Enhanced spectrofluorimetric determination of aflatoxin M1 in liquid milk after magnetic solid phase extraction

Article abstract of DOI:10.1016/j.saa.2014.02.108

A simple and sensitive method using magnetic solid phase extraction (MSPE) followed by spectrofluorimetric detection has been developed for separation and determination of aflatoxin M₁ (AFM₁) in liquid milk. The method is based on the extraction of AFM₁ on the modified magnetic nanoparticles (MMNPs) and subsequent derivatization of extracted AFM₁ to AFM₁ hemi-acetal derivative (AFM_2a) by reaction with trifluoroacetic acid (TFA) for spectrofluorimetric detection. Magnetic nanoparticles (MNPs) coated by 3-(trimethoxysilyl)-1-propantiol (TMSPT) and modified with 2-amino-5-mercapto-1,3,4-thiadiazole (AMT) were used as adsorbent in MSPE procedure. Influential parameters affecting the extraction efficiency were investigated and optimized. Under the optimum conditions the calibration curve for AFM₁ determination showed good linearity in the range 0.030-10.0 μg L^-1 (R² = 0.9991). The repeatability and reproducibility (RSD%) for 0.050 μg L^-1 of AFM₁ were 4.5% and 5.3%, respectively and limit of detection limit (S/N = 3) was estimated to be 0.010 μg L^-1. The developed method was successfully applied for extraction of AFM₁ from spiked liquid milk and natural contaminated liquid milk. The good spiked recoveries ranging from 91.6% to 96.1% were obtained. The results demonstrated that the developed method is simple, inexpensive, accurate and remarkably free from interference effects.

Full text of DOI:10.1016/j.saa.2014.02.108

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 128 (2014) 583–590
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Enhanced spectrofluorimetric determination of aflatoxin M1 in liquid
milk after magnetic solid phase extraction
a
b
Mahdi Hashemi a, , Zohreh Taherimaslak , Somayeh Rashidi
⇑
a Department of Analytical Chemistry, Faculty of Chemistry, Bu-Ali Sina University, Hamedan, Iran
b Department of Chemistry, Payam Noor University, Hamedan, Iran
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
ꢀ
Magnetic solid phase extraction-
spectrofluorimetry method is used for
AFM1 detection.
ꢀ
ꢀ
The proposed method is suitable for
determination of AFM1 in liquid milk.
Free antibody nanoparticles are used
for separation and preconcentration
of AFM1.
ꢀ
Trifluoroacetic acid was used as
derivatization reagent.
a r t i c l e i n f o
a b s t r a c t
Article history:
A simple and sensitive method using magnetic solid phase extraction (MSPE) followed by spectrofluori-
metric detection has been developed for separation and determination of aflatoxin M1 (AFM1) in liquid
milk. The method is based on the extraction of AFM1 on the modified magnetic nanoparticles (MMNPs)
and subsequent derivatization of extracted AFM1 to AFM1 hemi-acetal derivative (AFM2a) by reaction
with trifluoroacetic acid (TFA) for spectrofluorimetric detection. Magnetic nanoparticles (MNPs) coated
by 3-(trimethoxysilyl)-1-propantiol (TMSPT) and modified with 2-amino-5-mercapto-1,3,4-thiadiazole
(AMT) were used as adsorbent in MSPE procedure. Influential parameters affecting the extraction effi-
ciency were investigated and optimized. Under the optimum conditions the calibration curve for AFM1
Received 11 December 2013
Received in revised form 20 February 2014
Accepted 23 February 2014
Available online 4 March 2014
Keywords:
Aflatoxin M1
Modified magnetic nanoparticles (MMNPs)
Enhanced spectrofluorimetric detection
Solid phase extraction (SPE)
determination showed good linearity in the range 0.030–10.0
and reproducibility (RSD%) for 0.050
g LÀ1 of AFM1 were 4.5% and 5.3%, respectively and limit of detec-
tion limit (S/N = 3) was estimated to be 0.010
g LÀ1. The developed method was successfully applied for
l
g LÀ1 (R2 = 0.9991). The repeatability
l
l
extraction of AFM1 from spiked liquid milk and natural contaminated liquid milk. The good spiked recov-
eries ranging from 91.6% to 96.1% were obtained. The results demonstrated that the developed method is
simple, inexpensive, accurate and remarkably free from interference effects.
Ó 2014 Elsevier B.V. All rights reserved.
Introduction
frequently contaminate cereal crops, such as corn, beans, peanuts,
and dried fruit [1]. Among different AFs, AFB1 is the most frequent
Aflatoxins (AFs) are typically found as secondary metabolites of
Aspergillus flavus, Aspergillus parasiticus and Aspergillus nomius. AFs
produced mycotoxin, which is the most powerful carcinogen in
mammals. When AFB1 is ingested by cows, it is converted into its
monohydroxylated metabolites, which has been designated as
AFM1 [2,3]. AFM1 or milk toxin exhibits a high level of genotoxic
activity and certainly represents a health hazard because of its
⇑
Corresponding author. Tel.: +98 811 8282807; fax: +98 811 8257407.
