Validation of a liquid chromatography/tandem mass spectrometry method for the detection of aflatoxin B1 residues in broiler liver.

Aflatoxin B1 is a carcinogenic and mutagenic mycotoxin produced mainly by Aspergillus flavus and Aspergillus parasiticus. It is the predominant mycotoxin found in raw materials used for the manufacture of broiler feeds. The aim of the present study was to develop a new and optimized method for the detection and quantification of aflatoxin B1 (AFB1) residues in broiler liver using solid phase extraction (SPE) clean-up and liquid chromatography-electrospray ionization/tandem mass spectrometry (LC-ESI-MS/MS) detection. The method was validated for linearity, accuracy, precision, limit of detection (LOD) and limit of quantification (LOQ). The validation parameters indicated satisfactory linearity (r2>0.99), accuracy and precision (4.57% intra-day RSD; 14.65% inter-day RSD) a very high recovery (99±13%) and high sensitivity achieved for AFB1 in animal samples (LOD=0.017 and LOQ=0.050ng/g). The method was effective for the detection and quantification of AFB1 residues in broiler liver and could also be potentially used for detecting AFB1 in other edible animal tissues after natural or experimental AFB1 exposure with high sensitivity and precision.

Assessment of growth of Aspergillus spp. from agricultural soils in the presence of glyphosate.

Agriculture is one of the bases of the Argentine economy. Glyphosate is undoubtedly one of the most important herbicides used. The increasing consumption and the efficiency of glyphosate-based herbicides have encouraged several studies on their persistence in soils, their effects on soil microbiota and their degradation processes. Fungi have been reported as being the main herbicide-degrading microorganisms as well as the most tolerant to environmental stress conditions. This study evaluated the growth performance of Aspergillus section Flavi and Aspergillus niger aggregate strains on Czapek Dox media supplied with a commercial glyphosate formulation as sole source of carbon (CZC), phosphorus (CZP) or nitrogen (CZN). Six Aspergillus spp. strains were evaluated. Each medium was stab-inoculated with fungal spores from 7-day old cultures. Two measures of colony radii were taken daily. All of the Aspergillus section Flavi strains showed a significant increase (from 24 to 44%) in growth rate on the CZN medium, as compared to controls. The A. niger aggregate strains exhibited the same behavioral pattern under all the conditions tested, except on the CZN medium. Velutinous or slightly floccose colonies with abundant sporulation were observed on CZP. Moreover, the colonies produced sparse sporulation on CZC or CZN media, being their appearances completely different from those on the CZP medium. This study establishes that A. section Flavi and A. niger aggregate strains can grow in vitro in the presence of glyphosate, especially when it is used as a sole source of phosphorus or nitrogen.

Isolation of culturable mycobiota from agricultural soils and determination of tolerance to glyphosate of nontoxigenic Aspergillus section Flavi strains.

Glyphosate-based herbicides are extensively used in Argentina's agricultural system to control undesirable weeds. This study was conducted to evaluate the culturable mycobiota [colony forming units (CFU) g(-1) and frequency of fungal genera or species] from an agricultural field exposed to pesticides. In addition, we evaluated the tolerance of A. oryzae and nontoxigenic A. flavus strains to high concentrations (100 to 500 mM - 17,000 to 84,500 ppm) of a glyphosate commercial formulation. The analysis of the mycobiota showed that the frequency of the main fungal genera varied according to the analyzed sampling period. Aspergillus spp. or Aspergillus section Flavi strains were isolated from 20 to 100% of the soil samples. Sterilia spp. were also observed throughout the sampling (50 to 100%). Aspergillus section Flavi tolerance assays showed that all of the tested strains were able to develop at the highest glyphosate concentration tested regardless of the water availability conditions. In general, significant reductions in growth rates were observed with increasing concentrations of the herbicide. However, a complete inhibition of fungal growth was not observed with the concentrations assayed. This study contributes to the knowledge of culturable mycobiota from agricultural soils exposed to pesticides and provides evidence on the effective growth ability of A. oryzae and nontoxigenic A. flavus strains exposed to high glyphosate concentrations in vitro.

Influence of the pesticides glyphosate, chlorpyrifos and atrazine on growth parameters of nonochratoxigenic Aspergillus section Nigri strains isolated from agricultural soils.

