Determination Of Aflatoxin B1 Research Articles

Assessment Of The Conformity Of The Methods For Aflatoxin B1 And Deoxynivalenol Determination In Grain And Feeds By Method Of High-Performance Liquid Chromatography

Assessment Of The Conformity Of The Methods For Aflatoxin B1 And Deoxynivalenol Determination In Grain And Feeds By Method Of High-Performance Liquid Chromatography

Simultaneous determination of aflatoxin B1 and zearalenone by magnetic nanoparticle filled amino-modified multi-walled carbon nanotubes

We report a novel extractant based MSPE-HPLC-DAD method for simultaneous determination of aflatoxin B1 and zearalenone.

Rapid qualitative and quantitative analysis of aflatoxin B1 in Pu-erh tea by liquid chromatography-isotope dilution tandem mass spectrometry coupled with the QuEChERS purification method

An efficient method for the determination of aflatoxin B1 in Pu-erh tea by isotope dilution mass spectrometry coupled with QuEChERs was established.

Aflatoxin B1 residues in human livers and their relationship with markers of hepatic carcinogenesis in São Paulo, Brazil.

In this study, hepatic biopsies from autopsy cases in São Paulo, Brazil, showing hepatocellular carcinoma (HCC, n = 8), cirrhosis associated with viral hepatitis (VC, n = 20), cirrhosis associated with alcoholism (AC, n = 20), and normal livers (NL or controls, n = 10) were subjected to determination of aflatoxin B1 (AFB1) and its main metabolites, and of markers of hepatic carcinogenesis Only non-metabolized AFB1 was detected in 13 samples (27.1%, N = 48) of liver disorders (HCC, VC and AC), at levels between 10.0 and 418.0 pg/g (mean: 76.6 ± 107.7 pg/g). Immuno-labeling of p53, cyclin D1, p21, β-catenin, and Prohibitin (PB) increased mainly in HCC patients, in relation to the controls. AFB1+ samples of HCC presented higher expressions of p53, cyclin D1, p21, and β-catenin compared with AFB1-livers. In contrast, p27, p16, and Rb immuno-labeling decreased in HCC, VC, and AC samples, compared with NL, with lowest values in AFB1+ samples for all liver disorders. Compared with NL, gene expression of cyclin D1 and PB in AFB1+ samples of HCC and AC were also higher, along with higher gene expression of p21 in VC and AC AFB1+ livers. Results indicated that patients with liver disorders were exposed to dietary aflatoxins, and that residual AFB1 in liver negatively affected the p53 and protein Rb pathways in HCC. Moreover, the presence of AFB1 in cirrhotic livers warrants concern about the potential contribution of dietary aflatoxin to disease progression during VC and AC.

Enzyme-linked immunosorbent assay for aflatoxin B1using a portable pH meter as the readout

A simple and portable biosensor was developed for aflatoxin B1determination using a portable pH meter as the readout.

Potentiometric enzyme biosensor for aflatoxin B1 detection – Kinetic simulation

We have investigated the operation of a potentiometric biosensor based on reversible acetylcholinesterase inhibition for determination of aflatoxin B1. The biochemical reactions between enzyme, substrate, and inhibitor are described using rate equations. A sensitivity study of the biosensor response to the biochemical reaction rates was done by varying each rate constant. The response to substrate and inhibitor was used as a fitting target for evaluation of the biochemical reaction rates. Initial concentrations of the enzyme (2.0 × 10−5 M acetylcholinesterase), the substrate (4.0 × 10−3 M acetylcholine chloride), and the inhibitor (9.6 × 10−6 M aflatoxin B1) in the biosensor membrane act as the boundary conditions for the rate equations. This kinetic model allowed us to establish the effect of reaction rates on the biosensor response and reproduce the response to different inhibitor concentrations. The results may be used for biosensor design and optimization.

Photoinduced discharge of electrons stored in a TiO2-MWCNT composite to an analyte: application to the fluorometric determination of hydrogen peroxide, glucose and aflatoxin B1.

