Analytica Chimica Acta 829 (2014) 6874

Contents lists available at ScienceDirect

Analytica Chimica Acta

journal homepage: www.elsevier.com/locate/aca

Aatoxin B1 and M1 in milk

Scaglioni P.T a, *, Becker-Algeri T. b , Drunkler D. b , Badiale-Furlong E. a
a
Laboratrio de Cincia de Alimentos, Escola de Qumica e Alimentos, Universidade Federal do Rio Grande-FURG, Avenida Itlia, km 8, Bairro Carreiros, Rio
Grande do Sul 96203-900 Brazil
b
Programa de Ps Graduao em Tecnologia de Alimentos, Universidade Tecnolgica Federal do Paran-UTFPR, Avenida Brasil, Cmpus Medianeira, 4232 Parque Independncia, Medianeira, Parana 85884-000, Brazil

H I G H L I G H T S

G R A P H I C A L A B S T R A C T

The mobile phase chosen consists

acid:acetonitrile:methanol
acetic
(55:10:35).

We obtained a separation factor of
2.3 for AFLAB1 and AFLAM1.

The milk samples were contaminated by aatoxins above the legislated
limits.

Aatoxins tend to get attached to clot
after acid precipitation of milk.

A R T I C L E I N F O

A B S T R A C T

Article history:
Received 28 February 2014
Received in revised form 13 April 2014
Accepted 18 April 2014
Available online 26 April 2014

The aatoxin M1 (AFLAM1) is a mycotoxin that results from the hydroxylation of the aatoxin B1 (AFLAB1).
It contaminates the milk of animals fed with a diet containing its precursor. In this work, we determined
the occurrence of AFLAB1 and AFLAM1 in milk, as well as the chromatographic conditions to quantify
these mycotoxins. The extraction and quantication of AFLAB1 and AFLAM1 in naturally contaminated
and articially spiked milk samples which are produced and marketed in the state of RS were performed
using the AOAC ofcial method and UHPLC with uorescence detection. We obtained a separation factor
of 2.3 for AFLAB1 and AFLAM1 using a mobile phase consisting of 1% acetic acid:acetonitrile:methanol
(55:10:35). The analytical curves had a wide linearity range and the limit of quantication (LOQm)
concentrations of AFLAB1 and AFLAM1 were equal to 0.5 and 0.25 mg L1, respectively. Samples of
pasteurized and ultra-high-temperature processed (UHT) milk showed natural contamination, and the
levels for both aatoxins ranged from 0.7 to 1.5 mg L1. Raw and concentrated milk samples only
contained AFLAM1, with a maximum average concentration of 1.7 mg L1. These concentrations, higher
than permitted by legislation, conrm the existence of a health risk, as well as highlight the relevance of
searching for alternatives to reduce this contamination.
2014 Elsevier B.V. All rights reserved.

Keywords:
Aatoxin B1
Aatoxin M1
Analytical method
Milk
Liquid chromatography

1. Introduction

* Corresponding author. Tel.: +55 53 32935374/81011107; fax: +55 53 32338645.

E-mail addresses: priscilascaglioni@gmail.com, ptscaglioni@gmail.com
(P.T. Scaglioni).
http://dx.doi.org/10.1016/j.aca.2014.04.036
0003-2670/ 2014 Elsevier B.V. All rights reserved.

Milk is a food that provides macro and micronutrients for the

growth, development and maintenance of human health. However,
it may also be a vehicle of environmental and food contaminants,
causing various physiological alterations in individuals who
consume it. Microorganisms present in milk, as well as their
metabolites, are able to migrate in the uids and tissues of

