High-performance Thin-layer Chromatography Plates Research Articles

Development and Validation of HPTLC Method for the Estimation of Escitalopram oxalate and Flupentixol Dihydrochloride in Pharmaceutical Formulation

A simple, precise, accurate High-performance thin-layer chromatography (HPTLC) method has been developed and validated for the estimation of escitalopram oxalate (ESC) and flupentixol dihydrochloride (FLU) in pharmaceutical formulation. Separation was achieved on an aluminum HPTLC plate coated with a 0.2 mm layer of silica gel 60 F254 with chloroform: methanol 4:6 (v/v) as mobile phase. The densitometry analysis was carried out in absorbance mode at 254 nm. The Rf values of escitalopram and flupentixol were 0.24 and 0.44 respectively. The linearity of the method was achieved in the range 3-7 µg/spot for ESC with correlation coefficient value 0.998 and 0.3-0.7µg/spot for FLU with correlation coefficient value 0.999. The developed method was validated according to ICH guidelines and the results were statistically validated. The limit of detection and quantification for escitalopram oxalate and flupentixol dihydrochloride was found to be 500 ng/spot and 50 ng/spot, 1000 ng/spot, and 100 ng/spot respectively.

HPTLC determination of ergosterol in wheat and structure elucidation by NMR: Toward confirming method selectivity

Ergosterol (ERG) is considered as an excellent fungal biomarker, therefore it was utilized for the determination of the degree of fungi infection in wheat grains. A novel, fast, cost-effective and selective high performance thin layer chromatography (HPTLC) with reflectance/absorbance densitometry method was developed and validated for ERG determination. Separation was attained on pre-coated silica gel HPTLC plate with ethyl acetate: petroleum ether (6: 4 v/v) as a mobile phase. This system gave compact bands at retardation factor (Rf) of 0.45 ± 0.002 and quantification was performed at 282 nm over concentration range of 40–600 ng/band. The lower detectable limit was 10.79 ng per band which is three times more sensitive than the previously reported TLC method. The developed method could be utilized efficiently for the detection of ERG in pure powders and in fungi infected wheat samples. Resolution of ERG from other compounds in wheat grain extract allowed utilizing preparative TLC for separating and purifying ERG in a sufficient quantity for structure elucidation by nuclear magnetic resonance spectroscopy (NMR) for the first time. H1 and C13 NMR spectra indicated the purity of ERG extracted from the scrapped silica of preparative TLC plate at the Rf of the standard ERG verifying the method selectivity.

Quantification of biomarkers and evaluation of antioxidant, anti-inflammatory, and cytotoxicity properties of Dodonaea viscosa grown in Saudi Arabia using HPTLC technique

Abstract Dodonaea viscosa (Sapindaceae) was collected from Riyadh, Saudi Arabia. For the simultaneous measurement of quercetin and kaempferol, a validated high-performance thin-layer chromatography (HPTLC) approach was devised in D. viscosa leaf extract. The antioxidant activity was evaluated using diphenyl 1-picrylhydrazyl (DPPH) and 2,2′-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) assays. Moreover, the cytotoxic effect was tested against three cancer cell lines A549, HepG2, and MDA-MB-231. The potential anti-inflammatory properties of different fractions of D. viscosa were also evaluated using lipopolysaccharide (LPS)-induced THP-1 macrophages cells. The test samples include a crude extract of leaves and its solvent-soluble fractions of D. viscosa. The results showed that the crude extract and its fractions exhibited various significant biological activities, the fraction of chloroform demonstrated the highest free radical scavenging activity with IC50 values: 172.2 and 257.7 µg/mL for both DPPH and ABTS tests. Additionally, the chloroform fraction had the greatest cytotoxic activity against MDA-MB-231 (IC50 values: 24.6 ± 0.4 µg/mL). Moreover, the chloroform fraction exhibited the highest downregulation of the LPS-induced expression of TNF-α and IL-6. Quercetin and kaempferol were estimated concurrently in leaves crude extract using a validated technique on an HPTLC plate (10 cm2 × 10 cm2) with a combination of toluene–ethyl acetate–formic acid (5:4:0.2; v/v/v) as the mobile phase and a λ max of 254 nm. The amount of quercetin and kaempferol was found to be 31.8 and 15.01 mg/g of dried leaf extract, respectively. The presence of high levels of quercetin and kaempferol in D. viscosa leaves extract could explain its remarkable antioxidant, cytotoxic, and anti-inflammatory effects. The devolved HPTLC method can be used for routine analysis and standardization of D. viscosa crude plant material, extracts, and/or finished products using quercetin and kaempferol as appropriate markers.

