Development and validation of a method for the quantitative determination of aflatoxin contaminants in Maytenus ilicifolia by HPLC with fluorescence detection

A method for the quantification of aflatoxins B1, G1, B2 and G2 in the medicinal herb Maytenus ilicifolia was developed and validated. The method used immunoaffinity columns for sample clean-up and HPLC with fluorescence detection without any derivatisation step. The method showed good inter-day accuracy (bias values in the range 4.5-10.7%) and precision (5-16% RSD) when applied to the determination of levels of aflatoxins ranging from 7 to 20 ppb in the plant material. The detection limits for samples of the plant material spiked with aflatoxins were 3.5 ng/g for B1 and G1 and 0.1 ng/g for B2 and G2. The method was successfully applied to commercial samples of Maytenus ilicifolia for the screening of aflatoxin contaminants.     

Determination of aflatoxin levels in pistachio and cashew nuts using immunoaffinity column clean-up with HPLC and fluorescence detection

A technique for the detection of aflatoxins in pistachio and cashew nuts using immunoaffinity column clean-up with HPLC and fluorescent detection is presented. Recoveries were in the range of 79–99% for pistachio samples artificially contaminated with 10 g total aflatoxins kg–1 of food sample. For cashew samples recoveries ranged from 80–106%. This method is proposed as an accurate technique for aflatoxin detection in the range of g aflatoxins kg–1 nuts.     

Aflatoxins isolated by immunoaffinity chromatography from foods consumed in The Gambia, West Africa.

An aflatoxin-specific, monoclonal antibody-based immunoaffinity chromatography method has been developed for the rapid isolation of aflatoxins from human foods. Aflatoxins were isolated by immunoaffinity chromatography from a variety of cooked foods, including maize, rice, millets, groundnut sauces, and leaf sauces, collected in The Gambia, West Africa. The aflatoxins were measured by direct fluorescence or high-pressure liquid chromatography. The highest levels were found for groundnut sauces, mean 162 ppb (range 18 to 943 ppb) for 18 positive samples, but aflatoxins were found in other foods; e.g., maize, mean 9.7 ppb (range 2 to 35 ppb) for nine positive samples. The food analysis results were used with records of the amounts of cooked food to estimate a mean daily intake for an individual of the order of 3.5 micrograms of aflatoxins per day. This approach for exposure assessment is considered in relation to other biomarkers of aflatoxin exposure using biological fluids.     

Natural occurrence of aflatoxins,deoxynivalenol, fumonisins and zearalenone in maize from Entre Ríos Province,Argentina

In Entre Ríos, Argentina, corn is one of the most important cereal grains produced, being an important income for the regional economy. The aim of this work was to assess aflatoxins, zearalenone, deoxynivalenol (DON) and fumonisins (FB) in corn harvest in 2003 and 2004 in the most contaminated departments found in previous studies in selected sampling places. At the harvest time, when the trucks arrived to store plants, samples of corn were taken from seven different positions of the trucks and from five in the trailer. Composite samples were randomised reduced to 10 kg. The samples were analysed by immunological tests, by thin layer chromatography (TLC), high performance liquid chromatography (HPLC) and/or gas liquid chromatography-electron capture detector (GLC-ECD). In 2003 average contamination was 3.19 u.g/kg for aflatoxins, 118.5 μg/kg for deoxynivalenol, 230.8 μg/kg for zearalenone and 10200 μg/kg of total fumonisins (HPLC and ELISA quantification showed a linear correlation (r² =0.9618), but RIDASCREEN®FAST values were 1.7 higher than HPLC values); in 2004 deoxynivalenol and zearalenone were not detected and an average of 2.0 μg/kg for aflatoxins and 4700 μg/kg for total fumonisins was found.This province, with the earliest harvested corn in the country each summer, tends to display different contaminations from the rest of the provinces, probably due to climate characteristics, particularly hotter weather.     