The authors describe an analytical detection scheme based on the use of multiwalled carbon nanotubes (MWCNTs) that accept and store electrons upon contact with photo-irradiated TiO2 nanoparticles (TiO2-NPs). The Fermi level equilibration with photo-irradiated TiO2-NPs has a storage value of 0.35mM of electrons per 120mg·L-1 of MWCNTs. The stored electrons can be discharged on demand upon addition of electron acceptors to the TiO2-NP/MWCNT composite. These findings are applied to detect the quencher hydrogen peroxide. H2O2 also is produced on enzymatic action of glucose oxidase on glucose, and this enables glucose also to be quantified by using the TiO2-NP/MWCNT fluorescent nanoprobe. The wide scope of the method also is demonstrated by an assay for aflatoxin B1 that is making use of an FAM-labeled aptamer where the FAM fluorophore on the aptamer quenches the emission of the nanoprobe. The following analytical linear ranges and limits of detection are found: H2O2: 0.1-100μM and 15nM; glucose: 5-200μM and 0.5μM; aflatoxin: 0.1-40ng·mL-1 and 0.02ng·mL-1. The method was applied to the determination of glucose in human serum. Graphical abstract The assays demonstrated in (b) and (c) are based on the fluorescence quenching ability of MWCNTs-TiO2. In the presence of the target (analyte), the fluorescence is restored and the target concentration is determined from the percentage of fluorescence recovery.

Simultaneous Determination of Aflatoxin B1, Bisphenol A, and 4-Nonylphenol in Peanut Oils by Liquid-Liquid Extraction Combined with Solid-Phase Extraction and Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometry

An analytical method based on liquid-liquid extraction combined with solid-phase extraction and isotope dilution-ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was well developed for simultaneous determination of aflatoxin B1 (AFB1), bisphenol A (BPA), and 4-nonylphenol (4-NP) in peanut oil. After adding isotope internal standards, the samples were firstly diluted by normal hexane and then extracted by acetonitrile and Carb/PSA solid-phase extraction cartridge in sequence to obtain the extracted solution. All the extracted solution was merged and was subsequently dried to near dryness by a mild nitrogen stream. Three target analytes were separated on a Phenomenex Luna C18 chromatographic column, quantified by an internal standard method and detected by ESI positive (ESI+) and negative (ESI−) subsection acquisition modes under multi-reaction monitoring (MRM) conditions. Results demonstrated that the three target analytes exhibited excellent linearity in their corresponding concentration ranges of 0.1–100.0 μg/L with correlation coefficients all greater than 0.998. The corresponding method limits of quantitation (MLOQ, S/N = 10) of AFB1, BPA, and 4-NP were 0.2, 1.0, and 2.0 μg/kg, respectively. Moreover, the mean recoveries for negative samples spiked at three concentration levels were calculated between 87.7 and 105.1% with relative standard deviation (RSD, n = 6) ranging from 2.2 to 7.9% and the interday precision (n = 5) ranging from 5.0 to 8.7%. Finally, the method was successfully applied to analyze 52 peanut oil samples, and AFB1 and 4-NP were detected in 43 samples with the concentrations in the ranges of 0.5–69.4 and 9.3–77.8 μg/kg, respectively. None of BPA was detected in any samples.

Competitive horseradish peroxidase-linked aptamer assay for sensitive detection of Aflatoxin B1

Aflatoxin B1 (AFB1) is one of highly toxic mycotoxins and a known human carcinogen. The frequent contamination of AFB1 in food products and large health risk of AFB1 have raised global concerns. Sensitive detection of AFB1 is of vital importance and highly demanded. Herein, we reported a competitive horseradish peroxidase (HRP)-linked aptamer assay for AFB1, combining the advantages of aptamer for affinity binding and enzyme label for signal amplification. In this assay, free AFB1 in solution competed with a covalent conjugate of bovine serum albumin-AFB1 (BSA-AFB1) coated on the wells of microplate in binding to the HRP-labeled aptamer probe. HRP attached on BSA-AFB1 in the wells catalyzed the conversion of substrates into products, allowing the final detection of AFB1 through measurement of the generated products. When TMB (3,3′,5,5′-tetramethylbenzidine dihydrochloride) was used as substrate, absorbance analysis of the product of enzyme reaction enabled the detection of AFB1 at 0.2nM. We further lowered the detection limit of AFB1 to 0.01nM through chemiluminescence analysis by using chemiluminescence substrate of HRP. This assay enabled the detection of AFB1 in complex sample matrix, such as diluted white wine and maize flour. This assay provides a simple, sensitive and rapid method for AFB1 determination.