Multiplex planar bioassay detecting estrogens, antiestrogens, false-positives and synergists as sharp zones on normal phase

BackgroundPhytoestrogens are found in many plants used in traditional medicines. Increasingly, plant extracts (botanicals) are also being added to foods or marketed as dietary supplements. Especially such powder formulations are susceptible to adulteration and falsification, given the global processing chain. To detect estrogen-like compounds in such multicomponent mixtures, non-target screening for hormonally active or endocrine disrupting compounds in plant products is becoming more important. Unfortunately, the current planar yeast estrogen screen (pYES) is prone to zone diffusion on the normal-phase high-performance thin-layer chromatography (NP-HPTLC) plate due to long incubation times in the aqueous bioassay. PurposeThe present study aimed to reduce zone diffusion on NP plates, which provides the basis for extending pYES to a multiplex bioassay, offering 4 different biological activity principles, followed by targeted identification of active zones. Study design and methodsThe reduction of substance diffusion via a polyisobutyl methacrylate polymer coating was studied. After successful zone fixation (fix), a multiplex bioassay was developed, in which a 17β-estradiol-strip was applied along each sample track to detect synergists and antagonists (A), and for verification (V), a 4-methyl umbelliferone-strip to exclude false-positives. After multiplex bioassay screening of 68 botanicals, the zones with hormonal activities were heart-cut eluted to reversed-phase high-performance liquid chromatography−diode array detection−high-resolution tandem mass spectrometry (RP-HPLC–DAD–HESI-HRMS/MS). ResultsThe separated substances were successfully fixed by the chromatogram coating. The zone sharpness (achieved after the bioassay) made it possible to add two strips, the 17β-estradiol-strip for antagonistic and synergistic, and the 4-methyl umbelliferone-strip for false-positive effect detection, resulting in a multiplex bioassay. Using the 12D hyphenation NP-HPTLCfix–UV/Vis/FLD–pYAVES–FLD heart-cut RP-HPLC–DAD–HESI-HRMS/MS, it was possible to obtain information on estrogens, antiestrogens, false-positives, and synergists, and (tentatively) assign 17 hormonally active compounds, of which only 7 have been known to affect the human estrogen receptor, while another 4 had structural similarity to common phytoestrogens and antiestrogens. ConclusionsThe streamlined 12D hyphenation including a multiplex bioassay has been shown to differentiate hormonal effects, leading to new insights and better understanding. It can generally be used to identify unknown hormonally active compounds in complex samples.

Screening of orange peel waste on valuable compounds by gradient multiple development diode-array high-performance thin-layer chromatography

High-performance thin-layer chromatography (HPTLC), as the modern form of TLC (thin-layer chromatography), is suitable for detecting pharmaceutically active compounds over a wide polarity range using the gradient multiple development (GMD) technique. Diode-array detection (DAD) in conjunction with HPTLC can simultaneously acquire ultraviolet‒visible (UV‒VIS) and fluorescence spectra directly from the plate. Visualization as a contour plot helps to identify separated zones. An orange peel extract is used as an example to show how GMD‒DAD‒HPTLC in seven different developments with seven different solvents can provide an overview of the entire sample. More than 50 compounds in the extract can be separated on a 6-cm HPTLC plate. Such separations take place in the biologically inert stationary phase of HPTLC, making it a suitable method for effect-directed analysis (EDA). HPTLC‒EDA can even be performed with living organism, as confirmed by the use of Aliivibrio fischeri bacteria to detect bioluminescence as a measure of toxicity. The combining of gradient multiple development planar chromatography with diode-array detection and effect-directed analysis (GMD‒DAD‒HPTLC‒EDA) in conjunction with specific staining methods and time-of-flight mass spectrometry (TOF‒MS) will be the method of choice to find new chemical structures from plant extracts that can serve as the basic structure for new pharmaceutically active compounds.

