Abstract Background Alternaria toxins are ubiquitous contaminants in highly consumed food products. Therefore, they are candidates to be regulated by EU legislation. In this context, the availability of reliable analytical methods is a keystone both for protecting the health of citizens and smooth functioning of the European market. Objective This paper describes an advanced LC-MS/MS method based on isotope dilution quantification suitable for the determination of altenuene, alternariol, alternariol monomethyl ether, tenuazonic acid, and tentoxin in tomato puree, wheat, and sunflower seeds. Methods The method has been validated in an interlaboratory study that included the analysis of both spiked and naturally contaminated food commodities. Twenty-three participants contributed with analytical data. Results The average recoveries and relative standard deviations for repeatability and reproducibility obtained across the tested matrixes were: 97, 8.0, and 23%, for altenuene, respectively; 95, 9.2, and 17% for alternariol, respectively; 98, 6.4, and 13% for alternariol monomethyl ether, respectively; 97, 4.2, and 9.3% for tenuazonic acid, respectively; and 102, 5.6, and 15% for tentoxin, respectively. The method enabled the determination of all tested Alternaria toxins close to or below 1 µg/kg. Conclusion Overall, the method showed a satisfactory trueness and precision, complying with the requirements for the monitoring of mycotoxins in food in the EU. It is currently under evaluation by the European Committee for Standardization for adoption as a standard method. Highlights Isotope dilution mass spectrometry method for the determination of Alternaria toxins in food.
An interlaboratory comparison was carried out to evaluate the effectiveness of a method based on HPTLC in which reagent-free derivatization is followed by UV/fluorescence detection. The method was tested for the determination of sucralose (C12H19C13O8; (2R,3R,4R,5S,6R)-2-[(2R,3S,4S,5S)-2,5-bis(chloromethyl)-3,4-dihydroxyoxolan-2-yl]oxy-5-chloro-6-hydroxymethyl)oxane-3, 4-diol; CAS Registry No. 56038-13-2) in carbonated and still beverages at the proposed European regulatory limits. For still beverages, a portion of the sample was diluted with methanol-water. For carbonated beverages, a portion of the sample was degassed in an ultrasonic bath before dilution. Turbid beverages were filtered after dilution through an HPLC syringe filter. The separation of sucralose was performed by direct application on amino-bonded (NH2) silica gel HPTLC plates (no cleanup needed) with the mobile phase acetonitrile-water. Sucralose was determined after reagent-free derivatization at 190 degrees C; it was quantified by measurements of both UV absorption and fluorescence. The samples, both spiked and containing sucralose, were sent to 14 laboratories in five different countries. Test portions of a sample found to contain no sucralose were spiked at levels of 30.5, 100.7, and 299 mg/L. Recoveries ranged from 104.3 to 124.6% and averaged 112% for determination by UV detection; recoveries ranged from 98.4 to 101.3% and averaged 99.9% for determination by fluorescence detection. On the basis of the results for spiked samples (blind duplicates at three levels), as well as sucralose-containing samples (blind duplicates at three levels and one split level), the values for the RSDr ranged from 10.3 to 31.4% for determinations by UV detection and from 8.9 to 15.9% for determinations by fluorescence detection. The values for the RSDR values ranged from 13.5 to 31.4% for determinations by UV detection and from 8.9 to 20.7% for determinations by fluorescence detection.
This review highlights developments in mycotoxin analysis and sampling over a period between mid-2012 and mid-2013. It covers the major mycotoxins: aflatoxins, Alternaria toxins, ergot alkaloids, fumonisins, ochratoxins, patulin, trichothecenes and zearalenone. A wide range of analytical methods for mycotoxin determination in food and feed were developed last year, in particular immunochemical methods and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS)-based methods. After a section on sampling and sample preparation, due to the rapid spread and developments in the field of LC-MS/MS multimycotoxin methods, a separate section has been devoted to this area of research. It is followed by a section on mycotoxins in botanicals and spices, before continuing with the format of previous reviews in this series with dedicated sections on method developments for the individual mycotoxins.
