Eduardo Beltrán

Determination of mycotoxins in different food commodities by ultra‐high‐pressure liquid chromatography coupled to triple quadrupole mass spectrometry

A rapid multianalyte-multiclass method with little sample manipulation has been developed for the simultaneous determination of eleven mycotoxins in different food commodities by using ultra-high-pressure liquid chromatography coupled to triple quadrupole mass spectrometry (UHPLC/MS/MS). Toxins were extracted from the samples with acetonitrile/water (80:20, v/v) 0.1% HCOOH and, after a two-fold dilution with water, directly injected into the system. Thanks to the fast high-resolution separation of UHPLC, the eleven mycotoxins were separated by gradient elution in only 4 min. The method has been validated in three food matrices (maize kernels, dry pasta (wheat), and eight-multicereal babyfood (wheat, maize, rice, oat, barley, rye, sorghum, millet)) at four different concentration levels. Satisfactory recoveries were obtained (70-110%) and precision (expressed as relative standard deviation) was typically below 15% with very few exceptions. Quantification of samples was carried out with matrix-matched standards calibration. The lowest concentration successfully validated in sample was as low as 0.5 microg/kg for aflatoxins and ochratoxin A in babyfood, and 20 microg/kg for the rest of the selected mycotoxins in all matrices tested. Deoxynivalenol could be only validated at 200 microg/kg, due the poor sensitivity for this mycotoxin analysis. With only two exceptions (HT-2 and deoxynivalenol), the limits of detection (LODs), estimated for a signal-to-noise ratio of 3 from the chromatograms of samples spiked at the lowest level validated, varied between 0.1 and 1 microg/kg in the three food matrices tested. The method was applied to the analysis of different kinds of samples. Positive findings were confirmed by acquiring two transitions (Q quantification, q confirmation) and evaluating the Q/q ratio.

Faces of a Changing Climate: Semi-Quantitative Multi-Mycotoxin Analysis of Grain Grown in Exceptional Climatic Conditions in Norway

Recent climatological research predicts a significantly wetter climate in Southern Norway as a result of global warming. Thus, the country has already experienced unusually wet summer seasons in the last three years (2010–2012). The aim of this pilot study was to apply an existing multi-analyte LC-MS/MS method for the semi-quantitative determination of 320 fungal and bacterial metabolites in Norwegian cereal grain samples from the 2011 growing season. Such knowledge could provide important information for future survey and research programmes in Norway. The method includes all regulated and well-known mycotoxins such as aflatoxins, trichothecenes, ochratoxin A, fumonisins and zearalenone. In addition, a wide range of less studied compounds are included in the method, e.g., Alternaria toxins, ergot alkaloids and other metabolites produced by fungal species within Fusarium, Penicillium and Aspergillus. Altogether, 46 metabolites, all of fungal origin, were detected in the 76 barley, oats and wheat samples. The analyses confirmed the high prevalence and relatively high concentrations of type-A and -B trichothecenes (e.g., deoxynivalenol up to 7230 µg/kg, HT-2 toxin up to 333 µg/kg). Zearalenone was also among the major mycotoxins detected (maximum concentration 1670 µg/kg). Notably, several other Fusarium metabolites such as culmorin, 2-amino-14,16-dimethyloctadecan-3-ol and avenacein Y were co-occurring. Furthermore, the most prevalent Alternaria toxin was alternariol with a maximum concentration of 449 µg/kg. A number of Penicillium and Aspergillus metabolites were also detected in the samples, e.g., sterigmatocystin in concentrations up to 20 µg/kg.

Development of sensitive and rapid analytical methodology for food analysis of 18 mycotoxins included in a total diet study

A rapid and sensitive method for the determination of 18 mycotoxins in 24 different food matrices has been developed and validated. With the exception of beverages and oil samples, a simple extraction with acetonitrile:water 80:20 (0.1% formic acid) was applied. Fruit juice, wine and beer samples were simply diluted with water containing 0.1% formic acid. Oil samples were partitioned with acetonitrile/hexane in order to remove fats. Analyses were made by ultra-high performance liquid chromatography (UHPLC) coupled to tandem mass spectrometry with triple quadrupole. Validation was carried out in all selected matrices using blank samples spiked at two analyte concentrations. Extraction recoveries between 70 and 120% and relative standard deviations lower than 20% were obtained for the wide majority of analyte-matrix combinations. Matrix-matched calibration was used for a correct quantification in order to compensate for matrix effects. Limits of quantification were lower than maximum permitted levels for every regulated mycotoxin-matrix combination. The acquisition of three SRM transitions per compound allowed the unequivocal confirmation of positive samples, supported by the accomplishment of ion intensity ratios and retention time when compared with reference standards. The developed methodology was applied to the analysis of 240 samples within a total diet study performed at Comunidad Valenciana (Spain). The most frequently found mycotoxins were deoxynivalenol, fumonisin B1, ochratoxin A and zearalenone at low μg kg(-1) levels, mainly in bread, breakfast cereals and beer.

