Anal Chim Acta 1183 (2021) 338956. Demonstration of first on-surface adherent cell assays by investigating growth and viability of modified adherent human osteosarcoma U2OS cells (Cytotox CALUX® cell system) as a biodetector for the cytotoxicity evaluation of e.g. mycotoxins, cytostatics and plant extracts, on HPTLC plates. In the planar bioassay, the cytotoxicity of sample extracts was measured with the luciferase activity in the Cytotox CALUX® reporter cells. For direct bioresponse testing on the HPTLC plate, the U2OS cell suspension was pipetted along the the sample track. RP-HPTLC-CALUX® bioassays of Saussurea costus and Ginseng extracts on HPTLC RP-18 W with n-hexane - toluene - tetrahydrofuran 25:7:15 to 6 cm. After drying, immersion in citrate phosphate buffer pH 12, and several drying and moistening steps the cytotoxic zones were detected by bioluminescence inhibition. Two different test procedures proved Cytotox CALUX® cell viability on surface and in microtiter plate assay. The isolated cytotoxic zone was analyzed by HPTLC on RP-18 W with n-hexane - toluene - tetrahydrofuran 10:1:1 and one part of the  chromatogram cut in half was derivatized with sulfuric acid reagent and evaluated under white light, while the other part was subjected to the new planar Cytotox-CALUX® bioassay. The cytotoxic zone had the same hRF as standards costunolide and dehydrocostus lactone.

Talanta 223 (2021) 121701. Development of workflow for a bioanalytical multi-imaging screening based on HPTLC-UV/Vis/FLD-EDA-HESI-HRMS and application to 54 bark, leaf and seed extracts of Sri Lankan Abelmoschus moschatus (abelmosk) to find out the most bioactive individual compounds. HPTLC on silica gel with toluene - ethyl acetate - methanol 6:5:2 or toluene - ethyl acetate 7:3 up to 65 mm, evaluation in (A) UV 254 nm, (B) white light, (C) fluorescence 366 nm, and after derivatization with (D) primuline reagent at UV 366 nm, (E) p-anisaldehyde sulfuric acid reagent, (F) vanillin sulfuric acid reagent, (G) p-aminobenzoic acid reagent, (H) the latter in fluorescence evaluation at 366 nm, (I) diphenylamine aniline orthophosphoric acid reagent, (J) ninhydrin reagent, (K) Fast Blue B salt reagent. For (E-K) detection in white light after heating at 140°C for 5 min, and (L) natural product reagent in fluorescence 366 nm after air-drying. For effect-directed profiling, HPTLC on plates prewashed with methanol - water 3:1, drying for 15 min and immersing in the respective assay solution/suspension or spraying with it, then incubating, drying and evaluation in white light (A) for Gram-negative Aliivibrio fischeri bioassay, spraying the bacterial culture onto the chromatogram and recording the instant bioluminescence over a 30 min period with the positive controls of caffeine; (B) for Gram-positive Bacillus subtilis bioassay, dipping the chromatogram in the bacterial suspension for incubation at 37°C for 2 h, then dipping into a 0.2 % PBS-buffered MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-trazolium bromide) solution with positive control tetracycline; (C) for α-glucosidase inhibition assay, spraying with substrate solution, drying, pre-wetting and incubation by spraying with Fast Blue B salt solution with positive control acarbose, absorbance measurement at 546 nm and quantification via peak area; (D) for β-glucosidase inhibition assay, analog to (C), but using β-glucosidase and 2-naphthyl-β-D-glucopyranoside and longer incubation time, with positive control imidazole; (E) for tyrosinase inhibition assay, spraying with substrate solution, after drying with tyrosinase solution, incubated and dried with positive control kojic acid, absorbance measurement at 579 nm; (F) for radical-scavenging assay, immersing the chromatogram into 0.02 % methanolic 2,2-diphenyl-1-picrylhydrazyl solution, air-dried with the positive control ascorbic acid. The workflow provided comprehensive information about multi-potent compounds and sample diversity, which is elementary for product quality control in the field of botanicals, foods and medicinal plants.

