Food Chemistry 114 (2009) 1141–1146

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Analytical Methods

Determination of aflatoxins in eggs, milk, meat and meat products

using HPLC fluorescent and UV detectors
Saqer M. Herzallah *
Department of Nutrition and Food Science, Faculty of Agriculture, Mu’ta University, Karak, Jordan

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

Article history: Raw and pasteurised sheep’s, cow’s and goat’s milk, eggs, and beef samples from different local markets
Received 30 March 2008 in Jordan were collected during a period of 5 months (January through May 2007) and examined for afla-
Received in revised form 30 October 2008 toxins B1(AFB1), B2(AFB2), G1(AFG1), G2(AFG2), M1(AFM1) and M2(AFM2). The samples were analysed
Accepted 30 October 2008
with high performance liquid chromatography (HPLC) using UV and Fluorescent detectors. The analysed
samples of milk collected in January were found to contain 0.56 lg L 1 AFM1 and 0.1 lg L 1 AFM2 whilst,
the concentration of AFM1 and AFM2 was < 0.05 lg L 1 for milk samples collected between March and
Keywords:
May. The AFB1, AFB2, AFG1 and AFG2 contents in the analysed food products ranged from 1.10 to
Aflatoxin
M1 (AFM1)
8.32 lg L 1 and 0.15 to 6.36 lg L 1 in imported and fresh meat samples collected during March, respec-
M2 (AFM2) tively. The mean recovery for the HPLC method was 92% to 109% and the quantification levels were
B1 (AFB1) 50 ng L 1 for AFM1 and AFM2. The AFM1 was found in 10% of the tested samples with concentrations
B2 (AFB2) between 0.08 and 1.1 lg kg 1 and AFM2 was only found in 1.82% of the tested samples with a level of
G1 (AFG1) 0.1 lg kg 1. The AFM1 levels in the examined foods were higher than the maximum level of AFM1 in
G2 (AFG2) liquid milk set by the European Community and Codex Alimentarius of 50 ng L 1.
High performance liquid chromatography Ó 2008 Elsevier Ltd. All rights reserved.
(HPLC)

1. Introduction
Mycotoxins are natural food and feed toxic contaminants pro-
duced as a result of fungal growth on agricultural materials during
storage and/or transportation. These compounds diffuse into foods
and accumulate in the fat parts. Mycotoxins–aflatoxins (AF) in spe-
cific – are the main toxic secondary metabolites of the genus Asper-
gillus flavus and Aspergillus parasiticus (Ali et al., 2005; Asis,
Romina, Paola, & Mario, 2002; Rizzi, Simioli, Roncada, & Zaghini,
2003). Aflatoxins are considered the most serious threat to public
health due to their carcinogenic and hepatotoxic, teratogenic and
mutagenic effect in human and animals (Henry, Bosch, Troxell, &
Bloger, 1999; Maqbool, Ahmad, Anwar-Ul_Haq, & Iqbal, 2004;
Wang et al., 1996, 1998, 1999; Wild & Turner, 2002). Aflatoxins
AFB1, AFB2, AFG1 and AFG2 are metabolic products produced in
food and feed contaminated by Aspergillus species. The presence
of aflatoxins residues in food of animal origin such as meat, milk,
eggs and cheese may be a result of feed contamination (Peraica,
Domijan, Ana-Marija, Jurjevic, & Cvjetkovic, 2002). Aflatoxins M1
and M2 are a thermo-resistant hydroxylated metabolites produced
by lactating animals consuming aflatoxin contaminated feeds. The
ingested AFB1 and AFB2 are metabolized by livestock into AFM1
and AFM2, respectively, with estimated conversion ratio of 1–3%
* Tel.: +962 0795 80 1317; fax: +962 3 2323151.
E-mail address: saqermay@yahoo.com
between AFB1 and AFM1 (Ali, Hashim, & Yoshizawa, 1999; Barbi-
eri, Bergamini, Ori, & Pesca, 1994). The limits of AFB1 and total
AF in foods are 5 and 10 lg kg 1, respectively, in more than 75
countries around the world whilst they are 2 and 4 lg kg 1 in
the European Union (EU) (Lopez, Ramos, Ramadan, & Bulacio,
2003; Van Egmond & Jonker, 2004).
The aim of this study was to evaluate the quality of foods of ani-
mal origin retailed in Jordan in terms of its compliance with inter-
national aflatoxins limits using an optimised HPLC method.
2. Materials and methods
2.1. Chemical and reagent
Aflatoxins standards were purchased from Sigma (St. Louis, MO,
USA) with purity of 98%; standard stock solutions were prepared in
acetonitrile according to the AOAC method (International Agency
for Research on Cancer (IARC), 2002). The samples were cleaned
by passing them through solid phase extraction cyanopropyl car-
tridges (SPE-CN) purchased from Varian. The HPLC analysis was
performed on Si-column from Thermo. High-grade methanol, chlo-
roform, acetonitrile, ethyl acetate, acetic acid, formic acid, toluene,
and HPLC water were purchased from Tedia (Fairfield, OH, USA).
Diatomaceous earth (SiO2) was purchased from Sigma (St. Louis,
MO, USA) and Sodium sulphate anhydrous used (99.5%) was pur-
chased from American Chemical Society (ACS, Madrid, Spain).

