A bioimaging system combining human cultured reporter cells and planar chromatography to identify novel bioactive molecules.

For the first time, a human cancer cell line was shown to grow and be functionally active on the particulate porous adsorbent surface of separated sample mixtures. This allowed the novel combination of chromatographic separations with human cells as biological detector. As exemplary screening for cancer treatment drugs, cytotoxic substances were directly discovered in Saussurea costus and ginseng samples using the Cytotox CALUX® osteosarcoma cells (with luciferase expressing reporter gene) as detector. In addition, rosiglitazone and pioglitazone were detected as luminescent zones upon binding to the PPARγ receptor expressed in the respective CALUX cell line that was grown on the surface of the adsorbent. This demonstrates the ability to address receptor-mediated signaling with this method, and opens the perspective to use our novel bioimaging method to identify bioactive molecules targeting a wide range of pathways with toxicological, pharmaceutical and nutraceutical relevance. The new bioimaging directly pointed to individual effective compounds in multi-component mixtures. Furthermore, discovered effective compounds were directly characterized by online elution to high-resolution mass spectrometry and fragmentation.

Non-targeted detection and differentiation of agonists versus antagonists, directly in bioprofiles of everyday products.

Xenoestrogens exert antiandrogenic effects on the human androgen receptor. In the analytical field, such antagonists block the detection of testosterone and falsify results obtained by sum parameter assays. Currently, such agonistic versus antagonistic effects are not differentiated in complex mixtures. Oppositely acting hormonal effects present in products of everyday use can only be differentiated after tedious fractionation and isolation of the individual compounds along with subjection of each fraction/compound to the status quo bioassay testing. However, such long-lasting procedures are not suited for routine. Hence, we developed a fast bioanalytical tool that figures out agonists versus antagonists directly in complex mixtures. Exemplarily, 8 cosmetics and 15 thermal papers were analyzed. The determined antagonistic potentials of active compounds found were comparable to the ones of known antagonists (in reference shown for bisphenol A, 4-n-nonylphenol and four parabens). Relevant biological/chromatographic parameters such as cell viability, culture conditions, dose response curves, limits of biological detection/quantification and working range (shown for testosterone, dihydrotestosterone, nandrolone and trenbolone) were investigated to obtain the best sensitivity of the biological detection. The developed and validated method was newly termed reversed phase high-performance thin-layer chromatography planar yeast ant-/agonistic androgen screen (RP-HPTLC-pYAAS bioassay). Results were also compared with the RP-HPTLC-Aliivibrio fischeri bioassay (applied on RP plates for the first time). As proof-of-concept, the transfer to another bioassay (RP-HPTLC-pYES) was successfully demonstrated, analogously termed RP-HPTLC-pYAES bioassay detecting anti-/estrogens (exemplarily shown for evaluation of 4 pharmaceuticals used in breast cancer treatment). The new imaging concept provides (1) detection and differentiation of individual agonistic versus antagonistic effects in the bioprofiles, (2) bioanalytical quantification of their activity potential by scanning densitometry and (3) characterization of unknown bioactive compound zones by hyphenation to high-resolution mass spectrometry. Depending on the hormonal bioassay, 15 samples were analyzed in parallel within 5 h or 6 h (calculated as 20 or 24 min per sample). For the first time, piezoelectric spraying of the yeast cells was successfully demonstrated for the planar yeast-based bioassays.

Lovastatin in lactone and hydroxy acid forms and citrinin in red yeast rice powders analyzed by HPTLC-UV/FLD.