P.T. Scaglioni et al. / Analytica Chimica Acta 829 (2014) 6874

breeding mammals subsequently harming their health. There is an

especial concern for children and infants, who are more susceptible than adults to the toxic effects of contaminants such as
aatoxins present in milk [1].
Aatoxins are secondary metabolites produced by fungal species
such as Aspergillus avus, Aspergillus parasiticus and Aspergillus
nomius. These organisms contaminate agricultural products and
produce especially aatoxins B1 (AFLAB1), B2 (AFLAB2), G1 (AFLAG1)
and G2 (AFLAG2). Animals that ingest food contaminated with
AFLAB1 and AFLAB2 can hydroxylate these aatoxins and yield dairy
products contaminated with Aatoxins M1 (AFLAM1) and M2
(AFLAM2) [2]. Aatoxins are factors involved in the etiology of
human liver cancer. In animals, the ingestion of contaminated food
with a certain concentration of aatoxins was followed by
teratogenesis and, especially during the rst embryonic phase,
occurs the malformation of fetuses and the reabsorption of embryos
[3,4]. The AFLAB1 is classied as Group 1 (carcinogenic to humans),
while AFLAM1 is classied as Group 2B (possibly carcinogenic to
humans) [5]. The World Health Organization recommended the
reduction of the AFLAM1 levels in milk and dairy products to a
minimum, in order to minimize the potential risk it poses. The toxic
and carcinogenic effects have been extensively demonstrated in
several species, especially in young animals [6,7]. There is, therefore,
a great concern regarding the health of children, considering their
high consumption of milk and dairy products, their low body weight
and their great susceptibility to aatoxins [5].
The rst methods for determining AFLAM1 in samples of milk
and dairy products were developed in the mid 60s, based on the
uorescent property of the toxin when exposed to ultraviolet light.
Thus, the rst technique to identify and quantify was the thin layer
chromatography (TLC) [8]. Later, with the development of methods
for ultra high performance liquid chromatography (UHPLC), there
was a notable increase in the degree of precision of the
determinations, however with high costs [9]. The use of methods
for multitoxins is recommended to better assess the possibility of a
synergistic effect, as well as to circumvent the cost impact of
analytical determinations. These methods are especially important
when there is a metabolic relation between two analyzed toxins,
like in the case of AFLAM1 and AFLAB1. Both toxins can occur in
milk if the precursor AFLAB1 was abundant enough in the food to
surpass the metabolic capacity of the animal [10].
Reviews and validations of methods for the determination of
AFLAM1 are constantly made in order to rene the techniques of
extraction, purication and quantication, in order to improve the
reliability of the results concerning the impact of this compound in
milk and meet the current demand for minimum generation of
waste. However, there are few studies on adapting these methods
to determine the precursor toxin (AFLAB1) that may be circulating
in the uids of animals without being metabolized too.
In this study we sought to standardize a method to
simultaneously quantify the concentrations of AFLAB1 and AFLAM1
in milk destined to human consumption.
2. Materials and methods
2.1. AFLAB1 and AFLAM1 standards
The standards for AFLAB1 and AFLAM1 were purchased from
SigmaAldrich Chemical Company. They were resuspended in
benzene:acetonitrile (98:2), resulting in the desired concentrations for each analysis.
2.2. Milk sampling
The milk samples were collected from July to October 2012,
amounting to 40 samples. They were acquired from producers in

