ALTEX - Alternatives to animal experimentation, 38(3), 387-397 (2021). Samples were standards of food contact contaminants with genotoxicity (4-nitroquinoline-1-oxide (NQO), aflatoxin B1, hexachloroethane, nitroso-ethylurea, phenformin, PhIP) or negative controls (alosetron, mannitol), and extracts of coated tin cans (extracted with n-hexane – acetone at 25°C for 16 h or by heating at 60 °C with ethanol 95 % for 240 h). HPTLC on RP18W layer, pretreated to harden the binder by heating 1 h at 120 °C, prewashed with methanol and with ethyl acetate and dried 4 min in cold air stream after each development. Application areas were focused to their upper edges by a two-fold elution with ethyl acetate, followed by 1 min drying in cold air stream. Development with toluene – ethyl acetate 8:5, followed by 5 min drying, neutralization with citrate buffer (pH 12) and 4 min drying. Effect-directed analysis for genotoxicity (SOS response – UMU-C test, using NQO as positive control) by immersion (speed 3.5 cm/s, time 3 s) into Salmonella typhimurium suspension and, after 3 h incubation at 37 °C and 4 min drying in cold air stream, into one of two fluorogenic substrate solutions (methylumbelliferyl- vs. resorufin-galactopyranoside). After 1 h incubation at 37 °C, visualization of mutagenic compounds as (blue vs. red) fluorescent zones at FLD 366 nm, and densitometry performed with mercury lamp for fluorescence (at  366 / >400nm vs. 550 / >580 nm, respectively). Further validation experiments, including spiking extracts with NQO, were performed showing good mean reproducibility, no quenching or other matrix effects. Lowest effective concentration of NQO was 0.53 nM (20 pg/band), 176 times lower than in the corresponding microtiter plate assays.

Antioxidants, 10(1), 117 (2019). Samples were methanolic extracts of Camellia sinensis leaves or commercial black, white or green tea powdered extracts (Theaceae), as well as standards of caffeine (methylxanthine alkaloid), of flavonols (quercetin, rutin) and of flavanols (catechin, catechin-gallate, epicatechin, epicatechin-gallat, epigallocatechin, epigallocatechin-gallate, gallocatechin, and the thearubigin theaflavin). HPTLC on RP18-W phase (with classical irregular particles (SP1) vs. LiChrospher phase with spherical particles (SP2)), prewashed with methanol – water 4:1 and dried 20 min at 110 °C, developed with citric acid 0,295 % in acetonitrile – water 3:10 for SP1, with citric acid 0,17 % in acetonitrile – water 1:2 for SP2. Visualization under white light, UV 254 nm and 366 nm. Absorbance densitometry was performed at UV 275 nm (deuterium lamp). Derivatization with A) Fast Blue B salt reagent followed by 3 min heating at 100 °C, and by absorbance densitometry at 546 nm for flavanols (mercury lamp); B) natural product reagent (on the same plate), followed by fluorescence densitometry of flavonols at FLD 366/>400 nm (mercury lamp); C) anisaldehyde sulfuric acid reagent, followed by 2 min heating at 110 °C, to detect all flavonoids. Effect-directed analysis was performed using piezoelectric spraying: A) for free radical (DPPH•) scavengers (vs. gallic acid as positive control); B) for activity against Gram-negative Aliivibrio fischeri (bioluminescence assay, vs. caffeine) or Gram-positive Bacillus subtilis (vs. tetracycline); C) for enzymatic inhibition of acetyl-cholinesterase, α- and β-glucosidase, β-glucuronidase, tyrosinase (vs. rivastigmine, acarbose, imidazole, D–saccharolactone and kojic acid, respectively). When SP2 was used, previous neutralization was required through spraying of sodium bicarbonate buffer (2.5 %, pH 8). AChE inhibition assay was performed with indoxyl acetate (0.1 % in ethanol) as substrate, sprayed before the enzyme. After incubation (30min at 37°C), inhibition bands appeared indigo or blue under white light, but the substrate coloured theaflavin in yellow.

