Natalia Arroyo-Manzanares

Multiclass mycotoxin analysis in Silybum marianum by ultra high performance liquid chromatography–tandem mass spectrometry using a procedure based on QuEChERS and dispersive liquid–liquid microextraction

Ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) has been proposed for the determination of 15 mycotoxins in milk thistle (Silybum marianum), including aflatoxins, fumonisins, trichothecenes, ochratoxin A, citrinin, sterigmatocystin and zearalenone. The mycotoxins were detected by electrospray ionization in positive ion mode and multiple reaction monitoring (MRM), achieving the separation in about 4min. Sample treatment consisted of a modified method based on a first step using a QuEChERS-based procedure which allowed the determination of fumonisin B1, fumonisin B2, nivalenol, deoxynivalenol and fusarenon-X, and a subsequent clean-up based on dispersive liquid-liquid microextraction (DLLME) for the determination of the rest of mycotoxins. The method has been validated in extract and seeds of milk thistle, obtaining limits of quantification lower than those usually permitted by legislation in food matrices, with precisions lower than 10%. Recoveries were between 62.3% and 98.9%, except for zearalenone in seeds samples and citrinin in extract. The method was also applied for studying the occurrence of these mycotoxins in market samples (six samples of seeds, three of them purchased in bulk in a street vendor, and one natural extract of milk thistle), and HT-2, T-2 and zearalenone have been found in some of the samples. To the best of our knowledge, this is the first time that this type of treatment has been used for these complex food matrices, allowing the analyses of the most important mycotoxins.

Determination of ochratoxin A in wines by capillary liquid chromatography with laser induced fluorescence detection using dispersive liquid-liquid microextraction

A method based on reverse phase capillary high performance liquid chromatography (capillary HPLC) coupled to laser-induced fluorescence detection (LIF) has been proposed for the determination of ochratoxin A (OTA) in wine samples. An anionic micellar medium was added to the mobile phase for increasing the fluorescence intensity and peak efficiency. Dispersive liquid-liquid microextraction (DLLME) has been used as a simple and efficient sample pretreatment method for the analysis of OTA in wines, being optimised by means of experimental design. The limit of detection was 5.5 ng L(-1) (3 × S/N) and recoveries for different wines ranged from 91.7 to 98.1%. The proposed methodology could be classified as a green analytical chemistry alternative, combining the low organic solvent volumes required in the DLLME with the reduced consumption of mobile phase in capillary HPLC. The use of LIF as detector provided an extremely sensitive method for the determination of OTA in wines.

A new approach in sample treatment combined with UHPLC-MS/MS for the determination of multiclass mycotoxins in edible nuts and seeds

A sensitive, simple and rapid method for the determination of fourteen mycotoxins in nuts and seeds (including almonds, peanuts, sunflower seeds, pumpkin seeds, walnuts, macadamia nuts, pistachios, hazelnuts and pine nuts) has been developed using ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS). The sample treatment comprises a first step based on QuEChERS procedure for the determination of fumonisin B1, fumonisin B2, deoxynivalenol, fusarenon-X, T-2 and HT-2 toxin, citrinin, sterigmatocystin, zearalenone and ochratoxin A. A subsequent clean-up step based on the dispersive liquid-liquid microextraction (DLLME) was necessary for the determination of aflatoxins (B1, B2, G1 and G2), since their determination was not possible applying only the QuEChERS-based extraction. The method was validated for peanuts as representative matrix and was subsequently evaluated for the other eight matrices. Quantification limits obtained for aflatoxins, the unique mycotoxins legislated on these matrices, were lower than the maximum levels allowed by the current legislation, while quantification limits obtained for the other mycotoxins were lower than the limits usually permitted by the legislation in other food matrices. Precision of the method was always lower than 11%, and recoveries ranged between 60.7% and 104.3%.

