted in both ionisation modes: positive and
1.0 kV and at 1.0 kV in ESI- mode. negative. The analysis of the samples was carried out using elec-
trospray ionisation (ESI) and multiple reaction monitoring (MRM).
2.4. Method 2: other food matrices For each Alternaria toxin, at least two mass transitions were
measured for identifications, which is in agreement with SANCO
All samples, with the exception of cereal grains, were processed, Document 12571/2013 (SANCO, 2013). The general source settings
as follows: 2.5 g of sample were spiked, when needed, and then were as follows: source temperature 400  C, curtain gas 30 psi, ion
extracted with 10 mL of acetonitrile/water/formic acid (84/16/1 v/v/ source gas 50 psi, ion spray voltage 4000 V (negative) or þ4000 V
v). The mixture was shaken head-over-head for one hour and then (positive), entrance potential 10 V (negative) or þ10 V (positive).
centrifuged at 3000 rpm for 10 min. An aliquot of 0.5 mL of the Table 1b summarizes the obtained retention times, the ionisation
supernatant was taken and filtered using a mini-uniprep PTFE filter mode, the dwell time (DW), declustering potential (DP), collision
vial. The vial containing the extract was ready for analysis. energy (CE), collision cell exit potential (CXP) and the precursor and
The LC-MS/MS instrument was a Shimadzu ‘Prominence’ HPLC product ions for the five Alternaria toxins.
system, which consisted of a DGU-20A3 degasser, a SIL 20AC XR
autosampler, two LC-20 AD XR UFLC pumps, and a CTO-20AC col- 2.5. Sample analysis
umn oven. This system was coupled to an AB SCIEX QTRAP® 5500
mass spectrometer. The quantification of the Alternaria toxins was based on external
HPLC separation was performed by injecting 5 mL of the extract calibration using matrix-matched standards (MMS) when possible.
The MMS were prepared by dilution of standard solution in blank
matrix extract. Blank matrix extracts for each type of matrix under
Table 1 study (tomato, citrus, apple, fig, olive, wine, sunflower seed, apple
Retention times, the ionisation mode, the dwell time (DW), declustering potential
juices, tomato sauce and cereals) were first assessed to be free of
(DP) or cone voltage, collision energy (CE), collision cell exit potential (CXP) and the
precursor and product ions for the five Alternaria toxins. any detectable amount of the target mycotoxins. The concentration
levels of the calibration standards used for method 1 (cereals) were
a) Method 1: cereals
0.25, 0.5, 0.1, 2.5 and 5 ng mL1. The concentration levels of the
a
Mycotoxin ESI mode RT (min) Transition Cone voltage (V) CE (V) calibration standards for method 2 (other matrices) were 0, 1, 2.5,
ALT Positive 5.5 293.1 > 257.2 22 14 10, 25, 50 and 100 ng mL1 for AOH, AME, ALT and TEN and 0, 5,
293.1 > 239.1 20 12.5, 50, 125, 250 and 500 ng mL1 for TeA. When blank matrix
293.1 > 229.2 20 extracts were not available, as happened for TeA in figs and sun-
TeA Positive 5.6 198.1 > 125.1 40 16
flower seeds, and for AOH, AME and TeA in tomato sauces, cali-
198.1 > 139.0 14
198.1 > 153.0 12 bration by standard addition was conducted. For method 2,
AOH Negative 6.4 257.0 > 213.0 78 22 calibration curves from standards in solvent were also prepared.
257.0 > 147.1 32 The Alternaria toxins were identified by similarity of retention
257.0 > 185.0 26 times and ion ratios between the sample extract and the standard
TEN Positive 6.6 415.2 > 312.2 45 18
415.2 > 302.2 12
in matrix. The criteria used to ensure the correct identification of
415.2 > 132.1 38 the target mycotoxins were: (a) the retention times of the analyte in
AME Negative 7.4 271.0 > 228.0 62 30 the sample extract matched those of the matrix-matched standards
271.0 > 183.0 40 within a deviation of ±0.2 min; (b) the deviation of the ion ratio of
271.0 > 213.7 38
the sample extract fell within ±30% of the ion ratio for the matrix-
b) Method 2: other matrices matched standards (SANCO, 2013).
