Screening of α-amylase/trypsin inhibitor activity in wheat, spelt and einkorn by high-performance thin-layer chromatography.

α-Amylase/trypsin inhibitor proteins (ATI) are discussed as possible triggers for non-celiac gluten sensitivity. The potential of high-performance thin-layer chromatography (HPTLC) was studied for the first time to analyse the inhibitory properties of ATIs from flour of wheat, spelt, and einkorn. Inhibition by each flour of the digestive enzymes trypsin or α-amylase was determined by the reduction of released metabolisation products in comparison to non-digested flour, and positive (acarbose) and negative (water) controls. Firstly, amylolysis was carried out in miniaturized form on the HPTLC surface (HPTLC-nanoGIT) after in-vial pre-incubation of the amylase with the inhibitors from flour. α-Amylase inhibition was evident via the reduction of released saccharides, as analysed by normal phase HPTLC. A strong influence of the flour matrix on the assay results (individual saccharides) was evident, caused by an increased amylolysis of further polysaccharides present, making HPTLC analysis more reliable than currently used spectrophotometric sum value assays. The detection and visualization of such matrix influence helps to understand the problems associated with spectrophotometric assays. Only maltotriose was identified as a reliable marker of the amylolysis. The highest α-amylase inhibition and thus the lowest saccharide response was detected for maltotriose in refined spelt, whereas the lowest α-amylase inhibition and thus the highest saccharide response was detected for maltotriose in refined wheat. A comparison of refined and whole grain flours showed no clear trend in the responses. Secondly, trypsin inhibition and proteolysis were performed in-vial, and any inhibition was evident via the reduction of released peptides, analysed by reversed-phase HPTLC. Based on the product pattern of the proteolysis, einkorn and whole wheat showed the highest trypsin inhibition, whereas refined wheat and refined spelt showed the lowest inhibition. Advantageously, HPTLC analysis provided important information on changes in individual saccharides or peptides, which was more reliable and sustainable than spectrophotometric in-vial assays (only sum value) or liquid column chromatography analysis (targeting only the ATI proteins).

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.

2LabsToGo─Recipe for Building Your Own Chromatography Equipment Including Biological Assay and Effect Detection.

A complete recipe for building your own chromatography equipment from readily available materials is introduced. It combines sample separation (chemistry laboratory) with biological effect detection (biology laboratory). This hyphenation of two disciplines is necessary for prioritizing important compounds in complex samples. Among the thousands of compounds therein, it is often not clear which compounds are the important ones. On the same separation surface, additional detection of biological effects enables and guides substance prioritization. The newly developed open-source 2LabsToGo system for chemical and biological analysis is completely solvent-resistant and, due to miniaturization, environmentally friendly regarding the consumption of materials. It produces comparable results but is 10 times more compact (26 cm × 31 cm × 34 cm), 10 times lighter (6.8 kg), and 55 times less expensive (€ 1717) than current sophisticated commercial devices. As a proof of concept of the first 2LabsToGo system, the quality of different water samples was analyzed since clean water is becoming increasingly rare. In water, most of the thousands of substance signals or features can neither be identified nor classified toxicologically. However, methods that exploit this hyphenated strategy provide answers to such essential safety issues. Drinking or tap water did not show bioactive or toxic compounds, which was expected, whereas biogas or landfill water samples did. The hyphenated 2LabsToGo strategy is affordable and extremely useful for all laboratories with limited equipment but pressing challenges. It is ready to be used in various analytical tasks and applications.

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.

High-Performance Thin-Layer Chromatography Coupled with Electrospray Ionization Tandem Mass Spectrometry for Identifying Neutral Lipids and Sphingolipids in Complex Samples.

High-performance thin-layer chromatography was directly combined with electrospray mass spectrometry (ESI-MS) for structural identification issues below the level of lipid classes in complex samples through a portable, automated, elution-based interface. For samples as diverse as biodiesel and human plasma, separation conditions using Automated Multiple Development were selected in each case to provide lipid classes as zones narrow enough to ensure a direct transfer of them to ESI-MS. The respective zone of interest can be selected at will. ESI+ spectra of neutral lipids and sphingolipids showed sodium adducts when recorded from the plate. By using the described technique and ion-trap technology, the respective sodium adducts were fragmented. Sodium remained as the charge of the fragment ions and, thus, was useful for their structural identification through MSn. In this way, composition profiles of each class by ESI+-MS, and further identification of individual lipids and the molecular species belonging to each of them, were obtained by MS/MS and/or high-resolution MS. Thus, mono and diacylglycerides in ESI+ and fatty acids (in ESI-) were identified as low-concentration impurities in a fatty acid methyl ester-based biodiesel sample. Likewise, molecular species of sphingomyelins and globotriaosylceramides were unequivocally identified in human plasma samples.

Quantification of heterocyclic aromatic amines in fried meat by HPTLC/UV-FLD and HPLC/UV-FLD: a comparison of two methods.

A recently developed HPTLC/UV-FLD method was compared to the routinely used HPLC/UV-FLD method for the quantification of heterocyclic aromatic amines (HAA) formed at trace levels during the heating process of meat. For formation of these process contaminants under normal cooking conditions, beef patties were fried in a double-contact grill at 230 degrees C for five different frying times and extracted by solid-phase extraction. The HAAs most frequently found, that is, 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (PhIP), 2-amino-3,8-dimethylimidazo[4,5- f]quinoxaline (MeIQx), 2-amino-3,4,8-trimethylimidazo[4,5- f]quinoxaline (4,8-DiMeIQx), 9 H-pyrido[3,4- b]indole (norharman), and 1-methyl-9 H-pyrido[3,4- b]indole (harman), were quantified by two chromatographic methods, which were orthogonal to each other (normal versus reversed phase system). Both methods showed a similar performance and good correlation of the results ( R (2) between 0.8875 and 0.9751). The comparison of running costs and run time in routine analysis proved HPTLC/UV-FLD to be more economical (factor of 3) and faster (factor of 4) due to its capability of parallel chromatography. The HAA findings calculated by standard addition increased with the heating time from <1 to 33 microg/kg related to 3-6 min of frying time. The precision (RSD) was between 7 and 49% (HPTLC) and between 5 and 38% (HPLC) at these very low HAA levels formed.