Food Control 54 (2015) 74e78

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

Aflatoxins in animal feeds: A straightforward and cost-effective

analytical method

F. Huertas-Pe
Natalia Arroyo-Manzanares, Jose
rez, Ana M. García-Campan
~ a,

miz-Gracia *
Laura Ga
Department of Analytical Chemistry, Faculty of Sciences, University of Granada, Campus Fuentenueva s/n, E-18071 Granada, Spain

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

Article history: In this paper, a procedure for the determination of aflatoxin B1, B2, G1 and G2 in five different animal
Received 11 September 2014 feedstuffs, intended to feed different mammalians and fowls, has been developed and validated. The
Received in revised form method is based on a very simple solideliquid extraction with acetonitrile and subsequent analysis by
12 January 2015
high performance liquid chromatography coupled with fluorescence detection with post-column
Accepted 20 January 2015
Available online 29 January 2015
photochemical derivatization. The study carried out to optimize the extraction step, showed that us-
ing acetonitrile as extraction solvent provided not only satisfactory recoveries, but also extracts clean
enough to omit a further clean up step. The method has been fully validated on five different matrices,
Keywords:
Aflatoxins
and limits of quantification were below the allowed or recommended levels by European Union. Re-
Animal feed covery studies were carried out at three different concentration levels, with values ranging from 81 to
HPLC 105%; repeatability and intermediate precision showed relative standard deviations lower than 10% in all
Post-column photochemical derivatization cases.
Solideliquid extraction © 2015 Elsevier Ltd. All rights reserved.

1. Introduction In this context, fungi contamination and mycotoxin production

Over the past decades, animal feed has undergone a significant
development in whole Europe together with the livestock sector.
Specially, intensive livestock farming has involved a growth of an-
imal feed demand. From an economic point of view, feeding of
food-producing animals comprises the main production cost of
farms. For these reasons, animal nutritional needs have been
carefully studied in order to optimize production of livestock spe-
cies. Animal feed is the first link of food chain; therefore the risk of
contaminant carryover from contaminated feeds to animal tissues
and biological fluids, and eventually to products intended for hu-
man consumption (meat, milk and eggs) is a matter of concern. The
production of safe foods depends on the manufacturer compliance
with current legislation on one hand (The European Parliament,
2003; The European Parliament, 2005), and the use of safe feeds
by the farmers on the other hand. Therefore, special attention
should be paid to both, feed composition and contamination risk
while storing.
* Corresponding author. Tel.: þ34 958 248594; fax: þ34 958 243328.

miz-Gracia).
E-mail address: lgamiz@ugr.es (L. Ga
during pre- or post-harvest activities as well as storing are high risk
agents for the final consumer. Mycotoxins are secondary metabo-
lites produced primarily by fungi Aspergillus, Penicillium and Fusa-
rium, and they are considered the most dangerous contaminants of
cereals and animal feeds. Among other effects, mycotoxins produce
nutrients absorption decrease, decrease of animal productivity,
damage to vital organs, immunosuppression, changes on repro-
ductive ability and even death (Streit et al., 2012). Aflatoxins are
mycotoxins produced by molds of the Apergillus genera (especially
Aspergillus flavus, niger and parasiticus). They have been thoroughly
studied and have proved to be highly carcinogenic. Specifically,
aflatoxin B1 (AFB1) is already included as carcinogenic to humans by
the International Agency for Research on Cancer (IARC, 2013). Thus
maximum residue limits have been established for AFB1 in products
intended for animal feeding by Directive 2002/32/EC (European
Parliament, 2002) and the amended 2003/100/EC (The
Commission of the European Communities, 2003), as well as
guidance values for other mycotoxins by the recommendation
2006/576/EC (The Commission of the European Communities,
2006a). Furthermore, according to recent studies, mycotoxin
contamination of animal feeds is a frequent issue around whole
Europe, sometimes even above allowed or recommended levels
(Streit et al., 2012). Moreover, the last annual report of RASFF (The

http://dx.doi.org/10.1016/j.foodcont.2015.01.027
0956-7135/© 2015 Elsevier Ltd. All rights reserved.
 N. Arroyo-Manzanares et al. / Food Control 54 (2015) 74e78 75

