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Rapid analysis by coupling HPTLC with bioluminescence and mass spectrometry enables very fast response to bioactive substances in unknown samples. In this study marine sponges were screened for new bioactive compounds. After chromatographic separation of twelve methanolic marine sponge extracts the HPTLC plates were coated with bioluminescent bacteria ( Vibrio fischeri ) by a simple dipping procedure. If separated compounds were bioactive they inhibited or enhanced the bacterial luminescence and could be identified as dark zones on the luminescent background. This micro-biological detection revealed new compounds compared with physical (absorbance or fluorescence measurement) or chemical (microchemical derivatization) detection techniques. Effect-directed analysis turned out to be superior to target analysis in the search for natural products with a distinct effect. For identification of unknown bioactive zones the HPTLC system was coupled to a high-resolution mass spectrometer to obtain the exact masses of the unknowns. Thus, a Vibrio fischeri -bioactive zone was identified as avarone, a bioactive metabolite so far only known to be synthesized by the sponge Dysidea avara . This methodology proved very effective not only for detection but also for identification of unknown bioactive metabolites in sponges.

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We present a video-densitometric quantification method in combination with diode-array quantification for the methyl-, ethyl-, propyl-, and butylparaben in cosmetics. These parabens were separated on cyanopropyl bonded plates using water-acetonitrile-dioxane-ethanol-NH3 (25%) (8:2:1:1:0.05, v/v) as mobile phase. The quantification is based on UV-measurements at 255 nm and a bioeffectively-linked analysis using Vibrio fischeri bacteria. Within 5 min, a Tidas S 700 diode-array scanner (J&M, Aalen, Germany) scans 8 tracks and thus measures in total 5600 spectra in the wavelengths range from 190 to 1000 nm. The quantification range for all these parabens is from 20 to 400 ng per band, measured at 255 nm. In the V. fischeri assay a CCD-camera registers the white light of the light-emitting bacteria within 10 min. All parabens effectively suppress the bacterial light emission which can be used for quantifying within a linear range from 100 to 400 ng. Measurements were carried out using a 16-bit MicroChemi chemiluminescence system (biostep GmbH, Jahnsdorf, Germany), using a CCD camera with 4.19 megapixels. The range of linearity is achieved because the extended Kubelka-Munk expression was used for data transformation. The separation method is inexpensive, fast, and reliable.

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JPC - Journal of Planar Chromatography - Modern TLC
Authors:
Wolfgang Schulz
,
Wolfram Seitz
,
Stefan Weiss
,
Walter Weber
,
Martin Böhm
, and
Dirk Flottmann

Combination of HPTLC with the bioluminescence test using Vibrio fischeri enables effective screening for bioactive substances in unknown samples. This paper describes several applications in environmental testing using bioluminescence inhibition in HPTLC. The samples were extracted by solid-phase extraction or applied directly to the TLC plate. After chromatography of the samples the plates were immersed in a suspension of bioluminescence bacteria. The bioactive substances were identified as dark or light zones in comparison with the background of the plate. By the use of this method several types of sports field granule were compared for their toxicity toward Vibrio fischeri . It was also possible to compare the bioactivity of expressway waste water or landfill leachate by use of the method. To evaluate the digital images taken by a CCD camera, inhibition chromatograms over the developed sample tracks were created.

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Direct bioautography performed with luminescence gene-tagged bacteria enables almost real-time detection of antimicrobial compounds in plant extracts. This method for the detection of chamomile ( Matricaria recutita ) components with antibacterial effect against Bacillus subtilis soil bacteria was more sensitive than commonly used bioautographic visualization by staining with a tetrazolium salt. Some compounds had a strong inhibiting effect only via the bioluminescence measurement. Extraction of antibacterial components of chamomile flowers was most effective with 50% ethanol; slightly lower efficiency was achieved with acetone and methanol, and hexane was least effective. The results were confirmed by using luminescent Pseudomonas syringae pv. maculicola plant pathogen bacteria.

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713 721 G.K. Turner , in: K. Van Dyke (Ed.) Bioluminescence and Chemiluminescence: Instruments and Applications, CRC Press, Boca Raton, Florida, 43–78, 1985

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Sunscreen products are meant to protect people from damaging UVA and UVB radiation. However, in some formulations the UV filters they contain can react and form many photodegradation products. Their potential toxicity has not yet been investigated. In this study effect-specific analysis has been used to evaluate the bioactivity of photodegradation products in sunscreens. HPTLC-bioluminescence coupling with the luminescent bacterium Vibrio fischeri was used. Problems in method development were because of the sensitivity of the bacteria and the wettability of HPTLC plates. A separation system using HPTLC LiChrospher plates and automated multiple development (AMD) with tert -butyl methyl ether- n -hexane was chosen. Detection was by UV in addition to Vibrio fischeri . First, biodetection was performed on pure standard solutions of the UV filters. UV filters with molecular weight >400 had no bioactivity; these included all newer UV filters (not in use before 1998). Five commercially available sunscreens with different UV filter combinations were then analyzed. They were irradiated on microscope slides with artificial light and natural sunlight and on the skin with natural sunlight. For extraction, a mixture of ethanol and acetone was used. The bioactivity which can be indicative of (cyto)toxic effects of the photodegradation products was higher than that of the corresponding UV filter. In comparison of HPLC-DAD and LC-MS with detection with Vibrio fischeri , a high signal in chemical-physical detection did not always correspond to high bioactivity, and vice versa. It was shown that biodetection with Vibrio fischeri was a suitable method for examination of photodegradation products in sunscreens, making this bioassay a useful addition to conventional analytical methods.

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.) Bioluminescence and Chemiluminescence: Instruments and Applications, CRC Press, Boca Raton, Florida, 1985, pp. 43–78. Turner G.K. Bioluminescence and

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-exchange chromatography [ 13 , 14 ], thin-layer chromatography [ 15 , 16 ], different sensors [ 17 , 18 ], high-performance liquid chromatography [ 9 , 10 , 11 , 19 , 20 ], and bioluminescence methods [ 2 , 5 , 8 , 21 , 22 ]. The last two methods are the most

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