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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.

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.

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 β-zearalanol (BZAL) at the lowest haematocrit (26%) level and for the enniatins (ENNs) at the lowest volume (40 µL). 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 > 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.

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 (<50% and >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.

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 (AFB ), 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 AFB , which is a major fig contaminant.

This study is the first report of applying an ultra high performance liquid chromatography/tandem mass spectrometric (UHPLC-MS/MS) multi-mycotoxin method to identify and quantify the mycotoxins produced by pure fungal isolates grown on Yeast Extract Sucrose (YES) agar. The method developed concerns a triple extraction procedure based on methanol, dichloromethane and ethyl acetate. The total extract was chromatographically separated on an UHPLC BEH C18 column and analyzed with a triple quadrupole mass spectrometer. Performance characteristics (specificity, linearity, possible matrix effects, recovery, repeatability, reproducibility and limit of detection) were evaluated by spiking experiments with blank agar plugs and the analytes. Verrucarol was used as internal standard. Recovery percentages varied between 56 and 125%, whereas the limit of detection ranged from 1 to 1,500 ng g−1 with the exception of NIV, PAT and ZEA. The method was successfully applied for examining the in vitro mycotoxin production by Aspergillus fumigatus, A. flavus and A. niger. The mobile phases used for chromatographic separation were slightly modified when studying patulin-producing molds due to signal interference between this mycotoxin and an unknown metabolite. This modified method was successfully applied for Penicillium roqueforti, P. paneum and P. carneum grown on YES agar medium. Application of the multi-mycotoxin UHPLC-MS/MS method developed may be of great importance for studying the mycotoxin capacity of fungal isolates under varying growth conditions, in order to obtain a better insight into the conditions which induce or suppress mycotoxin production by pure fungal isolates or from a chemotaxonomic point of view.

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.

A reliable and practical multi-method was developed for the quantification of mycotoxins in plasma, urine, and feces of pigs, and plasma and excreta of broiler chickens using liquid chromatography–tandem mass spectrometry. The targeted mycotoxins belong to the regulated groups, i.e., aflatoxins, ochratoxin A and Fusarium mycotoxins, and to two groups of emerging mycotoxins, i.e., Alternaria mycotoxins and enniatins. In addition, the developed method was transferred to a LC-high resolution mass spectrometry instrument to qualitatively determine phase I and II metabolites, for which analytical standards are not always commercially available. Sample preparation of plasma was simple and generic and was accomplished by precipitation of proteins alone (pig) or in combination with removal of phospholipids (chicken). A more intensive sample clean-up of the other matrices was needed and consisted of a pH-dependent liquid–liquid extraction (LLE) using ethyl acetate (pig urine), methanol/ethyl acetate/formic acid (75/24/1, v/v/v) (pig feces) or acetonitrile (chicken excreta). For the extraction of pig feces, additionally a combination of LLE using acetone and filtration of the supernatant on a HybridSPE-phospholipid cartridge was applied. The LC-MS/MS method was in-house validated according to guidelines defined by the European and international community. Finally, the multi-methods were successfully applied in a specific toxicokinetic study and a screening study to monitor the exposure of individual animals.

This study applied multi-mycotoxin liquid chromatography with tandem mass spectrometric detection (LC-MS/MS) methods to determine the biomarkers of exposure in urine and serum samples from a dose-response study with pigs. The 24 studied pigs were divided into three groups: a control and two experimental ones (with different levels of feed contamination). They were exposed to feed prepared from cereals contaminated with deoxynivalenol (DON), zearalenone (ZEN), ochratoxin A (OTA) and citrinin (CIT) for 14 days. After that, both experimental groups received the same feed as the control group for the next 14 days to determine the kinetics of the disappearance of mycotoxin biomarkers. Urine samples were collected daily in the morning and blood samples—eight-times during the experiment. The study reported herein was the first prolonged exposure experiment for multiple mycotoxins like OTA and CIT in pigs. The urinary and serum levels of all biomarkers correlated well with the respective toxin intake; thereby demonstrating that they are suitable biomarkers of exposure in pigs. Urine is a good candidate to monitor DON, ZEN, OTA, CIT exposure while serum may be used to monitor DON, OTA and CIT. Additionally, OTA has even been quantified in both matrices in the experimental groups two weeks after changing the contaminated feed back to the control, this result differed from those produced by the other mycotoxins which were only quantified during the first two weeks. Therefore both matrices are suitable candidates to monitor prolonged OTA exposure in pigs.

Standard solutions of mycotoxins prepared in RP HPLC solvents from neat standards are usually used for analytical method development. Multi-mycotoxin HPLC-MS/MS methods necessitate stability estimation for the wide spectrum of fungal metabolites. The stability of individual diluted stock standard solutions of mycotoxins in RP-HPLC solvents and multi-analyte HPLC-MS/MS calibrants was evaluated under standard storage and analysis conditions. Individual stock standard solutions of aflatoxins, sterigmatocystin, A- and B-trichothecenes, zearalenone and its analogues, ochratoxin A, fumonisins, Alternaria toxins, enniatins and beauvericin, moniliformin, citrinin, mycophenolic, cyclopiazonic acids and citreoviridin were prepared in RP-HPLC solvents and stored at −18 °C for 14 months. UV-spectroscopy was utilized to monitor the stability of analytes, excluding fumonisins. The gradual degradation of α-, β-zearalenol and α-, β-zearalanol in acetonitrile was detected. Aflatoxins and sterigmatocystin, zearalenone, Alternaria toxins, enniatins and beauvericin, citrinin, mycophenolic, cyclopiazonic acids and citreoviridin can be referred to as stable. The concentration of the majority of trichothecenes should be monitored. Diluted multi-mycotoxin standard in water/methanol (50/50 v/v) solutions acidified with 0.1% formic acid proved to be stable in silanized glass at 23 °C exposed to light for at least 75 h (CV ≤ 10%). An unexpected manifestation of MS/MS signal suppression/enhancement was discovered in the course of multi-mycotoxin standard solution stability evaluation.