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.

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 %.

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.

Food Chem. 375, 131824 (2022). HPTLC of triacyclglycerols (1), diglycerides (2), monoglycerides (3) and medium and long chain free fatty acids (4) in an alternative
functional food through bulk compound chocolate on silica gel with hexane - ethyl ether - acetic acid 80:20:1. Detection by spraying with 50 % sulfuric acid, following by heating at 150 °C for 10-15 min. Quantitative determination by absorbance measurement at 500 nm.

Food Chem. 357, 129135 (2021). HPTLC of cinnamon on silica gel with toluene - ethyl acetate - methanol 6:5:3. Nine detection modes were used: 1) white light illumination, 2) UV 366 nm, 3) UV 254 nm, and six different derivatization reagents applied by immersion: 4) primuline reagent (100 mg primuline, 20 mL water and 80 mL acetone), 5) p-anisaldehyde sulfuric acid reagent (1 mL methoxy benzaldehyde, 140 mL methanol, 16 mL acetic acid and 8 mL sulfuric acid), 6) vanillin sulfuric acid reagent (1 g vanillin, 80 mL ethanol and 0.8 mL sulfuric acid), 7) diphenylamine aniline o-phosphoric reagent (2 % each of diphenylamine and aniline in 100 mL isopropanol plus 20 mL o-phosphoric acid), 8) Fast Blue B salt reagent (100 mg Fast Blue B salt in 100 mL ethanol, 70 %) and 9) natural product reagent (1 g 2-aminoethyl diphenyl borate in 100 mL ethanol), followed by heating at 110 °C (5), 120 °C (4, 6) or 140 °C (7, 8) for 3-5 min. Effect-directed profiling was performed through eight different assays: HPTLC–Aliivibrio fischeri bioassay, HPTLC–Bacillus subtilis bioassay, HPTLC–tyrosinase inhibition assay and densitometric evaluation, HPTLC–α–glucosidase and β–glucosidase inhibition assays, HPTLC–AChE and BChE inhibition assays, HPTLC–DPPH assay. Compounds were further characterized by heated electrospray ionization high–resolution mass spectrometry (HESI–HRMS).

Anal. Bioanal. Chem. 412, 7441-7451 (2020). HPTLC of mono- (1) and diacylglycerol (2) emulsifiers (E 471) in whipping creams on primuline impregnated silica gel with n-pentane - n-hexane - diethyl ether 9:9:22. Quantitative determination by fluorescence measurement at UV 366/> 400 nm. The hRF values for (1) and (2) were 10 and 52, respectively. Linearity was between 1.5 and 20 ng/zone for (1) and (2). Intermediate precision was below 7 % (n=4). The LOD and LOQ were 1.8 and 5.7 ng for (1) and (2). Recovery was between 95 and 105 % for (1) and 86 and 95 % for (2).