A collaborative study was conducted to evaluate the effectiveness of an immunoaffinity column cleanup liquid chromatography (LC) method for the determination of aflatoxin B1 and total aflatoxins at European regulatory limits. The test portion is extracted with methanol-water (8 + 2) for dried figs and paprika, and with methanol-water (8 + 2) plus hexane (or cyclohexane) for peanut butter and pistachios. The sample extract is filtered, diluted with phosphate buffer saline, and applied to an immunoaffinity column. The column is washed with water and the aflatoxins are eluted with methanol. Aflatoxins are quantitated by reversed-phase LC with post-column derivatization (PCD) involving bromination. PCD is achieved with either an electrochemical cell (Kobra cell) and addition of bromide to the mobile phase or pyridinium hydrobromide perbromide. Determination is by fluorescence. Peanut butter, pistachio paste, dried fig paste, and paprika powder samples, both naturally contaminated with aflatoxins and containing added aflatoxins, were sent to 16 collaborators in 16 European countries. Test portions of samples were spiked at levels of 2.4 and 9.6 ng/g for total aflatoxins which included 1.0 and 4.0 ng/g aflatoxin B1, respectively. Recoveries for total aflatoxins ranged from 71 to 92% with corresponding recoveries for aflatoxin B1 of 82 to 109%. Based on results for spiked samples (blind duplicates at 2 levels) as well as naturally contaminated samples (blind duplicates at 4 levels, including blank), the relative standard deviation for repeatability ranged from 4.6 to 23.3% for total aflatoxins and from 3.1 to 20.0% for aflatoxin B1. The relative standard deviation for reproducibility ranged from 14.1 to 34.2% for total aflatoxins, and from 9.1 to 32.2% for aflatoxin B1. The method showed acceptable within-laboratory and between-laboratory precision for all 4 matrixes, as evidenced by HORRAT values <1, at the low levels of determination for both total aflatoxins and aflatoxin B1.
The global occurrence of mycotoxins is considered to be a major risk factor for human and animal health. Contamination of different agricultural commodities with mycotoxins still occurs despite the most strenuous prevention efforts. As a result, mycotoxin contaminated feed can cause serious disorders and diseases in farm animals. A number of approaches, such as physical and chemical detoxification procedures, have been used to counteract mycotoxins. However, only a few of them have practical application. A recent and promising approach to protect animals against the harmful effects of mycotoxin contaminated feed is the use of substances for reduction of the contamination of feed by mycotoxins. These substances, so-called mycotoxin binders (MB), are added to the diet in order to reduce the absorption of mycotoxins from the gastrointestinal tract and their distribution to blood and target organs, thus preventing or reducing mycotoxicosis in livestock. Recently, the use of such substances as technological feed additives has been officially allowed in the European Union. The efficacy of MB appears to depend on the properties of both the binder and the mycotoxin. Depending on their mode of action, these feed additives may act either by binding mycotoxins to their surface (adsorption), or by degrading or transforming them into less toxic metabolites (biotransformation). Biotransformation can be achieved by mycotoxin-degrading enzymes or by microorganisms producing such enzymes. Various inorganic adsorbents, such as hydrated sodium calcium aluminosilicate, zeolites, bentonites, clays, and activated carbons, have been tested and used as MB. An interesting alternative to inorganic adsorbents for the detoxification of mycotoxins is the use of organic binders, such as yeast cell wall components, synthetic polymers (cholestyramine, polyvinylpyrrolidone), humic substances and dietary fibres. This paper gives an overview of the current knowledge and situation in the field of MB. The most important types of MB, mechanism of their action, and their application as a part of general strategy to counteract mycotoxins are described in this review. Recent advances in the use and study of MB, as well as data of their in vitro and in vivo effectiveness are given. Problems, potential, current trends and perspectives associated with the use of MB are discussed as well in the review.