Rapid Alert System for Food and Feed, European Commission, 2013)
stated that the second reason for notifying on feed was mycotoxin
contamination. Taking into account all these facts, accurate
methods are needed to guarantee feedstuff quality and to safeguard
animal and eventually consumer health. On the other hand, easier
analytical methodologies would enable an effective and smooth
transfer to routine laboratories.
Regarding aflatoxin determination on animal feeds, most of the
available methods are based on solideliquid extraction and clean
up with immunoaffinity columns (IACs) for sample treatment, fol-
lowed by high performance liquid chromatography (HPLC) with
fluorescence detection (FLD), including the method recommended
by the International Standard Organization (Beheshti & Asadi,
2014; ISO, 2006; Muscarella et al., 2009; Stroka, Von Holst,
Anklam, & Reutter, 2003).
Nevertheless, IACs are expensive, not recyclable and have a
limited storage time. Thus, solid phase extraction (SPE) using
multifunctional materials has also been applied for treatment of
feed samples prior to the determination of aflatoxins (Khayoon
et al., 2010) and the simultaneous determination of aflatoxins and
other mycotoxins (Lo
pez-Grío, Garrido-Frenich, Martínez-Vidal, &
Romero-Gonz
alez, 2010; Varzakas, Demopoulos, & Manolopoulou,
2012). However, simpler, more efficient and environmentally
friendly extraction systems are demanded. In this sense, several
novel microextraction techniques have been developed in order to
reduce the analysis time, increasing the sample throughput and
improving the quality and sensitivity of the analytical methods. In
this sense, QuEChERS-based extractions have been proposed for the
determination of mycotoxins (including aflatoxins) in cereals and
cereal-based products (Arroyo-Manzanares, Huertas-Pe
rez, García-
Campan ~ a, & G

