The leaves of Dichrostachys cinerea are used as laxative, diuretic, painkiller. It is also used in the treatment of gonorrhoea, boils, oedema, gout, veneral diseases and nasopharyngeal affections, etc.

Undirected modifications between food proteins and secondary plant metabolites can occur during food processing. The results of covalent interactions can alter the functional and biological properties of the proteins. The present work studied the extent of which covalent conjugation of the bioactive metabolite benzyl isothiocyanate (BITC; a glucosinolate breakdown product) to the whey protein α-lactalbumin affects the protein's allergenicity. Additional to the immunological analysis of native untreated and BITC-modified α-lactalbumin, the analysis of antigenic properties of proteolytically digested protein derivatives was also performed by high performance thin layer chromatography and immunostaining. As a result of the chemical modifications, structural changes in the protein molecule affected the allergenic properties. In this process, epitopes are destroyed or inactivated, but at the same time, buried epitopes can be exposed or newly formed, so that the net effect was an increase in allergenicity, in this case. Results from the tryptic hydrolysis suggest that BITC conjugation sterically hindered the cleavage sites for the enzyme, resulting in reduced digestibility and allergenicity. Residual antigenicity can be still present as short peptide fragments that provide epitopes. The desire to make food safer for allergy sufferers and to protect sensitized individuals from an allergenic reaction makes it clear that the detection of food antigens is mandatory; especially by considering protein interactions.

The global herbal products market has grown in recent years, making regulation of these products paramount for public healthcare. For instance, the common horsetail (Equisetum arvense L.) is used in numerous herbal products, but it can be adulterated with closely related species, especially E. palustre L. that can produce toxic alkaloids. As morphology-based identification is often difficult or impossible, the identification of processed material can be aided by molecular techniques. In this study, we explore two molecular identification techniques as methods of testing the purity of these products: a Thin Layer Chromatography approach (TLC-test) included in the European Pharmacopoeia and a DNA barcoding approach, used in recent years to identify material in herbal products. We test the potential of these methods for distinguishing and identifying these species using material from herbarium collections and commercial herbal products. We find that both methods can discriminate between the two species and positively identify E. arvense. The TLC-test is more cost- and time-efficient, but DNA barcoding is more powerful in determining the identity of adulterant species. Our study shows that, although DNA barcoding presents certain advantages, other established laboratory methods can perform as well or even better in confirming species' identity in herbal products.

Although numerous studies have been conducted on ginger extracts and fractions, the data on the pharmacological activity of single constituents of Zingiber officinale are still insufficient. To assess the antidementia properties of the plant, a thin layer chromatography (TLC)-based bioautography acetylcholinesterase inhibitory assay was performed on the Zingiber officinale diethyl ether extract. It led to the recognition of three active inhibitors among volatile constituents of the plant: ar-curcumene (A), α-sesquiphellandrene (B) and a-zingiberene (C). The identification of the components was possible thanks to the application of a TLC-HPLC-MS interface analysis of active zones and the GC-MS qualitative analysis of the tested samples. Based on the obtained results, the influence of several extraction techniques (hydrodistillation-HD, pressurized liquid extraction or accelerated solvent extraction-ASE, shaking maceration-SM, supercritical fluid extraction-SFE, and ultrasound-assisted extraction-UAE) on the recovery of the active metabolites from plant material was assessed to deliver enriched extracts. As a result, HD and SFE, were found to be the most efficient methods to recover the volatile components and the concentrations of A, B, and C reached 0.51 ± 0.025, 0.77 ± 0.045, and 1.67 ± 0.11 percent, respectively. Only HD and SFE were found to recover monoterpene hydrocarbons from the plant matrix. The remaining techniques provided extracts rich in more complex constituents, like sesquiterpenes.

