Thin-layer Chromatographic Systems Research Articles

Coupling thin layer chromatography with mass spectrometry for detection and identification of sertraline and its metabolite in the urine

The aim of the study was to detect sertraline and its metabolites in urine under standardized thin layer chromatography (TLC) screening conditions and identify the metabolites using the mass spectrometry method. Materials and methods. Urine samples, collected within 30 hours in portions of 20–50 mL, commenced from the seventh hour after the administration of a single therapeutic dose of the drug. The sample preparation process involved dilute acid hydrolysis, followed by the extraction of the native compound and metabolites with chloroform at a pH of 8–9. Thin layer chromatography studies of the extracts were conducted using four unified TLC systems recommended by The International Association of Forensic Toxicologists for general drug screening. Chromatograms were subjected to color reactions with a variety of chromogenic reagents. For the analysis of eluates from chromatograms, a Varian 1200 L mass spectrometer (Netherlands) equipped with a dual quadrupole mass analyzer was employed. Identification was performed through direct sample introduction into the ion chamber, electron-impact ionization (70 eV), and full ion scanning mode. Results. The spot of the native drug on the chromatogram was identified by the Rf, value. Metabolite of sertraline was identified as N-desmethyltrihydroxysertraline by the molecular ion peak in the mass spectrum. Conclusions. The study demonstrated the ability of TLC to detect sertraline and its metabolite, N-desmethyltrihydroxysertraline, in urine after the administration of a single therapeutic dose of the drug. The chromatographic mobility of the native compound and N-desmethyltrihydroxysertraline in the unified TLC screening systems, along with the results of their visualization using chromogenic reagents for toxicological drug screening, was determined. Furthermore, the potential of coupling TLC with mass spectrometry for the separation, detection, and confirmatory identification of sertraline and its metabolic products in urine was established.

Phytochemical Screening, Chromatographic and Quantitative Study of Phenols and Flavonoids in Leaves of Oroxylum indicum and Pongamia pinnata

Number of secondary compounds is produced by plants as natural antioxidants. The current study's goal was to identify the phytochemical components, thin-layer chromatographic profile, total phenol, and total flavonoid content in the leaves of Pongamia pinnata and Oroxylum indicum (O. indicum) (P. pinnata). The well-known test protocol described in the literature was used to determine the qualitative analysis of different phytochemical constituents, thin-layer chromatography, and quantitative analysis of total phenolics and flavonoids. By using the Folins Ciocalteau reagent method and the aluminium chloride method, respectively, phenolic and flavonoid quantities were analysed quantitatively. Both plants contained glycosides, flavonoids, diterpenes, phenol, proteins, saponins, and carbohydrates, according to a phytochemical analysis. Studies using thin-layer chromatography and various solvent systems revealed homogeneous spots with various Rf values. According to the findings of the current study, O. indicum and P. pinnata crude extracts are a rich source of secondary phytoconstituents that have significant antioxidant potential. The pharmaceutical industries, pharmacologists, and phytochemists will all benefit from the study's findings.

Development of rapid analysis of eye drops for monitoring during various technological operations

In this article, the composition and technology of combined eye drops were developed, including taurine as an anti-cataract agent and betaxolol hydrochloride as an anti-glaucoma agent. Butanol, toluene, isopropyl ether, chloroform, ammonia 25%, ethyl alcohol in various ratios were used as the mobile phase. However, these solutions and their combinations did not allow us to determine a system that makes it possible to register both active components simultaneously. Therefore, it was decided to select two thin layer chromatography systems, one of which made it possible to determine taurine, and the other betaxolol hydrochloride

Preparation of Patient Doses of [177Lu]Lu-DOTATATE and [177Lu]Lu-PSMA-617 with Carrier Added (CA) and No Carrier Added (NCA) 177Lu.