amiz-Gracia, 2014; Desmarchelier et al., 2010).
Concerning analytical methodologies, capillary electrophoresis
(Pen~ a, Alcaraz, Arce, Ríos, & Valca
rcel, 2002), HPLC coupled to
tandem mass spectrometry (MS/MS) (Lo
pez-Grío et al., 2010) and
ELISA (Maqbool, Ahmad, Haq, & Iqbal, 2004; Pleadin et al., 2012;
Rodríguez-Cervantes et al., 2013; Rossi et al., 2012) have been
applied for the determination of aflatoxins in feed. However, as
stated before, HPLC-FLD has been the most popular analytical
technique (ISO, 2006; Khayoon et al., 2010; Muscarella et al., 2009;
Stroka et al., 2003; Varzakas et al., 2012). Nevertheless, sensitive
fluorescent detection of AFB1 and aflatoxin G1 (AFG1) requires their
conversion into more highly fluorescent derivatives. Several
derivatization methods are available, including pre-column treat-
ment with trifluoroacetic acid (TFA) (Khayoon et al., 2010), post-
column derivatization with pyridinium hydrobromide per-
bromide or electrochemically generated bromine (ISO, 2006; Stroka
et al., 2003) and on-line photochemical derivatization (PCD)
(Muscarella et al., 2009). PCD does not need any chemical reagents,
additional pumps or electrochemical cells, making derivatization
quick and easy, with minimum intervention of the analyst. More-
over, PCD has been adopted in an official method for determination
of aflatoxin in several food matrices (AOAC International, 2005).
In this paper we present the optimization and validation of a
reliable and simple method to determine AFB1, aflatoxin B2 (AFB2),
AFG1 and aflatoxin G2 (AFG2) in different kinds of feedstuffs.
Different strategies for sample treatment based on the QuEChERS
method were studied, including extraction and clean up, but finally
2. Materials and methods
2.1. Chemicals and reagents
All reagents were of analytical reagent grade, solvents were of
HPLC grade and aflatoxins were of analytical standard grade.
Methanol (MeOH), carbon tetrachloride, tetrachloroethylene and
dibromomethane were obtained from SigmaeAldrich (St Louis,
MO, USA). Formic acid, tetrahydrofuran (THF) and acetone (ACO)
were supplied by Merck (Darmstadt, Germany) and MeCN, ethanol
(EtOH), dichloromethane and sodium dihydrogen phosphate
monohydrate were supplied by Panreac (Madrid, Spain). Chloro-
form was purchased from VWR BDH Prolabo (West Chester, Pen-
silvania, USA).
Individual standards of each aflatoxin were obtained from Sigma
Aldrich. When standards were provided in dry powder form, the
correct amount of solvent was injected through the septum vial.
From these stock solutions, a 1 mg mL1 intermediate working so-
lution in MeCN was prepared and used thorough all this work. This
solution was stored at 20  C and was stable at least three months.
Since aflatoxins are highly toxic compounds, some general pre-
cautions should be followed for their manipulation and solutions
preparation. Thus, safety glasses and disposable gloves were used
thorough the work. Decontamination of laboratory glassware and
laboratory surface was carried out by swabbing with 10% hypo-
chlorite solution using disposable paper towels. Contaminated
disposable material was properly stored and processed as
biohazard residues.
Ultrapure water (18.2 MU cm1, MillieQ Plus system, Millipore
Bedford, MA, USA) was used throughout all the work.
Kits SampliQ QuEChERS consisting of either buffered QuEChERS
extraction packed (4 g MgSO4, 1 g NaCl, 1 g sodium citrate and 0.5 g
disodium hydrogen citrate sesquihydrate) or non-buffered
QuEChERS extraction packed (4 g MgSO4, 1 g NaCl), C18 and pri-
mary/secondary amine (PSA) bulk materials were supplied by
Agilent Technologies Inc. (Wilmington, DE, USA).
Syringe filters (25 mm, 0.2 mm nylon membrane, Agela Tech-
nologies, DE, USA) were used for filtration of extracts prior to the
injection into the chromatographic system.
2.2. Instruments and equipment
All experiments were carried out using a modular HPLC system
consisted of a quaternary low pressure gradient pump (Model
PUe2089, Jasco, Tokyo, Japan); an autosampler with 100 mL loop
(Model AS-2055, Jasco); a UV derivatization module, which consists
on a photochemical reactor specific for the analysis of aflatoxins
with a 254 nm lamp (LCTech, Dorfen, Germany) placed between the
Table 1
Major ingredients (>9% w/w) and humidity of feedstuffs considered for method
validation studies.
Ingredient Feed intended for
Pig Rabbit Chicken Hen Partridge
Content [% w/w]