Microscale magnetic microparticle‐based immunopurification of cytokinins from Arabidopsis root apex

Cytokinins (CKs) are pivotal plant hormones that have crucial roles in plant growth and development. However, their isolation and quantification are usually challenging because of their extremely low levels in plant tissues (pmol g^?1 fresh weight). We have developed a simple microscale magnetic immunoaffinity‐based method for selective one‐step isolation of CKs from very small amounts of plant tissue (less than 0.1 mg fresh weight). The capacity of the immunosorbent and the effect of the complex plant matrix on the yield of the rapid one‐step purification were tested using a wide range of CK concentrations. The total recovery range of the new microscale isolation procedure was found to be 30–80% depending on individual CKs. Immunoaffinity extraction using group‐specific monoclonal CK antibodies immobilized onto magnetic microparticles was combined with a highly sensitive ultrafast mass spectrometry‐based method with a detection limit close to one attomole. This combined approach allowed metabolic profiling of a wide range of naturally occurring CKs (bases, ribosides and N⁹‐glucosides) in 1.0‐mm sections of the Arabidopsis thaliana root meristematic zone. The magnetic immunoaffinity separation method was shown to be a simple and extremely fast procedure requiring minimal amounts of plant tissue.     

Analytical methods for the determination of aflatoxins in various food matrices at concentrations regarding the limits set in european regulations: development, characteristics, limits

Methods for the determination of aflatoxins in paprika, peanut butter, pistachio paste, fig paste and baby food were developed. The methods employ an immunoaffinity cleanup step and reversed-phase liquid chromatography. All steps of the analysis were tested for their suitability for all matrices with focus on method robustness, simplicity, toxicology, environment, and user friendliness. Extraction procedures, chromatographic separation and post column derivatisation techniques were elaborated for this purpose. The methods were statistically validated in collaborative trials at currently established legal limits for aflatoxins and are in the process for adoption as official methods by CEN and AOAC.     

A limited survey of aflatoxins and fumonisins in retail maizebased products in the UK using immunoassay detection

A total of 27 maize-based products destined for human consumption were collected from retail outlets within the city of Glasgow in the UK and were analysed for the presence of aflatoxins using immunoaftinity column chromatography with fluorescence detection and for fumonisins by competitive ELISA. Aflatoxins were detected at a trace level below 4 in eight (30%) of the 27 samples tested, no sample contained aflatoxins at a high level although one sample of sweetcorn did contain aflatoxins at a level of 5-10 Fumonisins were detected in eight (30%) of the samples at levels from 1 to 8mgkg^-1 and a further eight samples contained fumonisin at a level below 1 mgkg^-1 but above the detectable level. The highest concentration of fumonisins was found in a sample of fine corn meal at 8-12mgkg^-1.     

Analysis of cocoa products for ochratoxin A and aflatoxins

Eighty-five samples of cocoa products sampled in Canada were analysed for ochratoxin A (OTA) and aflatoxins in 2011–2012. Inclusion of the aflatoxins in this survey required additional method development. Chocolate was extracted with methanol–water plus NaCl, while for cocoa two successive extractions with methanol and methanol–water were made. Extracts were cleaned on an AflaOchra immunoaffinity column (IAC). Determination was by reversed phase high performance liquid chromatography (HPLC). Detection of the aflatoxins was with a post-column photochemical reactor and of OTA by fluorescence detection. Mean limits of quantification (LOQ) of chocolate and cocoa powders were 0.16 ng/g (OTA) and 0.07 ng/g (aflatoxin B₁), respectively. Survey results showed that the incidences of OTA above the LOQ in natural cocoa were 15/15 (mean 1.17 ng/g), 20/21 for alkalized cocoa (mean 1.06 ng/g), 9/9 for baking chocolate (mean 0.49 ng/g), 20/20 for dark chocolate (mean 0.39 ng/g), 7/10 for milk chocolate (mean 0.19 ng/g), 5/5 for cocoa liquor (mean 0.43 ng/g), and 0/5 for cocoa butter. These results confirm our previous work with OTA. In the same samples, incidences of aflatoxin B₁ above the LOQ were 14/15 for natural cocoa (mean 0.86 ng/g), 20/21 for alkalized cocoa (mean 0.37 ng/g), 7/9 for baking chocolate (mean 0.22 ng/g), 16/20 for dark chocolate (mean 0.19 ng/g), 7/10 for milk chocolate (mean 0.09 ng/g), 4/5 for cocoa liquor (mean 0.43 ng/g), and 0/5 for cocoa butter. Both aflatoxins and OTA were confirmed by HPLC-MS/MS when OTA or aflatoxin levels found were above 2 ng/g in cocoa.     