Development of a High-Performance Thin-Layer Chromatography Method for the Quantification of Alkyl Glycerolipids and Alkenyl Glycerolipids from Shark and Chimera Oils and Tissues

Ether lipids are composed of alkyl lipids with an ether bond at the sn-1 position of a glycerol backbone and alkenyl lipids, which possess a vinyl ether bond at the sn-1 position of the glycerol. These ether glycerolipids are present either as polar glycerophospholipids or neutral glycerolipids. Before studying the biological role of molecular species of ether glycerolipids, there is a need to separate and quantify total alkyl and alkenyl glycerolipids from biological samples in order to determine any variation depending on tissue or physiopathological conditions. Here, we detail the development of the first high-performance thin-layer chromatography method for the quantification of total alkyl and alkenyl glycerolipids thanks to the separation of their corresponding alkyl and alkenyl glycerols. This method starts with a reduction of all lipids after extraction, resulting in the reduction of neutral and polar ether glycerolipids into alkyl and alkenyl glycerols, followed by an appropriate purification and, finally, the linearly ascending development of alkyl and alkenyl glycerols on high-performance thin-layer chromatography plates, staining, carbonization and densitometric analysis. Calibration curves were obtained with commercial alkyl and alkenyl glycerol standards, enabling the quantification of alkyl and alkenyl glycerols in samples and thus directly obtaining the quantity of alkyl and alkenyl lipids present in the samples. Interestingly, we found a differential quantity of these lipids in shark liver oil compared to chimera. We quantified alkyl and alkenyl glycerolipids in periprostatic adipose tissues from human prostate cancer and showed the feasibility of this method in other biological matrices (muscle, tumor).

New solvent systems to separate some estrogenically active compounds by high-performance thin-layer chromatography (HPTLC)

Two solvent mixtures for high-performance thin-layer chromatographic (HPTLC) separation of some compounds showing estrogenic activity in the yeast estrogen screen (YES) assay are presented. The new method, planar yeast estrogen screen (pYES) combines the YES assay and a chromatographic separation on silica gel HPTLC plates with the performance of the YES assay. For separation, the analytes were applied bandwise to HPTLC plates (10 × 20 cm) with fluorescent dye (Merck, Germany). The plates were developed in a vertical developing chamber after 30 min of chamber saturation over a separation distance of 70 mm, using cyclohexane‒methyl-ethyl ketone (2:1, V/V) or cyclohexane‒CPME (3:2, V/V) as solvents. Both solvents allow separation of estriol, daidzein, genistein, 17β-estradiol, 17α-ethinyl estradiol, estrone, 4-nonylphenol and bis(2-ethylhexyl) phthalate.

Nanomole-scaled high-throughput chemistry plus direct bioautography on the same chromatography plate for drug discovery

The powerful fusion of on-surface synthesis and effect-directed analysis was introduced as novel tool for synthetic drug discovery, all on the same high-performance thin-layer chromatography plate. Precise automated sample application allowed both, high-throughput chemistry of 60 reactions at once and reaction miniaturization down to the 15-nmol scale. The antibiotic activity of all on-surface synthesized compounds was evaluated on the same surface via the Gram-positive Bacillus subtilis bioassay. For one product, synthesis (reaction, purification and identification) took 5.3 min and semi-quantitative biological evaluation took 2.8 min. Out of 60 on-surface reactions 10 products (17%) were identified to be more active than a well-known antibiotic reference. The concept was transferred to the Gram-negative Aliivibrio fischeri bioassay. For the first time, a new analytical platform was shown for a streamlined workflow at the most miniaturized scale from synthesis, purification, identification and quantification to semi-quantitative biological activity evaluation (all on the same chromatography plate).

