Report of the VthWorkshop of the Spanish National Network on Mycotoxins and Toxigenic Fungi and Their Decontamination Processes (MICOFOOD), 10–11 December 2020

Spanish Ministry of Economy, Industry and Competitiveness AGL2017-90787-REDTFaculty of Pharmacy (University of Valencia, Spain)AIMPLAS (Plastic Technology Centre)AINIA Technology CentreIATA (Institute of Agrochemistry and Food Technology, CSIC)IMPORTAC

Application of LC–MS/MS in the Mycotoxins Studies

The editors are really grateful to all the authors who contributed their high-quality works to this Special Issue. Special thanks go to the expert peer reviewers, whose rigorous evaluations of all of the submitted manuscripts have, no doubt, improved this Special Issue. Last but not least, the editors really appreciate the good organization and constant support of the MDPI editorial team and staff

An integrated targeted and untargeted approach for the analysis of ergot alkaloids in cereals using UHPLC - hybrid quadrupole time-of-flight mass spectrometry

An ultra-high performance liquid chromatography hybrid quadrupole time of flight (Q-TOF) mass spectrometry (MS) method is described for the simultaneous quantitative determination of common ergot alkaloids and the screening, detection and identification of unexpected (less studied or novel) members of this class of toxic fungal secondary metabolites. The employed analytical strategy involves an untargeted data acquisition (consisting of full scan TOF MS survey and information dependent acquisition MS/MS scans) and the processing of data using both targeted and untargeted approaches. Method performance characteristics for the quantitative analysis of 6 common ergot alkaloids i.e. ergometrine, ergosine, ergotamine, ergocornine, ergocristine, ergokryptine and their corresponding epimers in rye were comparable to those previously reported for triple-quadrupole (QqQ) MS/MS. The method limits of quantification (LOQ) were in the range from 3 to 19 mu g/kg, and good linearity was observed for the different ergot alkaloids in the range from LOQ to 1000 mu g/kg. Furthermore, the method demonstrated good precision (relative standard deviations at 50 mu g/kg not higher than 14.6 and 16.2% for the intra-day and inter-day precision, respectively), and the trueness values at different concentration levels were all between 89 and 115%. The method was applied for the analysis of a set of 17 rye samples and demonstrated the presence of these ergot alkaloids in the range from <LOQ to 2,811 mu g/kg. Further mining of the same data based on a 'non-targeted peak finding' algorithm and the use of full MS and MS/MS accurate mass data allowed the detection and identification of 19 ergot alkaloids that are commonly not included in most analytical methods using QqQ instruments. Some of these alkaloids are reported for the first time in naturally contaminated samples

Occurrence of Ergot Alkaloids in Barley and Wheat from Algeria

The natural occurrence of six major ergot alkaloids, ergometrine, ergosine, ergotamine, ergocornine, ergokryptine and ergocristine, as well as their corresponding epimers, were investigated in 60 cereal samples (barley and wheat) from Algeria. Ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) and a QuEChERS extraction method were used for sample analysis. The results revealed that 12 out of 60 samples (20%) were contaminated with ergot alkaloids. Wheat was the most contaminated matrix, with an incidence of 26.7% (8 out of 30 samples). The concentration of total ergot alkaloids ranged from 17.8 to 53.9 µg/kg for barley and from 3.66 to 76.0 µg/kg for wheat samples. Ergosine, ergokryptine and ergocristine showed the highest incidences in wheat, while ergometrine was the most common ergot in barley.Spanish Ministry of Science, Innovation and Universities (Project Ref.: RTI2018-097043-B-I00

Ion mobility-mass spectrometry to extend analytical performance in the determination of ergot alkaloids in cereal samples

