J Chromatogr A 1638, 461895 (2021). Samples were sphingolipid-rich fractions of unproteinated blood plasma from healthy humans or from Fabry’s disease patients, as well as standards of sphingomyelin (SM) and of globotriaosylceramides (Gb3 = ceramide trihexosides), and related compounds (lyso-ceramide trihexosides, lactosyl ceramide, glucosyl ceramide). HPTLC on silica gel (Lichrosphere with spherical particles) by automated multiple development with a 9-step gradient, starting with pure methanol and ending with dichloromethane – methanol 9:1. Visualization and densitometry under UV 190 nm. Derivatization for Gb3 and derivatives (but not for SM) by immersion into orcinol solution (0.2 %, with sulfuric acid 10 %), followed by 15 min heating at 100 °C and by densitometry under visible light 550 nm. Bands of interest were directly eluted with methanol from underivatized plates into an ion-trap MS, through the oval head of a TLC-MS interface (with stainless steel frit to remove silica gel particles). Two different ionization processes were used: (A) electrospray ionization (ESI, capillary voltage 4 kV, endplate offset voltage -0.5 kV, nebulizer pressure 40 psi, drying gas 9 mL/min at 350 °C); (B) atmospheric pressure chemical ionization (APCI, capillary voltage 2–3 kV, current intensity 4.5 µA, nebulizer pressure 45 psi, drying gas 5 mL/min at 350 °C; vaporization at 450 °C). Full MS spectra were recorded up to m/z 1500 in positive ion mode. The relative ion intensities were used to quantify the detected species. Previous to this study, the precision of the elution head positioning was tested on Gb3 standard zones, comparing 3 positions for analyte elution: from the centre and from each higher or lower side of the band. The same main m/z peaks were observed in the 3 positions, but in different proportions. This was explained by the presence of coeluting Gb3 subclasses (the ceramide moiety CM being either saturated, mono-unsaturated fatty acyl with a slightly higher migration distance, or polar hydroxyl fatty acyl with the opposite effect on migration) and of coeluting Gb3 isoforms (the hexoside moiety consisting of glucose and/or galactose units). This resulted in the broadening and partial splitting of the standard band. In the plasma samples, 19 molecular species of Gb3 were identified (depending on the CM, the sugar isoforms being undistinguishable by MS): 5 with a saturated CM, 7 with two additional double bonds on the CM, 7 with a methylated CM. In case of Fabry’s disease, most Gb3 species with saturated CM were highly increased, whereas other species were decreased.

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.

J. AOAC Int. 91, 1237-1243 (2008). HPTLC of zinc bis(O,O’-diisobutyl dithiophosphate), zinc bis(O,O’-didodecyl dithiophosphate), and Aglamol 99 on RP-2 by automated multiple development with methanol - water - acetic acid 6:3:2 for 25 mm, then acetonitrile - water 11:9 for 60 mm, and again acetonitrile - water for 80 mm, or on silica gel with a 14-step gradient based on toluene. For derivatization, the plate was dipped in a solution of 0.05 % primuline in acetone - water 4:1 for 1 s and immediately dried in warm air. Quantitative determination by fluorescence measurement at 366/>400 nm and by absorbance measurement at 220 nm. HPTLC-ATR-IR and HPTLC-FTIR, as well as HPTLC/DART-MS and HPTLC/ESI-MS were applied for identification.

J. Chromatogr. A 1462, 134-145 (2016). Development of simple method for the separation of different colored pigment formulations used in the printing materials on food packaging to control the quality and safety of the package. HPTLC on silica gel by automated multiple development with a 9-step gradient based on ethyl acetate, methanol and water, and ending with toluene. Good resolution of differently soluble constituents of the pigment formulation like additives and coating materials. The results obtained by multi-detection allowed a first assignment of the differently detectable bands to particular chemical substance classes, enabled the comparison of different commercially available pigment batches and revealed substantial variations in the composition of the batches. Characterization of single unknown pigment constituents by HPTLC-MS and HPTLC combined with ATR-FTIR. The new HPTLC method for routine quality control for incoming pigment batches and monitoring of internal pigment production processes secures a consistent pigment composition, resulting in consistent ink quality. Hyphenation of HPTLC with the Aliivibrio fischeri bioassay revealed information on the toxicological potential of different pigment compounds which helps guarantee consumer safety, especially in regard to readily permeable pigment components.

J. Planar Chromatogr. 4, 255-257 (1991). HPTLC of progesterone, testosterone, testosterone hydrogen sulfate sodium salt, 20ß-hydroxy-4-pregnene-3-one, 2-methoxy estrone, coprostane, hydrocortisone on silica with a gradient consisting of methanol – ethyl acetate – chloroform – methylene chloride (first inverse gradient program) and of methanol – chloroform (second inverse gradient program). Detection under UV 254 nm. Quantification by densitometry.