Heliyon 8(8), e10103 (2022). Samples were a methanolic extract of Cymbopogon giganteus leaves (= C. caesius subsp. giganteus, Poaceae), as well as flavones as standards: isorhamnetin, luteolin and orientin (=luteolin 8-C-glucoside). HPTLC on silica gel with ethyl acetate – acetic acid – formic acid – water 100:11:11:26. Derivatization for flavones with Neu’s reagent (ethanolamine diphenylborate – PEG). Visualization under UV 365 nm. The standards (hRF 75, 70-72 and 96, respectively) were not detected in the extract. Some analytes detected by the reagent were scraped from the underivatized plate into a tube, and injected through a TLC-MS interface into a double-quadrupole – time-of-flight MS (electrospray ionization). Full mass scan spectra were recorded in positive and negative ionization modes in m/z range 150–550. For 3 of the compounds, isolated through MPLC columns, the HPTLC-MS results, combined to the NMR and HPLC-MS analyses, allowed the identification as epicatechin (hRF 86, a flavanol, not coloured by Neu’s reagent) and as luteolin 8-C- and 6-C-glucosides (hRF 67-70).

Anal. Bioanal. Chem. 414, 5991-6001 (2022). 2D-HPTLC fingerprint of Alpinia officinarum on silica gel in 384-well microplate array format (4.5 × 4.5 mm) matrix with trichloromethane - methanol - petroleum ether 97:3:25 in the first dimension and ethyl acetate - petroleum ether - acetic acid 15:35:1 in the second dimension. The paired chromatographic-based microassay array with the consistent chromatographic distribution was prepared by transferring a portion of the sample from the stock chromatographic-based microassay arrays to the corresponding array units of another 384-well microplate. A G‑quadruplex ligand bioassay was used to evaluate the ligand activity of the components in each array unit of the chromatographic-based microassay array. Further analysis by mass spectrometry. 

Anal. Bioanal. Chem. 414, 4481-4495 (2022). HPTLC of selected caffeine-containing standards and beverages (Red Bull, Coca-Cola, coffee, and black tea) on different stationary phases (silica gel, RP- and cyano-) with propan-2-ol - n-heptane - water 7:3:1. Direct surface analysis of the TLC plates with a flowing atmospheric pressure afterglow (FAPA) ambient desorption/ionization source (TLC-FAPA-MS). CN-HPTLC plates were the most efficient stationary phase, resulting in a significantly more intense caffeine signal.

Marine Drugs 17(3), 148 (2019). Samples were ethyl acetate extracts of seagrass Amphibolis antarctica (Cymodoceaceae), and of algae: Austrophyllis harveyana (Kallymeniaceae), Carpoglossum confluens, Cystophora harveyi, C. monilifera, C. pectinata and C. subfarcinata, Myriodesma integrifolium, Sargassum lacerifolium (Sargassaceae), Codium fragile subsp. tasmanicum (Codiaceae), Ecklonia radiata (Lessoniaceae), Hypnea valida, Rhodophyllis membaneacea (Cystocloniaceae), Hormosira banksii (Hormosiraceae), Perithalia caudata (Sporochnaceae), Phyllospora comoasa, Scytothalia dorycarpa (Seirococcaceae), Plocamium dilatatum (Plocamiaceae), and epiphytic brown algae. HPTLC on silica gel (pre-washed with methanol and heated 30 min at 100 °C) with n-hexane – ethyl acetate – acetic acid 15:9:1. Derivatization by immersion: A) into anisaldehyde – sulfuric acid reagent, followed by 10 min heating at 105 °C, for the detection of steroids and terpenes; B) into DPPH• (0.2 % in methanol), followed by 30 min incubation in the dark, for the detection of antioxydants; C) into Fast Blue B solution (0.1 % in 70 % ethanol) for detection of phenols (with alkylresorcinols detected as dark purple zones on colorless background). Effect-directed analyses were performed directly on the plates. D) α-Amylase inhibition assay by immersion into enzyme solution, incubation 30 min at 37 °C, immersion into substrate solution (starch 2 % in water), incubation 20 min at 37 °C and immersion into Gram’s iodine solution for detection (inhibition zones appear blue on white background). E) Acetylcholinesterase (AChE) inhibition assay (after neutralization by immersion into phosphate buffer) by immersion into enzyme solution, incubation 30 min at 37 °C, immersion into substrate solution (α-naphthyl acetate) and into dye reagent (Fast Blue Salt B). Densitometry through automated scanning, quantification expressed as equivalents to the respective standards used for calibration curves: A) β-sitosterol (LOQ 1.6 µg/band), B) gallic acid (LOQ 60 ng/band), D) acarbose (LOQ 173 µg/band), E) donepezil (LOQ 96 µg/mL). Alkylresorcinols were detected as antioxydant in C. harveyi and C. pectinata (hRF 88), and in C. subfarcinata (hRF 72, 81, 88). Enzymatic inhibitors in C. fragile were considered as a flavone (hRF 65) and a terpenoid (hRF 77), due to their absorption curves (densitometric scan in range 200-400 nm).

