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.

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.

Anal. Chim. Acta 1174 (2021) 3387022. Presentation of the next generation LabToGo system OCLab2 which was improved and made more robust e.g. by using print and media technologies in chromatography, so-called office chromatography (OC). The first LabToGo methods were developed in the field of food safety and quality for point-of-care or on-site usage. For application volume study,  HPTLC of different methanolic / propanolic Patent Blue V solutions on HPTLC silica gel with ethyl acetate - methanol - water - acetic acid 65:23:11:1 up to 50 mm. Evaluation in white light and  absorption measurement at 635 nm. For performance tests of plate imaging, HPTLC of benzoic acid and sorbic acid acid on HPTLC with ethyl acetate - toluene - acetic acid 25:25:1. For study of the illumination homogeneity, HPTLC of either sorbic acid, 7-hydroxy-4-methylcoumarin, or Patent Blue V on amino phase, evaluation at 254 nm (sorbic acid), 366 nm (7-hydroxy-4-methylcoumarin) and in white light (Patent Blue V), and in the OCLab2 at 278 nm, 365 nm, and white light, respectively, and scanning the plates at 254 nm 366 nm and 635 nm, respectively. For analysis of food dye mixture by OCLab2, HPTLC on silica gel with ethyl acetate - methanol - water - acetic acid 65:23:11:1 up to 5 cm, drying and capturing of plate images by the OCLab2 at 515-525 nm, 620-630 nm and 465-475 nm, video densitometry and evaluation using the open-source quanTLC software. For analysis of Stevia leave samples, the ethanolic extracts and four calibration levels of a steviol glycoside mixture were applied by OCLab2 for the hydrophilic interaction liquid chromatography (HILIC) separation on HPTLC silica gel with acetonitrile - water 5:1. Derivatization with 2-naphthol sulfuric acid reagent at evaluation in white light. For analysis of water and environmental samples, ink-printing the prepared samples on HPTLC silica gel MS-grade, focusing using acetone - water 4:1 up to 13 mm, drying,  developing with ethyl acetate - toluene 1:1 up to 70 mm, drying and capturing the plate images in white light, 254 nm and 366 nm. For biological detection, spraying the plate with A. fischeri bacteria suspension and capturing of images over 36 min with an exposure time of 60 s and a trigger interval of 3.0 min. 

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.