Enhancing the activity of zearalenone lactone hydrolase toward the more toxic α-zearalanol via a single-point mutation.

Zearalenone (ZEN) and its derivatives are estrogenic mycotoxins known to pose significant health threats to humans and animals. Especially, the derivative α-zearalanol (α-ZAL) is over 10 times more toxic than ZEN. Simultaneous degradation of ZEN and its derivatives, especially α-ZAL, using ZEN lactone hydrolases (ZHDs) is a promising solution to eliminate their potential hazards to food safety. However, most available ZHDs exhibit limited activity toward the more toxic α-ZAL compared to ZEN. Here, we identified a broad-substrate spectrum ZHD, named ZHDAY3, from Exophiala aquamarina CBS 119918, which could not only efficiently degrade ZEN but also exhibited 73% relative activity toward α-ZAL. Through rational design, we obtained the ZHDAY3(N153H) mutant, which exhibited the highest specific activity (253.3 ± 4.3 U/mg) reported so far for degrading α-ZAL. Molecular docking, structural comparative analysis, and kinetic analysis collectively suggested that the shorter distance between the side chain of the catalytic residue His242 and the lactone bond of α-ZAL and the increased binding affinity to the substrate were mainly responsible for the improved catalytic activity of ZHDAY3(N153H) mutant. This mechanism was further validated through additional molecular docking of 18 mutants and experimental verification of six mutants.IMPORTANCEThe mycotoxins zearalenone (ZEN) and its derivatives pose a significant threat to food safety. Here, we present a highly promising ZEN lactone hydrolase (ZHD), ZHDAY3, which is capable of efficiently degrading both ZEN and the more toxic derivative α-ZAL. Next, the ZHDAY3(N153H) mutant obtained by single-point mutation exhibited the highest specific activity for degrading α-ZAL reported thus far. We further elucidated the molecular mechanisms underlying the enhanced hydrolytic activity of ZHDAY3(N153H) toward α-ZAL. These findings represent the first investigation on the molecular mechanism of ZHDs against α-ZAL and are expected to provide a significant reference for further rational engineering of ZHDs, which will ultimately contribute to addressing the health risks and food safety issues posed by ZEN-like mycotoxins.

The Trp183 is essential in lactonohydrolase ZHD detoxifying zearalenone and zearalenols.

Lactonohydrolase ZHD can detoxify oestrogenic mycotoxin zearalenone and zearalenols through hydrolysis and decarboxylation. The detail mechanism, especially the role of Trp183, which interacts with substrate through p-π interaction and one hydrogen bond, is still unknown. The Trp183 mutants abolished activity to ZEN, α-ZOL and ß-ZOL, except that W183F mutant retained about 40% activity against α-ZOL. In two W183F-reactant complex structures the reactants still bind at the active position and it suggested that this p-π interaction takes responsible for the reactants recognization and allocation. Further, the ZHD-productant complex structures showed that the resorcinol ring of hydrolysed α-ZOL and hydrolysed ß-ZOL move a distance of one ring as compare to the resorcinol ring of reactant α-ZOL and ß-ZOL. The same movement also found in comparison of hydrolysed ZEN and ZEN. In the structure of W183F complex with hydrolysed α-ZOL the resorcinol ring of hydrolysed α-ZOL doesn't move as compare to the resorcinol ring of reactant α-ZOL. It suggested the Trp183 coordinated hydrogen bond takes responsible for the movement of the hydrolysed product. These functional and structural results suggested that Trp183 is essential for ZHD detoxifying zearalenone and zearalenols.

Identification of a Potent Enzyme for the Detoxification of Zearalenone.

The occurrence of mycotoxin zearalenone (ZEN) and its derivatives has been a severe global threat to food and animals. In addition to the chemical and physical degradation methods, a powerful biocatalyst is urgently required for the detoxification of ZEN. Here, an efficient ZEN-degrading lactonase from Gliocladium roseum, named ZENG, was identified for the first time. The recombinant ZENG exhibited the highest activity at pH 7.0 and 38 °C. In addition, the recombinant enzyme showed a high degrading performance toward ZEN and its toxic derivatives α-zearalenol (α-ZOL) and α-zearalanol (α-ZAL), with the specific activities as 315, 187, and 117 units/mg, respectively. To meet the industrial demands, attempts were also made to enhance the thermostability of ZENG using a structure-based modification. Three double-site mutants, including H134L/S136L, H134F/S136F, and H134I/S134I, in the position between the cap and core catalytic domain of ZENG were designed. Finally, the thermostability of both H134L/S136L and H134F/S136F displayed a significant improvement compared to the wild-type enzyme.

