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, 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).

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.

Braz. J. Biol. 45, e13764 (2021). HPTLC of filamentous fungi (Endomelanconiopsis endophytica, Myxospora musae, Neopestalotiopsis cubana and Fusarium pseudocircinatum) isolated from the digestive tract of Phylloicus sp, on silica gel with acetone - chloroform 5:3 and acetone - petroleum ether 5:2. Detection with iodine vapor, ferric chloride solution, sulfuric vanillin solution, green bromocresol solution and chromic acid solution. Qualitative identification under UV light at 254 and 366 nm. 

Anal. Bioanal. Chem. 414, 5991-6001 (2022). 2D-HPTLC fingerprint of Alpinia officinarum on silica gel in 384-well microplate array format (4.5 × 4.5 mm) matrix with trichloromethane - methanol - petroleum ether 97:3:25 in the first dimension and ethyl acetate - petroleum ether - acetic acid 15:35:1 in the second dimension. The paired chromatographic-based microassay array with the consistent chromatographic distribution was prepared by transferring a portion of the sample from the stock chromatographic-based microassay arrays to the corresponding array units of another 384-well microplate. A G‑quadruplex ligand bioassay was used to evaluate the ligand activity of the components in each array unit of the chromatographic-based microassay array. Further analysis by mass spectrometry. 

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.