One-pot green synthesis of ZIF-8/IgG composite for the precise orientation and protection of antibody and its application in purification and detection of aflatoxins in peanut oil.

This study presents a novel approach toward the one-pot green synthesis of ZIF-8/IgG composite, focusing on its precise orientation and protection of the anti-aflatoxins antibody. The antibody orientation is achieved through the specific binding of IgG to the Fc region of the antibody, while the antibody protection is accomplished by the structural change restriction of ZIF-8 framework to the antibody. Consequently, the antibody exhibits enhanced target capability and significantly improved tolerance to organic solvents. The ZIF-8/IgG/anti-AFT was employed for the purification and detection of AFTs by coupling with UPLC. Under optimized conditions, the recoveries of spiked AFTs in peanut oils are between 86.1% and 106.4%, with relative standard deviations (RSDs) ranging from 0.8% to 8.8%. The linearity range is 0.5-20.0 ng for AFB1 and AFG1, 0.125-5.0 ng for AFB2 and AFG2, the limit of detection is 0.1 ng for AFB1 and AFG1, 0.03 ng for AFB2 and AFG2.

Distribution of Aspergillus Fungi and Recent Aflatoxin Reports, Health Risks, and Advances in Developments of Biological Mitigation Strategies in China.

Aflatoxins (AFs) are secondary metabolites that represent serious threats to human and animal health. They are mainly produced by strains of the saprophytic fungus Aspergillus flavus, which are abundantly distributed across agricultural commodities. AF contamination is receiving increasing attention by researchers, food producers, and policy makers in China, and several interesting review papers have been published, that mainly focused on occurrences of AFs in agricultural commodities in China. The goal of this review is to provide a wider scale and up-to-date overview of AF occurrences in different agricultural products and of the distribution of A. flavus across different food and feed categories and in Chinese traditional herbal medicines in China, for the period 2000-2020. We also highlight the health impacts of chronic dietary AF exposure, the recent advances in biological AF mitigation strategies in China, and recent Chinese AF standards.

Genetic fingerprinting and aflatoxin production of Aspergillus section Flavi associated with groundnut in eastern Ethiopia.

BACKGROUND: Aspergillus species cause aflatoxin contamination in groundnut kernels, being a health threat in agricultural products and leading to commodity rejection by domestic and international markets. Presence of Aspergillus flavus and A. parasiticus colonizing groundnut in eastern Ethiopia, as well as presence of aflatoxins have been reported, though in this region, no genetic studies have been done of these species in relation to their aflatoxin production. RESULTS: In this study, 145 Aspergillus isolates obtained from groundnut kernels in eastern Ethiopia were genetically fingerprinted using 23 Insertion/Deletion (InDel) markers within the aflatoxin-biosynthesis gene cluster (ABC), identifying 133 ABC genotypes. Eighty-four isolates were analyzed by Ultra-Performance Liquid Chromatography (UPLC) for in vitro aflatoxin production. Analysis of genetic distances based on the approximately 85 kb-ABC by Neighbor Joining (NJ), 3D-Principal Coordinate Analysis (3D-PCoA), and Structure software, clustered the isolates into three main groups as a gradient in their aflatoxin production. Group I, contained 98% A. flavus, including L- and non-producers of sclerotia (NPS), producers of B1 and B2 aflatoxins, and most of them collected from the lowland-dry Babile area. Group II was a genetic admixture population of A. flavus (NPS) and A. flavus S morphotype, both low producers of aflatoxins. Group III was primarily represented by A. parasiticus and A. flavus S morphotype isolates both producers of B1, B2 and G1, G2 aflatoxins, and originated from the regions of Darolabu and Gursum. The highest in vitro producer of aflatoxin B1 was A. flavus NPS N1436 (77.98 µg/mL), and the highest producer of aflatoxin G1 was A. parasiticus N1348 (50.33 µg/mL), these isolates were from Gursum and Darolabu, respectively. CONCLUSIONS: To the best of our knowledge, this is the first study that combined the use of InDel fingerprinting of the ABC and corresponding aflatoxin production capability to describe the genetic diversity of Aspergillus isolates from groundnut in eastern Ethiopia. Three InDel markers, AFLC04, AFLC08 and AFLC19, accounted for the main assignment of individuals to the three Groups; their loci corresponded to aflC (pksA), hypC, and aflW (moxY) genes, respectively. Despite InDels within the ABC being often associated to loss of aflatoxin production, the vast InDel polymorphism observed in the Aspergillus isolates did not completely impaired their aflatoxin production in vitro.

