Evaluation of an alternative spectroscopic approach for aflatoxin analysis: Comparative analysis of food and feed samples with UPLC-MS/MS

Increasing research has highlighted the effects of changing climates on the occurrence and prevalence of toxigenic Aspergillus species producing aflatoxins. There is concern of the toxicological effects to human health and animal productivity following acute and chronic exposure that may affect the future ability to provide safe and sufficient food globally. Considerable research has focused on the detection of these toxins, based on the physicochemical and biochemical properties of the aflatoxin compounds, in agricultural products for human and animal consumption. As improvements in food security continue more regulations for acceptable levels of aflatoxins have arisen globally; the most stringent in Europe. These regulations are important for developing countries as aflatoxin occurrence is high significantly effecting international trade and the economy. In developed countries analytical approaches have become highly sophisticated, capable of attaining results with high precision and accuracy, suitable for regulatory laboratories. Regrettably, many countries that are affected by aflatoxin contamination do not have resources for high tech HPLC and MS instrumentation and require more affordable, yet robust equally accurate alternatives that may be used by producers, processors and traders in emerging economies. It is especially important that those companies wishing to exploit the opportunities offered by lucrative but highly regulated markets in the developed world, have access to analytical methods that will ensure that their exports meet their customers quality and safety requirements. This work evaluates the ToxiMet system as an alternative approach to UPLC–MS/MS for the detection and determination of aflatoxins relative to current European regulatory standards. Four commodities: rice grain, maize cracked and flour, peanut paste and dried distillers grains were analysed for natural aflatoxin contamination. For B1 and total aflatoxins determination the qualitative correlation, above or below the regulatory limit, was good for all commodities with the exception of the dried distillers grain samples for B1 for which no calibration existed. For B1 the quantitative R2 correlations were 0.92, 0.92, 0.88 (<250 μg/kg) and 0.7 for rice, maize, peanuts and dried distillers grain samples respectively whereas for total aflatoxins the quantitative correlation was 0.92, 0.94, 0.88 and 0.91. The ToxiMet system could be used as an alternative for aflatoxin analysis for current legislation but some consideration should be given to aflatoxin M1 regulatory levels for these commodities considering the high levels detected in this study especially for maize and peanuts. (Résumé d'auteur

Development and validation of a rapid multiplex ELISA for pyrrolizidine alkaloids and their N-oxides in honey and feed

Pyrrolizidine alkaloids (PAs) are a group of plant secondary metabolites with carcinogenic and hepatotoxic properties. When PA-producing plants contaminate crops, toxins can be transferred through the food chain and cause illness in humans and animals, most notably hepatic venoocclusive disease. Honey has been identified as a direct risk of human exposure. The European Food Safety Authority has recently identified four groups of PAs that are of particular importance for food and feed: senecionine-type, lycopsaminetype, heliotrine-type and monocrotaline-type. Liquid or gas chromatography methods are currently used to detect PAs but there are no rapid screening assays available commercially. Therefore, the aim of this study was to develop a rapid multiplex ELISA test for the representatives of three groups of alkaloids (senecionine, lycopsamine and heliotrine types) that would be used as a risk-management tool for the screening of these toxic compounds in food and feed. The method was validated for honey and feed matrices and was demonstrated to have a detection capability less than 25 μg/kg for jacobine, lycopsamine, heliotrine and senecionine. The zinc reduction step introduced to the extraction procedure allows for the additional detection of the presence of N-oxides of PAs. This first multiplex immunoassay for PA detection with N-oxide reduction can be used for the simultaneous screening of 21 samples for >12 PA analytes. Honey samples (n =146) from various origins were analysed for PA determination. Six samples were determined to contain measurable PAs >25 μg/kg by ELISAwhich correlated to >10 μg/kg by LC-MS/MS.JRC.D.5-Standards for Food Bioscienc

T-2 Toxin/HT-2 Toxin and Ochratoxin A ELISAs Development and In-House Validation in Food in Accordance with the Commission Regulation (EU) No 519/2014

