Accepted Manuscript Title: Magnetically assisted solid phase extraction using Fe3 O4 nanoparticles combined with enhanced spectrofluorimetric detection for aflatoxin M1 determination in milk samples Author: Zohreh Taherimaslak Mitra Amoli-Diva Mehdi Allahyary Kamyar Pourghazi PII: DOI: Reference:
S0003-2670(14)00579-0 http://dx.doi.org/doi:10.1016/j.aca.2014.05.007 ACA 233251
To appear in:
Analytica Chimica Acta
Received date: Revised date: Accepted date:
30-3-2014 1-5-2014 5-5-2014
Please cite this article as: Zohreh Taherimaslak, Mitra Amoli-Diva, Mehdi Allahyary, Kamyar Pourghazi, Magnetically assisted solid phase extraction using Fe3O4 nanoparticles combined with enhanced spectrofluorimetric detection for aflatoxin M1 determination in milk samples, Analytica Chimica Acta http://dx.doi.org/10.1016/j.aca.2014.05.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Magnetically assisted solid phase extraction using Fe3O4 nanoparticles combined with enhanced spectrofluorimetric detection for aflatoxin M1 determination in milk samples
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Zohreh Taherimaslaka, Mitra Amoli-Divab,*, Mehdi Allahyaryc, Kamyar Pourghazib Faculty of Chemistry, Bu-Ali Sina University, Hamadan, Iran
b
Faculty of Chemistry, Kharazmi (Tarbiat Moallem) University, Tehran, P.O. Box 15719-14911, Iran
c
Quality control laboratory, ARA quality research Co., Tehran, Iran
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a
Highlights
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► A new SPE technique coupled with spectrofluorimetry for extraction and determination of aflatoxin M1 has been developed. ► Modified magnetic nanoparticles were used as adsorbent. ► Β-cyclodextrin was used for fluorescence enhancement of AFM1. ► The method was applied to some commercial milk samples
Abstract
A novel, facile and inexpensive solid phase extraction (SPE) method using ethylene glycol bismercaptoacetate modified 3-(trimethoxysilyl)-1-propanethiol grafted Fe3O4 nanoparticles coupled with spectrofluorimetric detection was proposed for determination of aflatoxin M1 (AFM1) in liquid milk samples. The method uses the advantage fluorescence enhancement by βcyclodexterin complexation of AFM1 in 12% (v/v) acetonitrile-water and the remarkable properties of Fe3O4 nanoparticles namely high surface area and strong magnetization were utilized to achieve high enrichment factor (57) and satisfactory extraction recoveries (91-102%) using only 100 mg of magnetic adsorbent. Furthermore, fast separation time of about 15 min avoids many time-consuming column-passing procedures of conventional SPE. The main factors 1
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affecting extraction efficiency including pH value, desorption conditions, extraction/desorption time, sample volume, and adsorbent amount were evaluated and optimized. Under the optimal conditions, a wide linear range of 0.04-8 ng ml-1 with a low detection limit of 0.015 ng mL-1 was
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obtained. The developed method was applied for extraction and preconcentration of AFM1 in three commercially available milk samples and the results were compared with the official
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AOAC method.
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Keywords: Aflatoxin M1; Modified Fe3O4 nanoparticles; Magnetic solid phase extraction; β-
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cyclodexterin; spectrofluorimetry.
*Corresponding Author: Tel: +98-21-88848949; Fax: +98-21-88820993 E-mail: [email protected] (M. Amoli-Diva).
1. Introduction
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Aflatoxins are toxic secondary metabolites produced mainly by certain spices of Aspergillus, especially A. Flavus, A. Parasiticus and A. Nomius [1] which can colonize foodstuffs and feed. These compounds are widespread in many countries, especially in tropical and subtropical
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regions where temperature and humidity conditions are appropriate for moulds growth and toxin production. Today, the climate tropicalization makes aflatoxicos a global issue which receive
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great attention during the last three decades [2, 3]. The frequent incidence of these toxins in
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agricultural commodities has a potentially negative impact on the health and economies of the affected regions because they are potent cancer-promoting agents [4]. Four common aflatoxins in
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food and feed are aflatoxin B1, B2, G1 and G2. Aflatoxin B1 (AFB1), the most toxic compound in this group, has been found to be one of the most potent carcinogens occurring naturally. It was
(IARC) in 1987 [5].
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classified as group I human carcinogen by the International Agency of Research on Cancer
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Aflatoxin M1 (AFM1, Fig. 1) is the main monohydroxylated derivative of AFB1 which produces
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by means of cytochrome P450-associated enzymes in liver and also known as “milk toxin”. Mammals that ingest AFB1 contaminated diets excrete AFM1 into milk and subsequently can be found in a large variety of dairy products [6, 7]. AFB1 quickly absorbs from gastro-intestinal track and it appears as AFM1 in blood after just 15 min [8]. It can be detected in milk in 12-24 h after the first ingestion of AFB1 [9]. Thus, AFM1 concentration in milk and milk products depends on the level of exposure and the amount of AFB1 ingested. AFM1 is relatively stable in raw and processed milk products and is not affected by pasteurization (even those using UHT techniques) or cheese processing performed in dairy industry [10]. Due to the fundamental role of milk and milk products in human diets, especially for young children, findings of AFM1 in dairy products is regarded as significant hazard for food
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safety and public health [11]. Thus, the European community legislation imposes maximum AFM1 level in liquid, dried or processed milk products at 0.050 µg L-1 for adults and 0.025 µg L1
for infants [12].
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Determination of AFM1 is generally performed by different techniques such as thin layer chromatography (TLC) [13, 14], ultra-high performance liquid chromatography with mass
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detection [15, 16], electrochemical detection [17, 18] and chemiluminescence [19] and there are
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several recent reviews on the analytical methods used for determination of mycotoxins in different biological and food matrices [20, 21]. However, the “gold standard” for aflatoxin
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determination is HPLC followed by fluorimetric (FL) or mass spectroscopic (MS) analysis [2224] which is time-consuming and costly and mainly limited to laboratory uses.
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In the past few years, a number of rapid detection method based on immunoassays have been developed [25, 26] which utilize antibodies to capture aflatoxins selectively from sample
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solution. In spite of their high absorption selectivity and affinity, the antibodies are susceptible to
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denaturation and degradation, and therefore stringent testing conditions are necessary to ensure their performance. In addition, the production of antibodies requires living animals which is a complex and expensive process [27] and for legal purpose, positive results need confirmations by accepted reference methods.
On the other hand, food samples have complex matrices and concentration of aflatoxins is very low which makes their determination difficult. So, the methods normally used for aflatoxins analyses are based on the extraction with organic solvents, clean-up with solid phase extraction (SPE) or immunoaffinity columns (IAC), followed by concentration steps [28]. In the case of AFM1, C18 column, Carbograph-4, multifunctional clean-up column and IAC were reported to have appropriate clean-up/ preconcentration effect in dairy products [22, 29].
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Recently, application of nanostructures in SPE has shown a remarkable interest. One of the most popular strategies is focused on the use of magnetic nanoparticles (MNPs) taking into account many advantages arising from the inherent characteristics of magnetic particles [30]. Magnetic
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solid phase extraction (MSPE) methodology overcomes problems such as column packing and phase separation, which can be easily performed by applying an external magnetic field [31].
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Therefore, suspended MNPs tagged with analytes can be isolated from large volume samples
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using a strong magnet. Regarding AFM1, only a few examples of biosensors are reported on the use of MNPs and not any SPE procedure [6, 19, 32]. Starting from our previous work on the use
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of modified MNPs for extraction of mycotoxins [33], the present study reports a convenient and sensitive MSPE method for the determination of AFM1 in milk samples.
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In this study, ethylene glycol bis-mercaptoacetate grafted 3-(trimethoxysilyl)-1-propantiol modified Fe3O4 nanoparticles (EGBMA-MSPT-MNPs) were used as the magnetic adsorbent for
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extraction and preconcentration of AFM1 followed by enhanced spectrofluorimetric
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determination. The factors affecting extraction efficiency of the analyte were investigated and optimized. Finally, the proposed method was successfully applied to the extraction and preconcentration of AFM1 from commercial milk samples.
