SCREENING AND QUANTIFICATION OF AFLATOXINS AND

OCHRATOXIN A IN DIFFERENT CEREALS CULTIVATED IN

ROMANIA USING THIN-LAYER
CHROMATOGRAPHY-DENSITOMETRY

C. BRAICU1,4, C. PUIA2, E. BODOKI3 and C. SOCACIU1

1
Department of Chemistry and Biochemistry
University of Agricultural Sciences and Veterinary Medicine
400372 Cluj-Napoca, Romania
2
Department of Plant Protection
University of Agricultural Sciences and Veterinary Medicine
400372 Cluj-Napoca, Romania
3
Department of Analytical Chemistry
University of Medicine and Pharmacy “Iuliu Hatieganu”
400349, Cluj-Napoca, Romania

Accepted for Publication July 24, 2007

ABSTRACT

The main focus of our study was to implement a rapid, inexpensive and
reliable method that could be utilized to check the cereals for safety (i.e.,
screening for total aflatoxins, as well as individual B1, B2, G1, G2 aflatoxins
and ochratoxin A). We developed a protocol by which we were able to isolate
mycotoxins from cereals collected from different regions of Romania. After
extraction in chloroform, the mycotoxins were separated by thin-layer chro-
matography (TLC) and quantified using densitometry.
Forty-three samples of different cereals (wheat, maize, rye and Triticale)
were analyzed. Twenty-five of the 43 samples (58.14% of the total number)
were found to be contaminated with different mycotoxins in various concen-
trations: aflatoxin B1 (1.6–5.7 mg/kg), aflatoxin B2 (0.89–4 mg/kg), aflatoxin G1
(1.2–5.76 mg/kg), aflatoxin G2 (0.96–3.4 mg/kg) and/or 4.3–30 mg/kg ochra-
toxin A. The concentration of total aflatoxin contamination ranged from 11.2
to 10.8 mg/kg. Among the different cereals, the highest number of contami-
nated samples was found to be in the wheat samples (62.5%). The method
outlined in this study has been adopted already by our laboratory for current
analyses of mycotoxins. The results obtained using this method is in
4
Corresponding author. TEL: 0040-264-595825, ext. 213; FAX: 0040-264-593792; EMAIL:
cbraicu@usamvcluj.ro

Journal of Food Quality 31 (2008) 108–120. All Rights Reserved.

108 © 2008, The Author(s)
Journal compilation © 2008, Blackwell Publishing
 DETERMINATION OF AFLATOXINS AND OCHRATOXIN A 109

compliance with the strict regulatory guidelines developed both in Romania,

as well as in the European Union.

PRACTICAL APPLICATIONS

Thin-layer chromatography (TLC) is a rapid, inexpensive and convenient

method that can be used routinely to screen for mycotoxins. This article
describes the detailed procedures for the extraction, purification and quantifi-
cation of aflatoxins and ochratoxin A, using TLC. Using this method one
can identify concomitantly five different mycotoxins and by coupling it with
densitometry a quantitative determination is also possible. Therefore, this
could become a routine technique in regional laboratories responsible for
checking agrifood safety.

INTRODUCTION

Mycotoxins were first identified for more than 40 years as contaminants

frequently found in a variety of food products (seeds in particular) (Bennett
and Klich 2003; Castells et al. 2005). The aflatoxins are the most extensively
studied subclass of mycotoxins, and they can be identified in peanuts, pista-
chios, figs, paprika and corn, just to name a few of the most relevant foods
(Richard et al. 1993; Czerwiecki et al. 2005; Krska et al. 2005).
Aflatoxins are produced as secondary metabolites by the fungus Aspergil-
lus flavus and Aspergillus parasiticus and have been proven to be highly toxic,
carcinogenic, mutagenic and immunosuppressive agents (Mahfoud et al.
2002). Among the 20 known aflatoxins, four (designated as AB1, AB2, AG1 and
AG2) are the most frequently found in foods (Krska et al. 2005), and are
carcinogenic in both humans and animals (Smith et al. 1995; Bennett and
Klich 2003; Castells et al. 2005).
Ochratoxin A is produced by some species of Penicillium and Aspergillus
in cereal grains, but occurs naturally as well in a variety of plant products such
as cereals, coffee beans, beans, pulses and dried fruit (Bauer and Gareis 1987;
Czerwiecki 1994). It is also found in kidney, liver and blood from mammals by
absorption from food. Investigations of the frequency and levels of occurrence
of ochratoxin A in food and human blood samples indicate that foodstuffs are
frequently contaminated (Czerwiecki et al. 2005). Ochratoxin A possesses
carcinogenic, nephrotoxic, teratogenic and immunotoxic effects (Pohland
et al. 1992; Czerwiecki et al. 2005). It is also linked to nephropathy in humans
(Bauer and Gareis 1987; Pohland et al. 1992).
110 C. BRAICU ET AL.

