Chemosphere. 261, 127706 (2020). HPTLC of water samples from four riverbank filtration sites in Germany on silica gel with a gradient development consisting of methanol - formic acid 2000:1 (v/v), dichloromethane and n-hexane in 16 steps. Effect-directed analysis using four bioassays. Baseline toxicity was detected via inhibition of natural bioluminescence of Aliivibrio fischeri (AF). In Bacillus subtilis (BS) assay, metabolic activity of BS cells was detected via the yellow substrate 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT). Neurotoxic effects was analyzed by AChE assay, spraying the substrate indoxyl-acetate onto enzyme-incubated HPTLC plates. In YES assay, estrogenic effects are detected via Saccharomyces cerevisiae BJ3505 strain. 

Front. Pharmacol. 12, 755941 (2021). High-throughput eight-dimensional (8D) hyphenation of normal-phase HPTLC with multi-imaging by ultraviolet, visible and fluorescence light detection as well as effect-directed assay and heart-cut of the bioactive zone to orthogonal reversed-phase high-performance liquid chromatography-photodiode array detection-heated electrospray ionization mass spectrometry. The method allowed the analysis of 68 powdered plant extracts (botanicals) which are added to food products in food industry and the study of antibacterials, estrogens, antiestrogens, androgens, and antiandrogens, as well as acetylcholinesterase, butyrylcholinesterase, α-amylase, α-glucosidase, β-glucosidase, β-glucuronidase, and tyrosinase inhibitors in an array of 1,292 profiles.

J. Agric. Food Chem. 69, 12686-12694 (2021). HPTLC of 2Z,8Z- and 2E,8Z-matricaria esters in European goldenrod (Solidago virgaurea) and E- and Z-dehydromatricaria esters in grass-leaved goldenrod (Solidago graminifolia) and showy goldenrod (Solidago speciosa) on silica gel with n-hexane − acetone 17:3. Detection by dipping into mycelium suspension of F. avenaceum and B. sorokiniana, followed by incubation in a vapor chamber at 21 °C for 48−72 h. The lack of visible white (F. avenaceum) or dark gray (B. sorokiniana) fungal mycelia indicated the inhibition zones on the bioautograms. Compounds were further analyzed by high-resolution mass spectrometry and nuclear magnetic resonance spectroscopy.

Journal Chromatogr A, 1641, 461727 (2021). HPTLc of an ethanolic maceration of Solidago gigantea roots (Asteraceae) on silica gel with n-hexane – isopropyl acetate – acetone 16:3:1, or n-hexane – isopropyl acetate – acetic acid 40:9:1. With the second mobile phase, acid residues had to be eliminated by 20 min automated drying or by 2 h incubation with potassium hydroxide in the opposite twin trough (followed by 15 min cold air streaming); this latter mobile phase allowed to obtain higher hRF values, but some butyrylcholinesterase (BChE) inhibiting activities were lost. The chromatograms were documented at UV 254 nm and 365 nm and white light before and after A) derivatization with vanillin – sulfuric acid reagent; B) enzymatic reaction by immersion into acetylcholinesterase, BChE, glucosidase and amylase solutions; C) Aliivibrio fischeri and Xanthomonas euvesicatoria bioassays, to detect activity against Gram-negative bacteria; D) Bacillus subtilis bioassay to detect activity against Gram-positive bacteria; E) a new antifungal assay with Fusarium avenaceum. For this assay, the chromatograms were immersed 6 s into the isolated mycelium suspension (diluted to OD600 0.4-0.8) and incubated in a vapor chamber at 21 °C for 48-72 h. Inhibition zones were indicated by the lack of visible white fungal hyphae. An aqueous solution of iodonitrotetrazolium (INT, 1 mg/ml) was sprayed on the plate to enhance the contrast (bright zones on a purple background). Benomyl (a benzimidazole fungicide) was used as positive control. Eight clerodane diterpenes (including kingidiol, hautriwaic lactone, and solidagoic acids A and B) were identified from six multipotent zones by bioassay-guided purification through preparative flash chromatography and HPLC, followed by HRMS and NMR, as well as by HPTLC hyphenated to quadrupole-orbitrap HRMS: A) by eluting with methanol (flow 100 µL/min) the compounds from the plate through the oval elution head of an interface of heated electro-spray ionization (spray voltage 3.5 kV, capillary temperature 270 °C, nitrogen as sheath and auxiliary gas, full scan in negative and positive ionization modes in m/z range 50-750); B) without eluent with a DART interface (Direct Analysis in Real-Time, needle voltage 4 kV, grid voltage 50 V, helium as gas, temperature 500 °C, full scan in positive ionization mode in m/z range 100-750).