2. Experimental
2.1 Chemicals and reagents .
All chemicals and reagents were of analytical grade and used as supplied. Standard of AFM1 was purchased from Sigma chemical (St Louis, MO, USA). Ferrous chloride tetrahydrate (FeCl2.4H2O), ferric chloride hexahydrate (FeCl3.6H2O), sodium chloride, sodium hydroxide, β-
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cyclodexterin
(CD),
3-trimethoxysilyl-1-propanethiol
(MSPT),
ethylene
glycol
bis-
mercaptoacetate (EGBMA), ammonia solution (25% w/w), hydrochloric acid, glycerol, methanol and acetonitrile were purchased from Merck company (Darmstadt, Germany). Blank liquid milk
the samples contain less than 0.01 ppb of AFM1 using HPLC analysis.
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samples were supplied from the Institute of Standard and Industrial Research of Iran (ISIRI) and
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Aflatoxins are carcinogenic compounds. So, extracts and solutions should be handled with
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extreme care. Gloves and other protective clothing were worn as safety precaution during the
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handling of the analyte. The aflatoxin residues can be destroyed using 3% sodium hypochlorite.
2.2 Instrumentation fluorescence
spectra
were
recorded
by a
Varian
Cary Eclipse
fluorescence
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The
spectrophotometer (Palo Alto, CA, USA) equipped with a xenon lamp and a thermostat cell
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compartment. The scan rate was maintained at 1200 nm min-1 and the excitation and emission
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slits were both 5 nm. All measurements were performed in a 10 mm quartz microcell at 25± 0.1 ºC and the excitation and emission wavelengths were 365 and 460 nm respectively. The SEM images were obtained using a Hitachi S-4160 field emission scanning electron microscope (Tokyo, Japan). Chemical interactions were studied using a Perkin Elmer Spectrum one Bv5.3.0 FT-IR spectrometer in the range of 400-4000 cm-1 with KBr pellets (Waltham, Massachusetts, US). The X-ray diffraction (XRD) analysis of modified nanoparticles was performed by an Ital Structures APD 2000 X-ray diffractometer (Riva Del Garda, Italy) using Cu Kα radiation source λ 1.540598 Aº with reflection scan mode. A Metrohm 827 pH/mV meter (Herisau, Switzerland) with a combined glass electrode was used for the pH measurements.
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2.3 Preparation of magnetic adsorbent Magnetic adsorbent was prepared according to our previously reported method [33]. Briefly, FeCl2.4H2O (4.30 g) and FeCl3.6H2O (11.68 g) were dissolved in deionized water (200 mL)
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under nitrogen atmosphere with vigorous stirring at 85 ºC. Then, ammonia solution (45 mL, 25% w/w) was added and the solution was stirred for 15 min at the same conditions. After cooling
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down to the room temperature, the magnetic precipitate was isolated by applying an external
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magnet and the supernatant was removed. The precipitate was washed with deionized water (250 mL, three times) and 0.02 mol L-1 sodium chloride solution (100 mL, twice). Sodium chloride
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was used for the flocculation of magnetic nanoparticles. Furthermore, it could accelerate the magnetic separation particularly in alkaline solutions [34, 35]. Then, the suspension was
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transferred to a 250 mL round bottom flask and allowed to settle. The supernatant was removed and an aqueous solution of MSPT (10% v/v, 80 mL) was added followed by glycerol (60 mL).
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The mixture was stirred under nitrogen atmosphere at 90 ºC for 2 h. After cooling down slowly
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to the room temperature, the black precipitate (MSPT-MNPs) was easily isolated from the supernatant by magnetic decantation and the suspension was washed sequentially with deionized water (250 mL, three times), methanol (200 mL, twice), and deionized water (250 mL, once). In the next step, the supernatant was removed and the solid MSPT-MNPs were homogeneously dispersed to EGBMA solution (1.0% v/v, 150 mL). The solution was transferred to a 400 mL beaker and sonicated for 2 h. The resulting solid phase (EGBMA-MSPT-MNPs) was separated by magnetic decantation, washed with deionized water (3×250 mL) and methanol (2×200 mL) before it was dried in vacuum oven at 45 ºC for 24 h.
2.4 Recommended MSPE procedure
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Blank liquid milk sample (10 ± 0.1 gram) was accurately weighed and transferred into a 50 mL centrifuge tube. The sample was centrifuged at 4000 rpm for 15 min and the fat layer was removed. Then, the supernatant was spiked with appropriate amounts of AFM1 and diluted to 30
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mL with phosphate buffed solution (PBS, pH=7.5) in a capped container with intensive shaking. The diluted aqueous phase was transferred to a 50 mL beaker and the magnetic adsorbent (100
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mg) was added. The mixture was stirred for 5 min to facilitate adsorption of AFM1 to the
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modified MNPs. The adsorbent was collected at the bottom of the beaker by applying a supermagnet and the supernatant was removed. Afterwards, 3 mL of a mixture of acetone:
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acetonitrile: chloroform (1:2:1, v/v) was and the suspension was stirred for 4 min. After desorption, the eluent was separated by magnetic decantation and evaporated to dryness under a
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flow of nitrogen gas at room temperature. The dry residue was dissolved in 5 mL of 12 % (v/v) acetonitrile/water. Then, CD (70 mg) was added to the solution and heated at 50 ºC for 25 min.
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fluorescence spectra.
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The final solution was evaporated to 500 µL under nitrogen flow and used for taking
2.5 Method validation
The proposed MSPE-fluorimetric method was validated in terms of linearity, limit of detection (LOD), limit of quantitation (LOQ), accuracy, precision, and enrichment factor.
2.5.1 Linearity
Linearity of the proposed method was evaluated by analyzing different concentration of AFM1 (0.04, 0.1, 0.5, 1.25, 2.0, 4.0, 6.0 and 8.0 ng mL-1) and an eight-point calibration curve was constructed by plotting fluorescence intensity vs. AFM1 concentration (each point is the mean
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value of three replicates analyses). The slope, intercept and correlation coefficient were calculated using the least square regression method.
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2.5.2 LOD and LOQ The LOD and LOQ were calculated based on the standard deviation of blank (Sb) and slope of
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calibration curve (m) determined according to the following equations:
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LOD = 3Sb/m
(2)
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LOQ = 10Sb/m
(1)
2.5.3 Accuracy and precision
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The accuracy of the proposed method was determined by five replicates analyses of blank milk samples (Blank S1-S3) spiked with AFM1 at three concentration levels (0.05, 2.00 and 7.50 ng
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mL-1). The recovery was calculated by using the following equation:
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Recovery (%) = (Measured concentration/Nominal concentration) ×100
(3)
Repeatability or intra-day precision was evaluated by spiking blank samples with appropriate amounts of AFM1 at three different levels (0.05, 2.00 and 7.50 ng mL-1). Five replicates were performed and analyzed on the same day. In order to determine reproducibility or inter-day precision, five replicated samples which were spiked with the same amount of AFM1, were analyzed on five consecutive days. The intra and inter-day precisions were expressed as the percentage relative standard deviation (RSD %).
2.5.4 Enrichment factor The enrichment factor (EF) of the method was calculated using the following equation:
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EF = Vs/Ve× R%
(4)
Where Vs is the sample volume, Ve is elution volume, and R% is percent recovery.
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3. Results and discussion For AFM1 determination, low sensitivity and poor precision are the main problems arising when
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a simple solid phase extraction (SPE) was tried. Low recovery due to the well-known interaction
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between AFM1 and milk proteins and/or strong matrix effect due to co-extracted and co-eluted compounds [36] were probably responsible for this failure. On the other hand, one of our goals
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was to perform sample preparation without using the very expensive immunosorbent cartridges. Therefore, we resolved to use a modified MNPs-based SPE procedure. The synthesis of MNPs is
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based on a cost-effective and convenient co-precipitation method which Fe3O4 nanoparticles were synthesized from ferric and ferrous ions by addition of a base (ammonia solution here)
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under inert atmosphere at elevated temperature [37]. When the synthetic conditions are constant,
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the quality of nanoparticles is completely reproducible. However, naked nanoparticles suffer from aggregation. Ethaxysilyl or methaxysilyl compounds are the common modifiers solving this problem. Furthermore, they can provide favorable functional groups which can attack to the analyte molecules to improve their adsorption. We tried different modifiers for MNPs which have complete hydrophobic (oleic acid) or complete electrostatic (mercaptopropionic acid) skeleton and those with both of them (EGBMAMSPT-MNPs). The experimental results indicate that quantitative recoveries (˃ 90%) were obtained using the later one. This adsorbent provides both hydrocarbon skeleton and SH end group which are in the form of protonated SH2+ in extraction medium. On the other hand, AFM1 is a difurancoumarin and highly conjugated compound and according to the Pub Chem public
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chemical database (http://pubchem.ncbi.nml.gov/), its partition coefficient (Log P) is +0.5. This parameter is often used to measure how hydrophilic or hydrophobic a chemical substance is. The log P value shows that AFM1 is more hydrophobic [38]. Thus, hydrophobic interactions with the
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hydrocarbon skeleton of the adsorbent can play a role in the adsorption of AFM1. On the other hand, approaching the aromatic rings in AFM1 and SH2+ end groups of the adsorbent can also
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two mechanism, namely hydrophobic and cation-π interactions.