Manganese protects wheat from the mycotoxin zearalenone and its derivatives.

Searching for factors that reduce zearalenone (ZEN) toxicity is an important challenge in wheat production, considering that this crop is a basic dietary ingredient. ZEN, absorbed by cells, is metabolized into α-zearalenol and α-zearalanol, and this study focused on the function of manganese ions as potential protectants against the mycotoxins. Stress effects were invoked by an application of 30 µM ZEN and its derivatives. Manganese ions were applied at 100 µM, not stress-inducing concentration. Importance of the biomembrane structures in the absorption of the mycotoxins was demonstrated in in vitro wheat calli and on model membranes. ZEN showed the greatest and α-zearalanol the smallest stressogenic effect manifested as a decrease in the calli growth. This was confirmed by variable increase in antioxidant enzyme activity. Mn ions added to the toxin mixture diminished stressogenic properties of the toxins. Variable decrease in total lipid content and the percentage of phospholipid fraction detected in calli cells exposed to ZEN and its metabolites indicated significance of the membrane structure. An analysis of physicochemical parameters of model membranes build from phosphatidylcholine, a basic lipid in native membranes, and its mixture with the tested toxins made by Langmuir technique and verified by Brewster angle microscopy, confirmed variable contribution of ZEN and its derivatives to the modification of membrane properties. The order of toxicity was as follows: ZEN ≥ α-zearalenol > α-zearalanol. Manganese ions present in the hydrophilic phase interacted with polar lipid groups and reduced the extent of membrane modification caused by the mycotoxins.

Formation of Zearalenone Metabolites in Tempeh Fermentation.

Tempeh is a common food in Indonesia, produced by fungal fermentation of soybeans using Rhizopus sp., as well as Aspergillus oryzae, for inoculation. Analogously, for economic reasons, mixtures of maize and soybeans are used for the production of so-called tempeh-like products. For maize, a contamination with the mycoestrogen zearalenone (ZEN) has been frequently reported. ZEN is a mycotoxin which is known to be metabolized by Rhizopus and Aspergillus species. Consequently, this study focused on the ZEN transformation during tempeh fermentation. Five fungal strains of the genera Rhizopus and Aspergillus, isolated from fresh Indonesian tempeh and authentic Indonesian inocula, were utilized for tempeh manufacturing from a maize/soybean mixture (30:70) at laboratory-scale. Furthermore, comparable tempeh-like products obtained from Indonesian markets were analyzed. Results from the HPLC-MS/MS analyses show that ZEN is intensely transformed into its metabolites α-zearalenol (α-ZEL), ZEN-14-sulfate, α-ZEL-sulfate, ZEN-14-glucoside, and ZEN-16-glucoside in tempeh production. α-ZEL, being significantly more toxic than ZEN, was the main metabolite in most of the Rhizopus incubations, while in Aspergillus oryzae fermentations ZEN-14-sulfate was predominantly formed. Additionally, two of the 14 authentic samples were contaminated with ZEN, α-ZEL and ZEN-14-sulfate, and in two further samples, ZEN and α-ZEL, were determined. Consequently, tempeh fermentation of ZEN-contaminated maize/soybean mixture may lead to toxification of the food item by formation of the reductive ZEN metabolite, α-ZEL, under model as well as authentic conditions.

Insights into In Vivo Absolute Oral Bioavailability, Biotransformation, and Toxicokinetics of Zearalenone, α-Zearalenol, ß-Zearalenol, Zearalenone-14-glucoside, and Zearalenone-14-sulfate in Pigs.