Determination of 16 Mycotoxins in Maize by Ultrahigh-Performance Liquid Chromatography–Tandem Mass Spectrometry

ABSTRACTAn ultrahigh-performance liquid chromatography–tandem mass spectrometry method was developed and validated for the simultaneous determination of sterigmatocystin, verruculogen, enniatin A, fusarenon-X, fumonisins B1, B2, B3, aflatoxins B1, B2, G1, G2, ochratoxin A, deoxynivalenol, 3-acetyldeoxynivalenol, 5-acetyldeoxynivalenol, and zearalenone. The mycotoxins were extracted and cleaned up using a multitoxin column, separated on a C18 column, and then detected on a triple-quadrupole mass spectrometer. The limits of detection and quantification ranged within 0.2–2 µg/kg and 1–10 µg/kg, respectively. The recoveries ranged from 70.8 to 118.4%, with relative standard deviations below 15%. The method was used to analyze 80 samples obtained from Shandong Province in China. Fifty-eight samples were contaminated with 10 mycotoxins at concentrations ranging from 1.4 to 6566.1 µg/kg. Some samples exceeded the maximum limits in China and in European regulations for mycotoxins in unprocessed maize.

Separation of aflatoxin B1 from synthetic physiological fluids using talc and diatomite: Kinetic and isotherm aspects.

The objective of the study was to examine adsorption of the aflatoxin B1 from synthetic gastric fluid and synthetic intestinal fluid by talc, raw and calcined diatomite. The kinetic and equilibrium adsorption processes were studied in the batch adsorption experiments applying high performance liquid chromatography for the aflatoxin B1 determination. The kinetic study showed a very fast adsorption of the aflatoxin B1 onto the selected adsorbents from the both physiological fluids with reaching equilibrium within 1-15min. The aflatoxin B1 was almost completely adsorbed in initial linear step of the kinetic process that can be described well by the zero-order kinetics model. The experimental data of the equilibrium adsorption were characterized using the Langmuir and Freundlich isotherm models. The high adsorption effectiveness was found in a range of 90%-100% and 60%-100% for the diatomite samples and the talc respectively at the initial concentrations of the aflatoxin B1 as 31-300ng/mL. The possible mechanisms of the aflatoxin adsorption onto the used mineral adsorbents are also discussed in the work.

Broad-spectrum immunoaffinity cleanup for the determination of aflatoxins B1, B2, G1, G2, M1, M2 in Ophiocordyceps sinensis and its pharmaceutical preparations by ultra performance liquid chromatography tandem mass spectrometry.

An ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the simultaneous determination of aflatoxins B1, B2, G1, G2, M1 and M2 (AFB1, AFB2, AFG1, AFG2, AFM1 and AFM2) in Ophiocordyceps sinensis and its pharmaceutical preparations. A rapid and reliable immunoaffinity column containing a broad-spectrum monoclonal antibody for six aflatoxins was used for sample cleanup. Under the optimized conditions, the home-made immunoaffinity column capacity were about 315, 319, 292, 102, 444 and 369ng/mL gel for AFB1, AFB2, AFG1, AFG2, AFM1 and AFM2, respectively. Recoveries for all tested aflatoxins ranged from 79.28% to 103.42% with relative standard deviation less than 8%. The limits of quantitation were in the range of 0.008-0.045μg/kg. Among 31 real samples analyzed, one sample was contaminated with AFB1, AFB2 and AFM1 at levels of 0.483, 0.068 and 0.104μg/kg, respectively. The established method is simple, accurate, and can be effectively used to determine the aflatoxins in Ophiocordyceps sinensis and its pharmaceutical preparations.

Eu3+ -labeled IgG-based time-resolved fluoroimmunoassay for highly sensitive detection of aflatoxin B1 in feed.

