J Chromatogr A, 1611, 460602 (2020). Samples were methanolic root macerates of Euthamia graminifolia, Solidago canadensis, S. gigantea, S. rugosa and S. virgaurea (Asteraceae). HPTLC on silica gel with n-hexane – isopropyl acetate – acetone 16:3:1; or (for preparative TLC) on TLC silica gel with n-hexane – acetone 7:3, followed by scraping the layer and eluting with ethanol. When intended for MS experiments, layers were previously washed with methanol – water 4:1 and heated 20 min at 100 °C. Derivatization with vanillin – sulfuric acid reagent. Multivariate image analysis of the derivatized chromatograms allowed clear separation of samples according to species. Effect-directed analysis for: A) enzymatic inhibition by immersion into acetyl- and butyryl-cholinesterase, glucosidase and amylase solutions; B) activity against Gram-negative bacteria using Xanthomonas euvesicatoria chromogenic bioassay, and Aliivibrio fischeri and Pseudomonas syringae maculicola bioluminescence assays; C) activity against Gram-positive bacteria with Bacillus subtilis spizizenii bioassay. Two labdane diterpenes (solidagenone, hRF 47, and presolidagenone, hRF 55) in S. canadensis and two polyacetylenes (matricaria-esters = methyl-decadiene-diynoates, hRF 78 and 87 in HPTLC) in S. virgaurea were identified from multipotent zones by bioassay-guided purification through preparative TLC / HPLC, followed by HRMS and NMR, as well as by HPTLC hyphenated to quadrupole-orbitrap HRMS in 2 ways: A) by eluting with methanol the compounds from the plate through the oval elution head of a TLC-MS interface, with heated electro-spray ionization (HESI, spray voltage 3.5 kV, capillary temperature 270 °C, nitrogen as sheath and auxiliary gas, full scan in negative and positive ionization modes in m/z range 50-750); tandem mass spectra were acquired in parallel at fragmentation energy of 15-100 eV; B) without eluent with a DART interface (Direct Analysis in Real-Time, needle voltage 4 kV, grid voltage 50 V, helium as gas, temperature 500 °C, full scan in positive ionization mode in m/z range 100-750).

J Chromatogr A, 1609, 460438 (2020). HPTLC of defatted hydro-methanolic extract of Anarrhinum pubescens (= A. duriminium) aerial parts (Plantaginaceae) on silica gel with chloroform – methanol 9:2. When intended for MS experiments, layers were previously washed twice with methanol – water 4:1 and heated 20 min at 110 °C. Derivatization by automatic piezoelectric spraying of anisaldehyde sulfuric acid reagent, followed by heating 4 min at 105 °C. Effect-directed analysis for acetyl-cholinesterase (AChE) inhibitors was performed by successive piezoelectric sprayings with TRIS buffer, with AChE solution and (after 30 min incubation at 37 °C) with naphthyl acetate and Fast Blue salt B solution. White inhibiting zones on purple background were documented under white light, and densitometry was measured by scanning in fluorescence mode at 500 nm. One of the active bands was eluted from untreated layer with methanol through the oval elution head of a TLC-MS interface pump, into a quadrupole-Orbitrap mass spectrometer using heated electrospray ionization (HESI); a full scan mass spectrum (m/z 50−750) in the positive ionization mode was recorded, as well as HRMS/MS data across a range of collision energies (10–50 V). The compound was identified as foliamenthoyl-cinnamoyl-antirrhinoside. It was applied with two other active antirrhinosides (iridoids), all isolated from the extract through column chromatography, on an HPTLC layer without migration and submitted to AChE assay; their activity was expressed as equivalency towards rivastigmine tartrate as positive control. 

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 127, 13-15 (2021). HPTLC of rutin and quercetin in Ginkgo biloba (1), rosmarinic acid in rosemary (2) and oleuropein in olive oil (3) on silica gel with ethyl acetate - formic acid - acetic acid - water 100:11:11:26. Detection by spraying with NP reagent (1 g 2-aminoethyl diphenylborinate in 100 mL methanol) for (1) and (2) or with anisaldehyde reagent (0.5 mL p-anisaldehyde in 85 mL methanol, 10 mL acetic acid and 5 mL sulfuric acid), followed by heating at 100 °C for 3 min. Absorbance measurement at 254 nm and 238 nm for oleuropein and in fluorescence mode at 366>/400 nm for rosmarinic acid. The method showed the application of Quantified Reference Extracts for the identification of plant materials and quantification of markers by HPTLC.