69

the southern Brazil (raw milk), local shops (pasteurized milk and
ultra-high temperature-UHT) and a beneciation industry of the
region (raw, pasteurized, concentrated, powder and UHT).
Samples of raw milk were stored in polyethylene terephthalate
packs provided by the producer and kept under cooling temperature (4  C) until the time of analysis, which did not exceed 24 h
after the acquisition thereof.
The samples obtained in the local commerce were kept under
refrigeration from the time of acquisition (pasteurized milk) or
after opening the package (UHT). The sampling unit consisted of
sealed containers without apparent deformation, within the
validity period, containing 1 L of sample and from different
manufacturing batches. They were chosen considering the
availability of lots in major supermarkets in the city of Rio Grande,
RS, Brazil. In view of the geographical distance between the factory
and the laboratory where the analysis were performed, the
samples derived from the processing industry were inoculated
with an specic preservative to maintain the characteristics of the
materials unchanged, namely 2-bromo-2-nitropropane-1,3-diol
(Bronopol1).
2.3. Extraction of AFLAB1 and AFLAM1 from milk
Using a method described by AOAC [11], the volumes of both the
sample and the solvents were reduced three times in order to
minimize the generation of waste. After that, the extraction was
carried out using methanol and celite. A subsequent partition was
carried out using 4% sodium chloride and hexane. The extraction
and the removal of any remaining water in the extracts were
performed, respectively, with chloroform and anhydrous sodium
sulfate. The nal extracts were dried using nitrogen current and
stored at freezing temperature until the time of quantication.
2.4. Establishment of the chromatographic conditions for quantifying
AFLAB1 and AFLAM1 by UHPLC- FL
Tests were carried out to choose the mobile phase that would
result in the best chromatographic separation of aatoxins with
the lowest interference of the matrix components. These tests
were based on the solvent mixture established by AOAC [11],
namely 1% acetic acid:acetonitrile:methanol (40:35:25).
The run was performed on a ultra high-efciency liquid
chromatograph (UHPLC) consisting of a LC-10 AT pump, a DGU
degasser, a CBM-20A controller, a SPD-20A uorescence detector, a
20 mL 7725i manual injector, a LC Solution-Shimadzu software and
a Kromasil1 C18 5 mm 250  4.6 mm column. The standards and
extracts of the samples were eluted at 35  C with a ow rate of
1.0 mL min1. The detection of mycotoxins was performed in
wavelengths of 360 nm to 450 nm for absorption and emission,
with a loop of 20 mL.
The retention factor (k) was calculated dividing the corrected
retention time of each aatoxin by the dead time. The separation
factor (a) was determined by the kA/kB ratio, knowing that the
solute B is more retained in the column than the solute A [12,13].
Two standard curves were built, one for each aatoxin, using the
standard solutions of AFLAB1 and AFLAM1, which were diluted in the
solvent mixture that makes up the mobile phase, at increasing
concentrations (4.5 mg L1; 9 mg L1; 18 mg L1; 27 mg L1; 54 mg
L1; 81 mg L1; 108 mg L1; 162 mg L1 and 270 mg L1). Two other
curves, one for each aatoxin, were made from the fortication (in
triplicate) of milk samples with eight increasing concentrations of
aatoxins (0.25 mg L1; 0.5 mg L1; 4.5 mg L1; 18 mg L1; 54 mg L1;
81 mg L1; 108 mg L1 and 162 mg L1). These samples went through
the same extraction process as the others, according to the AOAC
method, and were subsequently quantied by UHPLC-FL. Then, the

70

P.T. Scaglioni et al. / Analytica Chimica Acta 829 (2014) 6874

range of linearity, the coefcients of determination and correlation,

as well as the limits of detection (LOD) and quantication (LOQ) for
each mycotoxin were determined.
The determination of the detection limit of the instrument
(LODi) was performed by injecting the standard mixture solution
of AFLAB1 or AFLAM1 diluted in the solvent mixture that makes up
the mobile phase. These injections were made at decreasing
concentrations, until the acquiring of a 3:1 ratio between the
analyte peak and the base line noise near the retention time of the
peak of aatoxins. To determine the quantication limit of the
instrument (LOQi), the analytical solutions prepared were injected
in the solvent until a 10:1 ratio between the analyte peak and the
baseline noise was obtained. To determine the detection limit
(LODm) and the quantitation limit of the method (LOQm), extracts
of the spiked samples were injected until the same relation to the
instrumental limits described was achieved [14].

2.5. Recovery test of the AOAC extraction method

The determination of the recovery percentage of the method
was performed by fortifying milk samples with the standards of
AFLAB1 and AFLAM1. The aliquots of each standard were
transferred to asks and, after evaporation of the solvent, aliquots
of milk were added. Six levels of fortication were used, based on
the legislated limits for milk AFLAM1 in Brazil (0.5 mg kg1) and
considering the LOQm estimated for each of the aatoxins. The
following concentrations were tested: 0.25 (only for AFLAM1);
0.5 mg L1; 3 mg L1; 6 mg L1; 8 mg L1 and 10 mg L1. The extraction and the quantication of the dried extract in 1 mL of the
mixture that consisted in the mobile phase in the UHPLC-FL were
performed by the AOAC method [11]. The percentage recovery was
estimated by relating the concentration of aatoxins found with
the expected concentration, as shown in Eq. (1).


C1  C2
(1)
 100
%R
C3
where %R-recovery percentage; C1-concentration determined in
the spiked sample; C2-concentration determined in the nonspiked sample; and C3-concentration of the standard used for the
fortication.

