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Wheat represents one of the most widely consumed cereals worldwide. Cultivated in winter and spring, it is vulnerable to an array of different pathogens, including fungi, which are managed largely through the in-field application of fungicides. During this study, a 4-year field investigation (2018–2021) was performed in France, aiming to assess the efficacy of fungicide treatment to reduce mycotoxin contamination in common and durum wheat. Several different commercially available fungicides were applied via sprayers. Concentrations of mycotoxins and fungal metabolites in wheat were determined using a multi-analyte liquid-chromatography–tandem-mass-spectrometry-based method. The highest contamination levels and strongest effects of fungicides were observed in 2018, followed by 2021. A significant fungicide-mediated reduction was observed for the trichothecenes deoxynivalenol, deoxynivalenol-3-glucoside, nivalenol, and nivalenol-3-glucoside. Furthermore, fungicide treatment also reduced levels of culmorin and its hydroxy metabolites 5- and 15-hydroxy-culmorin, as well as aurofusarin. Interestingly, the Alternaria metabolite infectopyron was increased following fungicide treatment. In conclusion, fungicide treatment was effective in reducing mycotoxin levels in wheat. However, as complete prevention of mycotoxin contamination was not achieved, fungicide treatment should always be combined with other pre- and post-harvest mycotoxin mitigation strategies to improve food and feed safety.

Mycotoxins and endocrine disruptors such as phytoestrogens can affect cattle health, reproduction, and productivity. Most studies of mycotoxins in dairy feeds in Mexico and worldwide have been focused on a few (regulated) mycotoxins. In contrast, less known fungal toxins, phytoestrogens, and other metabolites have been neglected and underestimated. This study analyzed a broad spectrum (>800) of mycotoxins, phytoestrogens, and fungal, plant, and unspecific secondary metabolites in whole-plant corn silages (WPCSs) and total mixed rations (TMRs) collected from 19 Mexican dairy farms. A validated multi-metabolite liquid chromatography/electrospray ionization–tandem mass spectrometric (LC/ESI–MS/MS) method was used. Our results revealed 125 of >800 tested (potentially toxic) secondary metabolites. WPCSs/TMRs in Mexico presented ubiquitous contamination with mycotoxins, phytoestrogens, and other metabolites. The average number of mycotoxins per TMR was 24, ranging from 9 to 31. Fusarium-derived secondary metabolites showed the highest frequencies, concentrations, and diversity among the detected fungal compounds. The most frequently detected mycotoxins in TMRs were zearalenone (ZEN) (100%), fumonisin B1 (FB1) (84%), and deoxynivalenol (84%). Aflatoxin B1 (AFB1) and ochratoxin A (OTA), previously reported in Mexico, were not detected. All TMR samples tested positive for phytoestrogens. Among the investigated dietary ingredients, corn stover, sorghum silage, and concentrate proportions were the most correlated with levels of total mycotoxins, fumonisins (Fs), and ergot alkaloids, respectively.

The availability of reliable sensitive multi-analyte methods for unambiguous determination of mycotoxins is crucial for ensuring food and feed safety, considering their adverse health effects and (co-)occurrence in various foods. Accordingly, a multi-mycotoxin confirmatory method for simultaneous determination of 11 mycotoxins regulated in cereals within the European Union (EU) using ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) was developed and in-house validated to fit the EU legislation requirements for analytical methods. A simple sample preparation was based on a solid–liquid extraction using a solvent mixture acetonitrile/water/formic acid (79/20/1, v/v/v) and a dilution of raw extract using water/acetonitrile/formic acid (79/20/1, v/v/v) before instrumental analysis. Average recoveries in all three validated cereal crop types (maize, wheat, and barley), spiked at multiple levels, were found acceptable for all analytes when matrix-matched calibration was used, ranging from 63.2% to 111.2% and also showing very good repeatability, with relative standard deviations below 20%. Matrix effect (SSE) evaluation revealed maize as the most complex of the three analyzed cereal matrices, with strong SSE (150%) recorded for all 11 analyzed mycotoxins. An additional method verification was performed through successful participation in proficiency testing schemes, with the achieved z-scores generally in the acceptable range of −2 ≤ z ≤ 2. The obtained validation results demonstrated the suitability of the developed confirmatory multi-mycotoxin UHPLC-MS/MS method based on a dilute-and-shoot principle for the simultaneous determination of low concentrations of 11 EU-regulated mycotoxins in cereals, including aflatoxins B1, B2, G1 and G2, deoxynivalenol, fumonisins B1 and B2, zearalenone, T-2 and HT-2 toxins, and ochratoxin A.