[177Lu]Lu-DOTATATE and [177Lu]Lu-PSMA-617 used for targeted radionuclide therapy are very often prepared in the hospital radiopharmacy. The preparation parameters vary depending upon the specific activity of the 177Lu used. The aim of this study was to develop optimized protocols to be used in the nuclear medicine department for the preparation of patient doses of the above radiopharmaceuticals. 177Lu (CA and NCA) were used for radiolabeling DOTATATE and PSMA-617. Parameters studied are 177Lu of different specific activity and different peptide concentrations and two different buffer systems. Paper and thin layer chromatography systems were used for estimating the radiochemical yield as well as radiochemical purity. Solid-phase extraction was used for the purification of the labeled tracers. [177Lu]Lu-DOTATATE was prepared with CA 177Lu (n = 13) and NCA177Lu (n = 6). Four batches each of [177Lu]Lu-PSMA-617 were prepared using CA and NCA 177Lu. Radiochemical yields > 80% and final product with less than < 1% radiochemical impurity could be obtained in all batches which were used for therapy. Robust protocols for the preparation of clinical doses of [177Lu]Lu-DOTATATE and [177Lu]Lu-PSMA-617 were developed and used for the preparation of clinical doses. The quality of the SPECT images of both the tracers are consistent with the expected uptake in respective diseases.

New System for Identification of Propranolol Tablet (Inderal)

Various new drugs are being introduced in the market. This proves a hectic problem for Toxicologist to identify the exact nature of the drug and its metabolites in the viscera for cases involving death due to drug poisoning. When a case is taken for drug analysis, we have to be more exhaustive in ruling out drugs. For identification of Inderal tablets, a new Thin Layer chromatography system has been developed. After systematic trials, the paper has found out a new solvent system with spray for eluting Inderal tablets.

Hydrophilic retention mechanism of imidazoline and serotonin receptor ligands in thin-layer and high-performance liquid chromatography systems

Hydrophilic retention mechanism of imidazoline and serotonin receptor ligands in thin-layer and high-performance liquid chromatography systems

Phytochemical Screening and TLC Profiling of Combination Extracts of Avocado (Persea americana Mill.) and Papaya (Carica papaya) Leaves from Timor Island

Traditionally in the village of Alor, East Nusa Tenggara Province, a combination of avocado and papaya leaves is often used to treat malaria. To be able to be developed as an ingredient in traditional medicine, it is necessary to conduct preliminary research to determine the content of secondary metabolites contained in the combination of avocado and papaya leaves. This study aims to determine the secondary metabolites contained in avocado and papaya leaves through phytochemical screening and Thin Layer Chromatography (TLC) analysis. Extraction was carried out using methanol solvent by maceration, and the extract yield was 91.4%. Testing the content of secondary metabolites in avocado and papaya leaf extracts was carried out by phytochemical screening. Furthermore, the TLC test was carried out to confirm the presence of a positive group of compounds on phytochemical screening, and to determine the chromatographic profile of the extract. After being tested by phytochemical and TLC analysis, the extract contains several metabolites, including flavonoids, alkaloids, tannins, saponins, and steroids. The TLC system used in this study can separate the phytochemical content and give positive results that confirm the results of the phytochemical screening.

Eutectic Thin-Layer Chromatography as a New Possibility for Quantification of Plant Extracts—A Case Study

Deep eutectic solvents (DES), compared to classic ones, have interesting properties, such as the ability to solubilize compounds differing in polarity or increased dissolution of selected chemical compounds. They also offer specific interactions between the mobile and stationary phases. Those features make them promising solvents in chromatographic techniques, including the use in the separation of complicated samples. The first quantitative analysis with eutectic thin-layer chromatography (TLC) is presented in the paper. As a case study, five alkaloids from Chelidonium maius were selected as target compounds. A wide range of terpene-based DESs was investigated to develop the chromatographic system, both pure and after dilution. Moreover, a novel approach was employed to adjust polarity, involving mixing DESs differing in chromatographic properties. This procedure has proved to be effective. The best results were obtained with a 2:1 (wt/wt) mixture of DESs: camphor + phenol and menthol + limonene, with a 20% addition of methanol. The chromatographic system was validated and checked on the real sample, which made it the first applicable and operational quantitative eutectic TLC system.