Two-dimensional high-performance thin-layer chromatography for the characterization of milk peptide properties and a prediction of the retention behavior – a proof-of-principle study

High-performance thin-layer chromatography (HPTLC) is a suitable method for the analysis of peptides and proteins due to a wide selection of stationary and mobile phases and various detection options. Especially, two-dimensional HPTLC (2D-HPTLC) enables a higher resolution compared to one-dimensional HPTLC in the separation of complex peptide mixtures. Similar to 2D electrophoresis, characteristic peptide patterns can be obtained, allowing a differentiation of ingredients based on varying protein origins.The aim of this study was to evaluate 2D-HPTLC with regard to its suitability for the characterization of proteins/peptides and to verify whether it is possible to predict the retention behavior of peptides based on their properties. As models, the five most abundant milk proteins α-lactalbumin, β-lactoglobulin, α-, β-, and κ-Casein were used. In order to determine the repeatability of the peptide separation by 2D-HPTLC, each tryptic protein hydrolyzate was separated eight times. The standard deviations of the retardation factors for the separated peptides varied between 1.0 and 11.1 mm for the x-coordinate and 0.5–7.3 mm for the y-coordinate. It was also shown that after the chromatographic separation, peptides of the individual protein hydrolyzates were located in specific areas on the HPTLC plate, so that a clustering could be obtained for the whey proteins‘ as well as the caseins‘ hydrolyzates. For establishing correlations between the properties of the peptides and their retardation factors, 51 of 85 selected peptides were identified by matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF-MS/MS). On this basis, statistically significant correlations (α = 0.05) between the retardation factors of the peptides and their isoelectric points, as well as the percentage of anionic and non-polar amino acids in the peptides were established. Finally, it was investigated, whether the retardation factors for peptides can be predicted on the basis of a linear regression of the percentage of non-polar amino acids in a peptide. For this purpose, a mixture of artifical (synthetic) peptides (n = 14) was separated by 2D-HPTLC and the measured retardation factors were compared with the corresponding retardation factors calculated. Absolute deviations of 0.3–17.9 mm were obtained. In addition, the universal applicability of the method to other protein sources other than milk proteins (animal protein) was tested using a mixture of pea peptides (plant protein, n = 3) resulting in absolute deviations of 0.7–8.6 mm.

Identification and quantification of glucose degradation products in heat-sterilized glucose solutions for parenteral use by thin-layer chromatography

During heat sterilization of glucose solutions, a variety of glucose degradation products (GDPs) may be formed. GDPs can cause cytotoxic effects after parenteral administration of these solutions. The aim of the current study therefore was to develop a simple and quick high-performance thin-layer chromatography (HPTLC) method by which the major GDPs can be identified and (summarily) quantified in glucose solutions for parenteral administration. All GDPs were derivatized with o-phenylenediamine (OPD). The resulting GDP derivatives (quinoxalines) were applied to an HPTLC plate. After 20 minutes of chamber saturation with the solvent, the HPTLC plate was developed in a mixture of 1,4-dioxane-toluene-glacial acetic acid (49:49:2, v/v/v), treated with thymol-sulfuric acid spray reagent, and heated at 130°C for 10 minutes. Finally, the GDPs were quantified by using a TLC scanner. For validation, the identities of the quinoxaline derivatives were confirmed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Glyoxal (GO)/methylglyoxal (MGO) and 3-deoxyglucosone (3-DG)/3-deoxygalactosone (3-DGal) could be identified and quantified in pairs, glucosone (2-KDG), 5-hydroxymethylfurfural (5-HMF), and 3,4-dideoxyglucosone-3-ene (3,4-DGE) each individually. For 2-KDG, the linearity of the method was demonstrated in the range of 1–50 μg/mL, for 5-HMF and 3,4-DGE 1–75 μg/mL, for GO/MGO 2–150 μg/mL, and for 3-DG/3-DGal 10–150 μg/mL. All GDPs achieved a limit of detection (LOD) of 2 μg/mL or less and a limit of quantification (LOQ) of 10 μg/mL or less. R2 was 0.982 for 3.4-DGE, 0.997 for 5-HMF, and 0.999 for 2-KDG, 3-DG/3-DGal, and GO/MGO. The intraday precision was between 0.4 and 14.2% and the accuracy, reported as % recovery, between 86.4 and 112.7%. The proposed HPTLC method appears to be an inexpensive, fast, and sufficiently sensitive approach for routine quantitative analysis of GDPs in heat-sterilized glucose solutions.