E-mail address: mhashemi@bacu.ac.ir (M. Hashemi).
http://dx.doi.org/10.1016/j.saa.2014.02.108
1386-1425/Ó 2014 Elsevier B.V. All rights reserved.

584
M. Hashemi et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 128 (2014) 583–590
possible accumulation and linkage to DNA. The toxicity of AFM1
was initially classified as a Group 2B agent, but it has now moved
to Group 1 by International Agency for Research on Cancer (IARC)
[4]. European Community legislation limits the concentration of
AFM1 for milk and processed milk products intended for adults
(H2O) were purchased from Sigma–Aldrich (St. Louis, MO, USA).
FeCl3Á6H2O, FeCl2Á4H2O, 3-(trimethoxysilyl)-1-propantiol (TMSPT),
2-amino-5-mercapto-1,3,4-thiadiazole(AMT)andotherusedchem-
icals were supplied by Merck (Darmstadt, Germany). Phosphate buf-
fered saline (PBS, pH = 7.4) was prepared by dissolving 0.20 g KCl,
0.20 g KH2PO4, 1.16 g Na2HPO4 and 8.00 g NaCl in 1L water. The pur-
ity of the used organic solvents was checked via fluorescence prior to
use. As safety notes, all used laboratory glassware were treated with
an aqueous solution of sodium hypochlorite (5%) before the discard-
ing to minimize health risks due to AFM1 contamination.
at 0.050
production at 0.025
l
g kgÀ1 [5] and for milk intended for infants or baby-food
l
g kgÀ1 [6]. AFM1 is relatively stable during
pasteurizations, storage, and preparation of various dairy products
[7]. Due to the widespread consumption of milk and dairy prod-
ucts, presence of AFM1 in these products has become a worldwide
concern. Therefore, accurate evaluation of AFM1 in milk is of great
interest.
Instrumentation
A
number of analytical methods have been reported for
determination of AFM1, such as ELISA [8,9], TLC [10], LC–MS [11],
HPLC–FD [12,13], UHPLC–MS–MS [14], fluorimetry [15,16],
electrochemical methods [17,18] and immunoassay methods
[19–21]. Among these methods, spectrofluorimety can be consid-
ered as a valuable method because of its simplicity, sensitivity, rel-
ative selectivity, low cost, and less time consuming [22–24].
However, there are a few reports about the application of direct
spectrofluorimetric determination of AFM1 [15,25]. In spectrofluo-
rimetric assay, chemical derivatization has been widely appreci-
ated due to increasing of selectivity and sensitivity.
Generally, determination of AFM1 in real samples requires a
clean-up or enrichment technique. Immunoaffinity column (IAC),
C18, carbograph-4 and multifunctional cleanup column were re-
ported to have preferable purification effect for AFM1 clean-up in
different dairy products [12,13,15,26–28]. IAC is the most common
clean-up method which allows a highly selective separation of ana-
lyte from a complex matrix. However, it is time consuming, tedious,
relatively expensive and commercially available as a single-use for-
mat [29]. Moreover, its collection efficiency has failed to provide
complete satisfaction for users. Recently, new SPE technique based
on the use of magnetic nanoparticles (MNPs), called magnetic solid
phase extraction (MSPE) has been introduced for separation and
preconcentration of organic and inorganic species from complex
matrices. In MSPE, the contact area between the adsorbent and
the analyte is large enough to ensure a fast mass transfer, which
is beneficial to guarantee high extraction efficiency for this method.
MNPs have been extensively used as adsorbent in MSPE due to its
super paramagnetism, high magnetic saturation, low toxicity, sim-
ple preparation process and low price. The stability and selectivity
of the MNPs can be significantly improved by the modification of
the surface of the adsorbent with special functional groups.
The aim of this study was to investigate the applicability of the
MSPE with modified magnetic nanoparticles (MMNPs) for en-
hanced spectrofluorimetric determination of AFM1 in milk. The
method is based on the separation of AFM1 by MSPE procedure
and subsequent derivatization of separated AFM1 to AFM1 hemi-
acetal derivative (AFM2a) by reaction with TFA and final spectroflu-
orimetric determination. To the best of our knowledge, this is the
first report about application of an antibody free adsorbent for sep-
aration and enhanced spectrofluorimetric determination of AFM1
in milk. All the experimental parameters affecting the extraction
were investigated in details and the analytical characteristics of
the method were evaluated. The method was demonstrated to be
applicable for the analysis of AFM1 in liquid milk samples.
The fluorescence measurements were performed using a Cary
Eclipse Fluorescence Spectrophotometer (Varian, USA) equipped
with a xenon lamp. All measurements were performed in 10 mm
quartz microcells, at room temperature. Spectra recording were
carried out in fluorescence scan mode with the slit widths of
5 nm. Chromatographic analysis was performed by Waters HPLC
system which consists of a Waters 474 fluorescence detector, a post
column derivatization reactor and a Waters C18 column. The mod-
ified magnetic nanoparticles were characterized by an S-4160 scan-
ning electron microscope (SEM) (Hitachi, Japan), APD2000 X-ray
Diffractometer (XRD) (Italstructures, Italy) and FT-IR Spectrometer
(Perkin Elmer, spectrum version 10.01.00, USA). A permanent mag-
net of Nd–Fe–B (100 mm  50 mm  40 mm, Model N48, China)
was used for magnetic separation. Ultrasonic bath (Uc-150 Sturdy
Industrial CO LTD, Taiwan) was used in modification step. An
Eppendorf 5810 centrifuge was used for centrifugation. A pH-meter
(Corning, Model 140, Switzerland) with a double junction glass
electrode was used to check the pH of the solutions.