0308-8146/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2008.10.077
1142 S.M. Herzallah / Food Chemistry 114 (2009) 1141–1146

2.2. Sampling
Two hundred and twenty samples from meat (poultry, lamb,
goat and beef), eggs, milk and feed were collected randomly during
the period January through May 2007 from different locations in
Jordan. Four samples from each feed of 2 kg, 1 kg from different
meat types, 15 eggs and three litres of milk (bovine and/or sheep’s
milk) were collected every week for 5 months. These samples were
tested for the presence of mycotoxins.
2.3. Aflatoxin working standards
the conditioned SPE-CN. The column then washed with 0.5 mL of
20% tetrahydrofuran (THF) in 0.5% aqueous acetic acid. This was
followed by passing 2 mL of hexane through the SPE tube, and then
dried under nitrogen. The SPE tube was washed with 3 mL of 25%
THF in hexane and dried for 1 min under N2. The retained aflatox-
ins by the SPE-CN column were eluted with 2  2 mL from 1% THF
in methylene chloride then dried over a stream of nitrogen in a
concentrator and reconstituted in 0.5 mL of toluene. 20 ll of the
reconstituted sample were injected in the HPLC using UV detector
at 365 nm and fluorescent detector at 365 and 425 nm for excita-
tion and emission, respectively.

Six working standard dilutions of 0, 0.05, 1, 3, 6, 12 and

24 lg kg 1 were prepared from aflatoxin standard (AFB1, AFB2,
AFG1, AFG2, M1 and M2) purchased from Sigma (St. Louis, MO,
USA).
2.4. Test protocol
Fifty gram from the homogenised food samples were mixed in a
blender (Waring, CA, USA) with 100 mL of acetone and 100 mL of
water for 10 min, 10 g of diatomaceous earth were added and stir-
red (gently) for 5 min and then filtered through fast filtering What-
man No. 1 filter paper. 100 mL of the filtrate were mixed with
50 mL of 5% NaCl and 50 mL of hexane and shaken (gently) on a
mechanical shaker (IKA, GmbH, Germany) for 5 min at 2400 rpm.
The hexane layer was discarded. Next, 50 mL of 5% NaCl and
150 mL of chloroform (3  50 mL) were added to the aqueous layer
and shaken (gently) for 5 min each time. The chloroform layer was
collected from the three extractions, dried over anhydrous sodium
sulphate and evaporated using rotary evaporator. The residues
were redissolved in 1 mL chloroform and cleaned over SPE-CN.
2.5. Clean-up procedure
The clean-up of the extract was carried out with SPE column
supelcoclean LC-CN 500 mg/3 mL. The SPE column was condi-
tioned with 2 mL of 0.5% aqueous acetic acid, then 1 mL of the fil-
tered extract followed by 4 mL of 0.5% acetic acid were loaded over
2.6. HPLC determination
The chromatography was carried out with Water HPLC
equipped with Water 1525 binary HPLC pump, column oven 5CH
(Waters Co., MA, USA), Waters 2487 UV/vis adjusted at 365 nm
and fluorescence detector (Model FL 2475) at wavelength 365
and 425 nm for excitation and emission, respectively. Control
and data analysis was performed using Breeze (3.3 Water soft-
ware) and manual injection Reodyne (Waters Co., MA, USA). The
column used was 250  4.6 mm Thermo LC-Si and the mobile
phase was composed of toluene, ethyl acetate, formic acid and
methanol (90:5:2.5:2.5). The column was kept in a column oven
at 40 °C at a flow rate of 2.0 mL per min to achieve the optimum
resolution of aflatoxins.
2.7. Recoveries of aflatoxins
A mixed aflatoxins (AF) standard was prepared by spiking AF-
free samples of milk and meat with AFB1, AFB2, AFG1, AFG2,
AFM1 and AFM2 to concentrations of 2, 5, 10, 15 and 20 lg kg 1
of each. The spiked samples were placed in darkness overnight at
room temperature and analysed for the aflatoxins. The concentra-
tion of AF was calculated using calibration curves that were pre-
pared from the standard aflatoxins in the range of 0.05–
24 ng mL 1. The standard curves were found to be linear with cor-
relation coefficient of 0.998.

