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Concept: Ochratoxin

232

Over the past 20 years, exposure to mycotoxin producing mold has been recognized as a significant health risk. Scientific literature has demonstrated mycotoxins as possible causes of human disease in water-damaged buildings (WDB). This study was conducted to determine if selected mycotoxins could be identified in human urine from patients suffering from chronic fatigue syndrome (CFS). Patients (n = 112) with a prior diagnosis of CFS were evaluated for mold exposure and the presence of mycotoxins in their urine. Urine was tested for aflatoxins (AT), ochratoxin A (OTA) and macrocyclic trichothecenes (MT) using Enzyme Linked Immunosorbent Assays (ELISA). Urine specimens from 104 of 112 patients (93%) were positive for at least one mycotoxin (one in the equivocal range). Almost 30% of the cases had more than one mycotoxin present. OTA was the most prevalent mycotoxin detected (83%) with MT as the next most common (44%). Exposure histories indicated current and/or past exposure to WDB in over 90% of cases. Environmental testing was performed in the WDB from a subset of these patients. This testing revealed the presence of potentially mycotoxin producing mold species and mycotoxins in the environment of the WDB. Prior testing in a healthy control population with no history of exposure to a WDB or moldy environment (n = 55) by the same laboratory, utilizing the same methods, revealed no positive cases at the limits of detection.

Concepts: Aflatoxin, Mycotoxin, Aspergillus, Chronic fatigue syndrome, Ochratoxin, Mycotoxins, Stachybotrys, Mold

152

Ruminant diets include cereals, protein feeds, their by-products as well as hay and grass, grass/legume, whole-crop maize, small grain or sorghum silages. Furthermore, ruminants are annually or seasonally fed with grazed forage in many parts of the World. All these forages could be contaminated by several exometabolites of mycotoxigenic fungi that increase and diversify the risk of mycotoxin exposure in ruminants compared to swine and poultry that have less varied diets. Evidence suggests the greatest exposure for ruminants to some regulated mycotoxins (aflatoxins, trichothecenes, ochratoxin A, fumonisins and zearalenone) and to many other secondary metabolites produced by different species of Alternaria spp. (e.g., AAL toxins, alternariols, tenuazonic acid or 4Z-infectopyrone), Aspergillus flavus (e.g., kojic acid, cyclopiazonic acid or β-nitropropionic acid), Aspergillus fuminatus (e.g., gliotoxin, agroclavine, festuclavines or fumagillin), Penicillium roqueforti and P. paneum (e.g., mycophenolic acid, roquefortines, PR toxin or marcfortines) or Monascus ruber (citrinin and monacolins) could be mainly related to forage contamination. This review includes the knowledge of mycotoxin occurrence reported in the last 15 years, with special emphasis on mycotoxins detected in forages, and animal toxicological issues due to their ingestion. Strategies for preventing the problem of mycotoxin feed contamination under farm conditions are discussed.

Concepts: Fungus, Aspergillus flavus, Aflatoxin, Mycotoxin, Aspergillus, Penicillium, Ochratoxin, Mycotoxins

28

Mycotoxins are toxic and carcinogenic metabolites produced by fungi that colonize food crops. The most agriculturally important mycotoxins known today are aflatoxins, which cause liver cancer and have also been implicated in child growth impairment and acute toxicoses; fumonisins, which have been associated with esophageal cancer (EC) and neural tube defects (NTDs); deoxynivalenol (DON) and other trichothecenes, which are immunotoxic and cause gastroenteritis; and ochratoxin A (OTA), which has been associated with renal diseases. This review describes the adverse human health impacts associated with these major groups of mycotoxins. First, we provide background on the fungi that produce these different mycotoxins and on the food crops commonly infected. Then, we describe each group of mycotoxins in greater detail, as well as the adverse effects associated with each mycotoxin and the populations worldwide at risk. We conclude with a brief discussion on estimations of global burden of disease caused by dietary mycotoxin exposure. Expected final online publication date for the Annual Review of Food Science and Technology Volume 5 is February 28, 2014. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.

