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Analytical Methods
A R T I C L E I N F O A B S T R A C T
Keywords: Graphene oxide wrapped silica nanocomposites were synthesized and selected as solid phase extraction ad-
Aflatoxins sorbents for high performance liquid chromatography analysis of aflatoxins. The major parameters affecting
Silica/graphene oxide nanocomposites extraction efficiency were optimized, including the amount of adsorbents, extraction time and desorption
Solid phase extraction conditions. The limit of detections and the limit of quantifications were from 0.1 to 0.3 µg/kg and from 0.3 to
High performance liquid chromatography
1.0 µg/kg, respectively. The recoveries of aflatoxins in the spiked maize and rice samples were in the range of
Cereal crops
76.8–104.7% and 81.1–106.9%, respectively, and with the RSDs less than 12.4%. The proposed method was
proven to be simple, rapid and reliable for routine analysis of aflatoxins in crops.
⁎
Corresponding authors at: Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences, Wuhan 430062, PR China.
E-mail addresses: yuli01@caas.cn (L. Yu), mafeicpu@163.com (F. Ma), peiwuli@oilcrops.cn (P. Li).
1
These authors contributed equally to this study.
https://doi.org/10.1016/j.foodchem.2017.11.070
Received 29 May 2017; Received in revised form 16 November 2017; Accepted 17 November 2017
Available online 21 November 2017
0308-8146/ © 2017 Elsevier Ltd. All rights reserved.
L. Yu et al. Food Chemistry 245 (2018) 1018–1024
balanced (HLB) and Sep-Pak cartridges were investigated as antibody- 2.3. Methods
free adsorbents for aflatoxins (Chen & Zhang, 2013; Li et al., 2014).
Different types of polymeric materials have also been synthesized to 2.3.1. Preparation of graphene oxide
purify aflatoxins, including 3-(trimethoxysilyl)-1-propanthiol (TMSPT) According to previous GO synthesis procedure (Marcano et al.,
modified with 2-amino-5-mercapto-1,3,4-thiadiazole, ethylene glycol 2010), 500 mg of GO was ultrasonicated in 1 L of water for 4 h at room
bis-mercaptoacetate modified TMSPT grafted Fe3O4 nanoparticles and temperature. Then, the GO solution was centrifuged at 10,000 rpm for
poly dopamine-coated magnetic nanoparticles (Hashemi, Taherimaslak, 10 min. The precipitate was discarded, and the unexfoliated GO sheets
& Rashidi, 2014; Liu et al., 2013; McCullum, Tchounwou, Ding, Liao, & could be removed from the GO solution. The final supernatant was
Liu, 2014). retained as GO precursor for the composite material.
Traditional carbon materials, graphitized carbon black, and acti-
vated carbon were mainly used to clean up. Recently, carbon nanotube 2.3.2. Pretreatment of silica particles
and graphene oxide (GO), with high specific surface area and good To activate the amino groups and remove the free amino groups, the
compatibility for organic and inorganic materials and solvents, play an pretreatment of aminopropyl silica particles was done before the
important role in sample extraction, especially as the adsorbents ap- synthesis of silica/GO nanocomposites. Aminopropyl silica particles
plied in the trace analysis of small molecular analytes (Es’haghi, (10.0 g) were swollen thoroughly in 100 mL of hydrochloric acid
Sorayaei, Samadi, Masrournia, & Bakherad, 2011; Yu et al., 2013). (0.05 mol/L), successively washed with 100 mL of pure water 100 mL of
According to our previously reported work (Yu et al., 2013), GO could methanol and 100 mL of pure water 3 times, and the final products
be an effective adsorbents for aflatoxins. However, there were several (9.0 g) were collected by vacuum freeze drying for assembly and named
problems associated with using GO as adsorbents directly for enrich- CK.
ment of aflatoxins. Although high-speed centrifugation was performed,
the existence of tiny sheets of GO could not be removed completely. To 2.3.3. Synthesis and characterization of silica/GO nanocomposites
avoid this problem, GO was bonded on silica nanoparticles as SPE ad- GO contains hydroxyl and carboxyl groups at the edge of the basal
sorbents for chlorophenols, polycyclic aromatic hydrocarbons and plane (Dreyer, Park, Bielawski, & Ruoff, 2010). The hydroxyl group was
heavy metals (Luo, Zhu, Li, Yuan, & Feng, 2013; Su, Chen, He, & Hu, negatively charged and could be attracted to the positively charged
2014; Xu, Feng, Li, Liu, & Jiang, 2012). To the best of our knowledge, aminopropyl silica. Aminopropyl silica particles could be easily
GO-wrapped silica nanocomposites coupled with HPLC-FLD have not wrapped in GO sheets by electrostatic interactions (Liu, Zhang, Chen, &
been applied to analyze total aflatoxins in cereal crops. Wang, 2011a). 3.5 g of the previously prepared aminopropyl silica
The aim of this work was to synthesize GO-wrapped silica nano- particles were added into 120 mL of GO solution (0.5 mg/mL) and
composites as solid phase adsorbents for the determination of aflatoxins gently shaken for 24 h at various temperatures (25 °C, 40 °C and 80 °C).