2.6. Occurrence of the AFLAB1 and AFLAM1 in milk samples and their
distribution in fractions of the raw material
A natural contamination by aatoxins was observed in milk
samples that underwent different processes such as, raw milk,
pasteurized, UHT, concentrated and powdered.
To investigate the distribution of aatoxins in raw material
during the processing of milk for cheese production, an acid
coagulation of milk was performed, by the method determined by
the Ministry of Agriculture, Livestock and Food Supply [15]. To

separate the milk serum from its clot, a solution of 10% glacial
acetic acid was added to a sample of pasteurized milk preheated to
35  C until the sample reached a pH of 4.6. The levels of AFLAB1 and
AFLAM1 and the protein content in both fractions were determined. This procedure was performed in samples of control milk
(without contamination by addition of the standard) and on
samples spiked with both aatoxins standards in the nal
concentration of 5 mg L1. For these determinations, we used
the extraction method of AOAC [11], as well as a subsequent
quantication by UHPLC-FL.
3. Results and discussion
To obtain an optimal chromatographic separation between
AFLAB1 and AFLAM1 and a reasonable run duration, different ratios
of mobile phase were tested (Table 1). Tests were made for the
retention factor, which indicates the relation between the time
each mycotoxin was retained in the stationary phase and the time
that it was being carried by the mobile phase. The separation factor
was also tested, indicating the selectivity of the chromatographic
system of mycotoxins eluting at adjacent peaks, both estimated for
the different elution gradients of their stationary phase (C18).
The proportion of the mobile phase used in Experiment 1 was
indicated by the ofcial method [11] using a mixture of patterns of
both aatoxins, at the same concentration (0.01 mg mL1), and
resulted in peaks eluted in less than 5 min of run, which could be
affected by other components of the sample eluted in the
beginning of the run. To increase the retention time, the plots of
1% acetic acid and methanol were increased, considering that the
polar character of these solvents can help a better retention of
aatoxins in the column. In Experiment 2, standards with
concentrations of 0.5 mg mL1 were used, resulting in a small
retention time. In the Experiments 4 and 5, the retention time of
AFLAB1 was too long, which promoted the generation of waste and
the reduction of the analytical efciency, resulting in a 25 min run.
Experiment 3 had the most suitable retention times for both
aatoxins, with a decreased risk of the initial interference in a total
running time of 15 min. To conrm the efciency of the separation
under these conditions, we tested it with the AFLAB1 standard
(0.5 mg mL1), the AFLAM1 standard (0.1 mg mL1) and a mixture of
both aatoxins (0.05 mg mL1), obtaining the same retention time
in all cases. In this gradient also occurred the largest separation
factor, indicating that the peaks are presented as far as possible
from each other, ensuring the best selectivity among the
performed tests and a sufciently small interval not to result in
peak broadening (Fig. 1).
The analytical curves for both aatoxins, as well as their
parameters (Table 2), show that the procedure was suitable for the
quantication of both aatoxins. Their indicators meet the
recommendations of analysis reliability, considering the recommendations of ANVISA [16] (a correlation coefcient of 0.99) and of
INMETRO [17] (values above 0.9). Furthermore, the linearity of the
curves showed a wide range of application, and the matrix curves

Table 1
Separation efciency of the mycotoxins eluted by different mobile phases.
Exp.

1
2
3
4
5

Acetic acid 1%:acetonitrile:methanol

40:35:25
50:15:35
55:10:35
60:05:35
65:10:25

Exp.-experiment, AFLAB1-aatoxin B1, AFLAM1-aatoxin M1.

Retention time (tr) (min)

Separation factor (a)

Retention factor
(k)

AFLAM1

AFLAB1

AFLAM1

AFLAB1

AFLAM1  AFLAB1

3.4
5.2
6.6
11.8
13.5

4.4
8.0
11.8
20.0
25.0

0.36
1.08
1.64
3.72
4.40

0.76
2.20
3.72
7.00
9.00

2.11
2.04
2.27
1.88
2.05

P.T. Scaglioni et al. / Analytica Chimica Acta 829 (2014) 6874

have the advantage of carrying the interferents inherent to the

sample extract.
The limits of detection and quantication for both the
instrument and the method were estimated by the signal-noise
ratio, which establishes the minimum concentration at which the
analyte can be easily detected. Botura [18] determined the LOD and
LOQ values for a modied method to determine AFLAM1 in goat
milk using UHPLC-FL as equal to 0.2 and 0.5 mg L1, respectively.
However, in order to validate a method to quantify AFLAM1 in
cows milk, Oliveira [19] determined LODm and LOQm values as
equal to 0.003 and 0.007 mg L1, respectively, using HPLCMS/MS.
The values of LODm and LOQm found in this study were among
those cited by the authors and were considered satisfactory,