Concepts: Epidemiology, Cancer, Nutrition, Fungus, Aflatoxin, Mycotoxin, Ochratoxin, Mycotoxins

28

It has previously been shown that the biosynthesis of the mycotoxins ochratoxin A and B and of citrinin by Penicillium is regulated by light. However, not only the biosynthesis of these mycotoxins, but also the molecules themselves are strongly affected by light of certain wavelengths. The white light and blue light of 470 and 455 nm are especially able to degrade ochratoxin A, ochratoxin B and citrinin after exposure for a certain time. After the same treatment of the secondary metabolites with red (627 nm), yellow (590 nm) or green (530 nm) light or in the dark, almost no degradation occurred during that time indicating the blue light as the responsible part of the spectrum. The two derivatives of ochratoxin (A and B) are degraded to certain definitive degradation products which were characterized by HPLC-FLD-FTMS. The degradation products of ochratoxin A and B did no longer contain phenylalanine however were still chlorinated in the case of ochratoxin A. Citrinin is completely degraded by blue light. A fluorescent band was no longer visible after detection by TLC suggesting a higher sensitivity and apparently greater absorbance of energy by citrinin. The fact that especially blue light degrades the three secondary metabolites is apparently attributed to the absorption spectra of the metabolites which all have an optimum in the short wave length range. The absorption range of citrinin is, in particular, broader and includes the wave length of blue light. In wheat, which was contaminated with an ochratoxin A producing culture of Penicillium verrucosum and treated with blue light after a pre-incubation by the fungus, the concentration of the preformed ochratoxin A reduced by roughly 50% compared to the control and differed by > 90% compared to the sample incubated further in the dark. This indicates that the light degrading effect is also exerted in vivo, e.g., on food surfaces. The biological consequences of the light instability of the toxins are discussed.

Concepts: Light, Electromagnetic radiation, Mycotoxin, Wavelength, Penicillium, Visible spectrum, Ochratoxin, Mycotoxins

28

Ochratoxin A (OTA) is a mycotoxin found in a wide range of food and feedstuffs. Intake of OTA-contaminated food causes health concern due to the harmful effects reported on humans and animals. Much effort is currently devoted to set up and optimise highly sensitive and accurate methods of OTA analysis. This work describes the comparison of fluorescence-based immunosensing strategies for the analysis of OTA. First, an indirect competitive fluoroimmunoassay was designed and optimised. The assay enabled the quantification of the toxin at the levels set by the European legislation. Then, a flow-immunoassay based on kinetic exclusion measurements was developed. It showed the theoretical lowest limit of detection enabled by the affinity of the anti-OTA antibody (IC(80)=12ngL(-1) in the assay solution). Wine and cereal samples were analysed using the optimised flow system. No significant matrix effects were observed after simple pre-treatment of wine and OTA extraction from corn-flakes samples. This simple and highly sensitive automated biosensing-system allows OTA quantification in food and beverages. It is envisaged as a powerful tool for rapid and reliable toxin screening.

Concepts: Scientific method, Energy, Measurement, Mycotoxin, Cereal, Search engine optimization, Ochratoxin, Ochratoxin A

28

A new sample preparation procedure, termed pH-controlled dispersive liquid-liquid microextraction (pH-DLLME), has been developed for the analysis of ionisable compounds in highly complex matrices. This DLLME mode, intended to improve the selectivity and to expand the application range of DLLME, is based on two successive DLLMEs conducted at opposite pH values. pH-DLLME was applied to determination of ochratoxin A (OTA) in cereals. The hydrophobic matrix interferences in the raw methanol extract (disperser, 1mL) were removed by a first DLLME (I DLLME) performed at pH 8 to reduce the solubility of OTA in the extractant (CCl(4), 400μL). The pH of the aqueous phase was then adjusted to 2, and the analyte was extracted and concentrated by a second DLLME (extractant, 150μL C(2)H(4)Br(2)). The main factors influencing the efficiency of pH-DLLME including type and volume of I DLLME extractant, as well as the parameters affecting the OTA extraction by II DLLME, were studied in detail. Under optimum conditions, the method has detection and quantification limits of 0.019 and 0.062μgkg(-1), respectively, with OTA recoveries in the range of 81.2-90.1% (n=3). The accuracy of the analytical procedure, evaluated with a reference material (cereal naturally contaminated with OTA), is acceptable (accuracy of 85.6%±1.7, n=5). The applicability of pH-DLLME to the selective extraction of other ionisable compounds, such as acidic and basic pharmaceutical products was also demonstrated. The additional advantages of pH-DLLME are a higher selectivity and the extension of this microextraction technique to highly complex matrices.