Synthesis of modified magnetic nanoparticles
The magnetic nanoparticles (MNPs) were prepared via im-
proved chemical co-precipitation method [30] and modified
according to Mashhadizadeh method [31]. FeCl3Á6H2O (11.68 g)
and FeCl2Á4H2O (4.30 g) were dissolved in 200 mL deionized water
under nitrogen atmosphere with vigorous stirring at 85 °C. Then,
20 mL of 30% aqueous ammonia was added to the solution. The col-
or of bulk solution changed from orange to black immediately. The
magnetic precipitates were washed twice with deionized water
and once with 0.02 mol LÀ1 sodium chloride solution. Then,
20 mL of prepared magnetic suspension was placed in a 250 mL
round-bottom flask and allowed to settle. The supernatant was re-
moved and coating of MNPs with 3-(trimethoxysilyl)-1-propanth-
iol (TMSPT) was carried with addition of an aqueous solution of
TMSPT (10%, v/v, 80 mL), followed by glycerol (60 mL). The mixture
was then stirred and heated at 85 °C for 2 h under a nitrogen atmo-
sphere. After cooling to room temperature, the suspension was
washed sequentially with deionized water (3 Â 200 mL), methanol
(3 Â 100 mL), and deionized water (5 Â 200 mL). The TMSPT-MNPs
composite was stored in deionized water at a concentration of
40 g LÀ1. TMSPT-MNPs prepared as described above (25 mL) were
washed with methanol (2 Â 100 mL) and then homogeneously dis-
persed into 150 mL of 1.0% aqueous solution of AMT. The solution
was transferred to a 500 mL beaker and ultrasonicated for 2 h.
After that, the resulting modified nanoparticles (AMT–TMSPT-
MNPs) were washed three times with deionized water and twice
with methanol and then dried in a vacuum oven at 45 °C for 2 h.
Experimental
Standards and materials
Analytical procedure
The standard solution of AFM1 (500
l
g LÀ1 in acetonitrile), TFA
Sample preparation and MSPE procedure
and all HPLC-grade solvents such as acetone (Me2CO), acetonitrile
(MeCN), dichloromethane (CH2Cl2), methanol (MeOH) and water
Liquid milk was accurately weighed (10 0.1 g) into 50 mL
centrifuge tube and centrifuged (4000 rpm) for 15 min. After

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585
centrifugation, fat layer was isolated and supernatant aqueous
phase diluted to 30 mL with PBS solution (pH = 7.4) in a capped
container and shacked intensively. Then, the diluted aqueous
phase was transferred to 50 mL vial and 100 mg of AMT–TMSPT-
MNPs was added to it. The solution was stirred for 5 min to
facilitate adsorption of the AFM1 on MMNPs. Then, the magnetic
adsorbent was collected using an external magnet and supernatant
was decanted. The adsorbed AFM1 were desorbed from surface of
adsorbent by addition of 2 mL Me2CO/MeCN/CH2Cl2 (1:2:2, v/v/v)
mixture and stirred for 3 min. Finally, the magnet was used again
to settle the nanoparticles. Desorption solvent was transferred to
5 mL vial and evaporated to dryness under a gentle nitrogen flow.
measurements. The selection of a suitable adsorbent is of great
importance for the optimization of MSPE process. A proper adsor-
bent must have several characteristics. It should have good stabil-
ity, suitable affinity for compound of interest, high surface area for
effective adsorption and can be easily separated from solution in a
short time. Bonding of special functional groups on the surface of
adsorbent can cause an increase in extraction efficiency for target
analytes. On the basis of these considerations, the usefulness of
MNPs modified with different functional groups including, carbox-
ylic group (3-mercaptopropionic acid modified silica coated
MNPs), amino group (3-aminopropyltriethoxysilane modified sil-
ica coated MNPs(, thiol group (TMSPT modified silica coated MNPs)
and both amino and thiol groups (AMT modified TMSPT coated
MNPs) were investigated in our preliminary studies. The best
adsorption efficiency was obtained with AMT–TMSPT-MNPs
(Fig. 2). As compared with other investigated adsorbent, AMT–
TMSPT-MNPs have both amino and thiol groups which can have
effective electrostatic interactions with carbonyl group of lactone
ring and –OH group of furfuran ring in AFM1. This type of interac-
tion has also been reported for adsorption of AFB1 which has same
structure, on some clay such as montmorillonite and smectite
[35,36].
