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.

Microbiol. Res. 267, 127260 (2023). HPTLC of lipids in a Glutamicum mutant ΔSYA, unable to synthesize trehalose constructed by deleting genes treS, treY and otsA in the three pathways of trehalose biosynthesis, on silica gel with chloroform - methanol - water - ammonia 65:25:1:3. Detection by spraying with 10 % sulfuric acid in methanol, followed by drying at 160 °C for 5 min. Further analysis by liquid chromatography mass spectrometry.

Microbiol. Res. 268, 127295 (2023). HPTLC of cardiolipin phospholipids in the deletion mutants of two cardiolipin synthetase genes, clsA (UM270 ΔclsA) and clsB (UM270 ΔclsB), in the rhizobacterium Pseudomonas fluorescens, on silica gel with chloroform - methanol - water 14:6:1 for the first dimension and chloroform - methanol - glacial acetic acid 13:5:2 for the second dimension. The lipid composition of UM270 wt, UM270 ΔclsA and UM270 ΔclsB mutant strains was determined by labeling with [1-¹⁴C] acetate. Detection by iodine staining and radioactive membrane lipids were visualized by exposure to autoradiography film or a Phosphor Imager screen. Individual lipids were quantified using an image software.

Trends Anal. Chem. 157, 116808 (2022). Review of recent advances in methods for the analysis of cardiolipin in different biological samples. The paper described analytical methods such as TLC and HPTLC for the study of cardiolipin abnormalities in neurological disorders, cancer, cardiovascular and metabolic diseases. Advantages and disadvantages of different techniques available to detect and quantify cardiolipin were also discussed.

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.

Food Chem. 334, 127562 (2021). HPTLC of five seaweed cultivars, namely three Saccharina japonica and two Undaria pinnatifida on silica gel with n-hexane - ethyl acetate - formic acid 30:50:1. Detection by spraying with anisaldehyde sulfuric acid reagent (1.5 mL of anisaldehyde was mixed with 210 mL of ethanol, 25 mL acetic acid and 13 mL conc. sulfuric acid), followed by heating at 120 °C for 3 min. Qualitative identification under UV light at 366 nm. HPTLC-bioautography antimicrobial assays by dipping into B. subtilis cell suspension, followed by incubation at 37 °C for 30 min and E. coli suspension, followed by incubation at 37 °C for 1 h. Visualization by dipping into a solution of MTT dye with triton X-100 (1 mg/mL). Stearidonic, eicosapentaenoic, and arachidonic acids were identified by HPLC-MS.

J Chromatogr A, 1640, 461929 (2021). Study of the impact of different strains, culture media and parameters (temperature, time, rotational speed, and glucide and amino-acid supply) on the metabolite profile of bacteria. Samples were cultivation broths of Bacillus subtilis, B. licheniformis, B. pumilus and B. amyloliquefaciens, as well as their respective supernatant liquid-liquid extracts (apolar solvents only or QuEChERS method with acetonitrile and MgSO4 – NaCl mixture 4:1). HPTLC on silica gel (normal phase and RP-18), either as bands (for small volumes of extracts) or as areas for supernatants and bigger volumes of extracts. Extract areas were focused with a three-step procedure (up to 20mm with acetone, and twice with methanol); unextracted supernatants were focused twice with methanol and once with tetrahydrofuran, but the application zone of the plate had to be cut before development, due to the high matrix load. Development with ethyl acetate – methanol – water at different ratios after activation of the plate surface with magnesium chloride (33% relative humidity), evaluation in white light and UV. Detection of antibacterial compounds with Aliivibrio fischeri bioassay. Derivatization with primuline (for lipophilic substances) and diphenylamine aniline sulfuric acid reagent (for saccharides). This method allowed a fast comparison: A) of the patterns of the different strains (presence /absence and intensity of detected or antibacterial bands); B) of cultivation parameters: the number of metabolites increased with time, rotational speed (oxygen level), and at 37°C (vs. 30°C), whereas a minimal medium allowed the detection of more metabolites, due to the lower matrix load; C) of the impact of the extraction parameters: choice of the solvents (QuEChERS method had no advantage here), solvent – supernatant ratio (1:3 showed richer patterns than 1:1); D) of the HPTLC parameters used (better separation and resolution with normal phase vs. RP18 layers).

Nature - Lab. Invest. 100, 1411–1424 (2020). Samples were chloroform – methanol 1:1 solutions of lipid standards and of liver tissue extracts from wild-type mice (1), from transgenic murine models of hepatic steatosis (2) (mice expressing HBs, hepatitis B virus surface protein), or of cholestasis (3) (mice totally knock-out for the gene of phospholipid translocator ABCB4, ATP-binding cassette subfamily B member 4), or of both (4) (hybrids of mice (2) and (3)). HPTLC on silica gel (preheated at 110°C for 15 min) with n-hexane – diethyl ether – acetic acid 20:5:1. (A) For qualitative analysis, visualization under white light after immersion into anisaldehyde 0.5 % (in sulfuric acid – acetic acid – methanol, 1:2:17), followed by heating at 110°C for 9 min. (B) Identification of lipids was confirmed by elution of the zones of interest with methanol from the HPTLC layer through a TLC-MS interface and a filter frit directly to a quadrupole-orbitrap MS (atmospheric pressure chemical ionization, full HR-MS scan in m/z range 100–1000). (C) For quantitative analysis, visualization at UV 366 nm after derivatization by immersion into primuline reagent (primuline 0.5 g/L in acetone – water 4:1); fluorescence was measured at UV 366 nm (mercury lamp, optical filter for wavelengths above 400 nm, scanning slit 6.0 mm × 0.2 mm, speed 20 mm/s). (A) and (B) allowed the separation and detection of cholesterol, cholesteryl oleate, methyl oleate, free fatty acids (FFA, expressed as oleic acid equivalents) and triacylglycerols (TAG, as triolein equivalents) in liver extracts. (C) showed that TAG was decreased and FFA increased in (3) and (4), compared to (1) and (2). Cholesterol and cholesteryl oleate had no significant changes between groups.