HPTLC Method for the Ultrasensitive Detection of Triamterene in Plasma.

A high-performance thin-layer chromatographic (HPTLC) method has developed for the selective detection of a diuretic drug, triamterene (TRIAM), in pure form, tablets and human plasma. The method was based on chromatographic separation of TRIAM using HPTLC plates, precoated with silica gel, and a mobile phase consisted of ethyl acetate: dimethylformamide: ammonia (7.0: 2.7: 0.3, by volume). The native fluorescence signal of TRIAM was detected at 440nm and used to quantify TRIAM using the proposed method, improving the method sensitivity to ~250-folds in comparison to that reported in previous HPTLC studies. The developed method enabled the detection of TRIAM in pure drug and biological samples (human plasma) within linear concentrations ranged from 0.8 to 60ng/band or 1.0 to 60ng/band for pure drug and plasma samples, respectively. Furthermore, the method was validated according to the official guidelines to permit its applicability in quality control and clinical laboratories.

A validated quantification of triclosan in toothpaste using high-performance thin-layer chromatography and a 48-bit flatbed scanner

We present a densitometric quantification method for triclosan in toothpaste, separated by high-performance thin-layer chromatography (HPTLC) and using a 48-bit flatbed scanner as the detection system. The sample was band-wise applied to HPTLC plates (10 × 20 cm), with fluorescent dye, Merck, Germany (1.05554). The plates were developed in a vertical developing chamber with 20 min of chamber saturation over 70 mm, using n-heptane–methyl tert-butyl ether–acetic acid (92:8:0.1, V/V) as solvent. The RF value of triclosan is hRF = 22.4, and quantification is based on direct measurements using an inexpensive 48-bit flatbed scanner for color measurements (in red, green, and blue) after plate staining with 2,6-dichloroquinone-4-chloroimide (Gibbs' reagent). Evaluation of the red channel makes the measurements of triclosan very specific. For linearization, an extended Kubelka–Munk expression was used for data transformation. The range of linearity covers more than two orders of magnitude and is between 91 and 1000 ng. The separation method is inexpensive, fast and reliable.

Stability indicating thin-layer chromatographic method for estimation of antidiabetic drug Remogliflozin etabonate

BackgroundA sensitive, precise, and stability-indicating high-performance thin-layer chromatographic (HPTLC) method has been developed for the analysis of Remogliflozin etabonate in tablet formulation. HPTLC plates precoated with silica gel 60 F254 were used as the stationary phase; methanol: ethyl acetate: toluene: NH3 (2:4:4:0.1, v/v/v) was used as mobile phase, and densitometry was used for the quantitative estimation of the drug. The proposed method was validated with respect to linearity, accuracy, precision, and robustness and applied for the estimation of drug in tablet dosage form.ResultsThe Rf value of Remogliflozin etabonate was observed to be 0.61. The densitometric estimation was performed in reflectance mode at 229 nm. The method was found to be linear in the range of 500–8000 ng/band for Remogliflozin etabonate. The possible degradation pathway was estimated by performing forced degradation studies. The degradant peaks were well resolved from the drug peak with acceptable resolution in their Rf value.ConclusionAn accurate and precise high-performance thin-layer chromatographic method has been developed for the quantification of Remogliflozin etabonate in tablets. Forced degradation studies were performed, and drug was found to be highly susceptible to acid, base hydrolysis, and oxidative stress degradation and gets converted into active drug Remogliflozin. Both Remogliflozin etabonate and Remogliflozin bands were well resolved. The method was applied for the analysis of drug in tablet formulation, and it can be used for routine quality control analysis, as well as for the analysis of stability samples.

Simultaneous Estimation of Cinnamaldehyde and Eugenol in Essential Oils and Traditional and Ultrasound-Assisted Extracts of Different Species of Cinnamon Using a Sustainable/Green HPTLC Technique.