Honey adulteration, where a range of sugar syrups is used to increase bulk volume, is a common problem that has significant negative impacts on the honey industry, both economically and from a consumer confidence perspective. This paper investigates High-Performance Thin Layer Chromatography (HPTLC) for the authentication and detection of sugar adulterants in honey. The sugar composition of various Australian honeys (Manuka, Jarrah, Marri, Karri, Peppermint and White Gum) was first determined to illustrate the variance depending on the floral origin. Two of the honeys (Manuka and Jarrah) were then artificially adulterated with six different sugar syrups (rice, corn, golden, treacle, glucose and maple syrup). The findings demonstrate that HPTLC sugar profiles, in combination with organic extract profiles, can easily detect the sugar adulterants. As major sugars found in honey, the quantification of fructose and glucose, and their concentration ratio can be used to authenticate the honeys. Quantifications of sucrose and maltose can be used to identify the type of syrup adulterant, in particular when used in combination with HPTLC fingerprinting of the organic honey extracts.

Aspalathin, the main polyphenol of rooibos (Aspalathus linearis), is associated with diverse health promoting properties of the tea. During fermentation, aspalathin is oxidized and concentrations are significantly reduced. Standardized methods for quality control of rooibos products do not investigate aspalathin, since current techniques of aspalathin detection require expensive equipment and expertise. Here, we describe a simple and fast thin-layer chromatography (TLC) method that can reproducibly visualize aspalathin in rooibos herbal tea and plant extracts at a limit of detection (LOD) equal to 178.7 ng and a limit of quantification (LOQ) equal to 541.6 ng. Aspalathin is a rare compound, so far only found in A. linearis and its (rare) sister species A. pendula. Therefore, aspalathin could serve as a marker compound for authentication and quality control of rooibos products, and the described TLC method represents a cost-effective approach for high-throughput screening of plant and herbal tea extracts.

Yohimbine is an indole alkaloid used as a promising therapy for erectile dysfunction. A number of methods were reported for the analysis of yohimbine in the bark or in pharmaceutical preparations.

The conditionally essential very-long-chain polyunsaturated fatty acids (VLC-PUFAs), such as eicosapentaenoic acid (EPA, C20:5 n-3), play a vital role in human nutrition. Their biological activity is thereby greatly influenced by the distinct glycerolipid molecule that they are esterified to. Here, microalgae differ from the conventional source, fish oil, both in quantity and distribution of VLC-PUFAs among the glycerolipidome. Therefore, the aim of this study was to develop a fast and reliable one-dimensional high-performance thin-layer chromatography (HPTLC)-based method that allows the separation and quantification of the main microalgal glycerolipid classes (e.g., monogalactosyldiacylglycerol (MGDG), sulfoquinovosyl diacylglycerol (SQDG), phosphatidylglycerol (PG)), as well as the subsequent analysis of their respective fatty acid distribution via gas chromatography (GC) coupled to mass spectrometry (MS). Following optimization, method validation was carried out for 13 different lipid classes, based on the International Conference on Harmonization (ICH) guidelines. In HPTLC, linearity was effective between 100 and 2100 ng, with a limit of quantification between 62.99 and 90.09 ng depending on the glycerolipid class, with strong correlation coefficients (R2 > 0.995). The recovery varied between 93.17 and 108.12%, while the inter-day precision measurements showed coefficients of variation of less than 8.85%, close to the limit of detection. Applying this method to crude lipid extracts of four EPA producing microalgae of commercial interest, the content of different glycerolipid classes was assessed together with the respective FA distribution subsequent to band elution. The results showed that the described precise and accurate HPTLC method offers the possibility to be used routinely to follow variations in the glycerolipid class levels throughout strain screening, cultivation, or bioprocessing. Thus, additional quantitative analytical information on the complex lipidome of microalgae can be obtained, especially for n-3 and n-6 enriched lipid fractions.

The plant species Arbutus unedo shows numerous beneficial pharmacological effects (antiseptic, antidiabetic, antidiarrheal, astringent, depurative, antioxidant, antihypertensive, antithrombotic, and anti-inflammatory). For the medicinal use, standardization of extracts is a necessity, as different compounds are responsible for different biological activities. In this paper, we analyze monthly changes in the content of quercitrin, isoquercitrin, hyperoside, and chlorogenic acid. Methanolic extracts of the leaves are analyzed by HPTLC for the identification and quantification of individual polyphenol, and DPPH test is used to determine antioxidant activity. Based on the results obtained, the leaves should be collected in January to obtain the highest concentrations of hyperoside and quercitrin (0.35 mg/g and 1.94 mg/g, resp.), in June, July, and October for chlorogenic acid (1.45-1.46 mg/g), and for the fraction of quercitrin and isoquercitrin in November (1.98 mg/g and 0.33 mg/g, resp.). Optimal months for the collection of leaves with the maximum recovery of individual polyphenol suggested in this work could direct the pharmacological usage of the polyvalent herbal drugs.

