Determination of multiple mycotoxins in paired plasma and urine samples to assess human exposure in Nanjing, China.

This study was conducted to investigate mycotoxin exposure in 260 rural residents (age 18-66 years) in Nanjing, China. Paired plasma and first morning urine samples were analyzed for 26 mycotoxin biomarkers, including 12 parent mycotoxins and 14 mycotoxin metabolites, by an ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method. Mycotoxins and their metabolites were detected in 95/260 (36.5%) plasma samples and 144/260 (55.4%) urine samples. The most prevalent mycotoxin in plasma was ochratoxin A (OTA), with the incidence of 27.7% (range 0.312-9.18 µg/L), while aflatoxin B1-lysine (AFB1-lysine) (incidence 19.6%, range 10.5-74.5 pg/mg albumin), fumonisin B1 (FB1) (incidence 2.7%, range 0.305-0.993 µg/L), deoxynivalenol (DON) (incidence 2.3%, range 1.39-5.53 µg/L), zearalenone (ZEN) (incidence 6.5%, range 0.063-0.418 µg/L) and zearalanone (ZAN) (incidence 1.2%, range 0.164-0.346 µg/L) were also detected in plasma samples. Deoxynivalenol-15-glucuronide (DON-15-GlcA) was the most frequently detected urinary mycotoxin, with the incidence of 43.8% (range 0.828-37.7 µg/L). DON (incidence 10.0%, range 1.39-14.7 µg/L), DON-3-glucuronide (DON-3-GlcA) (incidence 15.8%, range 0.583-5.84 µg/L), aflatoxin M1 (AFM1) (incidence 10.4%, range 0.125-0.464 µg/L), ZAN (incidence 7.7%, range 0.106-1.82 µg/L), ZEN (incidence 6.9%, range 0.056-0.311 µg/L), FB1 (incidence 3.1%, range 0.230-1.33 µg/L), T-2 toxin (incidence 2.3%, range 0.248-3.61 µg/L) and OTA (incidence 1.2%, range 0.153-0.557 µg/L) were also found in urine samples. Based on the plasma or urinary levels, the daily intakes of AFB1, FB1, ZEN, DON and OTA were estimated. The results showed that the investigated rural dwellers were exposed to multiple mycotoxins, especially to carcinogenic mycotoxin AFB1 with a mean daily intake of 0.41 µg/kg·bw/day, thereby underlining a potential public health concern. To the best of our knowledge, this is the first study to evaluate human exposure to mycotoxins with direct measurements of multiple mycotoxins in paired plasma and urine samples for over 200 subjects of a single population.

The role of roughage provision on the absorption and disposition of the mycotoxin deoxynivalenol and its acetylated derivatives in calves: from field observations to toxicokinetics.

A clinical case in Belgium demonstrated that feeding a feed concentrate containing considerable levels of deoxynivalenol (DON, 1.13 mg/kg feed) induced severe liver failure in 2- to 3-month-old beef calves. Symptoms disappeared by replacing the highly contaminated corn and by stimulating ruminal development via roughage administration. A multi-mycotoxin contamination was demonstrated in feed samples collected at 15 different veal farms in Belgium. DON was most prevalent, contaminating 80% of the roughage samples (mixed straw and maize silage; average concentration in positives: 637 ± 621 µg/kg, max. 1818 µg/kg), and all feed concentrate samples (411 ± 156 µg/kg, max. 693 µg/kg). In order to evaluate the impact of roughage provision and its associated ruminal development on the gastro-intestinal absorption and biodegradation of DON and its acetylated derivatives (3- and 15-ADON) in calves, a toxicokinetic study was performed with two ruminating and two non-ruminating male calves. Animals received in succession a bolus of DON (120 µg/kg bodyweight (BW)), 15-ADON (50 µg/kg BW), and 3-ADON (25 µg/kg) by intravenous (IV) injection or per os (PO) in a cross-over design. The absolute oral bioavailability of DON was much higher in non-ruminating calves (50.7 ± 33.0%) compared to ruminating calves (4.1 ± 4.5%). Immediately following exposure, 3- and 15-ADON were hydrolysed to DON in ruminating calves. DON and its acetylated metabolites were mainly metabolized to DON-3-glucuronide, however, also small amounts of DON-15-glucuronide were detected in urine. DON degradation to deepoxy-DON (DOM-1) was only observed to a relevant extent in ruminating calves. Consequently, toxicity of DON in calves is closely related to roughage provision and the associated stage of ruminal development.

