J. Ethnopharmacol. 320, 117404 (2024). HPTLC of lupeol and stigmasterol in the stem bark of Ficus benghalensis, Ficus glomerata, Ficus religiosa, Ficus infectoria, and Albizia lebbeck on silica gel with toluene - ethyl acetate - methanol - formic acid 38:5:5:20:1. Detection by spraying with anisaldehyde sulfuric acid. Qualitative identification under UV light at 525 nm.

Marine Drugs 21(1), 2 (2023). Samples were methanol extracts of cultivated marine bacteria Thalassomonas actiniarum, T. viridans and T. haliotis (Colwelliaceae),  as well as cholesterol, cholic acid, and deoxycholic acid as standards. TLC on silica gel with n-hexane – ethyl acetate – methanol – acetic acid 20:20:5:2. After drying at room temperature, visualization by spraying with phosphomolybdic acid (10 % in ethanol) and heating with a heat-gun. For isolation of cholic acid (hRF 80), present in all samples, preparative TLC on silica gel with the same mobile phase, the corresponding band was scraped off with a surgical blade and extracted with methanol overnight. The isolated cholic acid was identified by LC-MS.

Heliyon 7(2), e06116 (2021). Samples were a methanolic extract of a semi-solid ayurvedic conserve (ashwagandhadi lehyam) prepared with Withania somnifera roots (Solanaceae) and five other plants, as well as standards: withaferin A and withanolide A (= withaniol), two ergostane triterpene steroids with lactone cycle and epoxide. HPTLC on silica gel with toluene – ethyl acetate – formic acid 6:4:1. Visualization and densitometric scanning at UV 254 nm and 366 nm (deuterium lamps). Derivatization by immersion into vanillin – sulfuric acid reagent, followed by oven heating at 105 °C until optimal coloration. Documentation under white light and densitometry scanning at 540 nm (tungsten lamp). Both analytes (hRF 35 and 45 respectively) were shown at 254 nm and 540 nm (but not at 366 nm), in the standards and in the extract.

J Chromatogr A 1638, 461597 (2021). Samples were Isatis tinctoria (= I. indigotica) root extracts (Brassicaceae) and their fractions. Standards were oseltamivir acid (OA), a neuraminidase (NA) inhibitor; pinoresinol (PR, a lignan), β-sitosterol (SS, a sterol), and dihydro-neoascorbigen (DHNA, an alkaloid). HPTLC / TLC on silica gel with (1) petroleum ether – ethyl acetate – acetic acid 48:8:1 for petroleum ether extracts and SS, or 30:40:1 for ethyl acetate extracts, or 10:30:1 for PR; (2) with toluene – ethyl acetate – methanol – formic acid 16:3:1:2 or 10:4:1:2 also for ethyl acetate extracts and DHNA; (3) with n-butanol – acetic acid – water 25:4:3 for butanol extracts. OA was applied but not developed. RP-18, polyamide, cellulose, alumina layers were tested, but the resolution was lower. Derivatization by spraying with sulfuric acid (10 % in ethanol). Enzymatic assay by immersion of the plates into neuraminidase solution (6 U/mL), followed by 1 h incubation at 37 °C and by immersion into chromogenic substrate solution (1.75 mM 5-bromo-4-chloro-3-indolyl-α-D-N-acetylneuraminic acid). After 5 min, NA inhibitors were seen as white zones on blue background. The experiment was previously improved for the following parameters: incubation times, substrate and enzyme concentrations, followed by statistical evaluation and calculations using Box-Behnken design. Quantification by absorbance measurement (detection wavelength 605 nm, reference wavelength 420 nm). In optimal conditions, OA had LOD 300 ng/zone. Zones of interest on underivatized plates were directly submitted to MS, using EFISI (electrostatic-field-induced spray ionisation), as follows. Chromatograms were immersed 1–3 s into dimethicone – n-hexane 1:1 to form a hydrophobic film, and dried 30 min at room temperature; on the analyte spot, a hydrophilic droplet was formed with 5 µL methanol – water 1:1, extracting the analyte from the layer; the analyte was further attracted through a capillary tube (3–4 cm long, made of non-deactivated fused silica) under a strong electrostatic field, into the in-let orifice of the triple-quadrupole ­– linear ion-trap MS (induction voltage 4 kV; capillary voltage 40 V; tube lens voltage 100 V; capillary temperature 200 °C). Full-scan spectra were recorded in m/z range 50 – 1000, helium was used for collision-induced dissociation. 11 active compounds were identified in the extract: SS, 6 alkaloids (including cycloanthranilylproline, DHNA, hydroxy-indirubin, isatindigodiphindoside, isatindinoline A and), 3 lignans (including PR and isolariciresinol), 1 fatty acid (trihydroxy-octadecenoic acid).