This investigation was undertake to determine the effect of glyphosate, chlorpyrifos and atrazine on the lag phase and growth rate of nonochratoxigenic A. niger aggregate strains growing on soil extract medium at -0.70, -2.78 and -7.06 MPa. Under certain conditions, the glyphosate concentrations used significantly increased micelial growth as compared to control. An increase of about 30% was observed for strain AN 251 using 5 and 20 mg L(-1) of glyphosate at -2.78 MPa. The strains behaved differently in the presence of the insecticide chlorpyrifos. A significant decrease in growth rate, compared to control, was observed for all strains except AN 251 at -2.78 MPa with 5 mg L(-1). This strain showed a significant increase in growth rate. With regard to atrazine, significant differences were observed only under some conditions compared to control. An increase in growth rate was observed for strain AN 251 at -2.78 MPa with 5 and 10 mg L(-1) of atrazine. By comparison, a reduction of 25% in growth rate was observed at -7.06 MPa and higher atrazine concentrations. This study shows that glyphosate, chlorpyrifos and atrazine affect the growth parameters of nonochratoxigenic A. niger aggregate strains under in vitro conditions.

Influence of herbicide glyphosate on growth and aflatoxin B1 production by Aspergillus section Flavi strains isolated from soil on in vitro assay.

The effect of six glyphosate concentrations on growth rate and aflatoxin B1 (AFB1) production by Aspergillus section Flavi strains under different water activity (aW) on maize-based medium was investigated. In general, the lag phase decreased as glyphosate concentration increased and all the strains showed the same behavior at the different conditions tested. The glyphosate increased significantly the growth of all Aspergillus section Flavi strains in different percentages with respect to control depending on pesticide concentration. At 5.0 and 10 mM this fact was more evident; however significant differences between both concentrations were not observed in most strains. Aflatoxin B1 production did not show noticeable differences among different pesticide concentrations assayed at all aW in both strains. This study has shown that these Aspergillus flavus and A. parasiticus strains are able to grow effectively and produce aflatoxins in high nutrient status media over a range of glyphosate concentrations under different water activity conditions.

Comparative analysis of the mycobiota and mycotoxins contaminating corn trench silos and silo bags.

BACKGROUND: Silage is one of the most important feed sources for bovines. Mycotoxin contamination of feedstuffs is a worldwide concern. The aim of this study was to compare mycobiota and levels of aflatoxin B1 (AFB1), fumonisin B1 (FB1), deoxynivalenol (DON), zearalenone (ZEA) and patulin (PAT) in corn trench silos and silo bags. RESULTS: Dry matter was higher in trench silos. Counts varied from not detected to 108 CFU g⁻¹ in both trench silos and silo bags. Isolation frequencies of Aspergillus spp. and Fusarium spp. were higher in trench silos, whereas Penicillium spp. was higher in silo bags. Silo bags showed less diversity than trench silos. Strains isolated produced AFB1, FB1 and PAT. In trench silos, AFB1 was the only mycotoxin detected (1-160 µg kg⁻¹). In silo bags AFB1 levels varied from 5.8 to 47.4 µg kg⁻¹. DON was detected in two silo bag samples. CONCLUSION: When handling is adequate the reduction of mould and mycotoxin contamination in silo bags is considerable. This study will enable estimation of the mycotoxicological risk of different ensiling practices and determination of the most adequate method to minimize economic losses and reduce hazard to animal and human health.

Fumonisin occurrence in wheat-based products from Argentina.

In Argentina, wheat is the most consumed cereal by the human population. Since fumonisins occurence in wheat grains and wheat-based products have been reported worldwide, a survey was conducted in order to determine fumonisin contamination in 91 wheat-based products (white wheat flour samples, wheat flour used at bakery products and whole-wheat flour samples) collected from different retail stores of Rio Cuarto city in Argentina using HPLC-MS/MS. Sixty-seven samples (74%) showed contamination by fumonisins. From these samples, 16 showed fumonisin levels between LOD and LOQ (between 0.01 to 0.05 ng/g), while fumonisins (FB1 + FB2) in quantifiable samples ranged from 0.05 ng/g to 18.9 ng/g. Although FB1 was more prevalent, FB2 was foun3d in higher levels than FB1. Overall, fumonisin prevalence was high, but concentrations were far below EU or USA limits set for maize and maize-based products.

Distribution of amines in water/AOT/n-hexane reverse micelles: influence of the amine chemical structure.

