Among the analytical advances, hyphenated HPTLC offers great potential for solving pressing questions. It provides straightforward information about effects arising from individual compounds in ...complex or natural samples separated in parallel. The chromatographic separation is combined with effect-directed detection using enzymatic or biological assays. This helps to select from the thousands of compounds in a sample the important ones that need to be further characterized using high-resolution mass spectrometry. Unique benefits are discussed exemplarily arising from its super-hyphenation, minimum requirements for sample preparation, detection of multi-modulating compounds or agonistic versus antagonistic effects, and miniaturized on-surface metabolization. HPTLC stands for a versatile, creative and flexible open-format technique. As miniaturized open-source LabToGo system, it shows the potential to be applied as Citizen Science.
Display omitted
•Super-hyphenation suited to identify multi-modulating compounds in complex samples.•Important components are not overlooked by minimalistic sample preparation.•Simultaneous differentiation of agonistic/antagonistic effects provides understanding.•On-surface metabolization systems show (de)activation of conversion products.•Open-source LabToGo development miniaturizes status quo technology.
•Robust non-target bioanalytical screening for bioactive compounds was developed.•Eight-dimensional hyphenation: NP-HPTLC–UV/Vis/FLD–EDA–RP-HPLC–PDA–MS•Analyte trapping and online desalting reduced ...the transfer of bioassay salts to MS.•Orthogonal separations improved the separation power for complex samples.•Proof-of-principle was successfully made for active compounds in 18 cinnamon samples.
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.
Display omitted
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 17β-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.
Graphical Abstract
Display omitted
•All-in-one 2LabsToGo system for atline safety control of rye was introduced.•Sustainable, portable, miniaturized system is proposed for analysis in milling plants.•Cost-effective ...analysis of ergot alkaloids was developed, validated and verified.•Limit value for ergot alkaloids was well reached after minimal sample preparation.•2LabsToGo results were equivalent to those of status quo HPTLC instruments.
Ergot alkaloids, naturally occurring mycotoxins of Claviceps fungi, pose health risks. This necessitates accurate analysis methods to ensure food safety. This study explored the open-source miniaturized all-in-one 2LabsToGo system to analyze ergot alkaloids in whole rye samples. It is suited for sustainable atline analysis as it combines all planar chromatography tasks, allowing low-cost quality control in milling plants. The LOD and LOQ of ergocristine were determined to be 0.4 and 1.2 ng/zone, respectively. Detectability of ergot alkaloids was proven to be below the current maximum limit of 500 µg/kg for rye milling products. The repeatability (%RSD) was 4.1 % and the coefficient of determination of the analytical response (R2) was 0.9918 for ergocristine. The mean recovery rate of ergot alkaloids in spiked whole rye grain was close to 100 %. Results of screening whole rye for ergot alkaloids were successfully verified by comparison with those obtained by conventional status quo HPTLC instrumentation.
•New genotoxicity profiling was developed to analyze healthy oils for genotoxins.•Healthy oils containing unsaturated fatty acids were shown to be genotoxic.•Detoxification of genotoxins in healthy ...oils was demonstrated for the first time.•Since not S9-metabolized, topical application of healthy oils is deemed harmful.•Genotoxic effects were differentiated from opposing responses and cytotoxic effects.
Current techniques used in food analysis overlook genotoxic compounds. This urgently calls for a paradigm shift in analytics towards non-target planar genotoxicity profiling that can detect genotoxins. Up to eight different genotoxins (i.e., genotoxic compound zones) have been detected in 33 oils used for healthy diets. A comparison of fresh oils with oils stored open and closed for one month identified genotoxic degradation products. Characterization of genotoxic zones via high-resolution mass spectrometry revealed oxidized linolenic acid as a source of genotoxicity in all samples. Detoxification via on-surface S9 liver metabolization was investigated, which showed a reduction in most, but not all, genotoxins. Food, feed, dietary supplements, and cosmetics as sources of genotoxicity can now be identified by combining separation, effect detection and optionally simulated metabolization on the same surface. The application of the planar genotoxicity profiling will improve the understanding on food and its impact as well as risk assessment and derived recommendations.
To analyze a complex sample for endocrine activity, different tests must be performed to clarify androgen/estrogen agonism, antagonism, cytotoxicity, anti-cytotoxicity, and corresponding ...false-positive reactions. This means a large amount of work. Therefore, a six-fold planar multiplex bioassay concept was developed to evaluate up to the mentioned six endpoints or mechanisms simultaneously in the same sample analysis. Separation of active constituents from interfering matrix via high-performance thin-layer chromatography and effect differentiation via four vertical stripes (of agonists and end-products of the respective enzyme–substrate reaction) applied along each separated sample track were key to success. First, duplex endocrine bioassay versions were established. For the androgen/anti-androgen bioassay applied via piezoelectric spraying, the mean limit of biological detection of bisphenol A was 14 ng/band and its mean half maximal inhibitory concentration IC50 was 116 ng/band. Applied to trace analysis of six migrate samples from food packaging materials, 19 compound zones with agonistic or antagonistic estrogen/androgen activities were detected, with up to seven active compound zones within one migrate. For the first time, the S9 metabolism of endocrine effective compounds was studied on the same surface and revealed partial deactivation. Coupled to high-resolution mass spectrometry, molecular formulas were tentatively assigned to compounds, known to be present in packaging materials or endocrine active or previously unknown. Finally, the detection of cytotoxicity/anti-cytotoxicity and false-positives was integrated into the duplex androgen/anti-androgen bioassay. The resulting six-fold multiplex planar bioassay was evaluated with positive control standards and successfully applied to one migrate sample. The streamlined stripe concept for multiplex planar bioassays made it possible to assign different mechanisms to individual active compounds in a complex sample. The concept is generic and can be transferred to other assays.