A wide range of analytical techniques are reported for the determination of cinnamaldehyde (CCHO) and eugenol (EOH) in plant extracts and herbal formulations either alone or in combination. Nevertheless, sustainable/green analytical techniques for the estimation of CCHO and EOH either alone or in combination are scarce in the literature. Accordingly, the present research was carried out to establish a rapid, highly sensitive, and sustainable high-performance thin-layer chromatography (HPTLC) technique for the simultaneous estimation of CCHO and EOH in the traditional and ultrasound-assisted methanolic extracts of Cinnamomum zeylanicum, C. burmannii, and C. cassia and their essential oils. The simultaneous estimation of CCHO and EOH was performed through NP-18 silica gel 60 F254S HPTLC plates. The cyclohexane/ethyl acetate (90:10, v v−1) solvent system was optimized as the mobile phase for the simultaneous estimation of CCHO and EOH. The greenness score of the HPTLC technique was predicted using AGREE software. The entire analysis was carried out at a detection wavelength of 296 nm for CCHO and EOH. The sustainable HPTLC technique was observed as linear in the range 10–2000 ng band−1 for CCHO and EOH. The proposed technique was found to be highly sensitive, rapid, accurate, precise, and robust for the simultaneous estimation of CCHO and EOH. The content of CCHO in traditional methanolic extracts of C. zeylanicum, C. burmannii, and C. cassia was found to be 96.36, 118.49, and 114.18 mg g−1, respectively. However, the content of CCHO in ultrasound-assisted methanolic extracts of C. zeylanicum, C. burmannii, and C. cassia was found to be 111.57, 134.39, and 129.07 mg g−1, respectively. The content of CCHO in essential oils of C. zeylanicum, C. burmannii, and C. cassia was found to be 191.20, 214.24, and 202.09 mg g−1, respectively. The content of EOH in traditional methanolic extracts of C. zeylanicum, C. burmannii, and C. cassia was found to be 73.38, 165.41, and 109.10 mg g−1, respectively. However, the content of EOH in ultrasound-assisted methanolic extracts of C. zeylanicum, C. burmannii, and C. cassia was found to be 87.20, 218.09, and 121.85 mg g−1, respectively. The content of EOH in essential oils of C. zeylanicum, C. burmannii, and C. cassia was found to be 61.26, 79.21, and 69.02 mg g−1, respectively. The amounts of CCHO and EOH were found to be significantly higher in ultrasound-assisted extracts of all species compared to its traditional extraction and hence ultrasound extraction has been proposed as a superior technique for the extraction of CCHO and EOH. The AGREE analytical score of the present analytical technique was predicted as 0.75, suggesting excellent greenness profile of the proposed analytical technique. Based on all these observations and results, the proposed sustainable HPTLC technique can be successfully used for the simultaneous estimation of CCHO and EOH in different plant extracts and herbal products.

Coupling high-performance thin-layer chromatography with a battery of cell-based assays reveals bioactive components in wastewater and landfill leachates

Over the last two decades, effect-directed analysis (EDA) gained importance as a seminal screening tool for tracking biological effects of environmental organic micro-pollutants (MPs). As EDA using high-performance liquid chromatography and bioassays is costly and time consuming, recent implementations of this approach have combined high-performance thin-layer chromatography (HPTLC) with effect-based methods (EBMs) using cell-based bioassays, enabling the detection of estrogenic, androgenic, genotoxic, photosystem II (PSII)- inhibiting, and dioxin-like sample components on a HPTLC plate. In the present study, the developed methodologies were applied as a HPTLC-based bioassay battery, to investigate toxicant elimination efficiency of wastewater treatment plants (WWTPs), and to characterize the toxic potential of landfill leachates. Activity levels detected in untreated landfill leachates, expressed as reference compound equivalence (EQ) concentration, were up to 16.8 µg β-naphthoflavone-EQ L−1 (indicating the degree of dioxin-like activity), 1.9 µg estradiol-EQ L−1 (estrogenicity) and 8.3 µg diuron-EQ L‑1 (PSII-inhibition), dropping to maximal concentrations of 47 ng β-naphthoflavone-EQ L−1, 0.7 µg estradiol-EQ L−1 and 53.1 ng diuron-EQ L−1 following treatment. Bisphenol A (BPA) is suggested to be the main contributor to estrogenic activity, with concentrations determined by the planar yeast estrogen screen corresponding well to results from chemical analysis. In the investigated WWTP samples, a decrease of estrogenic activity of 6–100% was observed following treatment for most of the active fractions, except of a 20% increase in one fraction (Rf = 0.568). In contrast, androgenicity with concentrations up to 640 ng dihydrotestosterone-EQ L−1 was completely removed by treatment. Interestingly, genotoxic activity increased over the WWTP processes, releasing genotoxic fractions into receiving waters. We propose this combined HPTLC and EBM battery to contribute to an efficient, cheap, fast and robust screening of environmental samples; such an assay panel would allow to gain an estimate of potential biological effects for prioritization prior to substance identification, and its routine application will support an inexpensive identification of the toxicity drivers as a first tier in an EDA strategy.