A simple extraction method with no lipid removal for the determination of aflatoxins in almonds by liquid chromatography tandem-mass spectrometry (LC-MS/MS).

The present study describes a simple and rapid method for the determination of aflatoxins in almonds using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Aflatoxins were extracted using a modified QuEChERS method with little sample preparation, excluding the use of laborious purification procedures. Extracts were frozen overnight to separate the majority of lipids. The method was successfully validated for almonds. Linearity was demonstrated in the range 0.125-20 µg/kg. Limits of quantification (LOQ) ranged from 0.34 to 0.5 µg/kg. Matrix effect was not significant for the aflatoxins. Satisfactory recoveries were obtained at spike levels below 1 µg/kg and between 1 and 10 µg/kg. Relative standard deviations (RSDs) of repeatability and reproducibility were below 15%. The method was successfully tested with two proficiency tests in almond powder and peanut paste, with acceptable z-scores (-2 ≤ z ≤ 2). Only one of 11 local almond samples contained detectable aflatoxins, at concentrations below the maximum permitted level.

The AFLATOX® Project: Approaching the Development of New Generation, Natural-Based Compounds for the Containment of the Mycotoxigenic Phytopathogen Aspergillus flavus and Aflatoxin Contamination.

The control of the fungal contamination on crops is considered a priority by the sanitary authorities of an increasing number of countries, and this is also due to the fact that the geographic areas interested in mycotoxin outbreaks are widening. Among the different pre- and post-harvest strategies that may be applied to prevent fungal and/or aflatoxin contamination, fungicides still play a prominent role; however, despite of countless efforts, to date the problem of food and feed contamination remains unsolved, since the essential factors that affect aflatoxins production are various and hardly to handle as a whole. In this scenario, the exploitation of bioactive natural sources to obtain new agents presenting novel mechanisms of action may represent a successful strategy to minimize, at the same time, aflatoxin contamination and the use of toxic pesticides. The Aflatox® Project was aimed at the development of new-generation inhibitors of aflatoxigenic Aspergillus spp. proliferation and toxin production, through the modification of naturally occurring molecules: a panel of 177 compounds, belonging to the thiosemicarbazones class, have been synthesized and screened for their antifungal and anti-aflatoxigenic potential. The most effective compounds, selected as the best candidates as aflatoxin containment agents, were also evaluated in terms of cytotoxicity, genotoxicity and epi-genotoxicity to exclude potential harmful effect on the human health, the plants on which fungi grow and the whole ecosystem.

Development and validation of the one-step purification method coupled to LC-MS/MS for simultaneous determination of four aflatoxins in fermented tea.

Aflatoxin B1 is the potential chemical contaminant of most concern during the production and storage of fermented tea. In this work, a simple, fast, sensitive, accurate, and inexpensive method has been developed and validated for the simultaneous detection of four aflatoxins in fermented tea based on a modified sample pretreatment method and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Aflatoxins were extracted using acetonitrile and purified using mixed fillers (carboxyl multiwalled carbon nanotubes, hydrophilic-lipophilic balance, silica gel). Under optimum LC-MS conditions, the limits of quantification (LOQs) were 0.02-0.5 µg·kg-1. Recoveries from aflatoxins-fortified tea samples (1-12 µg·kg-1) were in the range of 78.94-105.23% with relative standard deviations (RSDs) less than 18.20%. The proposed method was applied successfully to determine aflatoxin levels in fermented tea samples.

The occurrence of aflatoxins and human health risk estimations in randomly obtained maize from some markets in Ghana.

