J Chromatogr A 1653, 462442 (2021). Samples were peptides obtained through tryptic hydrolysis of the 5 most abundant milk proteins: α-lactalbumin (α-LA), β-lactoglobulin (β-LG), α-, β- and κ-casein (CA). As standards, synthetic whey and pea (Pisum sativum, Fabaceae) peptides (selected based on the in silico tryptic digest of α-LA, β-LG, legumin A, and vicilin with one or zero miscleavages) were only used in the last assay for prediction of the RF values of peptides with known amino-acid (AA) sequences. Two-dimensional HPTLC on silica gel (pre-washed with methanol and activated 10 min at 100°), first with basic mobile phase sec-butanol – pyridine – ammonia – water 39:34:10:26, and (after 12h drying) in the orthogonal direction with acidic mobile phase sec-butanol – pyridine – acetic acid – water 11:8:2:5. Derivatization for peptides and proteins by immersion into fluorescamine (0.05 % in acetone); visualization under UV 254 nm and 365 nm. Computer-assisted determination of the x- and y-coordinates of the derivatized zones. Repeatability (n=8) of the 2D-HPTLC was statistically tested with the Kolmogorov-Smirnov test for normal distribution and with Dixon’s Q test for outliers. Relative standard deviation (RSD) for the RF values was 12.9 % for the first dimension (y-coordinates) and 16.5 % for the second dimension (x-coordinates). According to their higher intensity and sharpness, 15 – 20 detected zones from each protein hydrolyzate were selected, manually scraped from the derivatized layer, dissolved in formic acid solution (0.1 % in acetonitrile – water 3:2), mixed with an equal volume of matrix (dihydroxybenzoic acid 2 % in acetonitrile – water 3:7), crystallized on air on a ground steel target, before being desorbed by the laser beam of the MALDI-TOF-MS/MS (matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry). Direct hyphenation of HPTLC to MS was not performed, to avoid zone diffusion during plate coating with the matrix and to circumvent the stronger binding of polar peptides on the layer.  The MS spectra were acquired in positive reflector mode in m/z range 340 – 4000 (10 – 2500 for fragments), using an external peptide as calibration standard. Identification of 51 from the 85 selected peptides according to AA sequences was performed, using software programs allowing m/z calculation of protein fragments and estimation of cleavage sites. Correlation of the retention behaviour of the peptides with their properties (molecular weight MW, isoelectric point IEP, charges, polarity) was tested with Student’s two-sided t-test after calculation of Pearson’s correlation coefficients. The correlation was significant with IEP, percentages of anionic AA and of non-polar AA; but not with the following properties: MW, percentages of cationic AA and of uncharged polar AA. Finally, based on the correlation results, regression formulas were found to calculate the x- and y-coordinates of any known peptide from the percentage of non-polar AA (or vice-versa). The prediction power of these formulas was verified by repeating the complete 2D-HPTLC-MS experiment with the standard peptides of whey and of peas, and measuring the absolute and relative deviations between the actual x- and y-coordinates and the predicted values. The absolute deviations were higher in the lower RF zones. The average, relative RF value deviations (range 22.1 – 25.7 %) were not different between whey and pea peptides.

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, 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, 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).

Braz. J. Pharm. Sci. 58, e18691 (2022). HPTLC of paracetamol (1), diclofenac sodium (2), ibuprofen (3), and indomethacin (4) in wastewater effluents on silica gel with n-hexane - ethyl acetate - acetic acid 12:7:1. Quantitative determination by absorbance measurement at 254 nm. The hRF values for (1) to (4) were 18, 56, 69 and 44, respectively. Linearity was between 0.1 and 0.9 µg/zone for (1) to (4). Inter-day and intra-day precisions were below 1 % (n=3). Mean recovery was 99.7 % for (1), 99.8 % for (2), 99.7 % for (3) and 99.2 % for (4). 

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, 1305-1316 (2022). HPTLC of tamsulosin hydrochloride (1) and solifenacin succinate (2) along with their impurities on silica gel with ethyl acetate - butanol - glacial acetic acid 100:4:1. Quantitative determination by absorbance measurement at 225 nm. The hRF values for (1) and (2) were 61 and 26, respectively. Linearity was between 0.1 and 1.0 µg/zone for (1) and 1.0 and 15.0 µg/zone for (2). Inter-day and intra-day precisions were below 2 % (n=6). Mean recovery was 99.9 % for (1) and 99.8 % for (2).