This work evaluates the potential of ion mobility spectrometry (IMS) to improve the analytical perfor- mance of current liquid chromatography-mass spectrometry (LC-MS) workflows applied to the determi- nation of ergot alkaloids (EAs) in cereal samples. Collision cross section (CCS) values for EA epimers are reported for the first time to contribute to their unambiguous identification. Additionally, CCS values have been inter-laboratory cross-validated and compared with CCS values predicted by machine-learning mod- els. Slight differences were observed in terms of CCS values for ergotamine, ergosine and ergocristine and their corresponding epimers (from 3.3 to 4%), being sufficient to achieve a satisfactory peak-to-peak res- olution for their unequivocal identification. A LC-travelling wave ion mobility (TWIM)-MS method has been developed for the analysis of EAs in barley and wheat samples. Signal-to-noise ratio (S/N) was im- proved between 2.5 and 4-fold compared to the analog LC-TOF-MS method. The quality of the extracted ion chromatograms was also improved by using IMS.European Commission 795946MCIN/AEI (European Regional Development Fund-ERDF, a way to build Europe) PID2021-127804OB-I0

Non-aqueous capillary electrophoresis–time of flight mass spectrometry method to determine emerging mycotoxins

Las eniatinas (ENN) y la beauvericina (BEA) son micotoxinas emergentes que tradicionalmente se han determinado mediante cromatografía líquida acoplada a espectrometría de masas en tándem (LC-MS/MS). Sin embargo, hasta donde sabemos, hasta el momento no se han publicado métodos analíticos basados ​​en electroforesis capilar (CE)-MS/MS. Debido a su naturaleza no polar, en este trabajo se propone por primera vez un método de CE no acuosa (NACE) acoplada a MS de cuadrupolo-tiempo de vuelo para identificar y cuantificar estas micotoxinas. La determinación se logró en 4 min en condiciones óptimas: acetato de amonio 40 mM en acetonitrilo-metanol 80:20 (v/v) (tampón), 30 kV (voltaje), 80 cm (longitud del capilar), 20 °C (temperatura del capilar) y 50 mbar × 30 s (inyección). Se puede lograr una mayor selectividad en comparación con LC debido a la formación de aductos CE exclusivos como [M + CH3CH2NH3]+. Se seleccionó el modo de adquisición “All Ions” ya que permite la cuantificación de los ENN habituales, así como la identificación de las ENN menos comunes. El método fue validado para muestras de trigo, obteniendo límites de cuantificación de 4,0 a 8,3 μg/kg dependiendo de la micotoxina emergente, valores de recuperación superiores al 87,4 % y valores de precisión intra e interdiarios (RSD) inferiores al 15,1 % en todos los casos. Finalmente, se analizaron 29 muestras de trigo, encontrando 26 muestras con concentraciones de eniatina B superiores al límite de cuantificación (7,5-1480 μg/kg), 20 de eniatina B1 (5,2-550 μg/kg), 7 de eniatina A (10 –55 μg/kg), 4 para eniatina A1 (12,6-77 μg/kg) y 5 para BEA (9,2-16,4 μg/kg). Además, se identificaron tentativamente otras dos ENN. Enniatins (ENN) and beauvericin (BEA) are emerging mycotoxins that have been traditionally determined by liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS). However, to the best of our knowledge, no analytical methods based on capillary electrophoresis (CE)–MS/MS have been reported so far. Due to their non-polar nature, in this work, a non-aqueous CE (NACE) method coupled to quadrupole time-of-flight–MS is proposed for the first time to identify and quantify these mycotoxins. Determination was achieved in 4 min under optimum conditions: 40 mM ammonium acetate in 80:20 (v/v) acetonitrile-methanol (buffer), 30 kV (voltage), 80 cm (capillary length), 20 °C (capillary temperature) and 50 mbar × 30 s (injection). Higher selectivity can be achieved when compared with LC due to the formation of exclusive CE adducts such as [M + CH3CH2NH3]+. “All Ions” acquisition mode was selected as it allows the quantification of the usual ENNs, as well as the identity confirmation of less common ENNs. The method was validated for wheat samples, obtaining limits of quantification from 4.0 to 8.3 μg/kg depending on the emerging mycotoxin, recovery values higher than 87.4%, and intra- and inter-day precision values (RSDs) lower than 15.1% in all cases. Finally, 29 wheat samples were analyzed, finding 26 samples with concentrations of enniatin B higher than the limit of quantification (7.5–1480 μg/kg), 20 for enniatin B1 (5.2–550 μg/kg), 7 for enniatin A (10–55 μg/kg), 4 for enniatin A1 (12.6–77 μg/kg) and 5 for BEA (9.2–16.4 μg/kg). Moreover, two other ENNs were tentatively identified.Proyectos Ref.: RTI2018-097043-B-I00 y EQC2018-004453-P financiados por MCIN/AEI/10.13039/501100011033/FEDER “Una manera de hacer Europa” y Junta de Andalucía - Programa Operativo FEDER [B-AGR-202-UGR20]. M.M.D.P. agradece un contrato posdoctoral a la Junta de Andalucía (DOC_00230). Los autores agradecen al Dr. Choukri Khelifa por suministrar la muestras de cereales. Financiación para publicación en acceso abierto a cargo de Universidad de Granada/CBUA

