Some interferences in radioimmunoassay of aflatoxin B1

The specificity of radioimmunoassay of aflatoxin B₁ was tested. Relative cross-reactivity of used antiserum with aflatoxins B₁, B₂, G₁, G₂ and M₁ was found to be 100%, 24%, 44.2%, 10.3%, and 1.4%, respectively. The interference of coumarin, albumins, steroids and ethylvanilin was estimated also in radioimmunoassay of aflatoxin B₁. Thus these compounds may cause a false positive finding of aflatoxin B₁.     

Radioimmunoassay of aflatoxin M1

Radioimmunoassay for the differential determination of aflatoxin M₁ has been developed. It is based on the use of antiserum having almost the same affinity to both aflatoxins B₁ and M₁ /K_a for aflatoxin B₁=2.0×10⁹ and K_a for aflatoxin M₁=2.1×10⁹/ and making possible their simultaneous determination. Aflatoxin B₁ content is determined specifically by a commercial RIA, and aflatoxin M₁ concentration is calculated as the differences between these two assays.     

Stability of Aflatoxin B1 in Aqueous Solutions

Stability of aflatoxin B₁ in aqueous solutions at different temperatures and pH values was studied by thin-layer chromatography and fluorodensitometric measurements. The rate of deterioration was found to be increased with increasing temperature and also by changing pH from neutral to alkaline or acid. In acidic buffers the aflatoxin was converted to a new fluorescent product which has much lower R_f value than aflatoxin B₁ itself when chromatographed on silica gel thin-layer plates developed with chloroform-methanol(99:l, v/v). The UV absorption spectrum of this decomposition product has been determined.     

Selection of the separation step in the radioimmunoassay for aflatoxin B1 using125I as a marker

Aflatoxin B₁ was assayed by radioimmunoassay (RIA) using¹²⁵I-labelled antigen. The immunoreactivity of the radioligand applied is very close to the immunoreactivity of free aflatoxin B₁. The logit-log analysis was used to select the best separation of free and bound radioligand. The adsorption of the free radioligand on dextran-coated charcoal was found to be superior from the viewpoint of the assay sensitivity and accuracy. The detection limit of aflatoxin B₁ was about 10 pg per tube. The assay accuracy was estimated to 3.3% in intraassay and to 7.0% in interassay.     

Determination of aflatoxin B1 in alcoholic beverages: comparison of one- and two-photon-induced fluorescence

The qualitative and quantitative analysis of aflatoxin B₁ in a model system (water/ethanol), in different wines and in beer using one- and two-photon-induced fluorescence is discussed. The absorption and fluorescence properties of aflatoxin B₁ depend on the solvent and pH. The two-photon-absorption cross-section was calculated for aflatoxin B₁ in beer and wine (σ ₂ ∼ 25 GM) for excitation at 720 nm. A comparison of the one- and two-photon- induced fluorescence results showed that the disturbance due to background emission originating from matrix constituents is significantly reduced under two-photon-excitation conditions. The limit of detection for the one- and two-photon-induced fluorescence was determined.     

Certification of the aflatoxin B1 content in animal feed Reference Materials and intercomparison with supercritical fluid extraction (SFE)

Summary Intercomparison studies allowed an improvement in aflatoxin B₁ analysis and the development of procedural quality criteria. These led to a successful evaluation of the certification exercise on the aflatoxin B₁ content in five animal feed RMs. The 1st intercomparison study on supercritical fluid extraction (SFE) techniques gave very helpful explanations to solve matrix difficulties.     

Colloidal gold based immunochromatographic strip for the simple and sensitive determination of aflatoxin B1 and B2 in corn and rice

We have developed a simple and fast immunochromatographic test strip for the simultaneous quantitation of aflatoxin B₁ and aflatoxin B₂ in corn and rice. The strip contains three pads (sample, conjugate, and absorbing pad) and uses the respective polyclonal antibodies immobilized on gold nanoparticles. Matrix interferences were minimized by application of fugacity theory. Clean-up of samples and pre-treatment of strip pads is not required. The visual detection limit is 0.1 ng mL^?1, and the process can be completed within 5 min. Out of 113 natural samples, 16 rice and 27 corn samples (38% in total) were aflatoxin positive and the test results were confirmed by HPLC. The strip shows, however, high cross reactivity to aflatoxins G₁, G₂, and M₁. We consider this strip to possess wide applicability because of its ease of use, sensitivity, stability, and low cost.