Derivatization procedure
The derivatization procedure was according to the official
method 986.16 [32]. 200 lL of hexane and 200 lL of trifluoroacetic
acid were added to dried residue of desorption solvent and the
mixture was vortexed and allowed to sit at 40 °C for 20 min. The
mixture was again evaporated to dryness under a gentle nitrogen
flow. The final residue (AFM2a) was reconstituted in 300 lL of
12% (v/v) acetonitrile–water for final spectrofluorimetric detection.
The fluorescence intensity was measured at the emission wave-
length of 440 nm after excitation of solutions at 365 nm.
Characterization of the adsorbent
Results and discussion
To confirm that TMSPT and AMT are bonded to the Fe3O4 NPs,
the characterization was performed by FT-IR spectroscopy. The
FT-IR spectra for TMSPT-MNPs and AMT–TMSPT-MNPs are shown
in Fig. 3. The broad feature in the range 3441–3220 cmÀ1 is due to
O–H stretching vibration, which corresponds to the hydroxyl
groups attached by the hydrogen bonds to the iron oxide surface.
After initial coating step, the characteristic peaks at 1125–
1039 cmÀ1 are related to the O–Si stretching vibration, at 2931
and 2886 cmÀ1 are attributed to –C–H stretching and at
1442 cmÀ1 to the –CH2 bending. The transmittance wave band
from 690 to 580 cmÀ1 corresponds to the metal–oxygen bonds
(Fig. 3a). When the coated nanoparticles are modified with 2-ami-
no-5-mercapto-1,3,4-thiadiazole (AMT), at the first an increase
was observed for the broad feature (3441–3220 cmÀ1) that related
to –NH2 stretching band and two new vibrational bands appear at
1404 cmÀ1 and 1634 cmÀ1 (Fig. 3b). The characteristic peak
at 1404 cmÀ1 is attributed to the C–N stretching vibration and at
1634 cmÀ1 is assigned to the heterocyclic ring. These peaks indi-
cate the immobilization of AMT on the surface of TMSPT-MNPs
in modification step. Fig. 4a displays the SEM image of AMT–
TMSPT-MNPs, which illustrates the uniform size distribution. The
diameter of AMT–TMSPT-MNPs is about 30 nm, and most of parti-
cles are quasi-spherical in shape. Also, X-ray diffraction patterns of
AMT–TMSPT-MNPs was shown in Fig. 4b, representing the reflec-
tion patterns at peak position (2h) of about 30.2, 35.3, 43.2, 57.2,
62.7, and 74.2 which correspond to the reflection planes of 220,
The analysis of low concentration of contaminates in complex
matrices requires a sample clean-up and preconcentration step
to increase the selectivity and achieve low detection limits. In this
study, MSPE with modified nanoparticles was used as a separation
and preconcentration method for spectrofluorimetric determina-
tion of AFM1 in milk sample. AFM1 suffers a significant fluores-
cence quenching in aqueous mixtures and thus, shows low
fluorescence quantum efficiency. Therefore, the known reaction
of AFM1 with TFA (Fig. 1) was used for increasing of the fluores-
cence intensity. This reaction has been exploited as a pre-column
derivatization producer in some HPLC–FD methods for determina-
tion of AFs [33,34]. It is known that exposure of AFs to strong acids,
such as TFA, can cause their acid-catalyzed hydration to form an
emiacetalic group on the difuranic moiety. This transforms the
weakly fluorescent AFs in to their more polar and highly fluores-
cent hemi-acetal derivatives [34]. TFA can react with AFM1 with
good selectivity, no by-products and mild reaction conditions.
The effect of experimental parameters on the performance of
MSPE, such as the type of adsorbent, sample pH, amount of adsor-
bent, adsorption time, desorption time and the type of desorption
solvent were investigated. The intensity of the fluorescence peak
was used to assess the extraction efficiency under various condi-
tions. A univariate approach was employed to optimize influential
factors in this method and all results were average of five replicate
Fig. 1. The reaction of AFM1 with TFA.

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M. Hashemi et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 128 (2014) 583–590
particle size of AMT–TMSPT-MNPs adsorbent, based on the Scher-
rer equation, was approximately 10 nm corresponding to line
broadening of the 311-diffraction peak, which was observed at
2h of 35.3.
Effect of pH
The pH of the sample solution plays an important role in the
MSPE procedure. The pH of sample can change the nature of the
AMT–TMSPT-MNPs surface due to protonation of –NH and/or oxi-
dation of –SH groups. Also, in strong acidic and alkaline media, the
nature of AFM1 may change due to rupture of the lactonic ring and/
or hydrolysis reaction [38,39]. The effect of sample pH was inves-
tigated in the range 4.0–9.0 using 100 mg of MMNPs (see Fig. 5a).
As Fig. 5a shows, the highest extraction efficiency for AFM1 was ob-
tained over the pH range of 7.0–7.8. AFM1 is a neutral compound
and therefore a neutral environment is necessary to increase the
extraction efficiency. Hence, a pH value of 7.4 was selected for fur-
ther studies.
