Brominated Dioxins in Egg, Broiler, and Feed Additives: Significance of Bioassay-Directed Screening for Identification of Emerging Risks in Food.

Food authorities aim to safeguard our food. This requires sensitive analyses to guarantee detection of both banned and regulated substances at low concentrations. At the same time, broad screening methods are needed to identify new emerging risks. For this purpose, effect-based bioassays combined with mass spectrometric analyses offer an advantage. During the regular monitoring of dioxins in agricultural products, a discrepancy was observed between the results of the DR CALUX (Dioxin-Responsive Chemical Activated Luciferase gene Expression) bioassay and the confirmatory gas chromatographic high resolution mass spectrometric (GC-HRMS) analysis in egg and broiler fat samples. The response in the bioassay was high, suggesting a clear exceedance of the maximum limits of dioxins in these samples, yet regulated dioxins or dl-PCBs were not detected by GC/HRMS analysis. Ultimately, a broad screening analysis using GC-HRMS resulted in the identification of 2,3,7,8-tetrabromo-dibenzofuran (2,3,7,8-TBDF) in both egg and broiler fat. To investigate the potential source of this brominated furan contaminant, different samples were analyzed: bedding material, poultry feed, feed additives (choline chloride and l-lysine), and seaweed. The poultry feed and feed additives all contained 2,3,7,8-TBDF. Using a feed-to-food transfer model, it became clear that the poultry feed was probably the source of 2,3,7,8-TBDF in broilers and eggs through a feed additive like L-lysine or choline chloride. This study underlines the importance of using a combination of effect-based screening assays with sensitive analytical methods to detect potential new and emerging risks.

Comparison of Compound Identification Tools Using Data Dependent and Data Independent High-Resolution Mass Spectrometry Spectra.

Liquid chromatography combined with high-resolution mass spectrometry (LC-HRMS) is a frequently applied technique for suspect screening (SS) and non-target screening (NTS) in metabolomics and environmental toxicology. However, correctly identifying compounds based on SS or NTS approaches remains challenging, especially when using data-independent acquisition (DIA). This study assessed the performance of four HRMS-spectra identification tools to annotate in-house generated data-dependent acquisition (DDA) and DIA HRMS spectra of 32 pesticides, veterinary drugs, and their metabolites. The identification tools were challenged with a diversity of compounds, including isomeric compounds. The identification power was evaluated in solvent standards and spiked feed extract. In DDA spectra, the mass spectral library mzCloud provided the highest success rate, with 84% and 88% of the compounds correctly identified in the top three in solvent standard and spiked feed extract, respectively. The in silico tools MSfinder, CFM-ID, and Chemdistiller also performed well in DDA data, with identification success rates above 75% for both solvent standard and spiked feed extract. MSfinder provided the highest identification success rates using DIA spectra with 72% and 75% (solvent standard and spiked feed extract, respectively), and CFM-ID performed almost similarly in solvent standard and slightly less in spiked feed extract (72% and 63%). The identification success rates for Chemdistiller (66% and 38%) and mzCloud (66% and 31%) were lower, especially in spiked feed extract. The difference in success rates between DDA and DIA is most likely caused by the higher complexity of the DIA spectra, making direct spectral matching more complex. However, this study demonstrates that DIA spectra can be used for compound annotation in certain software tools, although the success rate is lower than for DDA spectra.

Bioassay-directed analysis-based identification of relevant pyrrolizidine alkaloids.

Pyrrolizidine alkaloids (PAs) are produced by various plant species and have been detected as contaminants in food and feed. Monitoring programmes should include PAs that are present in relevant matrices and that exhibit a high toxic potential. The aim of the present study was to use a bioassay-directed analysis approach to identify relevant PAs not yet included in monitoring programmes. To that end, extracts of Heliotropium europaeum and H. popovii were prepared and analysed with LC-MS/MS for the presence of 35 PAs included in monitoring programmes, as well as for genotoxic activity in the HepaRG/γH2AX assay. Europine, heliotrine and lasiocarpine were found to be the most abundant PAs. The extracts showed a higher γH2AX activity than related artificial mixtures of quantified known PAs, which might point to the presence of unknown toxic PAs. The H. europaeum extract was fractionated and γH2AX activities of individual fractions were determined. Fractions were further analysed applying LC-Orbitrap-MS analysis and Compound Discoverer software, identifying various candidate PAs responsible for the non-explained genotoxic activity. Altogether, the results obtained show that bioassay-directed analysis allows identification of candidate PAs that can be included in monitoring programmes.