Table 1
Aflatoxin recovery of AFB1, AFB2, AFG1, AFG2, M1 and M2 in spiked AF-free milk sample determined by HPLC method.

AFlg kg-1 % RecoveryA

AFB1 AFB2 AFG1 AFG2 M1 M2
2 88.32b ± 8.7 (9.8%) 90.21b ± 9.2 (10.2%) 90.20b ± 10.1(11.2%) 92.11c ± 9.8 (10.6%) 95.12b ± 11.1 (11.7%) 91.60b ± 9.7 (10.6%)
5 95.53ab ± 7.4 (7.7%) 96.22a ± 8.2 (8.5%) 96.6a ± 9.1 (9.4%) 95.31ab ± 7.8 (8.1%) 97.81ab ± 7.8 (7.9%) 96.70a ± 8.1 (8.3%)
10 97.14a ± 5.6 (5.8%) 98.25a ± 6.8 (6.9 %) 98.30a ± 7.5 (7.6%) 94.22b ± 5.9 (6.3%) 98.10a ± 6.3 (6.4%) 95.6ab ± 6.8 (7.1%)
15 98.21a ± 3.8(3.7%) 97.61a ± 5.2 (5.3 %) 96.30a ± 5.6 (5.8%) 98.12a ± 4.2 (4.3%) 99.10a ± 5.0 (5.0%) 96.50a ± 6.0 (6.2%)
20 97.22a ± 2.5(2.6%) 98.10a ± 3.8 (3.9%) 96.20a ± 4.9 (5.1%) 95.30ab ± 3.8 (3.9 %) 97.80ab ± 3.6 (3.7%) 95.22ab ± 5.4 (5.7%)
A
Results are represented as means ± SD and coefficient of variability (CV%) for 5 replicates (n = 5). Means within a column with different superscripts differ statistically
(P < 0.05).

Table 2
Aflatoxin recovery of AFB1, AFB2, AFG1, AFG2, M1 and M2 in spiked AF-free meat sample determined by HPLC method.

AFlg kg-1 % recoveryA

AFB1 AFB2 AFG1 AFG2 M1 M2
b b ab ab c
2 95.10 ± 9.7(10.2%) 96.10 ± 10.2(10.6%) 94.21 ± 11.1(11.8%) 93.50 ± 10.2(10.9%) 90.50 ± 12.2(11.9%) 88.90c ± 10.8 (12.1%)
5 96.20ab ± 8.3 (8.6%) 97.30ab ± 9.1 (9.4%) 92.40b ± 10.2 (9.4%) 91.70b ± 8.9 (9.7%) 94.91b ± 10.6(11.2%) 91.70b ± 9.1 (9.9%)
10 96.11ab ± 6.3 (6.6%) 99.20a ± 8.0 (8.1 %) 95.11a ± 8.4 (8.8 %) 94.20ab ± 7.4 (7.9%) 99.10a ± 8.2 (8.3%) 89.60c ± 6.8 (7.6%)
15 99.50a ± 5.8 (5.8%) 99.10a ± 6.9 (6.9 %) 95.20a ± 6.8 (7.1%) 96.10a ± 3.1 (3.2%) 98.70a ± 6.5(6.6%) 94.50ab ± 5.3 (5.6%)
20 98.20a ± 3.1 (3.2%) 98.10a ± 4.8(4.9 %) 96.70a ± 3.9 (4.0 %) 95.60a ± 2.5 (2.6 %) 97.30ab ± 4.3 (4.4%) 96.20a ± 4.9(5.1%)
A
Results are represented as means ± SD and coefficient of variability (CV%) for 5 replicates(n = 5). Means within a column with different superscripts differ statistically
(P < 0.05).
 S.M. Herzallah / Food Chemistry 114 (2009) 1141–1146 1143