The type A trichothecenes T-2 toxin (T-2) and HT-2 toxin (HT-2) are hazardous Fusarium products that contaminate many field crops growing in cold to temperate regions across the world. Toxicity studies in laboratory and farm animals have been used to derive a temporary tolerable daily intake (t-TDI) for the sum of T-2 and HT-2 of no more than 60 ng/kg body weight. To protect the consumers, it is now necessary to screen a large number of food samples for the presence of these poisonous fungal metabolites. Towards that goal, we discovered that the transcriptional apparatus of a human carcinoma cell line (MCF7) provides a sensitive biological sensor of type A trichothecenes. In fact, exposure of this easy-to-culture cell line to T-2 or HT-2 results in the regulation of >2,000 different transcripts with expression changes ranging from >5,000-fold gene inductions to >40-fold gene repressions. These transcriptional responses have been exploited to develop practical microchip and reverse transcription-polymerase chain reaction (RT-PCR) assays for the detection of type A trichothecenes at parts per billion levels.
A proficiency test was conducted by the Community Reference Laboratory for Mycotoxins with 33 European National Reference Laboratories (NRLs) for Mycotoxins and 2 Laboratory from candidate countries, thus a total of 35 participants. Test materials were a deoxynivalenol (DON) solution in acetonitrile and three cereal test materials. Laboratories determined the DON content by either enzyme linked immuno sorbent assay (ELISA), gas chromatography (GC) or reverse-phase high-performance liquid-chromatography (RP-HPLC). One NRL did not report any results. Applying the Horwitz equation as a basis for the target standard deviation (19% in the case of this proficiency test), 27 out of the remaining 34 laboratories reported values within the z-score limit of 2 after recovery correction of the result for the DON-positive sample. Twenty-five laboratories reported results within a z-score limit of 1. Thus, 79 % of the participating laboratories performed satisfactorily in the proficiency test. No z-scores were calculated for the blank material. How to obtain EU publications Our priced publications are available from EU Bookshop (http://bookshop.europa.eu), where you can place an order with the sales agent of your choice. The Publications Office has a worldwide network of sales agents. You can obtain their contact details by sending a fax to (352) 29 29-42758. The mission of the JRC is to provide customer-driven scientific and technical support for the conception, development, implementation and monitoring of EU policies. As a service of the European Commission, the JRC functions as a reference centre of science and technology for the Union. Close to the policy-making process, it serves the common interest of the Member States, while being independent of special interests, whether private or national. LA -N A -2387-EN -C
Alternaria toxins and citrinin are mycotoxins produced by fungi growing on different raw materials and agricultural commodities. Maximum levels of these toxins in foods are currently under consideration by the European Commission as a risk management measure. In this study, a new quantitative method is described for the determination of five Alternaria toxins and citrinin in tomato and tomato juice samples based on LC-MS/MS detection. Samples were extracted with pure methanol, followed by a derivatisation step with 2,4-dinitrophenylhydrazine to improve the determination of tenuazonic acid and to decrease the wide polarity difference between the compounds of interest. Samples were purified on hydrophilic-modified styrene polymer solid-phase extraction cartridges. High-performance liquid chromatographic columns packed with different core-shell materials were tested for the separation of toxins and a C-18 phase was in the final method applied to achieve sufficient separation of all relevant analytes. A key element of this approach was to prove successful transferability of the method to three different triple quadrupole mass spectrometers. A full single laboratory method validation was performed on two LC-MS/MS systems and performance characteristics met the predefined requirements. Moreover, the method was used in an international proficiency test and the satisfactory z-scores obtained (-0.1 to 0.8 in tomato juice samples) demonstrated the reliability of the approach described. The method will be validated in an inter-laboratory collaborative study and if the criteria for method precision are met, the method will be proposed as a new Work Item to the European Committee for Standardisation.
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