The aim of this study was to determine the toxicokinetic characteristics of ZEN and its modified forms, α-zearalenol (α-ZEL), ß-zearalenol (ß-ZEL), zearalenone-14-glucoside (ZEN14G), and zearalenone-14-sulfate (ZEN14S), including presystemic and systemic hydrolysis in pigs. Crossover pig trials were performed by means of intravenous and oral administration of ZEN and its modified forms. Systemic plasma concentrations of the administered toxins and their metabolites were quantified and further processed via tailor-made compartmental toxicokinetic models. Furthermore, portal plasma was analyzed to unravel the site of hydrolysis, and urine samples were analyzed to determine urinary excretion. Results demonstrate complete presystemic hydrolysis of ZEN14G and ZEN14S to ZEN and high oral bioavailability for all administered compounds, with further extensive first-pass glucuronidation. Conclusively, the modified-ZEN forms α-ZEL, ß-ZEL, ZEN14G, and ZEN14S contribute to overall ZEN systemic toxicity in pigs and should be taken into account for risk assessment.

Transfer of Fusarium mycotoxins from malt to boiled wort.

The fate of deoxynivalenol, deoxynivalenol-3-glucoside, 3- and 15-acetyl-deoxynivalenol, zearalenone, α- and ß-zearalenol and fumonisins (fumonisin B1 and fumonisin B2) through mashing and wort boiling was studied. Three different mycotoxin contamination scenarios were considered. In almost all samples an increase in the level of mycotoxins in wort was observed during mashing followed by a decrease after just 30 min of the process, with levels remaining constant until the end of boiling. Deoxynivalenol and its metabolites were reduced to their initial level contained in the malt before mashing, or even lower, however in none of the samples they were completely eliminated. Zearalenone was not quantitated at the end of boiling, although there was a significant initial level of ZEN. ß-Zearalenol remained unaltered during the process. Fumonisins were reduced by between 50 and 100 per cent during mashing and boiling.

Theoretical Study on Zearalenol Compounds Binding with Wild Type Zearalenone Hydrolase and V153H Mutant.

Zearalenone hydrolase (ZHD) is the only reported α/ß-hydrolase that can detoxify zearalenone (ZEN). ZHD has demonstrated its potential as a treatment for ZEN contamination that will not result in damage to cereal crops. Recent researches have shown that the V153H mutant ZHD increased the specific activity against α-ZOL, but decreased its specific activity to ß-ZOL. To understand whyV153H mutation showed catalytic specificity for α-ZOL, four molecular dynamics simulations combining with protein network analysis for wild type ZHD α-ZOL, ZHD ß-ZOL, V153H α-ZOL, and V153H ß-ZOL complexes were performed using Gromacs software. Our theoretical results indicated that the V153H mutant could cause a conformational switch at the cap domain (residues Gly161⁻Thr190) to affect the relative position catalytic residue (H242). Protein network analysis illustrated that the V153H mutation enhanced the communication with the whole protein and residues with high betweenness in the four complexes, which were primarily assembled in the cap domain and residues Met241 to Tyr245 regions. In addition, the existence of α-ZOL binding to V153H mutation enlarged the distance from the OAE atom in α-ZOL to the NE2 atom in His242, which prompted the side chain of H242 to the position with catalytic activity, thereby increasing the activity of V153H on the α-ZOL. Furthermore, α-ZOL could easily form a right attack angle and attack distance in the ZHD and α-ZOL complex to guarantee catalytic reaction. The alanine scanning results indicated that modifications of the residues in the cap domain produced significant changes in the binding affinity for α-ZOL and ß-ZOL. Our results may provide useful theoretical evidence for the mechanism underlying the catalytic specificity of ZHD.

[Determination of zearalenone and α-zearalenol in vegetable oil and grain products by C_(18)-Al_2O_3 solid phase extraction column purification coupled with ultra-performance liquid chromatography tandem mass spectrometry].