According to the European Commission’s Regulation, No 472/2002,

amending (EC) Regulation No 466/2001, the maximum level accepted (MLA)
for total aflatoxins is 4 mg/kg (for aflatoxin B1 is 2 mg/kg) and for ochratoxin
A is 3–5 mg/kg.
Different screening methods and advanced techniques for detection and
quantification of mycotoxins were reported in the last 20 years (Bauer
and Gareis 1987; Richard et al. 1993; Aziz et al. 1998; Castro and Vargas
2001).
Thin-layer chromatography (TLC) has been the most widely used method
since its development in the early 1960s, probably because of its low analysis
costs and accessibility (Stubblefield et al. 1967). This method is still recom-
mended by the Association of Official Analytical Chemists for mycotoxin
analysis (Grabarkiewicz-Saczesna et al. 1985; Castro and Vargas 2001). Ini-
tially, this technique was used as a qualitative method, but coupling with
densitometry increased its application for quantitative analysis.
Several advanced methods have been used during the last years for the
mycotoxin quantitative evaluation in food and cereals.
High Performance Liquid Chromatography (HPLC) separation is usually
achieved on C18 reversed-phase columns with methanol/water mixtures as
mobile phase (Hassan et al. 2001; Czerwiecki et al. 2005; Krska et al. 2005).
However, the technique is still expensive and it needs qualified personnel to
perform it (Amezqueta et al. 2004). HPLC is used more as a reference method
for mycotoxins quantification.
Enzyme-linked immunosorbent assays (ELISA) have recently been
developed also for quantitative analysis of some individual aflatoxins and have
been used also as a rapid screening technique. Cleanup is minimal, since these
methods are being used also for their quantification in less complex matrices
(Abouzied et al. 1991), but it is limited by the unavailability and the high price
of specific mycotoxin antibodies.
These advanced techniques require investments and expertise, and
because of their high cost, they are not recommended for screening and routine
analysis.
In comparison with HPLC and ELISA, previous studies have shown that
TLC is easy to perform, robust in its application and reliable in its results
(Stubblefield et al. 1967; Grabarkiewicz-Saczesna et al. 1985; Castro and
Vargas 2001). On this basis, it can be considered a more suitable method.
Coupled with densitometry, TLC can be optimized for a rapid screening of
mycotoxins found in cereals. The purification, separation and quantification of
four aflatoxins (AB1, AB2, AG1, AG2) and ochratoxin A, followed by quanti-
tative TLC analysis, was previously reported since 1967 and is still actual until
now (Stubblefield et al. 1967; Grabarkiewicz-Saczesna et al. 1985; Castro and
Vargas 2001; Braicu et al. 2005).
 DETERMINATION OF AFLATOXINS AND OCHRATOXIN A 111

There are only a few articles concerning the mycotoxin’s incidence in

Romania. The first study that investigates the natural occurrence of mycotox-
ins in feeding stuff was presented by Curtui et al. (1998). The present studies
are focused on using rapid, inexpensive and reliable methods for isolation and
screening of aflatoxin B1 (AB1), aflatoxin B2 (AB2), aflatoxin G1 (AG1), afla-
toxin G2 (AG2) and ochratoxin A in cereals (for feed and bakery) randomly
chosen from different regions of Romania.
This is a screening study for the occurrence of aflatoxins and ochratoxin
A in cereals, based on a TLC method developed in our laboratory (Braicu et al.
2005). Mycotoxins were extracted by specific protocols in chloroform, sepa-
rated by TLC and quantified using densitometry (Stubblefield et al. 1967;
Braicu et al. 2005).

