J Chromatogr A, 1624, 461239 (2020). Samples were chemical standards of acetylcholinesterase (AChE) inhibitors (azamethiphos, caffeine, donepezil, galanthamine, methiocarb-sulfoxide, paraoxon-ethyl) and of neurotoxic compounds, as well as drinking or contaminated water samples enriched through solid phase extraction. HPTLC on spherical silica gel (pre-washed twice by 20 min immersion in isopropanol, heated 20 min at 120 °C before and after pre-washing with acetonitrile). First separation (preparative TLC) with automated multiple development (16 steps). Effect-directed analysis for AChE inhibitors by immersion (speed 5 cm/s, time 1 s) into enzyme solution, incubation 5 min at 37 °C and immersion into substrate solution (indoxyl acetate 2 % in methanol); visualization under UV 366 nm. Active zones from untreated layers were eluted through the oval head of a TLC-MS interface to a second plate for a second separation with a panel of other mobile phases. Bands of interest were eluted from the second layer with water through the oval elution head of the TLC-MS interface pump, into a RP18 liquid chromatography guard column, followed by a quadrupole time-of-flight mass spectrometer. Full scan mass spectra (m/z 100–1200) were recorded in negative and positive modes using electrospray ionization (and collision-induced dissociation for MS2). Among the water contaminants, lumichrome (riboflavin photolysis product), paraxanthine and linear alkylbenzene sulfonates were identified as AChE inhibitors.

J Chromatogr A, 1684, 463582 (2022). Samples were concentrated filtrates of leachates of waste deposition sites, as well as testosterone, flutamide, bisphenol A (BPA) and nitroquinoline oxide (NQO) as standards. Automated Multiple Development on HPTLC silica gel (prewashed with methanol and dried 30 min at 110 °C) with 1) methanol up to 20 mm; 2A) chloroform – ethyl acetate –petroleum ether 11:4:5 or 2B) ethyl acetate – n-hexane 1:1 for flutamide and testosterone, up to 90 mm. Effect-directed analysis was performed by automated spraying 3 mL suspension of BJ1991 yeast (transfected Saccharomyces cerevisiae strain, pure for androgenic activity, with 50 ng/mL testosterone for anti-androgenic assay), followed by 20 h incubation at 30 °C in a closed chamber (90 % relative humidity), by 5 min drying under cold air stream, by spraying 2.5 mL MUG solution (4-methylumbelliferyl-galactopyranoside) and by 15 min incubation at 37 °C in an open chamber. Agonistic and antagonistic activities were detected qualitatively under UV 366 nm (light or dark blue bands, respectively, on blue background) and quantitatively documented using automated scanning at excitation wavelength 320 nm (deuterium lamp), with cut-off filter at 400 nm. Dose-response curves for model compounds were established by regression analysis. Anti-androgenic effective doses at 10 % were 28 ng/zone for flutamide and 20 ng/zone for BPA, without toxicity for the yeast. To exclude cytotoxicity where anti-androgenic activity was observed, the HPTLC layers (either without or after the spraying with MUG) were sprayed with 3 mL resazurin solution (0.01 % in water) and incubated 30 min at 30 °C and 90 % humidity. Cytotoxicity bands appeared as pink zones of resorufin on a colorless background (dihydroresorufin) under white light. Densitometric evaluation in absorption mode at 575 nm (under deuterium and halogen-tungsten lamps, no filter applied). NQO was cytotoxic at its lowest tested dose (1 ng/zone).

Frontiers in Marine Science 7, 266 (2020). Methanol extracts from marine sponge Haliclona cnidata (Chalinidae) submitted to different stresses (antibiotics and/or darkness) were separated on HPTLC silica gel with an automated 15-step gradient based on methanol, dichloromethane and n-hexane. Bioluminescence was recorded after immersing the HPTLC plates into Aliivibrio fischeri suspension. Antibacterial activity and quorum sensing enhancement were analysed on software, and Pearson’s similarity coefficient was applied to generate similarity matrices for cluster analysis (UPGMA, Unweighted Pair Group Method with Arithmetic Mean). Only slight differences were observed, especially in QS enhanced zones in stressed vs. control cultures.