Mycotoxin ESI mode RT (min) aTransition DW (ms) DP (V) CE (V) CXP (V)

ALT Positive 6.3 293.1 > 257.0 10 56 20 10

2.6. Method validation
293.1 > 239.1 10 60 30 10
TeA Negative 6.6 196.0 > 112.0 50 35 20 11 Method 1 and method 2 were assessed in terms of linearity,
196.0 > 139.0 50 35 20 11 matrix effects, LOQ, recovery and precision.
AOH Negative 6.8 257.0 > 215.0 10 5 30 15
Linearity of calibration curves was assessed for standards in
257.0 > 213.0 10 5 26 15
TEN Negative 6.8 413.0 > 141.0 10 60 26 11 matrix extracts of cereals within the range of 0.25e20 ng mL1; and
413.0 > 271.0 10 60 22 17 for fresh citrus, fresh apple, fresh tomato, dried fig, wine, sunflower
AME Negative 7.4 271.0 > 256.0 10 90 32 13 seed, olive, tomato sauce and apple juice, within the range of
271.0 > 227.0 10 90 50 9 1e100 ng mL1 for AOH, AME, ALT and TEN and 5e500 ng mL1 for
a
Quantification transition in bold. TeA. The linearity requirements were fulfilled when the correlation
 pez et al. / Food Control 60 (2016) 196e204
P. Lo
coefficient was greater than 0.99.
Matrix effects were evaluated by comparison of the slopes of
calibration curves from standards in solvent with those from
standards in matrix extract, at 95% confidence interval. For method
1, matrix effects from the MS measurements were determined by
comparison of the signals obtained from standards of 0.5 ng mL1
prepared in solvent and in matrix. For method 2, matrix effects
were assessed by comparison of the calibration slopes obtained
from standards prepared in solvent and from standards prepared in
matrix.
For method 1, recovery and precision expressed as repeatability
(RSDr) were calculated on the day of measurements. Cereals
(wheat) blank samples were spiked with TEN at 2 mg kg1 and with
ALT, AME and AOH at 20 mg kg1. For method 2, the combined re-
covery for all matrices and the within-lab reproducibility were
calculated at spiking levels of 10 mg kg1 for ALT, AME, AOH and TEN
and at 50 mg kg1 for TeA, based on the values obtained in each
series of measurements, which were carried out in 3 different days.
The method was considered compliant when both parameters
were in agreement with the values set by the European Regulation
519/2014 (EU., 2014) for the mycotoxins patulin and zearalenone at
concentrations close to the concentrations used in the present
work.
LOQ was defined as the concentration in a spiked sample, for
which the signal-to-noise (S/N) ratio of the most intense transition
(quantifier) was equal to ten, providing that the compound was
detectable, which means that the S/N ratio of the least intense
transition (qualifier) was equal to three. LOQs were estimated in
spiked samples at low levels.
Table 2 shows the performance parameters as regards recovery,
precision and LOQ for the different Alternaria toxins in the two
methods.
3. Results and dicussion
3.1. Chromatographic determination of TeA
Tenuazonic acid (pKa ¼ 3.5) is seldom included in multi-
mycotoxin analytical methods for the detection of mycotoxins
based on liquid chromatography-tandem mass spectrometry,
mainly due to its poor chromatographic behaviour. The methods
used in the past to determine TeA were based on HPLC with UV
detection (da Motta & Soares, 2001; Terminiello, Patriarca, Pose, &
Fernandez Pinto, 2006). As TeA is a strong acid and a metal
chelating compound, it shows irreproducible chromatographic
behaviour that is remedied by adding additives, e.g., Zn2SO4, to the
mobile phase (Stefan Asam, Liu, Konitzer, & Rychlik, 2011). Unfor-
tunately, this approach cannot be used when mass spectrometric
detection is applied. The derivation of TeA with 4-
dinitrophenylhydrazine (DNPH) (Siegel et al., 2009) and the use
Table 2
Limit of quantification (LOQ), precision and recovery data of the different methods
applied to determine Alternaria toxins.
