Development, validation, and application of a multimatrix UHPLC-MS/MS method for quantification of Datura-type alkaloids in food.

A quantitative ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated for the determination of tropane alkaloids (TAs), atropine and scopolamine, in a variety of food products. The sample preparation of cereal-based food, oilseeds, honey, and pulses consisted of a solid-liquid extraction with an acidified mixture of methanol and water, while an additional step of solid-phase extraction on a cation-exchange sorbent was introduced in the treatment of teas and herbal infusions, aromatic herbs, spices and food supplements. The limits of quantification of the method varied from 0.5 to 2.5 µg kg-1. Apparent recovery was in the range of 70-120%, and repeatability and intermediate precision were below 20%. The method was successfully applied in a proficiency testing exercise as well as in the analysis of various commercial foods. Only 26% of the analysed food samples contained one or both TAs. The mean concentrations for atropine and scopolamine amounted to 21.9 and 6.5 µg kg-1, respectively, while the maximum concentrations were 523.3 and 131.4 µg kg-1, respectively. Overall, the highest levels of TA sum were found in an herbal infusion of fennel and a spice mix containing fennel and anise seeds.

First study of bromophenols and hexabromocyclododecanes in seafood from North Africa (case of Bizerte Lagoon, Tunisia): occurrence and human health risk.

In spite of the fact that bromophenols (BPs) and hexabromocyclododecanes (HBCDs) are widely used as flame retardants, no data was available until now on the levels of these two chemicals in North Africa biota. Seafood products might represent one of the main sources of dietary exposure to persistent organic pollutants such as non-dioxin-like polychlorinated biphenyls (ndl-PCBs), brominated flame retardants (BFRs), and polycyclic aromatic hydrocarbons (PAHs). In this study, the concentrations of the ndl-PCBs, PAH4, and BFRs were determined in seafood products from a North African lagoon (Bizerte lagoon). Almost all the compounds were detected (15 out of 18) in the analyzed marine organisms. The accumulation of the contaminants followed the order BFRs > ndl-PCB > PAH4. Mean contaminants concentrations ranged from 0.35 to 28.7 ng g-1 ww for ∑ndl-PCBs; from below limit of quantification to 476 ng g-1 ww for ∑BFRs and from below limit of quantification to 5.30 ng g-1 ww for ∑PAH4. PCB 138, 153, and 180 were the most frequently detected ndl-PCB congeners due to their high resistance to metabolic degradation. 2,4-dibromophenol (2,4-DBP) was the predominant BFR. Chrysene (Chr) was found to be the main contributor to the total PAH4 concentration. Contaminant profiles varied significantly among seafood which may be due to the difference in lipid content, trophic level, feeding behavior, and metabolism. To assess the human health risks, the average daily dose exposure of ndl-PCBs, the dietary daily intake of PAHs and the estimated dietary intake of 3,3-,5,5-tetrabromobisphenol A (TBBPA) and HBCD from seafood were estimated. Findings indicated no adverse effects for human health from any of the analyzed contaminants, except for ndl-PCBs in eel.

A Targeted UHPLC-MS/MS Method Validated for the Quantification of Ergot Alkaloids in Cereal-Based Baby Food from the Belgian Market.

Following pending new legislation in the European Union setting a maximum of 20 ng g-1 for the total sum of ergot alkaloids in dry cereal-based baby food, a new UHPLC-MS/MS method was developed. It is suitable for the quantification of six ergot alkaloids: Ergocornine, ergocristine, ergometrine, ergosine, ergotamine, α-ergocryptine, and their corresponding epimers. The method is able to reliably detect individual ergot alkaloids at a level as low as 0.5 ng g-1. The method uses a modified QuEChERS extraction approach before UHPLC-MS/MS analysis. The method showed good sensitivity, accuracy, and precision. It has been applied to 49 samples from the Belgian market. In 26 samples, not a single ergot alkaloid was detected while in 23 out of 49 samples at least one ergot alkaloid was detected with 2 samples containing 12 ergot alkaloids. Ergometrine was the alkaloid most frequently detected i.e., 16 out of 49 samples. Only one sample, testing positive for all 12 ergot alkaloids, would be non-conforming to the newly proposed Maximum Residue Level (MRL).