Systematic assessment of acquisition and data-processing parameters in the suspect screening of veterinary drugs in archive matrices using LC-HRMS.

Monitoring strategies for veterinary drugs in products of animal origin are shifting towards a more risk-based approach. Such strategies not only target a limited number of predefined .substances but also facilitate detection of unexpected substances. By combining the use of archive matrices such as feather meal with suspect-screening methods, early detection of new hazards in the food and feed industry can be achieved. Effective application of such strategies is hampered by complex data interpretation and therefore, targeted data analysis is commonly applied. In this study, the performance of a suspect-screening data processing workflow using a suspect list or the online spectral database mzCloudTM was explored to facilitate detection of veterinary drugs in archive matrices. Data evaluation parameters specifically investigated for application of a suspect list were mass tolerance and the addition or omission of retention times. Application of a mass tolerance of 1.5 ppm leads to an increase in the number of false positives, as does omission of retention times in the suspect list. Different acquisition modes yielding different qualities of MS2 data were studied and proved to be a critical factor, where data-dependent acquisition is preferred when matching to the mzCloudTM database. Using this approach, it is possible to search for compounds on a dedicated suspect list based on the exact mass and retention times and, at the same time, detect unexpected compounds without a priori information. A pilot study was conducted and fourteen different antibiotics were detected (and confirmed by MS/MS). Three of these antibiotics were not included in the suspect list. The optimised suspect-screening method proved to be fit for the purpose of finding veterinary drugs in feather meal, which are not in the scope of the current monitoring methods and therefore, it gives added value in the perspective of a risk-based monitoring.

A single method to analyse residues from five different classes of prohibited pharmacologically active substances in milk.

In the European Union, the use of veterinary drugs belonging to the A6 group is prohibited in food-producing animals according to Commission Regulation (EU) No. 2010/37. The aim of this study was to improve the analytical control strategy by developing a single method to analyse residues of prohibited pharmacologically active substances in milk. For this, a single method was developed to analyse 16 prohibited pharmacologically active substances belonging to five different substance classes at required or recommended levels: nitroimidazoles at 3 µg kg-1, nitrofurans at 0.5 µg kg-1, chloramphenicol at 0.1 µg kg-1, dapsone at 5 µg kg-1 and chlorpromazine at 1 µg kg-1. Milk sample preparation started with an acid hydrolysis combined with a derivatisation. These steps were followed by a clean-up consisting of a dispersive solid-phase extraction and a liquid-liquid extraction. Finally, the sample extracts were analysed by liquid chromatography combined with tandem mass spectrometry, operating alternately in the positive and negative mode. The method was fully validated according to Commission Decision 2002/657/EC for bovine milk and additionally validated for caprine milk. The validation proved that the method is highly effective to detect and confirm all 16 substances in bovine and caprine milk and, additionally to quantify 15 of these substances in bovine milk and 13 of these substances in caprine milk. This study resulted in a new multi-class method to detect, quantify and confirm the identity of 16 prohibited pharmacologically active substances belonging to five different substance classes in two types of milk.

A new extraction procedure to abate the burden of non-extractable antibiotic residues in manure.

Through agricultural soil fertilization using organic manure, antibiotic residues can accumulate in the environment. In order to assess the risks of environmental pollution by veterinary drugs, monitoring of manure for antibiotic residues is necessary. As manure is a complex matrix, extraction of antibiotics proved to be challenging. In this study, 24 extraction solvents were assessed for the extraction of residues from manure representing ten antibiotics from the antibiotic classes tetracyclines, quinolones, macrolides, lincosamides and sulfonamides. Especially for the tetracyclines and quinolones the extraction solvent selection is critical, due to high fractions of non-extractable residues especially when using aqueous solvents (62-77% and 90-95% respectively when using milli-Q water). In contrast, sulfonamides can effectively be extracted with aqueous solvents. Overall, 0.125% trifluoroacetic acid in acetonitrile in combination with McIlvain-EDTA buffer proved to be the most effective extraction solvent. A longitudinal study pointed out that most antibiotics bind to solid manure particles instantaneously after addition. Trimethoprim is an exception, but because by using the optimal extraction solvent, the optimum fraction of bound residues is desorbed, this does not hamper quantitative analysis when using spiked manure quality control samples. Based on these new insights, the current in-house multi-residue LC-MS/MS method for manure analysis, containing 48 antibiotics, was revised, additionally validated and applied to 34 incurred manure samples.

