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Introduction Mycotoxins are harmful contaminants for human and animal health; their presence also impacts crop production and economies worldwide. They are frequently present in various African staple foods. Purpose In this study, a multi-mycotoxin analysis approach was implemented to reveal mycotoxin contamination of important staple foods in Kinshasa (Democratic Republic of the Congo). Methods A multi-mycotoxin analysis approach based on liquid chromatography tandem mass spectrometry (LC-MS/MS) with electrospray interface operated in positive mode (ESI+) and quadrupole mass analyzer was used. Twenty-five mycotoxins were investigated in 18 samples of staple foods collected in different markets in Kinshasa city, including maize flour (n=8), cassava flour (n=5), and peanut paste (n=5). Results Twenty mycotoxins were present in these samples at quantifiable levels. The maize flour samples were the most contaminated with aflatoxin B1 (AFB1) (87.5%), total aflatoxin (AFT) (100%), fumonisin B1 (FB1) (87.5%), deoxynivalenol (DON) (75%), and nivalenol (NIV) (75%). Emerging mycotoxins such as beauvericin (BEA), enniatin B (ENN B), alternariol (AOH), and alternariol mono-methyl ether (AME) were more frequent in maize flour. The study found that 25% of maize flour and 20% of peanut paste samples exceeded the European Commission’s regulatory limits for aflatoxin B1 (AFB1) and total aflatoxins (AFT). Additionally, ochratoxin A (OTA) was detected in one maize sample at a concentration above the regulatory limit. This could expose consumers to their genotoxic, teratogenic, and immunosuppressive effects. As for OTA, there are fears of its harmful effects, including nephrotoxicity and mutagenicity. The aflatoxin/fumonisin (AF/F) combination was more frequently observed (50% of all samples) than AF/DON, DON/F, and AF/OTA combinations. Conclusion Data from this exploratory study highlight a possible health risk for the population of Kinshasa through the consumption of maize flour, as well as the need for continuous monitoring of mycotoxins in this staple food that is increasingly being consumed across the Democratic Republic of the Congo.