Table 3
Aflatoxin recovery of AFB1, AFB2, AFG1, AFG2, M1 and M2 in spiked AF-feed samples determined by HPLC method.

AFlg kg-1 % recoveryA

AFB1 AFB2 AFG1 AFG2 M1 M2
2 96.30a ± 11.8(12.3%) 96.20a ± 12.1(12.6%) 97.20a ± 12.2(12.6%) 94.10b ± 11.7(12.4%) 95.10a ± 10.7(11.3%) 91.60a ± 12.5(13.6%)
5 97.50a ± 10.6 (10.9%) 97.20a ± 9.0 (9.3%) 98.60a ± 10.6(10.8%) 98.30a ± 10.0(10.2%) 93.8ab ± 8.1 (8.6%) 92.70a ± 11.4(12.3%)
10 98.10a ± 9.2(9.4%) 99.2a ± 7.2(7.3%) 98.30a ± 8.7 (8.9%) 97.20a ± 8.3(8.5%) 95.10a ± 6.8(7.1%) 92.60a ± 9.5(10.3%)
15 98.20a ± 8.6(8.8%) 97.60a ± 6.3(6.5%) 96.30a ± 6.1(6.3%) 98.10a ± 6.1(6.2%) 92.10b ± 6.1(6.6%) 93.50a ± 8.2(8.8%)
20 97.20a ± 6.4(6.6%) 97.10a ± 4.4(4.5%) 98.20a ± 4.2(4.3%) 98.30a ± 4.7(4.8%) 88.80c ± 5.7(6.4%) 86.20b ± 6.7(7.8%)
A
Results are represented as means ± SD and coefficient of variability (CV %) for 5 replicates (n = 5). Means within a column with different superscripts differ statistically
(P < 0.05).

Table 4
Comparison of results for AF analyzed by fluorescent (FL) and UV detectorA.

Sample name No. of samples Aflatoxins concentration (lg kg-1)B

UV /365 nm Flourescent (365/425)
AFB1 AFB2 AFG1 AFG2 M1 M2 AFB1 AFB2 AFG1 AFG2 M1 M2
Milk 10 0.21d <0.1 <0.1 <0.1 0.51b <0.1 0.3c 0.11b 0.18b 0.06b 0.62b 0.05b
Meat 10 0.30c <0.1 <0.1 <0.1 0.21c <0.1 0.36c 0.17b 0.08c <0.05 0.20c 0.06b
Eggs 10 1.23b <0.1 <0.1 <0.1 <0.1 <0.1 1.43b 0.12b <0.05 <0.05 0.06d <0.05
Spiked AF free Meat (2 lg kg-1) 1.91a 1.93a 1.90a 1.87a 1.82a 1.82a 2.02a 1.97a 1.93a 1.88a 2.03a 1.78a
Recovery % 95.5 96.5 95 93.5 91 90 101 98.5 96.5 94 101.5 89
A
Detection limit of Aflatoxins were 0.05 lg kg-1with fluorescent detector (P < 0.05) whereas, 0.1 with UV detector (P < 0.05).
B
Results are represented as means for 5 replicates (n = 5). Means within a column with different superscripts differ statistically (P < 0.05).

