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Gambacorta S.,CNR Institute of Sciences of Food Production | Solfrizzo H.,CNR Institute of Sciences of Food Production | Visconti A.,CNR Institute of Sciences of Food Production | Powers S.,Vicam | And 6 more authors.
World Mycotoxin Journal | Year: 2013

The multi-biomarker approach was used to validate urinary biomarkers in piglets administered boluses contaminated with mixtures of deoxynivalenol (DON), aflatoxin B1 (AFB1), fumonisin B1 (FB 1), zearalenone (ZEA) and ochratoxin A (OTA) at different concentrations. Boluses contaminated with mycotoxins were prepared by slurrying and freezedrying feed material fortified with culture extracts of selected toxigenic fungi. Piglets were individually placed in metabolic cages to collect urine before gavage and 24 h post dose. Urine samples were hydrolysed with β-glucuronidase and analysed by a multi-biomarker LC-MS/MS method developed and validated to identify and measure biomarkers of FB1, OTA, DON, ZEA and AFB1. Urinary levels of FB1, OTA, DON + de-epoxy-deoxynivalenol, ZEA + alphazearalenol and aflatoxin M1 were selected as biomarkers of FB1, OTA, DON, ZEA and AFB1, respectively. Mean percentages of dietary mycotoxins excreted as biomarkers in 24 h post dose urine were 36.8% for ZEA, 28.5% for DON, 2.6% FB1, 2.6% for OTA and 2.5% for AFB1. A good correlation was observed between the amount of mycotoxins ingested and the amount of relevant biomarkers excreted in 24 h post dose urine. Linear dose-response correlation coefficients ranged between 0.68 and 0.78 for the tested couples of mycotoxin/biomarker. The good sensitivity of the LC-MS/MS method and the good dose-response correlations observed in this study permitted to validate the selected mycotoxin biomarkers in piglets at dietary levels close to the maximum permitted levels reported in Commission Directive 2003/100/EC for AFB1 and the guidance values reported in Commission Recommendation 2006/576/EC for DON, ZEA, OTA and FB 1.


Lattanzio V.M.T.,CNR Institute of Sciences of Food Production | Ciasca B.,CNR Institute of Sciences of Food Production | Powers S.,Vicam | Visconti A.,CNR Institute of Sciences of Food Production
Journal of Chromatography A | Year: 2014

An improved method for the quantitative determination of aflatoxins (B1, B2, G1, G2), ochratoxin A, fumonisins (B1, B2), zearalenone, deoxynivalenol, nivalenol, T-2 and HT-2 toxins in cereals and derived products, at levels comparable with EU maximum permitted levels, was developed. The effective co-extraction of the mycotoxins under investigation was achieved in 4min by a double extraction approach, using water followed by methanol. Clean up of the extract was performed by a new multi-toxin immunoaffinity column. Analytical performance characteristics were evaluated through single laboratory validation. Raw wheat and maize, corn flakes and maize snacks were chosen as representative matrices for method validation. The validation assay was carried out at 50, 100 and 150% of EU maximum permitted levels for each mycotoxin. Statistical analysis of the results (ANOVA) provided the within laboratory reproducibility and the error contributions from repeatability, between day effects, and influences from different matrix composition. Recoveries generally higher than 70% were obtained for all tested mycotoxins with relative standard deviation (within laboratory reproducibility) lesser than 37%. Limits of quantification (calculated as the lowest amount of each analyte which could be determined with a precision of 10%) ranged from 1μg/kg to 30μg/kg. The trueness of generated data was assessed by analysis of reference materials. The proposed method was proven to be suitable to assess, with a single analysis, compliance of the selected cereal based foods with the EU maximum permitted or recommended levels for all regulated mycotoxins. © 2014 Elsevier B.V.


Lattanzio V.M.T.,CNR Institute of Sciences of Food Production | Ciasca B.,CNR Institute of Sciences of Food Production | Powers S.,Vicam | von Holst C.,European Commission
TrAC - Trends in Analytical Chemistry | Year: 2016

Recent advances in establishing harmonized guidelines for validation of screening methods are reviewed. Emphasis is given to guidelines set in the Regulation 519/2014/EU that specifies validation criteria for mycotoxin screening methods to be used for official control purposes. The application of this validation scheme is demonstrated through a case study. A commercial lateral flow immunoassay for deoxynivalenol has been and evaluated for its applicability to verify wheat compliance with EC maximum permitted level. The validation design provided information on the precision profile of the method, cut-off, false suspect and false negative rates of samples containing deoxynivalenol above the legal limit.The influence of the co-occurrence of major deoxynivalenol modified forms, i.e. 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, and deoxynivalenol-3-glucoside on test results, was evaluated by a factorial design. Finally, the applicability of the validated immunoassay was demonstrated by analysis of naturally contaminated wheat samples, and comparison with results obtained by a LC-MS/MS confirmatory method. © 2015 Elsevier B.V.


