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 4 min. 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 μg kg−1 level. Average recoveries (n = 5) ranged from 71% to 108%, with RSDs lower than 14

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

BACKGROUND: The Asian long-horned beetle [ALB; Anoplophora glabripennis (Motschulsky)] is an invasive, wood-boringinsect posing significant economic and ecological threats to the deciduous forests of North America. An efficacious andenvironmentally 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 familyof natural compounds derived f rom the neem tree, on both ALB larval and adult life stages. Growth inhibitory effects on ALBlarvae were greatest on early life stages. Significant effects on adults included inhibition of female feeding, oviposition effortand 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 adultlife stages of ALB, an exotic, invasive insect pest of critical importance in North America. Field efficacy studies are requiredto further understand dose acquisition by larval and adult ALB life stages following systemic injections to host trees undersemi-operational use scenarios. Such studies could also be used to test postulates regarding optimal deployment strategiesto meet objectives such as slowing the spread of this pest and protection of high-value deciduous forest resources

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

Study of cyanotoxin degradation and evaluation of their transformation products in surface waters by LC-QTOF MS

In the present work, the degradation of three cyanotoxins from the hepatotoxins group was investigated under laboratory-controlled experiments in water samples. Surface waters spiked with microcystin-LR (MC-LR), nodularin (NOD) and cylindrospermopsin (CYN) were subjected to hydrolysis, chlorination and photo-degradation, under both sunlight (SL) and ultraviolet (UV) radiation. A total of 12 transformation products (TPs) were detected and tentatively identified by liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (LC-QTOF MS). These comprised: 6 chlorination TPs (3 from CYN and 3 from MC-LR, 2 isomers); 4 UV TPs (all from CYN); and 2 sunlight TPs (one isomer from MC-LR and another from NOD). No TPs were observed under hydrolysis conditions. The chemical structures for all TPs were tentatively proposed based on the accurate-mass QTOF MS full-spectra. Analysis of real-world samples collected from the Peñol reservoir (Antioquia, Colombia) revealed the presence of MC-LR and CYN as well as a sunlight TP identified in the laboratory experiments. Data presented in this article will assist further research on TPs potentially formed in future tertiary degradation processes applied for the removal of organic micro-pollutants in water; as well as improving available knowledge on the toxic implications of cyanobacterial toxins TPs in surface waters

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 , 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 TreeAzinTM

Triacylglycerol Analysis in Human Milk and Other Mammalian Species: Small-Scale Sample Preparation, Characterization, and Statistical Classification Using HPLC-ELSD Profiles

In this work, a method for the separation of triacylglycerols (TAGs) present in human milk and from other mammalian species by reversed-phase high-performance liquid chromatography using a core–shell particle packed column with UV and evaporative light-scattering detectors is described. Under optimal conditions, a mobile phase containing acetonitrile/n-pentanol at 10 °C gave an excellent resolution among more than 50 TAG peaks. A small-scale method for fat extraction in these milks (particularly of interest for human milk samples) using minimal amounts of sample and reagents was also developed. The proposed extraction protocol and the traditional method were compared, giving similar results, with respect to the total fat and relative TAG contents. Finally, a statistical study based on linear discriminant analysis on the TAG composition of different types of milks (human, cow, sheep, and goat) was carried out to differentiate the samples according to their mammalian origin

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.EU Seventh Framework Programme by ARRAINA Project, 288925. project SAFE-PAP, Research Council Research and Development Project National Institute of Nutrition and Seafood Research (NIFES, Norway), 227387. Generalitat Valenciana, PROMETEO II/2014/023 PROMETEO II/2014/085 ISIC/2012/01

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 MSE 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.Generalitat Valenciana (research group of excellence PROMETEO II/2014/023; ISIC 2012/016), from Alcaldía Mayor de Bogotá (Special Agreement on Cooperation in Science and Technology No. 2215100-153-2013) and from Universidad Antonio Nariño Grant 2010246

Investigación de Toxinas Naturales en Aguas y Alimentos mediante Cromatografí­a Lí­quida acoplada a Espectrometrí­a de Masas en Tándem

