J. Planar Chromatogr. 35, 331-337 (2022). HPTLC of selected varieties of hop cultivars H. lupulus on silica gel with 8 % isopropanol in dichloromethane. Detection under UV light at 254 and 366 nm. Direct bioautography by dipping into Bacillus subtilis bacterial suspension for 10 s, followed by incubation at 37 °C for 17 h. TLC plates were covered with 0.2 % aqueous MTT tetrazolium dye solution (thiazolyl blue tetrazolium bromide, 98 %), followed by incubation at 37 °C for 30 min. 
 

J. Planar Chromatogr. 35, 273-279 (2022). HPTLC of sweetener saccharin (1), acesulfame-K (2), neohesperidin (3), aspartame (4), stevioside (5), rebaudioside A (6), sucralose (7), and Na-cyclamate (8) in food samples on silica gel with ethyl acetate - methanol - acetic acid 5:1:1. Detection by dipping into the following reagent sequece, followed each by plate heating and image documentation or densitometry: 1) Primuline reagent (100 mg primuline in 20 mL water and 80 mL acetone), followed by solvent evaporation and detection at 366 nm; 2) ninhydrin reagent (0.3 g ninhydrin dissolved in 95 mL isopropyl alcohol and 5 mL glacial acetic acid), followed by heating at 120 °C for 5 min and detection at white light; 3) 2-naphthol sulfuric acid reagent (1 g 2-naphthol dissolved in 90 mL ethanol and 6 mL 50 % sulfuric acid added dropwise), followed by heating at 120 °C for 5 min and detection at white light. Quantification by absorbance measurement at 200 nm for (1), 230 nm for (2), 290 nm for (3), 500 nm for (4) to (7) and 650 nm for (8).  Linearity was between 30 and 600 ng/zone for (5) and (6) and 800 and 1600 ng/zone for (8).   

Molecules 25 (17), E3928 (2020). Samples were curcumin (as standard) and methanolic extracts of Curcuma xanthorrhiza and C. aeruginosa (Zingiberaceae) rhizomes, both separately and in mixtures. Separation on TLC silica gel with chloroform – methanol – formic acid 94:3:3. Densitometry of curcumin (hRF 50) in absorption mode at UV 427 nm. This method was validated with curcumin standard for selectivity (vs. demethoxycurcumin hRF 32), linearity range (250 - 450 ng), LOD (21 ng) and LOQ (69 ng), accuracy and precision. Curcumin contents were between 0.74 and 1.23 % in pure C. xanthorrhiza extracts, but decreased when adulterated with C. aeruginosa.

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.

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.

Molecules, 26 (24), 7683 (2021). Samples were ultrasound-assisted extracts of fruit puree and juice (pre-treated with sulfur dioxide or ascorbic acid) of Ananas comosus (Bromeliaceae) and Mangifera indica (Anacardiaceae). HPTLC on silica gel with toluene – ethyl acetate – methanol – formic acid 120:90:35:3. Detection under white light, UV 254 nm and 366 nm, before and after  derivatization by immersion (2 s, 3 cm/s) into anisaldehyde sulfuric acid reagent and  diphenylamine aniline reagent, followed by heating at 110 °C for 5 min. Effect-directed analysis using automated immersion: A) for free radical (DPPH•) scavengers; B) for enzymatic inhibition (acetyl-cholinesterase, tyrosinase); C) for activity against Gram-negative (Aliivibrio fischeri bioluminescence assay) or Gram-positive bacteria (Bacillus subtilis bioassay). Active compounds were far more present in puree than in juice extracts, and differences were also seen between cultivars. Ascorbic acid (hRF 37), used as additive for the mango puree, was active as antioxidant and as transiently disruptive for A. fischeri metabolism and bioluminescence.

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.