Marine Drugs 20(12), 757 (2022). Samples were ethyl acetate macerates and diethyl ether Soxhlet extracts from invasive Caulerpa cylindracea and non-invasive C. lentillifera (Caulerpaceae), as well as caulerpine (bisindole alkaloid) as standard isolated from one of the extracts. TLC on silica gel with petroleum ether – diethyl ether 1:1. Quantitative evaluation by densitometry at 330 nm, quantification of caulerpine (hRF 41, LOD 20 ng/zone, LOQ 68 ng/zone). The concentrations of caulerpine in C. cylindracea extracts (96-112 µg/g) were higher than in C. lentillifera (0-8 µg/g).

J Chromatogr A, 1625, 461230 (2020). Samples were extracts of Chinese plants: Acorus tatarinowii (= Acorus calamus var. angustatus) rhizomes (Araceae / Acoraceae) (1), Angelica sinensis roots (Apiaceae) (2), Gynura japonica rhizomes (Asteraceae) (3), Phellodendron chinense bark (Rutaceae) (4), Picrasma quassioides twigs and leaves (Simaroubaceae) (5), Rheum sp. roots and rhizomes (R. palmatum, R. tanguticum and/or R. officinale) (Polygonaceae) (6), Sophora flavescens roots (Fabaceae) (7), Dendrobium stems (D. aphyllum, D. aurantiacum var. dennaeanum, D. chrysanthum, D. chrysotoxum, D. gratiosissimum, D. hercoglossum, D. thyrsiflorum, D. trigonopus and D. williamsonii) (Orchidaceae) (8). Standards were: gigantol (from D. sonia); methoxycarbonyl-β-carboline (MCC from (5)); caffeic acid, emodin; senecionine and β-asarone; crategolic acid (= maslinic acid), corosolic acid, oleanic acid, ursolic acid; sesquiterpenoids (atractylenolides I – III) from Atractylodes macrocephala (Asteraceae); flavonoids (baicalein, baicalin, daidzin, hesperidin, wogonin) from Scutellaria baicalensis roots (Lamiaceae). HPTLC on silica gel with 10 mobile phases, depending on the samples. Detection under UV 254 nm and white light. For (3), derivatization with Dragendorff’s reagent (bismuth potassium iodide solution) for visualization of alkaloids. Zones of interest on underivatized plates were identified by a triple-quadrupole ­– linear ion-trap MS, the compounds being removed from the layer by a continuous-wave (445 nm) diode laser pointer through a DART interface (Direct Analysis in Real-Time, helium as gas for plasma-based ambient ionization, discharge needle voltage 1.5 kV, grid voltage 350 V, capillary temperature 300 °C and voltage 40 V, full scan in positive ionization mode in m/z range 150-800). Pigment standards were used for validation of this laser-assisted HPTLC-DART-MS method: malachite green, crystal violet, chrysoidin, auramine O, rhodamine B, Sudan red I – IV, Sudan red G, dimethyl yellow. Afterwards, the same HPTLC-MS method was applied to the origin / species determination of Dendrobium samples, based on the presence of four bibenzyl compounds erianin, gigantol, moscatilin, tristin. Erianin was present only in D. chrysotoxum, whereas none of these were detected in D. hercoglossum. Several components of the extracts were thus identified: asarone (a phenylpropanoid) in (1); phthalide lactones (butenylphthalide, ligustilide and chuanxiong lactone) in (2); co-eluting pyrrolizidine alkaloids (senecionine and seneciphylline) in (3); benzylisoquinoline alkaloid berberine in (4); alkaloids (canthinone alkaloids and MCC) in (5); anthraquinones (rhein, aloe-emodin, emodin, emodin methyl ether, chrysophanol) and (in negative mode) caffeic acid (a hydroxycinnamic acid) and corosolic, maslinic and oleanic acids (triterpenoids) in (6); quinolizidine alkaloids (matrine, oxymatrine, oxysophocarpine, sophoridine) in (7).

J Chromatogr A, 1625, 461230 (2020). Samples were methanolic extracts of commercial supplements containing Magnolia sp. bark (Magnoliaceae), as well as honokiol (1) and magnolol (2) (biphenyl neolignans) as separated or mixed standards. TLC and HPTLC on silica gel with n-hexane – ethyl acetate – ethanol 16:3:1. Visualization under UV 254 nm. Quantification of (1) and (2) by densitometric scanning in absorbance mode at 290 nm (hRF were 34 and 39, LOQ 200 ng and 280 ng/spot, respectively). Variability between samples from the same brand supplement was also determined, as well as extraction yields. Effect-directed analysis with 3 assays: A) to detect radical scavengers, immersion into DPPH• 0.02 % solution; B) to detect activity against Gram-negative bacteria, immersion into Aliivibrio fischeri suspension, followed by recording the bioluminescence; C) to detect activity against Gram-positive bacteria, immersion into Bacillus subtilis, followed by incubation 2 h at 28 °C and immersion into MTT 1 g/L. Compounds (1) and (2) were active in all assays. Identification of zones of interest by eluting with methanol from untreated TLC layer through the oval elution head of a TLC-MS interface directly to a single Quadrupole MS (electrospray ionization, interface temperature 350°C, heat block temperature 400°C, desolvation line temperature 250°C, detector voltage 4.5kV). Full mass scan spectra were recorded in the positive and negative ionization modes in m/z range 150–800. Other molecules (from other ingredients) were identified: piperine (alkaloid) and/or its geometrical isomers (active on A, hRF 29-30); and daidzein (active on A and B, hRF 18), isoflavone from Pueraria montana root (Fabaceae). Stability was assessed through 2D-HPTLC, by repeating the same development method in the orthogonal direction 4 h or 20 h after the first separation. Degradation products of (1) and (2) appeared after 20 h (but not at 4 h), including a honokiol dimer (formed in tracks of (1) and of (2)).

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, 1621, 461044 (2020). Samples were five isoquinoline alkaloids (berberine, chelerythrine, chelidonine, coptisine, sanguinarine) either as standard mixture or present in a Chelidonium majus (Papaveraceae) herb extract obtained with HCl 0.05 M in methanol. Separation on TLC and HPTLC silica gel layers with a screening of mobile phases consisting of eutectic mixtures of chemicals and/or phytochemicals. These homogenous stable liquids called DES (deep eutectic solvents) were obtained either simply by mixing, or by mixing followed by heating at 50°C, or by mixing with water for dissolution followed by dehydratation through rotary evaporation. For polarity adjustment, the DES phases were tested pure or diluted with acetone, chloroform, diethyl ether, methanol, or water. Visualization under UV 366 nm. The best separation was obtained with menthol – phenol in equimolar mixture, with 35 % methanol added (hRF values of the selected alkaloids were 33, 39, 79, 20 and 52, respectively).

J. Planar Chromatogr. 35, 375-381 (2022). HPTLC of benzylisoquinoline alkaloids sanguinarine (1) and berberine (2) in Argemone mexicana on silica gel with benzene - ethanol 47:3 for (1) and n-butanol - water - ammonia 8:1:1 for (2). Quantitative determination by absorbance measurement at 365 nm. The hRF values for (1) and (2) were 36 and 32, respectively. Linearity was between 10 and 40 ng/zone for both (1) and (2). Interday and intra-day precisions were below 1 % (n=3). The LOD and LOQ were 1 and 4 ng/zone for both (1) and (2). Recovery was between 92.7 and 93.5 %.

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, 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.