This review summarises developments on the analysis of various matrices for mycotoxins that have been published in the period from mid-2018 to mid-2019. Analytical methods to determine aflatoxins, Alternaria toxins, ergot alkaloids, fumonisins, ochratoxins, patulin, trichothecenes, and zearalenone are covered in individual sections. Advances in sampling strategies are also discussed in a dedicated section. In addition, developments in multi-mycotoxin methods – including comprehensive mass spectrometric-based methods as well as simple immunoassays – are also reviewed. This critical review aims to briefly present the most important recent developments and trends in mycotoxin determination as well as to address limitations of the presented methodologies.

Forty-five samples of a landrace of sweet pepper (Capsicum annuum) widely cultivated in Basilicata (Italy) were screened for 17 mycotoxins and potential toxigenic fungal species. Two different LC-MS/MS methods were used for the determination of aflatoxins, ochratoxin A (OTA), Fusarium mycotoxins zearalenone (ZEA), fumonisins (FB1 and FB2), nivalenol (NIV), deoxynivalenol (DON), T-2 and HT-2 toxins and Alternaria mycotoxins altenuene (ALT), alternariol (AOH), alternariol monomethyl ether (AME), tentoxin (TTX) and tenuazonic acid (TeA). Frequency of potential toxigenic fungal species occurrence was: 87% Aspergillus Sect. Nigri; 58% Aspergillus Sect. Flavi; 38% Aspergillus Sect. Circumdati; 42% Alternaria spp.; 33% Penicillium spp. and 20% Fusarium spp. Frequency of mycotoxin occurrence and mean of positives were: 51% OTA, 29.5 µg/kg, 5 samples above the EU limit of 20 µg/kg; 31% aflatoxins, 12.8 µg/kg, two samples above the EU limit of 5 µg/kg for aflatoxin B1; 91% ZEA, 1.4 µg/kg; 78% FB2, 7.6 µg/kg; 58% FB1, 22.8 µg/kg; 38% NIV, 39.5 µg/kg; 36% DON, 6.9 µg/kg; 20% T-2 toxin, 5.6 µg/kg and 22% HT-2 toxin, 13.8 µg/kg. For the Alternaria mycotoxins, 100% of samples contained TeA, 4817.9 µg/kg; 93% TTX, 29.7 µg/kg; 56% AOH, 114.4 µg/kg; 33% AME, 13.0 µg/kg and 9% ALT, 61.7 µg/kg. Co-occurrence of mycotoxins in each sample ranged from 2 to 16 mycotoxins (mean 7). No statistical correlation was found between moulds and their mycotoxins occurrence. Within the four groups of peppers collected herein (fresh, dried, grounded and fried) higher percentages of contamination and mycotoxin levels were measured in grounded peppers, whereas much lower values were observed for fried peppers. The high percentages of positive samples and the high levels of some mycotoxins observed in this study confirm the susceptibility of peppers to mycotoxin contamination and claims for an improvement of the conditions used during production and drying process.

Dried blood spots (DBSs), a micro-sampling technique whereby a drop of blood is collected on filter paper has multiple advantages over conventional blood sampling regarding the sampling itself, as well as transportation and storage. This is the first paper describing the development and validation of a method for the determination of 23 mycotoxins and phase I metabolites in DBSs from pigs and broiler chickens using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The targeted mycotoxins belong to groups for which the occurrence in feed is regulated by the European Union, namely, aflatoxins, ochratoxin A and several Fusarium mycotoxins, and to two groups of unregulated mycotoxins, namely Alternaria mycotoxins and Fusarium mycotoxins (enniatins and beauvericin). The impact of blood haematocrit, DBS sampling volume and size of the analysed DBS disk on the validation results was assessed. No effects of variation in size of the analysed disk, haematocrit and spotted blood volume were observed for most mycotoxins, except for the aflatoxins and &beta, zearalanol (BZAL) at the lowest haematocrit (26%) level and for the enniatins (ENNs) at the lowest volume (40 &micro, L). The developed method was transferred to an LC-high resolution mass spectrometry instrument to determine phase II metabolites. Then, the DBS technique was applied in a proof-of-concept toxicokinetic study including a comparison with LC-MS/MS data from plasma obtained with conventional venous blood sampling. A strong correlation (r &gt, 0.947) was observed between plasma and DBS concentrations. Finally, DBSs were also applied in a pilot exposure assessment study to test their applicability under field conditions.