Maize and its products are most often prone to fungal contamination especially during cultivation and storage by toxigenic fungi. Aflatoxicosis still persist in Ghana despite the numerous education on several ways of its prevention at the farm as well as its adverse health implications which are food safety concerns. A random assessment and human risk analysis was conducted on 90 maize (72 white and 18 colored) samples from markets across all the regions of Ghana. Total aflatoxins (AFtotal) and the constitutive aflatoxins (AFB1, AFB2, AFG1, and AFG2) were analyzed by High-Performance Liquid Chromatography (HPLC). Out of a total of ninety (90) samples investigated, 72 (80%) tested positive for AFB1 and the contamination levels ranged from 0.78 ± 0.04 to 339.3 ± 8.6 µg kg-1. Similarly, AFG2 was detected in only 14 (15.5%) samples, and their values ranged between 1.09 ± 0.03 and 5.51 ± 0.26 µg kg-1 while AF total ranged between 0.78 ± 0.04 and 445.01 ± 8.9 µg kg-1 constituting approximately 72 (80%). Limits of AFB1 and total aflatoxins (AFtotal) for the Ghana Standards Authority (GSA) (5 and 10 µg kg-1) and the European Food Safety Authority (EFSA) (2 and 4 µg kg-1), were used as checks. A total of 33 (41.25%) samples were above the limits for both. Risk assessments recorded for Estimated Daily Intake (EDI), Hazard Quotient (H.Q), Hazard Index (H.I), Margin of Exposure (MOE), av. Potency, and population risks ranged 0.087-0.38 µg kg-1 bw day-1, 1.5-6.9, 0.0087-0.38, 3.64-12.09, 0-0.0396 ng Aflatoxins kg-1 bw day-1 and, 3.5 × 10-1-0.015 respectively for total aflatoxins. While ranges for aflatoxins B1 (AFB1) recorded were 0.068-0.3 µg Kg bw-1 day-1, 2.43-10.64, 0.0068-0.030, 4.73-20.51, 0-0.0396 ng Aflatoxins kg-1 bw day-1 and, 2.69 × 10-3-0.012 for Estimated Daily Intake (EDI), Hazard Quotient (H.Q), Hazard Index (H.I), Margin of Exposure (MOE), Av. potency, and population risks respectively. It was deduced that although there was some observed contamination of maize across the different ecological zones, the consumption of maize (white and colored) posed no adverse health effects on the population of Ghana since computed H.I was less than 1 (< 1).

Aflatoxins from the endophytic fungus Aspergillus sp. Y-2 isolated from the critically endangered conifer Abies beshanzuensis.

Two new [asperxins A (1) and B (2)] and four known (3-6) aflatoxins were isolated and identified from the endophytic fungus Aspergillus sp. Y-2, which was derived from the needles of the critically endangered conifer Abies beshanzuensis. Their structures were elucidated by extensive spectroscopic methods. Among the isolates, compounds 1 and 5 showed considerable cytotoxicities against the A549 and Hela human cancer cell lines, with IC50 values in the range of 7.5-16.8 µM.

One-step deep eutectic solvent strategy for efficient analysis of aflatoxins in edible oils.

BACKGROUND: Aflatoxins, a kind of carcinogen, have attracted increasing attention due to their toxicity and harmfulness to human health. Traditional methods for aflatoxins analysis usually involve tedious extraction steps with a subsequent derivatization process. Herein, a simple and efficient liquid-phase microextraction method based on deep eutectic solvents (DESs) for direct analysis of aflatoxins was developed. RESULTS: Adopting DESs as the extractant, we surprisingly found out that DESs could either achieve good extraction performance or play a similar role to the derivatization agent, achieving an enhancement of fluorescence intensity for direct analysis of aflatoxins by high-performance liquid chromatography combined with fluorescent detection. Under optimal conditions obtained by response surface methodology, the method provided satisfactory linear ranges (0.01-0.75 µg kg-1 for AFB1 and AFG1, 0.003-0.25 µg kg-1 for AFB2 and AFG2) with good determination coefficients (R2 > 0.9988), a low detection limit (0.0005-0.003 µg kg-1 ), and good recovery rates (72.05-113.54%). CONCLUSION: These results highlighted superiorities of the one-step DES strategy for analysis of aflatoxins in edible oils, providing insights for future development of efficient methods in food analysis. © 2020 Society of Chemical Industry.