J Chromatogr A, 1641, 462334 (2021). Validation of a newly built (using 3D-printing) and newly configurated on-surface multi-purpose autosampler, called “autoTLC−LC−MS system”, developed for orthogonal hyphenation of normal phase HPTLC with reversed phase HPLC and high-resolution MS. Details and protocols are given for the construction, installation and numerical control software programming of this autosampler. HPTLC of antibiotics cefoperazone (third-generation cephalosporin), clindamycin (lincosamide), erythromycin A (macrolide), ipronidazole (nitro-imidazole), nafcillin (penam), sulfaquinoxaline (sulphonamide), tiamulin (pleuromutilin), and trimethoprim (DHFR inhibitor) on silica gel without development. Bioassay with Bacillus subtilis: bacterial suspension was sprayed onto the plate, which was  horizontally incubated for 2 h at 37°C in a humid box; afterwards, the plate was sprayed with 0.2% MTT solution, incubated again for 30 min at 37°C, dried for 10 min at 50°C, and documented under white light. The image was uploaded as a template in the updated TLC–MS managing software, so that by clicking on the zones of the image showing antibacterial activity, the corresponding zones of the untreated plate, placed on a newly designed plate holder, were sequentially eluted by the round elution head of the automatic sampler into the RP-18 endcapped HPLC monolithic column connected to a Quadrupole-Orbitrap mass spectrometer. For the elution from the plate and HPLC separation, a gradient was used (flow rate 0.2 - 0.5 mL/min, depending on the step), with different proportions of two mobile phases: A) 0.1 % formic acid and 4 mM ammonium formate in water; B) 0.1 % formic acid and 4 mM ammonium formate in methanol. After separation in the column, antibiotics directly underwent electrospray ionization in positive mode (voltage 3.5 kV, capillary temperature 320 °C, probe heater temperature 350 °C) and were detected by HRMS. For validation, the achieved ranges were 2.1–14.1 % for intra-day and 2.5–16.1 % for inter-day precisions.

Anal Chim Acta 1182 (2021) 338950. On-surface synthesis and effect-directed analysis for quantitative nanomole-scaled high-throughput chemistry and biological response on a single HPTLC plate for up to 68 on-surface synthesis reactions. For generic on-surface synthesis, 1 mL or 15 nmol each of malononitrile and benzaldehyde candidates were automatically sprayed as 3-mm bands  on an HPTLC plate, dried, and oversprayed with  diketoester on the same zones, followed by drying. Development in anti-parallel with chloroform - tetrahydrofuran 6:1 up to 5 cm and detection in UV 254 nm, 366 nm and  white light. For yield calculation, four-point calibration with the preparatively synthesized standard ethyl 6-amino-5-cyano-2-methyl-4-(4-nitrophenyl)-4H-pyran-3-carboxylate, derivatization with ninhydrin collidine reagent, heating at 110°C, detection in UV 254 nm, 366 nm and white light and densitometric absorbance measurement at 430 nm. Online elution of the synthesis product zones with methanol to high-resolution mass spectroscopy and analysis by electrospray ionization MS. For the HPTLC-Bacillus subtilis bioassay 3 mm bands of ciprofloxacin solutions were developed in ethanol - water - hydrochloric acid 9:10:1, sprayed with the bacterial suspension, incubated at 37°C for 2 h, sprayed with phosphate buffered saline (PBS)-buffered thiazolyl blue tetrazolium bromide (MTT) solution, incubated at 37°C for 30 min, followed by drying. Detection of colorless (bright) inhibition zones in white light and by absorbance measurement of the blue-violet formazan background of the biodensitogram at 546 nm.

