Coupling of In Vitro Bioassays with Planar Chromatography in Effect-Directed Analysis

Abstract

Modern analytical test methods increasingly detect anthropogenic organic substances and their transformation products in water samples and in the environment. The presence of these compounds might pose a risk to the aquatic environment. To determine a possible (eco)toxicological risk, aquatic samples are tested using various bioassays, including sub-organismic assays such as the luminescent bacteria inhibition test, the acetylcholinesterase inhibition test, and the umu-test. The effect-directed analysis (EDA) combines physicochemical separation methods with biological (in vitro) tests. High-performance thin-layer chromatography (HPTLC) has proved to be particularly well suited for the separation of organic compounds and the subsequent analysis of effects by the application of the biotests directly on the surface of the HPTLC plate. The advantage of using HPTLC in comparison to high-performance liquid chromatography (HPLC) for EDA is that the solvent which is used as a mobile phase during chromatography is completely evaporated after the separation and therefore can no longer influence the applied bioassays.A prioritization during the complex identification process can be achieved when observed effects are associated with the separated zones in HPTLC. This increases the probability of identifying the substance responsible for an adverse effect from the multitude of organic trace substances in environmental samples. Furthermore, by comparing the pattern of biological effects of a separated sample, it is possible to track and assess changes in biological activity over time, over space, or in the course of a process, even without identifying the substance. HPTLC has already been coupled with various bioassays.Because HPTLC is a very flexible system, various detection techniques can be used and combined. In addition to the UV/Vis absorption and fluorescence measurements, TLC can also be coupled with a mass spectrometer (MS) for compound identification. In addition, detection of functional groups by means of derivatization reagents can support this identification. It is also possible to combine derivatization and HPLC-MS.Two case studies are used to illustrate the significance of HPTLC-EDA in investigating water quality: Study on a wastewater treatment plant Possible influence of an artificial turf surface on ground water.

Planar chromatography and immunodetection of hydrocarbons on polyvinylidene difluoride membranes

“…Here, we showed that PVDF membranes can also be used as the stationary phase in planar chromatographic assays of lipids and hydrocarbons of diverse origin, an application that, to our knowledge, has not been yet reported. Consequently, similar TLC applications and advantages are shared by PMC, such as the possibility of studying all nonvolatile analytes in a sample, applying different detection techniques and effect‐directed analysis to the same chromatographic run, reduced cost, high sample throughput, and overall simplicity compared to column chromatographic methods [2,4]. We showed that is possible to separate hydrocarbons and lipids from complex samples by PMC.…”

State-of-art and some ideas for future progress of bioassays with plants

“…Some publications report about the general physical methods: amperometry, voltametry, conductometry, potentiometry, and colorimetry (Hassan et al, 2016), as well as the general chemical methods: high-performance thin-layer chromatography (Weiss et al, 2017), high-resolution gas chromatography/high-resolution mass spectrometry (Kojima et al, 2018). The other research deal with the general biological methods, particularly utilizing diverse bioobjects in vitro (Agarwal et al, 2014;Leusch et al, 2017;Prinsloo et al, 2017;Weiss et al, 2017).…”

“…Firstly, the procedures of bioassays widely employ 1) microbiologic techniques, e. g. microbial fuel cells (Weiss et al, 2017); 2) biophysical analyses, namely bioluminescent (Essimbekova et al, 2014), fluorescent, and micrometric ones (Nishi et al, 2015;Weiss et al, 2017); 3) biochemical analyses, particularly based on ability of the inhibition of luminescent enzymes and acetylcholinesterase (Weiss et al, 2017).…”

“…1.1) atmosphere air (Kojima et al, 2018); 1.2) hydrosphere (Bürkholder et al, 2001;Weiss et al, 2017;Ghribi et al, 2019), including potable water (Leusch et al, 2017), wastewaters (Jirova et al, 2016); 1.3) soils (Essimbekova et al, 2014) and bottom sediments (Bürkholder et al, 2001);…”

Value and limitation ofin vitrobioassays to support the application of the threshold of toxicological concern to prioritise unidentified chemicals in food contact materials

“…Recently, this technique has been adapted to detect endocrine active chemicals (Klingelhofer and Morlock 2015), showing promising, high potential to detect very low levels. Attempts to combine the Ames test and SOS-umu test with TLC have been undertaken (Egetenmeyer and Weiss 2017;Weiss et al 2017). Further work is necessary to assess the value of such an approach and to validate it against a number of potent and less potent direct DNA-reactive mutagens in complex mixtures such as FCM migrates/extracts.…”