Performance Thin Layer Chromatography Research Articles

ANALYTICAL TECHNIQUES FOR DETERMINATION OF MIRABEGRON FROM BULK, PHARMACEUTICAL FORMULATION, AND BIOLOGICAL MATRICES: A CRITICAL REVIEW

Mirabegron is a beta-3 adrenergic receptor agonist and is specified for the treatment of overactive Bladder. This review covers analytical methods aimed at the identification and quantification of mirabegron in bulk, commercial dosage forms, and Biological fluids. Using various techniques such as UV-spectroscopy, spectro-fluorimetry, planer chromatography, High Performance-Thin Layer Chromatography (HPTLC), HPLC, High-Performance Liquid Chromatography-MS/MS (HPLC-MS/MS), Ultra-Pressure Liquid Chromatography-MS/MS (UPLC-MS/MS), and capillary electrophoresis. HPLC is the most used analytical technique for the identification and quantification of mirabegron in bulk and commercial dosage forms.

A Comparative Analysis of Churna and Ksheerapaka of Dried Root of Prishniparni- Desmodium Gangeticum (Linn.) DC. through High Performance Thin Layer Chromatography.

High Performance thin layer chromatography has become a potent analytical technique in the process of drug discovery. It serves as a tool for identification, authentication and quality control of herbal drugs due to its reliability and simplicity. Prishniparni-Desmodium gangeticum(Linn.) DC. is a significant and well-known drug expoundly discussed in the Ayurvedic classics. It is an erect diffusely branched undershrub with enriched therapeutic potential. The plant root is rich in flavonoids, iso flavonoids, cardiac glycosides, pterocarpans, alkaloids andsteroids which accounts for its multiple beneficial action in the therapeutics. It has strong antioxidant and cardioprotective properties. The churna(powder) and ksheerapaka(milk decoction) of dried root of Prishniparni was subjected to High Performancethin layer chromatography and comparative analysis of different peaks were obtained. The results suggest that change in phytochemical constituents and their quantity occur on changing the Kalpana (dosage form) from churna(powder) to ksheerapaka(milk decoction).

Simultaneous analysis of eprosartan and hydrochlorothiazide in tablet formulation by High- Performance thin layer chromatography with ultraviolet absorption densitometry

A new, simple, accurate, and precise high-performance thin-layer chromatographic method has been established for simultaneous analysis of Eprosartan and Hydrochlorothiazide from a tablet formulation. Standard and sample solutions of Eprosartan and Hydrochlorothiazide were applied to precoated 250 μm layer of silica gel G 60 F and the plates were developed with Chloroform: Acetonitrile: Glacial Acetic Acid (7:3:1,v/v/v) as mobile phase. Detection and evaluation of densitograms was performed densitometrically at 254 nm. The linear range was 200-700 ng/band with the retention factors of Eprosartan and Hydrochlorothiazide were 0.26± 0.02 and 0.44±0.02, respectively. The method was validated and successfully used for analysis of the drugs in tablets.

Quantitation of lupeol from stem bark extract of Betula alnoides Buch.-Ham. ex D.Don by two validated RP-HPLC and TLC-densitometric methods

To control the quality of Betula alnoides stem bark (BAS), lupeol, which is a promising compound found in BAS, has been determined by reverse phase high performance liquid chromatography (RP-HPLC) and thin-layer chromatography (TLC)-densitometry. These methods were developed and validated according to the International Council for Harmonization (ICH) and the Association of Official Agricultural Chemists (AOAC) guidelines. RP-HPLC was achieved within 10 min using C8 column under an isocratic elution of acetonitrile at a flow rate of 1.0 mL/min. Lupeol was detected at a UV wavelength of 200 nm. TLC-densitometry performed on TLC silica gel plates was developed by chloroform at 10 cm of distance. Lupeol was detected under visible wavelength at 525 nm after anisaldehyde-sulfuric acid derivatization. Both developed methods exhibited good linearity, specificity, limits of detection and quantitation, accuracy and precision, and robustness. The validated methods were effectively applied to determine lupeol content in BAS collected from three different locations. The amount of lupeol was found to be about 50–100 µg/g of dried powder. The results revealed no statistically significant differences between the two methods. These findings revealed the validated RP-HPLC and TLC-densitometry that were easily applied for routine quality control of BAS for the first time.