Aflatoxin B1 (AFB1 ) is a kind of toxic and carcinogenic mycotoxin. A time-resolved fluoroimmunoassay (TRFIA) was established for quantitative detection of AFB1 in feed using Eu3+ -labeled IgG as tracer. Monoclonal antibody (McAb) against AFB1 (9B11-D7) was prepared through immunization and cell fusion and was identified as high affinity, specificity and sensibility by enzyme-linked immunosorbent assay (ELISA). The 50% inhibition value (IC50 ) was 0.81 ng mL-1 , the limit of detection (LOD) was 0.10 ng mL-1 and detection range was 0.10-3.94 ng mL-1 . Goat anti-mouse immunoglobulin G (IgG) was modified by Eu3+ -DATT, generating Eu3+ -labeled IgG. Under optimal assay conditions, TRFIA was shown to be highly sensitive and specific in detection of AFB1 . The IC50 and LOD were 94.73 pg mL-1 and 3.55 pg mL-1 , respectively, and detection range was 3.55-1.11 × 103 pg mL-1 . Cross-reactivity with AFM1 , AFB2 , AFG1 and AFG2 was 31.26%, 37.6%, 127.46% and 35.74%, respectively, but zero with other analogues. In determination of AFB1 spiked in feed sample, TRFIA showed high accuracy and precision. The average recoveries ranged from 93.71% to 97.80%, and coefficient of variation was 1.25-3.73%. Good correlation between TRFIA and HPLC was demonstrated for determination of AFB1 in feeds, confirming the reliability of the developed method. The developed TRFIA exhibited good potential for employment in the ultrasensitive detection of AFB1 in feed and could be used to determine total aflatoxins. © 2017 Society of Chemical Industry.

Gold coated porous silicon nanocomposite as a substrate for photoluminescence-based immunosensor suitable for the determination of Aflatoxin B1

A rapid and low cost photoluminescence (PL) immunosensor for the determination of low concentrations of Aflatoxin B1 (AFB1) has been developed. This immunosensor was based on porous silicon (PSi) covered by thin gold layer (Au) and modified by antibodies against AFB1 (anti-AFB1). PSi layer was formed on silicon substrate, then the surface of PSi was covered by 30nm layer of gold (PSi/Au) using electrochemical and chemical deposition methods and in such ways PSi/Au(El.) and PSi/Au(Chem.) structures were formed, respectively. In order to find PSi/Au the most efficiently suitable for PL-based sensor design, structure several different PSi/Au(El.) and PSi/Au(Chem.) structures were designed while using different conditions for electrochemical or chemical deposition of gold layer. It was shown that during the formation of PSi/Au structure crystalline Au nanoparticles uniformly coated the surface of the PSi pores. PL spectroscopy of PSi/Au nanocomposites was performed at room temperature and it showed a wide emission band centered at 700nm. Protein A was covalently immobilized on the surface of PSi/Au(El.) and PSi/Au(Chem.) forming PSi/Au(El.)/Protein-A and PSi/Au(Chem.)/Protein-A structures, respectively. In the next step PSi/Au(El.)/Protein-A and PSi/Au(Chem.)/Protein-A structures were modified by anti-AFB1 and in such way a structures (PSi/Au(El.)/Protein-A/anti-AFB1 and PSi/Au(Chem.)/Protein-A/anti-AFB1) sensitive towards AFB1 were designed. The PSi/Au(El.)/Protein-A/anti-AFB1- and PSi/Au(Chem.)/Protein-A/anti-AFB1-based immunosensors were tested in a wide range of AFB1 concentrations from 0.001 upon 100ng/ml. Interaction of AFB1 with PSi/Au(El.)/Protein-A/anti-AFB1- and PSi/Au(Chem.)/Protein-A/anti-AFB1-based structures resulted PL quenching. The highest sensitivity towards AFB1 was determined for PSi/Au(El.)/Protein-A/anti-AFB1-based immunosensor and it was in the range of 0.01–10ng/ml. The applicability of PSi/Au-based structures as new substrates suitable for PL-based immunosensors is discussed.

Intra-laboratory Development and Evaluation of a Quantitative Method for Measurement of Aflatoxins B1, M1 and Q1 in Animal Urine by High Performance Liquid Chromatography with Fluorescence Detection.