Response surface methodology and artificial neural network modelling for optimization of solid-liquid extraction and rapid HPTLC analysis of glycyrrhizin in Glycyrrhiza glabra root

Glycyrrhizin, a major bioactive compound of Glycyrrhiza glabra has been used in traditional medicine system to treat several diseases. In the past few decades, its demand has been increased significantly for the active ingredient glycyrrhizin as well as for being a zero calorie sweetener. In the current study, extraction of glycyrrhizin from dry roots of G. glabra was established by reflux method. Quantitative estimation of glycyrrhizin content in the extracted samples was analysed by using high performance thin layer chromatography (HPTLC) system. In this study, response surface methodology (RSM) and artificial neural network (ANN) were used for the first time as a model to optimize the extraction conditions of glycyrrhizin from G. glabra roots in order to compare and establish effective prediction models. The extraction process variables including the time, temperature, solvent composition, particle size, solvent to solute ratio, pH and extraction steps were optimized using a Plackett-Burman design (PBD), Central Composite design (CCD), artificial neural network (ANN) and Multiple Layer Perceptron (MLP) on glycyrrhizin yield. The optimum extraction conditions were 55°C (extraction temperature), 45 min (extraction time), 55% (v/v) (ethanol concentration) and 30 ml/g (solvent to solute ratio) to obtain the maximum glycyrrhizin yield. The contributions of the quadratic model with high determination coefficient (R2=0.9806) were observed and showed good consistency between the experimental value 7.6 mg/g (0.76%) and predicted value 7.51 mg/g (0.751%). Response values were close to the predicted values with prediction accuracy as ANN > RSM, indicating that ANN model has higher prediction accuracy than RSM. The study suggests that RSM and ANN model system can be manipulated for the optimization and production of valuable bioactive compounds.

Determination of β-carotene in jute leaves by spectrophotometry and thin layer chromatography

β-carotene is a carotenoid pigment that converts to vitamin A in human body and plays crucial role in visual functions, reproductive performance and immune system. Jute leaves contain β-carotene but amounts in different varieties of jute leaves are not known. The study was undertaken to estimate the β-carotene content in fresh jute leaves of the cultivated jute species Corchorus olitorius L. and Corchorus capsularis L. by spectrophotometry and thin layer chromatography. β-carotene content varies in leaves of different varieties of two species. Leaves of some C. olitorius variety showed higher amount of β-carotene than the leaves of C. capsularis variety. Some varieties of C. olitorius showed similarity with some varieties of C. capsularis. Variety Robi-1 of C .olitorius showed the highest content of β-carotene 39.93mg/100g. O-72, O-4, O-3820, O-9897 and OM-1 of olitorius variety showed higher β-carotene than all the varieties of C. capsularis. JRO-524 and MG-1 of C. olitorius variety is comparable to CVL-1, D-154, BJC-2197 and deshi pat shak-1 variety of C. capsularis regarding β-carotene content. Lowest β-carotene was estimated 12.55mg/100g in O-795 of olitorius variety and 12.32mg/100g in BJC-83 and 11.34mg/100g in BJC-2142 of capsularis variety. Thin layer chromatography system silica gel on TLC Al foils as stationary phase and hexane as mobile phase performs good separation and identification of β-carotene in jute leaves.

Separation of some vitamins in reversed-phase thin-layer chromatography and pressurized planar electrochromatography with eluent containing surfactant

