Non-target estrogenic screening of 60 pesticides, six plant protection products, and tomato, grape, and wine samples by planar chromatography combined with the planar yeast estrogen screen bioassay.

For non-target residue analysis of xenoestrogens in food, sophisticated chromatographic-mass spectrometric techniques lack in biological effect detection. Various in vitro assays providing sum values encounter problems when opposing signals are present in a complex sample. Due to physicochemical signal reduction, cytotoxic or antagonistic effect responses, the resulting sum value is falsified. Instead, the demonstrated non-target estrogenic screening with an integrated planar chromatographic separation differentiated opposing signals, detected and prioritized important estrogenic compounds, and directly assigned tentatively the responsible compounds. Sixty pesticides were investigated, ten of which showed estrogenic effects. Exemplarily, half-maximal effective concentrations and 17ß-estradiol equivalents were determined. Estrogenic pesticide responses were confirmed in six tested plant protection products. In food, such as tomato, grape, and wine, several compounds with an estrogenic effect were detected. It showed that rinsing with water was not sufficient to remove selected residues and illustrated that, though not usually performed for tomatoes, peeling would be more appropriate. Though not in the focus, reaction or breakdown products that are estrogenic were detected, underlining the great potential of non-target planar chromatographic bioassay screening for food safety and food control.

Metabolic reprogramming of hepatocytes by Schistosoma mansoni eggs.

Background & Aims: Schistosomiasis is a parasitic infection which affects more than 200 million people globally. Schistosome eggs, but not the adult worms, are mainly responsible for schistosomiasis-specific morbidity in the liver. It is unclear if S. mansoni eggs consume host metabolites, and how this compromises the host parenchyma. Methods: Metabolic reprogramming was analyzed by matrix-assisted laser desorption/ionization mass spectrometry imaging, liquid chromatography with high-resolution mass spectrometry, metabolite quantification, confocal laser scanning microscopy, live cell imaging, quantitative real-time PCR, western blotting, assessment of DNA damage, and immunohistology in hamster models and functional experiments in human cell lines. Major results were validated in human biopsies. Results: The infection with S. mansoni provokes hepatic exhaustion of neutral lipids and glycogen. Furthermore, the distribution of distinct lipid species and the regulation of rate-limiting metabolic enzymes is disrupted in the liver of S. mansoni infected animals. Notably, eggs mobilize, incorporate, and store host lipids, while the associated metabolic reprogramming causes oxidative stress-induced DNA damage in hepatocytes. Administration of reactive oxygen species scavengers ameliorates these deleterious effects. Conclusions: Our findings indicate that S. mansoni eggs completely reprogram lipid and carbohydrate metabolism via soluble factors, which results in oxidative stress-induced cell damage in the host parenchyma. Impact and implications: The authors demonstrate that soluble egg products of the parasite S. mansoni induce hepatocellular reprogramming, causing metabolic exhaustion and a strong redox imbalance. Notably, eggs mobilize, incorporate, and store host lipids, while the metabolic reprogramming causes oxidative stress-induced DNA damage in hepatocytes, independent of the host's immune response. S. mansoni eggs take advantage of the host environment through metabolic reprogramming of hepatocytes and enterocytes. By inducing DNA damage, this neglected tropical disease might promote hepatocellular damage and thus influence international health efforts.

Validated Screening Method for 81 Multiclass Veterinary Drug Residues in Food via Online-Coupling High-Throughput Planar Solid-Phase Extraction to High-Performance Liquid Chromatography-Orbitrap Tandem Mass Spectrometry.

Current screening capabilities for veterinary drugs (VDs) in foods are limited, requiring time-consuming and expensive trace-level analyses. For the first time, a high-throughput planar solid-phase extraction (HTpSPE) cleanup, controlled by UV/vis/FLD imaging, was developed for screening 81 VDs from 6 different groups (glucocorticoids, anthelmintics, antiparasitics, coccidiostats, nonsteroidal anti-inflammatory drugs, and antibiotics) in 4 different matrices (honey, pig muscle, cow milk, and chicken eggs). It consumed 13 times less solvent and was more eco-friendly and 5 times faster than routine methods. The VDs were automatically eluted using the autoTLC-LC-MS interface, separated online on a high-performance liquid chromatography column via a 10-min gradient, and detected by Orbitrap high-resolution tandem mass spectrometry. The screening method was validated according to the latest European Commission Implementing Regulation 2021/808. Most VDs except penicillins and cephalosporins were detected at the 5-µg/kg level in pig muscle, cow milk, and chicken eggs and 25-µg/kg level in honey.

Potential of simple, rapid, and non-target planar bioassay screening of veterinary drug residues.

Veterinary drug residues in food samples of animal origin are currently analyzed by target analysis using high-performance liquid chromatography combined with sophisticated mass spectrometers. Since the results are only partially consistent with the microbiological results and positive findings occur rarely (in the per mil range in Germany), the potential of a simple planar bioassay screening was studied in the field of veterinary drug residue analysis. Using only a simple dilution of the milk for sample preparation, it was challenging to meet the maximum residue limits for antibiotic drug residues, exemplarily shown for the screening of two fluoroquinolones. However, the potential was evident for a simple, rapid, eco-friendly, and non-target screening without expensive instrumentation. Regardless of whether it is an active metabolite, contaminant, degradation product, or veterinary drug residue, the effect indicated on the planar surface due to bioassay detection will most likely also affect the human microbiome when consumed. The non-target screening of the milk samples revealed compounds with substantial antibacterial effects, which were not in the previous focus of interest. These antibacterial compounds will most likely also affect the human microbiome. Is it only the regulated antibiotic residues or generally all antibiotic compounds in a sample that count for consumer protection? The current prevailing understanding of food safety and antimicrobial resistance, based on the results of target (rather than effect) analyses, is being challenged. Non-target planar bioassay screening has been shown to fill a current gap by providing an understanding of inconsistencies and complementing routine target analysis of veterinary drug residues. As a highlight, it provides the full picture of the real levels of active compounds, regardless of the permitted limits of antibiotics.