71

considering that the LOQm presented a value below the legislated

for milk AFLAM1 in Brazil (0.5 mg kg1) [20].
According to the guideline of the European Commission [21],
which makes specic performance recommendations for essays
on mycotoxin recovery, when AFLAM1 is used at concentration
levels greater than 0.05 mg L1, the recommended recovery value is
70110%. Our results (Table 3) show recoveries that were
maintained within acceptable levels. Furthermore, when it is
necessary to recover mycotoxins with different polarities, the
efciency can be compromised due to a greater or lesser afnity of
each mycotoxin with the eluent. Such is the case in the
recuperation results, as the contamination levels increased. In
this study, AFLAM1 showed higher recoveries in comparison to

Fig. 1. Elution chromatograms of the AFLAB1 and AFLAM1 in UHPLC-FL using different mobile phase gradients composed of 1% acetic acid:acetonitrile:methanol. (1)
40:35:25; (2) 50:15:35; (3) 55:10:35; (4) 60:05:35; (5) 65:10:25.

72

P.T. Scaglioni et al. / Analytica Chimica Acta 829 (2014) 6874

Table 2
Analytical parameters evaluated in UHPLC-FL.
Analytical parameters

AFLAB1

AFLAM1

Solvent curves
Linearity (mg L1)
Coefcient of correlation
Coefcient of determination
LODi (mg L1)
LOQi (mg L1)

y = 2,947,476x 8261.30
4.5 a 108
0.9987
0.9974
1.5
4.5

y = 2,246,144 x 6973.83
4.5 a 270
0.9991
0.9983
1.5
4.5

Work curve
Linearity (mg L1)
Coefcient of correlation
Coefcient of determination
LODm (mg L1)
LOQm (mg L1)

y = 1,506,689x 14,579.84
0.5 a 162
0.9918
0.9836
0.2
0.5

y = 693,877.2x 5680.73
0.25 a 162
0.9933
0.9866
0.09
0.25

AFLAB1-aatoxin B1, AFLAM1-aatoxin M1, LOD1-instrument detection limit, LOQ1-instrument quantitation limit, LODm-method detection limit, LOQm-method quantitation
limit.

Table 3
Recovery of the AFLAB1 and AFLAM1 extracted from milk by the AOAC method.
Fortication (mg L1)

0.25
0.5
3.0
6.0
8.0
10.0
Average

Recovery (%) (CV%)

AFLAB1

AFLAM1

83 (3)
78 (16)
73 (14)
76 (21)
98 (12)
82

75 (29)
91 (15)
114 (15)
111 (33)
94 (11)
77 (19)
94

AFLAB1-alfatoxin B1, AFLAM1-aatoxin M1, CV%-coefcient of variation.

AFLAB1, a fact that was already expected, since the conditions of

the ofcial method were designed to quantify AFLAM1. However, it
was veried that AFLAB1 can also be quantied within reliable
analytical conditions.
Atanda et al. [22] evaluated the AFLAM1 contamination in milk
and ice cream consumed in Nigeria using the same extraction
method of this study [11], obtaining recovery values of 84.6, 85 and
88% for contamination levels equal to 20, 25 and 30 mg L1. Thus,
according to the ndings of the authors cited above, even with
contamination levels much larger than the legislated limits
(0.5 mg kg1) the method still presents a satisfactory recovery
for AFLAM1.
Samples of raw, pasteurized, UHT, concentrated and powdered
milk were analyzed in order to determine their natural contamination by AFLAB1 and AFLAM1 (Table 4). Fig. 2 shows chromatograms of pasteurized milk with and without natural contamination
samples. We note that the retention time of both aatoxins has not
changed in relation to the standard.
Other studies on the incidence of AFLAB1 in milk are scarce,
considering that the legislation of most countries only imposes

maximum concentrations to AFLAM1 in milk and its derivates.