Phylogeny of dracunculoid nematodes (Chromadorea: Rhabditida: Spirurina: Dracunculoidea) from some Eurasian freshwater fishes.

Sets of small ribosomal DNA (SSU rDNA) and large ribosomal DNA (LSU rDNA) sequences were obtained for Philometroides moraveci Vismanis Yunchis, 1994, Philometra kotlani (Molnár, 1969), Philometra rischta Skrjabin, 1923, Philometra cf. obturans (Prenant, 1886) (Philometridae), Sinoichthyonema amuri (Garkavi, 1972), Agrachanus scardinii (Molnár, 1966), Kalmanmolnaria intestinalis (Dogiel Bychowsky, 1934) and Skrjabillanus tincae Shigin Shigina, 1958 (Skrjabillanidae). Phylogenetic analysis of SSU rDNA data shows that dracunculoid nematodes are divided into two well-supported clades designated as Clade I and Clade II, respectively. Clade I includes the type species of the genus Philonema Kuitunen-Ekbaum, 1933, some species from the family Daniconematidae Moravec Køie, 1987 and two subfamilies of skrjabillanids, Skrjabillaninae Shigin Shigina, 1958 and Esocineminae Moravec, 2006. Clade II unites species from the families Dracunculidae Stiles, 1907, Micropleuridae Baylis Daubney, 1926 and Philometridae Baylis Daubney, 1926. Within the Philometridae, there are several well-supported groups of species, one of which unites freshwater Philometra spp. from the Palearctic cyprinids, identified as P. kotlani, P rischta, P. ovata (Zeder, 1803) and P. cyprinirutili (Creplin, 1825). However, the phylogenetic relationships of most philometrids are unresolved. An analysis of partial SSU and LSU rDNA sequences indicates that there is no direct phylogenetic relationship between Agrachanus Tikhomirova, 1971 (type species Skrjabillanus scardinii Molnár, 1966) and Skrjabillanus Shigin Shigina, 1958 (type species Sk. tincae), which means that the genus Agrachanus can be resurrected. Our study confirms that Philonematinae Ivashkin, Sobolev Khromova, 1971 should be elevated to the family rank. We formally establish the family Philonematidae Ivashkin, Sobolev Khromova, 1971 stat. nov. We also suggest combining the superfamilies Dracunculoidea Stiles, 1907 and Camallanoidea Railliet Henry, 1915 into the infraorder Camallanomorpha Roberts, Janovy Nadler, 2013.

Enantiomeric fraction of hexabromocyclododecanes in foodstuff from the Belgian market.

Diet is considered a major route of human exposure to hexabromocyclododecane, a chiral environmental contaminant. A previous study reported on the occurrence of hexabromocyclododecane diastereoisomers in food items of animal origin collected in Belgium. The present study reports further results on corresponding enantiomeric fractions of the same samples. None of the samples could be considered as racemic for the α-isomer suggesting that foodstuff contamination occurred prior to death of the corresponding producing animal and was not the result of the food item being in contact with technical HBCDD. Non-racemic chiral signatures were also observed for ß- and γ-isomers. We conclude that, depending on their dietary habits, different individuals might be overall exposed to non-racemic profiles. Considering that toxicological effects are enantiomer-dependent, this could modulate potential adverse effects.

Development and Validation of a UHPLC-ESI-MS/MS Method for Quantification of Oleandrin and Other Cardiac Glycosides and Evaluation of Their Levels in Herbs and Spices from the Belgian Market.