Development and validation of an LC-MS/MS method for the simultaneous determination of citrinin and ochratoxin a in a variety of feed and foodstuffs.

An ultra-performance liquid chromatography-electrospray tandem mass spectrometry (UPLC-ESI+/--MS/MS) method for the simultaneous analysis of citrinin (CIT) and ochratoxin A (OTA) in feed (chicken and pig) and food (cereal-based products, fruit, vegetable juices, nuts, seeds, herbs, spices, vegetarian and soy products, alcoholic beverages, baby food products and food supplements) was developed. The mycotoxins were extracted from these matrices using a QuEChERS-based extraction method without any further clean-up step. The samples were 5-fold concentrated. Final extracts were analyzed using a UPLC-MS/MS system and chromatographic separation was achieved by applying a gradient elution for a total run time of 10 min. Mycotoxins were quantified using an internal calibration via analyte/13C-labeled internal standard ratio. The developed method was validated according to the criteria described in Commission Regulation No. 401/2006/EC and Commission Decision No. 2002/657/EC. Specificity, linearity, apparent recovery, limit of detection and quantification, intraday and interday precision, measurement uncertainty, matrix effect, and extraction efficiency were the parameters studied. Finally, 90 Belgian chicken and pig feed samples were analyzed, revealing the simultaneous presence of CIT (

Microbial Detoxification of Deoxynivalenol (DON), Assessed via a Lemna minor L. Bioassay, through Biotransformation to 3-epi-DON and 3-epi-DOM-1.

Mycotoxins are toxic metabolites produced by fungi. To mitigate mycotoxins in food or feed, biotransformation is an emerging technology in which microorganisms degrade toxins into non-toxic metabolites. To monitor deoxynivalenol (DON) biotransformation, analytical tools such as ELISA and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) are typically used. However, these techniques do not give a decisive answer about the remaining toxicity of possible biotransformation products. Hence, a bioassay using Lemna minor L. was developed. A dose-response analysis revealed significant inhibition in the growth of L. minor exposed to DON concentrations of 0.25 mg/L and higher. Concentrations above 1 mg/L were lethal for the plant. This bioassay is far more sensitive than previously described systems. The bioassay was implemented to screen microbial enrichment cultures, originating from rumen fluid, soil, digestate and activated sludge, on their biotransformation and detoxification capability of DON. The enrichment cultures originating from soil and activated sludge were capable of detoxifying and degrading 5 and 50 mg/L DON. In addition, the metabolites 3-epi-DON and the epimer of de-epoxy-DON (3-epi-DOM-1) were found as biotransformation products of both consortia. Our work provides a new valuable tool to screen microbial cultures for their detoxification capacity.

A polyphasic approach for characterization of a collection of cereal isolates of the Fusarium incarnatum-equiseti species complex.

DNA-based phylogenetic analyses have resolved the fungal genus Fusarium into multiple species complexes. The F. incarnatum-equiseti species complex (FIESC) includes fusaria associated with several diseases of agriculturally important crops, including cereals. Although members of FIESC are considered to be only moderately aggressive, they are able to produce a diversity of mycotoxins, including trichothecenes, which can accumulate to harmful levels in cereals. High levels of cryptic speciation have been detected within the FIESC. As a result, it is often necessary to use approaches other than morphological characterization to distinguish species. In the current study, we used a polyphasic approach to characterize a collection of 69 FIESC isolates recovered from cereals in Europe, Turkey, and North America. In a species phylogeny inferred from nucleotide sequences from four housekeeping genes, 65 of the isolates were resolved within the Equiseti clade of the FIESC, and four isolates were resolved within the Incarnatum clade. Seven isolates were resolved as a genealogically exclusive lineage, designated here as FIESC 31. Phylogenies based on nucleotide sequences of trichothecene biosynthetic genes and MALDI-TOF MS (Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry) were largely concordant with phylogeny inferred from the housekeeping gene. Finally, Liquid Chromatography (Time-Of-Flight) Mass Spectrometry [LC-(TOF-)MS(/MS)] revealed variability in mycotoxin production profiles among the different phylogenetic species investigated in this study.

Use of UHPLC high-resolution Orbitrap mass spectrometry to investigate the genes involved in the production of secondary metabolites in Aspergillus flavus.

