J. Ethnopharmacol. 318, 116977 (2024). HPTLC of Oxalis corniculata on silica gel with chloroform - methanol - water 15:3:2. Detection by spraying with 2 % aluminium chloride. Qualitative analysis under UV light at 344 nm. Further analysis by mass spectrometry.

PLoS Neglected Tropical Diseases 17(09), e0011646 (2023). Samples were extracts rich in sphingolipids obtained from Trypanosoma cruzi epimastigotes, or from Leishmania major promastigotes (Trypanosomatidae), or from Chlorocebus sp. kidney Vero cells (Cercopithecidae), all cell lines incubated 2h before the extractions with ceramide N-hexanoyl-D-erythro-sphingosine coupled to fluorescent NBD-amine group (NBD = nitrobenzoxadiazolyle). Dried extracts were resuspended in chloroform – methanol (1:1) before application on TLC silica gel layers. Development with chloroform – methanol – potassium chloride 0.25 % aqueous solution 11:9:2. Visualization under automated laser scanner. Three sphingolipids were detected due to the NBD fluorescent group: sphingomyelin (hRF 42) was present in Vero cells only (negative control), whereas the targeted inositol-phosphorylceramide (IPC, hRF 70), was present in both L. major (positive control) and T. cruzi wild-type. It was absent in T. cruzi cell lines knock-out (KO) for the IPC-synthase (IPCS) gene, but present again in the add-back cell-lines (obtained with plasmide transfection of the IPCS gene into KO cells). An unknown lipid (hRF 78) was detected in all T. cruzi samples.

PLoS ONE 18(11), e0295012 (2023). TLC on silica gel to monitor the synthesis of 15 new camphene-based thiosemicarbazones produced by the reaction of camphene thiosemicarbazide either with benzaldehydes, or with acetophenones, or with one of the following molecules: benzophenone, cinnamic aldehyde, ethyl pyruvate, furaldehyde, menthone, pyrrole carboxaldehyde or thiophene-carboxaldehyde. Development with n-hexane – ethyl acetate 3:7 in the case of benzaldehydes, except vanillin; or 7:3 for the vanillin derivative and all others, followed by visualization of products with resublimated iodine. The aldehyde used for compound 15 is in fact vanillin.

J Chrom Sci, bmad055 (2022). Standards of antiglycemic drugs were metformin hydrochloride (S1, a biguanide), glibenclamide (S2 = glyburide, a sulfonylurea), pioglitazone hydrochloride (S3, a thiazolidinedione), repaglinide (S4, a glinide). Samples were methanolic solutions of commercial tablets of S1 with each of the other molecules. The following method was developed by a software-assisted AQbD approach (analytical quality by design): (1) Several TLC separations were tried with toluene together with other solvents and with acidic or basic modifiers, with also variations of 24 method or instrumental parameters. (2) Principal component analysis (PCA) was performed in order to identify two principal components (PCs) responsible for 98 % of the observed variations: namely, resolution and tailing factor. Three critical method parameters (CMPs) had a statistically significant impact on the PCs: mobile phase (MP) composition, ammonium acetate concentration in MP, and saturation time. (3) To optimize these CMPs, the Box–Behnken design was implemented in 15 software-proposed experiments; the impacts of the 3 CMPs on the 2 PCs were evaluated by ANOVA, multiple regression analysis, and 2D and 3D contour plots. (4) The optimal CMPs ranges were determined by defining a MODR (method operable design region) on the superposed contour plots, and one TLC condition was selected as analytical control point.
TLC on silica gel pre-washed with 10 mL methanol, dried and activated 10 min at 100° C. Separation with toluene – ethyl acetate – methanolic solution of 4 % ammonium acetate 7:7:6 after 15 min pre-saturation with 35 % relative humidity. Absorption emasurement at UV 254 nm. The hRF values were 13 for S1, 72 for S2, 82 for S3, 38 for S4. LOQ were 263, 387, 73 and 35 ng/zone, respectively. Linearity range was 25–75 µg/zone for S1, 100–300 ng/zone for S2 and S4, 750–2250 ng/zone for S3. Intermediate precision was below 2 %. For accuracy tests, recovery rates were between 97.6–101.4 %.

