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, 103-116 (2022). HPTLC of Daqinjiao standard decoction containing the following herbs: Gentianae Macrophyllae Radix (1), Paeoniae Radix Alba (2), Glycyrrhizae Radix et Rhizoma (3), Notopterygii Rhizoma et Radix (4), Saposhnikoviae Radix (5), Angelicae Pubescentis Radix (6), Angelicae Sinensis Radix and Chuanxiong Rhizoma (7), Scutellariae Radix (8) on silica gel with ethyl acetate - methanol - water 15:2:1 for (1) to (5), n-hexane - ethyl acetate 7:3 for (6) and (7) and toluene - ethyl acetate - methanol - formic acid 15:5:1.5:3 for (8). Detection by spraying with 5 % solution of vanillin in sulfuric acid, followed by heating at 105 °C for 5 min for (1) to (3), (5) and (6) and spraying with 2 % iron trichloride for (8). Qualitative identification under UV light at 254 and 366 nm. Reference standards were gentiopicroside for (1), paeoniflorin for (2), liquiritin for (3), nodakenin for (4), 5-O-methylvisammioside for (5), osthole for (6), ligustilide for (7) and baicalein and wogonin for (8). The hRF values for reference standards for (1) to (8) were 40, 35, 46, 28, 24, 37, 65 and 56, respectively.  

J. Planar Chromatogr. 35, 61-71 (2022). HPTLC of empagliflozin (1) in the presence of metformin HCl (2) on silica gel with toluene - methanol - ammonia - glacial acetic acid 72:26:1:1. Quantitative determination by absorbance measurement at 230 nm. The hRF values for (1) and (2) were 15 and 48, respectively. Linearity was between 11 and 112 ng/zone for (1) and 85 and 850 ng/zone for (2). Interday and intra-day precisions were below 2 % (n=3). The LOD and LOQ were 0.6 and 1,7 ng/zone for (1) and 5 and 15 ng/zone for (2), respectively. Recovery was found to be in the range of 99.4-101.0 % for (1) and 100.3-101.4 % for (2). Degradation products were characterized by liquid chromatography coupled with electrospray ionization‒quadrupole-time of flight‒tandem mass spectrometry (LC‒ESI‒QTOF‒MS/MS).

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).

J Chromatogr. A, 1652, 462377 (2021). Samples were vanilla tinctures, water − ethanol − ethyl acetate 1:1:1 extracts of vanilla-flavored food products and of natural Vanilla sp. (Orchidaceae) pods, oleoresin, paste and powders, as well as calibration standards of vanillin (1) and ethylvanillin (2). HPTLC on silica gel with n-hexane – ethyl acetate 1:1 for profiling, 3:2 for quantification. Other mobile phases were also tested and given in the supplement. Compounds (1) and (2) (hRF 68 and 82, respectively) were quantified by absorbance densitometry (at maximal wavelength 310 nm, deuterium lamp, scanning speed 10mm/s). Contents were found to be between 1 μg/g and 36 mg/g for (1) and null for (2) except in one tincture (62 µg/mL). Derivatizations performed for five assays: A) to detect radical scavengers, immersion (speed 3 cm/s, time 5 s) into DPPH• (0.5 mM in methanol), followed by drying for 90 s at room temperature and 30 s at 60 °C; B) to detect activity against Gram-negative bacteria, immersion (speed 2 cm/s, time 3 s) into Aliivibrio fischeri suspension, followed by recording the bioluminescence; C) to detect activity against Gram-positive bacteria, immersion (speed 3.5 cm/s, time 6 s) into Bacillus subtilis, followed by incubation 2 h at 37 °C, immersion in MTT solution, incubation for 30 min at 37 °C and heating for 5 min at 50 °C; D) to detect acetylcholinesterase (AChE) inhibitors, immersion (speed 2.5 cm/s, time 2 s) into AChE solution (666 units in TRIS buffer 0.05M, with bovine serum albumin 0.1 %, pH 7.8), incubation for 25 min at 37 °C and immersion into substrate solution (α-naphthyl acetate 0.1 % and Fast Blue salt B 0.18 % in ethanol – water, 1:2; E) to detect tyrosinase inhibitors, spraying with enzyme solution (400 unit/mL, in phosphate buffer 0.02 M, pH 6.8), followed by 2 min drying, immersion into substrate levodopa (18 mM in phosphate buffer, pH 6.8), 10 min incubation at room temperature and drying. For identification, zones of interest were transferred with methanol from underivatized HPTLC layer through a TLC-MS interface and a filter frit directly to a Quadrupole-Orbitrap MS (heated electrospray ionization, probe heater at 270°C, spray voltage 3.5kV, lock masses acetic acid for negative, dibutyl phthalate for positive ionization, mode full HR-MS scan in m/z range 50–750). Afterwards, the following substances assigned by MS were confirmed by using HPTLC comparison with standards: (1) and (2), vanillyl alcohol, vanillic acid, ethyl vanillyl ether, coumarin, 4-hydroxybenzoic acid, 4-methoxybenzoic acid, 4-hydroxybenzaldehyde, 4-allyl benzoic acid, oleamide, triacetin.

J. Planar Chromatogr. 34, 217-228 (2021). HPTLC of cordifolioside A (1), 20‑β‑hydroxyecdysone (2) and columbin (3) in stems of Tinospora cordifolia on silica gel with hexane - chloroform - methanol - formic acid 40:40:20:1. Detection by spraying with anisaldehyde‒sulfuric acid reagent. Quantitative determination by absorbance measurement at 254 nm for (1) and (2) and 600 nm for (3). The hRF values for (1) to (3) were 12, 43 and 85, respectively. Linearity was between 750 and 2250 ng/zone for (1) and (2) and 675 and 1875 ng/zone for (3). Intermediate precisions were below 2 % (n=3). The LOD and LOQ were 107 and 324 ng/zone for (1), 41 and 123 ng/zone for (2) and 54 and 163 ng/zone for (3). Recovery was between 99.0 and 101.4 % for (1), 98.1 and 101.6 % for (2) and 98.1 and 98.8 % for (3). Further analysis was performed by electrospray ionization‒tandem mass spectrometry.

Pharmacogn. Mag. 17, 233-239 (2021). HPTLC of esculin in the leaves of Launaea pinnatifida on silica gel with chloroform - methanol - formic acid 13:6:1. Quantitative determination by absorbance measurement at 343 nm. The hRF value of esculin was 70. Linearity was between 8 and 250 µg/mL. The LOD and LOQ were 8 and 25 µg/zone. Average recovery rate was 99.8 %.

J. Ethnopharmacol. 276, 114144 (2021). Review of analytical methods for the analysis of the main secondary metabolites detected and isolated from Ammi majus. The paper described TLC and HPTLC methods for the determination of coumarins in fruits.