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.

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, 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).