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, 1609, 460438 (2020). HPTLC of defatted hydro-methanolic extract of Anarrhinum pubescens (= A. duriminium) aerial parts (Plantaginaceae) on silica gel with chloroform – methanol 9:2. When intended for MS experiments, layers were previously washed twice with methanol – water 4:1 and heated 20 min at 110 °C. Derivatization by automatic piezoelectric spraying of anisaldehyde sulfuric acid reagent, followed by heating 4 min at 105 °C. Effect-directed analysis for acetyl-cholinesterase (AChE) inhibitors was performed by successive piezoelectric sprayings with TRIS buffer, with AChE solution and (after 30 min incubation at 37 °C) with naphthyl acetate and Fast Blue salt B solution. White inhibiting zones on purple background were documented under white light, and densitometry was measured by scanning in fluorescence mode at 500 nm. One of the active bands was eluted from untreated layer with methanol through the oval elution head of a TLC-MS interface pump, into a quadrupole-Orbitrap mass spectrometer using heated electrospray ionization (HESI); a full scan mass spectrum (m/z 50−750) in the positive ionization mode was recorded, as well as HRMS/MS data across a range of collision energies (10–50 V). The compound was identified as foliamenthoyl-cinnamoyl-antirrhinoside. It was applied with two other active antirrhinosides (iridoids), all isolated from the extract through column chromatography, on an HPTLC layer without migration and submitted to AChE assay; their activity was expressed as equivalency towards rivastigmine tartrate as positive control. 

J Chromatogr A, 1629, 461511 (2020). HPTLC of methanolic extracts of female inflorescences from ten hemp varieties (Cannabis sativa, Cannabaceae) on silica gel with toluene – ethyl acetate 1:1 or (for yeast assays) on RP-18W with toluene – ethyl acetate 7:3. When intended for MS experiments, layers were previously washed twice with methanol – formic acid 10:1, once with acetonitrile – methanol 2:1 and air-dried. Chromatograms were documented under white light, UV 254 nm and for fluorescence detection (FLD) at 366 nm. Afterwards, 6 derivatization assays were performed with the following reagents, either without heating: primuline; or requiring heating 5 min at 120 °C: p-aminobenzoic acid; anisaldehyde sulfuric acid; diphenylamine aniline phosphoric acid; ninhydrin; vanillin sulfuric acid. Besides, 8 effect-directed assays (EDA) were performed for free radical (DPPH•) scavengers, for antimicrobial compounds (Gram-positive Bacillus subtilis assay, Gram-negative Aliivibrio fischeri bioluminescence assay), for phytoestrogens (planar yeast estrogen assay), for inhibitors of the following enzymes: acetyl-cholinesterase (AChE), α- and β-glucosidase, tyrosinase. AChE assay was performed by immersion (speed 3.5 cm/s, time 5 s) into AChE solution (666 units in TRIS buffer 0.05 M, with bovine serum albumin 0.1 %, pH 7.8), incubation 25 min at 37 °C, spraying with substrate solution, and heating 2 min at 50 °C. Two AChE substrate solutions were used: A) α-naphthyl acetate 0.1 % and chromogenic reagent Fast Blue salt B 0.18 % in ethanol – water 1:2, giving white inhibition bands visible on purple background under white light; B) with 3-indoxyl-3-acetate, giving black inhibition bands on blue background under UV 254 nm, which was useful to prevent false negatives when Fast Blue Salt B formed colored bands with analytes. Two bands of multipotent compounds were eluted from normal-phase layer with methanol through the oval elution head of a TLC-MS interface pump, into a quadrupole-Orbitrap mass spectrometer. Full scan mass spectra (m/z 50−750) in the positive and negative ionization modes were recorded using heated electrospray ionization (HESI, spray voltage 3.5 kV, capillary temperature 270 °C). By comparison to literature and standards, they were identified as cannabidivarinic acid (hRF 55) and cannabidiolic acid (hRF 60-70).

J Chromatogr A, 1616, 461434 (2020). Samples were acetonic extracts of Malus domestica fruit peels (Rosaceae) and of Salvia officinalis, Thymus vulgaris and Origanum vulgare spice powders (Lamiaceae), as well as standards of maleic acid (dicarboxylic acid), carvacrol, thymol (phenolic monoterpenes), rosmanol (phenolic diterpene), betulinic acid, corosolic acid (CA), maslinic acid (MA), oleanolic acid (OA) and its isomer ursolic acid (UA) (triterpenes). HPTLC on silica gel, when intended for MS and NMR experiments, layers were prewashed twice with methanol – water 3:1, followed by 30 min drying at 120 °C. When intended for quantitative densitometry, start zones were submitted to prechromatographic derivatization with iodine solution (10 g/L in chloroform) allowed to migrate up to 12 mm, incubated 10 min at 27 °C and dried under cold air stream; this allowed separation of isomeric triterpenes. Separation with toluene – methanol – ethyl acetate 17:2:1 after 5 min chamber saturation at 50 % relative humidity. CA coeluted with MA, and OA with UA. Four hyphenations: A) Quantitative HPTLC densitometry for active analytes was performed by measuring absorption at 665 nm with a tungsten lamp after immersion of the chromatograms in anisaldehyde sulfuric acid reagent and heating 5 min at 110 °C. Linear range was obtained at 25 - 200 ng/band for OA and 100 - 400 ng/band for UA. B) Effect-directed analysis by immersing the chromatograms into Gram-positive Bacillus subtilis suspension for antibacterial activity and into acetyl-cholinesterase and tyrosinase solutions for enzymatic inhibition. C) Active bands were eluted with methanol through the oval elution head and in-line filter frit of a TLC-MS interface pump, into a quadrupole-Orbitrap mass spectrometer. Full scan mass spectra (m/z 100−1000) in the positive and negative ionization modes were recorded using heated electrospray ionization (HESI, spray voltage 3.5 kV, capillary temperature 270 °C, probe heater temperature 200 °C). D) With higher amounts applied, preparative HPTLC, by scraping the multipotent band corresponding to OA and UA, and dissolving these analytes in methanol, for NMR analyses (1H raw or deconvoluted, and 2D 1H–13C Heteronuclear Single Quantum Coherence). Both isomers were distinguished by their allylic H-18 protons and separately quantified by applying PULCON method (PUlse Length-based CONcentration). LOQ was 267 μM for OA and 173 μM for UA; optimal range was 300 – 4600 mM, corresponding to 126 - 2090 μg of triterpenes.