Mycotoxins negatively impact animal health. Aflatoxins (AFs) are the most common mycotoxins affecting both large and small animals and are a common cause of toxin-related pet food recalls. Definitive diagnosis of aflatoxicosis is constrained by a lack of validated ante-mortem analytical methods for detection and quantitation of AFs and their metabolites in biological specimens. Herein, we developed and evaluated a urine-based quantitative method for measurement of aflatoxin B1 (AFB1) and its metabolites aflatoxin M1 (AFM1) and aflatoxin Q1 (AFQ1) in animal urine. (Some of the results have been presented at 59th AAVLD conference, Greensboro, North Carolina, October 13-19th, 2016.) This method uses an immuno-affinity column for clean-up and pre-column derivatization followed by high performance liquid chromatography analysis with fluorescence detection. The method has high selectivity, recovery (>81%) and sensitivity with an instrument limit of detection of 0.20-1.02 pg; instrument limit of quantitation of 0.77-4.46 pg; and a method lower limit of quantitation of 0.30-2.5 ng/mL. The method has high accuracy, repeatability, and is rugged against minor changes. However, because of poor sensitivity of AFQ1 at low concentrations we recommend this method for quantitative determination of AFB1 and AFM1, and for qualitative measurement of AFQ1 in animal urine for diagnosis of aflatoxicosis.

A Novel Dispersive Nanomagnetic Particle Solid-Phase Extraction Method to Determine Aflatoxins in Nut and Cereal Samples

The application of a simple, fast, and cheap sample pre-concentration technique based on the dispersive magnetic Fe3O4 nanoparticle solid-phase extraction has been developed for extraction and determination of aflatoxins B1, B2, G1, and G2 in nut and cereals. Aflatoxins were extracted from samples with a mixture of methanol–water 8:2 (v/v) and then by magnetic Fe3O4 nanoparticles and a 500 μL of chloroform; pre-concentration and cleanup of the analytes simultaneously were done. HPLC with fluorescence detector and electrochemical post-column derivatization has been used for aflatoxin determination. The main parameters affecting the extraction efficiency, such as type and volume of extraction solvent, the volume of water, and sample volume, were investigated and optimized to achieve the best extraction efficiency. The LODs and LOQs of the proposed method for the AFs were determined, which LODs were 0.35 and 0.06 μg/kg, and LOQs were 1 and 0.2 μg/kg, for aflatoxin groups B and G, respectively. Acceptable recoveries were achieved (76.0–112.7%), and the low limits of detection (LOD <0.35 and 0.06 μg/kg), with proper precision (RSD <15%), were obtained. Therefore, the validated method represents simple and low-cost substitute to the conventional method and is suitable for screening of contamination in food samples.

Determination of Mycotoxins in Cereal-Based Porridge Destined for Infant Consumption by Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometry

An analytical method applying ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) for the determination of aflatoxins M2, M1, G2, G1, B2, B1, deoxynivalenol, ochratoxin A, fumonisins B1 and B2, hydrolyzed fumonisins B1 and B2, zearalenone and sterigmatocystin in cereal-based porridge destined for infant consumption was developed and validated. The mycotoxins were extracted using an adequate solvent ratio (n-hexane/3% formic acid solution/acetonitrile) under rapid shaking and sonication, without any further cleanup steps. Recoveries ranged from 63.5 to 113.2% and were considered satisfactory, with relative standard deviations lower than 20%. The limits of quantification ranged from 0.14 to 6.73 μg kg−1. The validated method was then applied to the determination of mycotoxins in 84 samples of cereal-based porridge destined for infant consumption obtained in the metropolitan region of Rio de Janeiro, RJ. Aflatoxins B1 and G1, fumonisins B1 and B2, hydrolyzed fumonisins B1 and B2, deoxynivalenol, and zearalenone were found, respectively, in 7.1, 2.4, 47.6, 3.6, 65.5, and 60.7% of the analyzed samples. The maximum permissible limits set by the Brazilian legislation were exceeded for at least one mycotoxin in 21 (25%) of the analyzed samples.

