Multiplex planar bioassay with reduced diffusion on normal phase, identifying androgens, verified antiandrogens and synergists in botanicals via 12D hyphenation.

Hormonal active compounds affecting health by altering the hormonal system are present in food. The planar yeast antagonist androgen screen (pYAAS) bioassay is a powerful tool to detect individual hormonal active compounds in complex samples separated by high-performance thin-layer chromatography (HPTLC). Previous methods lacked either detection sensitivity or zone sharpness. To overcome diffusion caused by long bioassay incubation on the normal-phase (NP) plate, zone fixation (fix) was achieved with a new polyisobutyl methacrylate coating, leading to enhanced zone sharpness. The exclusion of false-positive antagonists was integrated in the workflow, which allowed the verification (V) of true antagonists, apart from the detection of synergists. With the new multiplex bioassay providing information on 4 activities, 68 different botanicals were screened and hormonal active zones were identified by elution from the bioautogram to orthogonal reversed-phase high performance liquid chromatography with diode array detection and high-resolution mass spectrometry including fragmentation, resulting in the 12D hyphenation NP-HPTLCfix-UV/Vis/FLD-pYAVAS-FLD-heart cut-RP-HPLC-DAD-HRMS/MS.

Multiplex planar bioassay detecting estrogens, antiestrogens, false-positives and synergists as sharp zones on normal phase.

BACKGROUND: Phytoestrogens are found in many plants used in traditional medicines. Increasingly, plant extracts (botanicals) are also being added to foods or marketed as dietary supplements. Especially such powder formulations are susceptible to adulteration and falsification, given the global processing chain. To detect estrogen-like compounds in such multicomponent mixtures, non-target screening for hormonally active or endocrine disrupting compounds in plant products is becoming more important. Unfortunately, the current planar yeast estrogen screen (pYES) is prone to zone diffusion on the normal-phase high-performance thin-layer chromatography (NP-HPTLC) plate due to long incubation times in the aqueous bioassay. PURPOSE: The present study aimed to reduce zone diffusion on NP plates, which provides the basis for extending pYES to a multiplex bioassay, offering 4 different biological activity principles, followed by targeted identification of active zones. STUDY DESIGN AND METHODS: The reduction of substance diffusion via a polyisobutyl methacrylate polymer coating was studied. After successful zone fixation (fix), a multiplex bioassay was developed, in which a 17ß-estradiol-strip was applied along each sample track to detect synergists and antagonists (A), and for verification (V), a 4-methyl umbelliferone-strip to exclude false-positives. After multiplex bioassay screening of 68 botanicals, the zones with hormonal activities were heart-cut eluted to reversed-phase high-performance liquid chromatography-diode array detection-high-resolution tandem mass spectrometry (RP-HPLC-DAD-HESI-HRMS/MS). RESULTS: The separated substances were successfully fixed by the chromatogram coating. The zone sharpness (achieved after the bioassay) made it possible to add two strips, the 17ß-estradiol-strip for antagonistic and synergistic, and the 4-methyl umbelliferone-strip for false-positive effect detection, resulting in a multiplex bioassay. Using the 12D hyphenation NP-HPTLCfix-UV/Vis/FLD-pYAVES-FLD heart-cut RP-HPLC-DAD-HESI-HRMS/MS, it was possible to obtain information on estrogens, antiestrogens, false-positives, and synergists, and (tentatively) assign 17 hormonally active compounds, of which only 7 have been known to affect the human estrogen receptor, while another 4 had structural similarity to common phytoestrogens and antiestrogens. CONCLUSIONS: The streamlined 12D hyphenation including a multiplex bioassay has been shown to differentiate hormonal effects, leading to new insights and better understanding. It can generally be used to identify unknown hormonally active compounds in complex samples.

Effect-directed profiling of 32 vanilla products, characterization of multi-potent compounds and quantification of vanillin and ethylvanillin.

Food testing is of great importance to the food industry and organizations to verify the authenticity claims, to prove the quality of raw materials and products, and to ensure food safety. The market prices of vanilla differed by a factor of about 20 in the last three decades. Therefore the risk of adulteration and counterfeiting of vanilla products is high. Instead of commonly used target analyses and sum parameter assays, a complementary non-target multi-imaging effect-directed screening was developed, which provided a new perspective on the wide range of vanilla product qualities on the market. Planar chromatography was combined with effect-directed assays, and the obtained biological and biochemical profiles of 32 vanilla products from nine different categories revealed a variety of active ingredients. Depending on the region, typical vanilla product profiles and activity patterns were obtained for pods, tinctures, paste (inner part), oleoresin and powders. However, some vanilla products showed additional active compounds and a different intensity pattern. The vanilla product profiles substantially differed from those of vanilla aroma or products containing synthetic vanillin or vanilla-flavored food products. Bioactive compounds of interest were online eluted and further characterized via HPTLC-HRMS, which allowed their tentative assignment. After purchase of the standards, these were successfully confirmed by co-chromatography. Quantification of vanillin across nine different product categories revealed levels ranging from 1 µg/g to 36 mg/g with a mean repeatability of 1.9%. The synthetic ethylvanillin was not detected in the investigated samples in significant concentrations. The assessment of differences in the activity patterns pointed to highly active compounds, which were not detected at UV/Vis/FLD but first via the biological and enzymatic assays. This effect-directed profiling bridges the gap from analytical food chemistry to food toxicology, and thus, makes an important contribution to consumer safety. In the same way, it would accelerate investigations for Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) according to Regulation (EC) No. 1907/2006.