A sensitive, selective, precise and stability-indicating high-performance thin-layer chromatographic (HPTLC) method for analysis of repaglinide both as a bulk drug and in nanoemulsion formulation was developed and validated. The method employed TLC aluminum plates precoated with silica gel 60F-254 as the stationary phase. The solvent system consisted of chloroform/methanol/ammonia/glacial acetic acid (7.5:1.5:0.9:0.1, v/v/v/v). This system was found to give compact spots for repaglinide (R f value of 0.38 ± 0.02). Repaglinide was subjected to acid and alkali hydrolysis, oxidation, photodegradation and dry heat treatment. Also, the degraded products were well separated from the pure drug. Densitometric analysis of repaglinide was carried out in the absorbance mode at 240 nm. The linear regression data for the calibration plots showed good linear relationship with r (2)= 0.998 ± 0.032 in the concentration range of 50-800 ng. The method was validated for precision, accuracy as recovery, robustness and specificity. The limits of detection and quantitation were 0.023 and 0.069 ng per spot, respectively. The drug undergoes degradation under acidic and basic conditions, oxidation and dry heat treatment. All the peaks of the degraded product were resolved from the standard drug with significantly different R f values. Statistical analysis proves that the method is reproducible and selective for the estimation of the said drug. As the method could effectively separate the drug from its degradation products, it can be employed as a stability-indicating one. Moreover, the proposed HPTLC method was utilized to investigate the degradation kinetics in 1M NaOH.

[BMIM]OH was used in mobile and stationary phase of thin layer chromatography (TLC) to analyze berberine hydrochloride and tetrahydropalmatine for the first time. Supported imidazole ionic liquid with hydroxide ion on silica gel (SiO2·Im(+)·OH(-)) was synthesized through simple procedure and characterized by Fourier transform infrared spectroscopy (FT-IR), elemental analysis, and scanning electron microscope (SEM). Moreover, on the plates prepared by SiO2·Im(+)·OH(-), the contents of the above alkaloids in the Chinese patent medicine (CPM) of "Stomacheasy" capsule were successfully determined by TLC scanner. The key conditions and chromatographic behaviors were also investigated in detail. According to similar ways, ionic liquids (ILs) also can be used in other planar chromatographies in two modes. This study is expected to be helpful in expanding the application of IL and its bonded silica gel in TLC separation field.

Benzophenone-4 (BZ4) was separated from surfactants, dyes, preservatives, and other components of hair shampoos by thin-layer chromatography on silica gel 60 stationary phase, with ethyl acetate-ethanol-water-pH 6 phosphate buffer (15 : 7 : 5 : 1 v/v/v/v) as mobile phase. Densitometry scanning of chromatograms was performed at 285 nm. The densitometric calibration curve for BZ4 was nonlinear (second-degree polynomial), with R > 0.999. The limits of detection and quantification were ca. 0.03 and ca. 0.1 μg spot(-1), respectively. The results obtained by HPTLC-densitometry were compared to those obtained by zero and 2nd derivative UV spectrophotometry. In the case of spectrophotometric methods, calibration curves were linear with R > 0.9998. The chromatographic method was fully validated.

In substandard drugs enforcement, there is a need to develop reliable, fast, and inexpensive analytical methods. Due to its very characteristics, HPTLC offers opportunities for the development of methods that meet these requirements. This technique was used to develop and validate a method for the determination of sildenafil in pharmaceutical formulations from the licit and illicit supply chain in Burkina Faso. Taking into account optimization parameters such as measurement wavelength and mobile phase composition, the best elution quality is found at the maximum signals of spots on silica plates at 305 nm, using a mixture of dichloromethane-methanol mixture 9 : 1 (v/v) proportions. The method developed under these conditions was validated using the accuracy profile as a decision tool. The establishment of the response function curves allowed the choice of the polynomial function applied to the peak areas. This mathematical model provides a validity range between 0.4 and 0.6 mg/mL. The application of the developed and validated method to collected samples allowed the detection of two substandard drugs and confirmed the poor quality of drugs in the illicit market. More data using this approach in a variety of drug molecules could lead to the establishment of databases of counterfeit drugs in Burkina Faso.