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cause van der Waals force [39]. Thus the interaction of AFM1 with the adsorbent can occur by
In addition, AFM1 as a penahetrocyclic and highly conjugated compound, exhibits native
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fluorescence. So sensitive analytical methods for detection of AFM1 are based on either its native emission properties or on enhanced fluorescence after chemical complexation/
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drevitization. In this work, we used the inclusion of AFM1 within β-cyclodextrin which yields a
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sensitivity.
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significant enhancement to the molecular fluorescence of AFM1 and increases its determination
3.1 Characterization of adsorbent
The size and morphology of the prepared adsorbent were characterized by SEM images. As can be seen from Fig. 2, the prepared adsorbent have uniform size distribution with average diameter of 20 nm and most of the particles are quasi spherical in shape. The XRD pattern of the adsorbent (Fig. S1, supplemental materials) shows reflection peaks for 220, 311, 400, 511, 440 and 622 plans in peak position (2θ) of 30.2, 35.3, 43.2, 57.2, 62.7 and 74.2 respectively, which match well with those obtained by JCPDS for magnetite [40, 41]. The FT-IR spectra of naked MNPs, MSPT-MNPs and EGBMA-MSPT-MNPs adsorbent were shown in Fig. S2, supplementary materials. The adsorbent spectrum shows the characteristics absorption band of
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Fe-O band (for Fe3O4 nanoparticles) at 581 cm-1, two strong absorption bands for Si-O-H and SiO-Si stretching vibrations at 1122 and 1034 cm-1 respectively, the absorption band of alkyl chains at 2928 cm-1 and the absorption band of SH groups at 2552 cm-1, confirming the
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preparation of EGBMA-MSPT-MNPs.
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3.2 Effect of complexation conditions
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Among the various analytical techniques, fluorescence spectrophotometry remains one of the most interesting ones for the analysis and characterization of CD inclusion complexes, because
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of the significant analyte signal increment that is frequently induced. The fluorophore introduced into the CD internal cavity is isolated from the surrounding water molecules and its excited state
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is shielded from quenching processes. Indeed, larger is the signal increase provoked by the organized medium, more sensitive and accurate is the analytical measurement. The effect of
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complexation conditions on AFM1 fluorescence signal was investigated by adding different
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amounts of CD (10-100 mg) to the sample solutions. The experimental results (Fig. 3) showed a significant fluorescence enhancement with increasing CD amount and reached the maximum in 70 mg. The effect complexation time and temperature on the fluorescence signal of AFM1 was also investigated in the range of 5-50 min and 5-60 ºC respectively. The results (Figs. S3 and S4, supplemental materials) revealed that 25 min and 50 ºC were enough respectively for optimal complex formation of AFM1 and used for the next experiments. In addition, the effect of organic solvent (acetonitrile) in the predominantly aqueous CD solutions was investigated in the range of 0-30%. The experimental results revealed that the fluorescence signal of the complex was decreased for acetonitrile/water mixture in the 0-7% (v/v) range. Then, the AFM1-CD fluorescence intensity was increased with increasing
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acetonitrile percentage until 12% (v/v), and remains constant for larger amounts. The decrease of fluorescence intensity observed in the 0-7% (v/v) acetonitrile range is probably due to the competition of acetonitrile molecules with AFM1 for binding sites inside the protective CD
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cavity. This is in agreement with the decrease of cyclodextrin-analyte binding abilities noted when the concentration of urea [42] or several organic solvents [43-45] increases. Thus,
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complexation was performed by addition of 70 mg CD to the eluted AFM1 in 12% (v/v)
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acetonitrile/water at 50 ºC for 25 min.
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3.3 Extraction optimizations 3.3.1 Effect of pH
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An appropriate pH value is a key parameter improves adsorption efficiency and eliminates interferences. Therefore, it is necessary to adjust it prior to any SPE procedure. The effect of pH
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on the recovery of AFM1 was examined in the range of 3-9 while the other conditions were
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unchanged. The results were demonstrated in Fig. 4. As can be seen, recovery of the analyte increases with increase in pH value and reached maximum in 7.0-7.8 range. There was no change in the maximum emission wavelength within this pH range. It was known that aflatoxin molecules have been characterized to possess two potential site of weakness through which it is degraded by chemical agents. High proton concentration or oxidizing agents easily attack the double bond in the furo-furan ring while the lactone ring is hydrolyzed in alkaline environment [46]. When pH value decreases, a β-keto acid structure forms (catalyzed by the high acidic medium) and thus decreases conjugation and fluorescence properties. In high pH, a decrease in emission is also noted due to hydrolyzing lactone ring which reduces conjugation, as already
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reported for aflatoxin compounds [47]. Based on the results, pH 7.5 was selected for all subsequent experiments.
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3.3.2 Effect of desorption conditions An ideal elution solvent should be strong enough to elute all the target compounds from the
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sorbent. In order to achieve optimum desorption conditions, different organic solvents including
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methanol (MeOH), ethanol (EtOH), acetone (Me2OH), acetonitrile (MeCN), and chloroform (CH2Cl2) alone or in mixture were examined. The results (Fig. 5) showed that the maximum
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desorption was obtained with a mixture of acetone: acetonitrile: chloroform (1:2:1, v/v) and it was selected as the eluent for the next experiments.
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The volume of eluent was also studied in the range of 1-10 mL. Based on the results, the minimum volume of eluent required for quantitative desorption (>90%) was found to be 3 mL.
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for the subsequent experiments.
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Thus, 3 mL of acetone: acetonitrile: chloroform (1:2:1, v/v) was selected as the optimum eluent
3.3.3 Effect of sample volume
In order to explore the possibility of enriching low concentration of analyte from large volume, the maximum applicable sample volume must be determined. For this purpose, different sample volumes (10-80 mL) containing 10 ng of analyte were tested. The results indicated that the recoveries were quantitative within 10-50 mL. As mentioned before, the final volume of samples reached 500 µL. So, the theoretical enrichment factor of 60 can be obtained which demonstrates the feasibility of sample determination with different analyte concentration levels.
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3.3.4 Effect of extraction and desorption times A good dispersion of adsorbent should considerably increase the contact surface area between the adsorbent and analyte in sample, leading to a higher adsorption efficiency and faster
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extraction time. MNPs have higher surface area and can rapidly collect from sample solution by applying magnetically assisted separation, thus shorter extraction time can be achieved. Figs. S5
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and S6 show the recovery values of AFM1 as a function of extraction or desorption time. Based
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on the results, 5 min is sufficient for achieving appropriate adsorption and 4 min was enough for
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good desorption of AFM1 which are much lower than the traditional column-passing SPE.
3.3.5 Effect of adsorbent amount and reusability
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As mentioned before, nanoparticles based adsorbents compared to ordinary (micron-sized) adsorbents offer a significantly higher surface area-to-volume ratio and a short diffusion route,
d
which result in high extraction capacity, rapid extraction dynamics and high extraction
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efficiencies. Therefore, satisfactory results can be obtained with fewer amounts of these adsorbents. The effect of EGBMA-MSPT-MNPs amount was studied in the range of 20-160 mg. The experimental results (Fig. S7, supplemental materials) indicated that the maximum recovery was obtained by 100 mg of EGBMA-MSPT-MNPs and at higher amounts; the extraction efficiency was almost constant. Therefore, 100 mg of adsorbent was added to the sample solution in the subsequent experiments.
Reusability is one of the determinant factors for evaluation of an adsorbent’s performance. In order to determine regeneration capability of EGBMA-MSPT-MNPs, adsorbents which used in the general SPE procedure were washed with desorbing solvent (20 mL, once) and deionized water (50 mL, three times), then dried in oven at 45 ºC and reused. Experimental results revealed
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that the adsorbent can be reused at least up to ten times (RSD ˂3.6%) without sacrifice the analytical performance which makes it a suitable alternative to immunoaffinity columns.