the optimum selected procedure was based on the first step, con- Soy 21.0 e 24.7 24.8 35.7
sisting on a simple solideliquid extraction with acetonitrile Corn 22.2 e <9 36.0 29.9
Wheat e e 44.6 15.6 20.0
(MeCN). The extract is then analyzed by HPLC-FLD, with post-
Barley 50 11.8 11.5 9.9 <9
column PCD to increase significantly the fluorescence of AFB1 and Alfalfa e 37.5 e e e
AFG1. The method has been fully validated for five animal feed- Wheat bran e 33.9 <9 e e
stuffs, with different compositions and intended for the feeding of Sunflower e 13.8 e e <9
mammalians (such as pigs and rabbits) and fowls (such as chickens, CO3 2 <9 <9 <9 9.8 <9
Humidity 9.5 8.0 9.9 9.0 9.4
laying hens and partridges).
76 N. Arroyo-Manzanares et al. / Food Control 54 (2015) 74e78
C18 Kinetex separation column (150 mm  4.6 mm, 2.6 mm from
Phenomenex, Torrance, CA, USA) and a fluorescence detector
(Model FP 2020, Jasco).
A Universal 320R centrifuge (HettichZentrifugen, Tuttlingen,
Germany), a vortex-2 Genie (Scientific Industries, Bohemia, NY,
USA) and an evaporator system (System EVA-EC, from VLM GmbH,
Bielefeld, Germany) were also used for sample preparation.
ChromNAV software (1.09.03 version, Jasco) was used for data
acquisition and processing. The statistic software STATGRAPHICS
Centurion XV.II was used for data treatment.
2.3. Sample treatment
Feed samples intended for different kinds of livestock (pig,
laying hen, chicken, rabbit and partridge) were purchased from
local feed producers (Granada, Spain) and stored at room temper-
ature. Mayor ingredients of each feed can be seen in Table 1. Before
the analysis, 30-g samples were milled and homogenized using a
standard grinder (Moulinex, Alençon, Francia). For recovery ex-
periments, subsamples (2 g) were placed in 50-mL falcon tubes and
spiked with an adequate volume of the stock solution containing
aflatoxins (1 mg L1 in MeOH) to reach the desired concentration.
Then, they were homogenized by vortex agitation and stored
uncapped in the dark for at least 1 h, to allow evaporation of
solvent.
For extraction of analytes, a 2-g sample and 10 mL MeCN were
placed into a 50-mL screw cap test tube with conical bottom, and it
was shaken by vortex for 3 min. After that, the sample was
centrifuged at 4500 rpm for 5 min.
Then, 2 mL of the upper MeCN layer was transferred to a vial,
evaporated to near dryness under a gentle stream of N2 and
reconstituted with 1 mL of MeOH:H2O (50:50, v/v). The samples
were filtered with a 0.22-mm filter before injection into HPLC-FLD
system.
2.4. Chromatographic conditions
Chromatographic conditions were similar to those previously
reported with some modifications (Muscarella et al., 2009). The
reverse phase separation was developed in a C18 Kinetex separation
column (150 mm  4.6 mm, 2.6 mm), and the tertiary mobile phase
consisted on water (eluent A), MeOH (eluent B) and MeCN (eluent
C), using the following linear gradient elution: constant MeOH
composition of 27%, 0% C (0e3 min), 13% C (20 min) and 68% C
(21e23 min). The initial conditions were re-established by a 1 min
linear gradient, followed by an equilibration time of 6 min. The flow
rate was 1 mL min1 and the injection volume was 50 mL. The
temperature of the column was 30  C. The excitation and emission
wavelengths for the determination of the aflatoxins derivatives
were 365 and 460 nm, respectively. The fluorescence detector
operated at gain  100.
3. Results and discussion
3.1. Optimization of sample preparation
The optimization was carried out using pig feed as representa-
tive matrix (2 g of sample). Considering the good results reported
for the analysis of mycotoxins in other matrices using the extrac-
tion/partitioning process (first step of the QuEChERS procedure)
(Arroyo-Manzanares et al., 2014; Arroyo-Manzanares, García-
Campan ~ a, & Ga