The question of whether exosome lipids can be considered as potential cancer biomarkers faces our current limited knowledge of their composition. This is due to the difficulty in isolating pure exosomes, the variability of the biological sources from which they are extracted, and the uncertainty of the methods for lipid characterization. Here, we present a procedure to isolate exosomes and obtain a deep, repeatable, and rapid phospholipid (PL) composition of their lipid extracts, from embryonic murine fibroblasts (NIH-3T3 cell line) and none (B16-F1) and high (B16-F10) metastatic murine skin melanoma cells. The analytical method is based on High Performance Thin-Layer Chromatography with Ultraviolet and fluorescence densitometry and coupled to Electrospray (ESI)-tandem Mass Spectrometry (MS). Under the conditions described in this work, separation and determination of PL classes, (sphingomyelins, SM; phosphatidylcholines, PC; phosphatidylserines, PS; and phosphatidylethanolamines, PE) were achieved, expressed as µg PL/100 µg exosome protein, obtained by bicinchoninic acid assay (BCA). A detailed structural characterization of molecular species of each PL class was performed by simultaneous positive and negative ESI-MS and MS/MS directly from the chromatographic plate, thanks to an elution-based interface.

Diclofenac belongs to the drug class non-steroidal anti-inflammatory drugs widely used in Europe as well as all over the world. Thus, it is important to conduct research on its quality control of available pharmaceutical preparations like for example enteric coated tablets. Among various analytical techniques, thin-layer chromatography (TLC) is ideal for this task due to their short time analysis, ease of operation and low cost. Hence, the aim of this study was to develop the optimal conditions of analysis and quantitative determination of diclofenac sodium in enteric tablets by using TLC in combination with densitometry. Of all chromatographic systems tested, the best is the one which consists of silica gel 60F254 and cyclohexane: chloroform:methanol:glacial acetic acid (6:3:0.5:0.5 v/v) as the mobile phase, which allows the successful separation of examined diclofenac sodium as active component and the largest number (twelve) of its degradation products as potential impurities of its pharmaceutical products. This indicates that the newly developed method is more effective than previously reported assays by Starek and Krzek. Linearity range was found to be 4.00-18.00 μg/spot for diclofenac sodium. The results of the assay of enteric tablet formulations equals 98.8% of diclofenac sodium in relation to label claim is in a good agreement with pharmaceutical requirements.

Lipidomic analysis in diverse biological settings has become a frequent tool to increase our understanding of the processes of life. Cellular lipids play important roles not only as being the main components of cellular membranes, but also in the regulation of cell homeostasis as lipid signaling molecules. Yeast has been harnessed for biomedical research based on its good conservation of genetics and fundamental cell organisation principles and molecular pathways. Further application in so-called humanised yeast models have been developed which take advantage of yeast as providing the basics of a living cell with full control over heterologous expression. Here we present evidence that high-performance thin-layer chromatography (HPTLC) represents an effective alternative to replace cost intensive mass spectrometry-based lipidomic analyses. We provide statistical comparison of identical samples by both methods, which support the use of HPTLC for quantitative analysis of the main yeast lipid classes.

Spirulina platensis contains several biologically active compounds, some of them with antioxidant activity. Nevertheless, not all of these compounds have been identified to date. As a first step to achieving such identification, a methodology to perform two-dimensional thin layer chromatography bioautographies on silica gel thin layer chromatography plates was proposed. Starting with a reference binary system, 5 other binary systems were tested, in which the relative polarity was systematically increased. To further improve the separation behavior, a phase modifier (NH4OH) was used. The best separation results were obtained with the isopropyl alcohol/ethyl acetate/NH4OH ternary system. This experimental system allowed four well-resolved spots showing antioxidant activity as well as two additional areas with mixtures containing antioxidant compounds. Although the proposed methodology was designed with a specific application, it would be predictable that its field of use could be considerably greater, making the convenient modifications on the solvent polarity and "masking level" produced by the ammonium derivatives.

