PLoS ONE 18(11), e0294775 (2023). Sample was an Azadirachta indica seed extract (Meliaceae). TLC on silica gel with different mobile phases: (1) diethyl ether – methanol 49:1; (2) diethyl ether – acetone 2:1; (3) isopropanol – n-hexane 11:9; (4) dichloromethane – methanol – acetic acid 95:5:1. After 30 min hot air drying,detection under UV light. The hRF values of azadirachtin A (a limonoid) were 75, 42, 44, 55, respectively. Mobile phase (1) was therefore chosen as solvent for purification of azadirachtin A and for its quantification by Fourier transform infrared spectroscopy (FTIR).

Chin J Anal Sci 38 (4), 433-440 (2022). TLC of acetochlor, alachlor, metolachlor, butachlor and pretilachlor in the root vegetables onion and garlic on silica gel with hexane – acetone 4:1. Detection by spraying with 5 % iodized bismuth potassium in acidic aqueous solution. Then QuEChERS (quick, easy, cheap, effective, rugged, safe)-GC-MS/MS method in the selected ion monitoring mode for determination of residues of these compounds after extraction with acetonitrile and acetonitrile - acetic acid and purification by PSA , MWCNTs , GCB and C18 at optimized conditions. The linearity ranged from 0.02 to 2.0 μg/mL for the 5 compounds with the correlation coefficients greater than 0.99, the LOQ was 0.025 mg/ kg, the average recoveries ranged from 72.0 % to 102 % with the relative standard deviations from 0.5 % to 7.9 %.

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, 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, 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 Chromatogr A, 1608, 460415 (2019). Samples were acetonitrile extracts of Annona cherimola fruit peel, pulp and seeds (Annonaceae), as well as caffeic acid as standards. HPTLC on silica gel with chloroform – ethyl acetate – propanol 21:2:2 for peel extracts, with chloroform – methanol 9:1 for seed extracts. Derivatization by spraying Dragendorff’s reagent for alkaloids, secondary amines and non-nitrogenous oxygenated compounds.  Effect-directed assay was performed for inhibitors of α-glucosidase. Before sample application, plates were developed with enzyme substrate (2-naphthyl-α-D-glucopyranoside 0.1 % in methanol) and dried 20 min at 60 °C. Then, samples were applied and separated, and mobile phase was removed by heating 10 min at 60 °C. The chromatogram was sprayed with 4 mL enzyme solution (5 unit/mL in 100 mM phosphate buffer,  pH 7.4), liquid excess was removed under lukewarm air stream, the plate was incubated 10 min at 37 °C in a moisture box, followed by spraying chromogenic reagent Fast Blue salt B 0.1 % in water, giving after 2 min white inhibition bands visible on purple background under white light. Plate image was documented under illumination (reflectance mode) with white light. The bands of 3 inhibiting compounds were analyzed in a triple quadrupole mass spectrometer. 1) Full scan mass spectra (m/z 50−1000) in the positive ionization mode were recorded using electrospray ionization (ESI, spray voltage 3 kV, desolvation line temperature 250 °C, block temperature 400 °C) for compounds directly eluted with methanol – acetonitrile through the oval elution head of a TLC-MS interface pump. 2) Compounds were also isolated (either eluted directly from the plate into a vial through the same interface, or scraped from the plate and extracted with methanol – chloroform into a vial), dried, and submitted to HPLC-DAD-MS/MS; MS-MS spectra were recorded in the same conditions, using argon as collision gas and collision cell voltages from -20 and -40 V. Inhibitors were identified as phenolamides (phenylethyl cinnamides): moupinamide (hRF 66 in peels, 56 in seeds), N-trans-feruloyl phenethylamine (hRF 76 in peels), N-trans-p-coumaroyl tyramine (hRF 44 in seeds).