miz-Gracia, 2013; Arroyo-Manzanares, Huertas-
Pe
rez, Ga
miz-Gracia & García-Campan ~ a, 2013), it was tested for
feed samples. So, different solvent compositions with and without
the addition of different extraction kits were studied: a) 10 mL
MeOH; b) 10 mL of MeOH with 5% formic acid; c) 10 mL MeCN; d)
10 mL MeCN with 5% formic acid; e) 8 mL of H2O þ 10 mL of MeCN
with 5% formic acid þ non-buffered QuEChERS extraction kit (4 g
MgSO4, 1 g NaCl); f) 8 mL of H2O þ 10 mL of MeCN with 5% formic
acid þ buffered QuEChERS extraction kit (4 g MgSO4, 1 g NaCl, 1 g
sodium citrate, 0.5 g disodium hydrogen citrate sesquihydrate); g)
8 mL 30 mM NaH2PO4 buffer pH 7.1 þ 10 mL MeCN with 5% formic
acid þ buffered QuEChERS extraction kit. Although the recoveries
obtained were acceptable in all cases (>75%), the simplest extrac-
tions, that is only with MeOH or MeCN, provided cleaner extracts
with high recoveries, without requiring further clean up. As re-
coveries were slightly better with MeCN, this solvent was selected
for further experiments.
As can be seen, the final procedure is very simple and fast,
providing good recoveries for all the analytes and high reproduc-
ibility. As very clean extracts were obtained, the fluorescence de-
tector could be used with gain  100, therefore the sensitivity of the
method was significantly improved. The final procedure is sum-
marized in Section 2.3. A typical chromatogram corresponding to a
blank and a spiked pig feed sample free of aflatoxins and submitted
to the proposed method is shown in Fig. 1.
3.2. Characterization of the method
In order to check the suitability of the method for the deter-
mination of four aflatoxins in feed, an exhaustive characterization
was carried out. Selectivity of the method was confirmed for
samples from the five studied feeds, by injecting blank samples
submitted to the proposed method. Co-eluting peaks were not
observed at the retention times of aflatoxins, thus, selectivity was
considered adequate. Also, linear dynamic ranges, limits of quan-
tification (LOQs), precision and trueness were evaluated for each
matrix.
3.2.1. Calibration curves and performance characteristics
Matrix-matched calibration curves were obtained using feed
samples spiked with the following concentrations of aflatoxins: 1,
10, 20 and 50 mg kg1 (corresponding to concentrations of afla-
toxins in the final extracts of 0.4, 4, 8 and 20 mg L1, respectively).
Each concentration level was prepared in triplicate, submitted to
the subsequent extraction procedure and injected in triplicate.
The statistical parameters were calculated by least-square
regression, and LOQs were considered as the lowest concentra-
tion prepared and analysed in the calibration range (that is,
Fig. 1. Chromatogram of a pig feed sample subjected to the proposed method: sample
spiked with 1 mg kg1 of each aflatoxin (a); blank sample (b).
 N. Arroyo-Manzanares et al. / Food Control 54 (2015) 74e78 77

Table 2
Statistical and performance characteristics of the method for each matrix.

Animal feed Analyte Linear range Slope Standard error Intercept Standard error R2 Standard error
(mg kg1) of slope of intercept of the estimate

Pig AFG2 1e50 70,105 808 19,397 22,439 0.9956 87,990

AFG1 1e50 38,905 510 10,849 14,166 0.9944 55,550
AFB2 1e50 52,987 384 14,619 10,673 0.9983 41,853
AFB1 1e50 38,756 292 11,934 8,102 0.9981 31,771
Rabbit AFG2 1e50 70,757 508 11,271 13,901 0.9983 56,163
AFG1 1e50 39,154 303 95 8,286 0.9980 33,477
AFB2 1e50 51,666 389 8,061 10,662 0.9981 43,078
AFB1 1e50 37,184 284 7,430 7,789 0.9980 31,471
Chicken AFG2 1e50 66,659 339 41,717 9,295 0.9991 37,553
AFG1 1e50 36,713 254 32,236 6,969 0.9994 28,156
AFB2 1e50 47,964 204 17,962 5,601 0.9984 22,628
AFB1 1e50 34,990 157 10,250 4,312 0.9993 17,420
Hen AFG2 1e50 64,648 248 15,531 6,802 0.9995 27,483
AFG1 1e50 34,987 208 4,576 5,685 0.9988 22,970
AFB2 1e50 45,404 208 2,882 5,698 0.9993 23,020
AFB1 1e50 33,290 181 251 4,950 0.9990 20,000
Partridge AFG2 1e50 62,347 483 37,866 13,234 0.9980 53,469
AFG1 1e50 35,271 389 18,017 10,660 0.9959 43,070
AFB2 1e50 45,039 524 35,471 14,354 0.9954 57,994
AFB1 1e50 34,254 384 27,307 10,673 0.9959 41,855

Table 3
Precision study (%RSD of estimated concentration).