Determination of six microcystins and nodularin in surface and drinking waters by on-line solid phase extraction–ultra high pressure liquid chromatography tandem mass spectrometry

Microcystins and nodularin are cyclic peptides hepatotoxins produced by cyanobacterial genera (blue-green algae). Toxic cyanobacterial blooms are a worldwide problem, as reported in several countries, like China, Australia, or the United States. Therefore, it is necessary to develop sensitive and reliable analytical methodology to determine this type of toxins in water at parts per billion levels, or even lower. In this work, the potential of solid-phase extraction coupled on-line to ultra-high-pressure liquid chromatography/electrospray tandem mass spectrometry (SPE-UHPLC-MS/MS) has been investigated for the efficient quantification and confirmation of microcystins LR, RR, YR, LY, LW, LF and nodularin in surface and drinking water samples, at sub-ppb levels. The method developed involves the injection of only 1 mL of water sample into the on-line SPE-UHPLC-MS/MS system and allows the rapid determination of the compounds selected (8 min of chromatographic run), avoiding laborious sample treatment. The method was validated in surface and drinking water by means of recovery experiments at 0.25 and 1 μg L(-1). Average recoveries (n=5) ranged from 71 to 116%, with relative standard deviations (RSDs) lower than 15%. For microcystins LR, RR, YR and nodularin, a third level was also assayed (0.1 μg L(-1)) obtaining satisfactory data too. Limits of detection between 0.002 and 0.0405 μg L(-1) were estimated (0.0005 μg L(-1) for nodularin). The developed method was applied to the analysis of water samples collected in the province of Castellón (Spain). The acquisition of three MS/MS transitions for each compound allowed the unequivocal confirmation of positive samples, which was supported by the accomplishment of ion intensity ratios and retention time when compared with reference standards.

Determination of patulin in apple and derived products by UHPLC-MS/MS. Study of matrix effects with atmospheric pressure ionisation sources.

Sensitive and reliable analytical methodology has been developed for the measurement of patulin in regulated foodstuffs by using ultra-high-performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) with triple quadrupole analyser. Solid samples were extracted with ethyl acetate, while liquid samples were directly injected into the chromatographic system after dilution and filtration without any clean-up step. Chromatographic separation was achieved in less than 4min. Electrospray (ESI) and atmospheric pressure chemical ionisation (APCI) sources were evaluated, in order to assess matrix effects. The use of ESI source caused strong signal suppression in samples; however, matrix effect was negligible using APCI, allowing quantification with calibration standards prepared in solvent. The method was validated in four different apple matrices (juice, fruit, puree and compote) at two concentrations at the low μgkg(-1) level. Average recoveries (n=5) ranged from 71% to 108%, with RSDs lower than 14%.

Occurrence and potential transfer of mycotoxins in gilthead sea bream and Atlantic salmon by use of novel alternative feed ingredients

Plant ingredients and processed animal proteins (PAP) are suitable alternative feedstuffs for fish feeds in aquaculture practice, although their use can introduce contaminants that are not previously associated with marine salmon and gilthead sea bream farming. Mycotoxins are well known natural contaminants in plant feed material, although they also could be present on PAPs after fungi growth during storage. The present study surveyed commercially available plant ingredients (19) and PAP (19) for a wide range of mycotoxins (18) according to the EU regulations. PAP showed only minor levels of ochratoxin A and fumonisin B1 and the mycotoxin carry-over from feeds to fillets of farmed Atlantic salmon and gilthead sea bream (two main species of European aquaculture) was performed with plant ingredient based diets. Deoxynivalenol was the most prevalent mycotoxin in wheat, wheat gluten and corn gluten cereals with levels ranging from 17 to 814 and μg kg(-1), followed by fumonisins in corn products (range 11.1-4901 μg kg(-1) for fumonisin B1+B2+B3). Overall mycotoxin levels in fish feeds reflected the feed ingredient composition and the level of contaminant in each feed ingredient. In all cases the studied ingredients and feeds showed levels of mycotoxins below maximum residue limits established by the Commission Recommendation 2006/576/EC. Following these guidelines no mycotoxin carry-over was found from feeds to edible fillets of salmonids and a typically marine fish, such as gilthead sea bream. As far we know, this is the first report of mycotoxin surveillance in farmed fish species.