ALT AME AOH
Method 1. Cereals LOQ (mg kg ) 1
2.0 2.0 2.0
Recovery (%) 89 96 85
RSDr (%) 12 11 8 4
Method 2. Other matrices LOQ (mg kg1)
b
1.5 1.0 2.0
Recovery (%) 111 101 112
b
RSDR (%) 18 20 20
a
n.a.-not available.
b
Measured in 3 days: day 1 (n ¼ 3, fig, apple juice and tomato sauce), day 2 (n ¼ 4,
apple juice, tomato sauce, fresh tomato and wine), day 3 (n ¼ 5 fresh apple, fresh
citrus, sunflower seeds, dried figs and olives).
199
of stable isotope dilution assay (SIDA) with the addition of
[13C,15N]-TeA as internal standard (Stefan Asam et al., 2011) have
significantly improved the analysis and allowed the determination
of TeA in flour, bakery products and beer (Stefan Asam, Habler, &
Rychlik, 2013; Stefan Asam & Rychlik, 2013; Siegel, Merkel, Koch,
& Nehls, 2010). The main drawback of these methods, as opposed
to the dilute-and-shoot methods, is that they do not allow the
simultaneous determination of the other four Alternaria toxins,
they are more time-consuming and more laborious.
Method 1, which was used to monitor Alternaria toxins in ce-
reals, was an existing multi-mycotoxin method used for monitoring
and control purposes of food that already included AOH and AME.
For the purpose of this survey, the method was extended with the
Alternaria toxins ALT, TEN and TeA. The method proved to be suit-
able for ALT and TEN, but not for TeA, due to poor chromatographic
behaviour under the applied conditions (mobile phases containing
0.1% of formic acid). Hence, TeA was not included in the cereals
survey data.
Method 2, which was developed to determine Alternaria toxins
in other matrices, was based on an existing multi-mycotoxin
method for feed. The chromatographic method was modified for
the simultaneous detection of all five Alternaria toxins. First ex-
periments were carried out with the extraction mixture acetoni-
trile/water/formic acid (84/16/1 v/v/v) on a Restek (Interscience
B.V, Breda, the Netherlands) Ultra aqueous C18 (3 mm)
2.1  100 mm column with 1 mM ammonium bicarbonate in water/
methanol (95:5) as mobile phase A and methanol as mobile phase
B. These mobile phases had shown good peak shapes and repro-
ducible chromatography for TeA in plasma (Fraeyman et al., 2013).
However, in the present study, it resulted in acceptable chroma-
tography for TeA dissolved in extraction solvent, but not when the
TeA standard was prepared in matrix, as shown in Fig. 1a. Appar-
ently, the bicarbonate buffer (pKa 10.3) of the mobile phase could
not handle the low pH of the extraction solvent (pH around 2).
Therefore, the peak obtained from the standard in solvent corre-
sponded to the neutral form, while the peak obtained from the
standard in matrix corresponded to the charged form. Secondly,
mobile phases with 0.1% of acetic acid were tested with the same
analytical column. This resulted in broader than desired peak
shapes for TeA, although no displacement of peaks was observed, as
shown the Supplementary Information (Figure SS1a). Thirdly, the
Waters Atlantis HSS-T3 column, which provided paramount
retention for polar compounds and good peak shape across a wide
range of pH, was tested using mobile phases with 1% formic acid.
This chromatographic system provided the best peak shape for TeA
in both solvent and in matrix (Fig. 1B). The same column was tested
with mobile phases containing 0.1% of acetic acid, with less
acceptable results (Supplementary Information, Figure SS1b).
3.2. Method performance characteristics
The method performance characteristics linearity, matrix ef-
fects, LOQ, recovery and precision were assessed for both methods.
Overall average recovery, precision and LOQ data for all matrices
are summarized in Table 2.
TEN TeA Calibrations were linear in those ranges with a few exceptions:
2.0 a
n.a. 1e50 ng mL1 for ALT in fried figs, for AOH and AME in apple juices,
96 n.a. fresh tomatoes and figs and for TEN in fresh apples, olives, sun-
n.a. flower seeds, apple juices and tomato juices; 1e25 ng mL1 for TEN
2.5 5.0
in dried figs and wine and for ALT in wine; 5e250 ng mL1 for TeA
104 110
18 10 in dried figs and 5e125 ng mL1 for TeA in wine.