in cereal. The extraction parameters were optimized and successfully The silica particles were collected by centrifugation at 4500 rpm for
applied to quantify analytes by HPLC-FLD. 3 min and washed with deionized water several times until the super-
natant was clean. After vacuum freeze drying, the brown powders were
silica/GO nanocomposites RT, 40 and 80.
2. Experimental Owing to the presence of the carboxyl hydroxyl groups, GO could
also covalently bond with the aminopropyl silica particles by using EDC
2.1. Chemicals and standards and NHS as coupling agents (Liu et al., 2011b). 400 μL of 0.2 g/mL EDC
were added slowly into 160 mL of GO solution (0.5 mg/mL) with stir-
N-hydroxysuccinimide (NHS), 1-ethyl-3-(3-dimethylaminopropyl) ring for 20 min. Then, 400 μL of NHS solution (0.1 g/mL) was added
carbodiimide hydrochloride (EDC), AFB1, AFB2, AFG1, and AFG2 stan- into the mixture with continuous stirring for 1 h. Finally, 2.0 g of
dards were purchased from Sigma-Aldrich (St. Louis, MO, USA). aminopropyl silica particles were added into the same system and the
Methanol, acetonitrile and acetone of HPLC grade were obtained from mixture was gently shaken for 24 h at room temperature. The brown
Anhui Fulltime Specialized Solvents & Reagents Co., Ltd. (Anqing, product was collected by centrifugation at 4500 rpm for 3 min and
China). Hydrochloric acid (HCl), n-hexane, and trifluoroacetic acid washed with deionized water several times until the supernatant was
were purchased from Tianjin Kermel Chemical Reagent Co., Ltd. clean. After vacuum freeze drying, brown powders were obtained, and
(Tianjin, China). Unless otherwise stated, all other inorganic chemicals were named High for short. Under the otherwise identical experimental
and organic solvents were of analytical reagent grade or better. conditions, another two kinds of silica/GO nanocomposites were ob-
Ultrapure water (18 mΩ) was obtained from Milli-Q purification system tained by reducing the added volumes of EDC and NHS solution to
(Millipore Co., Ltd., Milford, MA, USA). Aminopropyl silica was sup- 200 μL and 40 μL, denoted as Mid and Low for short respectively. The
plied by Sphere Scientific Co., Ltd. (Wuhan, China). preparation scheme of silica/GO nanocomposites is shown in Fig. 1. The
The stock solutions of AFB1, AFB2, AFG1 and AFG2 were prepared by microstructures of silica/GO nanocomposites were observed using
accurately weighing out 10 ± 1 mg of each standard and dissolving it scanning electron microscopy (JSM-5610LV, 20 kV). The measurements
separately in 50 mL of methanol. A series of standard solutions were
prepared by diluting the stock solutions with methanol. All standard
solutions were sealed with parafilm, covered with aluminum foil and
stored in the dark at 4 °C until use. Certified reference material
(T04291QC Maize, FAPAS, UK) was used in the optimized procedures.
2.2. Instruments
1019
L. Yu et al. Food Chemistry 245 (2018) 1018–1024
1020
L. Yu et al. Food Chemistry 245 (2018) 1018–1024
Fig. 2. Optimized extraction conditions: (a) effect of the adsorbent amount on the recoveries of aflatoxins, (b) effect of extraction time on the recoveries of aflatoxins, (c) effect of
desorption solvent on the recoveries of aflatoxins, and (d) effect of desorption volume on the recoveries of aflatoxins.
GO nanocomposites (40) were selected as the extraction adsorbent for The effect of desorption solvent was evaluated using different types
further studies. of solvents, and high desorption recoveries were achieved when acet-
The SEM images of the prepared silica/GO nanocomposites and onitrile was selected (Fig. 2c). This phenomenon could be attributed to
aminopropyl silica particles were shown in Fig. 2s. The size of the the π – π electrostatic interactions between lower unoccupied mole-
prepared silica/GO nanocomposites were not changed basically after cular orbital and the higher occupied molecular orbital of the furan and
loading with GO. The presence of wrinkles on the matrix surface could coumarin rings of aflatoxins (Hii, Basheer, & Lee, 2009). Therefore,
be caused by the self-assembly of few layers of GO during the drying acetonitrile was adopted as the optimal desorption solvent. The deso-
process. In Fig. 2s (c and d), it showed that the surfaces of silica/GO rption volume was also investigated from 1 to 4 mL. As presented in
nanocomposites (40) were rougher than those of aminopropyl silica Fig. 2d, the results showed that the recoveries increased from 1 to 3 mL,
particles, and for the nanocomposites, GO nano sheet-like structures and no significant changes were observed when the volume was in-
(wrinkles and thin grooves) could be observed, indicating that GO creased. Consequently, 3 mL of acetonitrile was chosen in the following
sheets were attached to the surface of silica particles and enhances the experiments.
adsorption efficiency of GO.