Engineered anisotropic microstructures for ultrathin-layer chromatography.

The strong dependence of separation behavior on ultrathin-layer chromatography (UTLC) stationary phase microstructure motivates continued UTLC plate design optimization efforts. We fabricated 4.6-5.3 mum thick normal phase silica UTLC stationary phases with several types of in-plane macropore anisotropies using the glancing angle deposition (GLAD) approach to engineering nanostructured thin films. The separation behaviors of two new media, isotropic vertical posts and anisotropic bladelike films, were compared to that of anisotropic chevron media. Channel-like structures within the anisotropic media introduced preferential mobile phase flow directions that could be exploited to give separation tracks diagonal to the development direction. Extraction of chromatograms from these angled tracks required the development of a new analytical approach that involved a commercial flatbed film scanner and custom numerical image analysis software. GLAD stationary phase performance was quantified using the Dimethyl Yellow dye separated from a lipophilic dye mixture over migration distances less than approximately 10 mm. The limits of detection were 10 +/- 4 ng for the vertical posts and 11 +/- 3 ng for the bladelike media. We obtained theoretical plate heights that varied with film microstructure between 12 and 28 mum. Unoptimized separation performance was comparable to that of other planar chromatography media. Macropore anisotropies engineered by GLAD may expand the capabilities of future UTLC stationary phases.

In-process quality control of wine by planar chromatography versus micro planar chromatography.

High-performance thin-layer chromatography (HPTLC) methods were developed to determine glycerol, gluconic acid, amino acids and sugars for in-process quality control of wine. Twenty wine samples (Pfalz region, Germany) were diluted with methanol and used for quantitative analysis without any further sample preparation. The developed amino acid method provided quantitative and characteristic fingerprints of wine varieties. The amino acid assignments were verified by HPTLC-MS. The developed gluconic acid method was primarily used to control the threshold, indicating a Botrytis cinerea infection of grapes. However, this method also enabled the detection of further organic acids like malic, tartaric and citric acids. A glycerol method was developed for control of the grape must fermentation (spontaneous/regular) and for fraud detection (glycerol adulteration). The HPTLC results of the sugar contents in the wine samples were similar to those of the well-known Luff-Schoorl method. The combined use of these developed HPTLC methods allowed the fermentation control (e.g., alcoholic and malolactic fermentation) and the monitoring of the grapes' overall health status. Without modification, the HPTLC methods for sugar and amino acid analysis could be transferred to circular micro planar chromatography (µ-PLC), showing its potential and benefits in terms of an inexpensive alternative for wineries and distributors.

Effect-directed classification of biological, biochemical and chemical profiles of 50 German beers.

Biological and biochemical fingerprints were investigated for the first time for the feasibility of effect-directed classification, and thus, to allow the choice of a distinct beer with regard to beneficial health effects. A high-performance thin-layer chromatography method was newly developed and combined with in situ effect-directed analysis for profiling 50 German beers for multipotent active compounds, and thus, their health-related potential. Discovered multipotent active zones were online eluted and characterized by high resolution mass spectrometry. For example, isoxanthohumol, iso-α-ad/n-humulone or its isomers, desdimethyl-octahydro-isocohumulone and ad/n-humulone were proven as antimicrobial compounds, isoxanthohumol as an acetylcholinesterase inhibitor, and isoxanthohumol and iso-α-ad/n-humulone or its isomers as radical scavengers. Investigating multivariate data analysis of effect-directed fingerprints for the first time, the pattern recognition and classification results showed the power of clustering non-alcoholic beers from other types of beer, or it showed the differentiation of dark and non-alcoholic beers.

Effect-directed analysis of ginger (Zingiber officinale) and its food products, and quantification of bioactive compounds via high-performance thin-layer chromatography and mass spectrometry.