The distribution of different aliphatic and aromatic amines: n-butylamine (n-BA), isobutylamine (i-BA), tert-butylamine (t-BA), piperidine (PIP), N,N-dimethylaniline (DMA) and N-methylaniline (MA) in water/sodium 1,4-bis(2-ethylhexyl)sulfosuccinate(AOT)/n-hexane reverse micelles was investigated by steady-state fluorescence measurements. The partition constants were measured by an indirect method based on the effect that amine partitioning exert on the bimolecular rate of the reaction between a microphase incorporated fluorophore (Ru(bpy)2+(3)) and the quencher, (Fe(CN)3-(6)). For MA, that can act as a quencher of the fluorophore a direct method was used. The results show that primary amines have larger partition constants than the secondary ones. For tertiary amines the distribution constants were practically negligible. Laser flash photolysis experiments confirmed that tertiary amines, both aliphatic and aromatic, are not incorporated to the micellar pseudophase. The effect of the amine structure on the partition constant was analyzed through linear solvation free energy relationships (LSER) using solute parameters and compared with those obtained for alcohols. Hydrogen bond interactions with the AOT polar heads appear to be the main driving force for the distribution of amines between the organic and micellar pseudophases, whereas the size of the alkyl or aromatic group tends to hinder it.

Presence of Multiple Mycotoxins and Other Fungal Metabolites in Native Grasses from a Wetland Ecosystem in Argentina Intended for Grazing Cattle.

The aim of this study was to evaluate the occurrence of several fungal metabolites, including mycotoxins in natural grasses (Poaceae) intended for grazing cattle. A total number of 72 and 77 different metabolites were detected on 106 and 69 grass samples collected during 2011 and 2014, respectively. A total of 60 metabolites were found across both years. Among the few mycotoxins considered toxic for ruminants, no samples of natural grasses were contaminated with aflatoxins, ochratoxin A, ergot alkaloids, and gliotoxin, among others. However, we were able to detect important metabolites (toxic to ruminants) such as type A trichothecenes, mainly T-2 toxin and HT-2 toxin (up to 5000 µg/kg each), and zearalenone (up to 2000 µg/kg), all at very high frequencies and levels. Other fungal metabolites that were found to be prevalent were other Fusarium metabolites like beauvericin, equisetin and aurofusarin, metabolites produced by Alternaria spp., sterigmatocystin and its precursors and anthrachinone derivatives. It is important to point out that the profile of common metabolites was shared during both years of sampling, and also that the occurrence of important metabolites is not a sporadic event. Considering that this area of temperate grassland is used for grazing cattle all year long due to the richness in palatable grasses (Poaceae), the present work represents a starting point for further studies on the occurrence of multi-mycotoxins in natural grasses in order to have a complete picture of the extent of cattle exposure. Also, the present study shows that the presence of zeranol in urine of beef cattle may not be a consequence of illegal use of this banned substance, but the product of the natural occurrence of zearalenone and α-zearalenol in natural grasses intended for cattle feeding.

Sequence of Reactant Combination Alters the Course of the Staudinger Reaction of Azides with Acyl Derivatives. Bimanes. 30.

The Staudinger reaction of azides has now been followed by NMR and other spectroscopic techniques. syn-(Azidomethyl,methyl)(methyl,methyl)bimane (1) and Ph(3)P form a triazaphosphadiene intermediate 2 and then the bimane P-triphenyliminophosphorane 3. The iminophosphorane reacts with an acyl chloride to yield an iminophosphonium salt 4 which then forms the oxazaphosphetane 13. The latter undergoes an electrocyclic reversion to form the phosphine oxide and the chloroimines 7E and 7Z, the last being hydrolyzed to the (acylamido)bimane 6. This set of reactions constitutes the "iminophosphorane pathway". A significant diversion of the reaction path to an (N-alkylamino)phosphonium chloride 8 occurs through reaction of 4 with H(2)O present in the CDCl(3) and through reaction of 3 with HCl. A different azide (alpha-azido-o-xylene 1b) produces the (acylamido)-o-xylene as the sole product. A less sterically hindered phosphine (tri-2-furylphosphine) reacts more slowly to form the iminophosphorane 3a from the azidobimane 1. Reaction of the bimane P-tri-2-furyliminophosphorane with acyl chloride gives only the (acylamido)bimane 6. If the acyl chloride is mixed with 1, followed by addition of the Ph(3)P, the triazaphosphadiene adduct 5 is formed via the triazaphosphadiene. The adduct 5 is converted rapidly into a six-membered cyclic compound 11. The latter either loses nitrogen to yield 6 via 7Z and 7E and the phosphine oxide or loses chloride 10 through a novel chloride-induced elimination reaction from its protonated form. The change in procedure thus results in a dramatic change in the reaction pathway, a reaction set that constitutes the "triazaphosphadiene adduct pathway". In the case of alpha-azido-o-xylene, alpha-chloro-o-xylene (10b) is the only product. The reactions of the azides 1 or 1b with tri-2-furylphosphine also produce chlorides as the major products accompanied by some acetamido derivatives. The nucleophile-induced reaction explains a "surprising result" (formation of ester rather than amide) reported by Sahlberg et al. (Sahlberg, C.; Jackson, A. M.; Claesson, A. Acta Chem. Scand. 1988, B42, 556-562). The intramolecular "aza-Wittig" reaction may depend on the nucleophilicity of the triazaphosphadiene. A comprehensive mechanistic scheme for the Staudinger reaction of azides is conveniently divided into the following: (A) formation of the triazaphosphadiene (Scheme 1), (B) reactions of the triazaphosphadiene (Scheme 2), and (C) reactions via the iminophosphorane (Scheme 3). Some approximate kinetic parameters are reported for some of the reactions.