The separation of some water- and fat-soluble vitamins via micellar systems of reversed-phase high-performance thin-layer chromatography (HPTLC) and pressurized planar electrochromatography (PPEC) was subjected to research. Hence, the influence of the mobile phase composition (surfactant and acetonitrile concentration, eluent buffer pH) on the migration distances and zone separation of some vitamins (thiamine, riboflavin, niacin, pyridoxine, cyanocobalamin, folic acid, ergocalciferol and α-tocopherol) was investigated. Our results indicated that the applied technique has an impact on the solute order. Comparing the system capacity of HPLC and PPEC (measured as height of the theoretical plate) for the mobile phase systems with and without surfactant shows differences, especially for fat-soluble vitamin. The variances and reproducibilities (% RDS) values of the vitamin are less in PPEC than in TLC. Moreover, the migration distances of water-soluble vitamins are longer than fat-soluble ones. Overall, eluent consisting of 50% acetonitrile, 18.75 mM SDS, the buffer of pH 6.99 via the PPEC technique was most appropriate for determining the investigated vitamins in the artificial mixture and the two commercially available vitamin combinations.

Evaluation of Apium graveolens from different geographical origins based on TLC-fingerprint and chemometrics

Apium graveolens (celery) has various roles both in the food and medicine sectors. It grows very well in the tropical and subtropical areas of Africa and Asia, including Indonesia. This Apiaceae member contains a number of phytoconstituents, and geographical origin is known to significantly determine the type and concentration of phytochemicals in plant material. This study was carried out to validate and develop thin layer chromatography (TLC)-based fingerprinting combined with chemometrics, i.e., Principal Component Analysis (PCA) and Cluster Analysis (CA), to evaluate the quality of celery harvested from thirteen different geographical origins in Indonesia. The mobile phase was first optimized with a simplex axial design, resulting in 2-propanol, toluene, and dichloromethane (1:6:1) as the optimum mobile phase for a stable and precise TLC system in the celery sample analysis. When analyzed with chemometrics, the TLC-fingerprints could discriminate celeries from various origins. The PCA score plot of the first two principal components (PCs) and CA clearly distinguished the samples’ properties and classified them into four clusters. Samples grouped into one cluster were concluded to have comparable quality, while those in different clusters had different qualities.

MITRAGYNINE PERCENTAGES OF VARIOUS KRATOM VARIANTS SEIZED IN INDONESIA: A QUANTITATIVE ANALYSIS USING LIQUID CHROMATOGRAPHY-PHOTO DIODE ARRAY DETECTOR

Objective: This study details the determination of mitragynine in various kratom samples using the thin-layer chromatography (TLC) technique and validation of analytical methods for quantifying the concentration of mitragynine in various kratom samples using liquid chromatography with photodiode array detector (LC-PDA). Methods: TLC technique using n-hexane: ethyl acetate: ammonia 25% (30: 15: 1 v/v/v) was applied to isolate mitragynine from kratom samples. Several interesting spots obtained were visualized under UV light at 254 nm. Samples were also prepared with organic solvent extraction directly prior to LC analysis (non-isolation method of preparation) to quantify the concentration of mitragynine. Mobile phases used were acetonitrile (MP A) and 0.1% formic acid in water (MP B). Samples and standards were run by gradient elution with a flow rate of 0.3 ml/min, detection using PDA detector at 254 nm. Results: Mitragynine was successfully isolated from kratom samples in Rf 0.50 by TLC system applied. The validation of analytical methods for mitragynine passed the acceptance criteria as described by UNODC Guidance. The concentration of mitragynine in various kratom samples seized in Indonesia ranged from 0.37%-1.70% (%w/w). Conclusion: Both TLC and LC analytical methods could be applied to determine and quantify the concentration of mitragynine in each examined sample, respectively.

Validating and developing TLC-based fingerprinting for Curcuma longa L.

Context: Curcuma longa (turmeric) is extensively cultivated as spices and herbal medicines in tropical and sub-tropical countries. Geographical origin is known to significantly determine the quality of the herbs used and, consequently, the safety and efficacy of their products. Aims: To validate and develop TLC-fingerprint combined with chemometrics to differentiate C. longa collected from various origins. Methods: Thin Layer Chromatography (TLC) was employed together with chemometric methods, i.e., Principal Component Analysis (PCA) and Cluster Analysis (CA), to evaluate the quality of C. longa rhizomes collected from nine origins in Indonesia. Results: Chloroform, dichloromethane, and ethanol (64:64:1) were a suitable mobile phase for C. longa. The method used met the requirements for a stable and precise TLC system. As analyzed by the chemometric techniques, the TLC-fingerprints could discriminate C. longa from various origins. The PCA score plot of the first two principal components (PCs) and CA clearly distinguished two clusters of simples. Conclusions: When combined with PCA and CA, TLC-fingerprinting can discern the rhizomes of C. longa sourcedfrom various locations. TLC-fingerprints that are analyzed with chemometrics can be used as an alternative marker-oriented method for evaluating the quality of C. longa.