Determination of Δ-tetrahydrocannabinol from human saliva by tandem immunoaffinity chromatography-high-performance liquid chromatography

Smoking or ingestion of cannabis causes cognitive, perceptual and behavioural changes, which are responsible for impaired performance in driving motor vehicles. In this paper a novel liquid chromatographic assay for the selective quantification of Δ⁹-tetrahydrocannabinol, the major indicator of a present cannabis intoxication in saliva, is described. The method involves a column-switching procedure and requires an extremely simple pre-treatment of the sample. Deproteinized saliva was directly injected into the chromatographic system. The clean-up and enrichment procedure was performed in an immunoaffinity column, followed by the transfer of the antigens to an octylsilica analytical column. The immunoaffinity sorbent was obtained by covalent immobilization of specific antibodies on epoxy-activated silica. The mobile phase consisted of methanol-aqueous 0.15 mol/1 NaCl solution (elution programmed) and the analyte was detected by measuring the UV absorption at 220 nm. Using an injection volume of 4.5 ml (dilution 3:2, v/v) the limit of quantification was 20 ng/ml, at a signal-to-noise ratio of 5. Recoveries were estimated to be in the range of 70%. Both intra- and inter-day coefficients of variation were below 5%     

Determination of Δ9-tetrahydrocannabinol from human saliva by tandem immunoaffinity chromatography-high-performance liquid chromatography

Smoking or ingestion of cannabis causes cognitive, perceptual and behavioural changes, which are responsible for impaired performance in driving motor vehicles. In this paper a novel liquid chromatographic assay for the selective quantification of Δ⁹-tetrahydrocannabinol, the major indicator of a present cannabis intoxication in saliva, is described. The method involves a column-switching procedure and requires an extremely simple pre-treatment of the sample. Deproteinized saliva was directly injected into the chromatographic system. The clean-up and enrichment procedure was performed in an immunoaffinity column, followed by the transfer of the antigens to an octylsilica analytical column. The immunoaffinity sorbent was obtained by covalent immobilization of specific antibodies on epoxy-activated silica. The mobile phase consisted of methanol-aqueous 0.15 mol/1 NaCl solution (elution programmed) and the analyte was detected by measuring the UV absorption at 220 nm. Using an injection volume of 4.5 ml (dilution 3:2, v/v) the limit of quantification was 20 ng/ml, at a signal-to-noise ratio of 5. Recoveries were estimated to be in the range of 70%. Both intra- and inter-day coefficients of variation were below 5%     

Determination of deoxynivalenol in cereals by HPLC-UV

A simple method for determination of deoxynivalenol (DON) in cereal samples is described. DON was extracted with methanol, the solvent evaporated, and the residue redissolved with water. This extract was purified on immunoaffinity columns. DON was determined by HPLC with UV-detection. The limits of detection (LOD) and quantification (LOQ) were 10 and 50 μg/kg, respectively. Presented at the 25^th Mykotoxin Workshop in Giessen, Germany, May 19–21, 2003     

Production of ultrasensitive antibodies against aflatoxin B1

AIMS: To produce specific antibodies against the haptenic fungal toxin aflatoxin B1 (AFB1) and apply these antibodies in immunochemical assays for aflatoxins. METHODS AND RESULTS: Rabbits were immunized using an AFB1-bovine serum albumin conjugate and serum titres determined by double-antibody enzyme immunoassay. High titres of antibodies with very high affinity for AFB1 were obtained 15 and 4 weeks after the initial immunization and the first booster immunization respectively. The antibodies were employed in enzyme immunoassay (EIA) and immunoaffinity chromatography (IAC) methods for aflatoxins. With a detection limit of 15.8 pg ml(-1) for AFB1, the EIA employing these antibodies is the most sensitive test for AFB1 described so far. In IAC columns, these antibodies provided high binding capacity for all major aflatoxins, including AFB1, AFB2, AFG1 and AFG2. CONCLUSION: The antibodies described here are useful for the analysis of trace levels of aflatoxins. SIGNIFICANCE AND IMPACT OF THE STUDY: Polyclonal antibody-based EIA and IAC methods for aflatoxin analysis offer a suitable alternative to the more expensive monoclonal antibody-based methods.     