Aflatoxin Reduction in Maize by Industrial-Scale Cleaning Solutions.

Different batches of biomass/feed quality maize contaminated by aflatoxins were processed at the industrial scale (a continuous process and separate discontinuous steps) to evaluate the effect of different cleaning solutions on toxin reduction. The investigated cleaning solutions included: (i) mechanical size separation of coarse, small and broken kernels, (ii) removal of dust/fine particles through an aspiration channel, (iii) separation of kernels based on gravity and (iv) optical sorting of spatial and spectral kernel defects. Depending on the sampled fraction, dynamic or static sampling was performed according to the Commission Regulation No. 401/2006 along the entire cleaning process lines. Aflatoxin analyses of the water-slurry aggregate samples were performed according to the AOAC Official Method No. 2005.008 based on high-performance liquid chromatography and immunoaffinity column cleanup of the extracts. A significant reduction in aflatoxin content in the cleaned products, ranging from 65% to 84% with respect to the uncleaned products, was observed when continuous cleaning lines were used. Additionally, an overall aflatoxin reduction from 55% to 94% was obtained by combining results from separate cleaning steps. High levels of aflatoxins (up to 490 µg/kg) were found in the rejected fractions, with the highest levels in dust and in the rejected fractions from the aspirator and optical sorting. This study shows that a cleaning line combining both mechanical and optical sorting technologies provides an efficient solution for reducing aflatoxin contamination in maize.

An aptamer affinity column for purification and enrichment of aflatoxin B1 and aflatoxin B2 in agro-products.

We have developed an aptamer affinity column (AAC) for the purification and enrichment of trace aflatoxin B1 (AFB1) and aflatoxin B2 (AFB2) in genuine agro-products through the covalent conjugation of amino modified aptamer and NHS-activated Sepharose. The coupling and working conditions found to be suitable for this AFB-AAC were examined in regard to coupling time (2 min), loading volume (30 mL), and the methanol concentration (< 10%) used in the loading step. The performance of AFB-AAC was then further evaluated in terms of capacity (329.1 ± 13.7 ng for AFB1 and 162.5 ± 8.9 ng for AFB2), selectivity (excellent), reusability (twenty-three times for AFB1 and twelve times for AFB2), and repeatability (92.7% ± 2.9% for AFB1 and 71.5% ± 3.4% for AFB2). Furthermore, the AAC clean-up combined with HPLC-FLD demonstrated excellent linearity over a wide range, good sensitivity with an LOD of 50 pg mL-1 for AFB1 and 15 pg mL-1 for AFB2, and acceptable recovery with different spiking levels in different matrices. Finally, the AAC was successfully applied to analyte AFB1 and AFB2 in four types of agro-products as well as a maize flour reference material, and the results were found to be in accordance with those of commercial IACs. This study provides a reference for the analysis of other trace analytes by merely changing the corresponding aptamer and represents a strong contender for immune affinity columns. Graphical abstract An aptamer affinity column for purification and enrichment of aflatoxin B1 and aflatoxin B2 in agro-products with the aid of HPLC-FLD and a post-column photochemical derivatization reactor.

Integration of Fe3O4@UiO-66-NH2@MON core-shell structured adsorbents for specific preconcentration and sensitive determination of aflatoxins against complex sample matrix.

