Towards non-target proactive food safety: identification of active compounds in convenience tomato products by ten-dimensional hyphenation with integrated simulated gastrointestinal digestion.

Current strategies for non-target food screening focus mainly on known hazardous chemicals (adulterants, residues, contaminants, packaging migrants, etc.) instead of bioactive constituents in general and exclude the biological effect detection. To widen the perspective, a more proactive non-target effect-directed strategy is introduced to complement food safety in order to detect not only known but also unknown bioactive compounds. The developed 10-dimensional hyphenation included on-surface digestion (1D), planar chromatographic separation (2D), visualization using white light (3D), UV light (4D), fluorescence light (5D), effect-directed assay analysis (6D), heart-cut zone elution to an orthogonal reversed phase column chromatography including online desalting (7D) with subsequent diode array detection (8D), high-resolution mass spectrometry (9D), and fragmentation (10D). Metabolism, i.e., intestinal digestion of each sample, was simulated and integrated on the same adsorbent surface to study any changes in the compound profiles. As proof of principle, nine convenience tomato products and a freshly prepared tomato soup were screened via five different planar assays in a non-targeted mode. Non-digested and digested samples were compared side by side. In their effect-directed profiles, 14 bioactive compounds from classes of lipids, plant hormones, spices, and pesticides were identified. In particular, bioactive compounds coming from the lipid class were increased by gastrointestinal digestion, while spices and pesticides remained unaffected. With regard to food safety, the determination of the two dinitrophenol herbicides dinoterb and dinoseb in highly processed tomato products should be given special attention. The hyphenation covered a broad analyte spectrum and showed robust and reliable results.

Investigation of the estrogenic potential of 15 rosé, white and red wines via effect-directed ten-dimensional hyphenation.

Wine is consumed for thousands of years all over the world, however, its estrogenic potential is still underexplored. A non-target effect-directed screening was developed to reveal estrogen-like and antiestrogen-like compounds in 15 rosé, white and red wine samples of different origin and grape variety. Normal-phase high-performance thin-layer chromatography multi-imaging detection (NP-HPTLC-UV/Vis/FLD) was combined with the planar yeast estrogen screen (pYES) bioassay or the duplex planar yeast antagonist estrogen screen (pYAES) bioassay on the same adsorbent surface. Up to nine estrogen-like compound zones were detected and further characterized via heart-cut elution from the planar bioautogram to orthogonal reversed phase high-performance liquid chromatography (RP-HPLC) coupled with diode array detection (DAD) and high-resolution tandem mass spectrometry (HRMS/MS). Among the tentatively assigned estrogen-like substances, the HRMS/MS signals pointed to hexylresorcinol and diethyl esters from organic acids for the first time. This highlights the method suitability for non-target complex mixture screening and rapid dereplication. The 10D hyphenation NP-HPTLC-UV/Vis/FLD-pYAES-heart cut-RP-HPLC-DAD-HRMS/MS proved to be an efficient and powerful tool for detecting estrogens as well as antiestrogens in the matrix-rich wine samples. High-throughput capability and substantial reduction in the required resources for analysis were demonstrated by this straightforward hyphenation, if compared to bioassay-guided fractionation. The 10D information (via orthogonal chromatographic, versatile spectrometric and duplex endocrine activity data) obtained during a single chromatographic run for many samples in parallel was advantageous for the tentative molecule assignment.

Ten-dimensional hyphenation including simulated static gastro-intestinal digestion on the adsorbent surface, planar assays, and bioactivity evaluation for meal replacement products.

Meal replacement products are normally consumed in weight-loss interventions and the treatment of obesity and diabetes. Changing lifestyles and eating habits made meal replacement products in the forms of shakes and bars a good alternative as To-go-meals, promoted as balanced in its composition and thus healthier compared to other ready-to-eat meals. This study aimed to evaluate the bioactivity of six differently flavoured powdered meal replacement products. Their analysis was made by a ten-dimensional hyphenation composed of digestion on the adsorbent surface, followed by normal-phase high-performance thin-layer chromatographic separation, multi-imaging, and planar assay application (effect-directed analysis), and then heart-cut elution/transfer of bioactive compound zones to reversed-phase high-performance liquid chromatography, diode array detection, and high-resolution tandem mass spectrometry. The on-surface digestion of saccharides, fats, and proteins through intestinal enzymatic activity revealed new breakdown products. These exhibited bioactivity in their different effect-profiles obtained by the Gram-negative Aliivibrio fischeri bioassay as well as α-/ß-glucosidase and acetyl-/butyrylcholinesterease inhibition assays. The main bioactive compounds arising through simulated static pancreatic digestion were saturated and unsaturated free fatty acids. The synthetic sweetener sucralose was not influenced by simulated static intestinal digestion, but showed antimicrobial activity. In the prepared drinking meals with coffee and choco flavour, the acetylcholinesterase-inhibiting methylxanthines caffeine and theobromine were identified as bioactive compounds. Some other bioactive constituents could not be assigned to specific molecules and require further analyses. Although the studied meal replacement products showed health-beneficial properties through antimicrobial properties or inhibition of enzymes involved in the expression of the civilisation diseases, such as diabetes and Alzheimer's disease, plant foods, herbs and spices have been shown to be even richer and more versatile in bioactive compounds.

