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.

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, 1669, 462942 (2022). Samples were medroxyprogesterone acetate (MPA) as standards and commercial drug extracts, dissolved in dichloromethane. TLC on silica gel (preactivated by 30 min heating at 120 °C) with dichloromethane – ethyl acetate 10:1, followed by 30 min drying at 120 °C. Derivatization by spraying with sulfuric acid (50 % in ethanol). Visualization in a 3D-printed chamber designed especially for this purpose, blocking extraneous light and including a smartphone holder, a fluorescent lamp and an optical density step tablet. Pictures were taken with the smartphone digital camera, after spraying (6 background images) and after 10 min heating at 120 °C (6 foreground images). In the last case, MPA appeared as black spots (hRF 16–20). Using an image processing software program: (1) one averaged background image and one averaged foreground image were created by concatenation and were split into 3 colour channels; (2) the green colour channels were corrected to remove background noise, by subtraction of an averaged darkfield image (taken on blank plate without light) and by comparison ratio to an averaged blankfield image (taken on blank plate with light); (3) the pixel values of the MPA bands were converted to optical density values through the Robard’s function, by comparison to a reference image of a theoretical optical density step tablet; (4)  furthermore, the corrected background image was subtracted from the corrected (and denoised with a Gaussian Blur) foreground image; a triangle threshold algorithm was applied on the resulting image, and was converted to a mask (white spots on black background); (5) applying the binary mask to the original corrected images (obtained in (2)), the final integrated density values of MPA spots were obtained. This method was validated for linearity range (1.25–3.75 mg/mL), for precision, for reproducibility, for robustness, and for accuracy expressed as average recovery values (101 % overall mean) by comparison of TLC results with HPLC-DAD results.

J Chromatogr A 1638, 461830 (2021). The purpose was to find the first universal HPTLC mixture (UHM), a mixture of reference compounds that could be used for the system suitability test (SST) for the full RF range in all HPTLC experiments.
(Part 1) UHM composition: First, 56 organic molecules, detectable without derivatization, were tested on HPTLC silica gel with 20 different mobile phases (MP) belonging to different Snyder’s selectivity groups and with several polarity indices. Visualization under UV 254 nm and 366 nm. Densitometry scanning at 254 nm in absorption mode, and at 366 nm in a fluorescence mode (mercury lamp 366 nm, with wavelength filter <400 nm). For selected bands, spectra were recorded in absorbance-reflectance mode (wavelength range 190 – 450 nm, deuterium and tungsten lamp). This procedure allowed 8 molecules to be selected for their better spot resolution and for their specific RF values (at least 3 different values distributed throughout the full RF range for each MP). The final composition of UHM was: thioxanthen-9-one (0.001 %), guanosine (0.05 %), phthalimide (0.2 %), 9-hydroxyfluorene, octrizole, paracetamol, sulisobenzone and thymidine (each 0.1 %), in methanol.
(Part 2) UHM validation: Afterwards, UHM was submitted again to a panel of HPTLC assays with always two MP: (A) toluene – methanol – diethylamine 8:1:1; (B) ethyl acetate – formic acid – water 15:1:1; and for each MP, the means, standard deviation and 95 % confidence intervals of the RF values were calculated. (a) UHM was validated for intermediate intra-laboratory precision, as well as for inter-laboratory reproducibility, with ΔRF 0.045. (b) The capacity of UHM to detect small variations was demonstrated by significant changes in at least some RF values, when separation was deliberately performed at different levels of relative humidity (0 %, 33 %, 75 %, 100 %), or with smaller humidity variations (7 % compared to 0–5 %, and 49 % compared to 33 %), or when performing vs. omitting the 10min chamber pre-saturation, or when modifying the MP (+/-10% of one solvent at each time). These response characteristics (the opposite of robustness) made UHM a powerful tool for SST. (c) Finally, UHM stability was studied with UHM aliquots under several storage conditions (-78 °C, -20 °C, 4 °C, room temperature, 45 °C; or 40 °C with 75 % relative humidity) and durations (2 weeks or 2 months). The densitometric peak profiles at 254 nm were compared to those of the fresh compounds, qualitatively (RF value, UV spectrum) and quantitatively (peak area). UHM was stable at room temperature or below, for 2 months (at higher temperature, guanosine, phthalimide and paracetamol degraded).

J Chromatogr A 1638, 461895 (2021). Samples were sphingolipid-rich fractions of unproteinated blood plasma from healthy humans or from Fabry’s disease patients, as well as standards of sphingomyelin (SM) and of globotriaosylceramides (Gb3 = ceramide trihexosides), and related compounds (lyso-ceramide trihexosides, lactosyl ceramide, glucosyl ceramide). HPTLC on silica gel (Lichrosphere with spherical particles) by automated multiple development with a 9-step gradient, starting with pure methanol and ending with dichloromethane – methanol 9:1. Visualization and densitometry under UV 190 nm. Derivatization for Gb3 and derivatives (but not for SM) by immersion into orcinol solution (0.2 %, with sulfuric acid 10 %), followed by 15 min heating at 100 °C and by densitometry under visible light 550 nm. Bands of interest were directly eluted with methanol from underivatized plates into an ion-trap MS, through the oval head of a TLC-MS interface (with stainless steel frit to remove silica gel particles). Two different ionization processes were used: (A) electrospray ionization (ESI, capillary voltage 4 kV, endplate offset voltage -0.5 kV, nebulizer pressure 40 psi, drying gas 9 mL/min at 350 °C); (B) atmospheric pressure chemical ionization (APCI, capillary voltage 2–3 kV, current intensity 4.5 µA, nebulizer pressure 45 psi, drying gas 5 mL/min at 350 °C; vaporization at 450 °C). Full MS spectra were recorded up to m/z 1500 in positive ion mode. The relative ion intensities were used to quantify the detected species. Previous to this study, the precision of the elution head positioning was tested on Gb3 standard zones, comparing 3 positions for analyte elution: from the centre and from each higher or lower side of the band. The same main m/z peaks were observed in the 3 positions, but in different proportions. This was explained by the presence of coeluting Gb3 subclasses (the ceramide moiety CM being either saturated, mono-unsaturated fatty acyl with a slightly higher migration distance, or polar hydroxyl fatty acyl with the opposite effect on migration) and of coeluting Gb3 isoforms (the hexoside moiety consisting of glucose and/or galactose units). This resulted in the broadening and partial splitting of the standard band. In the plasma samples, 19 molecular species of Gb3 were identified (depending on the CM, the sugar isoforms being undistinguishable by MS): 5 with a saturated CM, 7 with two additional double bonds on the CM, 7 with a methylated CM. In case of Fabry’s disease, most Gb3 species with saturated CM were highly increased, whereas other species were decreased.

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).

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.

Marine Drugs 20(12), 757 (2022). Samples were ethyl acetate macerates and diethyl ether Soxhlet extracts from invasive Caulerpa cylindracea and non-invasive C. lentillifera (Caulerpaceae), as well as caulerpine (bisindole alkaloid) as standard isolated from one of the extracts. TLC on silica gel with petroleum ether – diethyl ether 1:1. Quantitative evaluation by densitometry at 330 nm, quantification of caulerpine (hRF 41, LOD 20 ng/zone, LOQ 68 ng/zone). The concentrations of caulerpine in C. cylindracea extracts (96-112 µg/g) were higher than in C. lentillifera (0-8 µg/g).