Quantitation of carcinogen bound protein adducts by fluorescence measurements

A highly significant correlation of aflatoxin B₁ serum albumin adduct level with daily aflatoxin B₁ intake was observed in a molecular epidemiological study of aflatoxin carcinogenesis which used conventional fluorescence spectroscopy methods for adduct quantitation. Synchronous fluorescence spectroscopy and laser induced fluorescence techniques have been employed to quantitate antibenzo[a]pyrene diol epoxide derived globin peptide adducts. Fast and efficient methods to isolate the peptide adducts as well as eliminate protein fluorescence background are described. A detection limit of several femtomoles has been achieved. Experimental and technical considerations of low temperature synchronous fluorescence spectroscopy and fluorescence line narrowing to improve the detection sensitivities are also presented.     

Determination of aflatoxin B1 by biomass derived porous carbon modified electrode with molecularly imprinted polymer

Aflatoxin determination is imperative among agricultural and food products as the pollution of the toxic aflatoxin could cause severe hazards towards animals and human beings. Here, we prepared Elaeagnus gum derived porous carbon material as electrode modified material. In situ electropolymerization of molecularly imprinted polymer utilizing phenol as monomer and aflatoxin B₁ as target template was further processed onto the porous carbon surface to achieve aflatoxin B₁ sensing ability. This sensitive MIP sensor has a detection range from 5 pM to 100 pM of aflatoxin B₁, an electrochemical sensitivity of 82.4 μA log(pM)⁻¹ cm⁻² with a detection limit of 1.7 pM, and a recovery rate of real sample measurement is 98.21 %. Good selectivity, fair repeatability and stability was confirmed. This work demonstrates the promising application of Elaeagnus gum derived porous carbon modified electrode sensor for food and drug monitoring.     

Production of ultrasensitive generic monoclonal antibodies against major aflatoxins using a modified two-step screening procedure

Monoclonal antibodies (McAbs) cross-reactive with four major aflatoxins were achieved using a modified two-step screening procedure. The first step was twice modified indirect enzyme-linked immunosorbent assay (ELISA) and resulted in positive hybridomas and hapten-specific antibodies. The modified indirect competitive ELISA (ciELISA) was the second step, in which the competition incubation time was decreased to 30 min, aflatoxin B₁, B₂, G₁ and G₂ were all used as competitors, the concentrations of four aflatoxins were gradiently decreased in each screening. 2-3 subclonings were performed after every modified fusion and resulted in eight hybridomas that secreted antibodies with good cross-reactivity and high affinity to four aflatoxins. Five McAbs were chosen for further analysis. Of the five, two antibodies had similar reaction efficiency with aflatoxin B₁, B₂ and G₁ but showed a weak cross-reaction to G₂. Another two had almost identical reaction capability with four aflatoxins. One clone 1C11 exhibited the highest sensitivity for all four aflatoxins. The concentrations of aflatoxin B₁, B₂, G₁ and G₂ at 50% inhibition for 1C11 were 1.2, 1.3, 2.2 and 18.0 pg mL⁻¹ respectively. This is the most sensitive for all four major aflatoxins described so far. The results indicated that the modified two-step screening procedure had superiority and these antibodies could be used for simultaneous analysis of total aflatoxins.     

Study of polyaniline conducting/electroactive polymer as sensor for some agricultural phosphorus pesticides

Abstract  

Due to their wide applications as insecticides, pesticides, chemical warfare agents, etc., detection of organophosphorus compounds is of great importance. This paper deals with application of polyaniline conducting polymer (PANi) as a sensor material for detection of some agricultural organophosphorus compounds that are commonly used as insecticides and pesticides. The compounds employed in this investigation are dichlorvos, trichlorophon, and chlorpyrifos. The effects of some important parameters such as concentration, thickness of the polymer film, etc., on electrical conductivity and conductivity stability for these compounds have also been investigated. In situ electrical conductivity measurements were performed using a standard direct-current (DC) four-point probe technique. It was found that adsorption and desorption of these compounds on PANi film cause measurable change in the resistance of conducting polymers.     