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3.4 Effect of interferences In real sample analysis, matrix effect caused by co-elution of matrix components can affect
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AFM1 determination. Since, the co-occurrence of mycotoxins such as aflatoxins (B1, B2, G1,
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G2 and M2), ochratoxin (OTA), deoxynivalenol (DON), and zearalenone (ZEN) rarely occur in milk [21, 48], the effect of interferences on the preconcentration and determination of AFM1 was
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studied by spiking these mycotoxins to the samples. Therefore, 10.0 g of blank samples were spiked with 100 ng of each aflatoxin, 500 ng of OTA, 100 ng of ZEN, and 5000 ng of DON.
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Then, the samples were spiked with 10 ng AFM1, diluted to 30 mL with PBS solution, and subjected to the proposed SPE procedure. The experimental results (table 1) revealed that all
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aflatoxins can quantitatively adsorb on the surface of EGBMA-MSPT-MNPs in these conditions
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and can interfere because they belong to the same chemical family as AFM1 and follow the same fluorescence pattern. However, co-occurrence of aflatoxins (except for M2) was rarely observed in milk [28, 49] unless for milk products (such as milk powder) with flour or cereals as part of ingredients which are not target samples here. Aflatoxin M2 (AFM2) level in milk is also estimated to be small. However, when it is exist, only the total AFM1 and AFM2 can be determined. OTA, DON and ZEN can also be found in milk in very low concentrations [15]. As can be seen from the table 1, the AFM1 recoveries were not affected by the presence of these compounds. Although the results revealed that they can be adsorbed on the surface of EGBMAMSPT-MNPs to some extent, it seems that the desorbing solvent cannot elute them from the
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adsorbent. Thus, AFM1 can be successfully determined by the proposed method without interference of them.
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3.5 Method validation With optimized MNPs-based SPE procedure, the calibration curve was found to be linear in the
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0.04-8 ng mL-1 range. The calibration data was subjected to linear least square regression
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analysis and calibration equation of Y=94.635(±1.3)C+15.007(±2.2) was obtained with correlation coefficient (R2) of 0.9989. The LOD and LOQ values were obtained 0.015 and 0.052
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ng mL-1 respectively. To evaluate the accuracy of the method, it was applied to the determination of AFM1 in blank milk and recovery studies were carried out by spiking the samples with
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different amounts of AFM1. As can be seen from table 2, good recoveries in the range of 91102% were obtained. The results for intra and inter-day precision analyzes were also summarized
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in Table 2. The RSDs were ranged from 3.0- 5.1% and 3.1- 6.3% for intra-day and inter-day
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variations respectively, which indicate good precision of the proposed method. Furthermore, by extracting 30 mL of sample solution containing analyte and collecting into a final volume of 500 µL (R=95%), the enrichment factor of 57 was achieved for AFM1 determination.
3.6 Analysis of real samples
The evaluated method was subsequently applied to determination of AFM1 in three different brands of commercially available milk samples widely used in Iran. Obtained results are summarized in table 3. Moreover, the AOAC standard method (IAC-HPLC-FL) was used to evaluate the accuracy of proposed method. The results exhibited that no significant difference was observed between the two methods and indicate the suitability of EGBMA-MSPT-MNPs
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adsorbent for extraction and preconcentration of trace amounts of AFM1 in real samples. Comparative information from some previously reported studies in the literature on the preconcentration of AFM1 is given in Table 4. In the present work, better analytical parameters
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were obtained in comparison with most of those methods. In addition, the cited methods are more complicated and use more reagents, besides application of the proposed method is simpler
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and takes less time.
4. Conclusion
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In this study, a novel MNPs-based solid phase extraction method coupled with enhanced fluorescence detection was successfully applied to determination of ultra-trace amounts of AFM1
separation,
providing
a
fast,
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in milk samples. The method combines high surface area of the nanoparticles and magnetic convenient,
effective,
and
sensitive
method
for
d
preconcentration/separation of AFM1 in liquid milk samples. In addition, reusability studies
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demonstrated the potential of the adsorbent for recyclable usage with compared to the immunoaffinity columns. The method is simple and inexpensive, and eliminates the need to use any auxiliary facilities to immobilize the adsorbent. What is more, usage is not limited to the applications demonstrated here; other detection systems can be used depending on the actual requirements.
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Ac ce pt e
Fig. 1 Chemical structure of AFM1.
d
Figure captions:
M
an
us
cr
[52]
ip t
Velasco-Garcia, D. Moscone, G. Palleschi, Anal. Chim. Acta 520 (2004) 141.
Fig. 2 SEM image of the prepared EGBMA-MSPT-MNPs adsorbent with mean diameter of 20 nm.
Fig. 3 Effect of CD amount on the fluorescence signal of AFM1. Solvent; 12% (v/v) acetonitrilewater, complexation time; 25 min, and complexation temperature 50 ºC. Fig. 4 Effect of pH on the recovery of AFM1. Sample volume; 30 mL, adsorbent amount; 100 mg, extraction time; 5 min, desorption time; 4 min, and eluent solvent; acetonitrile: chloroform (1:2:1, v/v). Fig. 5 Effect of eluent solvent on the recovery of AFM1. Sample volume; 30 mL, adsorbent amount; 100 mg, extraction time; 5 min, desorption time; 4 min, and pH; 7.5.
22
Page 22 of 28
Ac ce pt e
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d
M
an
us
cr
Fig. 1
Fig. 2
23
Page 23 of 28
cr
ip t Ac ce pt e
d
M
an
us
Fig. 3
Fig. 4
24
Page 24 of 28
ip t us
cr Table 1
Ac ce pt e
d
M
an
Fig. 5
Effect of mycotoxins interferences on the recovery of AFM1 adsorbed on EGBMA-MSPTMNPs.
Interference
Concentration (ng mL-1)
Recovery ± RSD (%)
Aflatoxins
3.4a
NCb
OTA
16.7
91 ± 2
ZEN
3.4
95 ± 3
25
Page 25 of 28
DON
166.7
97 ± 3
Concentration of each aflatoxin including B1, B2, G1, G2 and M2.
b
Not calculated because of interference effect of aflatoxins.
cr
ip t
a
us
Table 2
an
Results for recovery, intra-day and inter-day precision analyses for the proposed MNPs-based SPE procedure (n=5). Recovery
(ng mL-1)
(%)
101.01
Ac ce pt e
0.05
d
Blank S1
Intra-day variations
M
Spiked level Sample
Inter-day
(RSD%)
variations (RSD%)
5.08
6.18
2.00
91.21
3.45
3.61
7.50
97.73
3.03
3.13
0.05
92.15
5.01
6.29
2.00
102.22
3.52
3.74
7.50
96.53
3.12
3.33
0.05
90.62
5.07
6.10
2.00
93.44
3.32
3.81
Blank S2
Blank S3
26
Page 26 of 28
92.13
3.18
3.45
cr
ip t
7.50
us
Table 3
an
Comparison of AFM1 analyses in natural contaminated liquid milk samples by the proposed and official methods (mean ± SD, n=5).