Matrix effects result from co-eluting matrix components in the
extract and may affect the ionization efficiency of target analytes by
suppression or enhancement of the instrumental signal. Matrix
effects were evaluated by comparison of the slopes of calibration
200 pez et al. / Food Control 60 (2016) 196e204
P. Lo
Fig. 1. Optimization of the chromatographic conditions for TeA. A) Restek Ultra Aqueous C18, (3 mm), 2.1  100 mm column. Eluent A: 1 mM ammonium bicarbonate in water/
methanol (95:5), eluent B: methanol. B) Waters Atlantis HSS-T3, (1.8 mm), 2.1  100 mm column. Eluent A: water with 5 mM ammonium formate and 0.1% formic acid, eluent B:
methanol with 5 mM ammonium formate and 0.1% formic acid.
curves from standards in solvent with those from standards in
matrix extract, at 95% confidence interval. The results showed
(Supplementary Information Table SS1) that the matrix effect
depended on the combination of matrix and toxin. AOH was the
toxin most susceptible to the presence of matrix, especially in acidic
matrices such as fresh citrus (84% suppression) and tomato sauces
(70% suppression). These acidic matrices also influenced, at lower
levels, the ionization of the other Alternaria toxins. On the other
hand, TeA was the least affected by the presence of matrix, and only
significant matrix effect was observed in citrus (suppression), to-
mato sauces, olives and sunflower seeds (enhancement). Suppres-
sion of the signal was particularly significant for TEN, ALT and AOH
in acidic matrices, such as tomato sauces, wines, citrus and fresh
tomatoes, whereas enhancement was observed in sunflower seed
and cereal matrices for ALT and TEN.
Recoveries ranged from 85 to 96% and RSDr from 4 to 12%. As
observed for method 2, recoveries and within-lab reproducibility,
expressed as RSDR (%), ranged from 102 to 110% and from 11 to 20%,
respectively. Recoveries and precision values were compliant with
those set by Regulation 519/2014 (EU., 2014).
3.3. Occurrence of Alternaria toxins
The results from the survey are summarized in Table 3. The
concentrations of the five Alternaria toxins in each individual
sample are shown in Supplementary Information Table SS2.
ALT was not detected in any sample, while AME was detected in
5% of the samples (50% of tomato sauces and one wheat sample),
AOH in 7% of the samples (5 samples tomato juices, and in one
sample of wine, fresh apple and cereals) and TEN in 16% of the
samples, mainly in cereals. TeA was the most widely occurring toxin
and was quantified in all dried figs, sunflower seed and tomato
juice samples and in three samples of wine and one sample of
olives.
Alternaria toxins were not detected in any of the fresh citrus or
apple juice samples. One sample of fresh apple was positive for
AOH (29 mg kg1). None of the five Alternaria toxins under study
were detected in a recent survey performed in Italy in 2013. The
Italian apple juices hardly contained any of the mycotoxins: TeA
occurred in two samples out of 10 at 24.3 and 45.3 mg kg1 and ALT
in one sample at 45.6 mg kg1 (Prelle et al., 2013). Alternaria toxins
are usually produced when so-called Alternaria brown spots
appear; hence being associated to the decay of the products. The
results reported in the present study suggested that the collected
fresh products were not mouldy and, as a consequence, were
appropriate for consumption. There are not many data available on
the occurrence of Alternaria toxins in non-mouldy citrus fruits
(EFSA, 2011). In case of mouldy citrus, AOH and AME have been
proven to accumulate in flavedo tissues (the outer layer) and not in
the albedo tissues (the white spongy part of the citrus), therefore
the removal of the flavedo before consumption would decrease the
risk of mycotoxin intake (Magnani, De Souza, & Rodrigues, 2007).
AOH and AME were detected in apple juices from Canada (Lau et al.,
2003) and Germany (S. Asam, Konitzer, Schieberle, & Rychlik, 2009)
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Table 3
Results of the survey in food products in the Netherlands: concentration (mg kg1) of Alternaria toxins.