Rapid analysis of aflatoxins B1, B2, and ochratoxin A in rice samples using dispersive liquid–liquid microextraction combined with HPLC

A novel, simple, and rapid method is presented for the analysis of aflatoxin B₁, aflatoxin B₂, and ochratoxin A in rice samples by dispersive liquid–liquid microextraction combined with LC and fluorescence detection. After extraction of the rice samples with a mixture of acetonitrile/water/acetic acid, mycotoxins were rapidly partitioned into a small volume of organic solvent (chloroform) by dispersive liquid–liquid microextraction. The three mycotoxins were simultaneously determined by LC with fluorescence detection after precolumn derivatization for aflatoxin B₁ and B₂. Parameters affecting both extraction and dispersive liquid–liquid microextraction procedures, including the extraction solvent, the type and volume of extractant, the volume of dispersive solvent, the addition of salt, the pH and the extraction time, were optimized. The optimized protocol provided an enrichment factor of approximately 1.25 and with detection of limits (0.06–0.5 μg/kg) below the maximum levels imposed by current regulations for aflatoxins and ochratoxin A. The mean recovery of three mycotoxins ranged from 82.9–112%, with a RSD less than 7.9% in all cases. The method was successfully applied to measure mycotoxins in commercial rice samples collected from local supermarkets in China.     

A Guide to Thin-Layer Chromatographic Systems for the Separation of Aflatoxin B1, B2, G1 and G2

Abstract

The separation of aflatoxin B₁, B₂, G₁ and G₂ was compared on six commercial silica gel plates in twelve solvent systems. Two of the solvent systems, chloroform: acetone: ammonium hydroxide (90: 10: 0.25) and chloroform: acetone: hexane (85: 15: 20) resolved the four aflatoxins on all the tested plates. The solvent modifier played an important role in the resolution of these compounds. The effect of the hardness of the plate is also discussed.     

Simultaneous determination of aflatoxin B1, B2, G1, and G2 in corn powder,edible oil,peanut butter,and soy sauce by liquid chromatography with tandem mass spectrometry utilizing turbulent flow chromatography

A novel fully automated method based on dual column switching using turbulent flow chromatography followed by liquid chromatography with tandem mass spectrometry was developed for the determination of aflatoxin B₁, B₂, G₁, and G₂ in corn powder, edible oil, peanut butter, and soy sauce samples. After ultrasound‐assisted extraction, samples were directly injected to the chromatographic system and the analytes were concentrated into the clean‐up loading column. Through purge switching, the analytes were transferred to the analytical column for subsequent detection by mass spectrometry. Different types of TurboFlow^TM columns, transfer flow rate, transfer time were optimized. The limits of detection and quantification of this method ranged between 0.2–2.0 and 0.5–4.0 μg/kg for aflatoxins in different matrixes, respectively. Recoveries of aflatoxins were in range of 83–108.1% for all samples, matrix effects were in range of 34.1–104.7%. The developed method has been successfully applied in the analysis of aflatoxin B₁, B₂, G₁, and G₂ in real samples.     

Determination of aflatoxins B1, B2, G1 and G2 by high-performance liquid chromatography with electrochemical detection

A new approach to the determination of afiatoxins B₁, B₂, G₁ and G₂ is given; the method involves high-performance liquid chromatography with amperometric detection in the differential-pulse mode at the dropping mercury electrode with 1-s drop time. These aflatoxins can be determined simultaneously with good resolution but with some compromise in sensitivity. The detection limit of underivatized aflatoxin standards is around 5 ng. Average recoveries of aflatoxins from peanut butter by the Beebe method were G₂ 81%, G₁ 87%, B₂ 77% and B₁ 76%.     

AFB1–AP Conjugate for Enzyme Immunoassay of Aflatoxin B1 in Corn Samples

Abstract

This article describes the conjugation between aflatoxin B₁ (AFB₁), one of the major mycotoxins, and alkaline phosphatase (AP), one of the most used enzymes for immunoassays. In addition, an application of the ELISA method for aflatoxin B₁ determination in corn is presented. Three AFB₁–AP conjugates in different toxin–enzyme ratios were prepared and tested. The ELISA results, developed with the most effective conjugate obtained, showed a satisfactory working range between 2.4 and 4000 ng of toxin/g of corn. The detection limit was 2 ng/g in corn samples, and recoveries ranged from 105 to 120%.     

13C nuclear magnetic resonance spectra of aflatoxin B1 derived from acetate

The labeling distribution of the fungal metabolite, aflatoxin B₁, produced from [1- or 2- ¹³C] acetate was determined by ¹³C FT NMR. The results support the polyketide hypothesis for aflatoxin biogenesis.     