MATERIALS AND METHODS

Cereal Samples
A total of 202 different random samples of grains (wheat – 130, maize –
60, Triticale – 6 and rye – 6) were collected from seven districts of our country,
Romania (year: 2005). Each sample (1 kilo) was put in a sterilized bag and was
divided into two parts in the laboratory. One part was used for mycological
analysis and the second part was kept for mycotoxin analysis (stored at 4C
until the TLC analysis was performed).
The mycological analysis was conducted in five repetitions, using the
blotting paper method (Raicu and Baciu 1978) and the Ulster method on
agarised plates (Hulea et al. 1982) using 10 grains from each sample in three
or five repetitions. We used the Czapek–Dox agar medium with Streptomycin
(Sigma, Bucharest, Romania) as a bacteriostatic, added after sterilization
(Hulea 1969), and the identification of fungi was carried out according to
Raicu and Baciu (1978) and Domsch and Gams (1972).
Aliquots of 50 g from each sample were grounded and taken for analysis.
Some of the contaminated samples, as determined by mycological analyses,
were screened for the mycotoxins (aflatoxins and ochratoxin A) quantification
(43 samples). Each sample was analyzed in triplicate and the results were
expressed as an average of three repetitions.

Reagents
Standards: Total Aflatoxin Standard 1,000 ng/mL, containing 250 ng/mL
of each aflatoxin (AB1, AB2, AG1, AG2) and Ochratoxin A Standard 250 ng/mL
from Diagnostic (R-Biopharm Rhone, Ltd., Bucharest, Romania).
112 C. BRAICU ET AL.

Solvents provided from Merck GmbH (Bucharest, Romania) (chloro-

form, acetonitrile, acetone, toluene, ethyl acetate, anhydrous ethyl ether).
The reagents, formic acid, anhydrous sodium sulphate, sulphuric acid,
paper filter: Whatman 4 were provided by Merck GmbH.
Hamilton microsyringes were used for sample applications on the chro-
matographic plate (in 1 cm bands).

Simultaneous Extraction and Purification of Mycotoxins

The extraction was performed using a method described by Braicu et al.
(2005).
(1) Aliquots of 10 g cereal grains were finely ground and weighed into a
suitable flask;
(2) Extracted with 50 mL chloroform at 22–25C and then sonicated for
10 min;
(3) Then filtered under vacuum through paper filter Whatman 4;
(4) The chloroform extract was washed by distilled water and dried over 20 g
anhydrous sodium sulphate;
(5) The extracts were concentrated almost to dryness on a rotary evaporator
and the residue was resolved in 1 mL chloroform.
Known quantities of each standard were added to samples in order to
verify the percentage of mycotoxin’s recovery during the extraction.

TLC
Silica gel F254 10 ¥ 20 cm TLC plates with a thickness of 0.25 mm were
used (Merck). Samples (30 mL) of chloroform extracts and standard solution
of aflatoxins and ochratoxin were spotted in 1 cm bands. Three successive
developing solvent systems were used: chloroform : acetone (9:1, v/v) for first,
and toluene : ethyl acetate: 80% formic acid (6:3:1, v/v/v) for second and third
migration in a mobile phase vapor saturated chromatographic tank. This is a
method as described by Braicu et al. (2005). TLC plates were examined under
UV light at 366 nm before quantification.
Chemical tests to confirm mycotoxin presence were performed in order
to avoid mistakes. Several parameters were tracked in order to establish
the presence of mycotoxins: the Rf value, the fluorescence color
(lexcitation = 366 nm) and its change after treatment with spray reagents (AlCl3
and H2SO4) and heated 10 min at 110C.
When the plates were sprayed with 20% AlCl3 solution or 20% sulfuric
acid and heated 10 min at 110C the fluorescence intensity increased. This was
only applied to the confirmation of mycotoxins, and not for the densitometric
 DETERMINATION OF AFLATOXINS AND OCHRATOXIN A 113