C (mg kg1) ALT AME AOH TEN TeA

Fresh apples (N ¼ 11)

# positive/range 0 (<1.5) 0 (<1.0) 1 (<2.0e29) 0 (<2.5) 0 (<5.0)
Olives (N ¼ 10)
# positive/range 0 (<1.5) 0 (<1.0) 0 (<2.0) 0 (<2.5) 1 (<5.0e5.3)
Dried figs (N ¼ 5)
# positive/range 0 (<1.5) 0 (<1.0) 0 (<2.0) 0 (<2.5) 5 (25e2345)
a
Average e e e e 1043
a
Median e e e e 160
Sunflower seeds (N ¼ 5)
# positive/range 0 (<1.5) 0 (<1.0) 0 (<2.0) 1 (<2.5e5.0) 5 (85e449)
Average e e e e 223
Median e e e e 160
Fresh citrus (N ¼ 11)
# positive/range 0 (<1.5) 0 (<1.0) 0 (<2.0) 0 (<2.5) 0 (<5.0)
Fresh tomatoes (N ¼ 19)
# positive/range 0 (<1.5) 0 (<1.0) 0 (<2.0) 0 (<2.5) 0 (<5.0)
Apple juices (N ¼ 7)
# positive/range 0 (<1.5) 0 (<1.0) 0 (<2.0) 0 (<2.5) 0 (<5.0)
Wines (N ¼ 5)
# positive/range 0 (<1.5) 0 (<1.0) 1 (<2.0e11) 0 (<2.5) 3 (<5.0e46)
Average e e e e 25
Median e e e e 17
Tomato sauces (N ¼ 8)
# positive/range 0 (<1.5) 4 (<1.0e7.8) 4 (<2.0e25) 0 (<2.5) 8 (66e462)
Average e 3.8 16 e 202
Median e 3.0 17 e 143
Cereals (N ¼ 14)
# positive/range 0 (<1.4) 1 (<2.0e3.0) 1 (<2.0e5.2) 14 (2.0e14) Not in scope
Average e e e 6.0
Median e e e 5.1
Summary N ¼ 95 (except for TeA N ¼ 81)
%positives 0% 5% 7% 15% 22%
Minimum e 1.2 5.2 2.5 5.3
Maximum e 7.8 29 14 2345
a
Average and median of positive samples.

using SPE clean-up and LC-MS/MS analysis, at levels below the
LOQs reported in the present study. The levels of AOH and AME in
apple juices from Spain, which were analysed using the same
methodology as Canadians, were higher and ranged from 1.35 to
5.42 mg kg1 for AOH and 1.71 mg kg1 for AME (Delgado & Gomez-
Cordoves, 1998). Inmunochemical methods were applied in a sur-
vey performed in 2011 in Germany, which revealed that AOH
occurred in 20% of the apple juice samples at levels ranging from 1.7
to 3.5 mg kg1 (Ackermann et al., 2011).
TeA was detected in one olive sample at low level (5.3 mg kg1).
In literature, AOH, AME, ALT and TeA were reported at 2,900, 2,300,
1,400, and 260 mg kg1 respectively in severely damaged olive
samples collected in Italy (Visconti, Logrieco, & Bottalico, 1986). The
fungus A. alternate is often reported to contaminate olives, partic-
ularly if the fruits remain on the soil for a long time after ripening.
Physical surface damage of the olive fruit due to unfavourable
conditions, e.g. low temperature and insect damage, etc., is a major
precondition for fungal penetration into the fruit pulp and for
subsequent mycelial proliferation and mycotoxin production
(Logrieco, Moretti, & Solfrizzo, 2009).
The highest concentrations for TeA in the here presented study
were detected in dried figs samples, which confirmed the data
reported to EFSA by other authors (EFSA, 2011). Cyclopiazonic acid,
fumonisin B1, ochratoxin A and aflatoxins have been already re-
ported to naturally co-occur in dried figs. Fumonisin B1 was found
in higher amounts than the other mycotoxins (Heperkan, Guler, &
Oktay, 2012). Scarce literature is available on occurrence of Alter-
naria toxins, and more specifically TeA, in dried figs. This study has
demonstrated that TeA could be present at considerable high levels
in this fruit. However, more occurrence data are necessary to
properly evaluate the exposure of consumers to TeA through the
consumption of dried figs.