Solfrizzo M.,CNR Institute of Sciences of Food Production | Gambacorta L.,CNR Institute of Sciences of Food Production | Lattanzio V.M.T.,CNR Institute of Sciences of Food Production | Powers S.,Vicam | Visconti A.,CNR Institute of Sciences of Food Production
Analytical and Bioanalytical Chemistry | Year: 2011

Humans and animals can be simultaneously exposed through the diet to different mycotoxins, including aflatoxins, ochratoxin A, deoxynivalenol, zearalenone, and fumonisins, which are the most important. Evaluation of the frequency and levels of human and animal exposure to these mycotoxins can be performed by measuring the levels of the relevant biomarkers in urine. Available data on the toxicokinetics of these mycotoxins in animals suggest that aflatoxin M 1 (AFM 1), ochratoxin A (OTA), deoxynivalenol (DON)/de-epoxydeoxynivalenol (DOM-1), alpha-zearalenol (α-ZOL)/beta- zearalenol (β-ZOL), and fumonisin B 1 (FB 1) can be used as urinary biomarkers. A liquid chromatographic-tandem mass spectrometric method has been developed for simultaneous determination of these mycotoxin biomarkers in human or animal urine. Urine samples were purified and concentrated by a double cleanup approach, using a multitoxin immunoaffinity column and a reversed-phase SPE Oasis HLB column. Separation of the biomarkers was performed by reversed-phase chromatography using a multi-step linear methanol-water gradient containing 0.5% acetic acid as mobile phase. Detection and quantification of the biomarkers were performed by triple quadrupole mass spectrometry (LC-ESI-MS/MS). The clean-up conditions were optimised to obtain maximum analyte recovery and high sensitivity. Recovery from spiked samples was performed at four levels in the range 0.03-12 ng mL -1, using matrix-matched calibration curves for quantification. Mean recoveries of the biomarkers tested ranged from 62 to 96% with relative standard deviations of 3-20%. Enzymatic digestion with β-glucuronidase/sulfatase resulted in increased concentrations of the biomarkers, in both human and pig urine, in most samples containing measurable concentrations of DON, DOM-1, OTA, α-ZOL, or β-ZOL. A highly variable increase was observed between individuals. Co-occurrence of OTA and DON in human urine is reported herein for the first time. © 2011 Springer-Verlag.


Pascale M.,CNR Institute of Sciences of Food Production | Panzarini G.,CNR Institute of Sciences of Food Production | Powers S.,VICAM | Visconti A.,CNR Institute of Sciences of Food Production
Food Analytical Methods | Year: 2014

An ultra-performance liquid chromatography (UPLC®) method has been developed for the simultaneous determination of deoxynivalenol (DON) and nivalenol (NIV) in wheat. Ground sample was extracted with water and the filtered extract was cleaned up through an immunoaffinity column containing a monoclonal antibody specific for DON and NIV. Toxins were separated and quantified by UPLC® with photodiode-array detector (λ = 220 nm) in less than 3 min. Mean recoveries from blank wheat samples spiked with DON and NIV at levels of 100-2,000 μg/kg (each toxin) ranged from 85 to 95 % for DON and from 81 to 88 % for NIV, with relative standard deviations less than 7 %. Similar recoveries were observed from spiked samples when methanol/water (80:20, v/v) was used as extraction solvent. However, by using a wheat sample naturally contaminated with DON and NIV, the one-way analysis of variance (Student-Newman-Keuls test) between different extraction solvents and modes showed that water extraction provided a significant increase (P < 0.001) in toxin concentrations (mean values of six replicate analyses) with respect to methanol/water (80:20, v/v). No significant difference was observed between shaking (60 min) and blending (3 min). The limit of detection (LOD) of the method was 30 μg/kg for DON and 20 μg/kg for NIV (signal-to-noise ratio 3:1). The immunoaffinity columns showed saturation of DON/NIV binding sites at levels higher than 2,000 ng in blank wheat extracts spiked with the corresponding amount of mycotoxin, as single mycotoxin or sum of DON and NIV. The range of applicability of the method was from LOD to 4,000 μg/kg, as single mycotoxin or sum of DON and NIV in wheat. The analyses of 20 naturally contaminated wheat samples showed DON contamination in all analyzed samples at level ranging from 30 to 2,700 μg/kg. NIV was detected in two samples at negligible toxin levels (up to 46 μg/kg). This is the first UPLC® method using immunoaffinity column cleanup for the simultaneous and sensitive determination of DON and NIV in wheat. © 2013 Springer Science+Business Media New York.