Validation of a liquid chromatography/tandem mass spectrometry method for the detection of aflatoxin B1 residues in broiler liver / Validación de una técnica para detectar aflatoxina B1 en hígado de pollo por cromatografía líquida de alta performance acoplada a espectrómetro de masas en tándem

Aflatoxin B1 is a carcinogenic and mutagenic mycotoxin produced mainly by Aspergillus flavus and Aspergillus parasiticus. It is the predominant mycotoxin found in raw materials used for the manufacture of broiler feeds. The aim of the present study was to develop a new and optimized method for the detection and quantification of aflatoxin B1 (AFB1) residues in broiler liver using solid phase extraction (SPE) clean-up and liquid chromatography-electrospray ionization/tandem mass spectrometry (LC-ESI-MS/MS) detection. The method was validated for linearity, accuracy, precision, limit of detection (LOD) and limit of quantification (LOQ). The validation parameters indicated satisfactory linearity (r² >0.99), accuracy and precision (4.57% intra-day RSD; 14.65% inter-day RSD) a very high recovery (99 ±13%) and high sensitivity achieved for AFB1 in animal samples (LOD = 0.017 and LOQ= 0.050 ng/g). The method was effective for the detection and quantification of AFB1 residues in broiler liver and could also be potentially used for detecting AFB1 in other edible animal tissues after natural or experimental AFB1 exposure with high sensitivity and precision.

La aflatoxina B1 (AFB1) es una micotoxina carcinogénica y mutagénica producida principalmente por Aspergillus flavus y Aspergillus parasiticus. Es la principal toxina que contamina las materias primas utilizadas para la elaboración de alimentos balanceados destinados a la alimentación de pollos parrilleros. El objetivo de este trabajo fue desarrollar un método nuevo y optimizado para detectar y cuantificar bajos niveles de AFB1 en hígado de pollo, usando limpieza por extracción en fase sólida (SPE) y cromatografía líquida acoplada a detección por espectrometría de masa en tándem con ionización por electrospray (LC-ESI-MS/MS). Se validaron la linealidad, la exactitud, la precisión, el límite de detección (LOD) y el límite de cuantificación (LOQ). El método resultó tener linealidad (r²>0,99), exactitud y precisión muy satisfactorias (4,57% RSD intradía; 14,65% RSD interdía), un alto porcentaje de recupero (99 ± 13%) y la sensibilidad más alta lograda para la detección de AFB1 en muestras de origen animal (LOQ=0.050 ng/g y LOD = 0.017). El método fue muy efectivo para detectar y cuantificar bajos niveles de AFB1 en hígados de pollos parrilleros. Este método podría potencialmente utilizarse para la detección de esta toxina en otros tejidos y subproductos de origen animal luego de su exposición a AFB1 con una mayor sensibilidad y precisión.

Mycobiota and mycotoxins contamination in raw materials and finished feed intended for fattening pigs production in eastern Argentina.