OBJECTIVE: To develop a method for simultaneous determination of zearalenone( ZEN) and α-zearalenol( α-ZEL) in vegetable oil and grain products by solid phase extraction column purification coupled with ultra-performance liquid chromatography tandem mass spectrometry. METHODS: Firstly, ZEN and α-ZEL in grain products were extracted by hexane/ethyl acetate( 50 : 50, V/V), and then extracted as vegetable oil by acetonitrile-water solution( 90: 10, V/V), and purified by C_(18)-Al_2O_3 solid phase extraction column. ZEN and α-ZEL was separated by UPLC with acetonitrile-water gradient elution on C_(18) column( 2. 1 mm × 100 mm, 1. 6 µm), and qualified/quantified by mass spectrometry with ESI negative MRM mode with ~(13)C_(18)-zearalenone as internal standard. RESULTS: The linearity of ZEN and α-ZEL ranged from 1. 0-500 ng/mL. The limit of detection for ZEN and α-ZEL in vegetable oil and grain products was 0. 3 and 0. 2 µg/kg, respectively. The limit of quantification for ZEN and α-ZEL in vegetable oil and grain products was 1. 0 and 0. 5 µg/kg. The average recoveries of ZEN and α-ZEL for spiked samples of 1. 0-100 µg/kg were 93. 5%-108. 0% and 92. 0%-105. 0%. The relative standard deviations of ZEN and α-ZEL were 3. 2%-8. 5% and 4. 6%-7. 8%( n = 6). 55 samples sold in Hangzhou supermarkets were analyzed. ZEN was detected in all corn germ oil with median and maximum contents of 126. 2 and 453. 1 µg/kg. α-ZEL was detected in 50% corn germ oil with median and maximum contents of 2. 0 and 5. 0µg/kg. CONCLUSION: The method possesses several advantages including sensitivity, precision, good efficiency of purification, simplicity and economy, and it is applicable to the batch analysis of zearalenone and α-zearalenol in vegetable oil and grain products.

Interaction of α- and ß-zearalenols with ß-cyclodextrins.

Zearalenone (ZEN) is a mycotoxin produced by Fusarium fungi. ZEN primarily contaminates different cereals, and exerts a strong xenoestrogenic effect in animals and humans. ZEN is a fluorescent mycotoxin, although molecular interactions and microenvironmental changes significantly modify its spectral properties. During biotransformation, ZEN is converted into α-zearalenol (α-ZOL) and ß-zearalenol (ß-ZOL), the toxic metabolites of ZEN, which mimick the effect of estrogen. Cyclodextrins (CDs) are host molecules, and have been studied extensively; they can form stable complexes with several mycotoxins, including ZEN. However, information is limited regarding the interactions of CDs with ZOLs. Therefore, we studied the interactions of α- and ß-ZOLs with native and six chemically modified ß-CDs by fluorescence spectroscopy. Fluorescence enhancement during complex formation, as well as binding constants, were determined. To understand ZOL-CD interactions better, molecular modeling studies were also carried out. Both mycotoxin derivatives formed the most stable complexes with methylated and sulfobutylated CD-derivatives; however, the CD complexes of α-ZOL were significantly stronger than those of ß-ZOL. The data presented here indicate which of the chemically modified ß-CDs appear more suitable as fluorescence enhancers or as potential mycotoxin binders.

Reaction of zearalenone and α-zearalenol with allyl isothiocyanate, characterization of reaction products, their bioaccessibility and bioavailability in vitro.

This study investigates the reduction of zearalenone (ZEA) and α-zearalenol (α-ZOL) on a solution model using allyl isothiocyanate (AITC) and also determines the bioaccessibility and bioavailability of the reaction products isolated and identified by MS-LIT. Mycotoxin reductions were dose-dependent, and ZEA levels decreased more than α-ZOL, ranging from 0.2 to 96.9% and 0 to 89.5% respectively, with no difference (p⩽0.05) between pH 4 and 7. Overall, simulated gastric bioaccessibility was higher than duodenal bioaccessibility for both mycotoxins and mycotoxin-AITC conjugates, with duodenal fractions representing ⩾63.5% of the original concentration. Simulated bioavailability of reaction products (α-ZOL/ZEA-AITC) were lower than 42.13%, but significantly higher than the original mycotoxins. The cytotoxicity of α-ZOL and ZEA in Caco-2/TC7 cells was also evaluated, with toxic effects observed at higher levels than 75µM. Further studies should be performed to evaluate the toxicity and estrogenic effect of α-ZOL/ZEA-AITC.

Crocin protects human embryonic kidney cells (HEK293) from α- and ß-Zearalenol-induced ER stress and apoptosis.