Evidence that Indo-Pacific bottlenose dolphins self-medicate with invertebrates in coral reefs.

Indo-Pacific bottlenose dolphins (Tursiops aduncus) have been observed queueing up in natural environments to rub particular body parts against selected corals (Rumphella aggregata, Sarcophyton sp.) and sponges (Ircinia sp.) in the Egyptian Northern Red Sea. It was hypothesized that the presence of bioactive metabolites accounts for this selective rubbing behavior. The three invertebrates preferentially accessed by the dolphins, collected and analyzed by hyphenated high-performance thin-layer chromatography contained seventeen active metabolites, providing evidence of potential self-medication. Repeated rubbing allows these active metabolites to come into contact with the skin of the dolphins, which in turn could help them achieve skin homeostasis and be useful for prophylaxis or auxiliary treatment against microbial infections. This interdisciplinary research in behavior, separation science, and effect-directed analysis highlighted the importance of particular invertebrates in coral reefs, the urgent need to protect coral reefs for dolphins and other species, and calls for further vertebrate-invertebrate interaction studies.

Effect-Directed Profiling of Monofloral Honeys from Ethiopia by High-Performance Thin-Layer Chromatography and High-Resolution Mass Spectrometry.

Ethiopian honey is used not only as food but also for treatment in traditional medicine. For its valorization, bioactive compounds were analyzed in nine types of monofloral Ethiopian honey. Therefore, a non-target effect-directed profiling was developed via high-performance thin-layer chromatography combined with multi-imaging and planar effect-directed assays. Characteristic bioactivity profiles of the different honeys were determined in terms of antibacterial, free-radical scavenging, and various enzyme inhibitory activities. Honeys from Hypoestes spp. and Leucas abyssinica showed low activity in all assays. In contrast, others from Acacia spp., Becium grandiflorum, Croton macrostachyus, Eucalyptus globulus, Schefflera abyssinica, Vernonia amygdalina, and Coffea arabica showed more intense activity profiles, but these differed depending on the assay. In particular, the radical scavenging activity of Croton macrostachyus and Coffea arabica honeys, the acetylcholinesterase-inhibiting activity of Eucalyptus globulus and Coffea arabica honeys, and the antibacterial activity of Schefflera abyssinica honey are highlighted. Bioactive compounds of interest were further characterized by high-resolution mass spectrometry. Identifying differences in bioactivity between mono-floral honey types affects quality designation and branding. Effect-directed profiling provides new insights that are valuable for food science and nutrition as well as for the market, and contributes to honey differentiation, categorization, and authentication.

On-surface autosampling for liquid chromatography-mass spectrometry.

An on-surface multi-purpose autosampler was built for liquid chromatography-mass spectrometry (LC-MS) based on the autoTLC-MS interface, taking advantage of open-source hard- and software developments as well as 3D printing. Termed autoTLC-LC-MS system, it is introduced for orthogonal hyphenation of normal phase high-performance thin-layer chromatography with reversed phase high-performance LC (HPLC) and high-resolution MS (HRMS). For verification of its functionality, a multi-class antibiotic mixture was applied as a calibration band pattern on an adsorbent layer and detected by the Bacillus subtilis bioassay. This effect-image was uploaded as a template in the updated TLC-MS_manager software. The clicked-on antibiotic zones were sequentially eluted without intervention from the planar counterpart (without bioassay) via a monolithic HPLC column into the HRMS system. For elution of antibiotics of 7 structural classes at 5 different calibration levels, the new on-surface autosampler achieved intra-day precisions of 2.1-14.1%, while inter-day precisions ranged 2.5-16.1% (all n = 3). The new hyphenation offers potential for planar sample clean-up prior to HPLC, concentration of liquid samples, increase of peak capacity and proof of peak purity or isomers. The integrated autoTLC-LC-MS system enabled high sample throughput, efficiency and reproducibility for the first time through fully automated TLC-LC-MS sequence operation. Its contact-closure signal functionality, versatile 3D printed planar sample holder and open-source software made it readily adjustable for new analytical tasks. Undoubtedly, any planar material can be investigated for leachables, such as textiles, foils, papers and other packagings, as well as planar biological samples for ingredients.

High-throughput planar solid-phase extraction coupled to orbitrap high-resolution mass spectrometry via the autoTLC-MS interface for screening of 66 multi-class antibiotic residues in food of animal origin.

Antibiotic residues in food pose a major threat to the health of humans and animals worldwide. Their trace-level analysis is still too time- and cost-intensive to be adequately covered in routine analysis. Thus, a new high-throughput planar solid-phase extraction method has been developed for rapid screening of 66 antibiotics. Via simple clicks on the image, the autoTLC-MS interface automatically eluted the target analyte zones directly into an orbitrap high-resolution mass spectrometer operated in the variable data-independent acquisition mode. Muscle tissue, cow milk and chicken eggs were analyzed regarding nine different antibiotic classes, including sulfonamides, diaminopyrimidines, lincosamides, pleuromutilins, macrolides, cephalosporins, penicillins, amphenicols and nitroimidazoles. The planar clean-up took 7 min per sample, which is 5-fold faster than the routine state-of-the-art. The screening method has been validated for one representative of each class according to the European Commission Decision 2002/657/EC. Most analytes were successfully detected at half of their required maximum residue limit.