An improved method for the quantitative determination of aflatoxins (B1, B2, G1, G2), ochratoxin A, fumonisins (B1, B2), zearalenone, deoxynivalenol, nivalenol, T-2 and HT-2 toxins in cereals and derived products, at levels comparable with EU maximum permitted levels, was developed. The effective co-extraction of the mycotoxins under investigation was achieved in 4min by a double extraction approach, using water followed by methanol. Clean up of the extract was performed by a new multi-toxin immunoaffinity column. Analytical performance characteristics were evaluated through single laboratory validation. Raw wheat and maize, corn flakes and maize snacks were chosen as representative matrices for method validation. The validation assay was carried out at 50, 100 and 150% of EU maximum permitted levels for each mycotoxin. Statistical analysis of the results (ANOVA) provided the within laboratory reproducibility and the error contributions from repeatability, between day effects, and influences from different matrix composition. Recoveries generally higher than 70% were obtained for all tested mycotoxins with relative standard deviation (within laboratory reproducibility) lesser than 37%. Limits of quantification (calculated as the lowest amount of each analyte which could be determined with a precision of 10%) ranged from 1g/kg to 30g/kg. The trueness of generated data was assessed by analysis of reference materials. The proposed method was proven to be suitable to assess, with a single analysis, compliance of the selected cereal based foods with the EU maximum permitted or recommended levels for all regulated mycotoxins.


Liu J.,Vicam | Yang Y.,Tongji Medical College | Li X.,Second Affiliated Hospital of Chongqin Medical University | Zhang P.,Syracuse University | And 2 more authors.
Methods in Enzymology | Year: 2010

Protein O-mannose N-acetylglucosaminyltransferase 1 (POMGnT1) is an enzyme that catalyzes the transfer of N-acetylglucosamine to O-mannose of glycoproteins. It is involved in posttranslational modification of α-dystroglycan (α-DG). POMGnT1-null mice were generated by gene trapping with a retroviral vector inserted into exon 2 of the POMGnT1 gene. Expression of POMGnT1 was completely disrupted as evidenced by absence of its mRNA expression. POMGnT1 knockout mice were viable but with reduced fertility and variable lifespan. The functional glycosylated form of α-DG was markedly reduced in POMGnT1 knockout mice along with impaired α-DG-laminin binding activity. Multiple developmental defects in muscle, brain, and eye were observed. In addition, the knockout mice exhibited extensive abnormalities in the neocortex, including changed neuron distribution, presence of ectopic fibroblasts, and GFAP-positive reactive astrocytes. Analysis of POMGnT1 knockout neocortex at several developmental stages revealed that these defects were secondary to disruptions of the pial basement membrane. © 2010 Elsevier Inc.


Li W.,Texas A&M University | Powers S.,VICAM | Dai S.Y.,Texas A&M University
World Mycotoxin Journal | Year: 2014

Rapid test methods are widely used for measuring mycotoxins in a variety of matrices. This review presents an overview of the current commercially available immunoassay rapid test formats. Enzyme linked immune-sorbent assay (ELISA), lateral flow tests, flow through immunoassay, fluorescent polarisation immunoassay, and immunoaffinity columns coupled with fluorometric assay are common formats in the current market. The two existing evaluation programs for commercial testing kits by United State Department of Agricultural Grain Inspection, Packers & Stockyards Administration (USDA-GIPSA) and AOAC Research Institute are introduced. The strengths and weaknesses of these test kits are discussed with regard to the application scope, variance, specificity and cross reactivity, accuracy and precision, and measurement range. Generally speaking, the current commercially available testing kits meet research and industrial needs as 'fit-for-purpose'. Furthermore, quality assurance concerns and future perspectives are elaborated for broader application of commercial test kits in research, industry and regulatory applications. It is expected that new commercial kits based on advanced technologies such as electrochemical affinity biosensors, molecularly imprinted polymers, surface plasmon resonance, fluorescence resonance energy transfer, aptamer-based biosensors and dynamic light scattering might be available to users in the future. Meanwhile, harmonisation of testing kit evaluation, incorporation of more quality assurance into the testing kit utilisation scheme, and a larger variety of kits available at lower cost will expand the usage of testing kits for food safety testing worldwide.