Fig. 2. Effect of modified MNPs type on the fluorescence intensity. (A) 3-
Mercaptopropionic acid modified silica coated MNPs; (B) 3-aminopropyltriethoxy-
silane modified silica coated MNPs; (C) TMSPT modified silica coated MNPs; (D)
AMT modified TMSPT coated MNPs. Conditions: concentration of AFM1,
0.050
10 min; desorption time, 15 min; elution solvent volume and type, 2 mL of Me2CO/
MeCN/CH2Cl2 (1:2:2, v/v/v); reconstituting solvent volume and type, 300 L of
l
g kgÀ1; the pH of sample, 7.4; adsorbent amount, 120 mg; adsorption time,
l
acetonitrile–water 12% (v/v). Error bars represent the standard deviation for five
experiments.
Effect of sample volume
311, 400, 511, 440, and 622, respectively. The position and relative
intensity of all diffraction peaks are consistent with the standard
pattern of Fe3O4 according to the JCPDS card [37]. The average
In order to obtain a higher enrichment factor in MSPE proce-
dure, a larger volume of sample solution is required. The effect of
sample volume on the AFM1 extraction was investigated using
Fig. 3. FT-IR spectra of TMSPT-MNPs (a) and AMT–TMSPT-MNPs (b).

M. Hashemi et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 128 (2014) 583–590
587
Fig. 4. SEM image (a) and X-ray diffraction pattern (b) of AMT–TMSPT-MNPs.
different sample volumes in the range of 5–70 mL, which were
spiked, with 0.050
g LÀ1 of AFM1. The results showed that the
extraction efficiency was constant in the range of 5–40 mL and
then decreased at higher sample volumes due to the analyte loss
from the adsorbent surface. Thus, the volume of 30 mL was se-
lected for subsequent experiments.
l
The MMNPs amount
To study the effect of adsorbent amount on the extraction effi-
ciency, different amounts of adsorbent in the range 10–130 mg
were added to the analyte solution. The results showed that the
extraction efficiency increased with increasing amounts of adsor-
bent up to 100 mg and then leveled off (Fig. 5b). Therefore,
100 mg of adsorbent was selected for the further experiments.
a
Effect of adsorption time
Generally, sufficient contact time is required to achieve the
equilibrium between sample solution and adsorbents. For studying
the effect of adsorption time on extraction efficiency, adsorption
time was investigated in the range of 1–10 min, the results are
shown in Fig. S1 (Electronic Supplementary Material; ESM). It
was found that an adsorption time of 5 min was sufficient to attain
adsorption equilibrium. In fact, MNPs provide a large surface area
and a short diffusion rout which facilitate mass transfer of analyts
under vigorous stirring. Therefore, the equilibrium state is
achieved quickly and a short adsorption time is obtained.
b
Desorption conditions
Desorption of an extracted analyte from the surface of MNPs is a
rather critical step. The choice of desorption solvent is very impor-
tant. A suitable desorption solvent should effectively elute the ad-
sorbed analytes with the minimum volume and less interfering
impurities co-eluted. It also should not damage the nature of the
adsorbent surface. On the basis of the above considerations, the
usefulness of several types of desorption solvents was examined.
Fig. 5. Effect of the pH (a) and adsorbent amount (b). Extraction conditions:
Concentration of AFM1, 0.050
15 min; eluent solvent volume and type, 2 mL of Me2CO/MeCN/CH2Cl2 (1:2:2, v/v/
v); reconstituting solvent volume and type, 300 L of acetonitrile–water 12% (v/v).
Error bars represent the standard deviation for five experiments.
l
g LÀ1; adsorption time, 10 min; desorption time,
l

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M. Hashemi et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 128 (2014) 583–590
Results are shown in Fig. 6. As can be seen the best result was
found with the mixture of Me2CO/MeCN/CH2Cl2 (1:2:2, v/v/v).
Whereas MNPs’ surfaces have hydrophilic properties, the use of
acetone and acetonitrile could improve the dispersion efficiency
of MNPs in dichloromethane which acts as a hydrophobic solvent.
Also the effect of desorption time was investigated in the range of
1–10 min (Fig. S2, ESM). A duration time of 3 min appeared to be
sufficient for complete desorption and no significant effect was ob-
served when the time of desorption was greater than 3 min. The ef-
fect of eluent volume on AFM1 recovery was further investigated in
the range of 1–10 mL (Fig. S3, ESM). The maximum sensitivity was
obtained over the range 2–8 mL. Therefore, 2 mL of Me2CO/MeCN/
CH2Cl2 (1:2:2, v/v/v) was selected to ensure complete elution of
analytes for further experiments.