For the analysis of pigment-rich red yeast rice products, a fast quantitative high-performance thin-layer chromatography (HPTLC) method was newly developed and validated. The active ingredient lovastatin, present in lactone (LL) and hydroxy acid forms (LH), as well as the mycotoxin citrinin were analyzed in 19 red yeast rice products, including powders, dietary supplements, and Chinese proprietary medicines (Xuezhikang and Zhibituo). The HPTLC method including sample preparation allows a high throughput of matrix-rich samples (10 min per analysis) and is highly cost-efficient (running costs of 0.5 Euro per analysis). For a fast protocol, application volumes up to 10 µL were selected although higher application volumes will lower still the LODs, which were 30 mg/kg for LL and LH as well as 4 mg/kg for citrinin. Thanks to the minimalistic sample preparation, the overall mean recovery rate was good (109.9% ± 5.9%; repeated measurements of the three analytes per fresh sample preparation at three spike levels). Repeated calibrations (five per analyte) in the red yeast rice matrix showed highly satisfying determination coefficients (≥ 0.9991; mean 0.9996). For three analytes at three concentration levels, the obtained mean intermediate precisions in red yeast rice matrix analyzed over the whole procedure including sample preparation were highly satisfying (≤ 2.6%). Citrinin was not detectable in the samples down to the given LOD of 4.0 mg/kg for the 10-µL sample volume applied. The mean content of lovastatin in 15 RYR powders was 8.7 g/kg, with a rang of 1.5-26.2 g/kg. The content of lovastatin in Zhibituo tablets and Xuezhikang capsules was determined to be 2.7 and 11.1 g/kg, respectively. The two commercially available RYR dietary supplement samples showed the highest lovastatin contents of 40.7 and 41.4 g/kg. By these figures of merit, the HPTLC method was proven to be suited for the control of such matrix-rich, fermented food. Graphical abstract.

Bioprofiling of Surface/Wastewater and Bioquantitation of Discovered Endocrine-Active Compounds by Streamlined Direct Bioautography.

A direct bioautography has been used for the simultaneous determination of four estrogens [estrone (E1), 17ß-estradiol (E2), estriol (E3), and 17α-ethinylestradiol (EE2)] and two xenoestrogens [bisphenol A (BPA) and 4-n-nonyl-phenol (NP)] in surface water and wastewater samples from a sewage treatment plant. After either direct application or a liquid-liquid extraction of the water samples, the qualitative and quantitative detection of estrogen-effective compounds was performed with a planar yeast estrogen screen. The limits of detection were different for each compound, due to the specific receptor binding of individual (xeno)estrogens (1 ng/L to 15 µg/L). The mean recovery rate for all six substances at this ultratrace level was 88% [mean percent relative standard deviation (%RSD) of 17%, n = 3]. Over the whole procedure, precisions of three estrogens discovered in a wastewater sample were below 17%, n = 3. The identification of the detected bioactive compounds was performed by high-performance thin-layer chromatography-electrospray ionization mass spectrometry (HPTLC-ESI-MS) via the elution-head-based TLC-MS Interface. Whereas the estrogens E1 and E2 could always be detected in the influent of the treatment plant, E3 was detected occasionally. The concentrations of E1 and E2 ranged from 3 to 50 ng/L, and for E3 from 98 to 210 ng/L. EE2, BPA, and NP could not be detected at the given LOD. In every second surface water sample, E1 and E2 were detected, but not E3, EE2, BPA, and NP.

Maturity-related changes in venom toxicity of the freshwater stingray Potamotrygon leopoldi.

Aquatic venomous animals such as stingrays represent a largely untapped source for venom-based drug development. However, the major challenge for a potential drug development pipeline is the high inter- and intraspecific variability in toxicity and venom composition. As of today, little is known about maturity-driven changes in these traits in stingrays. The present study investigates the differences in toxicity and venom composition in different maturity stages of the freshwater stingray Potamotrygon leopoldi. This species can be found in the Xingú River basin (Brazil), where it mainly feeds on invertebrates, while being predated by other stingrays or large catfishes. P. leopoldi, as commonly known for stingrays, can cause severe injuries with the venomous dentine spine located at its tails. The toxicity of tissue extracts of juvenile and mature specimens was recorded on a myoblast cell culture bioassay. Venom composition and bioactivity of compounds were analyzed with planar chromatography linked to an Aliivibrio fischeri bioassay. Results revealed a decrease in venom toxicity during maturation, but no changes in venom composition. These findings may indicate that toxicity in mature specimens becomes evolutionary less important, probably due to a decrease in predation pressure.

Sharp-bounded zones link to the effect in planar chromatography-bioassay-mass spectrometry.