J Chrom Sci, bmad045 (2022). Standards were azilsartan medoxomil (AZL) and cilnidipine (CLN). Samples were acetonitrile solutions of commercial tablets of AZL and CLN, and purified human blood plasma as biological fluid spiked with AZL and CLN. The following method was developed by a software-assisted AQbD approach (analytical quality by design): (1) Taguchi orthogonal array design was implemented in 8 screening experiments in order to identify the 3 critical method variables (CMVs), which were: volume ratio of toluene – ethyl acetate, volume of methanol and saturation time. These CMVs had statistically significant impact (one-way ANOVA and Pareto charts) on the 3 critical analytical attributes (CAAs, they were: resolution between AZL and CLN and their hRF values). (2) To optimize these CMVs, the Box–Behnken design was implemented in 15 software-proposed experiments; the impacts of the 3 CMVs on the 3 CAAs were evaluated by ANOVA, multiple regression analysis, and 2D and 3D contour plots; the response surface analysis allowed the software to find a mathematical (quadratic or linear) equation for each CAA, based on the CMVs values. (3) The optimal CMVs ranges were determined by defining an analytical design space (ADS) on the superposed contour plots, and one TLC condition was selected as analytical control point.
TLC on silica gel pre-washed with 10 mL methanol, dried and activated 15 min at 110° C. Separation with toluene – ethyl acetate – methanol 13:3:4 after 15 min pre-saturation with 35 % relative humidity. Absorption measurement at UV 254 nm. The hRF values were 49–51 for AZL and 70–71 for LRT. Linearity range was 400–2000 ng/zone for AZL and 100–500 ng/zone for CLN. Intermediate precision was below 1.6 % (n=3). LOQ were 121 ng/zone for AZL and 34 ng/zone for CLN. Recovery rates were 99.3–99.7 % for AZL and 98.1–99.5 % for CLN. Recovery rates from spiked plasma were 83.3 % for both molecules.

J Chrom Sci, bmad025 (2023). Standards (separated and mixed) were montelukast sodium (MKT) and loratadine (LRT). Samples were methanolic solutions of commercial tablets, and purified blood plasma as biological fluid, from patients taking MKT or LRT as oral treatment. TLC on silica gel with ethyl acetate – ethanol 9:1. Visualization under UV 254nm. The hRF values were 80 for MKT and 71 for LRT. Densitometric absorbance measurement at 260 nm (20 mm/s scanning speed). System suitability was verified by resolution, selectivity, capacity and absence of tailing. The method was validated for linearity range (0.3–3.6 μg/zone for MKT, 0.2–4 µg/zone for LRT), for precision, for reproducibility, for robustness, and for accuracy expressed as average recovery values (100 % overall mean) at different concentrations. The TLC-densitometric method was also found statistically equivalent (Student’s t-test and F-test) to a previously described method (HPLC – spectrophotometry), but was better in terms of environmental and health impacts, using green analytical procedure index (GAPI) and analytical eco-scale (scores based on solvents/reagents, energy consumption, occupational hazard and waste generation). The TLC method was also compared to three (equally “green”) different analytical methods of spectrophotometry (without chromatography): response correlation, absorptivity-centering and LRT-MKT ratio derivatives. The TLC method was more sensitive (LOQ values were 82 ng/zone for MKT, 20 ng/zone for LRT).

J Chrom Sci, bmad042 (2023). Standards (separated and mixed) were antazoline (ANT) and tetryzoline (TET) hydrochlorides. Samples were one commercial ophthalmic solution containing both molecules (unspiked and spiked), and aqueous humour of untreated rabbits as biological fluid, spiked with various concentrations of ANT and TET. TLC on silica gel with ethyl acetate – ethanol 1:1. Visualization under UV 254 nm. Densitometric absorbance measurement at 220 nm (20mm/s scanning speed). The hRF was 47 for TET and 71 for ANT. System suitability was verified by resolution, selectivity, capacity and absence of tailing. The method was validated for linearity range (0.2 – 18 µg/band), for precision, for reproducibility, for robustness, and for accuracy expressed as average recovery values (100 % overall mean) at different concentrations. The method was also found statistically equivalent (Student’s t-test and F-test) to the official corresponding titrimetric methods of the European Pharmacopoeia. Finally, environmental and health impacts of the methods were qualitatively and quantitatively assessed better as the other described methods, using analytical greenness (AGREE), green analytical procedure index (GAPI), national environmental method index (NEMI), and analytical eco-scale (scores based on solvents/reagents, energy consumption, occupational hazard and waste generation).

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).