Nature - Sci. Rep. 11, 62 (2021). Samples were isopropylacetate – methanol 3:2 fractions of (1) n-hexane extracts of larvae of Apis mellifica carnica (Apidae) from hives exposed to different concentrations of neonicotinoid pesticide clothianidin in their food, as well as (2) worker jelly adsorbed from brood combs of the same hives on adsorptive filter strips (unused filter strip parts were kept as background control). HPTLC on silica gel with chloroform – methanol – water – ammonia 30:17:2:1 for (1), and with an 8-step gradient based on methanol, chloroform, toluene, and n-hexane for (2, see CBS 105: Optimization of an AMD 2 method for determination of stratum corneum lipids). Visualization at UV 366 nm before and after derivatization by immersion into primuline reagent (primuline 0.5 g/L in acetone – water 4:1). Furthermore, antibacterial activity of (2) was assessed by recording the bioluminescence on the HPTLC plates, neutralized after elution and immersed into Aliivibrio fischeri suspension. Semi-quantitative comparison showed that a higher exposure to clothianidin was correlated with a decrease in lipid composition as well as in antibacterial activity.

CBS 100, 2-5 (2008). HPTLC of photodegraded UV filters and sunscreen on silica gel LiChrospher prewashed with methanol. AMD 2 development of UV filter standards photodegradation products with diisopropylether - n-hexane in 6 steps over 50 mm without preconditioning, and of sunscreen samples photodegradation products with t-butylmethylether - n-hexane in 7 steps over 50 mm with preconditioning, followed by drying at 120 °C for 30 min. Detection at 254 and 366 nm, followed by biodetection via dipping the plate in a Vibrio fischeri solution for 1 s and evaluation with the Bioluminizer (exposure time 55 s). Densitometric evaluation by multi-wavelength scan at 200-400 nm.

CBS 113, 13-15 (2014). HPTLC of glycosylceramide Glc-d18:2 h16:0 from wheat germ and standards squalene, cholesteryl oleate, glyceryl trioleate, linoleic acid, ß-sitosterol, and ß-sitosterol glucoside on silica gel in the AMD 2 with a 18-step gradient modified from Opitz et al. (Chromatographia 73 (2011) 559), methanol replaced ethanol, and the mobile phase composition was changed slightly (pre-conditioning with 4 M acetic acid before each step, drying time 1.5 min, development duration 3 h and solvent consumption 200 mL). Detection by dipping in copper sulfate phosphoric acid reagent for 20 s and heating at 130 °C for 15 min revealed grey-brown bands. Densitometry evaluation by absorbance measurement at 546 nm. For Glc-d18:2 h16:0, regression analysis showed a polynomial relationship with coefficients of determination (R2) from 0.995 to 0.999 (n=3, 50 - 1000 ng/band). LOD (S/N 3) and LOQ (S/N 10) of Glc-d18:2_x000D_ h16:0 were 10 ng/band and 50 ng/band, respectively (n = 6).

J. Planar Chromatogr. 4, 106-110 (1991). Presentation of 3 examples of optimization of AMD gradients: 1 + 2: Universal gradients optimized for the separation of phenolic compounds spanning a wide polarity range on diol layers, 3: Optimization for the separation of organochlorine pesticides on silica spanning a low polarity range. Preliminary isocratic developments of selected standards were performed with binary solvent mixtures in order to set up the gradients. The retention data (as plots of Rm values against solvent composition) can be used for the choice of the gradient components and gradient boundaries.

Chromatographia 37, 365-373 (1993). TLC of title compounds on silica by AMD using two different solvent gradients. Identification by in-situ measurement of the UV spectra. Determination by densitometry at 280 nm. Discussion of the advantages of this TLC method.