J Chromatogr A, 1605, 460371 (2019). HPTLC of toluene – ethyl acetate extracts of Primula boveana leaves and of P. veris (Primulaceae) on silica gel with n-hexane – ethyl acetate 7:3. Visualization under white light, UV 254 nm and 366 nm. Derivatization by spraying with anisaldehyde sulfuric acid reagent, followed by heating for 4 min at 105 °C. Effect-directed analysis: A) for activity against Gram-negative (Aliivibrio fischeri bioluminescence assay) or Gram-positive bacteria (Bacillus subtilis bioassay) using automated immersion; B) for enzymatic inhibition (acetyl- and butyryl-cholinesterase) using piezoelectric spraying, with rivastigmine as standard, and absorbance spectra (500 nm) for P. boveana active bands measured by inverse scanning. Active bands were eluted from the untreated layer with methanol through the oval elution head of a TLC-MS interface pump, into a quadrupole-Orbitrap mass spectrometer to record full scan mass spectra (m/z 100−1000) using electrospray ionization (ESI voltage 3.5kV for P. boveana, -3kV for P. veris, source temperature 250°C). With the further help of preparative HPLC – NMR, they were identified as linoleic and linolenic acids in P. veris, and as flavone and its derivatives: hydroxyflavone, methoxyflavone and zapotin, in P. boveana.

J Chromatogr A, 1603, 355–360 (2019). Samples were ethyl acetate root macerates of fully flowered Tanacetum vulgare (Asteraceae). HPTLC on silica gel (classical irregular particles vs. Lichrosphere with spherical particles) previously washed with methanol, dried for 5 min at room temperature, perimeter-sealed with a polymer coat, and heated for 30 min at 100 °C. Separation with toluene or with toluene – n-hexane 7:3, in classical capillary flow or in OPLC (overpressured layer chromatography). For OPLC, off-line infusion was used (closed mobile phase (MP) outlet, automatically stopping development); external pressure 50 bar, rapid MP flush 175 and 350 µL, MP flow rate 250 and 500 µL/min, 1830 and 3475 µL MP, development time 446 s and 424 s. Derivatization by immersion into vanillin – sulfuric acid reagent, followed by 5 min heating at 110 °C; or into PABA reagent (500 mg p-aminobenzoic acid, 18 mL glacial acetic acid diluted, 20 mL water, 1 mL o-phosphoric acid, 60 mL acetone), followed by 5 min heating at 140 °C. Effect-directed analysis using automated immersion: A) for free radical (DPPH•) scavengers; B) for activity against Gram-negative bacteria using Aliivibrio fischeri bioluminescence assay; C) for activity against Gram-positive bacteria with Bacillus subtilis bioassay. Four active polyynes were identified as hexadiynylidene-epoxy-dioxaspiro-decane (1), pontica epoxyde (nonene-triynyl-vinyl-oxirane) (2), tetradeca-triine-en-one (3) and trans-(hexadiynylidene)-dioxaspiro-decene (4), by hyphenating OPLC to quadrupole-orbitrap HRMS without eluent, using a DART interface (Direct Analysis in Real-Time, needle voltage 4kV, grid voltage 50 V, helium as gas, temperature 500 °C, full scan in positive ionization mode in m/z range 100-750). Polyynes (3) and (4) were coeluting in HPTLC but not in OPLC, demonstrating that (4) is not produced by oxidation during the DART-MS procedure. Separation with OPLC compared to HPTLC was performed in a shorter time and with better resolution at the same time. Layers with spherical particles gave higher resolution; zone distortions occurring in OPLC due to dissolved air in MP were prevented by previous MP sonication.

J Chromatogr A, 1605, 460366 (2019). Samples were standards and complex mixtures of non-ribosomally synthesized cyclic lipopeptides (CLPs) from Bacillus strains (Bacillaceae) found in soil or in manure: B. amyloliquefaciens (SS-12.6, SS-13.1, SS-27.2, SS-38.4) and B. pumilus (SS-10.7). Two extraction methods were compared: ethyl acetate extraction (Ex1), and the acidic precipitation followed by methanol extraction (Ex2). HPTLC on silica gel with chloroform – methanol – water 65:25:4. Detection under white light, UV 254 nm and 366 nm. Absorption densitometry measured at 190 nm. Derivatization for peptides, amino acids and amino derivatives, by immersion into ninhydrin – collidine reagent (ninhydrin 0.3 %, collidine 5 %, acetic acid 5 %, in ethanol), followed by heating 5 min at 110 °C. Effect-directed analysis using automated immersion: A) for free radical (DPPH•) scavengers; B) for enzymatic inhibition (acetyl-cholinesterase, α-glucosidase); C) for activity against Gram-negative (Aliivibrio fischeri bioluminescence assay) or Gram-positive bacteria (Bacillus subtilis bioassay). Active bands were eluted 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 200−2000) in positive and in negative ionization modes were recorded using heated electrospray ionization (HESI, spray voltage 3.5 kV, capillary temperature 270 °C). Active zones were assigned to be CLPs: iturin A, surfactin dimethyl-ester, and surfactin, fengycin and kurstakin homologues. Ex1 provided richer extracts compared to Ex2. Standards were seen to contain a free radical scavenging impurity.