Talanta 219 (2020) 121306. Development of a workflow for compound characterization of coeluting compounds: employing an HPTLC-UV/Vis/FLD-EDA screening, followed by the characterization and identification of the most potent compounds by multi-imaging, heated electrospray ionization high-resolution mass spectrometry (HESI-HRMS) and hyphenated HPTLC-UV/Vis/FLD-HPLC-DAD-ESI-MS. HPTLC of methanolic Lemon balm leaf extract and standards oleanolic acid and ursolic acid on silica gel with n-hexane - ethyl acetate 7:3 up to 70 mm, drying for 2 min, evaluation in UV 254 nm (UV), UV 366 nm (FLD) and white light (Vis) after derivatization with a solution of vanillin (40 mg) and sulfuric acid (200 μL) in 10 mL ethanol and heating at 110°C for 5 min. Three additional chromatograms were prepared for the antibacterial assays (1) against B. subtilis, (2) A. fischeri and  (3) the α-glucosidase assay. (1) immersing in B. subtilis suspension, detectioon by immersion in aqueous 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide solution after incubation, followed by incubation until bright zones against a violet background appear, (2) immersing in Aliivibrio fischeri suspension and instantly monitoring in real-time for 30 min using 1 min exposure time in 5-min intervals and indicating the dark (or bright) active zones against the bioluminescent background, (3) immersing in α-glucosidase solution (10 units/mL) and 0.1 M sodium acetate buffer adjusted to pH 7.5 for incubation and immersing into substrate solution (1.2 mg/mL 2-naphthyl-α-D-glucopyranoside in ethanol) for further incubation at room temperature for 10 min and detection by immersion in aqueous Fast Blue Salt B solution (1 mg/mL) and drying, this revealed the enzyme inhibitors as bright zones against a violet background in white light. Online elution of zones of interest with methanol into the MS and full scan recording in the range of m/z 50–750 with a resolution of 280,000 in both negative and positive ionization modes. Expanding the HPLC-DAD-ESI-MS system by installing a TLC-MS interface enabling direct elution of HPTLC zones into the HPLC eluent.

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. Planar Chromatogr. 34, 447-453 (2021). HPTLC of gallic acid (1) and ellagic acid (2) in Triphala on silica gel with toluene - ethyl acetate - formic acid - methanol 15:15:5:1. Quantitative determination by absorbance measurement at 254 nm. The hRF values for (1) and (2) were 35 and 28, respectively. Linearity was between 200 and 2000 ng/zone for (1) and 50 and 400 ng/zone for (2). LOD and LOQ were 200 and 606 ng/zone for (1) and 50 and 151 ng/zone for (2), respectively. Intermediate precisions were below 2 % (n=3). Average recovery was 98.6 % for (1) and 99.1 % for (2).

J. Sep. Sci. 44, 3146-3157 (2021). HPTLC of  gallic acid (1), cinnamic acid (2), piperine (3), eugenol (4), and glycyrrhizin (5) in Divya-Swasari-Vati on silica gel with ethyl acetate - toluene - formic acid 10:9:1 for (1) to (4) and ethyl acetate - formic acid - acetic acid - water 100:10:10:23 for (5). Quantitative determination by absorbance measurement at 280 nm for (1), (2) and (4), 343 nm for (3) and 254 nm for (5). The hRF values for (1) to (5) were 31, 64, 53, 70 and 29, respectively. Linearity was between 400 and 800 µg/mL for (1), 5 and 25 µg/mL for (2), 600 and 1400 µg/mL for (3), 400 and 1200 µg/mL for (4) and 100 and 800 µg/mL for (5). Intermediate precision was below 2 % (n=18). The LOD and LOQ were 180 and 560 ng/g for (1), 3 and 10 ng/g for (2), 8 and 26 µg/g for (3), 3 and 11 µg/g for (4) and 300 and 920 ng/g for (5), respectively. Mean recovery was 96.0 % for (1), 92.3 % for (2), 94.2 % for (3), 96.2 % for (4) and 94.6 % for (5). 

J. Ethnopharmacol. 275, 114054 (2021). HPTLC of ellagic acid in Anogeissus latifolia on silica gel with toluene - ethyl acetate - formic acid 20:5:1. Detection by spraying with anisaldehyde - sulphuric acid reagent, followed by heating at 105 °C. The hRF value for ellagic acid was 38.

Chinese J. Food & Drug 23 (5), 411-415 (2021). Dashanzha tablet is a Chinese patent medicine with the function of appetizing and digestion, used for food accumulation caused by loss of appetite, indigestion, abdominal distension. For quality control, TLC of the petroleum ether extracts on silica gel with cyclohexane - methylene dichloride - ethyl acetate - glacial acetic acid 200:50:80:1. Detection by spraying with 10 % sulfuric acid in ethanol and heating at 105 ℃ until the zones are visible in daylight. Identification by fingerprint comparison with the standard ursolic acid and the standard ingredient drug undergone the same procedure in parallel. Satisfactory results were achieved by using plates from different manufactures and under varying temperature and humidity conditions.