Embelin: an HPTLC method for quantitative estimation in five species of genus Embelia Burm. f.

BackgroundThe plants belonging to the genus Embelia, a significant tropical genus with many biological activities, are benefiting because of their robust medicinal properties. Embelin is one of the principal bioactive molecules responsible for the medicinal properties of the genus Embelia. The quantification of the embelin compound among different species in this genus has not yet been investigated, so still uncertain which species and which part should be accepted. The present study was intended to establish a speedy and precise high-performance thin-layer chromatographic (HPTLC) method for quantitative study of embelin in various plant parts of Embelia ribes, Embelia tsjeriam-cottam, Embelia basaal, Embelia adnata, and Embelia gardneriana.ResultThis research confirmed the method as per the International Conference on Harmonization (ICH) guidelines. We achieved separation on silica gel 60 F254 HPTLC plates using propanol: butanol: ammonia (7:3:7 v/v/v) as a mobile phase. Densitometry scanning performed for detection and quantification at 254 nm and 366 nm. Among the species investigated, the highest amount of embelin was found in E. ribes fruits.ConclusionEmbelia ribes fruits are the best source of embelin. Embelin was first described in the endemic species, such as E. adnata and E. gardneriana. The method illustrated in this research may be applied for quantification of embelin and fingerprint analysis of other species within Embelia genus or described genera and chemo taxonomic studies of this genus.

Imaging high-performance thin-layer chromatography as powerful tool to visualize metabolite profiles of eight Bacillus candidates upon cultivation and growth behavior

Imaging high-performance thin-layer chromatography (HPTLC) was explored with regard to its ability to visualize changes in the metabolite profile of bacteria. Bacillus subtilis has become a model organism in many fields. The increasing interest in these bacteria is driven by their attributed probiotic activity. However, growth behavior and metabolism of Bacillus species have a considerable influence on their activity and secondary metabolite profile. On the HPTLC plate, cultivation broths of Bacillus species (B. subtilis, B. licheniformis, B. pumilus and B. amyloliquefaciens) and some B. subtilis strains of high genetic similarity up to 99.5% were applied directly and compared with their respective liquid-liquid extracts. The latter as well as the cultivation in a minimal medium reduced the matrix load and improved the zone resolution. Cultivation parameters such as nutrient supply, cultivation temperature, cultivation time and rotational speed (oxygen level) as well as medium change were shown to have a considerable influence on the growth behavior and resulting metabolite profiles. Imaging HPTLC turned out to be an efficient and affordable tool to visualize such influences of cultivation parameters on the metabolite profiles. It converts the complexity of reaction processes occurring during cell cultivation in easy-to-understand images, which are helpful to figure out factors of influence and understand activity changes. The results highlighted that optimal cultivation conditions need to be found for the intended bacterial application, and in particular, these conditions have to be kept constant. It must be ensured that small variations in cultivation parameters of bacteria do not change the specified (probiotic) effect on the health of animals and humans. The HPTLC metabolite profiles represented the cultivation conditions of specific bacteria and were found to be a proof of the activity of distinct bacteria. In addition, HPTLC can also be used to optimize and streamline the culture media. The quality control of cultivation or fermentation processes can benefit from such a powerful tool, as a picture is worth a thousand words.