However, more attention should be given to the study of AFLAB1,
considering that in Brazil it has been found in limits higher than
the ones legislated for its hydroxylated derivate (AFLAM1) in
pasteurized and UHT milk. Gurbay et al. [10] veried the
contamination by both aatoxins in 100% of the analyzed samples
of human milk originated from Turkey, nding levels from 0.095 to
4.1 mg L1 for AFLAB1 and 0.061 to 0.3 mg L1 for AFLAM1. Results
such as the ones presented in this work show that the aatoxin can
be excreted in its non hydroxilated form through the human and
animal organism. This fact suggests that the animal had a diet with
high mycotoxin levels, which surpassed the metabolic capacity of
the individual.
Another fact to be highlighted is that all of the samples with the
incidence of AFLAM1 had this mycotoxin in concentrations above
the legislated limit in Brazil. This enforces the necessity of a more
adequate control both in milk processing and in the handling of
animal producers. Only the powdered milk samples did not show
contamination by the aatoxins. This fact shows the need to study
the processing conditions that brought about this adequacy for
consumption. However, the collection period of the samples may
have inuenced this elevated incidence since, during winter,
granaries are the predominant source of the cattle due to pasture
scarcity. Therefore, due to its preparation conditions, this type of
food presented higher levels of contamination by mycotoxins.
Other recent studies show that the contamination by AFLAM1
occurs in different samples from many parts of the world. Duarte
et al. [23] veried the incidence of AFLAM1 in 40 samples of semiskimmed pasteurized and UHT milk commercialized in Portugal
and reported that 27.5% of the samples were contaminated with an
average concentration of 0.0234 mg L1. In addition, 5% of these
samples had values above the limits of the European legislation
(0.05 mg L1). Rahimi et al. [24] veried the presence of AFLAM1 in

Table 4
Incidence of AFLAB1 and AFLAM1 in different samples of milk.
Milk sample

AFLAB1
% incidence

Raw
0 (0/7)
Pasteurized
41.7 (5/12)
UHT
13.3 (2/15)
Concentrated 0 (0/3)
Powdered
0 (0/3)

AFLAM1
Average contamination (mg L1)
(CV%)

% above the
legislationa

%
incidence

Average contamination (mg L1)

(CV%)

% above the legislation b

ND
1.476 (51)
0.690 (20)
ND
ND

100
100

28.6
58.3
66.7
66.7
0 (0/3)

0.835 (9)
0.884 (42)
1.168 (27)
1.718 (8)
ND

100
100
100
100

ND = not detected, CV% = variation coefcient.

a
Number of contaminated samples/number of analyzed samples.
b
Brazilian Legislation for AFLAM1 in uid milk (0.5 mg kg1).

(2/7)
(7/12)
(10/15)
(2/3)

P.T. Scaglioni et al. / Analytica Chimica Acta 829 (2014) 6874

Fig.

73

2. Chromatograms of pasteurized milk without (1) and with (2) natural contamination by aatoxins.

79% of samples of raw cow milk, with an average concentration of

0.06 mg L1 (57.4). Among these samples, 36% had contamination
levels above the legislated limit of the European Community.
An acidic clotting of a pasteurized milk sample was performed
to verify the distribution of aatoxins and of the proteic content
between the serum and the clot (Table 5) of control and spiked
(5 mg L1) samples.
Both aatoxins adhered to the milk clot, with a recovery of 102%
for AFLAM1 in this fraction, when the control sample was coagulated.
A recovery of 100 and 111% was obtained for AFLAB1 and AFLAM1,
respectively, in the spiked sample clot. These results reinforce the
importance of establishing maximum permitted levels for dairy
products obtained by milk coagulation. They also highlight the
necessity of a rigid control of raw materials, considering that
currently the Brazilian legislation establishes only a maximum
tolerable limit for AFLAM1 in cheese (2.5 mg kg1).
Deveci [25] quantied AFLAM1 during the production and
storage of canned white cheese from milk samples spiked with the
mycotoxin in two concentrations (1.5 and 3.5 mg kg1). For both

concentrations, after the cheese was produced, about 60% of the

AFLAM1 remained in the clot cheese, while close to 30% was
transferred to the milk serum.
In a small cheese production from cheese articially contaminated with AFLAM1, Lpez et al. [26] veried, that 60% of the
AFLAM1 was found in the serum and 40% in the cheese. Hassanin
[27] investigated the stability of the AFLAM1 during the production
and storage of yogurt, cheese and acidied milk. The author
concludes that milk AFLAM1 is transmitted to its products, since
the presence of AFLAM1 in cheese can be due to the fact that the
toxin binds to casein and, on the other hand, that part of the milk
serum remains in the strands of the clot. Contrarily to these
studies, in the present study none of the aatoxins was detected in
the serum, indicating the total permanence of the mycotoxins in
the proteic fraction, when the acid condition was used for
coagulation. Thus, it was demonstrated that the contamination
between the serum and the clot depends on the coagulation
method used in the fractioning. This is an important subject to be
studied in order to develop recommendable strategies of