analysis, because postchromatographic derivatization potentially could have

given rise to errors in quantitative evaluation.
Densitometric Analysis
Desaga CD6 TLC Densitometer (Bucharest, Romania) equipped with
data acquisition and processing software was used for quantitative evaluation
of the plates. The separated mycotoxins were detected and quantified in fluo-
rescence mode (lexcitation = 365 nm, slit of 0.06 ¥ 4 mm for aflatoxins, and
lexcitation = 336 nm, slit of 0.2 ¥ 4 mm for ochratoxin A), with a scanning reso-
lution of 0.025 mm and 16 readouts/point.
The applied quantities of standards on the TLC plate ranged between
0.25–7.5 ng aflatoxin (AB1, AB2, AG1, AG2) and 1–30 ng for ochratoxin A.
The linear calibration curves were established representing the peak areas in
function of the applied quantities of separated mycotoxin standards.
Calculation of Mycotoxin Concentration in Cereals Samples
The concentration of mycotoxins was calculated according the formula:

c p = cs( vf vs )(1 m s )

where
cp – Concentration of mycotoxin (mg/kg) in the cereal sample
cs – Quantity determined by densitometry (ng mycotoxin/spot)
Vf – Final volume of the extract after concentration (1,000 mL)
vs – Volume of extract spotted on TLC plate (30 mL)
ms – Sample weight (10 g)

RESULTS AND DISCUSSION

Several pioneering studies of mycotoxins have been carried out using the
TLC method well before the general availability of HPLC and immunological
techniques. Nevertheless, not all laboratories are equipped with HPLC or GC,
particularly in the developing parts of the world. Therefore, there is an
increased need to monitor the level of mycotoxins in food (especially intended
for export). The TLC method provides reliable results for aflatoxins and
ochratoxin A screening from different matrices. So, in conclusion TLC can be
a good alternative to expensive techniques such as HPLC or ELISA.
Standards Curves of Five Mycotoxins and Densitometric Analysis
Fluorescent spots of pure aflatoxins and ochratoxin A standards were
visible under UV light (366 nm) used as a quantification method by densito-
114 C. BRAICU ET AL.

FIG. 1. SAMPLE TLC ANALYSIS IN PARALLEL WITH STANDARDS

A 1:1.25 ng from each aflatoxin (AB1, AB2, AG1, AG2); 2:2.5 ng from each aflatoxin (AB1, AB2,
AG1, AG2); 3:3.75 ng from each aflatoxin (AB1, AB2, AG1, AG2), 10 ng ochratoxin A; 4:5 ng from
each aflatoxin (AB1, AB2, AG1, AG2), 15 ng ochratoxin A; 5:20 ng ochratoxin A; 6:25 ng
ochratoxin A; 7, 8, 9: wheat samples. This figure appears in color in the online version of the article
[DOI:10.1111/j.1745-4557.2008.00187.x].

metry. In Fig. 1, the TLC separation of aflatoxin standards (AB1, AB2, AG1 and
AG2) and ochratoxin A bands, used for the calibration curve, are presented
parallel with three wheat samples.
The most important problem was the overlapping of bands with the
interference compounds on the TLC plate. Using a multiple developing solvent
system, we were able to separate all four aflatoxins and ochratoxin A without
interference compounds. The first developing system (chloroform : acetone,
9:1, v/v) proved to be very useful for the separation of mycotoxins. The second
and third migration offered a good separation of aflatoxins without overlap
while interference compounds were moved to higher Rf’s relative to aflatoxins.
The Rf values of aflatoxins were: 0.278 for AG2, 0.324 for AG1, 0.348 for AB1,
0.402 for AB2 and 0.584 for ochratoxin A.
Quantification by densitometry is based on peak area measurements and
calibration with standards for samples. The most difficult part of this proce-
dure was to find the right adjustment of the parameters necessary to obtain the
maximum intensity of fluorescence peaks, with minimal background noise.
A typical densitogram of aflatoxin standards was recorded, showing a good
baseline separation for each peak. The calibration curves were established based
on densitograms and considering the peak areas in function of the applied
quantities of separated mycotoxin standards on the following domain (0.25–
6.25 ng/spot for aflatoxins and 1–30 ng/spot for ochratoxin A). Data concerning
the calibration curve for the five mycotoxins are presented in Table 1.
The linear calibration curves show a high correlation between the ana-
lytical signal and the increasing quantities of mycotoxins (R2 > 0.993). The
method’s calculated limits of detection (ng/plate) were 0.14 ng AB1, 0.30 ng
AB2, 0.10 ng AG1, 0.13 ng AG2 and 0.88 ng ochratoxin A, respectively. The
calculated limits of quantification (ng/plate) are 0.47 ng AB1, 1.01 ng AB2,
0.33 ng AG1, 0.44 ng AG2 and 2.96 ng ochratoxin A, respectively. Individual
 DETERMINATION OF AFLATOXINS AND OCHRATOXIN A 115

