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 Chromatogr A, 1647, 462154 (2021). Hydromethanolic extracts of Cinnamomum verum and C. cassia (Lauraceae) were separated on MS-grade HPTLC silica gel (prewashed twice with methanol – water 4:1 and dried at 110 °C for 20 min) with toluene – ethyl acetate – methanol 6:3:1. Residual organic solvent was removed by drying under automated cold stream air for 20 min. Chromatograms were documented under white light, UV 254 nm and for fluorescence detection at 366 nm, and afterwards submitted to Aliivibrio fischeri bioassay: 2 mL of bacterial suspension were piezoelectrically sprayed on the plate and bioluminescence was measured every 3 min for 30 min (120 s exposure time). For the first time, analytes from a bioactive zone, isolated by the oval elution head of a TLC-MS interface pump, were trapped from the highly salted layer by a heart-cut elution (45 s, flow rate 0.1 mL/min) through a biocompatible in-line filter to different on-line desalting devices. Using a two-position switching valve, the desalted analytes were guided to a reverse-phase UPLC column and separated at 40 °C using a fast gradient (ca. 13 min, 0.6 mL/min) with methanol (from 2 - 90 %) and an ammonium acetate solution (2.5 mM, pH 4.5 adjusted with acetic acid). After HPLC separation, analytes were detected by photodiode array (PDA) and then ESI-MS in polarity switching mode (cone voltage of 10 V, ESI probe at 600 °C, ESI source at 120 °C). Identified active compounds were cinnamic acid, coumarin, as well as the in HPTLC coeluting cinnamaldehyde and 2-methoxycinnamaldehyde. Separately, proof-of-concept tests were also made for more polar phenolic acids (gallic, chlorogenic, caffeic, cinnamic, ferulic and coumaric acids) but without HPTLC separation.

J Chromatogr A, 1647, 462154 (2021). Hydromethanolic extracts of Cinnamomum verum and C. cassia (Lauraceae) were separated on MS-grade HPTLC silica gel (prewashed twice with methanol – water 4:1 and dried at 110 °C for 20 min) with toluene – ethyl acetate – methanol 6:3:1. Residual organic solvent was removed by drying under automated cold stream air for 20 min. Chromatograms were documented under white light, UV 254 nm and for fluorescence detection at 366 nm, and afterwards submitted to Aliivibrio fischeri bioassay: 2 mL of bacterial suspension were piezoelectrically sprayed on the plate and bioluminescence was measured every 3 min for 30 min (120 s exposure time). For the first time, analytes from a bioactive zone, isolated by the oval elution head of a TLC-MS interface pump, were trapped from the highly salted layer by a heart-cut elution (45 s, flow rate 0.1 mL/min) through a biocompatible in-line filter to different on-line desalting devices. Using a two-position switching valve, the desalted analytes were guided to a reverse-phase UPLC column and separated at 40 °C using a fast gradient (ca. 13 min, 0.6 mL/min) with methanol (from 2 - 90 %) and an ammonium acetate solution (2.5 mM, pH 4.5 adjusted with acetic acid). After HPLC separation, analytes were detected by photodiode array (PDA) and then ESI-MS in polarity switching mode (cone voltage of 10 V, ESI probe at 600 °C, ESI source at 120 °C). Identified active compounds were cinnamic acid, coumarin, as well as the in HPTLC coeluting cinnamaldehyde and 2-methoxycinnamaldehyde. Separately, proof-of-concept tests were also made for more polar phenolic acids (gallic, chlorogenic, caffeic, cinnamic, ferulic and coumaric acids) but without HPTLC separation.

CBS 125. HPTLC of estrogen-active compounds in a migrate of a food contact material on silica gel with chloroform - acetone - petroleum ether 11:4:5. Planar Yeast Estrogen Test (P-YES) was performed by spraying 2 mL yeast cells onto HPTLC plates, followed by incubation at 30 °C for 3 h and spraying with 2 mL 0.5 mg/mL 4-methylumbelliferyl-beta-D-galactopyranoside and incubation for 20 min at 37 °C. The method was compared with microtiter bioassay (L-YES). P-YES was more sensitive than L-YES and results can be repeated upto one year later.

Sens. Actuators B Chem. 299, 126902 (2019). Thin layer chromatography combined with surface-enhanced Raman spectroscopy (TLC-SERS) of melamine in milk on silica gel with acetone - chloroform - ammonia 14:1:4. 2 μL gold nanoparticles were drop casted onto the analyte spot, followed by measurement using a  Raman spectrometer equipped with a diode laser emitting at 785 nm wavelength for illumination over a 100 μm diameter was used to obtain the SERS signals. A parallel representation model of the triple-spectral data was constructed using a pure quaternion matrix. Quaternion principal component analysis (QPCA) was utilized to build a quantitative model using support vector regression (SVR) algorithm. The method allowed the determination of melamine-contaminated milk with concentrations from 1 to 250 ppm.

Chemosphere. 261, 127706 (2020). HPTLC of water samples from four riverbank filtration sites in Germany on silica gel with a gradient development consisting of methanol - formic acid 2000:1 (v/v), dichloromethane and n-hexane in 16 steps. Effect-directed analysis using four bioassays. Baseline toxicity was detected via inhibition of natural bioluminescence of Aliivibrio fischeri (AF). In Bacillus subtilis (BS) assay, metabolic activity of BS cells was detected via the yellow substrate 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT). Neurotoxic effects was analyzed by AChE assay, spraying the substrate indoxyl-acetate onto enzyme-incubated HPTLC plates. In YES assay, estrogenic effects are detected via Saccharomyces cerevisiae BJ3505 strain. 

Front. Pharmacol. 12, 755941 (2021). High-throughput eight-dimensional (8D) hyphenation of normal-phase HPTLC with multi-imaging by ultraviolet, visible and fluorescence light detection as well as effect-directed assay and heart-cut of the bioactive zone to orthogonal reversed-phase high-performance liquid chromatography-photodiode array detection-heated electrospray ionization mass spectrometry. The method allowed the analysis of 68 powdered plant extracts (botanicals) which are added to food products in food industry and the study of antibacterials, estrogens, antiestrogens, androgens, and antiandrogens, as well as acetylcholinesterase, butyrylcholinesterase, α-amylase, α-glucosidase, β-glucosidase, β-glucuronidase, and tyrosinase inhibitors in an array of 1,292 profiles.