Caryatin and 3’-O-methylcaryatin contents in edible yams (Dioscorea spp.)

The genus Dioscorea hosts a large number of yam species used medicinally and as a staple food by millions of people in tropical countries. All yams share a high antioxidant activity that correlates with their total phenolics contents. However, knowledge of edible species antioxidant phenolics remains incomplete. Bioassay-guided fractionation of the tubers of a yam species with high total phenolics content, D. nummularia, led to the isolation of two flavonols, caryatin and 3′-O-methyl caryatin, contributing over 92% of a stable methanol extract of the tuber in this species. Their structures were determined by high resolution mass spectrometry (HRMS), nuclear magnetic resonance (NMR) and a synthesized authentic sample. They displayed no bacterial toxicity. Their content was estimated in the tubers and bulbils of 411 landraces and hybrids belonging to eight food yam species using high performance-thin layer chromatography (HP-TLC). These were found to be highly variable at the intra- and interspecific levels to reach up to 1 030 and 457 μg/g DW respectively and added up to contents of already described antioxidant phenolics. This study provides additional understanding of the health benefits of a yam diet and constitutes the first phytochemical analysis of D. nummularia.

Statistical optimization of in vitro callus induction of wild and cultivated varieties of Mucuna pruriens L. (DC.) using response surface methodology and assessment of L-Dopa biosynthesis

Mucuna pruriens L. (DC.), is a valuable, economically significant medicinal crop, known worldwide for producing the anti-Parkinson's drug Levodopa (L-Dopa). Itching property of the plant poses certain constrains in its conventional breeding, but, in vitro cell culture systems offer advantage in which controlled conditions can be used for the production of valuable compounds. In the current investigation, a simple and efficient method for callus induction from the wild (var. pruriens) and cultivated (var. utilis) varieties of M. pruriens is described. Juvenile leaves and roots obtained from 30 day old aseptically grown seedlings were used as explants for callus induction. The leaves were found to be the best explants for obtaining maximum callus within a short period of time in 2.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) supplemented Murashige and Skoog (MS) medium. White compact root callus was produced from var. utilis in 2,4-D (2.0 mg/L) and indole 3-acetic acid (0.2 mg/L) + benzyl amino purine (2.0 mg/L) containing media. Whereas, callus formation was not observed from root explants of var. pruriens. Central composite design–response surface methodology (CCD-RSM) experiment was designed to analyze the linear-model and quadratic model (including pair-wise and interactive effects) of each variable to study the maximum biomass production from leaf and root explants. In this study, 1.5 mg/L concentrations of BAP as well as 2,4- D in plant tissue culture medium resulted in maximum callus biomass (2.378) and L-Dopa % yield (0.635 %). L-Dopa content in these varieties was investigated by high performance-thin layer chromatography (HP-TLC) using the methanolic extract prepared from callus cultures of both the varieties and compared with natural plant parts. L-Dopa content was found to be higher in seeds obtained from the naturally grown plants of var. pruriens followed by var. utilis (> 0.55 % and 0.494 % respectively). From the result obtained it can also be ascertained that root callus can serve as a source of L-Dopa extraction commercially.

Detection and isolation of flavonoid and aromatic acid from Cynara scolymus different parts cultivated in iraq

The target of this study was to study the natural phytochemical components of the head (capsule) of Cynara scolymus cultivated in Iraq. The head (capsule) of plant was extracted by maceration in70% ethanol for 72 hours, and fractioned by hexane, chloroform and ethyl acetate. Preliminary qualitative phytochemical screening was performed on the ethyl acetate fraction for capsule was revealed the presence of flavonoid and aromatic acids. These were examined by (high -performance liquid chromatography) (HPLC diodarray), (high- performance thin-layer chromatography)(HPTLC). Flavonoids were isolated by preparative layer chromatography and aromatic acid was isolated by preparative high-performance liquid chromatography HPLC from the ethyl acetate fraction of capsule. Then identified by High Performance Thin Layer Chromatography HPTLC, High performance liquid chromatography HPLC diode array , ultraviolet diode array UV-diode array and Liquid Chromatography /Mass Spectroscopy LC/MS. The chloroform fraction from the capsule was evaluated by Gas Chromatography//Mass Spectrometer(GC/MS). The different chromatographic and spectroscopic techniques revealed the presence of luteolin, apigenin and cinnamic acid in capsule of Cynara scolymus, also 9-octadecanoic acid (oleic acid), Oxalic acid, allyl tetradecyl ester, limonene, in chloroform of Cynara scolymus. The results of the current study proved the presence of luleolin, apigenin, and cinnamic acid in the ethyl acetate fraction of Cynara scolymus capsule.