Found (ng mL-1)
*
0.26 ± 0.03
Ac ce pt e
Sample 1
d
Proposed method
M
Milk sample
Official method*
0.31 ± 0.07
Sample 2
0.19 ± 0.04
0.17 ± 0.03
Sample 3
0.23 ± 0.09
0.28 ± 0.05
IAC-HPLC-FL
Table 4 Comparison of linear range and LOD for determination of AFM1 with some of the previously reported methods. 27
Page 27 of 28
Linear range
Detection limit
Time needed
Assay type
Reference -1
-1
a
(ng mL )
for assay
0.015-1
0.015
28 h
[50]
0.2-4
0.006
30 min
[29]
Immunochip
0.45-3.9
0.24
2.5 h
[25]
Flow-injection immunoassay
0.02-0.5
0.011
26 h
[51]
Competitive ELISAb
28-164
28
2.5 h
[52]
Indirect competitive ELISA
0.1-3.2
0.04
27.5 h
[8]
MNPs-based SPE
0.04-8
0.015
50 min
This work
Not mentioned exactly, at least
b
Enzyme-linked Immunosorbent Assay
cr
us
Ac ce pt e
d
a
an
HPLC-FL
M
Impedimetric immunosensor
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(ng mL )
28
Page 28 of 28
S0003-2670(14)00579-0 http://dx.doi.org/doi:10.1016/j.aca.2014.05.007 ACA 233251
To appear in:
Analytica Chimica Acta
Received date: Revised date: Accepted date:
30-3-2014 1-5-2014 5-5-2014
Please cite this article as: Zohreh Taherimaslak, Mitra Amoli-Diva, Mehdi Allahyary, Kamyar Pourghazi, Magnetically assisted solid phase extraction using Fe3O4 nanoparticles combined with enhanced spectrofluorimetric detection for aflatoxin M1 determination in milk samples, Analytica Chimica Acta http://dx.doi.org/10.1016/j.aca.2014.05.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Magnetically assisted solid phase extraction using Fe3O4 nanoparticles combined with enhanced spectrofluorimetric detection for aflatoxin M1 determination in milk samples
ip t
Zohreh Taherimaslaka, Mitra Amoli-Divab,*, Mehdi Allahyaryc, Kamyar Pourghazib Faculty of Chemistry, Bu-Ali Sina University, Hamadan, Iran
b
Faculty of Chemistry, Kharazmi (Tarbiat Moallem) University, Tehran, P.O. Box 15719-14911, Iran
c
Quality control laboratory, ARA quality research Co., Tehran, Iran
an
us
cr
a
Highlights
Ac ce pt e
d
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► A new SPE technique coupled with spectrofluorimetry for extraction and determination of aflatoxin M1 has been developed. ► Modified magnetic nanoparticles were used as adsorbent. ► Β-cyclodextrin was used for fluorescence enhancement of AFM1. ► The method was applied to some commercial milk samples
Abstract
A novel, facile and inexpensive solid phase extraction (SPE) method using ethylene glycol bismercaptoacetate modified 3-(trimethoxysilyl)-1-propanethiol grafted Fe3O4 nanoparticles coupled with spectrofluorimetric detection was proposed for determination of aflatoxin M1 (AFM1) in liquid milk samples. The method uses the advantage fluorescence enhancement by βcyclodexterin complexation of AFM1 in 12% (v/v) acetonitrile-water and the remarkable properties of Fe3O4 nanoparticles namely high surface area and strong magnetization were utilized to achieve high enrichment factor (57) and satisfactory extraction recoveries (91-102%) using only 100 mg of magnetic adsorbent. Furthermore, fast separation time of about 15 min avoids many time-consuming column-passing procedures of conventional SPE. The main factors 1
Page 1 of 28
affecting extraction efficiency including pH value, desorption conditions, extraction/desorption time, sample volume, and adsorbent amount were evaluated and optimized. Under the optimal conditions, a wide linear range of 0.04-8 ng ml-1 with a low detection limit of 0.015 ng mL-1 was
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obtained. The developed method was applied for extraction and preconcentration of AFM1 in three commercially available milk samples and the results were compared with the official
cr
AOAC method.
us
Keywords: Aflatoxin M1; Modified Fe3O4 nanoparticles; Magnetic solid phase extraction; β-
Ac ce pt e
d
M
an
cyclodexterin; spectrofluorimetry.
*Corresponding Author: Tel: +98-21-88848949; Fax: +98-21-88820993 E-mail: [email protected] (M. Amoli-Diva).
1. Introduction
2
Page 2 of 28
Aflatoxins are toxic secondary metabolites produced mainly by certain spices of Aspergillus, especially A. Flavus, A. Parasiticus and A. Nomius [1] which can colonize foodstuffs and feed. These compounds are widespread in many countries, especially in tropical and subtropical
ip t
regions where temperature and humidity conditions are appropriate for moulds growth and toxin production. Today, the climate tropicalization makes aflatoxicos a global issue which receive
cr
great attention during the last three decades [2, 3]. The frequent incidence of these toxins in
us
agricultural commodities has a potentially negative impact on the health and economies of the affected regions because they are potent cancer-promoting agents [4]. Four common aflatoxins in
an
food and feed are aflatoxin B1, B2, G1 and G2. Aflatoxin B1 (AFB1), the most toxic compound in this group, has been found to be one of the most potent carcinogens occurring naturally. It was
(IARC) in 1987 [5].
M
classified as group I human carcinogen by the International Agency of Research on Cancer
d
Aflatoxin M1 (AFM1, Fig. 1) is the main monohydroxylated derivative of AFB1 which produces
Ac ce pt e
by means of cytochrome P450-associated enzymes in liver and also known as “milk toxin”. Mammals that ingest AFB1 contaminated diets excrete AFM1 into milk and subsequently can be found in a large variety of dairy products [6, 7]. AFB1 quickly absorbs from gastro-intestinal track and it appears as AFM1 in blood after just 15 min [8]. It can be detected in milk in 12-24 h after the first ingestion of AFB1 [9]. Thus, AFM1 concentration in milk and milk products depends on the level of exposure and the amount of AFB1 ingested. AFM1 is relatively stable in raw and processed milk products and is not affected by pasteurization (even those using UHT techniques) or cheese processing performed in dairy industry [10]. Due to the fundamental role of milk and milk products in human diets, especially for young children, findings of AFM1 in dairy products is regarded as significant hazard for food
3
Page 3 of 28
safety and public health [11]. Thus, the European community legislation imposes maximum AFM1 level in liquid, dried or processed milk products at 0.050 µg L-1 for adults and 0.025 µg L1
for infants [12].
ip t
Determination of AFM1 is generally performed by different techniques such as thin layer chromatography (TLC) [13, 14], ultra-high performance liquid chromatography with mass
cr
detection [15, 16], electrochemical detection [17, 18] and chemiluminescence [19] and there are
us
several recent reviews on the analytical methods used for determination of mycotoxins in different biological and food matrices [20, 21]. However, the “gold standard” for aflatoxin
an
determination is HPLC followed by fluorimetric (FL) or mass spectroscopic (MS) analysis [2224] which is time-consuming and costly and mainly limited to laboratory uses.
M
In the past few years, a number of rapid detection method based on immunoassays have been developed [25, 26] which utilize antibodies to capture aflatoxins selectively from sample
d
solution. In spite of their high absorption selectivity and affinity, the antibodies are susceptible to
Ac ce pt e
denaturation and degradation, and therefore stringent testing conditions are necessary to ensure their performance. In addition, the production of antibodies requires living animals which is a complex and expensive process [27] and for legal purpose, positive results need confirmations by accepted reference methods.
On the other hand, food samples have complex matrices and concentration of aflatoxins is very low which makes their determination difficult. So, the methods normally used for aflatoxins analyses are based on the extraction with organic solvents, clean-up with solid phase extraction (SPE) or immunoaffinity columns (IAC), followed by concentration steps [28]. In the case of AFM1, C18 column, Carbograph-4, multifunctional clean-up column and IAC were reported to have appropriate clean-up/ preconcentration effect in dairy products [22, 29].
4
Page 4 of 28
Recently, application of nanostructures in SPE has shown a remarkable interest. One of the most popular strategies is focused on the use of magnetic nanoparticles (MNPs) taking into account many advantages arising from the inherent characteristics of magnetic particles [30]. Magnetic
ip t
solid phase extraction (MSPE) methodology overcomes problems such as column packing and phase separation, which can be easily performed by applying an external magnetic field [31].
cr
Therefore, suspended MNPs tagged with analytes can be isolated from large volume samples
us
using a strong magnet. Regarding AFM1, only a few examples of biosensors are reported on the use of MNPs and not any SPE procedure [6, 19, 32]. Starting from our previous work on the use
an
of modified MNPs for extraction of mycotoxins [33], the present study reports a convenient and sensitive MSPE method for the determination of AFM1 in milk samples.
M
In this study, ethylene glycol bis-mercaptoacetate grafted 3-(trimethoxysilyl)-1-propantiol modified Fe3O4 nanoparticles (EGBMA-MSPT-MNPs) were used as the magnetic adsorbent for
d
extraction and preconcentration of AFM1 followed by enhanced spectrofluorimetric
Ac ce pt e
determination. The factors affecting extraction efficiency of the analyte were investigated and optimized. Finally, the proposed method was successfully applied to the extraction and preconcentration of AFM1 from commercial milk samples.
2. Experimental
2.1 Chemicals and reagents .
All chemicals and reagents were of analytical grade and used as supplied. Standard of AFM1 was purchased from Sigma chemical (St Louis, MO, USA). Ferrous chloride tetrahydrate (FeCl2.4H2O), ferric chloride hexahydrate (FeCl3.6H2O), sodium chloride, sodium hydroxide, β-
5
Page 5 of 28
cyclodexterin
(CD),
3-trimethoxysilyl-1-propanethiol
(MSPT),
ethylene
glycol
bis-
mercaptoacetate (EGBMA), ammonia solution (25% w/w), hydrochloric acid, glycerol, methanol and acetonitrile were purchased from Merck company (Darmstadt, Germany). Blank liquid milk
the samples contain less than 0.01 ppb of AFM1 using HPLC analysis.
ip t
samples were supplied from the Institute of Standard and Industrial Research of Iran (ISIRI) and
cr
Aflatoxins are carcinogenic compounds. So, extracts and solutions should be handled with
us
extreme care. Gloves and other protective clothing were worn as safety precaution during the
an
handling of the analyte. The aflatoxin residues can be destroyed using 3% sodium hypochlorite.