Occurrence of aflatoxin M1 in conventional and organic milk offered for sale in Italy

In the present study, 58 samples of milk were analyzed for the presence of aflatoxin M₁ (AFM₁). The samples were purchased during the period April–May 2013 in a random manner from local stores (supermarkets, small retail shops, small groceries, and specialized suppliers) located in the surrounding of Bologna (Italy). The commercial samples of milk were either organic (n = 22) or conventional (n = 36); fresh milk samples and UHT milk samples, whole milk samples, and partially skim milk samples were present in both the two considered categories. For the quantification of AFM₁ in milk, the extraction-purification technique based on the use of immunoaffinity columns was adopted and analyses were performed using HPLC-FD. AFM₁ was detected in 35 samples, 11 from organic production and 24 from conventional production. No statistically (P > 0.05) significant differences were observed in the concentration of AFM₁ in the two categories of product. The levels of contamination found in the positive samples ranged between 0.009 and 0.026 ng mL^?1. No sample exceeded the limit defined at community level for AFM₁ in milk (0.05 μg kg^?1). This demonstrates the effectiveness of the checks before the placing on the market of these food products. Thus, the “aflatoxins” problem that characterized the summer of 2012 does not seem to have had effect on the contamination level of the considered milk samples.     

A multicomponent method for Fusarium toxins in cereal based food and feed samples using HPLC-MS/MS

A reliable, sensitive and selective multicomponent method has been developed to determine 12 differentFusarium mycotoxins (trichothecenes type A and B, zearalenone) simultaneously in cereal and grain samples using liquid chromatography tandem mass spectrometry (LC-ESI-MS/MS). The sample preparation based on a standard extraction step followed by two different kinds of solid phase clean-up (multifunctional MycoSep^® material) for trichothecenes, and an immuno-affinity purification which combined antibodies for aflatoxins, ochratoxin A and zearalenone (AOZ-IAC). For quantification of zearalenone (ZON) an internal standard (zearalanone, ZAN) was used, whereas for trichothecenes a recovery standard (verrucarol, VOL) was applied. The average recoveries for the trichothecenes ranged from 65% for nivalenol (NIV) up to 96% for deoxynivalenol (DON) and 89% for zearalenone. The limit of quantification is different for each of the individual trichothecenes and in the range of 1 ppb to 10 ppb.     

Determination of B-trichothecenes in wheat by post column derivatisation liquid chromatography with fluorescence detection (PCD-HPLC-FLD)

B-trichothecenes are one of the most common contaminants of cereals in Europe. Therefore, the use of fast and accurate methods is necessary to measure contamination levels and observe regulatory limits. At the moment, mostly gas chromatographic (GC) methods are used but HPLC-UV methods are also employed. Clean-up is commonly done either with immunoaffinity or Mycosep® columns. In the Christian Doppler Laboratory for Mycotoxin Research we have established an alternative HPLC method with post column derivatisation (PCD) as an alternative to existing chromatographic methods. This PCD-HPLC-FLD method uses a Mycosep® clean-up and allows the simultaneous detection and quantification of deoxynivalenol, nivalenol, fusarenon X, 3-acetyldeoxynivalenol and 15-acetyldeoxynivalenol. A validation with wheat gave for deoxynivalenol a limit of quantification ten times below the drafted European Union guideline level (500 µg.kg^?1) and a limit of detection of 8 µg.kg^?1. The relative standard derivation for DON was 10% (n=30). The obtained mean recovery rate for DON was 90% in a range from 50 to 1000 µg.kg^?1.     

Validation of an ELISA test kit for the detection of total aflatoxins in grain and grain products by comparison with HPLC

An ELISA Microtiter Plate, Total Aflatoxin Test called AgraQuant^® was validated to measure total aflatoxins in a range from 4 to 40 ppb in corn, corn meal, corn gluten feed, corn gluten meal, corn germ meal, corn/soy blend, popcorn, sorghum, wheat, milled rice, soybeans, peanuts and cottonseed. The test is performed as a solid phase direct competitive ELISA using a horseradish peroxidase conjugate as the competing, measurable entity. For the test method, aflatoxins are extracted from ground samples with 70 % methanol and sample extracts plus conjugate are mixed and then added to the antibody-coated microwells. After 15 min incubation at room temperature, the plate is washed and enzyme substrate is added and allowed to incubate for an additional 5 min. Stop solution is then added and the intensity of the resulting yellow color is measured optically with a microplate reader at 450 nm. Results obtained from internal validation studies assessing accelerated stability indicate a minimum of 1 year shelf life for the kits; accuracy and precision are comparable to HPLC in the range of 0–320 ppb and limit of detection in corn is 2.5 ppb. Comparison of the method to HPLC, ability to detect individual aflatoxins and ruggedness of the test kits at 18–30°C determined this test to be rugged, sensitive, accurate, precise and effective comparable to HPLC for measuring total aflatoxins ranging from 4 to 40 ppb in the commodities evaluated.     