Decision makers responsible for quality management along the food chain need to reflect on their analytical tools that should ensure quality of food and especially superfood. The "4ables" in target analysis (stable, extractable, separable, detectable) focusing on marker compounds do not cover all relevant information about the sample. On the example of ginger, a streamlined quantitative bioprofiling was developed for effect-directed analysis of 17 commercially available ginger and ginger-containing products via high-performance thin-layer chromatography (HPTLC-UV/Vis/FLD-bioassay). The samples were investigated concerning their active profile as radical scavengers, antimicrobials, estrogen-like activators and acetylcholinesterase/tyrosinase inhibitors. The [6]-gingerol and [6]-shogaol content of the different products ranged 0.2-7.4mg/g and 0.2-3.0mg/g, respectively. Further, multipotent compounds were discovered, characterized, and for example, assigned as [8]- and [10]-gingerol via HPTLC-ESI-HRMS. The developed bioprofiling is a step forward to new analytical methods needed to inform on the true product quality influenced by cultivation, processing, and storage.

Effect-directed fingerprints of 77 botanical extracts via a generic high-performance thin-layer chromatography method combined with assays and mass spectrometry.

Quantitative effect-directed profiles of 77 industrially and freshly extracted botanicals like herbs, spices, vegetables and fruits, widely used as food ingredients, dietary supplements or traditional medicine, gave relevant information on their quality. It allows the assessment of food, dietary supplements and phytomedicines with regard to potential health-promoting activities. In contrary to sum parameter assays and targeted analysis, chromatography combined with effect-directed analysis allows fast assignment of single active compounds and evaluation of their contribution to the overall activity, originating from a food or botanical sample. High-performance thin-layer chromatography was hyphenated with UV/Vis/FLD detection and effect-directed analysis, using the 2,2-diphenyl-1-picrylhydrazyl radical, Gram-negative Aliivibrio fischeri, Gram-positive Bacillus subtilis, acetylcholinesterase and tyrosinase assays. Bioactive compounds of interest were eluted using an elution head-based interface and further characterized by electrospray ionization (high-resolution) mass spectrometry. This highly streamlined workflow resulted in a hyphenated HPTLC-UV/Vis/FLD-EDA-ESI+/ESI--(HR)MS method. The excellent quantification power of the method was shown on three compounds. For rosmarinic acid, contents ranged from 4.5mg/g (rooibos) to 32.6mg/g (rosemary), for kaempferol-3-glucoside from 0.6mg/g (caraway) to 4.4mg/g (wine leaves), and for quercetin-3-glucoside from 1.1mg/g (hawthorn leaves) to 17.7mg/g (thyme). Three mean repeatabilities (%RSD) over 18 quantifications for the three compounds were ≤2.2% and the mean intermediate precision over three different days (%RSD, n=3) was 5.2%.

Effect-directed analysis of fresh and dried elderberry (Sambucus nigra L.) via hyphenated planar chromatography.

A healthy diet is an important factor in a healthy lifestyle that is becoming increasingly important in today's society. The fruits of European elder (Sambucus nigra L.) are a rich source of bioactive compounds like anthocyanins. In this study, dried and fresh fruits of four cultivated and six wild growing plants were investigated for their anthocyanin pattern and content as well as their bioactive compounds. After separation on HPTLC plates silica gel 60 F254 with a mixture of ethyl acetate, 2-butanone, formic acid and water, the plates were quantitatively evaluated by densitometry and also subjected to various (bio)assays to investigate the samples for compounds acting as radical-scavengers, antimicrobials, estrogens, and acetylcholinesterase or tyrosinase inhibitors. The mean contents for the two most abundant anthocyanins in European elderberries, confirmed by HPTLC-ESI-MS, ranged from 159 to 647mg/100g in fresh and from 166 to 2764mg/100g in dried fruits for cyanidin-3-sambubioside, and from 112 to 521mg/100g in fresh and 95 to 226mg/100g in dried fruits for cyanidin-3-glucoside. Additionally, the anthocyanin content was higher in berries of cultivars than of wild growing plants. The anthocyanins' radical scavenging activity and antimicrobial effect against Aliivibrio fischeri were confirmed. Further, a radical scavenging compound affecting A. fischeri and acting as acetylcholinesterase inhibitor was tentatively assigned by its protonated molecule at m/z 456 as either ursolic or oleanolic acid by HPTLC-ESI-MS. HPTLC hyphenated with bioassays and mass spectrometry was selected as method of choice for fingerprinting, pattern recognition, and bioprofiling of elderberry samples as well as quantitation and confirmation of bioactive compounds therein.

Analysis of pesticide residues in drinking water by planar chromatography.

Planar chromatography is described in the field of water analysis. The principle of automated multiple development (AMD) technique is mentioned, the strategy of the whole procedure which became a German standard is demonstrated and separations of pesticide mixtures, as well as water samples containing pesticides are presented. The suitability of this method was proved for 283 pesticides and the corresponding ISO Standard has been applied for.