Occurrence of Mycotoxins in Swine Feeding from Spain

A survey including 228 pig feed samples from Spain has been developed, exploring the occurrence of 19 mycotoxins (aflatoxins B1, B2, G1 and G2, ochratoxin A, fumonisins B1 and B2, citrinin, zearalenone, deoxynivalenol, fusarenon X, sterigmatocystin, T-2 toxin, HT-2 toxin, enniatins A, A1, B and B2, and beauvericin). The samples were analysed by solid-liquid extraction followed by liquid chromatography coupled with fluorescence or mass spectrometry detection. Enniatin B was found in 100% of the samples (up to 1200 ug/kg) and beauvericin in more than 90%. Moreover, 40% of samples were contaminated with more than five mycotoxins. This high occurrence is insurmountable and surpasses all previous studies, probably due to the inclusion of emerging mycotoxins, scarcely explored. The majority of the samples (96.9%) were in accordance with EU regulations, which do not address emerging mycotoxins or co-occurrence. These results show that in order to ensure mycotoxin absence, emerging mycotoxins should always be considered.This research was funded by SPANISH MINISTRY OF ECONOMY AND COMPETITIVENESS and EUROPEAN REGIONAL DEVELOPMENT FUND (MINECO/FEDER, UE), grant number AGL2015-70708-R

Multi-Mycotoxin Occurrence and Exposure Assessment Approach in Foodstuffs from Algeria

A survey on 120 cereal samples (barley, maize, rice and wheat) from Algerian markets has been carried out to evaluate the presence of 15 mycotoxins (ochratoxin A, deoxynivalenol, fumonisin B1 and B2, T-2 and HT-2 toxins, zearalenone, fusarenon X, citrinin, sterigmatocystin, enniatins A, A1, B and B1, and beauvericin). With this purpose, a QuEChERS-based extraction and ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) were used. Analytical results showed that 78 cereal samples (65%) were contaminated with at least one toxin, while 50% were contaminated with three to nine mycotoxins. T-2 toxin, citrinin, beauvericin and deoxynivalenol were the most commonly found mycotoxins (frequency of 50%, 41.6%, 40.8% and 33.3%, respectively). Fumonisins (B1 + B2), enniatins B and B1, deoxynivalenol and zearalenone registered high concentrations (289–48878 ug/kg, 1.2–5288 ug/kg, 15–4569 ug/kg, 48–2055 ug/kg and 10.4–579 ug/kg, respectively). Furthermore, concentrations higher than those allowed by the European Union (EU) were observed in 21, 8 and 1 samples for fumonisins, zearalenone and deoxinivalenol, respectively. As a conclusion, the high levels of fumonisins (B1 + B2) in maize and deoxynivalenol, zearalenone and HT-2 + T-2 toxins in wheat, represent a health risk for the average adult consumer in Algeria. These results pointed out the necessity of a consistent control and the definition of maximum allowed levels for mycotoxins in Algerian foodstuffs.This research was funded by SPANISH MINISTRY OF ECONOMY AND COMPETITIVENESS and EUROPEAN REGIONAL DEVELOPMENT FUND (MINECO/FEDER, UE) (Project ref: AGL2015-70708-R, MINECO/FEDER, UE). Choukri Khelifa Mahdjoubi would like to thanks the University of Granada for ERASMUS Mundus Doctoral Fellowship