J Chromatogr A, 1602, 458–466 (2019). HPTLC of caffeine, physostigmine (alkaloids) and hydroethanolic extract of Peganum harmala seeds (Nitrariaceae, Zygophyllaceae) on silica gel prewashed twice with methanol – water 3:1, followed by 1 h drying at 120 °C. Separation, after 5 min chamber saturation, with ethyl acetate – methanol – ammonia (25%) 85:11:4 (basic mobile phase) or ethyl acetate – toluene – formic acid – water 16:4:3:2 (acidic mobile phase, requiring neutralization with phosphate-citrate buffer). Derivatization with Dragendorff’s reagent and with anisaldehyde sulfuric acid. Effect-directed analysis by spraying A) with Gram-negative bioluminescent Aliivibrio fischeri suspension for antibacterial activity (caffeine was used as standard); B) with acetyl- and butyryl-cholinesterase (AChE / BChE) solutions for enzymatic inhibition. For AChE and BChE asssays, classical immersion into the enzyme solutions was also used for comparison, and inhibition densitometry for active analytes was performed by inverse scan measurement (fluorescence without optical filter) at 546 nm using a mercury lamp; activity was expressed as physostigmine equivalents. Active bands were eluted (only after basic MP) 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 positive ionization mode were recorded using heated electrospray ionization (HESI, spray voltage 3.5kV, capillary temperature 270°C). By comparison to literature, AChE inhibitors (also active against A. fischeri) were assigned to be harmine, harmaline and ruine (β-carboline alkaloids), and BChE inhibitors were harmol (same class) and vasicine and deoxyvasicine (quinazoline alkaloids, also called peganine and deoxypeganine). Piezoelectric spraying had the following advantages over automated immersion: (1) it covered the whole plate surface; (2) required much lower volumes of solutions; (3) applied always fresh enzyme or reagent solutions, thus avoiding gradual inactivation; (4) avoided zone distortions, shifts or tailings occurring during immersion or withdrawal of the plate, or due to the hydrophilicity of compounds.

Molecules, 26 (5), 1468 (2021). Summary: Samples were fortified extracts produced with iPowder technology (involving spray-drying of a rich first extract on a new batch of the same plant) from following plants: Camellia sinensis final bud and two leaves (Theaceae), Cynara scolumus leaves and Echinacea purpurea roots (Asteraceae), Eleutherococcus senticosus roots (Araliaceae), Equisetum arvense aerial part (Equisetaceae), Eschscholzia californica aerial parts (Papaveraceae), Humulus lupulus cones (Cannabaceae), Ilex paraguariensis leaves (Aquifoliaceae), Melissa officinalis aerial parts and Rosmarinus officinalis leaves (Lamiaceae), Passiflora incarnata aerial part (Passifloraceae), Raphanus sativus var. niger roots (Brassicaceae), Ribes nigrum leaves (Grossulariaceae), Spiraea ulmaria floral tops (Rosaceae), Valeriana officinalis roots (Caprifoliaceae), Vitis vinifera leaves or pomace (Vitaceae). HPTLC on silica gel with 1) ethyl acetate – toluene – formic acid – water 16:4:3:2,  or 2) cyclohexane – ethyl acetate – formic acid 30:19:1. Detection under white light, UV 254 nm and 366 nm. Extract stability after 2 years was also checked through HPTLC. Neutralization by spraying phosphate-citrate buffer, and drying in cold air stream. Effect-directed analysis using automated piezoelectrical spraying: A) for enzymatic inhibition (acetyl-cholinesterase, glucosidase, glucuronidase, tyrosinase); B) for activity against Gram-negative bacteria (Aliivibrio fischeri bioluminescence assay). Active bands of multipotent compounds were eluted from HPTLC layers 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 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). By comparison to literature, the following compounds were assigned: caffeine, catechins, carnosol, chlorogenic acid, cynaratriol, dicaffeoylquinic acid, feruloyl quinic acid, gallic acid, linoleic and linolenic acids, oleanic or ursolic acid, rosmarinic acid.

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).