Concepts: Acid, PH, Cereal, Extraction, PH indicator, Matrix, Ochratoxin, Ochratoxin A

27

Bisphenol A has been widely used in plastic containers and this has raised safety concerns for fetuses, infants, and young children. Aflatoxin B1, ochratoxin A, and patulin are among the most toxic regulated mycotoxins found as contaminants in agricultural crops and animal products. To facilitate the analysis of these chemicals for regulatory purposes, we have developed an analytical method enabling their simultaneous detection in beverages and food products.

Concepts: Fungus, Aflatoxin, Mycotoxin, Bisphenol A, Harvest, Aspergillus, Ochratoxin, Mycotoxins

26

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.

Concepts: Scientific method, Mycotoxin, Wheat, Cereal, Maize, Aspergillus, Ochratoxin, Mycotoxins

26

A sensitive, simple and rapid method for the simultaneous determination of 19 mycotoxins in biscuits (a dry matrix containing cereals and egg) has been developed using high performance liquid chromatography coupled to tandem mass spectrometry with electrospray source working in both positive and negative mode. Due to the matrix complexity and the high amount of contaminants, a solid phase extraction method using graphitized carbon black was optimized for an effective clean-up step. Accuracy was carried out in the selected matrix using blank samples spiked at three analyte concentrations. Recoveries between 63 and 107% and relative standard deviations lower than 12% were obtained. For all considered mycotoxin classes, i.e. thricotecenes A and B, zearalenone and its metabolites, fumonisins, ochratoxin A, enniatins and their structurally related beauvericin, the method was validated in terms of linearity, recovery, matrix effect, precision, limit of detection and limit of quantification. Matrix-matched calibration was used for quantification purposes, in order to compensate for matrix effect. The coefficients of determination obtained were in the range of 0.9927-1. The limits of quantification, ranging from 0.04μgkg(-1) for enniatin B1 to 80.2μgkg(-1) for nivalenol, were always lower than maximum permitted levels for every regulated mycotoxin by the current European legislation.

Concepts: Mass spectrometry, Mycotoxin, High performance liquid chromatography, Analytical chemistry, Liquid chromatography-mass spectrometry, Tandem mass spectrometry, Ochratoxin, Mycotoxins

26

In this paper we describe a rapid, simple, and cost-effective liquid chromatography-tandem mass spectrometric (LC-MS-MS) method for simultaneous analysis of aflatoxin B1, B2, G1, and G2, ochratoxin A, and sterigmatocystin in 25 traditional Chinese medicines (TCMs). The method is based on single extraction with 84:16 (v/v) acetonitrile-water then analysis of the diluted crude extract without further clean-up. Chromatographic separation was achieved on a C18 column, with a mobile phase gradient prepared from aqueous 4 mmol L(-1) ammonium acetate-0.1 % formic acid and methanol. Quantification of the analytes was by selective reaction monitoring (SRM) on a triple-quadrupole mass spectrometer in positive-ionization mode. Special focus was on investigating and reducing matrix effects to improve accuracy. The established method was validated by determination of linearity (r > 0.995), sensitivity (limits of quantification 1.6-25.0 ng L(-1)), apparent recovery (84.8-110.6 %), extraction recovery (83.6-106.1 %), and precision (relative standard deviation ≤9.9 %) for two representative TCMs, Semen Armeniacae Amarae and Radix Pseudostellariae. The applicability of the method to TCMs other than these was further investigated, and 23 other TCMs with acceptable matrix effects (80.2-118.6 %) were screened. The validated method was finally used to assess mycotoxin contamination of 244 samples of 25 TCMs collected from local hospitals and TCM pharmacies. Aflatoxin B1 and ochratoxin A were detected in 5.3 % of the samples. Sterigmatocystin, the most prevalent mycotoxin contaminant, was present in 26.2 % of the samples tested; this has not been reported previously. The results of this work imply greater attention should be devoted to evaluation of the potential hazard caused by sterigmatocystin in TCMs.

Concepts: Aflatoxin, Mycotoxin, Chromatography, Aspergillus, Traditional Chinese characters, Methanol, Ochratoxin, Mycotoxins