TeA was detected in all five samples of sunflower seeds. The
concentration of TeA varied between 85 and 449 mg kg1. The data
collected by EFSA evidenced that the highest concentration of AOH,
AME, TeA and TEN in the group of “Legumes, nuts and oilseeds”
corresponded to sunflower seeds (EFSA, 2011), which is not in
agreement with the findings of this survey since all sunflower seed
samples were negative for AOH and AME. TEN co-occurred with
TeA (308 mg kg1) in one sample, but in much lower amount
(5.2 mg kg1). The results on contamination of sunflower seeds from
this survey are lower than the reported concentrations in other
surveys performed in Argentina (AOH 792 mg kg1, AME
836 mg kg1, and TeA 31,600 mg kg1), in Italy (AOH 1840 mg kg1
and AME 129 mg kg1) (Scott, 2001) and in UK (AOH up to
570 mg kg1, AME up to 270 mg kg1 and TeA up to 5600 mg kg1)
(Nawaz, Scudamore, & Rainbird, 1997). Although the levels of
Alternaria toxins have been proved to decrease during the pro-
cessing of sunflower seeds to oil (Chulze et al., 1995), sunflower
seeds are commonly eaten with minimal processing. Therefore it is
important for consumer health to analyse whole sunflower seeds.
Unlike other studies (Ackermann et al., 2011; Noser et al., 2011;
Van de Perre et al., 2014), no Alternaria toxins were found in fresh
tomatoes. In contrast, TeA was the prevalent toxin in all tomato
sauces and processed tomato juices, ranging from 66 to
402 mg kg1, while AOH and AME occurred in half of these samples
at much lower levels (Fig. 2). Alternaria species have been observed
to grow best at moderate temperatures with an optimal tempera-
ture of 25  C for the production of AOH and AME, and of 21  C for
the production of TeA (Pose, Patriarca, Kyanko, Pardo, & Pinto,
202 pez et al. / Food Control 60 (2016) 196e204
P. Lo

Fig. 2. Occurrence of AOH, AME and TeA in one sample of tomato sauce.

2010). Therefore, the presence of Alternaria toxins in tomato
products might be indicative of the use of decaying tomatoes by the
processing plants. The highest value of TeA occurred for a sample of
concentrated juice (462 mg kg1), followed by an organic ketchup
sample (404 mg kg1), whereas the highest amount of AOH
(25 mg kg1) and AME (7.8 mg kg1) were detected in the concen-
trated juice and in an organic puree sample, respectively. TeA, AOH
and AME were observed to co-occur in four samples. The occur-
rence of Alternaria toxins in this study seems not to be related to
organic/non-organic production, although a more thorough
research with a much higher and representative number of samples
would be needed to confirm this fact. The findings presented here
for processed tomato sauces and juices are comparable with pre-
vious published data by other authors. In the survey performed by
Van de Perre in 2014, AOH and AME were found in tomato con-
centrates and purees, but not in more diluted samples like ketchup
or juices/soups (Van de Perre et al., 2014). In contrast, AOH and AME
were detected in around 15% of ketchups and 30% of juices from the
Swiss market (Noser et al., 2011) and in 100% of the ketchup
samples from Germany (Ackermann et al., 2011). The occurrence
data in the Netherlands are in agreement with these studies carried
out in Europe, but are much lower than those reported in Argentina
for tomato puree samples (Terminiello et al., 2006). The
quantification of these mycotoxins in tomato products might be
considered as an indicator of the quality of the used raw material.
TeA was also found in three out of the five samples of wine at
levels ranging 11e46 mg kg1, whereas AOH was only found at
11 mg kg1 in one wine sample, bottled in 2011. Although TeA has
been already reported to occur in Belgian beers at an average level
of 11 mg kg1, with Bock beers showing the highest average con-
centration (37 mg kg1) (Siegel et al., 2010), no literature on the
occurrence of TeA in wine samples could be found. TeA was neither
found in wine from Canada, which might be due to the high LOQ
(70 mg kg1) of the analytical method applied in that study (Ostry,
2008). AOH and AME were detected in wines (national and im-
ported) in Canada at levels ranging 0.03e19.4 mg kg1 and
0.01e0.23 mg kg1, respectively, but not in wines from Czech Re-
public (Ostry, 2008). The levels found for AME in the Canadian
wines were below the LOQ of the method presented here.
