J Chromatogr A, 1616, 460774 (2020). Methanolic extracts of leaves of Musa acuminata, M. balbisiana and M. sapientum (Musaceae), either from fields or from in vitro cultures or from the plantlets derived from in vitro culture and acclimatized in isolated warm room, were separated on HPTLC silica gel layers with toluene – ethyl acetate – methanol 6:3:1 or ethyl acetate – toluene – formic acid – water 34:5:7:5. When intended for MS experiments, layers were previously washed twice with methanol – formic acid 10:1, once with acetonitrile – methanol 2:1 and air-dried. Evaluation under white light, UV 254 nm and 366 nm. Derivatization by immersion (2s, 2cm/s) into natural product reagent preceded by heating at 110 °C for 5 min, or into anisaldehyde sulfuric acid reagent, diphenylamine aniline reagent, ninhydrin reagent, followed by the same heating procedure. Besides, plates were neutralized by cold air stream followed with phosphate buffer (8 %, pH 7.5) piezoelectrically sprayed on the plates and automated plate drying. Thereafter, 9 effect-directed assays (EDA) were performed for free radical (DPPH•) scavengers, for enzymatic inhibitors (α-amylase, acetyl- and butyryl-cholinesterase, α- and β-glucosidase), for antimicrobial compounds (Gram-positive Bacillus subtilis assay, Gram-negative Aliivibrio fischeri bioluminescence assay), and for mutagenic compounds (SOS response – UMU-C test using Salmonella typhimurium suspension and 4-nitroquinoline 1-oxide as positive control). The bands of 4 active compounds were eluted with methanol through a TLC-MS interface pump into a quadrupole-Orbitrap mass spectrometer. Full scan mass spectra (m/z 50−800) in the positive and negative ionization modes were recorded using electrospray ionization (ESI, spray voltage 3.3kV, capillary temperature 320°C, collision energy 35 eV). By comparison to a standard, one band present in all samples was identified as linolenic acid. For the other bands, only present in in vitro grown accessions, only raw molecular formulas and phytochemical classes were assigned (a pyrrolidine alkaloid, an amino-acid, a phenolic derivative).

J Chromatogr A, 1637, 461836 (2021). Successive ultrasonic macerates of Ficus religiosa leaves (Moraceae) were separated with toluene – ethyl acetate – methanol 6:3:1 on HPTLC silica gel or (for yeast and genotoxicity assays) on RP18W phase. For MS experiments, layers were previously washed twice with methanol – formic acid 10:1, once with acetonitrile – methanol 2:1 and air-dried. Chromatograms were documented under white light, UV 254 nm and 366 nm. Afterwards, 11 derivatization assays were performed with the following reagents, either without heating: Dragendorff’s reagent; Fast Blue B salt; ferric chloride; natural product reagent - PEG 400; primuline; or requiring heating for 5 min at 120 °C: anisaldehyde sulfuric acid; diphenylamine aniline phosphoric acid; 2-naphthol sulfuric acid; ninhydrin; Tillmans' reagent; vanillin sulfuric acid. Besides, 12 effect-directed assays (EDA) were performed for free radical (DPPH• and ABTS•) scavengers, for enzyme inhibitors (α-amylase, acetyl- and butyryl-cholinesterase, α- and β-glucosidase, tyrosinase), for antimicrobial compounds (Gram-positive Bacillus subtilis assay, Gram-negative Aliivibrio fischeri bioluminescence assay), for phytoestrogens (planar yeast estrogen assay) and genotoxicity (SOS response – UMU-C test by successive immersions into citric buffer, into Salmonella typhimurium suspension and into methylumbelliferyl-galactopyranoside solution, followed by FLD at 366nm of mutagenic compounds as blue fluorescent zones, using 4-nitroquinoline 1-oxide as positive control). No activity was found for the last two assays. Ethyl acetate extracts of all samples were the most active. After EDA, most active bands were scanned for semi-quantitative equivalence densitometry at 546 nm using mercury lamp, compared to the following standards: acarbose, gallic acid, imidazole, kojic acid, physostigmine, tetracycline, depending on the assay. The bands of 3 multipotent compounds were eluted with methanol through the oval elution head and in-line filter frit of a TLC-MS interface pump, into a quadrupole-Orbitrap mass spectrometer. Full scan mass spectra (m/z 50−750) in the positive and negative ionization mode were recorded using heated electrospray ionization (HESI, spray voltage 3.5 kV, capillary temperature 270 °C, probe heater temperature 200 °C). MS-MS spectra were recorded in the negative mode using HCD-NCE (higher-energy collisional dissociation –normalized collision energy, with stepped negative collision energies from 10 to 40 eV). The three active zones were assigned to palmitic acid, to linolenic acid and to its di-oxygenated derivative.