Table 5
Distribution of AFLAB1 and AFLAM1 between coagulated milk fractions.
Sample

Control

Spiked

Milk
Serum
Clot
Milk
Serum
Clot

Humidity
(%)
(CV%)

Proteins (%)
(CV%)a

AFLAB1 (mg L1) (CV%)

AFLAM1 (mg L1) (CV%)

88.9 (0.5)
94.8 (0.1)
27.3 (1.9)

2.7 (7.4)
0.6 (17.0)
11.7 (15.0)

ND
ND
ND
5.11 (9.5)
ND
5.42 (8.2)

1.20 (11.1)
ND
1.22 (6.4)
5.86 (5.7)
ND
6.48 (11.5)

AFLAB1-aatoxin B1, AFLAM1-aatoxin M1, CV%-variation coefcient, n = 3.

a
Estimated in humid basis.

74

P.T. Scaglioni et al. / Analytica Chimica Acta 829 (2014) 6874

processing to minimize the risk of contamination by milk product

consumption.
4. Conclusion
The chromatographic method validated for mycotoxins resulted
in a better proportion of the contents of the mobile phase, with a
separation factor of 2.3. In addition, the analytical parameters of the
method fullled the requirements of reliability, showing correlation
coefcients above 0.99, with LOQm values of 0.5 and 0.25 mg L1 for
AFLAB1 and AFLAM1, respectively. AFLAB1 was detected in 42% of the
pasteurized milk samples and 13% of the UHT milk samples, in levels
between 0.7 and 1.5 mg L1. AFLAM1 was present in 29% of the raw
milk samples, 58% of pasteurized, 67% of UHT and 67% of
concentrated, and all the samples that showed a natural contamination by both aatoxins were above the legislated limit in Brazil
(0.5 mg L1). Only the powdered milk samples did not show a natural
incidence of both aatoxins, considering the quantication limits of
the method. Acid milk coagulation promoted the accumulation of
contaminants in the clot
References
[1] A. Kwiatkowski, A.P.F. Alves, Importncia da deteco e do controle de
aatoxinas em alimentos, Sade e Biologia 2 (2007) 4453.
[2] E.E. Creppy, Update of survery, regulation and toxic effects of mycotoxins in
Europe, Toxicology Letters 127 (2002) 1928.
[3] V.M. Scussel, Micotoxinas em Alimentos, Editora Insular, Florianpolis, 1998.
[4] J. Zhao, R.B. Shirley, J.D. Dibner, F. Uraizee, M. Ofcer, M. Kitchell, M. VazquezAnon, C.D. Knight, Comparison of hydrated sodium calcium aluminosilicate
and yeast cell wall on counteracting aatoxicosis in broiler chicks, Poultry
Science 89 (2010) 21472156.
[5] International Agency of Research on Cancer- IARC, Monographs on the
Evaluation of Carcinogenic Risks to Humans, WHO, Lyon, 2002 v. 82.
[6] H.A. Partanen, H.S. El-Nezami, J.M. Leppnen, P.K. Myllynen, H.J. Woodhouse,
K.H. Vhkangas, Aatoxin B1 transfer and metabolism in human placenta,
Toxicological Sciences: an Ofcial Journal of the Society of Toxicology 113
(2010) 216225.
[7] F. Caloni, A. Stammati, G. Frigg, I.D. Angelus, Aatoxin, M1 absorption and
cytotoxicity on human intestinal in vitro model, Toxicon 47 (2006) 409415.
[8] OPS, Organizacin Panamericana de la Salud, Micotoxinas Critrios de Salud
Ambiental, OPS, Washington, 1983.
[9] R.D. Stubbleeld, H.P. Van-Egmond, Chromotographic methods of analysis for
aatoxin M1, in: VAN-EGMONDHP (Ed.), Mycotoxins in Dairy Products,
Elsevier Applied Science, London, 1989.

[10] A. Gurbay, A. Sabuncuoglu, G. Girgin, G. Sahin, S. Yigit, M. Yurdakok, G.