The fungus Aspergillus flavus is known for its ability to produce the toxic and carcinogenic aflatoxins in food and feed. While aflatoxins are of most concern, A. flavus is predicted to be capable of producing many more metabolites based on a study of its complete genome sequence. Some of these metabolites could be of great importance in food and feed safety. Here we describe an analytical methodology based on Orbitrap HRMS technology that allows the untargeted determination of fungal metabolites, in support of the study of the function of genes involved in secondary metabolism in fungi. The applied strategy implies the detection and identification of differentially expressed metabolites in extracts of wild-type and mutant fungal strains, using Orbitrap high-resolution mass spectrometry (HRMS) accurate mass data. The suitability of this approach was demonstrated by the confirmation of previously characterised genes involved in the aflatoxin biosynthetic pathway, namely a polyketide synthase (pksA), an oxidoreductase (ordA) and a methyltransferase (omtA) gene. Subsequently, the proposed methodology was applied for the detection and identification of metabolites produced by a yet uncharacterised gene cluster in A. favus, cluster 23. Comparative Orbitrap HRMS analysis of extracts of A. flavus wild-type strain and an over-expression mutant for the transcription factor of gene cluster 23 (lepE) demonstrated that this gene cluster is responsible for the production a set of 2-pyridone derivatives, the leporins. Besides the known derivatives leporin B and leporin B precursor that could be identified by automatic de-replication of the accurate mass data, five other compounds belonging to this class of fungal secondary metabolites were detected and identified for the first time, combining MS and multiple-stage MS data.

An Aspergillus flavus secondary metabolic gene cluster containing a hybrid PKS-NRPS is necessary for synthesis of the 2-pyridones, leporins.

The genome of the filamentous fungus, Aspergillus flavus, has been shown to harbor as many as 56 putative secondary metabolic gene clusters including the one responsible for production of the toxic and carcinogenic, polyketide synthase (PKS)-derived aflatoxins. Except for the production of aflatoxins, cyclopiazonic acid and several other metabolites the capability for metabolite production of most of these putative clusters is unknown. We investigated the regulation of expression of the PKS-non-ribosomal peptide synthetase (NRPS) containing cluster 23 and determined that it produces homologs of the known 2-pyridone leporin A. Inactivation and overexpression of a cluster 23 gene encoding a putative Zn(2)-Cys(6) transcription factor (AFLA_066900, lepE) resulted in downregulation of nine and up-regulation of 8, respectively, of the fifteen SMURF-predicted cluster 23 genes thus allowing delineation of the cluster. Overexpression of lepE (OE::lepE) resulted in transformants displaying orange-red pigmented hyphae. Mass spectral analysis of A. flavus OE::lepE extracts identified the known 2-pyridone metabolite, leporin B, as well as the previously unreported dehydroxy-precursor, leporin C. We provide strong evidence that leporin B forms a unique trimeric complex with iron, not found previously for other 2-pyridones. This iron complex demonstrated antiinsectan and antifeedant properties similar to those previously found for leporin A. The OE::lepE strain showed reduced levels of conidia and sclerotia suggesting that unscheduled leporin production affects fungal developmental programs.

Development and validation of an ultra-high-performance liquid chromatography tandem mass spectrometric method for the simultaneous determination of free and conjugated Alternaria toxins in cereal-based foodstuffs.

A UPLC-ESI+/--MS/MS method for the simultaneous determination of free (alternariol, alternariol monomethyl ether, altenuene, tenuazonic acid, tentoxin, altertoxin-I) and conjugated (sulfates and glucosides of alternariol and alternariol monomethyl ether) Alternaria toxins in cereals and cereal products (rice, oat flakes and barley) was developed. Optimization of the sample preparation and extraction methodology was achieved through experimental design, using full factorial design for extraction solvent composition optimization and fractional factorial design to identify the critical factors in the sample preparation protocol, which were in turn subjected to optimization. Final extracts were analysed using an Waters Acquity UPLC system coupled to a Quattro Premier XE mass spectrometer equipped with an electrospray interface operated in both positive and negative ionization mode. Chromatographic separation was achieved using an Acquity UPLC HSS T3 column, and the applied gradient elution programme allowed for the simultaneous determination of 10 Alternaria toxins in a one-step chromatographic run with a total run time of only 7min. Subsequently, the method, applying isotopically labelled internal standards ([2H4]-alternariol monomethyl ether and [13C6,15N]-tenuazonic acid), was validated for several parameters such as linearity, apparent recovery, limit of detection, limit of quantification, precision, measurement uncertainty and specificity (in agreement with the criteria mentioned in Commission Regulation No. 401/2006/EC and Commission Decision No. 2002/657/EC). During validation, quality of the bioanalytical data was improved by counteracting the observed heteroscedasticity through the application of weighted least squares linear regression (WLSLR). Finally, 24 commercially available cereal-based foodstuffs were subjected to analysis, revealing the presence of tenuazonic acid in both rice and oat flake samples (

Functional characterization of a veA-dependent polyketide synthase gene in Aspergillus flavus necessary for the synthesis of asparasone, a sclerotium-specific pigment.