Repeatability (n ¼ 3, injected in triplicate) Intermediate precision (n ¼ 5, injected in triplicate)

Animal feed Analyte Level 1 Level 2 Level 3 Level 1 Level 2 Level 3

(1 mg kg1) (10 mg kg1) (20 mg kg1) (1 mg kg1) (10 mg kg1) (20 mg kg1)

Pig AFG2 7.9 1.8 2.2 8.6 7.2 4.5

AFG1 7.6 1.8 3.2 8.1 9.2 5.7
AFB2 3.9 1.9 1.1 5.4 3.5 5.3
AFB1 4.0 2.5 3.2 5.3 6.0 5.2
Rabbit AFG2 2.9 1.7 2.2 3.2 9.2 7.7
AFG1 2.5 1.9 2.1 5.6 4.8 9.5
AFB2 5.9 1.8 1.3 9.9 3.5 3.9
AFB1 3.1 1.6 1.3 7.6 9.3 7.0
Chicken AFG2 3.8 4.4 1.2 3.9 6.0 4.5
AFG1 2.6 5.1 2.0 4.3 5.7 5.3
AFB2 3.8 1.5 1.2 6.6 9.4 3.9
AFB1 4.5 1.7 1.9 6.5 3.6 2.8
Hen AFG2 2.1 1.3 1.5 3.6 6.3 2.3
AFG1 3.9 0.6 1.3 7.2 6.5 3.1
AFB2 2.6 1.0 1.9 2.8 9.5 6.9
AFB1 9.7 1.7 1.6 5.9 9.9 2.9
Partridge AFG2 2.2 1.6 2.5 5.6 2.8 2.8
AFG1 4.0 2.7 2.9 4.7 6.7 3.7
AFB2 1.2 1.8 2.5 2.9 8.6 8.3
AFB1 1.5 3.4 2.1 4.8 5.8 8.6

1 mg kg1). Table 2 summarizes the results. Satisfactory determi-
nation coefficients confirmed that aflatoxin analytical responses
were linear over the studied ranges. Moreover, with the low LOQs
obtained, the four aflatoxins could be determined at concentrations
lower than the maximum contents established by current legisla-
tion in animal feeds (The Commission of the European
Communities, 2003).
3.2.2. Precision study
The precision of the whole method was evaluated in terms of
repeatability (intraday precision) and intermediate precision
(interday precision). Repeatability was assessed for samples from
the five studied matrices by application of the whole procedure on
the same day to three samples of each kind (experimental repli-
cates) spiked at three different concentration levels of each afla-
toxins, including the LOQ. Each processed sample was injected in
triplicate (instrumental replicates). Intermediate precision was
evaluated with a similar procedure, spiking and analysing five
different samples of each kind in five different days. The results,
expressed as %RSD of estimated concentrations are shown in
Table 3. In all cases RSD lower than 10% were obtained, in agree-
ment with current requirements for mycotoxins analyses (The
Commission of the European Communities, 2006b).
3.2.3. Recovery studies
In order to check the trueness of the proposed method, recovery
experiments were carried out for samples from the five studied
matrices, previously analysed in order to check the absence of
mycotoxins, using an LC-MS/MS method previously described by
Arroyo-Manzanares et al. (2014). None of them gave a positive
result above the LODs of the method. Recovery studies have been
assessed in reproducibility conditions. Samples from the five
studied matrices were spiked at three different concentration levels
(including the LOQ), processed as described previously and injected
in triplicate into the HPLC-FLD system. Recoveries were calculated
as (signal of a spiked sample/signal of a spiked extract) x 100. The
78 N. Arroyo-Manzanares et al. / Food Control 54 (2015) 74e78

Table 4 Arroyo-Manzanares, N., García-Campan ~ a, A. M., & Ga
miz-Gracia, L. (2013). Multi-