2.2 Instrumentation fluorescence
spectra
were
recorded
by a
Varian
Cary Eclipse
fluorescence
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The
spectrophotometer (Palo Alto, CA, USA) equipped with a xenon lamp and a thermostat cell
d
compartment. The scan rate was maintained at 1200 nm min-1 and the excitation and emission
Ac ce pt e
slits were both 5 nm. All measurements were performed in a 10 mm quartz microcell at 25± 0.1 ºC and the excitation and emission wavelengths were 365 and 460 nm respectively. The SEM images were obtained using a Hitachi S-4160 field emission scanning electron microscope (Tokyo, Japan). Chemical interactions were studied using a Perkin Elmer Spectrum one Bv5.3.0 FT-IR spectrometer in the range of 400-4000 cm-1 with KBr pellets (Waltham, Massachusetts, US). The X-ray diffraction (XRD) analysis of modified nanoparticles was performed by an Ital Structures APD 2000 X-ray diffractometer (Riva Del Garda, Italy) using Cu Kα radiation source λ 1.540598 Aº with reflection scan mode. A Metrohm 827 pH/mV meter (Herisau, Switzerland) with a combined glass electrode was used for the pH measurements.
6
Page 6 of 28
2.3 Preparation of magnetic adsorbent Magnetic adsorbent was prepared according to our previously reported method [33]. Briefly, FeCl2.4H2O (4.30 g) and FeCl3.6H2O (11.68 g) were dissolved in deionized water (200 mL)
ip t
under nitrogen atmosphere with vigorous stirring at 85 ºC. Then, ammonia solution (45 mL, 25% w/w) was added and the solution was stirred for 15 min at the same conditions. After cooling
cr
down to the room temperature, the magnetic precipitate was isolated by applying an external
us
magnet and the supernatant was removed. The precipitate was washed with deionized water (250 mL, three times) and 0.02 mol L-1 sodium chloride solution (100 mL, twice). Sodium chloride
an
was used for the flocculation of magnetic nanoparticles. Furthermore, it could accelerate the magnetic separation particularly in alkaline solutions [34, 35]. Then, the suspension was
M
transferred to a 250 mL round bottom flask and allowed to settle. The supernatant was removed and an aqueous solution of MSPT (10% v/v, 80 mL) was added followed by glycerol (60 mL).
d
The mixture was stirred under nitrogen atmosphere at 90 ºC for 2 h. After cooling down slowly
Ac ce pt e
to the room temperature, the black precipitate (MSPT-MNPs) was easily isolated from the supernatant by magnetic decantation and the suspension was washed sequentially with deionized water (250 mL, three times), methanol (200 mL, twice), and deionized water (250 mL, once). In the next step, the supernatant was removed and the solid MSPT-MNPs were homogeneously dispersed to EGBMA solution (1.0% v/v, 150 mL). The solution was transferred to a 400 mL beaker and sonicated for 2 h. The resulting solid phase (EGBMA-MSPT-MNPs) was separated by magnetic decantation, washed with deionized water (3×250 mL) and methanol (2×200 mL) before it was dried in vacuum oven at 45 ºC for 24 h.
2.4 Recommended MSPE procedure
7
Page 7 of 28
Blank liquid milk sample (10 ± 0.1 gram) was accurately weighed and transferred into a 50 mL centrifuge tube. The sample was centrifuged at 4000 rpm for 15 min and the fat layer was removed. Then, the supernatant was spiked with appropriate amounts of AFM1 and diluted to 30
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mL with phosphate buffed solution (PBS, pH=7.5) in a capped container with intensive shaking. The diluted aqueous phase was transferred to a 50 mL beaker and the magnetic adsorbent (100
cr
mg) was added. The mixture was stirred for 5 min to facilitate adsorption of AFM1 to the
us
modified MNPs. The adsorbent was collected at the bottom of the beaker by applying a supermagnet and the supernatant was removed. Afterwards, 3 mL of a mixture of acetone:
an
acetonitrile: chloroform (1:2:1, v/v) was and the suspension was stirred for 4 min. After desorption, the eluent was separated by magnetic decantation and evaporated to dryness under a
M
flow of nitrogen gas at room temperature. The dry residue was dissolved in 5 mL of 12 % (v/v) acetonitrile/water. Then, CD (70 mg) was added to the solution and heated at 50 ºC for 25 min.
Ac ce pt e
fluorescence spectra.
d
The final solution was evaporated to 500 µL under nitrogen flow and used for taking
2.5 Method validation
The proposed MSPE-fluorimetric method was validated in terms of linearity, limit of detection (LOD), limit of quantitation (LOQ), accuracy, precision, and enrichment factor.
2.5.1 Linearity
Linearity of the proposed method was evaluated by analyzing different concentration of AFM1 (0.04, 0.1, 0.5, 1.25, 2.0, 4.0, 6.0 and 8.0 ng mL-1) and an eight-point calibration curve was constructed by plotting fluorescence intensity vs. AFM1 concentration (each point is the mean
8
Page 8 of 28
value of three replicates analyses). The slope, intercept and correlation coefficient were calculated using the least square regression method.
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2.5.2 LOD and LOQ The LOD and LOQ were calculated based on the standard deviation of blank (Sb) and slope of
cr
calibration curve (m) determined according to the following equations:
us
LOD = 3Sb/m
(2)
an
LOQ = 10Sb/m
(1)
2.5.3 Accuracy and precision
M
The accuracy of the proposed method was determined by five replicates analyses of blank milk samples (Blank S1-S3) spiked with AFM1 at three concentration levels (0.05, 2.00 and 7.50 ng
d
mL-1). The recovery was calculated by using the following equation:
Ac ce pt e
Recovery (%) = (Measured concentration/Nominal concentration) ×100
(3)
Repeatability or intra-day precision was evaluated by spiking blank samples with appropriate amounts of AFM1 at three different levels (0.05, 2.00 and 7.50 ng mL-1). Five replicates were performed and analyzed on the same day. In order to determine reproducibility or inter-day precision, five replicated samples which were spiked with the same amount of AFM1, were analyzed on five consecutive days. The intra and inter-day precisions were expressed as the percentage relative standard deviation (RSD %).
2.5.4 Enrichment factor The enrichment factor (EF) of the method was calculated using the following equation:
9
Page 9 of 28
EF = Vs/Ve× R%
(4)
Where Vs is the sample volume, Ve is elution volume, and R% is percent recovery.
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3. Results and discussion For AFM1 determination, low sensitivity and poor precision are the main problems arising when
cr
a simple solid phase extraction (SPE) was tried. Low recovery due to the well-known interaction
us
between AFM1 and milk proteins and/or strong matrix effect due to co-extracted and co-eluted compounds [36] were probably responsible for this failure. On the other hand, one of our goals
an
was to perform sample preparation without using the very expensive immunosorbent cartridges. Therefore, we resolved to use a modified MNPs-based SPE procedure. The synthesis of MNPs is
M
based on a cost-effective and convenient co-precipitation method which Fe3O4 nanoparticles were synthesized from ferric and ferrous ions by addition of a base (ammonia solution here)
d
under inert atmosphere at elevated temperature [37]. When the synthetic conditions are constant,
Ac ce pt e
the quality of nanoparticles is completely reproducible. However, naked nanoparticles suffer from aggregation. Ethaxysilyl or methaxysilyl compounds are the common modifiers solving this problem. Furthermore, they can provide favorable functional groups which can attack to the analyte molecules to improve their adsorption. We tried different modifiers for MNPs which have complete hydrophobic (oleic acid) or complete electrostatic (mercaptopropionic acid) skeleton and those with both of them (EGBMAMSPT-MNPs). The experimental results indicate that quantitative recoveries (˃ 90%) were obtained using the later one. This adsorbent provides both hydrocarbon skeleton and SH end group which are in the form of protonated SH2+ in extraction medium. On the other hand, AFM1 is a difurancoumarin and highly conjugated compound and according to the Pub Chem public
10
Page 10 of 28
chemical database (http://pubchem.ncbi.nml.gov/), its partition coefficient (Log P) is +0.5. This parameter is often used to measure how hydrophilic or hydrophobic a chemical substance is. The log P value shows that AFM1 is more hydrophobic [38]. Thus, hydrophobic interactions with the
ip t
hydrocarbon skeleton of the adsorbent can play a role in the adsorption of AFM1. On the other hand, approaching the aromatic rings in AFM1 and SH2+ end groups of the adsorbent can also
us
two mechanism, namely hydrophobic and cation-π interactions.
