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, 1628, 461461 (2020). Samples were hydro-methanolic extracts of 100 genuine saffron samples (Crocus sativus stigmata, Iridaceae) from South Khorasan (SK) and Razavi Khorasan (RK) provinces (Iran), pure or mixed in several proportions with common vegetal adulterants: C. sativus style, Calendula officinalis petals (Asteraceae, Asteroideae), Carthamus tinctorius petals (Asteraceae, Carduoideae), Rubia tinctorum rhizomes (Rubiaceae). Commercial saffron samples (containing artificial adulterants) were also tested. TLC on silica gel with ethyl acetate – methanol – water – acetic acid 66:23:11:1. Evaluation at 254 nm, 366 nm, and 440 nm. Crocin (carotenoid, hRF 38) was used for optimization of extraction (parameters being first calculated by chemometry), using multilinear regression and ANOVA. Image data (pixel intensities and colors of each sample under the three selected wavelengths) were unfolded into a data matrix and transformed into a vector, used for multivariate image analysis of the chromatogram fingerprints. This allowed: A) separation of genuine samples by principal component analysis (PCA) into 2 clusters according to origin (cold climate in Northern half of RK vs. warm climate in SK and Southern part of RK) with 92 % prediction accuracy; B) separation of samples according to purity / vegetal adulterant groups by partial least squares – discriminant analysis (PLS-DA) with 98 % accuracy (if 10 µL extract applied); C) separation with 100 % prediction accuracy by PCA between genuine, mixed, and commercial samples.

J Chromatogr A, 1620, 460970 (2020). Samples were ethyl acetate extracts of Lavandula angustifolia herb and flowers and of aerial parts of other Lamiaceae (Ocimum basilicum, Origanum vulgare, Thymus vulgaris, Rosmarinus officinalis, Salvia officinalis), as well as standards. HPTLC on silica gel (pre-washed with methanol and heated 30 min at 105 °C) with n-hexane – ethyl acetate – acetic acid 70:27:3. Documentation at UV 254 nm and 365 nm and white light before and after A) derivatization with anisaldehyde – sulfuric acid reagent, followed by 10 min heating at 110 °C; B) spraying with DPPH• (0.2 % in methanol), followed by 30 min incubation in the dark; C) α-amylase inhibition assay by immersion into enzyme solution, incubation 30 min at 37 °C, immersion into substrate solution (starch 1 % in water), incubation 20 min at 37 °C and immersion into Gram’s iodine solution for detection (inhibition zones appear blue on white background). Quantification was performed on pictures using image processing software, and expressed as equivalents to the respective standards used for calibration curves: A) β-sitosterol (LOQ 1.5 µg/band), B) gallic acid (LOQ 60 ng/band), C) acarbose (LOQ 8 µg/band). An amylase inhibiting zone (hRF 68) present in all samples (except L. angustifolia), scraped from untreated plates and washed with ethyl acetate, was tentatively identified by ATR-FTIR analysis as oleanolic acid (pentacyclic triterpene).

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 Chromatogr A, 1598, 196-208 (2019). Samples were acertone – water 7:3 extracts of Reynoutria japonica (= Fallopia japonica = Polygonum cuspidatum) rhizomes (Polygonaceae) as well as flavanols (catechin, epicatechin, epicatechin gallate, epigallocatechin gallate) and procyanidins (A1, A2, B1–B3 and C1) as standards. HPTLC on diol silica gel with: (MP1) acetonitrile; (MP2) ethyl acetate; (MP3) ethyl acetate – formic acid 90:1; or (MP4) toluene – acetone – formic acid 3:6:1. Prewashing of the plates with mobile phase was needed only with MP1. After drying under hot air stream, derivatization by automated immersion into DMACA (dimethylaminocinnamaldehyde) – HCl solution (60 mg in 13 mL HCl + 187 mL ethanol), followed by 2 min drying under warm air stream. Visualization under UV 366 nm and white light, densitometry in absorption/reflectance mode at 280 nm (before derivatization) or 655 nm (10 min after derivatization). Bands of interest were eluted from layer with acetonitrile – methanol 2:1 through the oval elution head of a TLC-MS interface pump, into a RP18 liquid chromatography guard column, followed by a quadrupole ion trap mass spectrometer. Full scan mass spectra (m/z 150–2000) were recorded in negative mode using electrospray ionization (spray voltage 4 kV, capillary temperature 200◦C, capillary voltage -38.8 V). Monomer gallates to hexamer gallates were detected, separated with MP1, MP2 or MP4; monomers and oligomers (not gallates) were separated with MP3 (up to hexamers) and with MP1 and MP4 (up to decamers). Moreover, enhanced absorption of standards was also studied for influence of mobile phases, of layers (diol silica gel vs. classical silica gel vs. cellulose) and of luminosity (light vs. dark).

J Chromatogr A, 1675, 463167 (2022). Samples were ethanol extracts (and their flash chromatography fractions) of Prunus armeniaca leaves (Rosaceae), as well as betulinic, linolenic, maslinic (= crataegolic), oleanolic, ursolic acids and pygenic acids A (= corosolic acid) and B b as standards. When needed, to improve separation of triterpenoids, reversible pre-chromatographic derivatization was performed in situ by applying 10 µL iodine solution (2 % in chloroform) either before development on the deposit band, or for 2D-HPTLC after a first separation up to 60 mm and before a second orthogonal separation. Layers were covered 10 min with glass sheet after iodine application, and then dried 1 min under cold air stream. HPTLC on silica gel with chloroform – ethyl acetate – methanol 20:3:2, 85:9:6, or 15:2:3), followed by 5-10 min drying under cold air stream (eliminating iodine completely). Post-chromatographic derivatization by immersion (time 2 s, speed 3 cm/s) into vanillin – sulfuric acid (40 mg and 200µL, respectively, in 10 mL ethanol), followed by heating 5 min at 110 °C. Antibacterial effect-directed analysis was performed by immersion (time 8 s) into Bacillus subtilis suspension, followed by 2 h incubation at 37 °C, immersion in MTT solution and 30 min incubation at 37 °C. Active bands were eluted from layer with methanol through the oval elution head of a TLC-MS interface pump, into a single quadrupole mass spectrometer to record full scan mass spectra (m/z 200–1200 in both modes) using electrospray ionization (interface temperature 350°C, heat block temperature 400°C, desolvation line temperature 250°C, detector voltage 4.5kV). Five triterpenoids were identified: betulinic, corosolic, maslinic, oleanolic and ursolic acids, acid, as well as two fatty acids: linolenic and palmitic acid.

J. Planar Chromatogr. 35, 439-451 (2022). HPTLC of withanoside IV (1), withanoside V (2), withaferin A (3), and kaempferol-based glucoside (4) in the roots and aerial parts of Withania somnifera on silica gel with ethyl acetate - chloroform - methanol - water 40:15:22:9. Detection of (1) to (3) by spraying with anisaldehyde sulfuric acid reagent, followed by heating at 100 °C for 3 min. Quantitative determination by absorbance measurement at 540 nm for (1) to (3) and 254 nm for (4). The hRF values for (1) to (4) were 33, 42, 62 and 21, respectively. Linearity was between 200 and 1000 ng/zone for (3) and (4) and 400 and 2000 ng/zone for (1) and (2). Interday and intra-day precisions were below 4 % (n=6). The LOD and LOQ were 180 and 544 ng/zone for (1), 215 and 652 ng/zone for (2), 170 and 516 ng/zone for (3) and 48 and 144 ng/zone for (4). Recovery was between 95.9 and 99.6 % for (1) and (4).