intensity and C, l
g LÀ1 of AFM1 standard solution) and correlation
coefficient of 0.9991 (Fig. S5, ESM). Limit of detection (LOD = 3.3
Sb/m, where Sb is the standard deviation for ten blank measurements
and m is the slope of the calibration curve) was found to be
0.010 l
g LÀ1. Also to investigate the possible matrix effect on the
AFM1 determination in real sample, matrix-matched limit of detec-
tion (LOD) was evaluated from matrix-matched calibration. Solu-
tions for matrix-matched calibration were prepared by spiking
appropriate amounts of AFM1 working solutions to the none-con-
taminated milk sample and following the MSPE procedure and fluo-
rescence measurement. Matrix-matched LOD was obtained to be
0.011 l l
g LÀ1 (equivalent to 0.010 g kgÀ1 content in sample). This
result indicated that sample matrix cannot significantly affect the
AFM1 determination. The obtained LOD was lower than the maxi-
mum levels (MLs) imposed by current EU regulation for liquid milk
intended for direct human consumption (0.050 l
g kgÀ1 of AFM1)
Reconstituting solvent effect
[5]. The precision of the method was evaluated through investiga-
tion the repeatability and reproducibility (expressed as RSD%). The
repeatability was evaluated over five replicates spiked at concentra-
Solvent polarity has a remarkable effect on the fluorescence
intensity of AFs. The fluorescence intensity of AFs increases with
increasing of solvent polarity [40,41]. Since the solubility of AFM2a
in water is low, so it is necessary to use organic solvent mixed with
water for increasing of solubility. In this study acetonitrile was se-
lected for increasing of AFM2a solubility and the effect of acetoni-
trile content was investigated on the fluorescence intensity in the
range of 1–25% (v/v). The best compromise between AFM1 solubil-
ity and fluorescence intensity was achieved using 12% (v/v) aceto-
nitrile–water.
tion level 0.050 l
g LÀ1 of AFM1 within one day (n = 5). The repro-
ducibility was evaluated over five daily replicates, spiked at same
level per work day, over a period of three days (n = 15). The repeat-
ability and reproducibility were 4.5% and 5.3%, respectively. Also, by
extracting 30 mL of sample solution into 300 lL of reconstituting
solvent (Recovery = 96.5%), the pre-concentration factor of 96.5
was achieved for AFM1 determination by the developed method.
Selectivity study
Reusability of adsorbent
Although, AFM1 is the most common mycotoxin found in milk,
other mycotoxins such as deoxynivalenol (DON), zearalenone
(ZEN) and ochratoxin A (OTA) may exist in milk. For evaluation
of the method selectivity for determination of AFM1, the possible
interference effects of deoxynivalenol (DON), zearalenone (ZEN)
and ochratoxin A (OTA) was studied by co-existing of them alone
and in mixture. The obtained results (Table 1) showed that the
recoveries were not significantly affected by the presence of
In order to investigate the recycling of the adsorbent under
optimized conditions, the adsorbent were rinsed sequentially with
2 mL of Me2CO/MeCN/CH2Cl2 (1:2:2, v/v/v) and deionized water
(2 Â 5 mL) before application in the next time. No obvious changes
were observed in the recoveries after 10 times (Fig. S4, ESM). The
results of this study indicate that the adsorbent is reusable without
a considerable loss in it adsorption efficiency during extraction
procedure.
Analytical parameters
Table 1
Effect of possible interferences on the determination of AFM1 (0.050 l
g kgÀ1).
Under the selected experimental conditions, a linear calibration
graph was obtained over the range 0.030–10.0 l
g LÀ1 of AFM1 with
Interferences
Concentration (
l
g kgÀ1
)
Recovery RSD (%)
the linear regression equation If = 93.615 C + 18.912 (If, fluorescence
OTA
ZEN
DON
Mixture
0.50
0.50
0.50
Total
94.1 4.1
93.9 4.3
94.4 3.9
93.6 4.1
Table 2
Results (mean SD based on five replicate analysis, n = 3) of determination of AFM1
by the solid phase extraction with MMNPs in spiked samples of liquid milk.
Milk sample
Sample 1
Add (
l
g kgÀ1
)
Found (
l
g kgÀ1
)
Recovery (%)
0.000
0.050
0.100
0.500
NDa
0.046 0.002
0.093 0.004
0.467 0.009
–
91.6
93.1
93.4
Sample 2
Sample 3
0.000
0.050
0.100
0.500
NDa
0.048 0.002
0.094 0.003
0.473 0.009
NDa
0.047 0.002
0.096 0.004
0.463 0.009
–
95.6
94.4
94.6
Fig. 6. Effect of desorption solvent type. (A) Me2CO, (B) MeOH, (C) MeCN, (D)
1Me2CO + 1CH2Cl2, (E) 1MeCN + 1CH2Cl2, (F) 1Me2CO + 1MeCN + 1CH2Cl2, (G)
1Me2CO + 1MeCN + 2CH2Cl2, (H) 1Me2CO + 2MeCN + 2CH2Cl2, (I) 2Me2CO +
0.000
0.050
0.100
0.500
–
1MeCN + 2CH2Cl2. Conditions: concentration of AFM1, 0.050
sample, 7.4; adsorbent amount, 100 mg; adsorption time, 5 min; desorption time,
3 min; eluent solvent volume, 2 mL; reconstituting solvent volume and type, 300
l ; the pH of
g LÀ1
93.2
96.1
92.6
l
L
of acetonitrile–water 12% (v/v). Error bars represent the standard deviation for five
experiments.
a
ND, not detected.