The filamentous fungus, Aspergillus flavus, produces the toxic and carcinogenic, polyketide synthase (PKS)-derived family of secondary metabolites termed aflatoxins. While analysis of the A. flavus genome has identified many other PKSs capable of producing secondary metabolites, to date, only a few other metabolites have been identified. In the process of studying how the developmental regulator, VeA, affects A. flavus secondary metabolism we discovered that mutation of veA caused a dramatic down-regulation of transcription of a polyketide synthase gene belonging to cluster 27 and the loss of the ability of the fungi to produce sclerotia. Inactivation of the cluster 27 pks (pks27) resulted in formation of greyish-yellow sclerotia rather than the dark brown sclerotia normally produced by A. flavus while conidial pigmentation was unaffected. One metabolite produced by Pks27 was identified by thin layer chromatography and mass spectral analysis as the known anthraquinone, asparasone A. Sclerotia produced by pks27 mutants were significantly less resistant to insect predation than were the sclerotia produced by the wild-type and more susceptible to the deleterious effects of ultraviolet light and heat. Normal sclerotia were previously thought to be resistant to damage because of a process of melanization similar to that known for pigmentation of conidia. Our results show that the dark brown pigments in sclerotia derive from anthraquinones produced by Pks27 rather than from the typical tetrahydronapthalene melanin production pathway. To our knowledge this is the first report on the genes involved in the biosynthesis of pigments important for sclerotial survival.

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

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

In vitro glucuronidation of ochratoxin a by rat liver microsomes.

Ochratoxin A (OTA), one of the most toxic mycotoxins, can contaminate a wide range of food and feedstuff. To date, the data on its conjugates via glucuronidation request clarification and consolidation. In the present study, the combined approaches of ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS), UHPLC-Orbitrap-high resolution mass spectrometry (HRMS) and liquid chromatography-multiple stage mass spectrometry (LC-MS(n)) were utilized to investigate the metabolic profile of OTA in rat liver microsomes. Three conjugated products of OTA corresponding to amino-, phenol- and acyl-glucuronides were identified, and the related structures were confirmed by hydrolysis with ß-glucuronidase. Moreover, OTA methyl ester, OTα and OTα-glucuronide were also found in the reaction solution. Based on these results, an in vitro metabolic pathway of OTA has been proposed for the first time.

Human exposure to mycotoxins and their masked forms through cereal-based foods in Belgium.

In the present study, a quantitative dietary exposure assessment of mycotoxins and their masked forms was conducted on a national representative sample of the Belgian population using the contamination data of cereal-based foods. Cereal-based food products (n=174) were analysed for the occurrence of deoxynivalenol, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, zearalenone, α-zearalenol, ß-zearalenol, T-2-toxin, HT-2-toxin, and their respective masked forms, including, deoxynivalenol-3-glucoside, zearalenone-4-glucoside, α-zearalenol-4-glucoside, ß-zearalenol-4-glucoside and zearalenone-4-sulfate. Fibre-enriched bread, bran-enriched bread, breakfast cereals, popcorn and oatmeal were collected in Belgian supermarkets according to a structured sampling plan and analysed during the period from April 2010 to October 2011. The habitual intake of these food groups was estimated from a national representative food intake survey. According to a probabilistic exposure analysis, the mean (and P95) mycotoxin intake for the sum of the deoxynivalenol-equivalents, zearalenone-equivalents, and the sum of HT-2-and T-2-toxin for all cereal-based foods was 0.1162 (0.4047, P95), 0.0447 (0.1568, P95) and 0.0258 (0.0924, P95) µg kg(-1)body weight day(-1), respectively. These values were below the tolerable daily intake (TDI) levels for deoxynivalenol, zearalenone and the sum of T-2 and HT-2 toxin (1.0, 0.25 and 0.1 µg kg(-1)body weight day(-1), respectively). The absolute level exceeding the TDI for all cereal-based foods was calculated, and recorded 0.85%, 2.75% and 4.11% of the Belgian population, respectively.