Cardiac glycosides (CGs) are naturally occurring plant secondary metabolites that can be toxic to humans and animals. The aim of this work was to develop a targeted analytical method utilizing liquid chromatography-tandem mass spectrometry (LC-MS/MS) for quantification of these plant toxins in a herbal-based food and human urine. The method included oleandrin, digoxin, digitoxin, convallatoxin, and ouabain. Samples of culinary herbs were extracted with acetonitrile and cleaned using Oasis® MAX solid-phase extraction (SPE), while samples of urine were diluted with acidified water and purified on Oasis® HLB SPE cartridges. Limits of quantification were in the range of 1.5-15 ng/g for herbs and 0.025-1 ng/mL for urine. The mean recovery of the method complied with the acceptable range of 70-120% for most CGs, and relative standard deviations were at maximum 14% and 19% for repeatability and reproducibility, respectively. Method linearity was good with calculated R² values above 0.997. The expanded measurement uncertainty was estimated to be in the range of 7-37%. The LC-MS/MS method was used to examine 65 samples of culinary herbs and herb and spice mixtures collected in Belgium, from supermarkets and local stores. The samples were found to be free from the analyzed CGs.

Development and validation of a quantitative UHPLC-MS/MS method for selected brominated flame retardants in food.

An ultra-high performance liquid chromatography - tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated (in-house) for the quantification of selected brominated flame retardants (BFRs), including tetrabromobisphenol A (TBBPA), hexabromocyclododecanes (HBCDs), tetrabromobisphenol S (TBBPS) and bromophenols (BPs), in various food matrices. The sample preparation consisted of extraction of TBBPS with acidified acetonitrile followed by a fast dispersive solid-phase extraction (dSPE) clean-up and extraction of the other BFRs with a mixture of hexane and dichloromethane (1:1, v/v) with subsequent clean-up using acidified silica (44%, w/w). The limits of quantification of the method varied widely for the types of food matrices and the different classes of BFRs from 4 pg g-1 wet weight (ww) to 8 ng g-1 ww. For most of the analytes the apparent recovery was in the range 70-120%, and the method precision (under repeatability conditions) was below 20%. The method was successfully applied in proficiency testing exercises as well as for analysis of various food items. Only 25% of the collected food samples contained BFRs, with 4-bromophenol and α-HBCD as the only detected compounds. The contaminated foodstuffs were fish and eggs with concentrations in the range from 48 to 305 pg g-1 ww.

Occurrence of selected halogenated flame retardants in Belgian foodstuff.

This paper reports on the occurrence of halogenated flame retardants (HFRs), namely PBDEs, HBCDs, TBBPA, brominated phenols (BrPhs), dechlorane plus (DP) and emerging FRs in a variety of Belgian foodstuffs. A total of 183 composite food samples were analyzed by GC-MS and LC-MS/MS techniques for the presence of HFRs. The analyses revealed that 72% of the samples was contaminated with HFRs to some extent. The highest number of contaminated samples was observed within the group 'Potatoes and derived products', 'Fish and fish products' and 'Meat and meat products', while the least contaminated group was 'Food for infants and small children'. The total HFR content ranged from

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.

Heth impalutiensis n. sp. (Nematoda: Ransomnematoidea: Hethidae) a millipede parasite from Central Mindanao, Philippines.

The nematode Heth impalutiensis n. sp. is described from an unidentified spirostreptid millipede (Harpagophoridae) from the Bukidnon Province of Mindanao, the Philippines. Based on morphological characters, H. impalutiensis n. sp. is closest to Asian-Pacific representatives of the genus. Females of H. impalutiensis n. sp. are close to H. dimorphum and H. vietnamensis in body size and form of the lateral lappets, but can be distinguished by the significantly longer tail. Males of H. impalutiensis n. sp. strongly resemble that of H. xaniophora by the presence of such a rare character combinations as mammiform papillae and a bursa-like cuticular fold, but can be easily differentiated by the numbers of genital papillae (7 vs 6 pairs, respectively). Heth impalutiensis n. sp. can be distinguished from all nominal species by hypertrophy of the anterior anal lip in females which overlaps the anal aperture. Phylogenetic analysis based on the newly obtained set of sequences did not provide an evidence of infraorder Rhigonematomorpha monophyly as two superfamilies Ransomnematoidea and Rhigonematoidea formed independent clades in the frames of ascaridid-spirurid-oxyurid super clade (Clade III of Nadler et al., 2007).

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.

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.