Silages constitute a major component of the feed ration for dairy cows, being a potential source of mycotoxins due to the possible contamination by filamentous fungi capable of producing these toxic compounds. In this study, samples of different kinds of silages collected from farms located in four regions of Spain, were analysed to evaluate the occurrence of aflatoxins (AFs) and Fusarium mycotoxins. Lactic acid bacteria and fungal populations as well as pH and water activity were also studied. Penicillium, Geotrichum and Monascus were the main fungi identified in all the silages examined. The incidence of AFs was low (10 % of positive samples). Fusarium mycotoxins were detected in 40 % of the samples and fumonisins (FBs) were the most commonly detected. Maize silage was the most heavily contaminated type of silage. Levels of mycotoxins detected in positive samples did not exceed the EU guidance values. The lack of relationship between Fusarium counts and its mycotoxin concentrations suggested that mycotoxin production possibly occurred pre-ensiling or immediately post-ensiling. Outcomes showed that mould growth and mycotoxin contamination in silages should be regularly monitored in order to minimize the exposure of dairy cows to contaminated feed. This work was supported by the Spanish Ministry of Economy and Competitiveness (project AGL2014-55379-P). María Rodríguez-Blanco thanks the University of Lleida for a pre-doctoral grant. The authors would like to thank Montserrat Prim for her technical assistance.

The aim of this explorative study was to investigate how effective drying preservation methods are in reducing mycotoxin content in figs. Dried autochthonous varieties of white and dark figs (Petrovača Bijela and Šaraguja, respectively) were analysed for mycotoxins using an LC-MS/MS “dilute and shoot” method capable of determining 295 fungal and bacterial secondary metabolites. Before drying in a cabinet dryer the figs were preserved with 0.5 % citric acid solution or 0.5 % ascorbic acid solution or 0.3 % L-cysteine solution or 0.2 % chestnut extract solution or 0.15 % Echinacea extract solution by immersion. We found nine metabolites: aflatoxin B1 (AFB1), ochratoxin A, ochratoxin alpha, kojic acid, emodin, altenuene, alternariol methyl ether, brevianamide F, and tryptophol. The most efficient preserver was L-cysteine (15 % reduction), while ascorbic acid favoured mycotoxin production (158 % increase). However, all pretreatment solutions reduced AFB1, which is a major fig contaminant.

The aim of this explorative study was to investigate how effective drying preservation methods are in reducing mycotoxin content in figs. Dried autochthonous varieties of white and dark figs (Petrovača Bijela and Šaraguja, respectively) were analysed for mycotoxins using an LC-MS/MS “dilute and shoot” method capable of determining 295 fungal and bacterial secondary metabolites. Before drying in a cabinet dryer the figs were preserved with 0.5 % citric acid solution or 0.5 % ascorbic acid solution or 0.3 % L–cysteine solution or 0.2 % chestnut extract solution or 0.15 % Echinacea extract solution by immersion. We found nine metabolites: aflatoxin B1 (AFB1), ochratoxin A, ochratoxin alpha, kojic acid, emodin, altenuene, alternariol methyl ether, brevianamide F, and tryptophol. The most efficient preserver was L-cysteine (15 % reduction), while ascorbic acid favoured mycotoxin production (158 % increase). However, all pretreatment solutions reduced AFB1, which is a major fig contaminant., Za određivanje pojavnosti mikotoksina u osušenim autohtonim sortama bijelih i tamnih smokava petrovača bijela i šaraguja korištena je metoda LC-MS / MS “razrijedi i mjeri”, kojom je moguće analizirati 295 gljivičnih i bakterijskih sekundarnih metabolita. Smokve su sušene u pilot-pogonu uz korištenje različitih predtretmana za održavanje sušenog voća: uranjanje u 0,5 %-tnu otopinu limunske kiseline, 0,5 %-tnu otopinu askorbinske kiseline, 0,3 %-tnu otopinu L-cisteina, 0,2 %-tnu otopinu ekstrakta kestena i 0,15 %-tnu otopinu ekstrakta Echinaceae. Ovim se istraživanjem otkrilo devet različitih metabolita u koncentracijama iznad njihove granice detekcije: aflatoksin B1, okratoksin A, okratoxin α, kojična kiselina, emodin, altenuen, alternariolmetileter, brevianamid F i triptofol. Sveukupno, najbolji antimikotoksigenski učinak postignut je predtretmanom s L-cisteinom (prosječno smanjenje od 15 %), a tretman s askorbinskom kiselinom pokazao je najveći učinak na indukciju mikotoksina (povećanje od 158 %). Svi predtretmani smanjili su koncentracije aflatoksina, glavnog kontaminanta smokava.