Alternative sample treatments for the determination of sulfonamides in milk by HPLC with fluorescence detection

This paper presents two sample treatments, dispersive liquid-liquid microextraction (DLLME) and QuEChERS for the determination of 9 sulfonamides, regulated by the EU Council in milk samples. Both methods represent useful alternatives to conventional procedures based mainly in solid-phase extraction, in terms of simplicity, reduction of organic solvents, sample throughput and effectiveness for cleaning-up complex samples. They have been evaluated and compared in terms of efficiency, trueness, sensitivity and precision, using HPLC with fluorescence detection employing a previous derivatisation step with fluorescamine. Clean extracts were obtained with recoveries between 90.8-104.7% and 83.6-104.8% for DLLME and QuEChERS, respectively. Matrix-matched calibration curves were established for both methods using milk samples spiked at four concentration levels. LODs (3xS/N) lower than 1.21μgL(-)(1) and 2.73μgL(-)(1) for DLLME and QuEChERS, respectively, were obtained in all cases. The precision, in terms of repeatability and intermediate precision, was lower than 10% in all cases.

Transcriptome Analysis of Aspergillus flavus Reveals veA-Dependent Regulation of Secondary Metabolite Gene Clusters, Including the Novel Aflavarin Cluster

ABSTRACT The global regulatory veA gene governs development and secondary metabolism in numerous fungal species, including Aspergillus flavus. This is especially relevant since A. flavus infects crops of agricultural importance worldwide, contaminating them with potent mycotoxins. The most well-known are aflatoxins, which are cytotoxic and carcinogenic polyketide compounds. The production of aflatoxins and the expression of genes implicated in the production of these mycotoxins are veA dependent. The genes responsible for the synthesis of aflatoxins are clustered, a signature common for genes involved in fungal secondary metabolism. Studies of the A. flavus genome revealed many gene clusters possibly connected to the synthesis of secondary metabolites. Many of these metabolites are still unknown, or the association between a known metabolite and a particular gene cluster has not yet been established. In the present transcriptome study, we show that veA is necessary for the expression of a large number of genes. Twenty-eight out of the predicted 56 secondary metabolite gene clusters include at least one gene that is differentially expressed depending on presence or absence of veA. One of the clusters under the influence of veA is cluster 39. The absence of veA results in a downregulation of the five genes found within this cluster. Interestingly, our results indicate that the cluster is expressed mainly in sclerotia. Chemical analysis of sclerotial extracts revealed that cluster 39 is responsible for the production of aflavarin.

Method optimization and validation for the determination of eight sulfonamides in chicken muscle and eggs by modified QuEChERS and liquid chromatography with fluorescence detection.

A simple, effective and reliable method for the determination of eight sulfonamide antibiotics (sulfadiazine, sulfapiridine, sulfamerazine, sulfamethazine, sulfachloropiridazine, sulfamethoxazole, sulfadoxine, sulfadimethoxin) in chicken muscle and eggs by liquid chromatography and fluorescence detection has been developed and validated. Sulfonamides do not present native fluorescence, however their direct determination was achieved by on-line post-column photochemical derivatization by UV irradiation. Sample treatment was based on QuEChERS with several modifications depending on the matrix. Egg extracts were cleaned-up using PSA for the dispersive solid phase extraction step. On the other hand, a new clean-up sorbent, Supel™ QuE Z-Sep(+), has been successfully applied in chicken muscle extract and has proved to be effective for interference removal from this matrix. Under optimum conditions, recoveries from 65.9 to 88.1%, relative standard deviations lower than 10% (except for sulfachloropiridazine), and limits of quantification (LOQs) from 14 to 85 μg kg(-1) were achieved. Thus, the method complies with current European requirements.

Simple and efficient methodology to determine mycotoxins in cereal syrups.

