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).

Chinese Medicine 15, 76 (2020). This review compared the 2020 editions of Chinese (ChP) and European Pharmacopoeas (EuP) in different aspects of quality control of traditional Chinese medicinal plants (73 of which drugs were common to both, but with differences in species or organs for 17 of them). Discussed points included history, identification, plant origin and processing, sample preparation, marker selection, tests and assays, as well as advanced analytical techniques for quality control and for the establishment of comprehensive quality standard. TLC was discussed in relation to its following aspects: purposes, markers/references, techniques and result description.
(A) The main uses of TLC and HPTLC were (1) chemical-based identification of the plant in a more accurate and precise method than by macroscopic and microscopic observation only, and in a more direct and easily interpretation than HPLC, and allowing the simultaneous analysis of multiple samples in parallel; (2) control of possible adulterants; (3) quantification of active compounds. Both uses (1) and (2) were combined in some EuP monographs: as example were given the roots of Angelica dahurica, A. pubescens, A. sinensis, using TLC for identification of the species and of adulterants from other species (Angelica, Levisticum and Ligusticum).
(B) In ChP, identification through TLC was in most cases achieved by fingerprint comparison to an official reference extract or herb (herbal reference substance). At the opposite, EuP often indicated analytical markers, irrespective of any pharmacological activity, but chosen only for analytical purposes in TCM identification and quantification. Examples were: aescin and arbutin as analytical markers for TLC identification of Anemarrhena asphodeloides rhizome and Panax notoginseng root.
For the TLC system suitability assessment tests, ChP used the same intensity markers or active markers that were chosen for the identification or assay; whereas EuP often used other specific references, e.g. isoeugenol and methyleugenol in the case of Ophiopogon japonicus roots.
(C) For the techniques, conventional separations and chemical derivatizations were used. Hyphenations of TLC to other analytical methods (e.g. MS) were absent. Only one monograph applied an effect-directed analysis directly on TLC chromatogram (free DPPH• radical scavenging assay for TLC identification of Rehmannia glutinosa root, in ChP).
Sometimes, the TLC methods were different between both reference books for the same species. Example was given for Belamcanda chinensis (=Iris domestica) rhizome: in EuP, development on silica gel with cyclohexane – ethyl acetate – acetic acid 20:80:1, detection under UV 254 nm, comparison to standards coumarin and irisflorentin; whereas in ChP, development on polyamide layer with chloroform – butanone – methanol (3:1:1), detection under UV 365nm after derivatization with aluminium chloride, comparison to a reference rhizome powder.
 (D) Finally, the results in ChP were described as a text stating the similarity of sample profile with the profile of the chosen reference, whereas the results in EuP were described with a schematic box indicating the positions of bands of interest.

Chinese Medicine 10, 13 (2015). Samples were root and rhizome extracts of Panax notoginseng (Araliaceae), either raw or in the form of commercial granules. Standards were ginsenosides Rg1, Rb1, Rd, Re and Rg2, notoginsenoside NR1. TLC on silica gel with chloroform – ethyl acetate – methanol – water 15:40:22:9, followed by 10 min air drying. Derivatization for ginsenosides by immersion into sulfuric acid (10 % in ice cold methanol), followed by 10 min air drying and 5 min heating at 100 °C. Quantification by densitometric fluorescence measurement (deuterium and tungstene lamp, 366 nm). For each standard the linear range was 0.05-1 mg/mL (LOQ comprised between 38 and 431 µg/µL). As NR1 and Re (ratio ca. 2:1) had almost the same hRF, they were quantified together as one substance. Multivariate analysis through hierarchical (HCA) and principal component analyses (PCA) was used to order the samples into two clusters, according to the analyte concentrations, the raw plant extracts being richer than most of the commercial products. This TLC method was compared to quantification through UPLC-PDA (Ultra-performance liquid chromatography with photo diode array), which was more sensitive (LOQ between 10 and 49 µg/µL) but did not allow the separation between Rg1 and Re (ratio ca. 6:1).

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).

J Chromatogr A, 1619, 460945 (2020). Samples were lamotrigin as standard, or extracted with an oil-in-water microemulsion (10 µL butyl acetate, 4 mL n-butanol, 925 mg sodium dodecyl sulphate, 8.6 mL water) either from patients’ raw plasma (for separation from blood proteins) after spiking, or from commercial tablets dissolved in methanol. TLC on silica gel with a water-in-oil microemulsion of 9 mL butyl acetate, 1 mL n-butanol, 250 mg sodium dodecyl sulphate, 250 µL water. Both optimal microemulsions were predicted using Taguchi orthogonal array and Plackett-Burman design. Evaluation in UV 254 nm, quantification from the digital picture using four image processing software programs. For lamotrigin (hRF 24), limits of quantification were 170 ng for pure drug and 10 ng for spiked plasma. Linearity (in range 20–200 ng/spot) was directly obtained for the calibration curve in spiked plasma; however, for pure drug, linearity was obtained only when using log values of the calculated densities (300–3000 ng/spot).

J Chromatogr A, 1621, 461044 (2020). Samples were five isoquinoline alkaloids (berberine, chelerythrine, chelidonine, coptisine, sanguinarine) either as standard mixture or present in a Chelidonium majus (Papaveraceae) herb extract obtained with HCl 0.05 M in methanol. Separation on TLC and HPTLC silica gel layers with a screening of mobile phases consisting of eutectic mixtures of chemicals and/or phytochemicals. These homogenous stable liquids called DES (deep eutectic solvents) were obtained either simply by mixing, or by mixing followed by heating at 50°C, or by mixing with water for dissolution followed by dehydratation through rotary evaporation. For polarity adjustment, the DES phases were tested pure or diluted with acetone, chloroform, diethyl ether, methanol, or water. Visualization under UV 366 nm. The best separation was obtained with menthol – phenol in equimolar mixture, with 35 % methanol added (hRF values of the selected alkaloids were 33, 39, 79, 20 and 52, respectively).