Anthraquinones (yellow dyes) were extracted from Japanese knotweed rhizomes with twelve extraction solvents (water; ethanol(aq) (20%, 40%, 60%, 70% and 80%), ethanol, 70% methanol(aq), methanol, 70% acetone(aq), acetone and dichloromethane). The obtained sample test solutions (STSs) were analyzed using high-performance thin-layer chromatography (HPTLC) coupled to densitometry and mass spectrometry (HPTLC-MS/MS) on HPTLC silica gel plates. Identical qualitative densitometric profiles (with anthraquinone aglycones and glycosylated anthraquinones) were obtained for STSs in all the solvents except for the STS in dichloromethane, which enabled the most selective extractions of anthraquinone aglycones emodin and physcion. The highest extraction efficiency, evaluated by comparison of the total peak areas in the densitograms of all STSs scanned at 442 nm, was achieved for 70% acetone(aq). In STS prepared with 70% acetone(aq), the separation of non-glycosylated and glycosylated anthraquinones was achieved with developing solvents toluene-acetone-formic acid (6:6:1, 3:6:1 and 3:3:1 v/v) and dichloromethane-acetone-formic acid (1:1:0.1, v/v). Non-glycosylated anthraquinones were separated only with toluene-acetone-formic acid, among which the best resolution between emodin and physcion gave the ratio 6:6:1 (v/v). This solvent and dichloromethane-acetone-formic acid (1:1:0.1, v/v) enabled the best separation of glycosylated anthraquinones. Four HPTLC-MS/MS methods enabled the identification of emodin and tentative identification of its three glycosylated analogs (emodin-8-O-hexoside, emodin-O-acetyl-hexoside and emodin-O-malonyl-hexoside), while only the HPTLC-MS/MS method with toluene-acetone-formic acid (6:6:1, v/v) enabled the identification of physcion. Changes of the shapes and the absorption maxima (bathochromic shifts) in the absorption spectra after post-chromatographic derivatization provided additional proof for the detection of physcion and rejection of the presence of chrysophanol in STS.

Acetyl phosphate (acetyl-P) serves critical roles in coenzyme A recycling and ATP synthesis. It is the intermediate of the Pta-AckA pathway that inter-converts acetyl-coenzyme A and acetate. Acetyl-P also can act as a global signal by donating its phosphoryl group to specific two-component response regulators. This ability derives from its capacity to store energy in the form of a high-energy phosphate bond. This bond, while critical to its function, also destabilizes acetyl-P in cell extracts. This lability has greatly complicated biochemical analysis, leading in part to widely varying acetyl-P measurements. We therefore developed an optimized protocol based on two-dimensional thin layer chromatography that includes metabolic labeling under aerated conditions and careful examination of the integrity of acetyl-P within extracts. This protocol results in greatly improved reproducibility, and thus permits precise measurements of the intracellular concentration of acetyl-P, as well as that of other small phosphorylated molecules.

A rapid and simple high-performance thin layer chromatography (HPTLC) method with densitometry at 230 nm was developed and validated for simultaneous determination of diphenhydramine hydrochloride (DPH) and naproxen sodium (NPS) from pharmaceutical preparation. The separation was carried out on aluminum plates precoated with silica gel 60 F254 using mobile phase toluene:methanol:glacial acetic acid (7.5:1:0.2, v/v/v). The linearity range lies between 200 and 1200 ng/band for DPH and 1760 and 10,560 ng/band for NPS with correlation coefficients of 0.994 and 0.995, respectively. The R f value for DPH is 0.20 ± 0.05 and for NPS is 0.61 ± 0.06. % Recoveries of DPH and NPS was in the range of 99.70%-99.95% and 99.63%-99.95%, respectively. Limit of detection value for DPH was 13.21 ng/band and for NPS was 8.03 ng/band. Limit of quantitation value for DPH was 40.06 ng/band and for NPS was 24.34 ng/band. The developed method was validated as per ICH guidelines. In stability testing, DPH was found unstable to acid and alkaline hydrolysis, and DPH and NPS were found unstable to oxidation, whereas both the drugs were stable to neutral and photodegradation. The proposed method was successfully applied for the routine quantitative analysis of dosage form containing DPH and NPS.