Recovery study (%RSD of recoveries given in parentheses, n ¼ 5, injected in class mycotoxin analysis in Silybum marianum by ultra high performance liquid
triplicate). chromatography tandem mass spectrometry using a procedure based on
quechers and dispersive liquid-liquid microextraction. Journal of Chromatog-
Animal feed Analyte Concentration level raphy A, 1282, 11e19.
Arroyo-Manzanares, N., Huertas-Pe
rez, J. F., Ga
miz-Gracia, L., & García-
1 mg kg1 10 mg kg1 20 mg kg1 ~ a, A. M. (2013). A new approach in sample treatment combined with
Campan
Pig AFG2 88.4 (8.6) 92.3 (7.2) 93.6 (4.5) UHPLC-MS/MS for the determination of multiclass mycotoxins in edible nuts
AFG1 85.2 (8.1) 89.9 (9.2) 90.4 (5.7) and seeds. Talanta, 115, 61e67.
Arroyo-Manzanares, N., Huertas-Pe
rez, J. F., García-Campan ~ a, A. M., & Ga
miz-
AFB2 95.5 (5.4) 100.2 (3.5) 98.0 (5.3)
Gracia, L. (2014). Simple methodology for the determination of mycotoxins in
AFB1 88.3 (5.3) 91.3 (6.0) 93.9 (5.2)
pseudocereals, spelt and rice. Food Control, 36, 94e101.
Rabbit AFG2 88.5 (3.2) 91.5 (9.2) 92.4 (7.7)
Beheshti, H. R., & Asadi, M. (2014). Aflatoxins in animal feed in Iran. Food Additives &
AFG1 91.9 (5.6) 94.1 (4.8) 90.0 (9.5)
Contaminants: Part B: Surveillance, 7, 40e42.
AFB2 81.0 (9.9) 104.8 (3.5) 100.5 (3.9) Desmarchelier, A., Oberson, J. M., Tella, P., Gremaud, E., Seefelder, W., & Mottier, P.
AFB1 86.3 (7.6) 88.9 (9.3) 92.1 (7.0) (2010). Development and comparison of two multiresidue methods for the
Chicken AFG2 97.9 (3.9) 98.8 (6.0) 99.5 (4.5) analysis of 17 mycotoxins in cereals by liquid chromatography electrospray
AFG1 86.9 (4.3) 95.1 (5.7) 97.3 (5.3) ionization tandem mass spectrometry. Journal of Agricultural and Food Chem-
AFB2 93.0 (6.6) 98.0 (9.4) 96.8 (3.9) istry, 58, 7510e7519.
AFB1 83.0 (6.5) 102.2 (3.6) 96.5 (2.8) IARC. International Agency for Research on Cancer. (2013). Available on: http://
Hen AFG2 95.3 (3.6) 100.9 (6.3) 99.9 (2.3) www.iarc.fr.
AFG1 94.1 (7.2) 99.5 (6.5) 99.8 (3.1) ISO. International Standard Organization. (2006). Animal feeding stuffs. Determina-
AFB2 95.4 (2.8) 100.6 (9.5) 101.0 (6.9) tion of aflatoxin B1. ISO 17375:2006.
AFB1 95.5 (5.9) 96.6 (9.9) 95.0 (2.9) Khayoon, W. S., Saad, B., Yan, C. B., Hashim, N. H., Ali, A. S. M., Salleh, M. I., et al.
(2010). Determination of aflatoxins in animal feeds by HPLC with multifunc-
Partridge AFG2 98.2 (5.6) 93.2 (2.8) 97.5 (2.8)
tional column clean-up. Food Chemistry, 118, 882e886.
AFG1 102.1 (4.7) 91.4 (6.7) 92.8 (3.7)