J. Food Biochem. 44, e13137 (2020). HPTLC of kaempferol-3-O-rutinoside (1) and rutin (2) in Musa acuminata Colla on silica gel with ethyl acetate - toluene - formic acid - water 34:5:7:5. Detection by dipping into natural products reagent (0.5 % methanolic solution of 2-aminoethyl diphenylborinate), followed by dipping into a 5 % methanolic polyethylene glycol 400 solution. Qualitative identification under UV light at 366 nm. Radical scavenging activity was studied by dipping into a 0.2% methanolic DPPH* solution. The hRF values for (1) and (2) were 32 and 25, respectively. 

Journal Chromatogr A, 1641, 461727 (2021). HPTLc of an ethanolic maceration of Solidago gigantea roots (Asteraceae) on silica gel with n-hexane – isopropyl acetate – acetone 16:3:1, or n-hexane – isopropyl acetate – acetic acid 40:9:1. With the second mobile phase, acid residues had to be eliminated by 20 min automated drying or by 2 h incubation with potassium hydroxide in the opposite twin trough (followed by 15 min cold air streaming); this latter mobile phase allowed to obtain higher hRF values, but some butyrylcholinesterase (BChE) inhibiting activities were lost. The chromatograms were documented at UV 254 nm and 365 nm and white light before and after A) derivatization with vanillin – sulfuric acid reagent; B) enzymatic reaction by immersion into acetylcholinesterase, BChE, glucosidase and amylase solutions; C) Aliivibrio fischeri and Xanthomonas euvesicatoria bioassays, to detect activity against Gram-negative bacteria; D) Bacillus subtilis bioassay to detect activity against Gram-positive bacteria; E) a new antifungal assay with Fusarium avenaceum. For this assay, the chromatograms were immersed 6 s into the isolated mycelium suspension (diluted to OD600 0.4-0.8) and incubated in a vapor chamber at 21 °C for 48-72 h. Inhibition zones were indicated by the lack of visible white fungal hyphae. An aqueous solution of iodonitrotetrazolium (INT, 1 mg/ml) was sprayed on the plate to enhance the contrast (bright zones on a purple background). Benomyl (a benzimidazole fungicide) was used as positive control. Eight clerodane diterpenes (including kingidiol, hautriwaic lactone, and solidagoic acids A and B) were identified from six multipotent zones by bioassay-guided purification through preparative flash chromatography and HPLC, followed by HRMS and NMR, as well as by HPTLC hyphenated to quadrupole-orbitrap HRMS: A) by eluting with methanol (flow 100 µL/min) the compounds from the plate through the oval elution head of an interface of heated electro-spray ionization (spray voltage 3.5 kV, capillary temperature 270 °C, nitrogen as sheath and auxiliary gas, full scan in negative and positive ionization modes in m/z range 50-750); B) without eluent with a DART interface (Direct Analysis in Real-Time, needle voltage 4 kV, grid voltage 50 V, helium as gas, temperature 500 °C, full scan in positive ionization mode in m/z range 100-750).

J. Planar Chromatogr. 35, 161-167 (2022). HPTLC of catechin in the seeds of Parkia roxburghiion silica gel with ethyl acetate - acetic acid - formic acid - water 40:4:3:4. Quantitative determination by absorbance measurement at 302 nm. The hRF value for catechin was 61. Interday and intra-day precisions were below 1 % (n=3). The LOD and LOQ were 12 and 37 ng/zone, respectively. Recovery was found in the range of 99.1-99.5 %.

J. Planar Chromatogr. 35, 127-138 (2022). HPTLC of fresh fruit peels of some species of the genus Citrus L.: Citrus aurantium L. (bitter orange), C. unshiu Marc. (satsuma mandarin), C. reticulata Blanco (Robinson mandarin), C. sinensis L. (Washington Navel orange), C. limon Brum. (lemon), C. limetta (sweet lime), C. bergamia (bergamot), C. Medica (citron), C. paradisi Macf. (starruby grapefruit), C. maxima (pomelo) and C. maxima x C. paradisi (oroblanco) on silica gel with ethyl acetate - formic acid - acetic acid - water 100:11:11:26. Detection by dipping into a solution of NP (0.5% diphenylborinic acid aminoethyl ester in ethyl acetate), followed by dipping into a PEG (5 % PEG400 in dichloromethane). Tyrosinase inhibition analysis by dipping into 32 % ammonia, followed by drying and spraying with enzyme solution and incubation at room temperature for 10 min. DPPH radical scavenging activity by dipping into a solution of 0.05 % DPPH in methanol, followed by incubation for 30 min in darkness.