cr
cause van der Waals force [39]. Thus the interaction of AFM1 with the adsorbent can occur by
In addition, AFM1 as a penahetrocyclic and highly conjugated compound, exhibits native
an
fluorescence. So sensitive analytical methods for detection of AFM1 are based on either its native emission properties or on enhanced fluorescence after chemical complexation/
M
drevitization. In this work, we used the inclusion of AFM1 within β-cyclodextrin which yields a
Ac ce pt e
sensitivity.
d
significant enhancement to the molecular fluorescence of AFM1 and increases its determination
3.1 Characterization of adsorbent
The size and morphology of the prepared adsorbent were characterized by SEM images. As can be seen from Fig. 2, the prepared adsorbent have uniform size distribution with average diameter of 20 nm and most of the particles are quasi spherical in shape. The XRD pattern of the adsorbent (Fig. S1, supplemental materials) shows reflection peaks for 220, 311, 400, 511, 440 and 622 plans in peak position (2θ) of 30.2, 35.3, 43.2, 57.2, 62.7 and 74.2 respectively, which match well with those obtained by JCPDS for magnetite [40, 41]. The FT-IR spectra of naked MNPs, MSPT-MNPs and EGBMA-MSPT-MNPs adsorbent were shown in Fig. S2, supplementary materials. The adsorbent spectrum shows the characteristics absorption band of
11
Page 11 of 28
Fe-O band (for Fe3O4 nanoparticles) at 581 cm-1, two strong absorption bands for Si-O-H and SiO-Si stretching vibrations at 1122 and 1034 cm-1 respectively, the absorption band of alkyl chains at 2928 cm-1 and the absorption band of SH groups at 2552 cm-1, confirming the
ip t
preparation of EGBMA-MSPT-MNPs.
cr
3.2 Effect of complexation conditions
us
Among the various analytical techniques, fluorescence spectrophotometry remains one of the most interesting ones for the analysis and characterization of CD inclusion complexes, because
an
of the significant analyte signal increment that is frequently induced. The fluorophore introduced into the CD internal cavity is isolated from the surrounding water molecules and its excited state
M
is shielded from quenching processes. Indeed, larger is the signal increase provoked by the organized medium, more sensitive and accurate is the analytical measurement. The effect of
d
complexation conditions on AFM1 fluorescence signal was investigated by adding different
Ac ce pt e
amounts of CD (10-100 mg) to the sample solutions. The experimental results (Fig. 3) showed a significant fluorescence enhancement with increasing CD amount and reached the maximum in 70 mg. The effect complexation time and temperature on the fluorescence signal of AFM1 was also investigated in the range of 5-50 min and 5-60 ºC respectively. The results (Figs. S3 and S4, supplemental materials) revealed that 25 min and 50 ºC were enough respectively for optimal complex formation of AFM1 and used for the next experiments. In addition, the effect of organic solvent (acetonitrile) in the predominantly aqueous CD solutions was investigated in the range of 0-30%. The experimental results revealed that the fluorescence signal of the complex was decreased for acetonitrile/water mixture in the 0-7% (v/v) range. Then, the AFM1-CD fluorescence intensity was increased with increasing
12
Page 12 of 28
acetonitrile percentage until 12% (v/v), and remains constant for larger amounts. The decrease of fluorescence intensity observed in the 0-7% (v/v) acetonitrile range is probably due to the competition of acetonitrile molecules with AFM1 for binding sites inside the protective CD
ip t
cavity. This is in agreement with the decrease of cyclodextrin-analyte binding abilities noted when the concentration of urea [42] or several organic solvents [43-45] increases. Thus,
cr
complexation was performed by addition of 70 mg CD to the eluted AFM1 in 12% (v/v)
us
acetonitrile/water at 50 ºC for 25 min.
an
3.3 Extraction optimizations 3.3.1 Effect of pH
M
An appropriate pH value is a key parameter improves adsorption efficiency and eliminates interferences. Therefore, it is necessary to adjust it prior to any SPE procedure. The effect of pH
d
on the recovery of AFM1 was examined in the range of 3-9 while the other conditions were
Ac ce pt e
unchanged. The results were demonstrated in Fig. 4. As can be seen, recovery of the analyte increases with increase in pH value and reached maximum in 7.0-7.8 range. There was no change in the maximum emission wavelength within this pH range. It was known that aflatoxin molecules have been characterized to possess two potential site of weakness through which it is degraded by chemical agents. High proton concentration or oxidizing agents easily attack the double bond in the furo-furan ring while the lactone ring is hydrolyzed in alkaline environment [46]. When pH value decreases, a β-keto acid structure forms (catalyzed by the high acidic medium) and thus decreases conjugation and fluorescence properties. In high pH, a decrease in emission is also noted due to hydrolyzing lactone ring which reduces conjugation, as already
13
Page 13 of 28
reported for aflatoxin compounds [47]. Based on the results, pH 7.5 was selected for all subsequent experiments.
ip t
3.3.2 Effect of desorption conditions An ideal elution solvent should be strong enough to elute all the target compounds from the
cr
sorbent. In order to achieve optimum desorption conditions, different organic solvents including
us
methanol (MeOH), ethanol (EtOH), acetone (Me2OH), acetonitrile (MeCN), and chloroform (CH2Cl2) alone or in mixture were examined. The results (Fig. 5) showed that the maximum
an
desorption was obtained with a mixture of acetone: acetonitrile: chloroform (1:2:1, v/v) and it was selected as the eluent for the next experiments.
M
The volume of eluent was also studied in the range of 1-10 mL. Based on the results, the minimum volume of eluent required for quantitative desorption (>90%) was found to be 3 mL.
Ac ce pt e
for the subsequent experiments.
d
Thus, 3 mL of acetone: acetonitrile: chloroform (1:2:1, v/v) was selected as the optimum eluent
3.3.3 Effect of sample volume
In order to explore the possibility of enriching low concentration of analyte from large volume, the maximum applicable sample volume must be determined. For this purpose, different sample volumes (10-80 mL) containing 10 ng of analyte were tested. The results indicated that the recoveries were quantitative within 10-50 mL. As mentioned before, the final volume of samples reached 500 µL. So, the theoretical enrichment factor of 60 can be obtained which demonstrates the feasibility of sample determination with different analyte concentration levels.
14
Page 14 of 28
3.3.4 Effect of extraction and desorption times A good dispersion of adsorbent should considerably increase the contact surface area between the adsorbent and analyte in sample, leading to a higher adsorption efficiency and faster
ip t
extraction time. MNPs have higher surface area and can rapidly collect from sample solution by applying magnetically assisted separation, thus shorter extraction time can be achieved. Figs. S5
cr
and S6 show the recovery values of AFM1 as a function of extraction or desorption time. Based
us
on the results, 5 min is sufficient for achieving appropriate adsorption and 4 min was enough for
an
good desorption of AFM1 which are much lower than the traditional column-passing SPE.
3.3.5 Effect of adsorbent amount and reusability
M
As mentioned before, nanoparticles based adsorbents compared to ordinary (micron-sized) adsorbents offer a significantly higher surface area-to-volume ratio and a short diffusion route,
d
which result in high extraction capacity, rapid extraction dynamics and high extraction
Ac ce pt e
efficiencies. Therefore, satisfactory results can be obtained with fewer amounts of these adsorbents. The effect of EGBMA-MSPT-MNPs amount was studied in the range of 20-160 mg. The experimental results (Fig. S7, supplemental materials) indicated that the maximum recovery was obtained by 100 mg of EGBMA-MSPT-MNPs and at higher amounts; the extraction efficiency was almost constant. Therefore, 100 mg of adsorbent was added to the sample solution in the subsequent experiments.