Front. Pharmacol. 12, 678611 (2021). HPTLC of withanolide S in Withania somnifera and myristicin in Myristica fragrans on silica gel with toluene - ethyl acetate - acetic acid 5:4:1. Detection of myristicin under UV 254 nm. Detection of withanolide S by spraying with 5 % anisaldehyde sulfuric acid, followed by visualization under UV 540 nm. Screening of anticholinesterase active metabolites by spraying with DTNB/ATCI reagent (1 mmol/L 5,5-dithiobis-(2- nitrobenzoic acid) and 1 mmol/L acetyl thiocholine iodide). 

J. Sep. Sci. 45, 1616-1635 (2022). HPTLC of gallic acid (1), protocatechuic acid (2), vanillic acid (3), cinnamic acid (4), piperine (5), guggulsterone-E (6), and guggulsterone-Z (7) in Mahayograj Guggul on silica gel with toluene - ethyl acetate - formic acid 10:9:2 for (1) to (3) and toluene - acetone 9:1 for (4) to (7). Quantitative determination by absorbance measurement at 250 nm for (6) and (7), 280 nm for (1), (2), (3) and (4) and 343 nm for (5). The hRF values for (1) to (7) were 30, 41, 47, 15, 33, 41 and 45, respectively. Linearity was between 100 and 1000 µg/mL for (1), 5 and 60 µg/mL for (2), 10 and 80 µg/mL for (3), (4) and (7), 20 and 100 µg/mL for (5) and 40 and 120 µg/mL for (6). Inter-day and intra-day precisions were below 4 % (n=18). The LOD and LOQ were 4 and 14 µg/g for (1), 7 and 21 µg/g for (2), 24 and 72 µg/g for (3), 0.8 and 2.4 µg/g for (4), 12 and 35 µg/g for (5), 2 and 6 µg/g for (6) and 4 and 14 µg/g for (7), respectively. Recovery was between 86.6 and 102.0 % for (1) to (7).

J. Food Compos. Anal. 114, 104763 (2022). HPTLC of ergosterol in wheat, on silica gel with ethyl acetate - petroleum ether 3:2. Quantitative determination by absorbance measurement at 282 nm. The hRF value for ergosterol was 45. Linearity was between 40 and 600 ng/zone. Inter-day and intra-day precisions were below 4 % (n=6). The LOD and LOQ were 11 and 36 ng/zone. Recovery was between 103.7 and 107.7 %. 

Marine Drugs 19(3), 161 (2021). Samples were a standard mix (tripalmitin, palmitic acid, cholesterol, phosphatidylcholine) and lipid-enriched extracts of zebrafish larvae (Danio rerio, Cyprinidae), that were anesthetized with tricaine after having being treated with 11 extracts of cyanobacteria strains and/or with a green fluorescent lipid analogue of fatty acids (BODIPY-C16, bore-dipyrromethene derivative). HPTLC on nano silica gel in 3 steps: 1) and 2) with chloroform – methanol – water 12:6:1 (twice up to 4 cm); 3) hexane – diethyl ether – acetic acid 160:40:3 (once up to 9 cm). Derivatization of lipids by spraying primuline solution (0.01 % in acetone – water, 3:2). Quantification based on fluorescence peak area intensity, was performed using image software on pictures taken through a green fluorescence imager. Triglycerides were decreased in the case of larvae treated with 2 extracts of Synechocystis strains (Merismopediaceae), but the levels of other lipid classes were not affected. No treatment significantly affected the incorporation of BODIPY-C16 into any of the lipid classes of the larvae.