TABLE 1.
REPORTING SLOPE, INTERCEPT AND R2 VALUES USED FOR QUANTIFICATION OF
AB1, AB2, AG1, AG2 OCHRATOXIN A STANDARDS

Nr. Mycotoxin Slope Intercept R2

1 AB1 62.525 -1.9821 0.9988

2 AB2 120.89 9.9426 0.9966
3 AG1 45.251 -0.6142 0.9984
4 AG2 104.1 13.897 0.9931
5 Ochratoxin A 26.689 -4.5405 0.9971

recovery values for densitometric analysis varied from 83.4 to 105.5% for all
aflatoxins and from 67.5 to 101% for ochratoxin A. The recovery of aflatoxins
from cereals achieved in the present study is similar with the results reported
by Castro and Vargas (2001) and Kamimura et al. (1985).

Quantification of Mycotoxins Isolated from Cereals

The mycotoxin quantification was based on TLC coupled with densito-
metry and is presented in Fig. 1. Quantification was accomplished by com-
parison to the reference standards on the TLC plate and fluorescence
densitometry.
The proposed method can quantify with good precision, amounts as low
as 0.25 ng/spot for each of AG1, AG2, AB1, AB2 and 1 ng/spot for ochratoxin
A, respectively. The lowest limit of quantification for mycotoxin contamina-
tion in cereals were 0.83 mg/kg AG1, AG2, AB1, AB2 and 3.33 mg/kg for
ochratoxin A, being similar with other citations (Richard et al. 1993; Mahfoud
et al. 2002). Castro and Vargas (2001) reported a similar method, using TLC/
visual and densitometric analysis, and obtained aflatoxin levels as low as
1.0 mg/kg of AB1, 0.3 mg/kg of AB2 and 0.6 mg/kg of AG2 and AG1 in maize.
In Table 2 we present the natural occurrence of mycotoxins analyzed by TLC
coupled with densitometry from different Romanian districts. From the 43
samples taken for analysis, 16 samples contained mycotoxin levels higher than
MLA. In a total of 11 samples, we obtained higher amounts of AB1 and total
aflatoxins than the MLA. In 11 samples, the concentration of B1 was higher
than MLA. In a total of six samples, ochratoxin A was identified in one maize
sample (lower than MLA) and two rye samples.
AB1 was identified in nine wheat samples, in three maize samples and in
one rye sample. AB2 was identified in nine wheat samples, in one maize
sample and two rye samples. AG1 was identified in four wheat samples, in one
maize sample, in four Triticale samples. AG2 was identified in four wheat
samples. In general, the cereal contamination ranged between 0.93 and
 TABLE 2.
NATURAL OCCURRENCE OF MYCOTOXINS IN CEREALS ANALYZED BY TLC COUPLED WITH DENSITOMETRY 116
FROM DIFFERENT ROMANIAN DISTRICTS

Samples District No. of samples Ochratoxin Aflatoxins mg/kg

A mg/kg
Analyzed Contaminated AB1 AB2 AG1 AG2 Total

Wheat BC 2 1 0 5.29 0 5.56 0 10.85

BN 4 4 0 4.29 1.43 0 0 5.72
6.57 4.99 1.49 0 0 6.48
0 4.8 2.0 0 0 6.8
0 3.15 1.8 4.95
BV 3 3 31.3 0 0 5.6 0 5.6
30.0 0 0 2.0 1.8 3.8
0 0 0 0 1.2 1.2
BH 2 1 0 0 0 4.22 0.99 5.21
HD 3 2 0 3.89 0.89 0 0 4.78
0 5.7 2.6 0 0 8.3
CJ 2 0 0 0 0 0 0 0
MM 4 1 0 0 0 0 0.93 0.93
GR 2 0 0 0 0 0 0 0
SB 2 2 0 5.6 2.7 0 0 8.3
0 5.1 2.9 0 0 8.0
C. BRAICU ET AL.

