Anal Chim Acta 1087 (2019) 131-139. Presentation of versatile devices for quantitative inkjet application on self-printed, binder-free HPTLC layers for submicromole-scaled analytical 1H NMR spectroscopy, providing the freedom to influence the composition of the slurry and directly access to control the printed quantity of HPTLC silica gel adsorbent to be constant, thus pledging the quality of the layer. To make spectroscopy reveal the cleanest proton spectra with the lowest background signals and most pronounced analyte signals, and to enable the identification of a compound from one 80-mm band on a single HPTLC layer by a 1H NMR spectrum in the full spectral range, the plate quality was improved by pre-developing silica gel plates twice with formic acid - methanol 1:10, then once with acetonitrile -  methanol 2:1. Silica gel particles for self-printed plates were puryfied under solvent pressure using a HPLC pump. For HPTLC,  the homogeneous slurry prepared by stirring 5 g cleaned-up/pre-eluted silica gel particles in 15 mL water - 2-propanol 2:1 was printed on 10 cm x 10 cm glass plates purified with 2-propanol and methanol, and the wet layers were heated to 80°C until dry. Inkjet printing of the solutions as 80-mm band and sample application using a 3-hydroxy-2-naphthoic acid solution (10 mg/mL water – methanol 1 : 9), spraying a methanolic 3-hydroxy-2-naphthoic acid solution (5 mg/mL) as 80-mm band (20mL/band) on the HPTLC plate. After plate drying and chamber pre-conditioning for 7 min with 2 N ammonia solution (10 mL), development with methanol - ethyl acetate - toluene 2:7:1 (5 mL). The deuterated methanol solution of the samples extracted from the analyte area scraped off the analytical plate was measured by NMR with the methanol signal as reference, documentation at UV 366 nm and data evaluation with an open-source video densitometry software (quan TLC). The results showed that HPTLC separated zones had better resolution and less matrix interference with the NMR analyte signal, and thus opened the avenue for submicromole-scaled analytical 1H NMR spectroscopy, which allows a faster structure elucidation of unknown compounds and easier signal interpretation.

Anal Chim Acta 1129 (2020) 76-84. Demonstration of a new hyphenated HPTLC-S9-AChE assay for detection of neurotoxic chemicals, including metabolic activation, at levels consistent with the threshold of toxicological concern (TTC) for organophosphates (OPs). The high sensitivity allows for the direct application of packaging migrates or extracts on the HPTLC plate without additional requirement steps. HPTLC of the ethanolic standards chlorpyrifos (CP), quinalphos (QP), eserine (ER), parathion (PT), nonylphenol (NP) and tris(nonylphenyl) phosphite (TNPP) at five different levels in the range of 0.5-10 mL/band (overall range for all six chemicals 0.1-1000 ng/band) on silica gel. After application, drying and pre-wetting the application bands by immersion up to 10 mm in water, then spraying the S9 mixture (7 mL each, 0.1 mg/mL) immediately on top of the start zones, followed by incubation at room temperature for 30 min. After drying plates for 20 min and 5 min chamber saturation, development with ethyl acetate - methanol - water 5:5:2 for ER; n-hexane - ethyl acetate - ethanol 16:3:1 for QP and PT; n-hexane - toluene - ethyl acetate 5:4:1 for CP, NP and TNPP. Evaluation in 254 nm, 366 nm and white light. Detection by immersion in AChE solution (6.6 U/mL plus 1 mg/mL bovine serum albumin in Tris-HCl buffer, 0.05 M, pH7.8) for 2 s, and incubating at 37 °C for 25 min, then immersing into the substrate-chromogenic solution (ethanolic solution of 1-naphthyl acetate  - aqueous solution of Fast Blue B salt 1:2 , 3 mg/mL each) for 1 s, drying for 10 min. Evaluation in white light, absorbance measurement at 546 nm (mercury lamp) using an inverse scan. The advantages of this straightforward workflow were demonstrated by comparison with the status quo microtiter plate assay. The method is a pragmatic new tool in risk assessment in general and can be transferred to further toxicities of interest and any other category of complex sample mixtures.