Aflatoxins have been a hot topic in the field related into public health and ecosystem protection, and great effort has been made in developing of adsorptive materials for effective probing the target aflatoxins. Conventional materials, like metal-organic frameworks (MOFs) showed promising application in separation science. However, the cumbersome separation process, competitive adsorption are also major challenges. Regarding this, a novel magnetic micro-composite denoted as Fe3O4@UiO-66-NH2@MON with core-shell structure was constructed. The core of Fe3O4 microspheres was coated with MOFs crystals, and then microporous organic network (MON) was introduced onto the surface of Fe3O4@UiO-66-NH2 through a sonogashira coupling reaction. It exhibited good magnetic separation ability, which effectively simplified the pre-treatment steps. The proposed method possessed excellent selectivity and sensitivity, with detection limits in the range of 0.15-0.87 µg L-1 combination with HPLC analysis. More importantly, the MON coating significantly improved the hydro-stability of whole adsorbents, thus enhancing the adsorption efficiency and favoring the practical application of the materials. The developed Fe3O4@UiO-66-NH2@MON-based solid extraction method has been well-applied for real sample analysis, with the recovery of 87.3%-101.8%. We believe the newly-constructed hybrid nano-adsorbents hold great potential in further application in various analytical methods for different target analytes.

A facile one-pot green synthesis of ß-cyclodextrin decorated porous graphene nanohybrid as a highly efficient adsorbent for extracting aflatoxins from maize and animal feeds.

In this paper, ß-cyclodextrin (ß-CD) supported on porous graphene nanohybrid (ß-CDPG) was obtained by self-assembly of functionalized graphene nanosheets into a three-dimensional network in the presence of ascorbic acid via an in situ graphene oxide reduction and ß-CD functionalization process during a hydrothermal reaction. The prepared supramolecular nanohybrid was further packed into a reusable syringe filter holder and applied as an adsorbent for solid phase extraction of four aflatoxins (B1, B2, G1, G2). Under optimal conditions, the detection limits and linear dynamic ranges were achieved in the range of 0.0075-0.030 µg kg-1 and 0.025-100 µg kg-1, respectively and the relative standard deviations were less than 6.1%. Good recoveries were observed for analyzing target AFs in maize and cereal-based chicken feed samples ranged from 90.5 to 105%. The method offered simultaneous advantages of high supramolecular recognition and enrichment capability of ß-CD and the high specific surface area of the porous graphene.

Integrated in-syringe magnetic sheet solid-phase extraction and dispersive liquid-liquid microextraction for determination of aflatoxins in fresh and moldy breads.

BACKGROUND: Magnetic three-dimensional graphene-based nanoadsorbents have unique characteristics such as large surface area, good thermal and chemical stability, and high adsorption capacity that make them efficient materials in sorbent-based extraction techniques. In this study, four aflatoxins (AFs) were analyzed in bread samples using magnetic three-dimensional graphene as the adsorbent phase in dispersive micro solid-phase extraction. RESULTS AND CONCLUSIONS: In-syringe magnetic sheet solid-phase extraction based on magnetic three-dimensional graphene in tandem with dispersive liquid-liquid microextraction was used for the extraction and preconcentration of the target AFs. The effect of significant parameters of the method was investigated and the optimum conditions were determined as follows: adsorbent dosage, 20 mg; desorption/disperser solvent (methanol) volume, 700 µL; desorption solvent flow rate, 0.7 mL min-1 ; pH, neutral; salt (NaCl) concentration, 10% (w/v); extraction solvent (chloroform) volume, 250 µL; and centrifugation rate (and time), 4000 rpm (5 min). The limits of detection and quantification were in the ranges 0.043-0.083 and 0.14-0.28 µg kg-1 , respectively. The extraction method was followed by the HPLC technique with fluorescence detection and applied to the determination of the AFs in four different Iranian fresh and moldy bread samples. The relative recoveries were in the range 84-107% with relative standard deviations of 3.9-8.6%. © 2019 Society of Chemical Industry.

An amino-functionalized zirconium-based metal-organic framework of type UiO-66-NH2 covered with a molecularly imprinted polymer as a sorbent for the extraction of aflatoxins AFB1, AFB2, AFG1 and AFG2 from grain.