La presencia de contaminantes de origen natural en productos destinados al consumo humano ha suscitado una gran preocupación en los últimos años debido al mayor conocimiento de sus efectos nocivos para la salud humana. Por este motivo, los organismos responsables de velar por la salud de los ciudadanos, han decretado los niveles máximos permitidos de dichos contaminantes en alimentos y aguas. Se requiere pues metodología analítica que permita la determinación fiable de dichos contaminantes en las matrices correspondientes a los bajos niveles requeridos por la legislación. En la presente Tesis Doctoral se ha estudiado el potencial analítico y la aplicabilidad del acoplamiento cromatografía líquida-espectrometría de masas en tándem (LC-MS/MS) con analizador de triple cuadrupolo (QqQ), en el campo del análisis de tóxicos de origen natural en muestras alimenticias y mediambientales. El elevado potencial de esta técnica ha permitido el desarrollo de metodología analítica rápida, sensible, selectiva y fiable para la determinación de dichas toxinas. Además, todos los métodos de análisis desarrollados se han validado siguiendo los criterios de calidad recogidos en las guías europeas, desde el punto de vista cualitativo y cuantitativo, con el fin de que los datos obtenidos presenten elevada fiabilidad. La investigación realizada en esta Tesis Doctoral se estructura en dos bloques, estudiándose en el primero de ellos las toxinas correspondientes al grupo conocido como micotoxinas, mientras que el segundo está dedicado a las ficotoxinas. Esta estructura se ha elegido para agrupar los trabajos según los tóxicos y el tipo de matrices estudiadas. En el primer bloque se han desarrollado metodologías para el análisis de micotoxinas en alimentos en su mayoría de origen agrícola. En el primer trabajo se ha estudiado el potencial de la cromatografía líquida de ultra resolución acoplada a espectrometría de masas en tándem (UHPLC-MS/MS) para el desarrollo de un método multianalito que permita la determinación simultánea de las micotoxinas legisladas, con propiedades físico-químicas muy diferentes, de manera rápida, sensible y selectiva. Se evaluó las diferentes opciones que permitieran realizar una correcta cuantificación de las muestras, seleccionando el calibrado en matriz como la opción más favorable. Esta metodología se validó y aplicó al análisis de diferentes muestras pertenecientes a cereales y alimentos infantiles. En el segundo trabajo se amplió el número de analitos, introduciendo micotoxinas estructuralmente relacionadas. Se estudió el potencial de esta técnica para el análisis de un elevado número de matrices utilizando la aproximación “dilute and shoot† con el objetivo de poder determinar simultáneamente todos los compuestos seleccionados en una única inyección. Se consiguió validar la metodología analítica para todos los compuestos legislados a concentraciones inferiores a las establecidas por el reglamento de la Comisión Europea. Esta metodología se aplicó al análisis de muestras pertenecientes a un estudio de dieta total (EDT) de la Comunidad Valenciana mediante el cual se pretendió determinar la ingesta de este tipo de contaminantes en los productos de consumo más habituales en la población de esta región. El tercer y cuarto trabajo abordan distintos problemas analíticos surgidos en el desarrollo de la metodología multianalito. En el tercer trabajo se puso especial énfasis en aumentar la sensibilidad de la metodología analítica con el fin de determinar concentraciones sub-µg/kg de determinadas aflatoxinas y ochratoxina A en muestras de alimentos destinadas a bebés y niños de corta edad, según se establece en la regulación establecida por la Comunidad Europea. Se evaluó el uso de la extracción en fase sólida (SPE) como la opción que permite la preconcentración de los analitos y purificación de los extractos, comparando el uso de diferentes materiales. Se observó que los mejores resultados se obtenian mediante el uso de cartuchos de inmunoafinidad, destacando su elevada selectividad para las micotoxinas objeto de interés. En la última parte de este primer bloque se desarrolló metodología analítica para la determinación de la micotoxina patulina, la cual, debido a sus propiedades físico-químicas, no pudo ser incluida en los métodos multianalito. Debido a su elevada polaridad y bajo peso molecular se requirió una cuidadosa optimización del tratamiento de muestra, así como de las condiciones cromatográficas y espectrométricas. Se comparó el uso de dos fuentes de ionización a presión atmosférica, la ionización electrospray (ESI) y la ionización química a presión atmosférica (APCI). Mediante el uso de la APCI se consiguió minimizar los problemas de supresión iónica debidos a la matriz observados en la fuente ESI. De este modo se consiguió la determinación de este compuesto en productos derivados de manzana, tanto sólidos como líquidos. En el caso de los productos sólidos se realizó una extracción sólido-líquido, mientras que en el caso de las muestras líquidas se llevó a cabo la inyección directa en el sistema cromatográfico tras una etapa de dilución. El segundo bloque aborda el análisis de ficotoxinas en muestras ambientales y alimenticias. En el primer trabajo de este bloque se desarrolló metodología analítica para la determinación de microcistinas y nodularina, toxinas procedentes de algas cianobacterianas, en aguas superficiales y de consumo. En este caso, al igual que en las micotoxinas, se pretendía desarrollar un método multianalito que permitiera la detección simultánea de la nodularina y las seis microcistinas seleccionadas. Se estableció el objetivo de poder determinar cada una de las toxinas seleccionadas a concentraciones inferiores a 1 µg/L (límite máximo establecido en la legislación Española para la microcistina LR). El procedimiento desarrollado incluyó una etapa de pre-concentración de los analitos en línea con el sistema UHPLC-MS/MS, el cual, con la inyección de 1 mL de muestra y de una manera totalmente automatizada, consiguió alcanzar las concentraciones deseadas. Para finalizar, en el segundo trabajo de este bloque se desarrolló metodología analítica basada en cromatografía líquida de alta resolución acoplada a espectrometría de másas en tándem (HPLC-MS/MS) para la determinación simultánea de toxinas marinas pertenecientes a diferentes grupos o familias. Este hecho aumentó la dificultad de la tarea ya que se pretendía la determinación de compuestos iónicos simultáneamente con compuestos de mayor apolaridad mediante cromatografía de fase reversa. Para ello se hizo uso de cromatografía de pares iónicos con el objetivo de aumentar la retención de los compuestos iónicos. Esta metodología se aplicó al análisis de muestras de vieiras contaminadas naturalmente en un “bloom† de algas tóxicas y sometidas posteriormente a un proceso de depuración, en las que se detectó la presencia de ácido domoico. El estudio de la concentración de ácido domoico en muestras tomadas a diferentes días posteriores a su contaminación permitió establecer la cinética de depuración de este contaminante en este tipo de organismos bivalvos

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