α-zearalenol (α-ZOL) and ß-zearalenol (ß-ZOL) are the major metabolites of Zearalenone (ZEN) and are known to induce many toxic effects. In the present study, we investigated the involvement of endoplasmic reticulum (ER) stress in α- and ß-ZOL-mediated toxicity in human kidney cells (HEK293) and evaluated the effect of a common dietary compound Crocin (CRO), from saffron. We show that α- and ß-ZOL treatment induces ER stress as evidenced by the upregulation of the 78 kDa glucose-regulated protein (GRP78) and the Growth arrest and DNA damage-inducible protein (GADD34). Activation of the ER stress response is associated with activation of the mitochondrial pathway of apoptosis. This apoptotic process is characterized by an increase in ROS generation and lipid peroxidation, a loss of mitochondrial transmembrane potential (ΔΨm) and activation of caspases. We also demonstrate that the antioxidant properties of CRO help to prevent ER stress and reduce α- and ß-ZOL-induced apoptosis in HEK293 cells. Our results suggest that saffron consumption might be helpful to prevent α- and ß-ZOL-induced ER stress and toxicity.

Physiologically-based toxicokinetic modeling of zearalenone and its metabolites: application to the Jersey girl study.

Zearalenone (ZEA), a fungal mycotoxin, and its metabolite zeranol (ZAL) are known estrogen agonists in mammals, and are found as contaminants in food. Zeranol, which is more potent than ZEA and comparable in potency to estradiol, is also added as a growth additive in beef in the US and Canada. This article presents the development and application of a Physiologically-Based Toxicokinetic (PBTK) model for ZEA and ZAL and their primary metabolites, zearalenol, zearalanone, and their conjugated glucuronides, for rats and for human subjects. The PBTK modeling study explicitly simulates critical metabolic pathways in the gastrointestinal and hepatic systems. Metabolic events such as dehydrogenation and glucuronidation of the chemicals, which have direct effects on the accumulation and elimination of the toxic compounds, have been quantified. The PBTK model considers urinary and fecal excretion and biliary recirculation and compares the predicted biomarkers of blood, urinary and fecal concentrations with published in vivo measurements in rats and human subjects. Additionally, the toxicokinetic model has been coupled with a novel probabilistic dietary exposure model and applied to the Jersey Girl Study (JGS), which involved measurement of mycoestrogens as urinary biomarkers, in a cohort of young girls in New Jersey, USA. A probabilistic exposure characterization for the study population has been conducted and the predicted urinary concentrations have been compared to measurements considering inter-individual physiological and dietary variability. The in vivo measurements from the JGS fall within the high and low predicted distributions of biomarker values corresponding to dietary exposure estimates calculated by the probabilistic modeling system. The work described here is the first of its kind to present a comprehensive framework developing estimates of potential exposures to mycotoxins and linking them with biologically relevant doses and biomarker measurements, including a systematic characterization of uncertainties in exposure and dose estimation for a vulnerable population.

Sorption and mineral-promoted transformation of synthetic hormone growth promoters in soil systems.

This work examines the fate of synthetic growth promoters (trenbolone acetate, melengestrol acetate, and zeranol) in sterilized soil systems, focusing on their sorption to organic matter and propensity for mineral-promoted reactions. In organic-rich soil matrices (e.g., Pahokee Peat), the extent and reversibility of sorption did not generally correlate with compound hydrophobicity (e.g., K(ow) values), suggesting that specific binding interactions (e.g., potentially hydrogen bonding through C17 hydroxyl groups for the trenbolone and melengestrol families) can also contribute to uptake. In soils with lower organic carbon contents (1-5.9% OC), evidence supports sorption occurring in parallel with surface reaction on inorganic mineral phases. Subsequent experiments with pure mineral phases representative of those naturally abundant in soil (e.g., iron, silica, and manganese oxides) suggest that growth promoters are prone to mineral-promoted oxidation, hydrolysis, and/or nucleophilic (e.g., H2O or OH(-)) addition reactions. Although reaction products remain unidentified, this study shows that synthetic growth promoters can undergo abiotic transformation in soil systems, a previously unidentified fate pathway with implications for their persistence and ecosystem effects in the subsurface.

Zearalenone (ZEN) metabolism and residue concentrations in physiological specimens of dairy cows exposed long-term to ZEN-contaminated diets differing in concentrate feed proportions.