1021
L. Yu et al. Food Chemistry 245 (2018) 1018–1024
Table 2
Inter-day
Precision (RSD, %)b
(n = 4)
Results for the determination of aflatoxins in cereal samples.
6.4
3.2
2.5
3.9
Intra-day Sample Number of samples Analytes (µg/kg)a Detection rate %
(n = 5)
AFG1 AFB1 AFG2 AFB2
3.9
2.0
1.7
0.5
Rice 22 NDb ND ND ND –
(2.7) Maize 1 20 ND 4.1 ND ND 25%
(4.3)
(6.4)
(7.6)
Maize 4 ND 2.5 ND ND
5 µg/kg
Spiked
Maize 5 ND 3.8 ND ND
88.3
93.6
94.8
81.1 Maize 12 ND 1.8 ND 0.8
Maize 16 ND 1.2 ND ND
(6.8)
(4.8)
(5.1)
(6.9)
2 µg/kg
Spiked
a
Mean value of aflatoxins were analyzed in cereal samples.
94.6
95.1
93.4
97.5
b
Aflatoxins were no detect in samples.
106.9 (8.5)
maize samples contained AFB1 and AFB2, and the amounts of aflatoxins
Recovery (%) ± RSD (%, n = 3)a
96.4
95.7
95.3
76.8
were all under the MRLs set by EU and China. AFB1 was detected in five
maize samples, ranging from 1.2 µg/kg to 4.1 µg/kg, and AFB2 was
(12.4)
(7.5)
(9.4)
(6.7)
and the results were illustrated in Table 3 (Andrade, da Silva, & Caldas,
2013; Hashemi et al., 2014; Khayoon et al., 2012; Liu et al., 2013; Ma
et al., 2013). The developed method showed good sensitivity and ex-
(µg/kg)
LOQ
synthetic steps for magnetic solid phase adsorbents and polymers. The
LOQs in the developed SPE HPLC-FLD method were comparable with
(µg/kg)
LOD
2014). Moreover, this is the first time that GO-wrapped silica nano-
composites were applied to extract aflatoxin in cereal crops, which
Y = 21.10335X + 3.66836 (0.99746)
4. Conclusions
1.0–20.0
0.5–20.0
0.8–20.0
0.3–20.0
(µg/kg)
Compound
Fig. 3. Typical chromatograms of aflatoxins analysis in maize (a) and rice (b) samples
Table 1
AFG1
AFG2
AFB1
AFB2
(dash line-unspiked samples; solid line-samples spiked with aflatoxins at the concentra-
b
a
1022
L. Yu et al. Food Chemistry 245 (2018) 1018–1024
Table 3
Comparison of sample preparation procedures and recoveries among different methods.
Matrix Absorbents Sample pretreatment Derivatisation Determination Recovery (%) LOQs (µg/kg) Ref.
condition technique
Breast milk – Liquid–liquid extraction with Post-column HPLC-FLD 73.0–99.5 0.005–0.03 Andrade et al.
low temperature purification derivatisation (2013)
Agri-product mAbs-amino silica gel Immunoaffinity Pre-column HPLC-FLD 90.1–104.4 0.10–0.30 Ma et al.
microparticles chromatography cleanup derivatisation (2013)
Chilli, peanut ISOLUTE-multimode Solid phase extraction Pre-column HPLC-FLD 86.3–104.5 Peanut 0.23–0.58 Khayoon et al.
and rice absorbents derivatisation Rice 0.27–0.62 (2012)
Chilli 0.64–5.59
Coffee and malt C8 sorbents Porous membrane-protected – HPLC-MS/MS 86.0–109.5 Malt 0.40–1.11 Khayoon et al.
beverage micro-solid phase extraction Coffee 0.76–2.52 (2014)
Grains and grain Oasis HLB and Bond Elut Solid phase extraction Pre-column HPLC-FLD 77.9–111.6 Corn 0.13–0.18 Chen and
products cartridges derivatisation Wheat 0.11–0.15 Zhang (2013)
Cereal Hyperbranched Solid phase extraction – HPLC-FLD 82.7–103.0 0.04–0.40 Liu et al.
