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, 1608, 460415 (2019). Samples were acetonitrile extracts of Annona cherimola fruit peel, pulp and seeds (Annonaceae), as well as caffeic acid as standards. HPTLC on silica gel with chloroform – ethyl acetate – propanol 21:2:2 for peel extracts, with chloroform – methanol 9:1 for seed extracts. Derivatization by spraying Dragendorff’s reagent for alkaloids, secondary amines and non-nitrogenous oxygenated compounds.  Effect-directed assay was performed for inhibitors of α-glucosidase. Before sample application, plates were developed with enzyme substrate (2-naphthyl-α-D-glucopyranoside 0.1 % in methanol) and dried 20 min at 60 °C. Then, samples were applied and separated, and mobile phase was removed by heating 10 min at 60 °C. The chromatogram was sprayed with 4 mL enzyme solution (5 unit/mL in 100 mM phosphate buffer,  pH 7.4), liquid excess was removed under lukewarm air stream, the plate was incubated 10 min at 37 °C in a moisture box, followed by spraying chromogenic reagent Fast Blue salt B 0.1 % in water, giving after 2 min white inhibition bands visible on purple background under white light. Plate image was documented under illumination (reflectance mode) with white light. The bands of 3 inhibiting compounds were analyzed in a triple quadrupole mass spectrometer. 1) Full scan mass spectra (m/z 50−1000) in the positive ionization mode were recorded using electrospray ionization (ESI, spray voltage 3 kV, desolvation line temperature 250 °C, block temperature 400 °C) for compounds directly eluted with methanol – acetonitrile through the oval elution head of a TLC-MS interface pump. 2) Compounds were also isolated (either eluted directly from the plate into a vial through the same interface, or scraped from the plate and extracted with methanol – chloroform into a vial), dried, and submitted to HPLC-DAD-MS/MS; MS-MS spectra were recorded in the same conditions, using argon as collision gas and collision cell voltages from -20 and -40 V. Inhibitors were identified as phenolamides (phenylethyl cinnamides): moupinamide (hRF 66 in peels, 56 in seeds), N-trans-feruloyl phenethylamine (hRF 76 in peels), N-trans-p-coumaroyl tyramine (hRF 44 in seeds).

J Chromatogr A, 1611, 460602 (2020). Samples were methanolic root macerates of Euthamia graminifolia, Solidago canadensis, S. gigantea, S. rugosa and S. virgaurea (Asteraceae). HPTLC on silica gel with n-hexane – isopropyl acetate – acetone 16:3:1; or (for preparative TLC) on TLC silica gel with n-hexane – acetone 7:3, followed by scraping the layer and eluting with ethanol. When intended for MS experiments, layers were previously washed with methanol – water 4:1 and heated 20 min at 100 °C. Derivatization with vanillin – sulfuric acid reagent. Multivariate image analysis of the derivatized chromatograms allowed clear separation of samples according to species. Effect-directed analysis for: A) enzymatic inhibition by immersion into acetyl- and butyryl-cholinesterase, glucosidase and amylase solutions; B) activity against Gram-negative bacteria using Xanthomonas euvesicatoria chromogenic bioassay, and Aliivibrio fischeri and Pseudomonas syringae maculicola bioluminescence assays; C) activity against Gram-positive bacteria with Bacillus subtilis spizizenii bioassay. Two labdane diterpenes (solidagenone, hRF 47, and presolidagenone, hRF 55) in S. canadensis and two polyacetylenes (matricaria-esters = methyl-decadiene-diynoates, hRF 78 and 87 in HPTLC) in S. virgaurea were identified from multipotent zones by bioassay-guided purification through preparative TLC / HPLC, followed by HRMS and NMR, as well as by HPTLC hyphenated to quadrupole-orbitrap HRMS in 2 ways: A) by eluting with methanol the compounds from the plate through the oval elution head of a TLC-MS interface, with heated electro-spray ionization (HESI, 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); tandem mass spectra were acquired in parallel at fragmentation energy of 15-100 eV; 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, 1647, 462154 (2021). Hydromethanolic extracts of Cinnamomum verum and C. cassia (Lauraceae) were separated on MS-grade HPTLC silica gel (prewashed twice with methanol – water 4:1 and dried at 110 °C for 20 min) with toluene – ethyl acetate – methanol 6:3:1. Residual organic solvent was removed by drying under automated cold stream air for 20 min. Chromatograms were documented under white light, UV 254 nm and for fluorescence detection at 366 nm, and afterwards submitted to Aliivibrio fischeri bioassay: 2 mL of bacterial suspension were piezoelectrically sprayed on the plate and bioluminescence was measured every 3 min for 30 min (120 s exposure time). For the first time, analytes from a bioactive zone, isolated by the oval elution head of a TLC-MS interface pump, were trapped from the highly salted layer by a heart-cut elution (45 s, flow rate 0.1 mL/min) through a biocompatible in-line filter to different on-line desalting devices. Using a two-position switching valve, the desalted analytes were guided to a reverse-phase UPLC column and separated at 40 °C using a fast gradient (ca. 13 min, 0.6 mL/min) with methanol (from 2 - 90 %) and an ammonium acetate solution (2.5 mM, pH 4.5 adjusted with acetic acid). After HPLC separation, analytes were detected by photodiode array (PDA) and then ESI-MS in polarity switching mode (cone voltage of 10 V, ESI probe at 600 °C, ESI source at 120 °C). Identified active compounds were cinnamic acid, coumarin, as well as the in HPTLC coeluting cinnamaldehyde and 2-methoxycinnamaldehyde. Separately, proof-of-concept tests were also made for more polar phenolic acids (gallic, chlorogenic, caffeic, cinnamic, ferulic and coumaric acids) but without HPTLC separation.