News Article | November 1, 2016
Site: www.businesswire.com

MILFORD, Mass.--(BUSINESS WIRE)--Waters Corporation (NYSE: WAT) today announced VICAM®, a Waters® Business, now offers a single extraction method for the detection of aflatoxins and fumonisins in corn and grain. The Afla-V® AQUA™ and Fumo-V® AQUA™ provide quantitative results in only five-minutes (after extraction/sample prep). The simple, one-step, water-based process eliminates solvent use completely. Extraction is performed using a simple, environmentally safe solution which requires no special handling, mixing or blending. Traditional testing methods often require individual extraction procedures for each toxin, some requiring the use of costly chemicals such as methanol, a chemical solvent which requires special storage and handling. In addition, specialized shipping often adds to the expense and complexity of on-site mycotoxin detection. In contrast, the Afla-V® AQUA™ and Fumo–V® AQUA™ tests can be run on the same sample extract, reducing time, effort and consumables cost. “Afla-V® AQUA™ and Fumo-V® AQUA™ are designed to make mycotoxin monitoring more accessible by enabling early detection and quantitation while also eliminating the dependence on chemicals that may be difficult to obtain, store and use in a safe manner,” said Marjorie Radlo-Zandi, Managing Director, VICAM. “All this is accomplished while delivering a performance that meets quality and regulatory standards that safeguard food and feed products worldwide.” Afla-V® AQUA™ and Fumo-V® AQUA™ are now both available worldwide. About Aflatoxins Aflatoxins are naturally occurring chemical byproducts of mold species, which are natural inhabitants of crop soils. Severe rain and drought can encourage aflatoxin production, and once released, aflatoxin and other mycotoxins are difficult to manage and nearly impossible to destroy. Aflatoxin B1 is classified as a Group I carcinogen and is highly toxic even at very low concentrations. The USDA, FDA, EU, and other international government agencies impose specific regulations and guidelines for acceptable levels of mycotoxins. Producers and exporters use on-site and laboratory testing to ensure regulatory compliance and to protect human and animal health from illnesses associated with mycotoxins, including cancer and feed refusal in animals. About Fumonisins Fumonisins are mycotoxins produced by the fungus Fusarium moniliforme. F. moniliforme is a frequent, almost universal, inhabitant of corn. Fumonisin B1, B2 and B3 are present in most corn samples tested, often totaling greater than 1 ppm. Fumonisin is thought to cause equine leukoencephalomalacia in horses, swine pulmonary edema, and human esophageal cancer. The FDA/USDA Working Group on Fumonisin advises less than 4 ppm for products for human consumption, less than 5 mg/kg in horse feed, less than 10 mg/kg for swine feed, and less than 50 mg/kg for cattle feed. VICAM is a world-leading provider of mycotoxin testing solutions. Since 1985, VICAM has been dedicated to developing USDA- and AOAC-approved rapid tests for mycotoxins. VICAM’s mycotoxin test kits offer a choice of rapid qualitative screening and quantitative test methods for a wide variety of mycotoxins. Around the world, VICAM products are used with confidence by customers who trust our commitment to extending the value of those products with unmatched service and support. We guarantee satisfaction with expert technical assistance and top-of-the-line customer care through a global scientific and distribution network that develops, markets, sells, and services our products in more than 100 countries. For more information, please visit www.vicam.com or call +1.508.482.4935. Waters Corporation (NYSE:WAT) develops and manufactures advanced analytical and material science technologies for laboratory dependent organizations. For more than 50 years, the company has pioneered a connected portfolio of separations science, laboratory information management, mass spectrometry, and thermal analysis systems.


Sato T.,Shinshu University | Higashihara K.,Shinshu University | Sasaki A.,Shinshu University | Toth D.,Vicam | Goto T.,Shinshu University
World Mycotoxin Journal | Year: 2010

Citrinin (CIT) is a nephrotoxic secondary metabolite produced by several fungal species. CIT contamination has been reported in many cereals. Recently CIT contamination of red mold rice, used as an artificial colour, received attention because a CIT producing Monascus species was used in its production. Therefore, we developed a relatively simple method, immunoaffinity column clean-up followed by high performance liquid chromatographic (IAC-HPLC) detection, to determine CIT in red mold rice and several other commodities, and performed a single laboratory validation of the method. CIT was extracted by shaking for 30 min with 70% methanol, after which the extract was purified using an immunoaffinity column. The isolated CIT was quantitated by HPLC with fluorescence detection. Recoveries of CIT from red mold rice and koji-rice at levels of 50-2,500 μg/kg were 81-88%. RSDs, estimated with naturally contaminated red mold rice at 50, 200 and 400 μg/kg were 5.1%, 2.8% and 4.1%, respectively. Recoveries of CIT from cereals and mushroom samples at 50, 200 and 400 μg/kg were 60-123%, 69-88% and 73-100% with RSDs of 2.1-7.8%, 1.7-9.2% and 0.6-5.3%, respectively. © 2010 Wageningen Academic Publishers.

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