Display omitted
•Six-fold multiplex bioassay integrated cytotoxicity and false-positive antagonists on the same surface.•Non-target HPTLC−Vis/FLD−pYAVCytotox−pYAVAS/pYAVES bioassay screening.•Up to 19 migrating endocrine-disrupting compounds detected with duplex bioassay.•Dose-response-curve, IC50 and LOBD of bisphenol A and further standards determined.•Molecule formulas assigned to endocrine active compounds via HPTLC−HRMS.
On the basis of open-source packages, 3D printing of thin silica gel layers is demonstrated as proof-of-principle for use in planar chromatography. A slurry doser was designed to replace the plastic ...extruder of an open-source Prusa i3 printer. The optimal parameters for 3D printing of layers were studied, and the planar chromatographic separations on these printed layers were successfully demonstrated with a mixture of dyes. The layer printing process was fast. For printing a 0.2 mm layer on a 10 cm × 10 cm format, it took less than 5 min. It was affordable, i.e., the running costs for producing such a plate were less than 0.25 Euro and the investment costs for the modified hardware were 630 Euro. This approach demonstrated not only the potential of the 3D printing environment in planar chromatography but also opened new avenues and new perspectives for tailor-made plates, not only with regard to layer materials and their combinations (gradient plates) but also with regard to different layer shapes and patterns. As such an example, separations on a printed plane layer were compared with those obtained from a printed channeled layer. For the latter, 40 channels were printed in parallel on a 10 cm × 10 cm format for the separation of 40 samples. For producing such a channeled plate, the running costs were below 0.04 Euro and the printing process took only 2 min. All modifications of the device and software were released open-source to encourage reuse and improvements and to stimulate the users to contribute to this technology. By this proof-of-principle, another asset was demonstrated to be integrated into the Office Chromatography concept, in which all relevant steps for online miniaturized planar chromatography are performed by a single device.
Display omitted
•Coffee beans roasted differently and coffees prepared differently were studied for effects.•Storage conditions influenced the bioactive potential of the coffee brews.•Antioxidative ...components were identified using HPTLC-EDA-MS.•Chlorogenic acid, caffeine, caffeoyl-β-d-glucopyranoside and melanoidines were highly active.•Caffeine and chlorogenic acid as important quality parameters were exemplarily quantified.
Coffee is an inherent part of our daily nutrition and seems to have protective effects against diseases, whereby it is often not fully understood, which ingredients are responsible for the observed effect. Hence, a non-targeted bioactivity profiling was developed to investigate 27 hand-filtered coffee brews of differently roasted coffee beans and 14 differently prepared and stored coffee brews. After separation, multi-imaging, and densitometry, six planar effect-directed assays were performed to reveal individual antioxidative, antibacterial, anti-cholinesterase, anti-diabetic, and estrogenic effects. Individual compounds were mainly responsible for the observed effects, e.g. 5-O-caffeoylquinic acid regarding antioxidative potential and α-glucosidase inhibition, while coffee brews made by a fully automated coffee machine showed the highest antioxidative potential. Unlike preparation and storage conditions, applied roasting conditions and origin of coffee samples played a less important role. Therefore, the way we daily consume our coffee has an impact on the magnitude of potential health effects.
•Set up a cost-effective automated piezoelectric spraying for biological and enzymatic assays.•Bioprofiling of Peganum harmala seed extract via piezoelectric spaying versus immersion.•Selective ...cholinesterase assays (AChE/BChE) to find the active ingredients.•Cholinesterase inhibitory equivalency referred to physostigmine via HPTLC-AChE/BChE.•In situ HRMS measurement of the active zones in the P. harmala seed extract.
Bioanalytical questions are more and more solved by bioassays directly in situ the planar separation. If compared to chemical derivatization in situ, several reagent applications on the same chromatogram make the workflow for enzymatic and biological assays more complex. Hence, if compared to piezoelectric spraying of chemical derivatization reagents, an assay transfer to the piezoelectric spraying technique was much more challenging. Important aspects were investigated, i.e., plate pre-wetting, spraying nozzle type and applied volumes for microorganism suspension as well as enzyme and substrate-chromogenic solutions. Finally, with the newly developed piezoelectric spraying procedures for the application of biological (Aliivibrio fischeri) and enzymatic (acetyl- and butyrylcholinesterase) assays, several obstacles of the state-of-the-art automated immersion were avoided such as the (1) required high volumes of solutions, (2) tailing of highly water-soluble zones upon slow plate withdrawal, (3) zone distortion or shift observed after previous buffer salt applications or long/slow immersion times/speeds, (4) gradual inactivation of the enzyme solution along with its ongoing re-use, and (5) lack of covering the whole plate surface. The benchmarking of both techniques also showed that simplicity remains the key argument for immersion. As proof of concept, piezoelectrically sprayed autograms were compared with those of immersion, by taking the example of Peganum harmala (P. h.) seed extract. The plate background and thus homogeneity of the applied solutions were found to be almost comparable. Three bands among the pronounced fluorescent bands were responsible for the most antibacterial activity of P. h. seed extract in the A. fischeri bioassay and were also inhibiting the AChE. These AChE and three further BChE inhibitors were detected, whereby the AChE inhibition was twice as strong as the BChE inhibition. By their in situ HRMS spectra, the active zones in the P. h. seed extract were assigned to be the AChE-inhibiting β-carboline alkaloids, harmine, harmaline and ruine, as well as the BChE-inhibiting quinazoline alkaloids, vasicine and deoxyvasicine, and the β-carboline alkaloid harmol. For the first time, the found inhibitors were calculated equivalently to the well-known ChE-inhibitor physostigmine, and thus, piezoelectric spraying was proven to be suited for quantifications.
PDF