Talanta 223 (2021) 121701. Development of workflow for a bioanalytical multi-imaging screening based on HPTLC-UV/Vis/FLD-EDA-HESI-HRMS and application to 54 bark, leaf and seed extracts of Sri Lankan Abelmoschus moschatus (abelmosk) to find out the most bioactive individual compounds. HPTLC on silica gel with toluene - ethyl acetate - methanol 6:5:2 or toluene - ethyl acetate 7:3 up to 65 mm, evaluation in (A) UV 254 nm, (B) white light, (C) fluorescence 366 nm, and after derivatization with (D) primuline reagent at UV 366 nm, (E) p-anisaldehyde sulfuric acid reagent, (F) vanillin sulfuric acid reagent, (G) p-aminobenzoic acid reagent, (H) the latter in fluorescence evaluation at 366 nm, (I) diphenylamine aniline orthophosphoric acid reagent, (J) ninhydrin reagent, (K) Fast Blue B salt reagent. For (E-K) detection in white light after heating at 140°C for 5 min, and (L) natural product reagent in fluorescence 366 nm after air-drying. For effect-directed profiling, HPTLC on plates prewashed with methanol - water 3:1, drying for 15 min and immersing in the respective assay solution/suspension or spraying with it, then incubating, drying and evaluation in white light (A) for Gram-negative Aliivibrio fischeri bioassay, spraying the bacterial culture onto the chromatogram and recording the instant bioluminescence over a 30 min period with the positive controls of caffeine; (B) for Gram-positive Bacillus subtilis bioassay, dipping the chromatogram in the bacterial suspension for incubation at 37°C for 2 h, then dipping into a 0.2 % PBS-buffered MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-trazolium bromide) solution with positive control tetracycline; (C) for α-glucosidase inhibition assay, spraying with substrate solution, drying, pre-wetting and incubation by spraying with Fast Blue B salt solution with positive control acarbose, absorbance measurement at 546 nm and quantification via peak area; (D) for β-glucosidase inhibition assay, analog to (C), but using β-glucosidase and 2-naphthyl-β-D-glucopyranoside and longer incubation time, with positive control imidazole; (E) for tyrosinase inhibition assay, spraying with substrate solution, after drying with tyrosinase solution, incubated and dried with positive control kojic acid, absorbance measurement at 579 nm; (F) for radical-scavenging assay, immersing the chromatogram into 0.02 % methanolic 2,2-diphenyl-1-picrylhydrazyl solution, air-dried with the positive control ascorbic acid. The workflow provided comprehensive information about multi-potent compounds and sample diversity, which is elementary for product quality control in the field of botanicals, foods and medicinal plants.

Anal Chim Acta 1129 (2020) 76-84. Demonstration of a new hyphenated HPTLC-S9-AChE assay for detection of neurotoxic chemicals, including metabolic activation, at levels consistent with the threshold of toxicological concern (TTC) for organophosphates (OPs). The high sensitivity allows for the direct application of packaging migrates or extracts on the HPTLC plate without additional requirement steps. HPTLC of the ethanolic standards chlorpyrifos (CP), quinalphos (QP), eserine (ER), parathion (PT), nonylphenol (NP) and tris(nonylphenyl) phosphite (TNPP) at five different levels in the range of 0.5-10 mL/band (overall range for all six chemicals 0.1-1000 ng/band) on silica gel. After application, drying and pre-wetting the application bands by immersion up to 10 mm in water, then spraying the S9 mixture (7 mL each, 0.1 mg/mL) immediately on top of the start zones, followed by incubation at room temperature for 30 min. After drying plates for 20 min and 5 min chamber saturation, development with ethyl acetate - methanol - water 5:5:2 for ER; n-hexane - ethyl acetate - ethanol 16:3:1 for QP and PT; n-hexane - toluene - ethyl acetate 5:4:1 for CP, NP and TNPP. Evaluation in 254 nm, 366 nm and white light. Detection by immersion in AChE solution (6.6 U/mL plus 1 mg/mL bovine serum albumin in Tris-HCl buffer, 0.05 M, pH7.8) for 2 s, and incubating at 37 °C for 25 min, then immersing into the substrate-chromogenic solution (ethanolic solution of 1-naphthyl acetate  - aqueous solution of Fast Blue B salt 1:2 , 3 mg/mL each) for 1 s, drying for 10 min. Evaluation in white light, absorbance measurement at 546 nm (mercury lamp) using an inverse scan. The advantages of this straightforward workflow were demonstrated by comparison with the status quo microtiter plate assay. The method is a pragmatic new tool in risk assessment in general and can be transferred to further toxicities of interest and any other category of complex sample mixtures.