polymers (2013)
Cereal product AMT-TMSPT MNPs Magnetic solid phase – HPLC-FLD 90.0–97.0 Rice 0.215 (B1) & Hashemi et al.
nanoparticles extraction 0.046 (B2) (2014)
Corn 0.219 (B1) &
0.048 (B2)
Peanut Graphene oxide Solid phase extraction Pre-column HPLC-FLD 85.1–100.8 0.08–0.65 Yu et al.
adsorbents derivatisation (2013)
Cereal crops Silica/graphene oxide Solid phase extraction Pre-column HPLC-FLD 76.5–119.0 Rice 0.30–1.0 This work
nanocomposites derivatisation
electrostatic interactions. This is the first report about the character- December 2006 setting maximum levels for certain contaminants in foodstuffs.
istics of silica/GO nanocomposites in extracting the aflatoxins in cereal Official Journal of the European Union, L2364, 2005–2024.
EC (2010). European Commission. Commission Regulation (EC) no. 165/2010 of 26
samples. Under the optimized conditions, the method for quantification February 2010 amending Regulation (EC) no. 1881/2006 setting maximum levels for
of aflatoxins in rice and maize samples was established. The validation certain contaminants in foodstuffs. Official Journal of the European Union, L2050,
results indicated that the developed method was suitable for the de- 2011–2018.
Es’haghi, Z., Sorayaei, H., Samadi, F., Masrournia, M., & Bakherad, Z. (2011). Fabrication
termination of aflatoxins in cereals. Owing to its simplicity, rapidity, of a novel nanocomposite based on sol-gel process for hollow fiber-solid phase mi-
and economical nature, the proposed method represents a valuable croextraction of aflatoxins: B1 and B2, in cereals combined with high performance
method for the routine analysis and survey of aflatoxins and its deri- liquid chromatography-diode array detection. Journal of Chromatography B, 879(28),
3034–3040.
vatives in protein-based food.
Hashemi, M., Taherimaslak, Z., & Rashidi, S. (2014). Application of magnetic solid phase
extraction for separation and determination of aflatoxins B1 and B2 in cereal products
Acknowledgments by high performance liquid chromatography-fluorescence detection. Journal of
Chromatography B, 960(1), 200–208.
Hii, T. M., Basheer, C., & Lee, H. K. (2009). Commercial polymeric fiber as sorbent for
This work was supported by the National Natural Science solid-phase microextraction combined with high-performance liquid chromatography
Foundation of China (31601576, 31540048 & 31201447), the Natural for the determination of polycyclic aromatic hydrocarbons in water. Journal of
Science Foundation of Hubei Province – China (2016CFB612), the Chromatography A, 1216(44), 7520–7526.
Iqbal, S. Z., Asi, M. R., Hanif, U., Zuber, M., & Jinap, S. (2016). The presence of aflatoxins
Fundamental Research Funds for Central Non-profit Scientific and ochratoxin A in rice and rice products; and evaluation of dietary intake. Food
Institution, the National Key Research and Development Program of Chemistry, 210(1), 135–140.
China (2016YFF0201901) and the International Science & Technology Khayoon, W. S., Saad, B., Lee, T. P., & Salleh, B. (2012). High performance liquid chro-
matographic determination of aflatoxins in chilli, peanut and rice using silica based
Cooperation Program of China (2016YFE0112900). monolithic column. Food Chemitry, 133(2), 489–496.
Khayoon, W. S., Saad, B., Salleh, B., Manaf, N. H., & Latiff, A. A. (2014). Micro-solid phase
Conflict of interest extraction with liquid chromatography-tandem mass spectrometry for the determi-
nation of aflatoxins in coffee and malt beverage. Food Chemistry, 147(15), 287–294.
Lattanzio, V. M. T., Ciasca, B., Powers, S., & Visconti, A. (2014). Improved method for the
The authors declare no conflict of interest. simultaneous determination of aflatoxins, ochratoxin A and Fusarium toxins in cer-
eals and derived products by liquid chromatography-tandem mass spectrometry after
multi-toxin immunoaffinity clean up. Journal of Chromatography A, 1354(8),
Appendix A. Supplementary data
139–143.
Li, X., Li, P. W., Zhang, Q., Li, R., Zhang, W., Zhang, Z. W., ... Tang, X. Q. (2013). Multi-
Supplementary data associated with this article can be found, in the component immunochromatographic assay for simultaneous detection of aflatoxin
B1, ochratoxin A and zearalenone in agro-food. Biosensors and Bioelectronics, 49(15),
online version, at http://dx.doi.org/10.1016/j.foodchem.2017.11.070.
426–432.
Li, C., Xie, G., Lu, A. X., Ping, H., Ma, Z. H., Luan, Y. X., & Wang, J. H. (2014).
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