N-Acetylcysteine boosts xenobiotic detoxification in shellfish

Water pollution represents a threat of increasing importance to human health. Bivalve mollusks are filter-feeding organisms that can accumulate chemical and microbiological contaminants in their tissues from very low concentrations in the water or sediments. Consumption of contaminated shellfish is one of the main causes of seafood poisoning. Thus, marine bivalves are normally depurated in sterilized seawater for 48 h to allow the removal of bacteria. However, this depuration time might be insufficient to eliminate chemical contaminants from their tissues. We have developed a novel technology that accelerates up to fourfold the excretion rate of xenobiotics in bivalves by treatment with the antioxidant and glutathione (GSH) pro-drug N-acetylcysteine (NAC) during the depuration period. NAC improved dose-dependently the detoxification of the organophosphate (OP) pesticide fenitrothion in the mussel Mytilus galloprovincialis, diminishing its levels up to nearly a hundred fold compared to conventional depuration, by enhancing the glutathione S-transferase (GST) activity and inducing the GSH anabolism (GSH synthesis and reduction by glutathione reductase). Notably, this induction in GSH anabolism and GST activity was also observed in uncontaminated bivalves treated with NAC. As the GSH pathway is involved in the detoxification of many pollutants and biotoxins from harmful algal blooms, we validated this proof of principle in king scallops (Pecten maximus) that naturally accumulated the amnesic shellfish poisoning (ASP) toxin domoic acid. We illustrate here a method that enhances the elimination of organic contaminants in shellfish, opening new avenues of depuration of marine organisms.

Identification of mycotoxins by UHPLC-QTOF MS in airborne fungi and fungi isolated from industrial paper and antique documents from the Archive of Bogotá.

Mold deterioration of historical documents in archives and libraries is a frequent and complex phenomenon that may have important economic and cultural consequences. In addition, exposure to toxic fungal metabolites might produce health problems. In this work, samples of broths of fungal species isolated from the documentary material and from indoor environmental samples of the Archive of Bogotá have been analyzed to investigate the presence of mycotoxins. High resolution mass spectrometry made possible to search for a large number of mycotoxins, even without reference standards available at the laboratory. For this purpose, a screening strategy based on ultra-high pressure liquid chromatography coupled to quadrupole-time of flight mass spectrometry (UHPLC-QTOF MS) under MS(E) mode was applied. A customized home-made database containing elemental composition for around 600 mycotoxins was compiled. The presence of the (de)protonated molecule measured at its accurate mass was evaluated in the samples. When a peak was detected, collision induced dissociation fragments and characteristic isotopic ions were also evaluated and used for tentative identification, based on structure compatibility and comparison with literature data (if existing). Up to 44 mycotoxins were tentatively identified by UHPLC-QTOF MS. 34 of these tentative compounds were confirmed by subsequent analysis using a targeted LC-MS/MS method, supporting the strong potential of QTOF MS for identification/elucidation purposes. The presence of mycotoxins in these samples might help to reinforce safety measures for researchers and staff who work on reception, restoration and conservation of archival material, not only at the Archive of Bogotá but worldwide.

Application of liquid chromatography/mass spectrometry in assessment of potential use of azadirachtins (TreeAzin™) against Asian longhorned beetle

Azadirachtins are natural triterpenoid compounds derived from Neem tree extracts with potential for use as systemic insecticides against invasive wood-boring insect pests. In this work, a sensitive and selective analytical method has been developed for the simultaneous determination of azadirachtin A and azadirachtin B (3-tigloylazadirachtol) in foliage and twigs of various tree species. Samples were mixed with C18 and primary-secondary amine (PSA), and extracted with acetonitrile. Then, an aliquot of the raw extract was 10-fold diluted with water and directly analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS). The method was validated in foliage and twig matrices of four different tree species (London Plane Tree, Red/Freemani Maple, Norway Maple and Sugar Maple) that are known hosts of the exotic invasive insect pest – Asian Longhorn Beetle (ALB). Analytical results for replicate (N = 5) samples, fortified at 0.01, 0.1 and 1 mg kg−1, showed good recoveries (86–119%) and precision (<20% RSD). The methodology was successfully applied to the analysis of 200 samples taken from a field experiment designed to investigate uptake, translocation and expression of azadirachtins in representative high-value urban trees followed by stem injection with TreeAzin™.

Inhibition of larval growth and adult fecundity in Asian long-horned beetle (Anoplophora glabripennis) exposed to azadirachtins under quarantine laboratory conditions: Azadirachtin effects on ALB

BACKGROUND The Asian long-horned beetle [ALB; Anoplophora glabripennis (Motschulsky)] is an invasive, wood-boring insect posing significant economic and ecological threats to the deciduous forests of North America. An efficacious and environmentally acceptable chemical control technique is a requirement of a comprehensive, integrated response strategy. RESULTS Results of this study demonstrate statistically significant, concentration-dependent effects of azadirachtins, a family of natural compounds derived from the neem tree, on both ALB larval and adult life stages. Growth inhibitory effects on ALB larvae were greatest on early life stages. Significant effects on adults included inhibition of female feeding, oviposition effort and fecundity for adults exposed to azadirachtins via maturation feeding on systemically loaded twigs. CONCLUSION These quarantine laboratory experiments verify multi-mechanistic, deleterious effects on both larval and adult life stages of ALB, an exotic, invasive insect pest of critical importance in North America. Field efficacy studies are required to further understand dose acquisition by larval and adult ALB life stages following systemic injections to host trees under semi-operational use scenarios. Such studies could also be used to test postulates regarding optimal deployment strategies to meet objectives such as slowing the spread of this pest and protection of high-value deciduous forest resources.