Analysis of Mycotoxins Contamination in Poultry Feeds Manufactured in Selected Provinces of South Africa Using UHPLC-MS/MS.

A total of 105 different types of poultry feed samples from South Africa were simultaneously analysed for the presence of 16 mycotoxins using ultra-high-performance liquid chromatography coupled to a triple quadrupole mass spectrometer (UHPLC-MS/MS). The data revealed the presence of 16 mycotoxins in the various poultry feed samples. Fumonisin B1 (FB1) was the most dominant recovered from 100% of samples analysed at concentrations ranging between 38.7 and 7125.3 µg/kg. This was followed by zearalenone (ZEN) (range: 0.1-429 µg/kg) and deoxynivalenol (DON) (range: 2.5-154 µg/kg). Samples were also found to be contaminated with fumonisin B2 (FB2) (range: 0.7-125.1 µg/kg), fumonisin B3 (FB3) (range: 0.1-125.1 µg/kg), α-zearalenol (α-ZEL) (range: 0.6-20 µg/kg ), ß-zearalenol (ß-ZEL) (range: 0.2-22.1 µg/kg), 3-acetyldeoxynivalenol (3-ADON) (range: 0.1-12.9 µg/kg) and 15-acetyldeoxynivalenol (15-ADON) (range: 1.7-41.9 µg/kg). Alternaria mycotoxin, i.e., Alternariol monomethyl ether (AME) was recovered in 100% of samples at concentrations that ranged from 0.3-155.5 µg/kg. Aflatoxins (AFs) had an incidence rate of 92% with generally low concentration levels ranging from 0.1-3.7 µg/kg. Apart from these metabolites, 2 type A trichothecenes (THs), i.e., HT-2 toxin (HT-2) (range: 0.2-5.9 µg/kg) and T-2 toxin (T-2) (range: 0.1-15.3 µg/kg) were also detected. Mycotoxin contamination in South African poultry feed constitutes a concern as correspondingly high contamination levels, such as those observed herein are likely to affect birds, which can be accompanied by severe health implications, thus compromising animal productivity in the country. Such exposures, primarily to more than one mycotoxin concurrently, may elicit noticeable synergistic and or additive effects on poultry birds.

Development and Characterization of a Multimycotoxin Reference Material.

Background: Matrix-matched reference materials (RMs) are critical for adequate quality assurance of extraction, digestion, separation, and/or detection processes for analytes of interest in foods and dietary supplements. The accurate determination of mycotoxins in foods is an international concern. While RMs for mycotoxins are available from a variety of RM producers, these mainly address a single mycotoxin or group of mycotoxins and therefore require the use of multiple RMs for multitarget methods. Objective: To address the increasing needs of laboratories moving toward LC-MS-based multimycotoxin analysis, the U.S. National Institute of Standards and Technology (NIST) collaborated with the U.S. Food and Drug Administration (FDA) to produce a naturally incurred RM for multiple mycotoxins in corn. Methods: Homogeneity of the RM has been assessed using a stratified random sampling of the final product based on mycotoxin mass fractions measured by the FDA and NIST. Multiple sample sizes were evaluated to maximize homogeneity in the obtained results. The mycotoxin levels in the final materials have been evaluated via interlaboratory comparison and isotope dilution LC-tandem MS measurements made at the FDA and NIST. The final value assignment combined results from these data sets. Conclusions: The study successfully developed a certified RM, SRM 1565 Mycotoxins in Corn, and a workflow for the future development of multimycotoxin RMs in different matrices.

Certified Reference Materials and Metrological Traceability for Mycotoxin Analysis.

In the past decade, with the signing of the joint declaration by the International Bureau of Weights and Measures (BIPM), the International Organisation of Legal Metrology, the International Laboratory Accreditation Cooperation, and the International Organization for Standardization, and the issuing of new international standards such as ISO/IEC 17025 and ISO 13528, metrological traceability has become more and more important for the mutual recognition of measurement results. It is found that the important factors for ensuring the accuracy of results are not only relevant to calibrants but also to the analysis procedure. This paper focuses on the analysis of key factors that affect measurement results, such as analytical methods, reference materials (RMs), and proficiency testing of mycotoxins. The Capacity Building and Knowledge Transfer Programme for mycotoxins, led by the BIPM, which aims to establish a global measurement system traced to the Système International d'Unités (SI), is well illustrated. Furthermore, this paper suggests to stakeholders that great effort is needed to promote the effective use of certified RM resources that are traced to the SI and globally recognized by the International Committee for Weights and Measures to finally realize "one measurement, global recognition."

Identifying Challenges and Risks Associated with the Analysis of Major Mycotoxins in Feed and Botanicals.