High-performance thin-layer chromatography in combination with a yeast-based multi-effect bioassay to determine endocrine effects in environmental samples.

Effect-directed analysis (EDA) that combines effect-based methods (EBMs) with high-performance thin-layer chromatography (HPTLC) is a useful technique for spatial, temporal, and process-related effect evaluation and may provide a link between effect testing and responsible substance identification. In this study, a yeast multi endocrine-effect screen (YMEES) for the detection of endocrine effects is combined with HPTLC. Simultaneous detection of estrogenic, androgenic, and gestagenic effects on the HPTLC plate is achieved by mixing different genetically modified Arxula adeninivorans yeast strains, which contain either the human estrogen, androgen, or progesterone receptor. Depending on the yeast strain, different fluorescent proteins are formed when an appropriate substance binds to the specific hormone receptor. This allows to measure hormonal effects at different wavelengths. Two yeast cell application approaches, immersion and spraying, are compared. The sensitivity and reproducibility of the method are shown by dose-response investigations for reference compounds. The spraying approach indicated similar sensitivities and higher precisions for the tested hormones compared to immersion. The EC10s for estrone (E1), 17β-estradiol (E2), 17α-ethinylestradiol (EE2), 5α-dihydrotestosterone (DHT), and progesterone (P4) were 95, 1.4, 10, 7.4, and 15pg/spot, respectively. Recovery rates of E1, E2, EE2, DHT, and P4 between 88 and 120% show the usability of the general method in combination with sample enrichment by solid phase extraction (SPE). The simultaneous detection of estrogenic, androgenic, and gestagenic effects in wastewater and surface water samples demonstrates the successful application of the YMEES in such matrices. This promising method allows us to identify more than one endocrine effect on the same HPTLC plate, which saves time and material. The method could be used for comparison, evaluation, and monitoring of different river sites and wastewater treatment steps and should be tested in further studies.

The comprehensive analysis of sage leaves by high-performance thin-layer chromatography: simultaneous identification of flavonoids and phenylpropanoids, quantitative determination and the test for the minimal content of rosmarinic acid

Aim. To develop methods for the comprehensive analysis of sage (Salvia officinalis L.) leaves using high-performance thin-layer chromatography (HPTLC): identification of flavonoids and phenylpropanoids, quantitative determination and the test for the minimal content (MCT) of rosmarinic acid. Materials and methods. The study was performed using HPTLC procedure (2.8.25, SPhU) in the HPTLC automatic system; the results were processed using VisionCats Software, CAMAG, Switzerland.Results. Flavonoids and phenylpropanoids fingerprints of six samples of sage leaves were evaluated on the same HPTLC plate. The specific zones and their chromatographic profiles useful for identification were determined. In the same mobile phase, the method for quantifying the marker – rosmarinic acid, as well as MCT of rosmarinic acid (NLT 0.4 %) was proposed. According to the results, the quantity of rosmarinic acid in sage leaves was in the range of 5.83-15.94 mg/g (scanner detection) and 5.99-16.27 mg/g (visualizer detection). All six samples of sage contained rosemary acid in an amount greater than the proposed minimum content (0.4 %), while S. aethiopis and S. nemerosa had a lower content of rosmarinic acid and failed the test.Conclusions. The approach proposed for evaluating the quality of sage using a comprehensive analysis of HPTLC flavonoid and phenylpropanoid fingerprints allows simultaneous identification, quantitative determination and the test for the minimal content for several samples on the same plate. It can be an alternative to the current Pharmacopoeia methods.

Detection reagents used for on-plate identification of organic pesticides in biological samples with preliminary separation by TLC/HPTLC

Many specific and non-specific chromogenic spray reagents have been used to detect organic pesticides (organophosphorus, organochlorine, carbamates, and pyrethroids) on thin-layer chromatographic (TLC)/high-performance thin-layer chromatographic (HPTLC) plates. To realize high sensitivity and improved selectivity, several chromogenic reagents have been introduced. The physical properties of organic pesticides reported so far are also presented in tabular form. The colors produced on TLC plates due to reactions taking place between pesticides and spray reagents are illustrated in the form of chromatograms, and reaction mechanism is also presented.