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 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.

Talanta 219 (2020) 121306. Development of a workflow for compound characterization of coeluting compounds: employing an HPTLC-UV/Vis/FLD-EDA screening, followed by the characterization and identification of the most potent compounds by multi-imaging, heated electrospray ionization high-resolution mass spectrometry (HESI-HRMS) and hyphenated HPTLC-UV/Vis/FLD-HPLC-DAD-ESI-MS. HPTLC of methanolic Lemon balm leaf extract and standards oleanolic acid and ursolic acid on silica gel with n-hexane - ethyl acetate 7:3 up to 70 mm, drying for 2 min, evaluation in UV 254 nm (UV), UV 366 nm (FLD) and white light (Vis) after derivatization with a solution of vanillin (40 mg) and sulfuric acid (200 μL) in 10 mL ethanol and heating at 110°C for 5 min. Three additional chromatograms were prepared for the antibacterial assays (1) against B. subtilis, (2) A. fischeri and  (3) the α-glucosidase assay. (1) immersing in B. subtilis suspension, detectioon by immersion in aqueous 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide solution after incubation, followed by incubation until bright zones against a violet background appear, (2) immersing in Aliivibrio fischeri suspension and instantly monitoring in real-time for 30 min using 1 min exposure time in 5-min intervals and indicating the dark (or bright) active zones against the bioluminescent background, (3) immersing in α-glucosidase solution (10 units/mL) and 0.1 M sodium acetate buffer adjusted to pH 7.5 for incubation and immersing into substrate solution (1.2 mg/mL 2-naphthyl-α-D-glucopyranoside in ethanol) for further incubation at room temperature for 10 min and detection by immersion in aqueous Fast Blue Salt B solution (1 mg/mL) and drying, this revealed the enzyme inhibitors as bright zones against a violet background in white light. Online elution of zones of interest with methanol into the MS and full scan recording in the range of m/z 50–750 with a resolution of 280,000 in both negative and positive ionization modes. Expanding the HPLC-DAD-ESI-MS system by installing a TLC-MS interface enabling direct elution of HPTLC zones into the HPLC eluent.

Anal Chim Acta 1087 (2019) 131-139. Presentation of versatile devices for quantitative inkjet application on self-printed, binder-free HPTLC layers for submicromole-scaled analytical 1H NMR spectroscopy, providing the freedom to influence the composition of the slurry and directly access to control the printed quantity of HPTLC silica gel adsorbent to be constant, thus pledging the quality of the layer. To make spectroscopy reveal the cleanest proton spectra with the lowest background signals and most pronounced analyte signals, and to enable the identification of a compound from one 80-mm band on a single HPTLC layer by a 1H NMR spectrum in the full spectral range, the plate quality was improved by pre-developing silica gel plates twice with formic acid - methanol 1:10, then once with acetonitrile -  methanol 2:1. Silica gel particles for self-printed plates were puryfied under solvent pressure using a HPLC pump. For HPTLC,  the homogeneous slurry prepared by stirring 5 g cleaned-up/pre-eluted silica gel particles in 15 mL water - 2-propanol 2:1 was printed on 10 cm x 10 cm glass plates purified with 2-propanol and methanol, and the wet layers were heated to 80°C until dry. Inkjet printing of the solutions as 80-mm band and sample application using a 3-hydroxy-2-naphthoic acid solution (10 mg/mL water – methanol 1 : 9), spraying a methanolic 3-hydroxy-2-naphthoic acid solution (5 mg/mL) as 80-mm band (20mL/band) on the HPTLC plate. After plate drying and chamber pre-conditioning for 7 min with 2 N ammonia solution (10 mL), development with methanol - ethyl acetate - toluene 2:7:1 (5 mL). The deuterated methanol solution of the samples extracted from the analyte area scraped off the analytical plate was measured by NMR with the methanol signal as reference, documentation at UV 366 nm and data evaluation with an open-source video densitometry software (quan TLC). The results showed that HPTLC separated zones had better resolution and less matrix interference with the NMR analyte signal, and thus opened the avenue for submicromole-scaled analytical 1H NMR spectroscopy, which allows a faster structure elucidation of unknown compounds and easier signal interpretation.