TEN was found in all surveyed cereals samples at levels ranging
2.5e14 mg kg1. It only co-occurred with AOH (5.2 mg kg1) and
AME (3.0 mg kg1) in one wheat sample (Fig. 3). The occurrence data
and the proportion of positive samples for AME and AOH are in
agreement with the data collected by EFSA for wheat grains (EFSA,
2011). However, to the best of our knowledge, this is the first time
that such a high frequency of positive samples for TEN in wheat

Fig. 3. Occurrence of AOH, AME and TEN in one wheat sample.

 pez et al. / Food Control 60 (2016) 196e204
P. Lo
intended for food has been reported. TEN had been already found at
levels ranging 0.7e29 mg kg1 in most of wheat silage feed in Israel
(Shimshoni et al., 2013), hence the lack of TEN occurrence data
might be due to the fact that this mycotoxin was not included in the
scope of most of the surveys. A survey in Czech Republic in
2003e2005 showed the presence of AOH and ALT in cereals at
levels ranging 6.3e44 mg kg1 and 6.3e41 mg kg1, respectively
(Ostry, 2008). The average levels found in 64 cereal samples in
Argentina for AOH (645e1385 mg kg1), AME (566e7451 mg kg1)
and TeA (1000e8814 mg kg1) were much higher because weather-
damaged grains were also included (Azcarate, Patriarca,
Terminiello, & Pinto, 2008). Unlike other mycotoxins, the pres-
ence of Alternaria toxins in cereals was largely ignored both in
Europe and overseas and was omitted in national monitoring
programmes. Recently, a retrospective study on the occurrence of
Alternaria toxins in wheat over 10 years was carried out in north-
east Germany. The results revealed that 30.3% of the cereal samples
were naturally contaminated with TeA at a maximum concentra-
tion of 4224 mg kg1, 8.1% with AOH at a maximum concentration of
832 mg kg1, 3.1% with AME at a maximum concentration of
905 mg kg1 and 2.6% with ALT at a maximum concentration of
197 mg/kg. TEN was not considered in the scope of the survey
(Muller & Korn, 2013). TeA was not monitored in the wheat samples
from this survey study since its analysis requires not only strong
acid extraction conditions, but also low pH of mobile phase to
displace the equilibrium of TeA to the acid form. Nevertheless, and
taken into account other surveys, the scope of the method pre-
sented here for cereals must be extended to incorporate TeA.
4. Conclusions
The data from this survey showed that tenuazonic acid, which is
seldom included in multi-mycotoxin methods, is the prevalent
Alternaria mycotoxin in processed tomato products, dried figs,
sunflower seeds and wines and can occur at high levels, up to a
maximum of 2345 mg kg1. The presence and concentration levels
of these mycotoxins in processed tomato products might be
considered as an indicator of the quality of the raw material used.
Tentoxin was detected in all cereals samples at levels ranging from
2.5 to 14 mg kg1. A more extended survey, focussing on those
products with high occurrence of Alternaria toxins (sunflower
seeds, dried figs and tomato products for TeA and cereal and cereal-
based products for TEN), is required to increase insight into their
occurrence in these food products and the subsequent exposure of
consumers to Alternaria toxins.
Multi-mycotoxin methods are validated and applied in national
monitoring programmes and for exposure assessment purposes.
These studies, as far as Alternaria toxins are concerned, are often
limited to the determination of the occurrence of AOH and AME.
The results provided by this study indicate that the scope of multi-
mycotoxin methods must be extended to include TeA and TEN.
Acknowledgements
The authors gratefully acknowledge Alwin Kruijt and Jacques
Boonstra (NVWA) for their contribution to analyse the cereal
samples.
This study was carried out in WOT project-02-001-061 ‘Method
development and surveys on mycotoxins in food’ at RIKILT Wage-
ningen UR, on behalf of and funded by the Netherlands Ministry of
Economic Affairs.
Appendix A. Supplementary information
Supplementary information related to this article can be found
203
at http://dx.doi.org/10.1016/j.foodcont.2015.07.032.
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