J Chromatogr A, 1616, 460774 (2020). Methanolic extracts of leaves of Musa acuminata, M. balbisiana and M. sapientum (Musaceae), either from fields or from in vitro cultures or from the plantlets derived from in vitro culture and acclimatized in isolated warm room, were separated on HPTLC silica gel layers with toluene – ethyl acetate – methanol 6:3:1 or ethyl acetate – toluene – formic acid – water 34:5:7:5. When intended for MS experiments, layers were previously washed twice with methanol – formic acid 10:1, once with acetonitrile – methanol 2:1 and air-dried. Evaluation under white light, UV 254 nm and 366 nm. Derivatization by immersion (2s, 2cm/s) into natural product reagent preceded by heating at 110 °C for 5 min, or into anisaldehyde sulfuric acid reagent, diphenylamine aniline reagent, ninhydrin reagent, followed by the same heating procedure. Besides, plates were neutralized by cold air stream followed with phosphate buffer (8 %, pH 7.5) piezoelectrically sprayed on the plates and automated plate drying. Thereafter, 9 effect-directed assays (EDA) were performed for free radical (DPPH•) scavengers, for enzymatic inhibitors (α-amylase, acetyl- and butyryl-cholinesterase, α- and β-glucosidase), for antimicrobial compounds (Gram-positive Bacillus subtilis assay, Gram-negative Aliivibrio fischeri bioluminescence assay), and for mutagenic compounds (SOS response – UMU-C test using Salmonella typhimurium suspension and 4-nitroquinoline 1-oxide as positive control). The bands of 4 active compounds were eluted with methanol through a TLC-MS interface pump into a quadrupole-Orbitrap mass spectrometer. Full scan mass spectra (m/z 50−800) in the positive and negative ionization modes were recorded using electrospray ionization (ESI, spray voltage 3.3kV, capillary temperature 320°C, collision energy 35 eV). By comparison to a standard, one band present in all samples was identified as linolenic acid. For the other bands, only present in in vitro grown accessions, only raw molecular formulas and phytochemical classes were assigned (a pyrrolidine alkaloid, an amino-acid, a phenolic derivative).

J Chromatogr A, 1637, 461836 (2021). Successive ultrasonic macerates of Ficus religiosa leaves (Moraceae) were separated with toluene – ethyl acetate – methanol 6:3:1 on HPTLC silica gel or (for yeast and genotoxicity assays) on RP18W phase. For MS experiments, layers were previously washed twice with methanol – formic acid 10:1, once with acetonitrile – methanol 2:1 and air-dried. Chromatograms were documented under white light, UV 254 nm and 366 nm. Afterwards, 11 derivatization assays were performed with the following reagents, either without heating: Dragendorff’s reagent; Fast Blue B salt; ferric chloride; natural product reagent - PEG 400; primuline; or requiring heating for 5 min at 120 °C: anisaldehyde sulfuric acid; diphenylamine aniline phosphoric acid; 2-naphthol sulfuric acid; ninhydrin; Tillmans' reagent; vanillin sulfuric acid. Besides, 12 effect-directed assays (EDA) were performed for free radical (DPPH• and ABTS•) scavengers, for enzyme inhibitors (α-amylase, acetyl- and butyryl-cholinesterase, α- and β-glucosidase, tyrosinase), for antimicrobial compounds (Gram-positive Bacillus subtilis assay, Gram-negative Aliivibrio fischeri bioluminescence assay), for phytoestrogens (planar yeast estrogen assay) and genotoxicity (SOS response – UMU-C test by successive immersions into citric buffer, into Salmonella typhimurium suspension and into methylumbelliferyl-galactopyranoside solution, followed by FLD at 366nm of mutagenic compounds as blue fluorescent zones, using 4-nitroquinoline 1-oxide as positive control). No activity was found for the last two assays. Ethyl acetate extracts of all samples were the most active. After EDA, most active bands were scanned for semi-quantitative equivalence densitometry at 546 nm using mercury lamp, compared to the following standards: acarbose, gallic acid, imidazole, kojic acid, physostigmine, tetracycline, depending on the assay. The bands of 3 multipotent compounds were eluted with methanol through the oval elution head and in-line filter frit of a TLC-MS interface pump, into a quadrupole-Orbitrap mass spectrometer. Full scan mass spectra (m/z 50−750) in the positive and negative ionization mode were recorded using heated electrospray ionization (HESI, spray voltage 3.5 kV, capillary temperature 270 °C, probe heater temperature 200 °C). MS-MS spectra were recorded in the negative mode using HCD-NCE (higher-energy collisional dissociation –normalized collision energy, with stepped negative collision energies from 10 to 40 eV). The three active zones were assigned to palmitic acid, to linolenic acid and to its di-oxygenated derivative.

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.