M. Hashemi et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 128 (2014) 583–590
589
method for the determination of AFM1 in two contaminated real
samples was checked by the AOAC standard method (IAC–HPLC–
FD) [42]. The results are presented in Table 3. The statistical anal-
ysis of the results using Student’s t-test showed that there are no
significant differences between results obtained by two methods
at 95% confidence level. A comparison of the analytical feature
achieved by the proposed method and other methods for AFM1
determination is presented in Table 4. The presented method has
distinct advantages in term of low detection limit, wide linear
range, ease of operation and simplicity.
b
a
Conclusions
This study describes a new and simple method for determina-
tion of AFM1 in liquid milk using solid phase extraction of AFM1
on MMNPs and subsequent derivatization of extracted AFM1 to
AFM2a for enhanced spectrofluorimetric detection. The solid phase
extraction with MMNPs integrates sample clean up, extraction, and
pre-concentration. Also, the proposed method had many advanta-
ges including simplicity, inexpensive, wide linearity, good sensitiv-
ity and precision, for determination of AFM1 in complex matrix of
milk samples. The good spiked recoveries of AFM1 in real samples
and the inherent high sensitivity and selectivity of spectrofluori-
metric method showed that the present method was applicable
for determination of AFM1 in liquid milk samples.
Fig. 7. The typical spectra of non-spiked liquid milk (blank) (a(and spiked liquid
milk(b). Conditions: concentration of AFM1, 0.050
g kgÀ1; the pH of sample, 7.4;
adsorbent amount, 100 mg; adsorption time, 5 min; desorption time, 3 min; elution
solvent volume and type, 2 mL of Me2CO/MeCN/CH2Cl2 (1:2:2, v/v/v); reconstitut-
l
ing solvent volume and type, 300 lL of acetonitrile–water 12% (v/v).
Table 3
Determination AFM1 in contaminated liquid milk samples by proposed method and
IAC–HPLC–FD method (all results: mean SD, n = 3).
Milk sample
Proposed method
AFM1
g kgÀ1
IAC–HPLC–FDa
AFM1 )
g kgÀ1
(
l
)
(
l
Sample 1
Sample 2
0.496 0.009
0.514 0.009
0.459 0.008
0.575 0.009
Appendix A. Supplementary material
a
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.saa.2014.02.108.
IAC–HPLC–FD analysis by AOAC standard method [42].
References
Table 4
Comparison of diverse methods for the determination of AFM1.
[1] H.S. Hussein, J.M. Brasel, Toxicology 167 (2001) 101–134.
[2] J.L. Richard, Int. J. Food Microbiol. 119 (2007) 3–10.
[3] M. Carvajal, F. Rojo, I. Mendez, A. Bolanos, Food Addit. Contam. 20 (2003)
1077–1086.
Method
Linear
range
LOD
Recovery
Reference
(l
g LÀ1) (%)
(l
g LÀ1)
[4] IARC, International Agency for Research on cancer, Monograph on the
Evaluation of Carcinogenic Risk to Humans, vol. 82, World Health
Organization, Lyon, France, 2002, pp. 171–174.
[5] European Commission Regulation, No. 466/2001/EC of 8 March 2001, Setting
Maximum Levels for Certain Contaminants in Foodstuffs, Official Journal of
European Communities L077, 2001, pp. 1-13.
[6] European Commission Regulation, No. 683/2004/EC of 13 April 2004,
Amending Regulation (EC) No. 466/2001 as Regards Aflatoxins and
Ochratoxin A in Foods for Infants and Young Children, Official Journal of
European Communities L106, 2004, pp. 3–5.
[7] L. Stoloff, J. Food Prot. 43 (1989) 226–230.
[8] Sh. Ch. Pei, Y.Y. Zhang, S.A. Eremin, W.J. Lee, Food Control 20 (2009) 1080–
1085.
[9] D. Guan, L. Peiwu, Q. Zhang, W. Zhang, D. Zhang, J. Jiang, Food Chem. 125
(2011) 1359–1364.
[10] O. Atanda, A. Oguntubo, O. Adejumo, J. Ikeorah, I. Akpan, Chemospere 68
(2007) 1455–1458.
Direct fluorimetry
Fluorimetric sensor
HPLC–FD
–
0.050
97.0
–
115.6–
117.9
78–108
84.6–88.0
87.9–128.3 [8]
91–110
90–101
[15]
[16]
[12]
0.00–0.125 0.050
0.010–
0.200
0.020–1
–
0.040–5
–
0.030–
0.240
1–14
0.010
LC–MS
TLC
ELISA
Ultra-sensitive ELISA
Electrochemical
immunosensor
Impedimetric biosensor
Potentiometric
immunosensor
0.010
2
0.040
0.030
0.025
[11]
[10]
[9]
[18]
1
107
74–136
[21]
[19]
0.125–2
0.040
Enhanced spectrofluorimetry 0.030–10.0 0.010
91.6–96.1
This work
[11] L.K. Sørensen, T.H. Elbæk, J. Chromatogr. B 820 (2005) 183–196.
[12] A. Gurbay, S. Aydin, G. Girgin, A.B. Engin, G. Sahin, Food Control 17 (2006) 1–4.
[13] Y. Wang, X. Liu, Ch. Xiao, Zh. Wang, J. Wang, H. Xiao, L. Cui, Q. Xiang, T. Yue,
Food Control 28 (2012) 131–134.