After India and the USA, Pakistan is the third country leading in global dairy production, a sector of very high socioeconomic relevance in Asia. Mycotoxins can affect animal health, reproduction and productivity. This study analysed a broad range of co-occurring mycotoxins and fungal secondary metabolites derived from Alternaria, Aspergillus, Fusarium, Penicillium and other fungal species. To complete this, a validated multi-metabolite liquid chromatography/electrospray ionization-tandem mass spectrometric (LC/ESI–MS/MS) method was employed, detecting 96 of > 500 tested secondary fungal metabolites. This first preliminary study demonstrated that total mixed rations (TMRs) (n = 30) from big commercial dairy cattle farms (> 200 lactating cows) in Punjab, Pakistan, presented ubiquitous contamination with mixtures of mycotoxins. The mean of mycotoxins per sample was 14, ranging from 11 to 20 mycotoxins among all TMR samples. Metabolites derived from other fungi and Fusarium spp. showed the highest levels, frequency and diversity among the detected fungal compounds. Among the most prevalent mycotoxins were Fusarium toxins like fumonisins B1 (FB1) (93%), B2 (FB2) (100%) and B3 (FB3) (77%) and others. Aflatoxin B1 (AFB1) was evidenced in 40% of the samples, and 7% exceeded the EU maximum limit for feeding dairy cattle (5 µg/kg at 88% dry matter). No other mycotoxin exceeds the EU guidance values (GVs). Additionally, we found that dietary ingredients like corn grain, soybean meal and canola meal were related to increased contamination of some mycotoxins (like FB1, FB2 and FB3) in TMR from the province of Punjab, Pakistan. Among typical forage sources, the content of maize silage was ubiquitous. Individually, the detected mycotoxins represented relatively low levels. However, under a realistic scenario, long-term exposure to multiple mycotoxins and other fungal secondary metabolites can exert unpredictable effects on animal health, reproduction and productivity. Except for ergot alkaloids (73%), all the groups of metabolites (i.e. derived from Alternaria spp., Aspergillus spp., Fusarium spp., Penicillium spp. and other fungi) occurred in 100% of the TMR samples. At individual levels, no other mycotoxins than AFB1 represented a considerable risk; however, the high levels of co-occurrence with several mycotoxins/metabolites suggest that long-term exposure should be considered because of their potential toxicological interactions (additive or synergistic effects). [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The responses to artificial spike inoculation with Fusarium culmorum were compared in 11 Tritordeum lines, two durum wheat cultivars and one naked barley cultivar. Inoculation of Tritordeum spikes led to a significant decrease in spike weight, kernel weight per spike, and kernel weight (by 18, 28, and 16 %, respectively). Durum wheat responded most strongly to inoculation, particularly with regard to spike weight and kernel weight per spike (decrease of 42 % and 53 %, respectively). Inoculation induced a significant increase in the total concentration of trichothecenes (9902 vs 558 μg/kg in non-inoculated control) and other Fusarium toxins (40,207 vs 3250 μg/kg in non-inoculated control) in Tritordeum grain. The content of three Alternaria toxins was not significantly modified by inoculation. The principal component analysis (PCA) of all fungal metabolites supported the discrimination of control and inoculated grain, and the results were used to divide the examined Tritordeum lines into two groups with different mycotoxin profiles. The first group (five lines) was more similar to naked barley, whereas the second group (six lines) showed greater similarity to durum wheat. The analyzed Tritordeum lines responded differently to inoculation, which suggests that lines with a low propensity to accumulate Fusarium toxins in grain can be selected from the existing gene pool. The study also demonstrated that Tritordeum grain accumulates significantly smaller amounts of mycotoxins than durum wheat grain. • Tritordeum grain accumulates significantly smaller amounts of mycotoxins than durum wheat grain. • The examined Tritordeum lines can be divided into two groups that differ in mycotoxin profiles. • Some Tritordeum lines accumulate low amounts of Fusarium mycotoxins. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Fungi and mycotoxins in silage can have detrimental consequences for both cattle and human health. This pilot study identified, via the routinary direct plating method, the dominant cultivable fungi in mouldy grass silages (GS) (n = 19) and maize silages (MS) (n = 28) from Austria. The profiles of regulated, modified, and emerging mycotoxins together with other fungal metabolites were analysed via LC-(ESI)MS/MS. Penicillium roqueforti, Saccharomyces spp., Geotrichum candidum, Aspergillus fumigatus and Monascus ruber were the most frequent fungal organisms identified. Other species including Mucor circinelloides, Fusarium spp. and Paecilomyces niveus were detected at lower frequencies. The presence of complex mixtures of toxic and potentially toxic compounds was evidenced by high levels and occurrences (≥ 50%) of Penicillium-produced compounds such as mycophenolic acid (MPA), roquefortines (ROCs), andrastins (ANDs) and marcfortine A. Mouldy silages contained toxins commonly produced by genus Fusarium (e.g. zearalenone (ZEN) and trichothecenes), Alternaria (like tenuazonic acid (TeA) and alternariol (AHO)) and Aspergillus (such as sterigmatocystin (STC)). Compared to those in GS, mouldy spots in MS presented significantly higher fungal counts and more diverse toxin profiles, in addition to superior levels of Fusarium spp., Penicillium spp. and total fungal metabolites. Generally, no correlation between mould counts and corresponding metabolites was detected, except for the counts of P. roqueforti, which were positively correlated with Penicillium spp. metabolites in mouldy MS. This study represents a first assessment of the fungal diversity in mouldy silage in Austria and highlights its potential role as a substantial contributor to contamination with complex mycotoxin mixtures in cattle diets. [ABSTRACT FROM AUTHOR]

Twenty-seven mycotoxins in unprocessed cereals (n = 110) and pulses (n = 23) harvested in Latvia were analysed by nanoflow liquid chromatography combined with Orbitrap high-resolution mass spectrometry. One or more mycotoxins were found in 99% of the cereals and 78% of the pulses. Deoxynivalenol, zearalenone and T-2 and HT-2 toxins were prevalent in 9 to 86% of the cereals, mostly below their maximum levels as set by the European regulations. Non-regulated type A and B trichothecenes were prevalent in 5 to 87% of the cereals, at concentrations of 0.27–83 µg kg−1 and 1.7–4,781 µg kg−1, respectively. Quantification of emerging mycotoxins was also provided. Enniatins were detected in 94% of the cereals (3.5–2,073 µg kg−1) and 13% of the pulses (4.4–17 µg kg−1). Alternaria toxins were prevalent in 94% of the cereals at concentrations of 0.72–307 µg kg−1 and in 39% of the pulses at 0.69–10 µg kg−1. [ABSTRACT FROM AUTHOR]