Changing weather conditions have heightened the risk of growth of mycotoxigenic molds on crops and various agricultural commodities. Mycotoxins, which are linked to carcinogenic and nephrotoxic effects in animals and humans, have been traditionally analyzed by immunoassays, gas, and LC techniques with spectrophotometric detectors. This review discusses the current techniques and challenges in commercial settings associated with the analysis of mycotoxins in unique matrices such as animal feeds, herbal products, and dietary supplements containing botanicals. Because of the advantages and growing acceptance of LC-tandem MS (MS/MS) over traditional approaches, discussion is mainly based on LC-MS/MS-based approaches. Considering the impact of sample preparation on accuracy of quantitative results, discussion about pros and cons of recently introduced sample preparation techniques is integrated with analytical methods. A section of the review explains the importance and availability of reference materials for mycotoxins. The present discussion provides good insight into the current challenges and developments during mycotoxin analysis of feed and botanicals and addresses the need for researchers in terms of an official MS-based method.

Assessing the mycotoxicological risk from consumption of complementary foods by infants and young children in Nigeria.

This study assessed, for the first time, the mycotoxicological risks from consumption of complementary foods by infants and young children in Nigeria. Molds belonging to Aspergillus aculeatinus, A. flavus, A. luchuensis, A. tubingensis, A. welwitschiae and Geotrichum candidum were recovered from the complementary foods. Twenty-eight major mycotoxins and derivatives, and another 109 microbial metabolites including chloramphenicol (a bacterial metabolite), were quantified in 137 food samples by LC-MS/MS. Aflatoxins and fumonisins co-contaminated 42% of the cereal- and nut-based food samples, at mean concentrations exceeding the EU limits of 0.1 and 200 µg/kg set for processed baby foods by 300 and six times, respectively. Milk contained mainly beauvericin, chloramphenicol and zearalenone. The trichothecenes, T-2 and HT-2 toxins, were quantified only in infant formula and at levels three times above the EU indicative level of 15 µg/kg for baby food. Chronic exposure estimate to carcinogenic aflatoxin was high causing low margin of exposure (MOE). Exposures to other mycotoxins either exceeded the established reference values by several fold or revealed low MOEs, pointing to important health risks in this highly vulnerable population. The observed mycotoxin mixtures may further increase risks of adverse health outcomes of exposure; this warrants urgent advocacy and regulatory interventions.

Development of a high performance liquid chromatography tandem mass spectrometry based analysis for the simultaneous quantification of various Alternaria toxins in wine, vegetable juices and fruit juices.

An analytical method based on high performance liquid chromatography (HPLC) and tandem mass spectrometry (MS/MS) detection for the simultaneous quantification of 12 Alternaria toxins in wine, vegetable juices and fruit juices was developed. Excellent chromatographic performance was demonstrated for tenuazonic acid (TeA) in a multi-analyte method. This comprehensive study is also the first to report the determination of TeA, alternariol (AOH), alternariol monomethyl ether (AME), tentoxin (TEN) and altenuene (ALT), altertoxin I (ATX-I), altertoxin II (ATX-II), altenuisol (ATL), iso-altenuene (isoALT), altenuic acid III (AA-III) and the AAL toxins TB1 und TB2 in samples from the German market. Several types of HPLC columns were tested for the liquid chromatographic separation of the toxins of interest that widely differ in their polarities. The focus was on gaining suitable retention while avoiding derivatization steps especially for TeA and AA-III. Three atmospheric pressure ionization techniques used with liquid chromatography (electrospray, chemical and photo ionization) were tested to obtain the best selectivity and sensitivity. Samples were diluted with sodium hydrogen carbonate buffer and extracted on a diatomaceous earth solid phase extraction cartridge. Method validation was carried out by using tomato juice, citrus juice and white wine as blank matrices. Limits of detection ranged from 0.10 to 0.59µgL(-1) and limits of quantification ranged from 0.4-3.1µgL(-1) depending on the toxin and matrix. Recoveries were around 100±9% for all toxins except stemphyltoxin III (STTX-III) and altenusin (ALS) due to instability during sample clean up. Matrix-induced effects leading to ion suppression especially for ATX-I, ATX-II and AA-III were investigated. Relative standard deviations of repeatability (RSDr) and intermediate reproducibility (RSDR) were ≤9.3 and ≤17.1, respectively, for the toxins in different matrices at levels of 5 and 30µgL(-1). Finally, 103 commercially obtained wine and juice samples from the German market in 2015 were analysed. TeA was found most frequently (68% of all analysed samples) in concentrations of up to 60.0µgL(-1). AOH, AME and TEN were detected in fewer samples (37%, 16% and 30%) at lower concentrations of up to 8.2, 1.5 and 10.3µgL(-1), respectively. AA-III and ATL were detected for the first time in 3% and 17% of food all samples, in concentrations of up to 6.0µgL(-1) and 5.9µgL(-1), respectively.