Marine Drugs 18(4), 184 (2020). A new alginate lyase (AlgSH7), isolated from marine bacterium Microbulbifer sp. strain SH1 (Alteromonadaceae), was incubated (24 h at 40 °C) with sodium alginate from brown algae (1 % in TRIS-HCl buffer, pH 9), or with related mannuronate and guluronate polymers (polyM and polyG), or with related saccharides with different polymerisation degrees (PD 1 – 4). TLC of reaction products as well as saccharides, on silica gel with n-butanol ­– acetic acid – water 3:2:2. Derivatization by spraying sulfuric acid (10 % in ethanol), followed by 5 min heating at 130 °C. The enzyme was active only on alginate and on polyM, cleaving them into oligomeric fragments (PD 2 – 4); it was inactive on polyG or on oligomers.

J Chromatogr A, 1647, 462153 (2021). Samples were extracts of Pittosporum angustifolium leaves (Pittosporaceae), either pure or fermented 1-4 weeks in NaCl solution, as well as acarbose, gallic acid, β-sitosterol, caffeic and chlorogenic acids, as standards. HPTLC on silica gel (prewashed with methanol and dried 15 min at 105 °C) with n-hexane – ethyl acetate – acetic acid 15:9:1. Derivatization by immersion (speed 5 cm/s, time 1 s): (A) into DPPH• 0.2 % solution, to detect radical scavengers; (B) into neutralized ferric chloride (3 % in ethanol), followed by 5 min heating at 110 °C, for detection of phenolic compounds; (C) into anisaldehyde – sulphuric acid reagent, followed by 10 min heating at 110 °C, to detect terpenes and steroids. Effect-directed analysis (EDA) for α-amylase inhibition assay (D) by immersion into enzyme solution, incubation 15 min at 37 °C, immersion into substrate solution (starch 2 % in water), incubation 20 min at 37 °C and immersion into Gram’s iodine solution for detection (inhibition zones appear blue on white background). In all cases, visualization under white light. Quantification was performed on pictures using image processing software, and expressed as equivalents to the respective standards used for calibration curves: (A) and (B) gallic acid (LOQ 250 and 740 ng/band, respectively), (C) β-sitosterol (LOQ 1.5 µg/band), (D) acarbose (LOQ 8 µg/band). Zones of interest, scraped from untreated plates and washed with ethyl acetate, were submitted by ATR-FTIR analysis. An amylase inhibiting zone (hRF 85) present in all extracts was identified as fatty acid esters: ethyl palmitate in unfermented and methyl linoleate in fermented extracts. Moreover, fermented extracts contained antioxidant zones (hRF 15 – 20), identified as monomers and oligomers (including hydroxycinnamic, guaiacyl, syringyl derivatives) from decomposed lignin.

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)).