Holistic approach based on high resolution and multiple stage mass spectrometry to investigate ergot alkaloids in cereals.

A holistic approach based on high resolution and multiple stage mass spectrometry was developed for identification of less studied or novel ergot alkaloid derivatives. Initially, the fragmentation of nine known ergot alkaloids was studied to establish a strategy for the identification of novel ergot alkaloids. Ions with m/z 223 and m/z 251 were found to be common for all ergopeptines, ergoamides and ergopeptams. Subsequently, parent scan experiments using these ions were performed to screen grain samples for the presence of possible ergot alkaloid derivatives. Besides the six most common ergot alkaloids and their corresponding epimers (for which reference standards were available), eleven other ergot alkaloid derivatives were identified following the proposed strategy.

A systematic assessment of the variability of matrix effects in LC-MS/MS analysis of ergot alkaloids in cereals and evaluation of method robustness.

The study presents for the first time a systematic investigation of matrix effects in the LC-MS/MS analysis of ergot alkaloids in cereals. In order to assure the accuracy of the results, several approaches to minimize/eliminate matrix effects were investigated including variation of ionization techniques, chromatography and sample preparation on different grain types and grain varieties. It was revealed that the use of UPLC and careful choice of sample preparation might reduce signal suppression/enhancement. In general, ergometrine was found to be the most susceptible among the ergot alkaloids studied, but none of the used approaches suggested a total elimination of matrix effects; only less than half of its MS signal could be recovered. The late-eluting compounds were less affected by matrix components in all conditions tested. Further, the robustness of the applied LC-MS method was checked by means of a fractional factorial design. The results indicate that small changes to the sample preparation parameters, namely pH and concentration of extraction buffer, shaking time, drying temperature and extraction volumes, did not significantly (α = 0.05) affect the recoveries of ergot alkaloids.

Skin penetration enhancing properties of the plant N-alkylamide spilanthol.

ETHNOPHARMACOLOGICAL RELEVANCE: Plants are often used for skin diseases in different ethnopharmacological systems. Local and systemic effects of topically applied compounds can be significantly increased by plant constituents having skin penetration enhancers. MATERIALS AND METHODS: In this study, we examined the proposed penetration enhancing properties of spilanthol, an N-alkylamide abundantly present in several Asteraceae plants like Spilanthes acmella L., on three model drugs (caffeine, testosterone and ibuprofen). Moreover, as plants are frequently contaminated with toxic environmental substances, the mutual influence on the transdermal behavior between spilanthol and six model mycotoxins (aflatoxin B1, ochratoxin A, fumonisin B1, citrinin, zearalenone, T-2 toxin) was investigated. RESULTS: Spilanthol exhibits component and concentration dependent penetration enhancing effects. No significant penetration enhancing effect for ibuprofen has been observed, but with increasing spilanthol concentration (from 0 up to 1w/V%), the permeability of caffeine increased, resulting in an enhancing ratio (ER) of 4.60. For testosterone, a maximal penetration enhancing concentration of 0.5% spilanthol was found (ER=4.13). Next to its beneficial applicability to increase local as well as systemic pharmacological effects of dermally co-administrated drug, this N-alkylamide negatively influences human health risk if spilanthol containing formulations are polluted with mycotoxins: the presence of spilanthol (0.3w/V%) induced a significant increase of permeability coefficient Kp of five investigated mycotoxins, with ER values ranging between 1.57 and 6.37. On the other hand, mycotoxins themselves do not significantly influence the transdermal behavior of spilanthol. CONCLUSIONS: The existence of a significant mutual influence of compounds towards skin penetration should always be considered during the development or as part of the functional quality evaluation of topical products.

Improved positive electrospray ionization of patulin by adduct formation: usefulness in liquid chromatography-tandem mass spectrometry multi-mycotoxin analysis.