A surface imprinted polymer of type UiO-66-NH2@MIP was prepared by combining molecular imprinted polymers (MIPs) and an amino-functionalized zirconium-based metal-organic framework. Quercetin is used as the virtual template, UiO-66-NH2 acts as the carrier to which the monomer acrylamide can be copolymerized. The material was characterized by scanning electron microscopy, transmission electron microscopy, X-ray diffraction, and Fourier transform infrared spectroscopy. It was used as a sorbent in a solid-phase extraction column. The extraction conditions were optimized. The adsorption capacities for aflatoxins AFB1, AFB2, AFG1 and AFG2 by this SPE and by the commercial SPE were compared. The method was successfully applied to quantify the aflatoxins in grain. Figures of merit include (a) good linearity (range from 0.20-45 µg·kg-1) with R2 (range from 0.9986-0.9994), (b) low detection limits (90-130 ng·kg-1), (c) acceptable reproducibility (1.0-5.9%; for n = 6), and (d) relatively satisfactory recovery rates (74.3-98.6%). The new sorbent has good selectivity and reusability. Graphical abstractUiO-66-NH2@MIPs were synthesized with modified UIO-66-NH2 as core and quercetin as pseudo template. A cartridge was prepared with the polymers as the sorbent, and its performance was compared with different commercial SPE cartridges.

Extraction of aflatoxins by using mesoporous silica (type UVM-7), and their quantitation by HPLC-MS.

A solid-phase extraction procedure has been developed by using a sorbent derived from UVM-7 mesoporous silica. The sorbent was applied to the extraction of aflatoxins B1, B2, G1 and G2 from tea samples followed by HPLC with mass spectrometric detection. The sorbent was characterized by transmission electron microscopy, nuclear magnetic resonance, X-ray diffraction and nitrogen adsorption-desorption. UVM-7 is found to be the best solid phase. The amount of solid-phase, type and volume of eluent, pH value and ionic strength and breakthrough volume were optimized. Following the recommended procedure, recoveries between 96.0 and 98.2% were achieved, with RSD values of <5.1%, and the limits of detection are in the range from 0.14 to 0.7 µg·kg-1. The material is reusable. The method was applied to the analysis of real tea samples. A low matrix effect is found, and recoveries are >88%. The results were compared with those obtained by immunoaffinity columns as a reference method. Only low concentrations of aflatoxin G2 were found in some samples, and results obtained with both methods are shown to be statistically sound and comparable. Graphical abstractSchematic representation of a mesoporous silica sorbent (type UVM-7) for the extraction of aflatoxins (AF) from tea by solid-phase extraction (SPE), and its determination by liquid chromatography. The morphology of the material allows to retain the analytes very well.

Dual-Purpose Materials Based on Carbon Xerogel Microspheres (CXMs) for Delayed Release of Cannabidiol (CBD) and Subsequent Aflatoxin Removal.

The main objective of this study is to develop a novel dual-purpose material based on carbon xerogel microspheres (CXMs) that permits the delayed release of cannabidiol (CBD) and the removal of aflatoxin. The CXMs were prepared by the sol-gel method and functionalized with phosphoric acid (CXMP) and melamine (CXMN). The support and the modified materials were characterized by scanning electronic microscopy (SEM), N2 adsorption at -196 °C, X-ray photoelectron spectroscopy (XPS), and zeta potential. For the loading of the cannabidiol (CBD) in the porous samples, batch-mode adsorption experiments at 25 °C were performed, varying the concentration of CBD. The desorption kinetics was performed at two conditions for simulating the gastric (pH of 2.1) and intestinal (pH of 7.4) conditions at 37 °C based on in vitro CBD release. Posteriorly, the samples obtained after desorption were used to study aflatoxin removal, which was evaluated through adsorption experiments at pH = 7.4 and 37 °C. The adsorption isotherms of CBD showed a type I(b) behavior, with the adsorbed uptake being higher for the support than for the modified materials with P and N. Meanwhile, the desorption kinetics of CBD at gastric conditions indicated release values lower than 8%, and the remaining amount was desorbed at pH = 7.4 in three hours until reaching 100% based on the in vitro experiments. The results for aflatoxin showed total removal in less than 30 min for all the materials evaluated. This study opens a broader landscape in which to develop dual-purpose materials for the delayed release of CBD, improving its bioavailability and allowing aflatoxin removal in gastric conditions.

Separation and Purification of Aflatoxins by Centrifugal Partition Chromatography.