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.

Is Our Natural Food Our Homeostasis? Array of a Thousand Effect-Directed Profiles of 68 Herbs and Spices.

The beneficial effects of plant-rich diets and traditional medicines are increasingly recognized in the treatment of civilization diseases due to the abundance and diversity of bioactive substances therein. However, the important active portion of natural food or plant-based medicine is presently not under control. Hence, a paradigm shift from quality control based on marker compounds to effect-directed profiling is postulated. We investigated 68 powdered plant extracts (botanicals) which are added to food products in food industry. Among them are many plants that are used as traditional medicines, herbs and spices. A generic strategy was developed to evaluate the bioactivity profile of each botanical as completely as possible and to straightforwardly assign the most potent bioactive compounds. It is an 8-dimensional hyphenation of normal-phase high-performance thin-layer chromatography with multi-imaging by ultraviolet, visible and fluorescence light detection as well as effect-directed assay and heart-cut of the bioactive zone to orthogonal reversed-phase high-performance liquid chromato-graphy-photodiode array detection-heated electrospray ionization mass spectrometry. In the non-target, effect-directed screening via 16 different on-surface assays, we tentatively assigned more than 60 important bioactive compounds in the studied botanicals. These were antibacterials, estrogens, antiestrogens, androgens, and antiandrogens, as well as acetylcholinesterase, butyrylcholinesterase, α-amylase, α-glucosidase, ß-glucosidase, ß-glucuronidase, and tyrosinase inhibitors, which were on-surface heart-cut eluted from the bioautogram or enzyme inhibition autogram to the next dimension for further targeted characterization. This biological-physicochemical hyphenation is able to detect and control active mechanisms of traditional medicines or botanicals as well as the essentials of plant-based food. The array of 1,292 profiles (68 samples × 19 detections) showed the versatile bioactivity potential of natural food. It reveals how efficiently and powerful our natural food contributes to our homeostasis.

Non-target bioanalytical eight-dimensional hyphenation including bioassay, heart-cut trapping, online desalting, orthogonal separations and mass spectrometry.

It is still a challenge to discover and identify individual bioactive compounds directly in multicomponent mixtures. Current workflows are too tedious for routine use. Hence, the hyphenation of separation and detection techniques is a powerful tool to maximize the information obtained by a single sample run. A robust eight-dimensional (8D) hyphenation was developed. Orthogonal separations, biological assay detection, analyte trapping, desalting, and physico-chemical detections were arranged in the following order, i.e. 1) normal phase high-performance thin-layer chromatography (NP-HPTLC) separation, 2) Vis detection, 3) UV detection, 4) fluorescence detection (FLD), 5) bioassay for effect-directed analysis (EDA), 6) heart-cut trapping/desalting/elution to reversed phase high-performance liquid chromatography (RP-HPLC) separation, 7) photodiode array (PDA) and 8) mass spectrometry (MS) detection. For the first time, the hyphenation exploited online analyte trapping to desalt the eluted bioactive zone from the plate containing highly salted bioassay media. Subsequent valve switching guided the trapped analyte(s) to the main column, followed by multiple detection. As proof-of-principle, cinnamon samples were analyzed by NP-HPTLC-UV/Vis/FLD-EDA-RP-HPLC-PDA-MS, whereby a bioactive zone was separated into two distinct peaks detected by PDA and MS to be 2-methoxy cinnamaldehyde and cinnamaldehyde. The developed 8D hyphenation is applicable for routine, allowing the non-target high-throughput screening of complex samples for individual bioactive compounds.