Development of mycotoxin reference materials

Summary The analytical difficulties in mycotoxin determination have given rise to the development of mycotoxin reference materials by the European Community Bureau of Reference (BCR) in cooperation with several European Laboratories, among them the National Institute of Public Health and Environmental Protection. The first project undertaken concerned four milk powders certified for their aflatoxin M₁ content (concentrations 0, 0.1, 0.3 and 0.8 g/ kg). Projects that are well-advanced include two peanut meals (aflatoxin B₁ contents 0 and ca. 200 g/kg) and two compounded animal feedingstuffs (aflatoxin B₁ contents 0 and 10 g/kg). Mycotoxin reference materials are also under development for zearalenone in wheat and maize, and for ochratoxin A in wheat.The general approach followed in the development and certification of mycotoxin reference materials (preparation, testing for homogeneity and stability, intercomparisons of methods and certification exercise) will be discussed and exemplified with the help of the completed project on aflatoxin M₁ in milk powder.

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Entwicklung von Mycotoxin-Referenzmaterialien

     

Development of a multi‐mycotoxin liquid chromatography/tandem mass spectrometry method for sweet pepper analysis

A multi‐mycotoxin method was developed for the simultaneous determination of trichothecenes (nivalenol, deoxynivalenol, 3‐acetyldeoxynivalenol, 15‐acetyldeoxynivalenol, neosolaniol, fusarenon‐X, diacetoxyscirpenol, HT‐2 toxin, T‐2 toxin), aflatoxins (aflatoxin‐B₁, aflatoxin‐B₂, aflatoxin‐G₁ and aflatoxin‐G₂), Alternaria toxins (alternariol, alternariol methyl ether and altenuene), fumonisins (fumonisin‐B₁, fumonisin‐B₂ and fumonisin‐B₃), ochratoxin A, zearalenone, beauvericin and sterigmatocystin in sweet pepper. Sweet pepper was extracted with ethyl acetate/formic acid (99:1, v/v). After splitting up the extract, two‐thirds of the extract was cleaned up using an aminopropyl column followed by an octadecyl column. The remaining part was cleaned up using a strong anion‐exchange column. After recombination of both cleaned parts of the sample extract, the combined solvents were evaporated and the residue was dissolved in mobile phase; 20 µL was injected into the chromatographic system, so only one run was used to separate and detect the mycotoxins in positive electrospray ionization using selected reaction monitoring. The samples were analyzed with a Micromass Quattro Micro triple quadrupole mass spectrometer (Waters, Milford, MA, USA). The mobile phase consisted of variable mixtures of water and methanol, 1% acetic acid and 5 mM ammonium acetate. The limits of detection of the multi‐mycotoxin method varied from 0.32 µg.kg^?1 to 42.48 µg.kg^?1. The multi‐mycotoxin liquid chromatography/tandem mass spectrometry (LC/MS/MS) method fulfilled the method performance criteria required by the Commission Regulation (EC) No 401/2006. Sweet peppers inoculated by Fusarium species were analyzed using the developed method. Beauvericin (9–484 µg.kg^?1) and fumonisins (fumonisin‐B₁ up to 4330 µg.kg^?1, fumonisin‐B₂ up to 4900 µg.kg^?1, and fumonisin‐B₃ up to 299 µg.kg^?1) were detected. Copyright © 2008 John Wiley & Sons, Ltd.     

Simultaneous determination of aflatoxins B1, B2, G1, G2 in Fructus Bruceae by high‐performance liquid chromatography with online postcolumn photochemical derivatization

A simple, reliable, and highly sensitive method for the simultaneous determination of aflatoxin B₁, B₂, G₁, G₂ in Fructus Bruceae was developed using high‐performance liquid chromatography coupled to online postcolumn photochemical derivatization and fluorescence detection. Aflatoxins were first extracted by a methanol/water mixture and then cleaned up with an AflaTest? immunoaffinity column. Different clean‐up and derivatization methods were compared and optimized. The established method was extensively validated to show satisfactory performance of linearity (R² ≥ 0.9997), recovery (74.3–100.8%), and precision (RSDs ≤ 2.8%) for the investigated aflatoxins. This proposed method was also applied to 11 F. Bruceae samples and the results showed that 10 out of 11 were contaminated with aflatoxins ranging from 0.26 to 27.52 μg/kg and the occurrence of aflatoxin B₁, the most toxic one, was as high as 91% in all the samples, highlighting the severe contamination and the necessity to set legal limits for aflatoxins in F. Bruceae.