Reusability is one of the determinant factors for evaluation of an adsorbent’s performance. In order to determine regeneration capability of EGBMA-MSPT-MNPs, adsorbents which used in the general SPE procedure were washed with desorbing solvent (20 mL, once) and deionized water (50 mL, three times), then dried in oven at 45 ºC and reused. Experimental results revealed
15
Page 15 of 28
that the adsorbent can be reused at least up to ten times (RSD ˂3.6%) without sacrifice the analytical performance which makes it a suitable alternative to immunoaffinity columns.
ip t
3.4 Effect of interferences In real sample analysis, matrix effect caused by co-elution of matrix components can affect
cr
AFM1 determination. Since, the co-occurrence of mycotoxins such as aflatoxins (B1, B2, G1,
us
G2 and M2), ochratoxin (OTA), deoxynivalenol (DON), and zearalenone (ZEN) rarely occur in milk [21, 48], the effect of interferences on the preconcentration and determination of AFM1 was
an
studied by spiking these mycotoxins to the samples. Therefore, 10.0 g of blank samples were spiked with 100 ng of each aflatoxin, 500 ng of OTA, 100 ng of ZEN, and 5000 ng of DON.
M
Then, the samples were spiked with 10 ng AFM1, diluted to 30 mL with PBS solution, and subjected to the proposed SPE procedure. The experimental results (table 1) revealed that all
d
aflatoxins can quantitatively adsorb on the surface of EGBMA-MSPT-MNPs in these conditions
Ac ce pt e
and can interfere because they belong to the same chemical family as AFM1 and follow the same fluorescence pattern. However, co-occurrence of aflatoxins (except for M2) was rarely observed in milk [28, 49] unless for milk products (such as milk powder) with flour or cereals as part of ingredients which are not target samples here. Aflatoxin M2 (AFM2) level in milk is also estimated to be small. However, when it is exist, only the total AFM1 and AFM2 can be determined. OTA, DON and ZEN can also be found in milk in very low concentrations [15]. As can be seen from the table 1, the AFM1 recoveries were not affected by the presence of these compounds. Although the results revealed that they can be adsorbed on the surface of EGBMAMSPT-MNPs to some extent, it seems that the desorbing solvent cannot elute them from the
16
Page 16 of 28
adsorbent. Thus, AFM1 can be successfully determined by the proposed method without interference of them.
ip t
3.5 Method validation With optimized MNPs-based SPE procedure, the calibration curve was found to be linear in the
cr
0.04-8 ng mL-1 range. The calibration data was subjected to linear least square regression
us
analysis and calibration equation of Y=94.635(±1.3)C+15.007(±2.2) was obtained with correlation coefficient (R2) of 0.9989. The LOD and LOQ values were obtained 0.015 and 0.052
an
ng mL-1 respectively. To evaluate the accuracy of the method, it was applied to the determination of AFM1 in blank milk and recovery studies were carried out by spiking the samples with
M
different amounts of AFM1. As can be seen from table 2, good recoveries in the range of 91102% were obtained. The results for intra and inter-day precision analyzes were also summarized
d
in Table 2. The RSDs were ranged from 3.0- 5.1% and 3.1- 6.3% for intra-day and inter-day
Ac ce pt e
variations respectively, which indicate good precision of the proposed method. Furthermore, by extracting 30 mL of sample solution containing analyte and collecting into a final volume of 500 µL (R=95%), the enrichment factor of 57 was achieved for AFM1 determination.
3.6 Analysis of real samples
The evaluated method was subsequently applied to determination of AFM1 in three different brands of commercially available milk samples widely used in Iran. Obtained results are summarized in table 3. Moreover, the AOAC standard method (IAC-HPLC-FL) was used to evaluate the accuracy of proposed method. The results exhibited that no significant difference was observed between the two methods and indicate the suitability of EGBMA-MSPT-MNPs
17
Page 17 of 28
adsorbent for extraction and preconcentration of trace amounts of AFM1 in real samples. Comparative information from some previously reported studies in the literature on the preconcentration of AFM1 is given in Table 4. In the present work, better analytical parameters
ip t
were obtained in comparison with most of those methods. In addition, the cited methods are more complicated and use more reagents, besides application of the proposed method is simpler
us
cr
and takes less time.
4. Conclusion
an
In this study, a novel MNPs-based solid phase extraction method coupled with enhanced fluorescence detection was successfully applied to determination of ultra-trace amounts of AFM1
separation,
providing
a
fast,
M
in milk samples. The method combines high surface area of the nanoparticles and magnetic convenient,
effective,
and
sensitive
method
for
d
preconcentration/separation of AFM1 in liquid milk samples. In addition, reusability studies
Ac ce pt e
demonstrated the potential of the adsorbent for recyclable usage with compared to the immunoaffinity columns. The method is simple and inexpensive, and eliminates the need to use any auxiliary facilities to immobilize the adsorbent. What is more, usage is not limited to the applications demonstrated here; other detection systems can be used depending on the actual requirements.
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18
Page 18 of 28
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Fig. 1 Chemical structure of AFM1.
d
Figure captions:
M
an
us
cr
[52]
ip t
Velasco-Garcia, D. Moscone, G. Palleschi, Anal. Chim. Acta 520 (2004) 141.
Fig. 2 SEM image of the prepared EGBMA-MSPT-MNPs adsorbent with mean diameter of 20 nm.
Fig. 3 Effect of CD amount on the fluorescence signal of AFM1. Solvent; 12% (v/v) acetonitrilewater, complexation time; 25 min, and complexation temperature 50 ºC. Fig. 4 Effect of pH on the recovery of AFM1. Sample volume; 30 mL, adsorbent amount; 100 mg, extraction time; 5 min, desorption time; 4 min, and eluent solvent; acetonitrile: chloroform (1:2:1, v/v). Fig. 5 Effect of eluent solvent on the recovery of AFM1. Sample volume; 30 mL, adsorbent amount; 100 mg, extraction time; 5 min, desorption time; 4 min, and pH; 7.5.
22
Page 22 of 28
Ac ce pt e
ip t
d
M
an
us
cr
Fig. 1
Fig. 2
23
Page 23 of 28
cr
ip t Ac ce pt e
d
M
an
us
Fig. 3
Fig. 4
24
Page 24 of 28
ip t us
cr Table 1
Ac ce pt e
d
M
an
Fig. 5
Effect of mycotoxins interferences on the recovery of AFM1 adsorbed on EGBMA-MSPTMNPs.
Interference
Concentration (ng mL-1)
Recovery ± RSD (%)
Aflatoxins
3.4a
NCb
OTA
16.7
91 ± 2
ZEN
3.4
95 ± 3
25
Page 25 of 28
DON
166.7
97 ± 3
Concentration of each aflatoxin including B1, B2, G1, G2 and M2.
b
Not calculated because of interference effect of aflatoxins.
cr
ip t
a
us
Table 2
an
Results for recovery, intra-day and inter-day precision analyses for the proposed MNPs-based SPE procedure (n=5). Recovery
(ng mL-1)
(%)
101.01
Ac ce pt e
0.05
d
Blank S1
Intra-day variations
M
Spiked level Sample
Inter-day
(RSD%)
variations (RSD%)
5.08
6.18
2.00
91.21
3.45
3.61
7.50
97.73
3.03
3.13
0.05
92.15
5.01
6.29
2.00
102.22
3.52
3.74
7.50
96.53
3.12
3.33
0.05
90.62
5.07
6.10
2.00
93.44
3.32
3.81
Blank S2
Blank S3
26
Page 26 of 28
92.13
3.18
3.45
cr
ip t
7.50
us
Table 3
an
Comparison of AFM1 analyses in natural contaminated liquid milk samples by the proposed and official methods (mean ± SD, n=5).
Found (ng mL-1)
*
0.26 ± 0.03
Ac ce pt e
Sample 1
d
Proposed method
M
Milk sample
Official method*
0.31 ± 0.07
Sample 2
0.19 ± 0.04
0.17 ± 0.03
Sample 3
0.23 ± 0.09
0.28 ± 0.05
IAC-HPLC-FL
Table 4 Comparison of linear range and LOD for determination of AFM1 with some of the previously reported methods. 27
Page 27 of 28
Linear range
Detection limit
Time needed
Assay type
Reference -1
-1
a
(ng mL )
for assay
0.015-1
0.015
28 h
[50]
0.2-4
0.006
30 min
[29]
Immunochip
0.45-3.9
0.24
2.5 h
[25]
Flow-injection immunoassay
0.02-0.5
0.011
26 h
[51]
Competitive ELISAb
28-164
28
2.5 h
[52]
Indirect competitive ELISA
0.1-3.2
0.04
27.5 h
[8]
MNPs-based SPE
0.04-8
0.015
50 min
This work
Not mentioned exactly, at least
b
Enzyme-linked Immunosorbent Assay
cr
us
Ac ce pt e
d
a
an
HPLC-FL
M
Impedimetric immunosensor
ip t
(ng mL )
28
Page 28 of 28