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. Planar Chromatogr. 35, 153-159 (2022). HPTLC of glucuronic acid in gum samples of Sterculia urens on silica gel with 1-propanol - water 7:3. Detection by spraying with napthoresorcinol sulfuric acid reagent, followed by heating at 105 °C for 5 min. Quantitative determination by absorbance measurement at 580 nm. The hRF value for glucuronic acid was 43. Linearity was between 300 and 700 ng/zone. Interday and intra-day precisions were below 2 % (n=3). Recovery was between 100.4 and 102.3 %.

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).

Journal of Chromatography B, 1184, 122956 (2021). Test for acetyl- and butyrylcholinesterase (AChE and BChE) inhibition without development of piperin (standard inhibitor of AChE and BChE) and ethanol – water (3:2) extracts of Iranian plants, on HPTLC silica gel prewashed twice with methanol – water 3:2 and dried 60 min at 120°C. After sample application the plate was immersed (speed 3.5 cm/s, time 2 s) into enzyme solution (6.6 units/mL AChE or 3.3 units/mL BChE in TRIS buffer 0.05 M, with bovine serum albumin 0.1 %, pH 7.8), incubation 25 min at 37°C and immersion (speed 3.5 cm/s, time 1 s) into chromogenic substrate solution (α-naphthyl acetate 0.1 % and Fast Blue salt B 0.2 % in ethanol – water, 1:2). Seven mobile phases were tested for the active samples. Best separation was obtained with toluene – ethyl acetate – formic acid – water 4:16:3:2 and with toluene – ethyl acetate – methanol 6:3:1. Before enzymatic assay, plates developed with acidic mobile phases were neutralized by spraying 3 mL citrate phosphate buffer (Na2HPO4 8 %, citric acid q.s. ad pH 7.5) followed by 10 min of automatic drying. Enzymatic assay was performed using a piezoelectric spraying device: a) pre-wetting by spraying 1 mL TRIS buffer (0.05 M, pH 7.8); b) spraying 3 mL of the enzyme solution; c) incubation 25 min in a humid box at 37°C; d) spraying 0.5 mL substrate solution; e) 5 min drying at room temperature, and then 10 min of automatic drying. By spraying, zone shift and zone diffusion, which occurred with plate immersion, were avoided. For development control, derivatization was done by piezoelectrically spraying 4 mL of sulfuric anisaldehyde reagent (anisaldehyde – sulfuric acid – acetic acid – methanol, 1:10:20:170), followed by heating 3 min at 110°C. For identification of zones of interest, direct elution with methanol from underivatized HPTLC plates through a TLC-MS interface directly to a MS. Identified zones were 3-O-acetyl-β-boswellic acid (triterpenoid) from Boswellia carteri gum-resin (Burseraceae), pimpinellin and psoralen (furocoumarins) from Heracleum persicum flowers (Apiaceae), oleuropein (seco-iridoid) from Olea europaea leaves (Oleaceae), harmine, harmaline, vasicine, deoxyvasine (alkaloids) from Peganum harmala seeds (Zygophyllaceae), costic acid (sesquiterpene) from Nardostachys jatamansi hypocotyl (Valerianaceae), elaidic, linoleic, palmitic, palmitoleic acids (fatty acids) from Pistacia atlantica fruits (Anacardiaceae).

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.

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.