The aim of the present study was to determine the mycobiota and natural levels of mycotoxins such as zearalenone, fumonisin B(1), aflatoxin B(1) and ochratoxin A present in raw materials and finished fattening pig feed. Samples were examined for total fungi and genera distribution. Zearalenone, FB(1), AFB(1) and OTA contamination were determined using high pressure liquid chromatography and thin layer chromatography. Milled maize and finished feed samples showed fungal contamination over than 1 × 10(4) CFU/g. All samples contained at least one of the main mycotoxigenic genera Aspergillus, Fusarium and Penicillium. A. flavus and F. verticillioides were the most prevalent species. Only some Aspergillus section Nigri strains from suckling pig to growing pig samples were able to produce OTA. A. flavus strains from milled maize, wheat bran, suckling pig to growing pig samples were able to produce AFB(1). All samples were positive for FB(1). Sucking pig, piglet, growing and boar feed samples showed ZEA natural contamination. AFB(1) and OTA contamination were not detected. There was a 100% correlation between FB(1) and ZEA contamination in sucking pig, piglet, growing and boar feed samples; 50% piglet samples and 67% suckling pig samples showed ZEA levels over the recommended limits. The present study has shown the occurrence of two mycotoxins, FB(1) and ZEA in feed intended for fattening pig consumption. In animal production, the simultaneous presence of toxicogenic fungi and low dietary levels of mycotoxins in field conditions can cause possible health impacts and lost performance in pigs from feeding spoiled feeds.

Occurrence of Fusarium spp. and fumonisin in durum wheat grains.

A survey was carried out to determine Fusarium species and fumonisin contamination in 55 durum wheat (Triticum turgidum L. var. durum) samples collected during two harvest seasons (2007 and 2008) using HPLC and further LC-MS/MS confirmation. All samples showed Fusarium contamination with infection levels ranging from 8 to 66%, F. proliferatum being the species most frequently isolated during 2007 and the second most frequently isolated one during the 2008 harvest season, respectively. Natural contamination with fumonisins was found in both harvest seasons. In 2007, 97% of the samples showed total fumonisin (FB(1) + FB(2)) levels ranging from 10.5 to 1245.7 ng/g, while very low levels of fumonisins were detected in samples collected during 2008. These results could be explained by differences in the amount of rainfall during both periods evaluated. A selected number (n = 48) of F. proliferatum isolates showed fumonisin production capability on autoclaved rice. This is the first report of the presence of natural fumonisins in durum wheat grains.

Occurrence of ochratoxin A and ochratoxigenic mycoflora in corn and corn based foods and feeds in some South American countries.

Cereals and cereal- derived products constitute the base of human and animal feeding in South American countries. This review attempts to give an overview of the ochratoxin A (OTA) occurrence and potential sources of OTA contamination in those products. The environmental conditions as humidity and temperature in the colonization of the substrates by Aspergillus section Nigri isolated from corn kernels were also discussed. The available information on the ochratoxigenic mycoflora and OTA presence in corn, corn based food and feed is limited. Only few surveys have been carried out in Argentina, Ecuador and Brazil; which showed that Aspergillus niger aggregate and A. ochraceus species would be the main source of OTA. It's possible to emphasize that, the species A. carbonarius has not been isolated from these substrates and Penicillium verrucosum was isolated only from pig feeds of Argentinean samples in low percentage. Studies about the ecophysiology of ochratoxigenic fungi and OTA occurrence are in progress in Latin America to reduce the impact of this toxin in the food chain.

Kinetics of the reaction between 2-phenylpropionitrile and 2-chloro-5-nitrotrifluoromethylbenzene under phase-transfer catalysis.

Phase-transfer catalysis (PTC) is a widely accepted methodology in organic synthesis. Although a great number of organic syntheses were reported in PTC conditions, systematic kinetic studies are scarce. In the present report, a detailed study of the kinetics of the reaction between 2-chloro-5-nitrotrifluoromethylbenzene (CNTFB) and 2-phenylpropionitrile anion (HPP-), under PTC, was performed under several conditions. The reaction was carried out either in toluene or chlorobenzene as the organic phase, in the presence of a concentrated aqueous solution of NaOH using tetraalkylammonium (Q+X-) salts as phase-transfer catalysts. The major product was 2-(4-nitro-2-trifluoromethylphenyl)-2-phenylpropionitrile, and its yield depends on the experimental conditions. Different aspects of the mechanism are discussed and quantified. Kinetic data were explained by means of an interfacial mechanism that involves the deprotonation of the adsorbed nucleophile precursor followed by its catalyst-mediated extraction to the organic phase. A multicomponent Langmuir-type interface was assumed. Although the extraction of OH- by catalyst to the organic phase is usually disregarded, the formation of the substrate hydrolysis product that leads to catalyst poisoning was also investigated. The influence of this side reaction on the yield of the main product was established. A discussion about the influence of this side process on the main reaction and the operating mechanism is presented.