Aflatoxins are mycotoxins that are produced by several species of filamentous fungi. In the European Union, the concentration limits for this group of mycotoxins in food and feed products are very low (on the order of parts per billion). Thus, relatively high amounts of these substances in their pure forms are required as reference standards. Chromatographic techniques based on solid stationary phases are generally used to purify these molecules; however, liquid-liquid chromatographic separations may be a promising alternative. Therefore, this study proposes a liquid-liquid chromatographic method for the separation of four aflatoxins and impurities. To optimise the method, numerous biphasic solvent systems (chloroform-, acetone- and acetic acid-based systems) were tested and evaluated in terms of their effectiveness at partitioning aflatoxins; the toluene/acetic acid/water (30:24:50, v/v/v/%) system was found to be the most efficient for application in centrifugal partition chromatographic instrument. Using liquid-liquid instrumental separation, the four aflatoxins, namely B1 (400 mg), B2 (34 mg), G1 (817 mg) and G2 (100 mg), were successfully isolated with 96.3%-98.2% purity from 4.5 L of Aspergillus parasiticus fermented material in a 250 mL centrifugal partition chromatography column. The identities and purities of the purified components were confirmed, and the performance parameters of each separation step and the whole procedure was determined. The developed method could be effectively used to purify aflatoxins for analytical applications.

Determination of aflatoxin biomarkers in excreta and ileal content of chickens.

Aflatoxins are carcinogenic secondary metabolites frequently detected in food and feed stuff based on maize and other crops susceptible to infection with the fungal pathogen Aspergillus flavus. We investigated the metabolization of aflatoxins in chickens by analyzing excreta and ileal content and developed and validated a biomarker method for detection of aflatoxins and their metabolites in these matrices. Analysis of ileal content served to distinguish between urinary and fecal excretion combined in the excreta samples. During a 3-wk animal trial, one hundred sixty-eight 1-day-old chicks were randomly allocated to 24 pens with 7 chicks per pen and subjected to different feed regimens with: A) toxin-free feed, B) feed supplemented with 18 ng of total aflatoxins/g, and C) feed supplemented with 515 ng of total aflatoxins/g. Chicken excreta and ileal content were sampled after 7, 14, and 21 D. An analytical method based on liquid chromatography coupled to tandem mass spectrometry was validated for the determination of aflatoxin B1, B2, G1, G2, M1, P1, Q1, and aflatoxin B1-N7-guanine (AFB1-N7-Gua) in chicken's samples. Comparing chicken excreta, which contain urine and feces, to ileal content, which contains no urine, we explored the secretion pathway of aflatoxin metabolites. The AFB1-N7-Gua was only detected in excreta, whereas aflatoxin M1 (AFM1) was detected both in ileal content and excreta. Aflatoxin M1 was detected in excreta in concentrations 5 times higher than in ileal content, suggesting primary excretion via urine. Although chickens are relatively resistant to aflatoxins, contamination of feed can lead to adverse effects and thus economic losses in farming. Therefore, a biomarker method to estimate the exposure of chickens to aflatoxins can play an important role to monitor the animals' health.

Electrospun polymer composite nanofiber-based in-syringe solid phase extraction in tandem with dispersive liquid-liquid microextraction coupled with HPLC-FD for determination of aflatoxins in soybean.

A fast method based on in-syringe solid phase extraction combined with dispersive liquid-liquid microextraction was developed for extraction of aflatoxins prior to HPLC-FD. Electrospun polyurethane nanofibers doped with graphene oxide were collected on a thin metal net sheet without using a binder, placed into a filter holder between filter papers on a syringe tip and used as an efficient adsorbent for the first time. The major parameters affecting whole extraction efficiency were investigated and optimized. Under the optimum conditions, the limits of detection and the limits of quantification were in the range of 0.09-0.15 and 0.3-0.5 µg kg-1, respectively. The linear dynamic range was 0.3-1000 µg kg-1 with determination coefficients of 0.9946-0.9965. The inter- and intra-day precisions were lower than 4.3 and 7.2%, respectively. The method was successfully applied for the determination of aflatoxins B1, B2, G1, and G2 in soybeans and satisfactory relative recoveries of 76-101% were achieved.