In this study, fifty-four wheat flour and wheat-based products available on the Hungarian market were assessed for twelve mycotoxins. Prior to analysis, a multi-mycotoxin method using high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) was developed and validated for wheat and wheat-based products. A simple extraction with acetonitrile/water/formic acid (79/20/1 v/v%) was used for sample preparation. The limits of quantitation (LOQ) were between 0.02 and 161 μg kg−1. Good linearity (r2 > 0.995) was achieved for all mycotoxins investigated. Recoveries varied between 88 and 120% at three concentration levels. Based on the low relative matrix effect (RSD < 0.15%) of the different wheat flour samples, matrix-matched calibration was used, which also proved its suitability in proficiency testing (z-scores: −0.6 for DON; +1.5 for OTA; −0.5 for ZEA). DON was the predominant mycotoxin, which contaminated 84% of the investigated samples. Metabolised forms of DON were found in spelt, durum flour and some wheat-based products (D3G in 4 samples, 15Ac-DON in 7 samples). T-2 and HT-2 were the second most frequently detected mycotoxins. All investigated samples complied with current European/Hungarian legislation. [ABSTRACT FROM AUTHOR]

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. • Analysis of Fusarium mycotoxins in silages by HPLC-MS/MS multi-mycotoxin method. • Analysis of aflatoxins in silages by UHPLC-FLD. • Maize silages was the most heavily contaminated type of silage. • Fumonisins were the most abundant mycotoxins analysed. • Fusarium mycotoxins were probably synthetized before ensiling. [ABSTRACT FROM AUTHOR]

The occurrence of 12 mycotoxins has been analysed by liquid chromatography – time of flight mass spectrometry in the batch of 60 herbal teas purchased from drugstores in Latvia. Among the dry tea samples, 90% were positive for one to eight mycotoxins. Enniatin B and deoxynivalenol (DON) were the most frequently detected mycotoxins in 55% and 45% of the samples, respectively. DON reached the highest level, from 129 µg kg−1 in herbal blend to 5,463 µg kg−1 in wormwood tea. Ochratoxin A (OTA) and aflatoxin B1 (AFB1) were found in 10% and 20% of the samples at the concentrations ranged between 2.99–30.3 µg kg−1 and 3.40–23.7 µg kg−1. Studies of the tea infusion process indicated that 32–100% of DON and zearalenone present in dry teas were extracted into the infusions. Dietary exposure assessment was performed, using the determined mycotoxin levels and the available consumption data. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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.

A sensitive and reliable multi-mycotoxin-based method was developed to identify and quantify several carcinogenic mycotoxins in human blood and urine, as well as edible animal tissues, including muscle and liver tissue from swine and chickens, using liquid chromatography–tandem mass spectrometry (LC–MS/MS). For the toxicokinetic studies with individual mycotoxins, highly sensitive analyte-specific LC–MS/MS methods were developed for rat plasma and urine. Sample purification consisted of a rapid ‘dilute and shoot’ approach in urine samples, a simple ‘dilute, evaporate and shoot’ approach in plasma samples and a ‘QuEChERS’ procedure in edible animal tissues. The multi-mycotoxin and analyte-specific methods were validated in-house: The limits of detection (LOD) for the multi-mycotoxin and analyte-specific methods ranged from 0.02 to 0.41 μg/kg (μg/L) and 0.01 to 0.19 μg/L, respectively, and limits of quantification (LOQ) between 0.10 to 1.02 μg/kg (μg/L) and 0.09 to 0.47 μg/L, respectively. Apparent recoveries of the samples spiked with 0.25 to 4 μg/kg (μg/L) ranged from 60.1% to 109.8% with relative standard deviations below 15%. The methods were successfully applied to real samples. To the best of our knowledge, this is the first study carried out using a small group of patients from the Chinese population with hepatocellular carcinoma to assess their exposure to carcinogenic mycotoxins using biomarkers. Finally, the multi-mycotoxin method is a useful analytical method for assessing exposure to mycotoxins edible in animal tissues. The analyte-specific methods could be useful during toxicokinetic and toxicological studies. [ABSTRACT FROM AUTHOR]

