J Chromatogr A 1666, 462863 (2022). Theoretical discussion on the factors determining the RF value of a given substance in a chromatographic system: A) the stationary phase (SP); B) the mobile phase (MP), the composition of which can be different from the solvent mixture prepared because of evaporation, saturation and liquid or gas adsorption effects over migration time; C) the difference of the free energies for the analyte transfer from SP to MP; D) external parameters like temperature and humidity. The universal HPTLC mixture (UHM) is a mixture of reference compounds that can be used for the system suitability test (SST) for the full RF range in all HPTLC experiments. Its composition is: thioxanthen-9-one (0.001 %), guanosine (0.05 %), phthalimide (0.2 %), 9-hydroxyfluorene, octrizole, paracetamol, sulisobenzone and thymidine (each 0.1 %), in methanol. The purpose was to study the potential of UHM to replace SST (described with specific markers in European Pharmacopoeia monographs) and to assess the quality of HPTLC results. TLC and HPTLC silica gel on different support (aluminium, glass) or with different granulometries and binders (classic, Durasil, Adamant), of the UHM, an acetonitrile extract of Abelmoschus manihot flowers (Malvaceae), a methanol extract of Sambucus canadensis flowers (Adoxaceae), and essential oils of Lavandula angustifolia, of Mentha × piperita (Lamiaceae) and of Myristica fragrans (Myristicaceae), as well as the following specific markers (standards): borneol, bornyl acetate, linalool, linalyl acetate (terpenoids), isoeugenol, isoeugenol acetate, chlorogenic acid (phenylpropanoids), gossypin (flavone), gossypetin-glucuronide, hyperoside (flavonol heterosides). Development (after 20 min plate conditioning with a saturated MgCl2 solution) with one of the following mobile phases: (MP1) toluene – ethyl acetate 19:1, especially for essential oils; (MP2) ethyl acetate – butanone – formic acid – water 5:3:1:1, especially for S. canadensis; (MP3) ethyl acetate – acetic acid – formic acid – water 100:11:11:26, especially for A. manihot. Documentation in UV 254 nm and 350 nm, and with white light (reflection + transmission), before and after derivatization. RF values were determined by scanning densitometry at 254 nm in absorption mode (for octrizole, at 366 nm in fluorescence mode with mercury lamp and optical filter K400 nm). For each HPTLC condition, intra-laboratory precision assay of UHM separation was performed (at least 5 analyses) with average RF values and 95 % prediction intervals, and calculating RF differences between pairs of UHM constituents and 95 % confidence intervals, which were max. +/-0.012 of the RF values for all UHM and markers. The sensitivity of UHM, and thus its usefulness as generic SST was demonstrated by repeating the HPTLC experiments with modifying by 10 % the quantity of one of the solvent each time. There were always significant changes in RF values of UHM components and/or in RF differences between pairs of UHM bands; it was often but no always the case with the official specific markers. UHM underwent also significant changes (although less than A. manihot extract) when several silica gel phases were compared under the same HPTLC conditions. This property is crucial to verify the right stationary phase before doing any RF correlations, and could make UHM a universal tool to identify discrepancies between different analyses. Finally, the use of UHM for a computer-supported evaluation of HPTLC results was discussed, either for zone identification and RF corrections (within confidence intervals), or for correlations of entire fingerprints as first step to implement machine learning algorithms.

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).

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).

Biotecnologia en el Sector Agropecuario y Agroindustrial. 20, 152-154 (2022). HPTLC of propolis on silica gel with toluene - ethyl acetate - formic acid 30:12:5. Detection under UV light at 254 and 366 nm. The method allowed the identification of apigenin, naringenin, quercetin, caffeic acid, galangin, feruloyl and derivatives of coumaric acid.

J. Food. Biochem. 45, e13608 (2022). HPTLC of kaempferol-3-O-β-D-glucoside in florets of Brassica oleracea on silica gel with toluene: acetone: formic acid 15:5:1. Quantitative determination by absorbance measurement at 240 nm. The hRF value for kaempferol-3-O-β-D-glucoside was 40.

J. Food. Biochem. 46, e13855 (2022). HPTLC of Amomum subulatum dry fruits on silica gel with toluene - ethyl acetate - methanol - formic acid 14:6:2:1. Qualitative identification under UV light at 254 and 366 nm. 

J. Food. Biochem. 46, e13852 (2022). HPTLC of gallic acid (1), caffeic acid (2), quercetin (3) and ferulic acid (4) in the leaves of Aegle marmelos on silica gel with toluene - ethyl acetate - formic acid 9:10:1. Quantitative determination by absorbance measurement at 254 nm. The hRF values for (1) to (4) were 19, 37, 45 and 48, respectively. The LOD and LOQ were 9 and 24 ng/zone for (1), 6 and 17 ng/zone for (2), 8 and 23 ng/zone for (3) and 6 and 17 ng/zone for (4). Recovery was in the range of 97.9-101.3 % for (1), 100.6-104.0 % for (2), 99.2-110.4 % for (3) and 98.0-99.9 % for (4).

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).