Fast and sensitive detection of mycotoxins in wheat using microfluidics based Real-time Electrochemical Profiling.

The objective of the study has been the development of a new sensing platform, called Real-time Electrochemical Profiling (REP) that relies on real-time electrochemical immunoassay detection. The proposed REP platform consists of new electrode arrays that are easy to fabricate, has a small imprint allowing microfluidic system integration, enables multiplexed amperometric measurements and performs well in terms of electrochemical immunoassay detection as shown through the deoxynivalenol detection assays. The deoxynivalenol detection has been conducted according to an optimised REP assay protocol using deoxynivalenol standards at varying concentrations and a standard curve was obtained (y=-20.33ln(x)+124.06; R(2)=0.97) with a limit of detection of 6.25 ng/ml. As both ELISA and REP detection methods use horse radish peroxidase as the label and 3.3',5.5'-Tetramethylbenzidine as the substrate, the performance of the REP platform as an ELISA reader has also been investigated and a perfect correlation between the deoxynivalenol concentration and the current response was obtained (y=-14.56ln(x)+101.02; R(2)=0.99). The calibration curves of both assays have been compared to conventional ELISA tests for confirmation. After assay optimisation using toxin spiked buffer, the deoxynivalenol detection assay has also been performed to detect toxins in wheat grain.

Development and certification of a reference material for Fusarium mycotoxins in wheat flour.

Deoxynivalenol (DON), nivalenol (NIV) and zearalenone (ZEN) are toxic secondary metabolites produced by several species of Fusarium fungi. These mycotoxins are often found together in a large variety of cereal-based foods, which are regulated by maximum content levels of DON and ZEN. To date, suitable certified reference materials (CRM) intended for quality control purposes are lacking for these Fusarium mycotoxins. In order to overcome this lack, the first CRM for the determination of DON, NIV and ZEN in naturally contaminated wheat flour (ERM®-BC600) was developed in the framework of a European Reference Materials (ERM®) project. This article describes and discusses the whole process of ERM®-BC600 development, including material preparation, homogeneity and stability studies, and an interlaboratory comparison study for certification. A total of 21 selected expert laboratories from different European countries with documented expertise in the field of mycotoxin analysis took part in the certification study using various gas and liquid chromatographic methods. The certified values and their corresponding expanded uncertainties (k = 2) were assigned in full compliance with the requirements of ISO Guide 35 and are as follows: 102 ± 11 µg kg(-1) for DON, 1000 ± 130 µg kg(-1) for NIV and 90 ± 8 µg kg(-1) for ZEN.

Co-occurrence of aflatoxins B1, B2, G1 and G2, ochratoxin A, zearalenone, deoxynivalenol, and citreoviridin in rice in Brazil.

A total of 230 samples of processed rice and its sub-products or derived products were analysed to establish the co-occurrence of several mycotoxins. Samples were analysed in the period 2007-2009 due to the outbreak of beriberi associated with the consumption of rice stored in inappropriate conditions in Brazil. According to data from the Ministry of Health, 323 cases of disease were registered in 2006, of which at least 47 cases resulted in death. The occurrence of total aflatoxin (AFT) (aflatoxin B(1) + B(2) + G(1) + G(2)), ochratoxin A (OTA), zearalenone (ZON), deoxynivalenol (DON), and citreoviridin (CTV) was 58.7%, 40.0%, 45.2%, 8.3% and 22.5%, respectively. From 166 rice samples analysed, 55% had levels <0.11 µg kg(-1) for AFT. For OTA and ZON, of 165 rice samples analysed, 28% and 29% were contaminated with levels from 0.20 to 0.24 µg kg(-1) and from 3.6 to 290.0 µg kg(-1), respectively. One sample (0.6%) was contaminated with 4872.0 µg kg(-1) of ZON. A total of 91% of rice samples (n = 165) did not contain detectable DON (<30.00 µg kg(-1)), although the highest level of contamination was found to be 244 µg kg(-1). From the total of 65 samples analysed, 94% had no detectable CTV (<0.9 µg kg(-1)), with a range from 0.9 to 31.1 µg kg(-1) in 6% of the samples. The highest levels of contamination were found in rice sub-products or derived products from the husk and rice bran. Co-occurrence was observed for AFT and ZON in 17.0%, AFT and OTA in 24.2%, AFT and CTV in 6.2%, OTA and CTV in 4.6%, and ZON and CTV in 3.1%. These fractions were also the major contributors for the co-occurrence. The results found show the necessity of monitoring rice production.