• A novel method is reported for multi-mycotoxin analysis in 5 Indian medicinal herbs. • Sample preparation involved extraction by methanol+water & immunoaffinity cleanup. • The target analytes included aflatoxins, ochratoxin A, zearalenone, DON, T-2 & HT-2. • The LC-MS/MS & LC-fluorescence techniques provided high throughput analyses. • The method-performance complied with the analytical quality control guidelines. While medicinal plants are in high demand worldwide for their therapeutic properties, they can constitute a health concern to consumers when contaminated with mycotoxins. The unavailability of standardised methods for multiclass mycotoxin analysis to assess health risks has thus been realised. This study reports a simple, robust and precise method to estimate nine regulated mycotoxins in a range of Indian medicinal plant matrices including giloy (Tinospora cordifolia) , ashwagandha (Withania somnifera), safed musli (Chlorophytum borivilianum), satavari (Asparagus racemosus) and tulsi (Ocimum sanctum). The sample preparation method involved extraction of homogenised matrices (12.5 g) using methanol:water (8:2, 100 mL) followed by cleanup through a multi-mycotoxin immunoaffinity column (IAC), which significantly reduced matrix interferences. The method was initially developed and validated using liquid chromatography tandem mass spectrometry (LC-MS/MS) for the simultaneous analysis of aflatoxins (B 1 , B 2 , G 1 , G 2), ochratoxin A, zearalenone, deoxynivalenol, T-2 and HT-2 toxin. Later, it was validated using LC-fluorescence (LC-FLD) for aflatoxins, ochratoxin A and zearalenone. The optimised sample preparation protocol and analytical method provided acceptable results. Compared to LC-FLD, it was possible to attain a lower limit of quantification (LOQ) with LC-MS/MS for all the tested analytes except aflatoxins. However, LOQs of both instruments were lower than the maximum limits (MLs), with recoveries ranging between 71 and 110% and precision (RSD) of ≤10% across matrices. Despite matrix-induced signal suppressions in LC-MS/MS analysis, the matrix-matched calibrations corrected all recoveries. Considering its accuracy, reliability, robustness and time-effectiveness, this method is recommended for regulatory testing purposes. [ABSTRACT FROM AUTHOR]

Abstract: A sensitive and reliable multi-mycotoxin method was developed for the identification and quantification of several toxicological important mycotoxins such as deoxynivalenol (DON), deepoxy-deoxynivalenol (DOM-1), T-2 toxin (T-2), HT-2 toxin (HT-2), zearalenone (ZON), zearalanone (ZAN), α-zearalenol (α-ZOL), β-zearalenol (β-ZOL), α-zearalanol (α-ZAL), β-zearalanol (β-ZAL), ochratoxin A (OTA), fumonisin B1 (FB1) and aflatoxin B1 (AFB1) in pig plasma using liquid chromatography combined with heated electrospray ionization triple quadrupole tandem mass spectrometry (LC–h-ESI-MS/MS). Sample clean-up consisted of a deproteinization step using acetonitrile, followed by evaporation of the supernatant and resuspension of the dry residue in water/methanol (85/15, v/v). Each plasma sample was analyzed twice, i.e. once in the ESI+ and ESI− mode, respectively. This method can be used for the assessment of animal exposure to mycotoxins and in the diagnosis of mycotoxicoses. For the performance of toxicokinetic studies with individual mycotoxins, highly sensitive analyte-specific LC–MS/MS methods were developed. The multi-mycotoxin and analyte-specific methods were in-house validated: matrix-matched calibration graphs were prepared for all compounds and correlation and goodness-of-fit coefficients ranged between 0.9974–0.9999 and 2.4–15.5%, respectively. The within- and between-run precision and accuracy were evaluated and the results fell within the ranges specified. The limits of quantification for the multi-mycotoxin and analyte-specific methods ranged from 2 to 10ng/mL and 0.5 to 5ng/mL, respectively, whereas limits of detection fell between 0.01–0.52ng/mL and <0.01–0.15ng/mL, respectively. [Copyright &y& Elsevier]