A direct assessment of mycotoxin biomarkers in human urine samples by liquid chromatography tandem mass spectrometry.

Detection of mycotoxin biomarkers in urine of humans and animals provides a direct approach for assessing exposure to these mycotoxins as opposed to the indirect approach of food analysis, which in most cases is affected by the heterogeneity of the toxin in the food samples. Seven (7) mycotoxins and their metabolites (total 18 analytes) were selected and an LC-MS/MS method for their determination in human urine was developed and validated. The method consisted of direct analysis of two mycotoxin conjugates, deoxynivalenol-glucuronide and zearalenone-glucuronide without beta glucuronidase digestion of the urine samples. Since high method sensitivity is of utmost importance in such study, critical factors which could improve the analyte recovery and method sensitivity were investigated by a D-optimal experimental design. Urine samples (10 mL) were first extracted with 15 mL ethyl acetate/formic acid (99/1, v/v) followed by SAX SPE clean-up of the acidified aqueous fraction. Both extracts were combined and analyzed using an LC-MS/MS system operated in the positive ionization mode. A total run time of 28 min was adopted with all the 18 analytes eluting within 15 min. The method was validated by taking into consideration the guidelines specified in Commission Decision 2002/657/EC and 401/2006/EC. Forty samples obtained from volunteers within the laboratory research group were analyzed as part of a pilot study. All results were expressed per mg creatinine. A total of 9 samples were found contaminated with one or more of the following analytes: DON, OTA, OTα, 4-OH OTA, ZEN, CIT and ß-ZOL. One-eighth (5/40) of the samples were contaminated with DON in the range of 3.7-67 ng mg(-1) creatinine. Samples with detectable levels of DON did not show any co-occurrence of DON-3Glu. One sample was found to be contaminated with 4-OH OTA (

A validated multianalyte LC-MS/MS method for quantification of 25 mycotoxins in cassava flour, peanut cake and maize samples.

This study was designed to develop a sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the simultaneous detection and quantification of 25 mycotoxins in cassava flour, peanut cake and maize samples with particular focus on the optimization of the sample preparation protocol and method validation. All 25 mycotoxins were extracted in a single step with a mixture of methanol/ethyl acetate/water (70:20:10, v/v/v). The method limits of quantification (LOQ) varied from 0.3 µg/kg to 106 µg/kg. Good precision and linearity were observed for most of the mycotoxins. The method was applied for the analysis of naturally contaminated peanut cake, cassava flour and maize samples from the Republic of Benin. All samples analyzed (fifteen peanut cakes, four maize flour and four cassava flour samples) tested positive for one or more mycotoxins. Aflatoxins (total aflatoxins; 10-346 µg/kg) and ochratoxin A (

Determination of hypericin in human plasma by high-performance liquid chromatography after intravesical administration in patients with transitional cell carcinoma of the bladder.

In the present study, the systemic absorption of hypericin was investigated after intravesical instillation of the compound in nine patients with superficial transitional cell carcinoma (TCC) bladder tumors. Hypericin (8 microM) was instilled in the bladder for 2-3 h before photodynamic diagnosis of bladder tumors. Blood was then collected from a peripheral vein 1 h after termination of the instillation. Solid phase extraction with ammonium acetate buffer and methanol was used to extract hypericin from the plasma. A reversed-phase high performance liquid chromatographic method with fluorescence detection was used to identify and quantify hypericin in the extracts from plasma samples. Analysis of standard plasma samples, which were spiked with known amounts of hypericin, indicated that the pH of the buffer was a determining factor in the extraction yield. The results obtained using ammonium buffer (pH 3.5) and methanol showed the mean extraction recovery of hypericin to be 64% (RSD=12%, n=6). The limits of detection and quantification were 6 and 20 nM, respectively. Extraction and analysis of the plasma of patients after intravesical administration showed hypericin concentrations below the detection limit (<6 nM). In addition, photodynamic treatment of in vitro cultured HeLa cells incubated with 1-100 nM hypericin concentrations showed that lower concentrations (1-20 nM) of hypericin do not induce significant photocytotoxic effects. Taken together, these results imply that photosensitization or other systemic side effects in patients are not to be expected after photodynamic diagnosis of TCC bladder tumors with hypericin.