A long-term feeding experiment with dairy cows was performed to investigate the effects of feeding a Fusarium toxin contaminated (FUS) and a background-contaminated control (CON) ration with a mean concentrate feed proportion of 50% during the first 11 weeks after parturition (Groups FUS-50, CON-50, Period 1), and with concentrate feed proportions of 30% or 60% during the remaining 17 weeks (Groups CON-30, CON-60, FUS-30 and FUS-60, Period 2), on zearalenone (ZEN) residue levels in blood serum, milk, urine and bile. ZEN, α-zearalenol (α-ZEL) and ß-zearalenol (ß-ZEL), zearalanone (ZAL), α-zearalanol (α-ZAL) and ß-zearalanol (ß-ZAL) were determined by HPLC with fluorescence detection. The ZEN concentrations of the rations fed to Groups CON-50, FUS-50 (Period 1), CON-30, CON-60, FUS-30 and FUS-60 (Period 2) amounted to 53.1, 112.7, 35.0, 24.4, 73.8 and 72.5 µg/kg dry matter, respectively. The concentrations of ZEN, α-ZEL, ß-ZEL, ZAN, α-ZAL and ß-ZAL in serum, urine and milk were lower than 1, 1, 4, 100, 50 and 200 ng/g, respectively, while ZEN, α-ZEL and ß-ZEL were detected in bile. Their levels changed with oral ZEN exposure in the course of the experiment and in a similar direction with concentrate feed proportion (Period 2 only). Thus the proportions of the individual ß-ZEL, α-ZEL and ZEN concentrations of their sum varied only in narrow ranges of 68-76%, 6-13% and 12-20%, respectively. Interestingly, the bile concentrations of ß-ZEL, α-ZEL and ZEN of Groups CON-60 and FUS-60 amounted to only approximately 50%, 45% and 62%, respectively, of those of Groups CON-30 and FUS-30 despite a similar or even lower ZEN exposure. The results indicate that conversion of ZEN to its detectable metabolites was not changed by different dietary concentrate feed proportions while their absolute levels were decreased. These findings might suggest concentrate feed proportion-dependent and rumen fermentation-mediated alterations in ZEN/metabolite degradation, and/or liver associated alterations in bile formation and turnover.

Changes in Th1 and Th2 cytokine concentrations in ileal Peyer's patches in gilts exposed to zearalenone.

GALT induces tolerance to foreign food antigens and plays an important role in the development of food allergies and the inflammatory bowel disease. The immune function of GALT is significantly influenced by an equilibrium between Th1 and Th2 subpopulations and the cytokines they produce. Th1 cytokines participate in the induction of a cell-mediated immune response, whereas Th2 cytokines induce powerful antibody-mediated responses. Changes in Th1/Th2 cell polarization of an immune response are associated with susceptibility to autoimmune and infectious diseases. This experiment investigated changes in cytokine levels produced by Th1 and Th2 cells in ileal Payer's patches in gilts exposed to ZEN doses below the NOEL (approximately 8 microg kg(-1) BW) for 14, 28 and 42 days. A significant linear increase in IL-4 (40.32 +/- 1.55 ng mg(-1)--137.60 +/- 29.96 ng mg(-1)), and IL-10 (5.99 +/- 0.15 ng mg(-1)--16.39 +/- 1.11 ng mg(-1)) concentrations was observed. An increase in Th1 (IL-2 and IFN-gamma) cytokine levels was also noted in the experimental group, but it was not statistically significant. An HPLC analysis of Peyer's patches in group E animals revealed a linear increase in ZEN concentrations (3.65 +/- 0.91 ng g(-1)--4.72 +/- 1.85 ng g(-1)) and an absence of alpha-ZEL. IL-4 stimulates monocytes and macrophages, it induces the production of proinflammatory cytokines and it may directly and indirectly contribute to the development of inflammatory foci. Higher IL-4 levels could shift polarization toward Th2 cells, stimulate B cells to undergo class switching to produce IgE and contribute to the development of allergies.

Simultaneous determination of six resorcylic acid lactones in feed using liquid chromatography-tandem mass spectrometry and multi-walled carbon nanotubes as a dispersive solid phase extraction sorbent.