Several sensitive methods have been developed for patulin determination; however, mass spectrometric (MS) detection of this toxin in the positive electrospray ionization (ESI(+)) mode is not straightforward. Furthermore, the combined determination of patulin with other mycotoxins in one single run has not been reported yet. The present paper demonstrates the formation and use of a methanol adduct of patulin in ESI(+). A study of the fragmentation pathway confirmed the authenticity of the patulin adduct, while the use of ion trap and high resolution Orbitrap mass spectrometry allowed reliable assignment of the patulin fragment ions. Exploiting the formation of the methanol adduct, patulin has been successfully included in a single run multi-mycotoxin liquid chromatography tandem mass spectrometric (LC-MS/MS) method in support of ex vivo-in vitro biomedical studies.

Identification of volatile markers for indoor fungal growth and chemotaxonomic classification of Aspergillus species.

Microbial volatile organic compounds (MVOCs) were collected in water-damaged buildings to evaluate their use as possible indicators of indoor fungal growth. Fungal species isolated from contaminated buildings were screened for MVOC production on malt extract agar by means of headspace solid-phase microextraction followed by gas chromatography-mass spectrometry (GC-MS) analysis. Some sesquiterpenes, specifically derived from fungal growth, were detected in the sampled environments and the corresponding fungal producers were identified. Statistical analysis of the detected MVOC profiles allowed the identification of species-specific MVOCs or MVOC patterns for Aspergillus versicolor group, Aspergillus ustus, and Eurotium amstelodami. In addition, Chaetomium spp. and Epicoccum spp. were clearly differentiated by their volatile production from a group of 76 fungal strains belonging to different genera. These results are useful in the chemotaxonomic discrimination of fungal species, in aid to the classical morphological and molecular identification techniques.

Human skin penetration of selected model mycotoxins.

Dermal exposure data for mycotoxins are very scarce and fragmentary, despite their widespread skin contact and hazard toxicity. In this study, the transdermal kinetics of aflatoxin B1 (AFB1), ochratoxin A (OTA), fumonisin B1 (FB1), citrinin (CIT), zearalenone (ZEA) and T-2 toxin (T-2) were quantitatively evaluated, using human skin in an in vitro Franz diffusion cell set-up. All mycotoxins penetrated through the skin, except for FB1, which showed concentrations in the receptor fluid below the LoD, resulting in a K(p)<3.24×10(-6)cm/h. OTA showed the highest permeation (K(p)=8.20×10(-4)cm/h), followed by CIT (K(p)=4.67×10(-4)cm/h). AFB1 and ZEA showed lower permeability rates (K(p)=2.11 and 2.33×10(-4)cm/h, respectively). T-2 was found to have the lowest permeability (K(p)=6.07×10(-5)cm/h). From literature-based mycotoxin-concentrations, dermal contact surface, exposure time and apparent K(p)'s obtained in this study, the daily dermal exposure (DDE) in two industrial and one residential scenario was estimated. Dermal exposure to the DNA-reactive genotoxic carcinogenic AFB1 can lead to a health risk for agricultural workers which are exposed to a mycotoxin contaminated solution in a worst case situation. For all the other investigated mycotoxins, no significant health risk is calculated after dermal contact in neither agricultural nor residential environments.

Cattiena fansipanis n. sp. (Nematoda: Rhigonematida: Carnoyidae) from a millipede (Myriapoda: Diplopoda: Spirobolida) in North Vietnam.

A new species of Cattiena Hunt & Spiridonov, 2001 from a diplopod (Spirobolida: Pseudospirobolellidae Brolemann) collected near Sa Pa, Lao Cai Province, Vietnam, is described. Females of Cattiena fansipanis n. sp. are closely related to females of two other known species of the genus, C. trachelomegali Hunt & Spiridonov, 2001 and C. trigoniuli Hunt & Spiridonov, 2001, but can be distinguished by the distinctly more anterior position of the vulva, abrupt constriction of the body at the vulval level, presence of two swollen portions of the oviducts, and longer body and tail. Males of new species differ by having a rounded cephalic region followed by 13-14 annules which are larger than those which follow them, a different size and shape of the spicules and gubernaculum, and body and tail length. Three size groups of juveniles were found in the host gut lumen, presumably representing second, third and fourth juvenile stages. The morphology of the juvenile stages is described.