Polyaniline modified silica gel coupled with green solvent as eco favourable mobile phase in thin layer chromatographic analysis of organic dyes

This article studies a new green eco-friendly TLC (thin layer chromatography) using silica gel and polyaniline modified silica gel as stationary phase in combination with ethyl acetate (EA), n-butyl acetate (BA) and butane-1-ol (BO) solutions as mobile phase for the comparative study of migration behaviour of organic dyes to identify the most suitable thin layer chromatographic system for the resolution of co-existing dyes. Better separation efficiency was observed by modifying silica gel with polyaniline as compared to pure silica stationary phase. Densitogrpahic presentation of separations achieved on polyaniline modified silica gel Pani@SG-EB1 was also presented. The thin layer chromatographic system comprising of polyaniline modified silica gel Pani@SG-EB1 as stationary phase and n-butyl acetate:DDW, 5:5 as green mobile phase was observed to be the most favourable for the separation of various combinations of three or four-component mixtures of organic dyes viz. methyl thymol blue, tartrazine, carmoisine, rose bengal, amidoblack 10B, bromopyrogallol red and 4-nitrobenzene dizonium tetrafluoroborate. The effect of presence of cations and anions on separation trend was also examined and the limits of detection of the separated organic dyes were estimated. Fourier transform infrared spectroscopy (FTIR), x-ray diffraction (XRD), scanning electron microscopy (SEM) and transmission electron micrograph (TEM) studies were undertaken to characterize silica gel and modified silica gel (stationary phase). The developed method has been successfully applied for the identification of carmoisine in Solvin cold DS syrup and tartrazine in MefastTM syrup.

Pressurized planar electrochromatography of DNS amino acids derivatives in silica gel and silanized silica gel systems with formic acid addition to the water mobile phase

Pressurized planar electrochromatography (PPEC) of dansyl (DNS) derivatives of amino acids in normal- and reversed-phase systems is presented. The results have been obtained for mobile phases with different acetonitrile (ACN) concentrations (0–85%). The data obtained show differences in separation selectivity between high-performance thin-layer chromatography (HPTLC) and PPEC systems. These differences originate from the electrophoretic effect which is involved in the PPEC system, contrary to the HPTLC one.

Predictive retention study of β-blockers in different chromatographic systems as potential tool for lipophilicity screening based on QSRR approach

The chromatographic analysis of a number of β-blockers on different thin-layer chromatography (TLC) systems was carried out. The analysis was done on three different systems, namely normal phase, paraffin oil impregnated in methylene chloride and n-hexane as two types of reversed phase systems. This represents an economic and environmental approach pathway for the determination of four solvents systems of two components were used to get the retention parameters and the pH was adjusted using ammonia and glacial acetic acid. Quantitative structure–retention relationship (QSRR) was applied to the different phases and 315 molecular descriptors has been calculated for each β-blocker. A contingency analysis has been used to declare the highest correlation between the chromatographic retention and the calculated descriptors, which resulted in obtaining different models for each system used. The produced models correlate the predicted retention to the experimental one in a significant way which could be useful for the future behavior prediction of this group of β-blockers against normal and reversed phase TLC systems.

HPTLC study to determine the antioxidant activity of dried leaves of Portulaca oleracea L.