An automated, handheld biosensor for aflatoxin

A new immunoaffinity fluorometric biosensor has been developed for detecting and quantifying aflatoxins, a family of potent fungi-produced carcinogens that are commonly found in a variety of agriculture products. They have also been cited as a biological agent under weapons development. The handheld, self-contained biosensor is fully automatic, highly sensitive, quick, quantitative, and requires no special storage. Approximately 100 measurements can be made before refurbishment is required, and concentrations from 0.1 parts per billion (ppb) to 50 ppb can be determined in <2 min with a 1 ml sample volume. The device operates on the principles of immunoaffinity for specificity and fluorescence for a quantitative assay. The analytic procedure is flexible so that other chemical and biological analytes could be detected with minor modifications to the current device. Advances in electro-optical components, electronics, and miniaturized fluidics were combined to produce this reliable, small, and versatile instrument.     

The mycotoxin research in foodstuffs in the Czech Republic in the 90th Years

Mycotoxins are naturally occurring secondary metabolites produced by several toxigenic microscopic fungi on a variety of crops, especially cereal grains and further foodstuffs. Series of experimental research projects on the determination of mycotoxins (aflatoxins, cyclopiazonic acid, ochratoxin A, patulin, deoxynivalenol, fumonisin B₁, T-2 toxin, zearalenone, sterigmatocystin, alternaria toxins) in several foods were realized in the National Reference Centre for Microscopic Fungi and Mycotoxins in the 90^th years. The aim of our work was an estimation of dietary exposure to mycotoxins and risk assessment. The method of a solid phase extraction (SPE), liquid — liquid extraction and immunoaffinity chromatography (f. e. R-Biopharm, VICAM) were used to elaborate for sample analyses of mycotoxins in our projects. The mycotoxins were detected most frequently by chromatographic methods (HPTLC, HPLC, GC) and immunochemical methods (ELISA). Average dietary exposure has been calculated by multiplying of concentration data for specific foods with their consumption rates per 1 kg of b. w. per day. The estimation of the dietary exposure dose of mycotoxins for the Czech population is presented.     

Immunoaffinity Purification of Indole-3-acetamide Using Monoclonal Antibodies

A single-step immunoaffinity purification procedure using monoclonalantibodies was developed to isolate indole-3-acetamide fromplant extracts. Antibodies from a selected clone, raised againstIAA-Cl'-BSA, with pronounced ability to recognize indole-3-acetamide(IAM) were used to prepare an immunoaffinity absorbent. Antibodiespurified by thiophilic interaction chromatography were immobilizedon divinylsulfoneactivated agarose. This column shows a veryhigh selectivity towards IAM compared to IAA. This single stepof immunoaffinity purification gave plant extracts of sufficientpurity for direct quantification by on-line spectrofluorimetryafter an analytical ionsuppression-HPLC run. Successive approximationby a second analytical ion-pairing-HPLC run confirmed the validityof this analytical technique. (Received November 15, 1986; Accepted May 7, 1987)     

Determination of beta-sympathomimetics in liver and urine by immunoaffinity chromatography and gas chromatography—mass-selective detection

A specific and sensitive method for the determination of several β-agonistic drugs in liver and urine is described. Following clean-up by immunoaffinity chromatography and two different derivatizations, gas chromatography—mass spectrometry with electron-impact ionization is performed. The immunoaffinity chromatography columns were packed with Sepharose-immobilized polyclonal antibodies raised against the β-agonist clenbuterol. Owing to the high clean-up efficiency of the immunoaffinity column large sample volumes can be used (up to 100 ml urine or 25 gram liver). The immunoaffinity sample pretreatment is highly specific and no further sample pretreatment was necessary. Due to the combination of two different derivatizations only GC—MS with electron-impact ionization is necessary to fulfil legal requirements. The first confirmation step consists of a derivatization reaction between the hydroxyl group of the parent compound and trimethylsilane. The second confirmation method is a derivatization to a cyclic derivative with the hydroxyl group and the aliphatic nitrogen group. Limits of determination in liver as well in urine are at the 10 ng/kg or ng/l (ppt) level with acceptable signal-to-noise ratio. The method is suitable for identification and quantification of trace amounts of several similar β-agonistic drugs either used separately or in combination and can be used also for quantification of clenbuterol in liver with regard to levels exceeding the maximum residue limit (MRL) of 1 μg/kg (ppb).