pez-Grío, S. J., Garrido-Frenich, A., Martínez-Vidal, J. L., & Romero-Gonz

Lo alez, R.
AFB2 99.8 (2.9) 96.5 (8.6) 101.8 (8.3)
(2010). Determination of aflatoxins B1, B2, G1, G2 and ochratoxin a in animal
AFB1 90.2 (4.8) 96.0 (5.8) 100.8 (8.6) feed by ultra high-performance liquid chromatography-tandem mass spec-
trometry. Journal of Separation Science, 33, 502e508.
results are shown in Table 4 and as can be seen, very good re- Maqbool, U., Ahmad, M., Haq, A. U., & Iqbal, M. M. (2004). Determination of aflatoxin
B1 in poultry feed and its components employing enzyme-linked immunosor-
coveries were obtained (between 81.0% and 104.8%), fulfilling the bent assay (ELISA). Toxicological & Environmental Chemistry, 86, 213e218.
current demands for mycotoxins analysis (The Commission of the Muscarella, M., Iammarino, M., Nardiello, D., Lo Magro, S., Palermo, C., Centonze, D.,
European Communities, 2006b). et al. (2009). Validation of a confirmatory analytical method for the determi-
nation of aflatoxins B1, B2, G1 and G2 in foods and feed materials by HPLC with
on-line photochemical derivatization and fluorescence detection. Food Additives
& Contaminants: Part A, 26, 1402e1410.
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agents. The method has been fully validated for five animal et al. (2012). Immunoassay based on monoclonal antibody for aflatoxin
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feedstuffs, with different compositions and intended for the Streit, E., Schatzmayr, G., Tassis, P., Tzika, E., Marin, D., Taranu, I., et al. (2012).
feeding of mammalians (such as pig and rabbit) and fowls (such as Current situation of mycotoxin contamination and co-occurrence in animal feed
chicken, laying hen and partridge). Calibration curves were estab- e focus on Europe. Toxins, 4, 788e809.
Stroka, J., Von Holst, C., Anklam, E., & Reutter, M. (2003). Immunoaffinity column
lished in the presence of matrix and the low LOQs obtained allowed cleanup with liquid chromatography using post-column bromination for
determining the four aflatoxins at concentrations lower than limits determination of aflatoxin B1 in cattle feed: collaborative study. Journal AOAC
established by current legislation in animal feed. International, 86, 1179e1186.
The Commission of the European Communities. (2003). Commission Directive
The precision (repeatability and intermediate precision) and the 2003/100/EC amending Annex I to Directive 2002/32/EC of the European
recoveries obtained were satisfactory for all the samples studied. Parliament and of the Council on undesirable substances in animal feed. Official
The proposed method could be easily implemented as a simple and Journal of the European Union, L285, 33e37.
The Commission of the European Communities. (2006a). Commission recommen-
high throughput alternative for routine analysis in the quality dation on the presence of deoxynivalenol, zearalenone, ochratoxin A, T-2 and
control of feedstuffs. HT-2 and fumonisins in products intended for animal feeding. Official Journal of
the European Union, L229, 7e9.
The Commission of the European Communities. (2006b). Commission Regulation
Acknowledgements (EC) No. 401/2006 of laying down the methods of sampling and analysis for the
official control of the levels of mycotoxins in foodstuffs. Official Journal of the
European Union, L70, 12e34.
NAM thanks the “Junta de Andalucía” for a postdoctoral grant. The European Parliament and the Council of the European Union. (2002). Directive
JFHP thanks the Ministry of Economy and Competitiveness of the 2002/32/EC on undesirable substances in animal feed. Official Journal of the
Spanish Government for a Juan de la Cierva postdoctoral contract. European Communities, L140, 10e21.
The European Parliament and the Council of the European Union. (2003). Regula-
The authors gratefully acknowledge the financial support of the tion (EC) no. 1831/2003 on additives for use in animal nutrition. Official Journal
“Campus de Excelencia Internacional BioTic Granada” (Ref: of the European Union, L268, 29e43.
CEI2013-MP-17) and the Junta de Andalucía (Proyecto de Excelencia The European Parliament and the Council of the European Union. (2005). Regula-
tion (EC) no. 183/2005 laying down requirements for feed hygiene. Official
Ref. P12-AGR-1647). Journal of the European Union, L35, 1e22.
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mission. Available on http://ec.europa.eu/food/safety/rasff/docs/rasff_annual_
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