2.8. Statistical Analysis
Results are presented as means (±SD), CV%, of measurements of
n = 5. ANOVA was also used in the general linear model procedure
in PC-SASÒ version 7.0 (SAS, 2000). Variable means for measure-
ments showing significant differences in the ANOVA were com-
pared using the least significant difference procedure. Values
were judged to be significantly different (LSD) if P < 0.05.
3. Results and discussion
Aflatoxins recoveries from milk samples as determined by HPLC
are presented in Table 1. The recovery rate was 88.3% at 2 lg kg 1
compared to 98.2% and 97.2% for B1 at concentration 15 and
20 lg kg 1 respectively. Also, the recovery for M1 was high at
15 lg kg 1 (99.1%) when compared to the recovery at 2 lg kg 1
(95.5%). The recoveries in all concentrations were found to be more
than 88.3% regardless of the spiking concentration from the same
food matrices (milk samples). Furthermore, there was a significant
(P < 0.05) difference found between the recoveries for the concen-
tration 2 lg kg 1 and those greater than 2 lg kg 1.
The recoveries of the aflatoxins (B1, B2, G1, G2, M1 and M2)
with the HPLC with UV detector were evaluated and compared
with HPLC fluorescent (FL) detector. The results indicate that the
proper method of extraction, as well as the clean-up process, en-
hanced the detection limit of aflatoxins – in particular for M1
and M2. The optimised condition of the used method, as applied,
was found to be of importance to help the less sophisticated labo-
ratories with HPLC instruments equipped with UV detector to de-
tect AF-toxins with a precision that complies with the international
guidelines and regulations. The results revealed that the recoveries
of the four aflatoxins and their metabolites (M1 and M2) were very
high and the interference effects were small. These results were in
agreement with the previous research, i.e. at the lower concentra-
tion of AF distribution within the sample, the losses increase and
the recovery decreases (Garner, Whattam, Taylor, & Stow, 1993;
International Agency for Research on Cancer (IARC), 2002; Stroka,
Anklam, Joerissen, & Gilbert, 2000). Lower recovery percentage
was also reported by Gobel and Lusky (2004) and Nakajima
(2004) as a result of using Immunoaffinity clean-up column.
The recoveries of aflatoxins and their metabolites from meat
and feed samples that were determined by the HPLC fluorescent
detector are shown in Tables 2–4. The recoveries for aflatoxins
(AFB1, AFB2, AFG1, AFG2, M1 and M2) from meat samples spiked
with different levels before extraction and analysed by the opti-
mised HPLC method in this study were 95.10%, 96.10%, 94.21%,
93.50%, 90.50% and 88.90%, respectively. The low recoveries for
aflatoxins metabolites M1 and M2 were noticed (90.50% and
88.9%, respectively) when compared with other aflatoxins. Table
3 shows the recoveries of aflatoxins from spiked feed samples at
levels range from 2 to 20 lg kg 1. Moreover, the recoveries from
the three matrices (milk, meat and Feed) were found to be more
than 86.20%. The incidence level of aflatoxins in the analysed sam-
ples was high, which must be considered as one of the potential
sources of contamination with AF-toxins (Ali et al., 2005). The
recoveries from meat samples were between 90% and 96.5% and
between 94% and 101% for UV and fluorescent detector of the HPLC
methods, respectively. This indicates that the two detector results
were still within the value reported by CEN Report (1999), which
states that the percentage recovery for AFB1 and total aflatoxins
should be between 50% and 120%. The recovery of AF and their
metabolites were in compliance with Ventura et al. (2004), that
is, the recovery of AF is matrix dependent. The results of the tested
samples indicate that most of the samples were contaminated with
aflatoxins at levels exceeding the permitted limits of 2 lg kg 1 for
B1 and 4.2 lg kg 1 for total aflatoxins (B1 + B2 + G1 + G2). Thus, it
can be easily predicted that these food sources pose potential car-
cinogenic effect to the consumers (Grong et al., 2002) and that afla-
toxins levels in animal food sources must be monitored because of
their harmful effects which might pose threat to human health
(FAO/NACA/WHO, 1998). The results obtained by HPLC-UV give
lower value when compared with HPLC-FL, this implies that HPLC
with FL is more sensitive in aflatoxin determination when com-
pared to HPLC-UV, especially at lower AF levels. For example, a va-
lue of 1.23 lg kg 1 was obtained from eggs contaminated with
aflatoxins B1 by HPLC-UV and 1.43 lg kg 1 when measured by
HPLC-FL (Table 4 and Fig. 1). These values obtained in this study
are higher than those obtained by Gobel and Lusky (2004) and
Nakajima (2004) without triflouroacetic acid derivatization (TFA)
and reported by Ali et al. (1999) and Akiyama, Goda, Tanaka, and
1144 S.M. Herzallah / Food Chemistry 114 (2009) 1141–1146