AFLATOXINS CONTAMINATION IN FEED AND PRODUCTS OF ALABIO DUCK (Anas platyrinchos borneo) COLLECTED FROM SOUTH KALIMANTAN, INDONESIA

A limited survey was conducted to determine aflatoxins contaminations in feed and products of Alabio duck. A total of 271 feed samples, 48 liver sample, 42 meat samples, and 38 egg samples were analyzed for determination of aflatoxin B1 (AFB1) and aflatoxin M1 (AFM1) using Enzyme-Linked Immuno-Sorbent Assay (ELISA) tests. Results showed high prevalence of AFB1 contamination in feed sample, especially in feed concentrate, dried fish, and rice hulls (100%). AFB1 concentrations were higher than tolerable limit for AFB1 in feed for laying duck according to Indonesia National Standard (SNI), being 20 ppb, except for sago pit. This survey also showed high prevalence and levels of aflatoxin residues in the products of Alabio duck. AFB1 was found in all liver samples, with concentrations ranging from 4 to 12 ppb (average: 7 ppb). AFM1 was found in all of liver, meat, and egg samples. The highest level of AMF1 was found in liver which was ranging from 105 to 1,215 ppt (average: 304 ppt). High level of AFM1 was also found in meat, namely between 71 to 128 ppt (averaged: 91 ppt). Although found at low level, AFM1 was detected in egg, which was ranging from 10 to 36 ppt (average: 19 ppt). This survey showed high contaminations of aflatoxins in the liver, meat and egg of Alabio duck collected from the area of survey and their concentrations of aflatoxins were harmful for the consumer.

Impedimetric immunoassay for aflatoxin B1 using a cysteine modified gold electrode with covalently immobilized carbon nanotubes

The authors describe a label-free electrochemical biosensor based on CNTs for picogram range detection of the food carcinogen aflatoxin B1 (AFB1). A gold electrode was modified with a cysteine (Cys) self-assembled layer, and carboxyl-functionalized carbon nanotubes were covalently attached to Cys for the subsequent tethering of antibody against AFB1 (anti-AFB1). Topographical images of the biosensor surface were acquired by atomic force microscopy throughout the modification and assay procedure. Upon exposure to samples containing AFB1, its binding to anti-AFB1 will result in a change in electrical conductivity. The use of CNT warrants enhanced electrical properties, and the charge-transfer resistance (Rct) can be related to the concentration of AFB1. Biosensor selectivity was tested by using samples containing ochratoxin A. The electrode displays a limit of detection as low as 0.79 pg·g−1, and response is linear in the 0.1 to 20 pg·g−1 concentration range. The assay was applied to the determination of AFB1 in contaminated corn flour at concentrations so low that they cannot be quantified by established ELISAs. In our perception, this method represents a viable point-of-care probe for detection of AFB1.

Determination of mycotoxins in biscuits, dried fruits and fruit jams: an assessment of human exposure

ABSTRACTA reliable, fast and simple method using UHPLC-MS/MS was developed for the determination of aflatoxins B1 (AFB1), G1 (AFG1), B2 (AFB2) and G2 (AFG2), ochratoxin A (OTA), deoxynivalenol (DON), zearalenone (ZEA), HT-2 toxin and T-2 toxin in crude extracts of biscuits with fruit filling, cookies, dried fruits and fruit jams. The method was successfully demonstrated on 39 samples of biscuits with fruit filling, 34 cookies, 14 dried fruits and 10 fruit jams. The mycotoxins detected in biscuits samples were ZEA, OTA, T-2 and AFB1 with an average concentrations of positive samples of 2.64, 4.10, 8.13 and 1.32 µg kg−1, respectively; while the mycotoxins detected in jam samples were AFB1, OTA, T-2 and AFB2 with an average concentrations of positive samples of 2.00, 17.7, 4.37 and 1.15 µg kg−1, respectively. The results showed that the majority of samples were in compliance with relevant regulations. However in eight samples of biscuits and three samples of fig jam the contents of OTA were higher than the existing OTA limits. The combined dietary exposure of selected mycotoxins was estimated for the first time for children, adolescents and adults. The estimated combined dietary exposures were all lower than the proposed value assumed to predict a possible risk scenario.