Tekinalp, Exposure of newborns to aatoxina M1 and B1 from mothers breast
milk in Ankara, Turkey, Food and Chemical Toxicology 48 (2010) 314319.
[11] W. Horwitz, Ofcial Methods of Analysis International, 17th ed., Association of
Ofcial Analytical Chemists AOAC, USA, 2000.
[12] C.H. Collins, G.L. Braga, P.S. Bonato, Fundamentos de Cromatograa, Editora da
Unicamp, Campinas, 2006.
[13] L.V. Soares, Curso Bsico de Instrumentao Para Analistas de Alimentos e
Frmacos, Editora Manole Ltda, Baueri-SP, 2006.
[14] M. Ribani, C.B.G. Bottoli, C.H. Collins, C.S.F. Jardim, L.F.C. Melo, Validao em
mtodos cromatogrcos e eletroforticos, Qumica Nova 27 (2004) 771780.
[15] A.C.R. Krolow, M.E.R. Ribeiro, BRASIL, MAPAMinistrio da Agricultura,
Pecuria e Abastecimento. Obteno de leite com qualidade e elaborao
de derivados, Embrapa Clima Temperado 154 (2006) Documentos, ISSN 18069193.
[16] BRASIL, ANVISA. Agncia Nacional de Vigilncia Sanitria, Resoluo RE n 899,
de 29/05/2003. Disponvel em: www.anvisa.gov.brwww.anvisa.gov.br. Acesso
em: dez; 2013.
[17] Brasil, INMETRO. Instituto nacional de metrologia, normalizao e qualidade
industrial, orientaes sobre validao de mtodos de ensaios qumicos. DOQCGCRE -008; 2003.
[18] M.B. Botura, Otimizao de Mtodos Analticos Para Determinao de
Aatoxinas em Raes e Leite de Cabra, e sua Ocorrncia no Estado da Bahia.
Dissertao (Mestrado em Medicina Veterinria Tropical), Escola de Medicina
Veterinria da Universidade Federal da Bahia, Bahia, 2005, pp. 2005.
[19] M.S. Oliveira, Validao de Metodologia Analtica Para Anlise de Aatoxina
M1 e sua Ocorrncia em leite Bovino Comercializado no sul do Brasil.
Dissertao (Mestrado em Cincia e Tecnologia de Alimentos), Universidade
Federal de Santa Maria, Santa Maria, 2010.
[20] BRASIL. ANVISA-Agncia Nacional de Vigilncia Sanitria, Resoluo RDC n 7,
de 18/02/2011. Disponvel em: www.anvisa.gov.brwww.anvisa.gov.br. Acesso
em: dez; 2013.
[21] COMISSO EUROPIA - Regulamento (CE) n 401/2006 da Comisso, de 23 de
Fevereiro de 2006. Mtodos de amostragem e de anlise para o controle ocial
dos teores de micotoxinas nos gneros alimentcios. Disponvel em http://eurlex.europa.eu/pt/index.htm Acesso: dez; 2013.
[22] O. Atanda, A. Oguntubo, O. Adejumo, J. Ikeorah, I. Akpan, Aatoxin M1
contamination of milk and ice cream in Abeokuta and Odeda local governments of Ogun state, Nigeria, Chemosphere 68 (2007) 14551458.
[23] S.C. Duarte, A.M. Almeida, A.S. Teixeira, A.L. Pereira, A.C. Falco, A. Pena, C.M.
Lino, Aatoxin M1 in marketed milk in Portugal: assessment of human and
animal exposure, Food Control 30 (2013) 411417.
[24] E. Rahimi, M. Bonyadian, M. Rafei, H.R. Kazemeini, Occurrence of aatoxina M1
in raw milk of ve dairy species in Ahvaz, Iran, Food and Chemical Toxicology
48 (2010) 129131.
[25] O. Deveci, Changes in the concentration of aatoxin M1 during manufacture
and storage of white pickled cheese, Food Control 18 (2007) 11031107.
[26] C. Lpez, L. Ramos, S. Ramadn, L. Bulacio, J. Perez, Distribution of aatoxin M1
in cheese obtained from milk articially contaminated, International Journal
of Food Microbiology 64 (2001) 211215.
[27] N.I. Hassanin, Stability of aatoxin M1 during manufacture and storage of
yoghurt, yoghurt-cheese and acidied milk, Journal of the Science of Food and
Agriculture 65 (1994) 3134.