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 C 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 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. [ABSTRACT FROM AUTHOR]

• An LC–MS/MS method for mycotoxin analysis in beer was developed and validated. • Focus of the multi-method was set on analysis of several modified mycotoxins. • Validation data were consistent with guideline requirements. • Application on beers revealed contamination especially with modified mycotoxin D3G. A fast high performance liquid chromatography tandem mass spectrometry multi-method based on an ACN-precipitation extraction was developed for the analysis of 41 (modified) mycotoxins in beer. Validation according to the performance criteria defined by the European Commission (EC) in Commission Decision no. 657/2002 revealed good linearity (R 2 > 0.99), repeatability (RSDr < 15%), reproducibility (RSDR < 15%), and recovery (79–100%). Limits of quantification ranging from 0.04 to 75 µg/L were obtained. Matrix effects varied from −67 to +319% and were compensated for using standard addition. In total, 87 beer samples, produced worldwide, were analyzed for the presence of mycotoxins with a focus on modified mycotoxins, whereof 76% of the samples were contaminated with at least one mycotoxin. The most prevalent mycotoxins were deoxynivalenol-3-glucoside (63%), HT-2 toxin (15%), and tenuazonic acid (13%). Exposure estimates of deoxynivalenol and its metabolites for German beer revealed no significant contribution to intake of deoxynivalenol. [ABSTRACT FROM AUTHOR]

Here, a reliable and sensitive method for the determination of 38 (modified) mycotoxins was developed. Using a QuEChERS-based extraction method [acetonitrile/water/formic acid (75:20:5, v/v/v)], followed by two runs of high performance liquid chromatography tandem mass spectrometry with different conditions, relevant mycotoxins in cereals were analyzed. The method was validated according to the performance criteria defined by the European Commission (EC) in Commission Decision no. 657/2002. Limits of quantification ranged from 0.05 to 150 μg/kg. Good linearity ( R 2 > 0.99), recovery (61-120%), repeatability (RSDr < 15%), and reproducibility (RSDR < 20%) were obtained for most mycotoxins. However, validation results for Alternaria toxins and fumonisins were unsatisfying. Matrix effects (-69 to +59%) were compensated for using standard addition. Application on reference materials gave correct results while analysis of samples from local retailers revealed contamination, especially with deoxynivalenol, deoxynivalenol-3-glucoside, fumonisins, and zearalenone, in concentrations up to 369, 58, 1002, and 21 μg/kg, respectively.

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. [ABSTRACT FROM AUTHOR]

Ultrahigh-performance liquid chromatography using positive electrospray ionization and quadrupole orbital ion trap high-resolution mass spectrometry was evaluated for analyzing mycotoxins in finished cereal and nut products. Optimizing the orbital ion trap mass analyzer in full-scan mode using mycotoxin-fortified matrix extracts gave mass accuracies, δM, of < ± 2.0 ppm at 70,000 full width at half maximum (FWHM) mass resolution (RFWHM). The limits of quantitation were matrix- and mycotoxin-dependent, ranging from 0.02 to 11.6 μg/kg. Mean recoveries and standard deviations for mycotoxins from acetonitrile/water extraction at their relevant fortification levels were 91 ± 10, 94 ± 10, 98 ± 12, 91 ± 13, 99 ± 15, and 93 ± 17% for corn, rice, wheat, almond, peanut, and pistachio, respectively. Nineteen mycotoxins with concentrations ranging from 0.3 (aflatoxin B1 in peanut and almond) to 1175 μg/kg (fumonisin B1 in corn flour) were found in 35 of the 70 commercial grain and nut samples surveyed. Mycotoxins could be identified at δM < ± 5 ppm by identifying the precursor and product ions in full-scan MS and data-dependent MS/MS modes. This method demonstrates a new analytical approach for monitoring mycotoxins in finished grain and nut products.