A simple and cost-effective pre-treatment procedure was developed for six resorcylic acid lactones (RALs) in feed using dispersive solid phase extraction (dSPE) with multi-walled carbon nanotubes (MWCNTs). The sample was analysed after purification by ultra-high performance liquid chromatography-negative electrospray ionisation tandem mass spectrometry (UHPLC-ESI-MS/MS). After extraction with acetonitrile/water (80:20, v/v) and dilution with water, a dSPE procedure was carried out with MWCNTs. The pH value of the extract, the extraction time for MWCNTs, the type and amount of MWCNTs and the type of eluent were optimised to increase the sample throughput and the sensitivity. The samples were quantified using the internal standard zearalenone-D6. The absolute recoveries of the target compounds from feed samples were most efficient when using 100mg of MWCNTs with an outer diameter of less than 8nm and a length of 10-30µm, and ethyl acetate was shown to be the most suitable solvent for desorbing the target compounds from the MWCNTs. The mean recoveries from fortified swine mixed feed samples ranged from 95.3% to 107.2% and had relative standard deviations lower than 10%; the limits of detection and quantification for RALs were in the ranges of 0.20-0.29µg/kg and 0.54-0.78µg/kg, respectively.

An optimised method for the accurate determination of zeranol and diethylstilbestrol in animal tissues using isotope dilution-liquid chromatography/mass spectrometry.

Isotope dilution-liquid chromatography/mass spectrometry (ID-LC/MS) has been established as a candidate reference method for the accurate determination of growth promoters (zeranol, taleranol, and diethylstilbesterol) in raw meat samples. Sample preparation processes including an enzymatic hydrolysis, extraction, and SPE clean-up were optimised. The sensitivity difference of trans- and cis-diethylstilbestrol (isomerizing in sample preparation processes) by the LC/MS was measured by running a trans/cis mixture (ratio measured by a quantitative NMR) with and without sample matrices, and applied for the determination of total diethylstilbestrol. Validity, repeatability, and reproducibility of the analytical method were tested by measuring gravimetrically fortified samples (chicken breast, bovine muscles, and porcine muscle) in a number of different time periods. Measurement results agreed with the fortified values within their uncertainties. The method provided accurate results of the target analytes in the range of 0.05-15 µg/kg with the relative expanded uncertainty of 2-15%.

Simultaneous preparation of α/ß-zearalenol glucosides and glucuronides.

An improved and reproducible procedure for the preparation of four different glycosides of the mycotoxins α- and ß-zearalenol (α,ß-ZEL), both metabolites of the Fusarium toxin zearalenone (ZEN), is reported. These conjugated or masked mycotoxins are formed during phase II metabolism in plants (glucosides) or animals and humans (glucuronides). Improved regioselective Königs-Knorr glucuronidation was applied to ZEN followed by reduction of the keto group of the mycotoxin, leading to α- and ß-configuration of ZEL and also to a partial reduction of the glucuronic acid methyl ester to obtain the corresponding glucosides. After deprotection of the sugar moiety, α- and ß-zearalenol-14-ß,D-glucuronide as well as the corresponding glucosides were isolated at once using preparative HPLC. The reduction step was studied under different reaction conditions to finally develop an optimized and also tunable procedure for the first simultaneous preparation of both, glucosides and glucuronides of a xenobiotic substance in reasonable amounts to be used as reference materials for bioanalytical and toxicological investigations.

Nanoporous PtCo-based ultrasensitive enzyme-free immunosensor for zeranol detection.

Nanoporous PtCo alloy was designed as an antibody carrier for preparation of a highly sensitive immunosensor. The immunosensor was constructed by assembling the capture zeranol antibody on thionine decorated graphene nanosheets modified glassy carbon electrode. With an enzyme-free immunosensor mode, the nanoporous PtCo alloy, synthesized by dealloying method, had shown strong electrocatalytic activity toward antigen-antibody reaction. The use of PtCo alloy carrier offered a high amount of antibody on each immunoconjugate, hence amplified the detectable signal from the electro-reaction of dissolved oxygen. Cyclic voltammetry and electrochemical impedance spectroscopy were used to characterize the recognition of zeranol. Due to the poor conductivity of zeranol, a small amount of zeranol immobilized onto the electrode could result in great change in the electron-transfer resistance. Some factors that would affect the performance of the immunosensor were studied, such as concentration of PtCo, pH, and the ratio of TH to GS. With zeranol concentration range (0.05 to 5.0 ng/mL), the immunosensor exhibited a highly sensitive response to zeranol with a detection limit of 13 pg/mL. The immunosensor was evaluated for bovine urine sample, receiving satisfactory results.