The traditional direct bioautography workflow was substantially altered to yield narrow, sharp-bounded effective zones. For the first time, microorganisms quantitatively detected the single effective compounds in complex samples, separated in parallel on a planar chromatogram. This novel effect-directed workflow was demonstrated and optimized for the discovery of endocrine disrupting compounds (EDCs) reacting with the human estrogen receptor down to the femtogram-per-zone range, like 250fg/zone for 17ß-estradiol (E2). For application volumes of up to 0.5mL, estrogen-effective compounds could directly be detected in complex samples at the ultratrace level (ng/kg-range). Sharp-bounded, estrogen-effective zones discovered were further characterized by direct elution into the mass spectrometer. HPTLC-ESI-MS mass spectra of (xeno)estrogens were shown for the first time. Owed to the substantially improved zone resolution, compound assignment was reliable and a comparison of the receptor affinities was conducted for six (xeno)estrogens. Also, long-term cell cultivation of the genetically modified yeast was demonstrated on the HPTLC plate. The optimized HPTLC-pYES workflow was proven for real food samples, exemplarily shown for beer. The general applicability of generating sharp-bounded zones was successfully proven by transfer of the fundamentally improved workflow to the Bacillus subtilis bioassay used for discovery of antibiotics in plant extracts. This new era of quantitative direct bioautography in combination with mass spectrometry will accelerate the scientific understanding in a wide application field via the streamlined access to fast and reliable information on effective components in complex samples.

Liquid chromatography-bioassay-mass spectrometry for profiling of physiologically active food.

Complex samples like food contain thousands of single compounds. In the past, only known target compounds were looked for; however, most bioactive compounds in food are unknown. On the contrary, nontarget analyses face the challenge of determining the thousand peaks' identities, but it remains largely unclear which peaks are bioactive. Here, we show a novel effect-directed food profiling, as food and food supplements can be unknowingly physiologically active. By the combination of planar chromatography, using water-wettable reversed phase high-performance thin-layer chromatography (HPTLC RP18 W) plates, with detection by specific microorganisms, endocrine compounds in food were quantitatively detected as sharp-bounded zones and further characterized by mass spectrometry. This analytical workflow allowed frequent food intakes to be identified as risky with regard to estrogen-effective compounds, in discussion for their potential involvement in foodborne pathogenesis and for use in personalized health care. Using this accelerated workflow with its comprehensive detection potential, unknown endocrine compounds can be discovered. Exemplarily, the discovery of up to six endocrine disrupting compounds was shown in seven propolis samples and in four spices. For example, microorganisms quantitatively detected an estrogen-effective compound in the range of 0.07-0.24% in seven propolis samples, which was assigned to be caffeic acid phenethyl ester by mass spectrometry. This streamlined nontarget analysis detected modes of action, followed by targeted characterization of newly discovered effective compounds. Also, drug discovery or analysis of traditional medicines may profit from this effect-directed profiling of complex samples.

Comparison of an HPTLC method with the Reflectoquant assay for rapid determination of 5-hydroxymethylfurfural in honey.

5-Hydroxymethylfurfural (HMF) was analyzed in 17 botanical varieties of honey from 12 countries. A recently developed high-performance thin-layer chromatographic (HPTLC) method was limited because of increased matrix effects at higher honey sample loading. Therefore, the method was modified to achieve higher sensitivity and eliminate matrix interference by use of rectangular application combined with a focusing step. The HPTLC results were compared with results from the new spectrophotometric Reflectoquant hydroxymethylfurfural assay. Both methods had quantification limits of 4 mg kg(-1) and were suitable for rapid quantification of HMF in honey at the strictest regulated level of 15 mg kg(-1). Comparable results were obtained for the 17 honey samples, with a mean deviation of 2.9 mg kg(-1) (15%). The optimized HPTLC method was proved to be highly matrix-robust and was validated for the 17 different honey matrices (correlation coefficients ≥0.9994 (n = 6), mean intra-day precision 3.2% (n = 3 within a plate; n = 2 repeated within a day), mean inter-day precision 3.7% (n = 3), mean reproducibility over the whole procedure including sample preparation 4.1% (n = 2), and mean recovery 106.9% (n = 5 different concentrations; n = 4 different honey matrices). Recovery for a range of different application volumes, and thus for different honey matrix loading, differed by only ≤4.2%. HMF results when calculated by use of external calibration and by use of the standard addition method varied by 8.8%. Both revealed that any matrix effect was minor and that the original matrix interference problem was successfully solved.