Consumption of cereal syrups is increasing nowadays. Mycotoxins may be found in syrups resulting from the use of contaminated raw material or invading microorganisms in the final manufactured product. However, these matrices have been scarcely explored regarding their mycotoxin content. A sensitive, simple and rapid method for the determination of ten mycotoxins (ochratoxin A, fumonisin B1, fumonisin B2, deoxynivalenol, fusarenon-X, T-2 and HT-2 toxin, citrinin, sterigmatocystin and zearalenone) in cereal syrups (rice, wheat and barley) has been developed and characterised using ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) and a sample treatment based on QuEChERS procedure. Matrix-matched calibration curves were established and limits of quantification were below the limits usually established by current legislation in different foodstuff. The relative standard deviation of the whole analytical method was lower than 12% in all cases, while recoveries ranged from 70.2% to 100.6%, therefore fulfilling the current requirements for mycotoxins analysis.

On-line preconcentration for the determination of aflatoxins in rice samples by micellar electrokinetic capillary chromatography with laser-induced fluorescence detection.

MEKC coupling with LIF detection has been used for the determination of four aflatoxins (B1, B2, G1 and G2). Separations were performed in an uncoated fused‐silica capillary (70 cm×75 μm id, 55 cm effective length), using 20 mM borate buffer with 30 mM SDS (pH 8.5) and 7% ACN. In order to increase sensitivity, an on‐line preconcentration procedure was applied, based on sweeping, using the same separation buffer without SDS as solvent of the sample. The precision of the method was evaluated in terms of repeatability and intermediate precision and the results were acceptable in all cases (RSD<12%). With the on‐line preconcentration LODs (obtained as 3×S/N) were as low as 0.11, 0.52, 0.04 and 0.10 μg/L for G2, G1, B2 and B1, respectively. Recovery studies were developed with extracts of rice samples spiked with aflatoxins, being in the range between 93.0 and 105.4%. The method has also been applied to the determination of aflatoxins in rice samples, and the results compared with those obtained by a standard method, being in good agreement.

Holistic approach based on high resolution and multiple stage mass spectrometry to investigate ergot alkaloids in cereals.

A holistic approach based on high resolution and multiple stage mass spectrometry was developed for identification of less studied or novel ergot alkaloid derivatives. Initially, the fragmentation of nine known ergot alkaloids was studied to establish a strategy for the identification of novel ergot alkaloids. Ions with m/z 223 and m/z 251 were found to be common for all ergopeptines, ergoamides and ergopeptams. Subsequently, parent scan experiments using these ions were performed to screen grain samples for the presence of possible ergot alkaloid derivatives. Besides the six most common ergot alkaloids and their corresponding epimers (for which reference standards were available), eleven other ergot alkaloid derivatives were identified following the proposed strategy.

Identification of novel metabolites from Aspergillus flavus by high resolution and multiple stage mass spectrometry

The filamentous fungus Aspergillus flavus is one of the most important species in the Aspergillus genus and is distributed worldwide as a prevalent aflatoxin-producing food and feed contaminant. A. flavus contains more than 55 gene clusters that are predicted to encode proteins involved in secondary metabolite production. One of these, cluster 27, contains a polyketide synthase (pks27) gene that encodes a protein that is highly homologous to the aflatoxin cluster PKS. Comparative metabolomics, using ultra-high performance liquid chromatography (UHPLC) coupled to high resolution Orbitrap mass spectrometry (MS) was used to detect metabolites differentially expressed in the A. flavus wild-type and ∆pks27 mutant strains. Metabolite profiling was aided by a statistical differential analysis of MS data using SIEVE software. This differential analysis combined with accurate mass data from the Orbitrap and ion trap multiple stage MS allowed four metabolites to be identified that were produced only by the wild-type culture. These included asparasone A (358 Da), an anthraquinone pigment, and related anthraquinones with masses of 316, 340 and 374 Da. These latter three compounds had similar fragmentation patterns to that of asparasone A. The 316 Da anthraquinone is particularly interesting because it is most likely formed by incorporation of seven malonyl-CoA units rather than the eight units required for the formation of asparasone A. The 340 and 374 Da metabolites are the dehydration and an oxy-derivative of asparasone A, respectively. Asparasone A was also identified in extracts from several other Aspergillus species. Graphical Abstract