This present study involves the assessment of the anti-oxidant activity study of the sample which was obtained from the methanolic extracts of dried leaves of Portulaca Oleracea L.(common name Purslane). Purslane is a rich source of Vitamin A, Vitamin-C and some other B-complex vitamins like riboflavin, niacin, pyridoxine and carotenoids which are known powerful natural anti-oxidants. Anti-oxidants are compounds that inhibit oxidation. This methanolic extract of leaves was evaluated for the determination of its anti-oxidant efficiency by using 1,1–diphenyl-2-picryl-hydrazyl (DPPH) by using Silica TLC plates on Camag High-Performance Thin Layer Chromatography (HPTLC) system using visionCATS software. Densitograms and chromatographs obtained show the presence of anti-oxidant activity. It is a rapid, inexpensive and straightforward method to measure anti-oxidant properties of substances after separation by HPTLC. It involves the use of the free radical, 2, 2-Diphenyl-1- picrylhydrazyl (DPPH) which is widely used to test the ability of compounds to act as free radical scavengers or hydrogen donors and to evaluate anti-oxidant activity. When Anti-oxidants substances react with DPPH, which is a stable free radical becomes paired off in the presence of a hydrogen donor (e.g., a free radical scavenging anti-oxidant) and is reduced to the DPPHH. As a consequence, the absorbance's decreased from the DPPH.

Thin-Layer Chromatography.

Lipid extracts from plants represent a mixture of polar membrane lipids and nonpolar lipids. The main constituents of the polar lipid fraction are glycerolipids, that is, galactolipids, sulfolipid, and phospholipids. In addition, betaine lipids are found in pteridophytes, bryophytes, and algae. Nonpolar lipids include the storage lipid triacylglycerol, wax esters, diacylglycerol and free fatty acids. The complex lipid mixtures from plant tissues can be separated by thin-layer chromatography (TLC) into different lipid classes. In most cases glass plates coated with a silica gel are used as stationary phase and an organic solvent as mobile phase. Different solvent systems are required to separate polar membrane lipids or nonpolar lipids by TLC. Depending on the complexity of the lipid mixture, lipids are separated using one- or two-dimensional TLC systems. Different dyes and reagents allow the visualization of all lipid classes, or the selective staining of glycolipids or phospholipids. Lipids can be isolated from the TLC plate for subsequent analysis, provided that nondestructive methods are used for visualization.

An investigation on the enzyme inhibitory activities, phenolic profile and antioxidant potentials of Salvia virgata Jacq

The genus Salvia is regarded as an important medicinal plant and also as spice worldwide. Current report might be counted as one of the first investigations to reveal the enzyme inhibitory and antiglycation activities, together with phenolic and antioxidant profiles of 80% methanolic extract prepared from the aerial parts of S.virgata (SVM). Furthermore, the bioavailability parameters of major phenolic compounds in SVM were evaluated. An in vitro human digestion simulation model composed of both gastric and intestinal phases was used for this purpose. Total phenolic, phenolic acid and flavonoid components of each of these phases were calculated in order to assess the phenolic profile of the samples. In addition, quantitative analysis of major bioactive compounds (rutin and rosmarinic acid), before and after the simulation, was practiced by employing High Performance Thin Layer Chromatography (HPTLC) system. Moreover, the potential of non-digested (ND) and bioavailable (IN) phases on the inhibition of diabetes-related enzymes (α-amylase and α-glucosidase) was determined. Also, the inhibitory effect of the samples on the advanced glycation end products (AGEs), formed due to hyperglycemia, was detected. In addition, cholinesterase enzyme inhibition activity of both ND and IN samples was determined. Since, the role of oxidative stress on diabetes and neurodegenerative diseases is well-known, the antioxidant potential of the samples was also estimated by different methods: DPPH and DMPD tests for free radical scavenging activity, FRAP and CUPRAC tests for metal reducing activity and total antioxidant capacity test, as well. The results showed that total phenolic contents, antioxidant potential and diabetes-related and cholinesterase enzymes inhibition of the bioavailable phase were lower than non-digested sample. Besides, in vitro human digestion simulation system had a declining influence on the concentrations of the major bioactive metabolites (rutin and rosmarinic acid).