T-2 toxin/HT-2 toxin (T-2/HT-2) and ochratoxin A (OTA) are mycotoxins that can contaminate a variety of agricultural commodities. To protect consumers’ health, indicative limits for T-2/HT-2 and maximum limits for OTA have been set by the European Commission, requiring food business operators and controlling agencies to conduct routine checks for the presence of these harmful contaminants. Screening methods are increasingly used for monitoring purposes. Due to the demand for new and improved screening tools, two individual detection methods, T-2/HT-2 and OTA enzyme-linked immunosorbent assays (ELISAs), were developed in this study. The T-2/HT-2 ELISA was based on a T-2 monoclonal antibody with an IC50 (50% inhibitory concentration) of 0.28 ng/mL and 125% cross-reactivity with HT-2. As regards the OTA ELISA, a new sensitive monoclonal antibody specific to OTA with an IC50 of 0.13 ng/mL was produced. Both developed ELISA tests were then validated in agricultural commodities in accordance with the new performance criteria guidelines for the validation of screening methods for mycotoxins included in Commission Regulation (EU) No 519/2014. The T-2/HT-2 ELISA was demonstrated to be suitable for the detection of T-2/HT-2 in cereals and baby food at and above the screening target concentration (STC) of 12.5 μg/kg and 7.5 μg/kg, respectively. The OTA ELISA was shown to be applicable for the detection of OTA in cereals, coffee, cocoa and wine at and above the STC of 2 μg/kg, 2.5 μg/kg, 2.5 μg/kg and 0.4 ng/mL, respectively. The accuracy of both ELISAs was further confirmed by analysing proficiency test and reference samples. The developed methods can be used for sensitive and high-throughput screening for the presence of T-2/HT-2 and OTA in agricultural commodities

Detection of illegal food dyes using immunochemical techniques

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Multiplex Detection of Plant Pathogens Using a Microsphere Immunoassay Technology

Plant pathogens are a serious problem for seed export, plant disease control and plant quarantine. Rapid and accurate screening tests are urgently required to protect and prevent plant diseases spreading worldwide. A novel multiplex detection method was developed based on microsphere immunoassays to simultaneously detect four important plant pathogens: a fruit blotch bacterium Acidovorax avenae subsp. citrulli (Aac), chilli vein-banding mottle virus (CVbMV, potyvirus), watermelon silver mottle virus (WSMoV, tospovirus serogroup IV) and melon yellow spot virus (MYSV, tospovirus). An antibody for each plant pathogen was linked on a fluorescence-coded magnetic microsphere set which was used to capture corresponding pathogen. The presence of pathogens was detected by R-phycoerythrin (RPE)-labeled antibodies specific to the pathogens. The assay conditions were optimized by identifying appropriate antibody pairs, blocking buffer, concentration of RPE-labeled antibodies and assay time. Once conditions were optimized, the assay was able to detect all four plant pathogens precisely and accurately with substantially higher sensitivity than enzyme-linked immunosorbent assay (ELISA) when spiked in buffer and in healthy watermelon leaf extract. The assay time of the microsphere immunoassay (1 hour) was much shorter than that of ELISA (4 hours). This system was also shown to be capable of detecting the pathogens in naturally infected plant samples and is a major advancement in plant pathogen detection

Evaluation of Mass Sensitive Micro-Array biosensors for their feasibility in multiplex detection of low molecular weight toxins using mycotoxins as model compounds

Mycotoxins produced by Fusarium species including trichothecenes, zearalenone and fumonisins, can co-contaminate food and feed throughout the supply chain, including cereal grains and animal feeds. There is an increasing demand to enhance global food security by improving the monitoring of mycotoxins throughout our food supply chain. For time and cost-efficient analysis, rapid tests capable of detecting multiple toxins from a single sample are ideal. Considering these current trends in mycotoxin testing, this project examined the feasibility of using both a portable and non-portable mass-based biosensor for multiplex mycotoxin detection. The biosensor was a mass sensitive microarray (MSMA) which consisted of 4 × 16 miniaturized mass sensitive transducer pixels based on solidly mounted resonator (SMR) technology. Functionalisation of individual pixels on the sensor surface using nano-spotting technology for the simultaneous and semi-quantitative detection of three regulated mycotoxins: T2-toxin (T2) zearalenone (ZEN), and fumonisin B1 (FumB1) was examined. With the integration of portable and non-portable microfluidic devices for antibody and standard sample injections, competitive inhibition assays were developed followed by singleplex and multiplex calibration curves. The characteristics and performance of the MSMA were evaluated including sensitivity which was determined as the concentration causing 50% inhibition. Sensitivity of singleplex assays using the portable microfluidic device (PMD) were 1.3 ng/ml, 2.0 ng/ml and 6.8 ng/ml for T2, FumB1 and ZEN, respectively. Sensitivity of the multiplex assay again using the PMD was 6.1 ng/ml, 3.6 ng/ml and 2.4 ng/ml for T2, FumB1 and ZEN, respectively. The PMD was an easy to use and highly sensitive screening tool which has been demonstrated for the multiplex detection of three regulated mycotoxins. Analysis was in real time and results were fully digital. Since detection of analytes was by mass it was both a label-free and cost-efficient method proposed method of analysis for mycotoxins.</p