Global monitoring of antimicrobial resistance based on metagenomics analyses of urban sewage.

Antimicrobial resistance (AMR) is a serious threat to global public health, but obtaining representative data on AMR for healthy human populations is difficult. Here, we use metagenomic analysis of untreated sewage to characterize the bacterial resistome from 79 sites in 60 countries. We find systematic differences in abundance and diversity of AMR genes between Europe/North-America/Oceania and Africa/Asia/South-America. Antimicrobial use data and bacterial taxonomy only explains a minor part of the AMR variation that we observe. We find no evidence for cross-selection between antimicrobial classes, or for effect of air travel between sites. However, AMR gene abundance strongly correlates with socio-economic, health and environmental factors, which we use to predict AMR gene abundances in all countries in the world. Our findings suggest that global AMR gene diversity and abundance vary by region, and that improving sanitation and health could potentially limit the global burden of AMR. We propose metagenomic analysis of sewage as an ethically acceptable and economically feasible approach for continuous global surveillance and prediction of AMR.

QSAR-based molecular signatures of prenylated (iso)flavonoids underlying antimicrobial potency against and membrane-disruption in Gram positive and Gram negative bacteria.

Prenylated flavonoids and isoflavonoids are phytochemicals with remarkable antibacterial activity. In this study, 30 prenylated (iso)flavonoids were tested against Listeria monocytogenes and Escherichia coli (the latter in combination with an efflux pump inhibitor). Minimum inhibitory concentrations of the most active compounds ranged between 6.3-15.0 µg/mL. Quantitative structure-activity relationships (QSAR) analysis was performed and linear regression models were proposed with R2 between 0.77-0.80, average R2m between 0.70-0.75, Q2LOO between 0.66-0.69, and relatively low amount of descriptors. Shape descriptors (related to flexibility and globularity), together with hydrophilic/hydrophobic volume and surface area descriptors, were identified as important molecular characteristics related to activity. A 3D pharmacophore model explaining the effect of the prenyl position on the activity of compounds was developed for each bacterium. These models predicted active compounds with an accuracy of 71-88%. With regard to the mode of action, good antibacterial prenylated (iso)flavonoids with low relative hydrophobic surface area caused remarkable membrane permeabilization, whereas those with higher relative hydrophobic surface area did not. Based on the QSAR and membrane permeabilization studies, the mode of action of antibacterial prenylated (iso)flavonoids was putatively rationalized.

Multiple heart-cutting two dimensional liquid chromatography quadrupole time-of-flight mass spectrometry of pyrrolizidine alkaloids.

Pyrrolizidine alkaloids (PAs) and their and the corresponding N-oxides (PAs-ox) are genotoxic plant metabolites which can be present as unwanted contaminants in food products of herbal origin like tea and food supplements. PAs and PAs-ox come in a wide variety of molecular structures including many structural isomers. For toxicity assessment it is important to determine the composition of a sample and to resolve all isomeric PAs and PAs-ox, which is currently not possible in one liquid or gas chromatographic (LC or GC) run. In this study an online two dimensional liquid chromatography quadrupole time-of-flight mass spectrometry (2D-LC QToF-MS) method was developed to resolve isomeric PAs and PAs-ox. After comprehensive column and mobile phase selection a polar endcapped C18 column was used at pH 3 in the first dimension, and a cross-linked C18 column at pH 10 in the second dimension. Injection solvents, column IDs, flow rates and temperatures were carefully optimized. The method with column selection valve switching described in this study was able to resolve and visualize 20 individual PAs/PAs-ox (6 sets of isomers) in one 2D-LC QToF-MS run. Moreover, it was shown that all isomeric PAs/PAs-ox could be unambiguously annotated. The method was shown to be applicable for the determination and quantification of isomeric PAs/PAs-ox in plant extracts and could be easily extended to include other PAs and PAs-ox.

Glyceollins and dehydroglyceollins isolated from soybean act as SERMs and ER subtype-selective phytoestrogens.