Anal Chim Acta 1125 (2020) 288-298. Development and validation of a reversed phase HPTLC planar yeast ant-/agonistic androgen screen (RP-HPTLC-pYAAS bioassay), as the basis for the creation of the new imaging concept for discovery and differentiation of agonistic and antagonistic ingredients in everyday products. HPTLC of parabens in cosmetics, additives in thermal papers and standards testosterone (T), dihydrotestosterone (DHT), nandrolone (ND) and trenbolone (TB) on pretreated RP-18 W layers, with methanol - water 1:1 for parabens in cosmetics, n-hexane - ethanol - ethyl acetate 40:3:3 for additives in thermal papers and with n-hexane - tetrahydrofuran - ethyl acetate 6:4:1 for androgens T, DHT, ND and TB. Immersing the developed HPTLC plates in citrate buffer for neutralization, followed by drying. For RP-HPTLC-pYAS bioassay, immersing in or spraying  with yeast cell suspension and incubating in horizontal position for 4 h at 30°Cand 100 % humidity, and after drying, spraying with or immersing in substrate solution of 4-methylumbelliferyl-b-D-galactopyranoside (MUG). Evaluation at UV 366 nm and by fluorescence measurement at 366/>400 nm. Determination of the limits of biological detection (LOBD, S/N 3) and quantification (LOBQ, S/N 10) via peak area signal-to-noise ratio and calculated by linear interpolation. For RP-HPTLC-pYES bioassay the same workflow as for RP-HPTLC-pYAS was used, except for incubation. For pYAAS bioassay, overspraying the antagonists bisphenol A (BPA), 4-n-nonylphenol (NP), Methyl 4-hydroxybenzoate (ME), ethyl 4-hydroxybenzoate (EE), propyl 4-hydroxybenzoate (PE), butyl 4-hydroxybenzoate (BE) and testosterone (T) solution in an overlapped mode on the RP-HPTLC layer (not developed), resulted in a three-parted 30-mm band (1-10 mm antagonist, 11-20 mm antagonist plus T, and 21-30 mm T). Immersion of the plates in the cell suspension for incubation, and densitometry with macro slit dimension. Extending both assays, pYAS and pYES, by the antagonistic effect analysis (pYAAS/pYAES). Application of the cosmetic or thermal paper samples as 12-mm band, developed to 65 mm, and application of the androgen T or Estradiol (E2) partially over-lapping. Densitometry with micro slit dimension after the respective bioassay. For RP-HPTLC-Aliivibrio fischeri bioassay, immersing in or spraying with the cell suspension and recording bioautograms. Characterization of found antagonistic zones by RP-HPTLC-HESI-HRMS. Minimalistic sample preparation allows a fast screening. Confirmation of the results by comparison with the more universally detecting HPTLC-Aliivibrio fischeri bioassay proved the potential of the new concept.

Anal Chim Acta 1154 (2022). Development of a compact and miniaturized integrated nanoGIT+active system developed for food systems. It enables on-surface metabolization, immediate separation, multiple imaging and effect-directed detection on the same adsorbent surface and also shows bioactivity changes. Presentation of a scheme for simulation of on-surface digestion, separation and detection in successive steps: (1) food sample (e.g., starch, caseine protein and rape seed oil) in contact with enzyme system (HSA: human saliva α-amylase, PPep: porcine pepsin and Panc: porcine pancreatin plus bile extract), (2) start of metabolization by moistening of the HPTLC silica gel layer and digest including effect-directed detection ion over the incubation period, (3) HPTLC development of the conversion products with 2-propanol - ethyl acetate - water 3:3:1 for starch, 1-butanol - acetic acid - water 4:1:1 for casein, petroleum ether - diethyl ether - acetic acid 80:20:1 for rapeseed oil, and petroleum ether - ethyl acetate - cyclohexane 25:11:14 for turmeric, each followed by drying for 3 min. Documentation in white light, UV 254 nm, and fluorescence detection at 366 nm. Detection of metabolites from starch, caseine protein and rapeseed oil by immersion with 2g in 180 mL ethanol plus dropwise 8 mL 50 % sulfuric acid (followed by plate heating at 120°C, 5 min for saccharides degraded from starch) and densitometry at 434 nm and 475 nm. Detection of the amino acids and peptides degraded from the caseine protein by spraying with ninhydrin reagent (500 mg in 230 mL ethanol and 20 mL acetic acid) followed by heating at 110°C for 5 min and densitometry at 490 nm and 520 nm. Detection of the fatty acids degraded from rapeseed oil by immersion in primuline reagent (250 mg in 50 mL water plus 200 mL acetone) and densitometry by fluorescence measurement at 366/>400 nm. Effect-directed detection of the turmeric separation with the tyrosinase assay using levodopa as substrate, positive control kojic acid, evaluation by densitometry 546 nm with a specialized software. Verification by comparison with state-of-the-art in vitro assays. The developed method is more comprehensive and cost-/time-efficient, and attractive for nutritional, health and pharmaceutical sciences, drug development, medicinal research and for lean laboratories.

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.

Chromatography Research International 2014, 626801 (2014). HPTLC of extracts of Gymnema sylvestre (Apocynaceae) in tablets, as well as standards for calibration, on silica gel (prewashed with methanol and activated at 120°C for 15 min) with toluene – ethyl acetate – methanol 65:25:14. Derivatization by immersing into sulfuric acid (5 % in methanol) and heating at 110°C for 4 min. Densitometric evaluation within 25 min in absorbance mode at 423 nm, which was the optimal wavelength for quantifying simultaneously the triterpenoid gymnemagenin (hRF 27, linearity range 100–1200 ng/band, LOD 32 ng/band, LOQ 53 ng/band) and β-sitosterol (hRF 78, linearity range 200–1200 ng/band, LOD 97 ng/band, LOQ 159 ng/band). Interday and intra-day precisions as well as recovery rates provided relative deviation values below 1 %. This method was used to determine the analyte contents in the tablets (0.041 % gymnemagenin and 0.138 % β-sitosterol), as well as to confirm the stability of the analytes in solution at room temperature after 48h.   

Frontiers in Marine Science 7, 266 (2020). Methanol extracts from marine sponge Haliclona cnidata (Chalinidae) submitted to different stresses (antibiotics and/or darkness) were separated on HPTLC silica gel with an automated 15-step gradient based on methanol, dichloromethane and n-hexane. Bioluminescence was recorded after immersing the HPTLC plates into Aliivibrio fischeri suspension. Antibacterial activity and quorum sensing enhancement were analysed on software, and Pearson’s similarity coefficient was applied to generate similarity matrices for cluster analysis (UPGMA, Unweighted Pair Group Method with Arithmetic Mean). Only slight differences were observed, especially in QS enhanced zones in stressed vs. control cultures.