Marine Drugs 20(2), 106 (2022). TLC was used to monitor the fractionation of hydro-methanolic extracts of Rhodophytes: Gracilaria gracilis (Gracilariaceae) (A), Porphyra sp. (Bangiaceae) (B), Spongoclonium pastorale (Ceramiaceae / Wrangeliaceae) (C); and to assess the purity of two isolated mycosporine-like amino acids: porphyra-334 and shinorine.  TLC on silica gel with n-butanol - acetic acid - water 3:1:1. Derivatization by spraying ninhydrin reagent for the detection of peptides and amino-acids; or by spraying anisaldehyde - sulfuric acid for most phytochemicals; in both cases, followed by 5 min heating at 100 °C. Visualization under white light and at 366 nm. Porphyra-334 (hRF 28) was isolated pure from (B) and (C). Shinorine (hRF 25), isolated from (A) and (B), contained a coeluting sugar (hRF 48), which was absent after further purification on solid phase extraction, and, only when isolated from (B), a coeluting peptide or amino-acid (hRF 35), which was absent after further purification on cyclodextrane column chromatography.

J. AOAC Int. 104, 1719-1725 (2021). HPTLC of diflunisal (1) and its impurity biphenyl-4-ol (2) on silica gel with toluene - acetone - acetic acid 7:13:2. Quantitative determination by absorbance measurement at 254 nm. The hRF values for (1) and (2) were 59 and 79, respectively. Linearity was between 0.5 and 3 µg/zone for (1) and 0.3 and 1.7 µg/zone for (2). Inter-day and intra-day precisions were below 1 % (n=3). The LOD and LOQ were 131 and 398 ng/zone for (1) and 72 and 219 ng/zone for (2), respectively. Mean recovery was 100.2 % for (1) and 100.5 % for (2). 

J. AOAC Int. 104, 1430-1441 (2021). HPTLC of eight different fixed-dose combination products of aspirin (1) with ramipril (2), caffeine (3), metoprolol (4), paracetamol (5), atorvastatin (6) and simvastatin (7) on silica gel with toluene - ethyl acetate - 1 % formic acid in methanol 35:65:3. Quantitative determination by absorbance measurement at 229 nm. The hRF values for (1) to (7) were 88, 15, 34, 49, 59, 66 and 78, respectively. Linearity was between 2000 and 6000 ng/zone for (1) and (5), 100 and 300 ng/zone for (2), 600 and 1800 ng/zone for (3), 1000 and 3000 ng/zone for (4), 200 and 600 ng/zone for (6) and 800 and 2400 ng/zone for (7). Inter-day and intra-day precisions were below 2 % (n=3). The LOD and LOQ were 107 and 323 ng/zone for (1), 32 and 98 ng/zone for (2), 17 and 51 ng/zone for (3), 3 and 10 ng/zone for (4), 9 and 27 ng/zone for (5), 109 and 331 ng/zone for (7), respectively. Recovery ranged 99.2-100.8 % for (1), 99.2-100.2 % for (2), 98.2-101.3 % for (3), 99.4-100.6 % for (4), 100.2-101.4 % for (5), 98.7-101.3 % for (6) and 99.6-101.3 % for (7).

J. Liq. Chromatogr. Relat. Technol. https://doi.org/10.1080/10826076.2022.2159977 (2021). HPTLC of Ophiopogonis Radix from different habitats on silica gel with toluene - methanol - glacial acetic acid 800:50:1. Detection under UV light at 254 nm. 

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)).

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.