Seven prenylated 6a-hydroxy-pterocapans and five prenylated 6a,11a-pterocarpenes with different kinds of prenylation were purified from an ethanolic extract of fungus-treated soybean sprouts. The activity of these compounds toward both human estrogen receptors (hERα and hERß) was determined in a yeast bioassay and the activity toward hERα was additionally tested in an U2-OS based hERα CALUX bioassay. In the yeast bioassay, compounds with chain prenylation showed in general an agonistic mode of action toward hERα, whereas furan and pyran prenylation led to an antagonistic mode of action. Five of these antagonistic compounds had an agonistic mode of action in the U2-OS based hERα CALUX bioassay, implying that these compounds can act as SERMs. The yeast bioassay also identified 8 ER subtype-selective compounds, with either an antagonistic mode of action or no response toward hERα and an agonistic mode of action toward hERß. The ER subtype-selective compounds were characterized by 6a-hydroxy-pterocarpan or 6a,11a-pterocarpene backbone structure. It is suggested that either the extra D-ring or the increase in length to 12-13.5Å of these compounds is responsible for an agonistic mode of action toward hERß and, thereby, inducing ER subtype-selective behavior.

Prenylation and Backbone Structure of Flavonoids and Isoflavonoids from Licorice and Hop Influence Their Phase I and II Metabolism.

In vitro liver metabolism of 11 prenylated flavonoids and isoflavonoids was investigated by determining their phase I glucuronyl and sulfate metabolites using pork liver preparations. One hundred metabolites were annotated using RP-UHPLC-ESI-MS(n). A mass spectrometry-based data interpretation guideline was proposed for the tentative annotation of the position of hydroxyl groups, considering its relevance for estrogenic activity. To relate structure to metabolism, compounds were classified on the basis of three criteria: backbone structure (isoflavene, isoflavan, or flavanone), number of prenyl groups (0, 1, or 2), and prenyl configuration (chain or pyran). Glucuronidation was most extensive for isoflavenes and for unprenylated compounds (yield of 90-100%). Pyran and chain prenylation gave more complex hydroxylation patterns with 4 or more than 6 hydroxyl isomers, respectively, as compared to unprenylated compounds (only 1 hydroxyl isomer). Moreover, the number of hydroxyl isomers also increased with the number of prenyl groups.

Involvement of a Hydrophobic Pocket and Helix†11 in Determining the Modes of Action of Prenylated Flavonoids and Isoflavonoids in the Human Estrogen Receptor.

Six prenylated (iso)flavonoids were purified from a licorice root extract and subjected to competition experiments with six commercially available (iso)flavonoids. The agonistic and antagonistic activities of these compounds towards both hERα (human estrogen receptor alpha) and hERß were determined. Differences in the modes of action (agonist or antagonist) were observed for the various compounds tested. In general, each compound had the same mode of action towards both ERs. In silico modeling was performed in order to study the differences in estrogenicity observed between the compounds. It is suggested that prenyl chains fit into a hydrophobic pocket present in the hER, resulting in an increased agonistic activity. In addition, it was shown that an increase in length (≈1.7 Å) of pyran prenylated isoflavonoids resulted in an antagonistic mode of action. This might be caused by collision of the pyran ring with helix 11 in the ligand binding cavity of the hER.

Structural changes of 6a-hydroxy-pterocarpans upon heating modulate their estrogenicity.

The isoflavonoid composition of an ethanolic extract of fungus-treated soybean sprouts was strongly altered by a combined acid/heat treatment. UHPLC-MS analysis showed that 6a-hydroxy-pterocarpans were completely converted to their respective, more stable, 6a,11a-pterocarpenes, whereas other isoflavonoids, from the isoflavone and coumestan subclasses, were affected to a much lesser extent (loss of ∼15%). Subsequently, mixtures enriched in prenylated 6a-hydroxy-pterocarpans (pools of glyceollin I/II/III and glyceollin IV/VI) or prenylated 6a,11a-pterocarpenes (pools of dehydroglyceollin I/II/III and dehydroglyceollin IV/VI) were purified, and tested for activity on both human estrogen receptors (ERα and ERß). In particular, the response toward ERα changed, from agonistic for glyceollins to antagonistic for dehydroglyceollins. Toward ERß a decrease in agonistic activity was observed. These results indicate that the introduction of a double bond with the concomitant loss of a hydroxyl group in 6a-hydroxy-pterocarpans extensively modulates their estrogenic activity.