10-fold excesses of investigated species, indicating good selectivity
for determination of AFM1 in milk.
[14] E. Beltrán, M. Ibáñez, J.V. Sancho, M.Á. Cortés, V. Yusà, F. Hernández, Food
Chem. 126 (2011) 737–744.
Analysis of real samples
[15] T.J. Hansen, J. Food Prot. 53 (1990) 75–77.
´
[16] C. Cucci, A. G Mignani, R. DallAsta, R. Pela, A. Dossena, Sens. Actuators, B 126
(2007) 467–472.
To evaluate the applicability of the proposed method in real
samples, it was applied to the determination of AFM1 in milk sam-
ples. Recovery studies were carried out by spiking the samples
with different amounts of AFM1. Results (Table 2) showed that
the recovery values were in the range of 91.6–96.1%. Also, Fig. 7
[17] B.H. Nguyen, L.D. Tran, Q.P. Do, H.L. Nguyen, N.H. Tran, P.X. Nguyen, Mater. Sci.
Eng. C 33 (2013) 2229–2234.
[18] L. Micheli, R. Grecco, M. Badea, D. Moscone, G. Palleschi, Biosens. Bioelectron.
21 (2005) 588–596.
[19] S. Rameil, P. Schubert, P. Grundmann, R. Dietrich, E. Märtlbauer, Anal. Chim.
Acta 661 (2010) 122–127.
[20] G. Bacher, S. Pal, L. Kanungo, S. Bhand, Sens. Actuators, B 168 (2012) 223–230.
[21] E. Dinckaya, Ö. Kınık, M.K. Sezgintürk, C. Altug˘, A. Akkoca, Biosens. Bioelectron.
26 (2011) 3806–3811.
shows the typical spectra of the spiked (0.050 l
g kgÀ1 of AFM1)
and non-spiked milk sample at optimum working conditions.
Comparison of the spectra and acceptable recoveries demonstrated
that the matrices of liquid milk sample had no effects on the per-
formance of the presented method. Accuracy of the developed
[22] K.J. Huang, Q. Sh. Jing, C.Y. Wei, Y.Y. Wu, Spectrochim. Acta A 79 (2011) 1860–
1865.
[23] L.L. Wang, Sh. Yu, M. Yu, Spectrochim. Acta A 98 (2012) 337–342.

590
M. Hashemi et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 128 (2014) 583–590
[24] P.M.S. Maria, F.B.F. Rezende, A.D. Pereira Netto, F.F.C. Marques, Spectrochim.
Acta A 100 (2013) 127–130.
[25] I. Hussain, J. Anwar, Food Control 19 (2008) 393–395.
[26] Ch. Cavaliere, P. Foglia, E. Pastorini, R. Samperi, A. Lagan, J. Chromatogr. A 1101
(2006) 69–78.
[33] D.M. Takahashi, J. Chromatogr. A 131 (1997) 147–156.
[34] G. Shephard, Anal. Bioanal. Chem. 395 (2009) 1215–1224.
[35] Q. Desheng, L. Fan, Y. Yanhu, Z. Niya, Poult. Sci. 84 (2004) 959–961.
[36] Y. Deng, A.L. Barrientons Velazquez, F. Billes, J.B. Dixon, Appl. Clay Sci. 50
(2010) 92–98.
[27] A.C. Manetta, L. Di Giuseppe, M. Giammarco, I. Fusaro, A. Simonella, A.
Gramenzi, A. Formigoni, J. Chromatogr. A 1083 (2005) 219–222.
[28] C.-Y. Chen, W.-J. Li, K.-Y. Peng, J. Agric. Food Chem. 53 (2005) 8474–8480.
[29] M. Castegnarol, M. Tozlovanul, Ch. Wild, A. Molini, A. Sylla, A. Pfohl-
Leszkowicz, Mol. Nutr. Food Res. 50 (2006) 480–487.
[37] JCPDS card 19-0629, Joint Committee on Powder Diffraction Standards,
Swarth-more, 1989.
[38] F.K. Saalia, R. Dixon Phillips, Food Control 21 (2010) 1066–1069.
[39] I. Nicolas-Vazquez, A. Mendez-Albores, E. Moreno-Martınez, R. Miranda, M.
Castro, Arch. Environ. Contam. Toxicol. 59 (2010) 393–406.
[40] M. Aghamohammadi, N. Alizadeh, J. Lumin. 127 (2007) 575–582.
[41] J. Hashemi, Gh. Asadi Kram, N. Alizadeh, Talanta 75 (2008) 1075–1081.
[42] Official Methods of Analysis of AOAC International, Official Method 2000.08,
18th ed., Gaithersburg, MD, USA, 2005, pp. 20877–2417.
[30] H.C.Z. Huang, B. Hu, J. Sep. Sci. 31 (2008) 760–767.
[31] M.H. Mashhadizadeh, Z. Karami, J. Hazard. Mater. 190 (2011) 1023–1029.
[32] Official Method of Analysis of AOAC International, Method 986.16, 17th
edition, Gaithersburg, MD,USA, 2000, pp. 40–42.