Mass spectrometry-based strategy for direct detection and quantification of some mycotoxins produced by Stachybotrys and Aspergillus spp. in indoor environments.

Dampness in buildings has been linked to adverse health effects, but the specific causative agents are unknown. Mycotoxins are secondary metabolites produced by molds and toxic to higher vertebrates. In this study, mass spectrometry was used to demonstrate the presence of mycotoxins predominantly produced by Aspergillus spp. and Stachybotrys spp. in buildings with either ongoing dampness or a history of water damage. Verrucarol and trichodermol, hydrolysis products of macrocyclic trichothecenes (including satratoxins), and trichodermin, predominately produced by Stachybotrys chartarum, were analyzed by gas chromatography-tandem mass spectrometry, whereas sterigmatocystin (mainly produced by Aspergillus versicolor), satratoxin G, and satratoxin H were analyzed by high-performance liquid chromatography-tandem mass spectrometry. These mycotoxin analytes were demonstrated in 45 of 62 building material samples studied, in three of eight settled dust samples, and in five of eight cultures of airborne dust samples. This is the first report on the use of tandem mass spectrometry for demonstrating mycotoxins in dust settled on surfaces above floor level in damp buildings. The direct detection of the highly toxic sterigmatocystin and macrocyclic trichothecene mycotoxins in indoor environments is important due to their potential health impacts.

Quantification of estrogenic mycotoxins at the ng/L level in aqueous environmental samples using deuterated internal standards.

Because of their pronounced estrogenicity, resorcyclic acid lactones (RALs) are of concern in aqueous environments even at the low ng/L level. Therefore, we developed an accurate, precise and sensitive HPLC-MS/MS method to detect these mycotoxins in different aqueous environmental samples. The compounds investigated included zearalenone (ZON), alpha- and beta-zearalenol, zearalanone as well as alpha- and beta-zearalanol. The use of isotope labelled internal standards (in this case deuterated RAL-analogues) ensured an accurate quantification of the target analytes, independent of matrix compounds interfering with the analytes during ionisation and analyte losses occurring during sample preparation. Sample enrichment was carried out by solid-phase extraction (SPE) using Supelclean Envi-18 cartridges. Absolute method recoveries for all analytes ranged from 95 to 108%, 70 to 102%, and 76 to 109%, method detection limits from 0.5 to 2.1 ng/L, 0.4 to 1.1 ng/L, and 0.8 to 12.4 ng/L and precision from 3 to 14%, 2 to 13% and 4 to 16% in drainage water, river water and wastewater treatment plant (WWTP) effluent, respectively. The method was applied to verify the emission of RALs from a Fusarium graminearum infested crop field into the drainage system. Zearalenone was present in drainage water in concentrations up to 30 ng/L. So far, none of the other five investigated compounds have been detected.

Methods and method evaluation for mycotoxins.

Mycotoxins are metabolites of molds frequently found on and in agricultural commodities, food and feeds. Owing to their demonstrated acute, sub-acute and, in some cases, chronic toxicity, an effort has been made, worldwide, to control human and animal exposure to these toxic chemicals. This effort depends upon the availability of validated analytical methods for their detection and quantitation. This paper outlines the methodology available, and the procedures used to validate, i.e. evaluate, these methods based on the use of interlaboratory collaborative studies and the application of the HORRAT.

Safety issues associated with processing soybeans in an enclosed habitat intended for long-duration space missions.

Soybeans have been selected to be grown in a habitat (BIO-Plex, Bioregenerative Planetary Life Support Systems Test Complex) designed to evaluate advanced life support systems for long-duration space missions. Soymilk and soy bread will be incorporated into this nutritious, plant-based food system. Because all consumables will be recycled and reused, food safety is a particular concern. Critical control points were identified to control microbiological hazards, particularly mycotoxins, and chemical hazards from antinutrients and volatiles emitted during processing of soymilk and soy bread. Volatile compounds, evolved during the manufacturing of soymilk and soy bread, were quantified by GC/MS to assess their impact on this closed loop system. All concentrations of volatiles evolved during soymilk production were below the 24-h Space Craft Maximum Allowable Concentration (SMAC), while acetaldehyde surpassed the SMAC criteria for soy bread. Recommendations were made for processing of soybeans in such environments to minimize risk to crew member health.

A new monoclonal antibody detecting ochratoxin A at the picogram level.

A monoclonal antibody against ochratoxin A was produced after immunization of Balb/c mice with ochratoxin A-BSA. This antibody was of the IgG1 heavy chain subclass with a kappa type light chain. The 50% inhibition value was 0.45 ng ml-1 in a direct competitive ELISA and the detection limit was 42 pg ml-1. This antibody is very specific, cross-reacting only with ochratoxin B (9.3%).