Fig. 1. Aflatoxins chromatograms for analysed samples and standards using UV detector (a) blank, (b) extracted milk sample containing B1, B2 and M1; (c) extracted meat
samples with B1 detected, (d) standard containing B1, B2, G1, G2, M1 and M2 toxins (5, 5, 4, 1, 0.5 and 5 lg kg 1) measured by FL detector and (e) aflatoxins standard
measured by UV-detector.

Toyoda (2001). Also, the results obtained in the present study show
a minimum effect of interfering substances and matrix effect on
aflatoxins elution and separation (Fig. 1). These results show an
improvement in the clean-up and extraction procedures, which
minimises the effect of matrix on aflatoxin determination indi-
cated by previous studies (Akiyama et al., 2001; MacDonald & Cas-
tle, 1996). The occurrence of aflatoxins and their metabolites
(Table 5) indicate that the level of aflatoxins were higher in the
tested samples in the winter. The imported meat also showed that
the degree of contamination with aflatoxins was high. This result
could be attributed to the effect of the type of feed intake on the
residual levels of aflatoxins in meat and eggs during winter as a re-
sult of low feed quality compared with the amount of toxins, and
their metabolite during spring and summer when the green pas-
ture composed the major part of their feeds. For example, the inci-
dence levels in meat samples were 13.3% and 6.7% for winter and
spring, respectively. On the other hand, the incidence levels were
60% and 30% for feeds and imported beef meat, respectively. Thus,
the increase in the incidence levels between winter and spring
could be a result of feed types that were fed for most animals dur-
ing the winter, whereas, the natural pasture was the major feed
constituent during the spring. The concentrations of M1 and M2
during the spring were found to be lower than those in the winter.
This result could be correlated to the amount of B1 and B2 in feeds
that were fed to animals, which is considered the precursor for M1
and M2. The effects of contaminated feed on AF residues in animal
 S.M. Herzallah / Food Chemistry 114 (2009) 1141–1146 1145

products were in agreement with previous studies on the effect of

<0.05
<0.05
<0.05
<0.05

<0.05
<0.05
<0.05
<0.05
<0.05
0.10a
B1 concentration on broiler chickens (Del Bianchi, Oliveira, Albu-

M2
querque, Guerra, & Correa, 2005).

<0.05
<0.05

<0.05
0.56b
0.08d

0.08d
0.14c

0.15c
0.12c
1.1a
M1
4. Conclusion

In this study, the optimised HPLC method that was used for afla-

0.31ab

0.15bc
<0.05

<0.05
<0.05

<0.05
0.21b
0.51a

0.09c

0.10c
AFG2

toxins determination is accurate and precise for both the UV and

fluorescent detector. This will provide an accurate and reliable afla-
Average AF concentration lg kg-1

toxins analytical method with the UV detector with low running

0.32ab
<0.05

0.13b cost. The use of the HPLC in determination of aflatoxins and their
0.14b
0.10b
1.08a
0.73a

0.87a
0.08c

0.08c
AFG1

metabolites showed higher levels of accuracy and lower detection

limits when using SPE-CN or IAC regardless of the HPLC detectors
used. Furthermore, the assessment of the quality of foods using
0.60ab
<0.05
0.36b
0.21b

0.27b
0.18b
1.23a

1.27a

1.03a
0.12c
AFB2

this method provides an acceptable, accurate, and alternative

method to establish guidelines and to evaluate the status of afla-
toxins contaminated foods.
2.27cd

2.53cd

2.85cd
<0.05

1.45d
4.02b

4.50b
7.10a

3.25c

3.46c
AFB1

Acknowledgment

This work was supported by the grant from the Deanship of

Incidence

Academic Research at Mu’tah University.

16.7

13.3
6.7
(%)

25

30
60

30
20
5
0

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