SM 1 1 0 0 3.99 0 0 3.99
SJ 1 0 0 0 0 0 0 0
Maize BC 2 2 0 1.76 0 0 0 1.76
0 3.92 1.39 0 0 5.31
BV 1 0 0 0 0 0 0 0
MM 1 0 0 0 0 0 0 0
SB 2 2 0 0 0 3.33 0 3.33
0 0 0 0 3.4 3.4
HD 2 1 4.39 1.67 0 0 0 1.67
GR 2 0 0 0 0 0 0 0
SM 1 0 0 0 0 0 0 0
Triticale BV 4 3 0 0 0 5.76 0.89 6.65
0 0 0 1.12 0 1.12
0 0 0 5.76 0 5.76
0 0 0 1.4 2.2 3.6
Rye SB 2 2 25.6 4.65 2.76 0 0 7.41
24.1 0 3.1 0 0 3.1
Limits by European Commissions of Regulation (EC) no. 472/2002 3–5 <2 <4 <4
Limits by Romanian legislation <20 Unspecified Unspecified Unspecified

TLC, thin-layer chromatography.

 DETERMINATION OF AFLATOXINS AND OCHRATOXIN A 117

10.85 mg/kg for total aflatoxins and 1.67–5.6 mg/kg AB1, 0.89–3.99 mg/kg
aflatoxin B2, 1.12–5.76 mg/kg aflatoxin G1, 0.89–3.4 mg/kg AG2. The range for
ochratoxin A was 4.39–31.3 mg/kg.
In the present study concerning ochratoxin A, only 12.5% of wheat and
9% of maize samples were contaminated. In the case of Triticale, the ochra-
toxin A concentration was at very low or “zero level” and it was not detected
in any of those samples. The present study reports the natural occurrence of
ochratoxin A and co-occurrence of aflatoxins. We have to mention that two
samples of wheat exceeded the limits established by Romanian legislation,
which is very permissive (20 mg/kg); this is less than 7.7%.
The incidence of total aflatoxins in wheat samples was about 62.5% of
wheat samples. The incidence of total aflatoxins is higher than MLA in 45.83%
of wheat contaminated samples. Concerning the maize samples contamination
with total aflatoxins, 45% of those were under MLA and 20% higher than
MLA.
AB1 was identified in 37.5% of wheat samples, in which all were higher
than MLA, and in 27.7% of maize samples, in which 9% were higher than
MLA. The most potent and most frequently occurring of the mycotoxins
analyzed is AB1.
The highest number of contaminated samples was identified in wheat
samples, which may be due to culture condition or possibly weather condition
of a particularly wet summer. This emphasizes the importance of climate
condition, crop and storage on fungal development and mycotoxin production.
Our dates are in reasonable agreement with the incidence found in wheat
and maize reported by Curtui et al. (1998) based on a study of 25 wheat and
30 maize samples. It was reported that one maize sample contained 140 mg/kg
AB1, and ochratoxin A was found in one wheat sample (37 mg/kg).
The same author (Curtui et al. 2001) emphasized the necessity for screen-
ing of mycotoxins in foodstuffs. Moreover, Curtui et al. (2001) reported the
accumulation of mycotoxins in animal blood and kidneys, sampled from
slaughtered pigs.
Taking into consideration all of the above concerns and the presence of
diseases with unknown etiologies (like liver cancer and leukemia), compre-
hensive studies, including a periodical survey on natural co-occurrence of
mycotoxins in cereals, are needed in order to have a broader perspective on the
problems involved by mycotoxin contamination.

CONCLUSIONS

Our studies reveal that TLC coupled with densitometry assures good
resolution, precision and reliability in the quantitative determination of afla-
118 C. BRAICU ET AL.

toxins and ochratoxin A extracted from cereals. Its sensitivity enables the
accurate quantification of lower levels of mycotoxins than the MLA estab-
lished by the European Commission. Once the contaminated samples are
identified, they can be further analyzed by a more selective or sensitive
method.
The proposed TLC-densitometric method, due to its simplicity, cost-
effectiveness and high sample throughout, represents the optimal rapid screen-
ing method in spite of its slightly lower sensitivity as compared with HPLC
and ELISA testing methods.
This method has been adopted by our laboratory for the current analysis
of mycotoxins, and it complies with the strictest regulatory guidelines and
limits established by Romanian and European Commission directives.

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