LIPIDS, FATTY ACIDS AND PIGMENTS OF LEAVES DICTAMNUS CAUCASICUS FISCH. EX GROSSH. (RU-TACEAE)

Plants of the genus Dictamnus are a source of chemical compounds with different biological activity – alkaloids, bitterness, furocoumarins, saponins, essential oils. In the present work, the qualitative and quantitative composition of components lipids, fatty acids, and pigments isolated from the leaves of Dictamnus caucasicus Fisch. ex Grossh. – is presented for the first time, also of interest for medicine and pharmacology. Plants were selected in the Samara region, on the territory of which D. caucasicus is currently under threat of extinction. Plant extracts were analyzed by high performance thin-layer and gas-liquid chromatography. Identification of polar lipids was carried out using specific reagents – anthrone reagent, molybdenum blue, Dragendorff’s reagent, 0.2% ninhydrin solution. Neutral lipids were identified using an mixture of standards – triacylglycerol (TAG), oleic acid (FFA), cholesterol (ST), diacyglycerol (DAG). It was shown that the main group of lipids contains carbohydrate fragments – glycolipids as structural components. Among phospholipids, a high content of phosphatidylglycerol was found, and in neutral lipids – free and esterified forms of sterols. Polyunsaturated linolenic acid (C18: 3n3) dominates in the composition of fatty acids, and chlorophyll a dominates in the pigment pool.

Development of Validated Stability Indicating HPTLC Method for the Estimation of Teriflunomide in Bulk and Tablet Dosage Form

Background: Teriflunomide is an immunosuppressive agent. Immunosuppressive agents are drugs that inhibit or prevent activity of immune system. Teriflunomide was investigated as a medication for multiple sclerosis (MS). Various studies have reported the HPLC, UPLC, LC/MS methods for the estimation of teriflunomide. However, till date stability indicating HPTLC analysis method has not been reported for the estimation of teriflunomide in bulk and tablet dosage form. Objective: Objective of the present work was to develop and validate stability indicating high performance thin layer chromatography method for the determination of teriflunomide in bulk and tablet dosage form. Methods: Chromatography was performed on aluminium plates coated with silica gel 60F254 using toluene, methanol and acetic acid (7.5: 2.5: 0.05 v/v/v) as mobile phase. Densitometric analysis was performed at 254 nm. The method was validated with different parameters such as linearity, precision, accuracy, specificity, robustness, limit of detection (LOD) and limit of quantitation (LOQ). The RF value of teriflunomide was 0.56 ± 0.03. The method is sensitive (limit of quantification 17.83 ng/band), precise (RSD ≤ 1.34%), accurate (drug recovery 98.49–99.53 %), and linear over the range 100–600 ng/band (r2 0.998). Results: Degradation products were found in stress conditions did not interfere with the detection of teriflunomide; therefore, the proposed technique can be considered stability-indicating. Teriflunomide did not degrade under alkaline hydrolysis, thermal and photolytic conditions but showed degradation under acid hydrolysis and oxidation with about 14.5 and 13.8 % decompositions respectively. Conclusion: The developed method was satisfactorily applied for the analysis of pharmaceutical preparations and proved to be specific and accurate for quality control of the cited drugs in their tablet dosage form.

Amelioration of Dementia and Antioxidant Activity of Holarrhena antidysenterica Bark in Mice

Background: Holarrhena antidysenterica is a deciduous shrub/small-tree having bioactive alkaloids such as conessine, known for astringent, antidysenteric, anthelmintic, stomachic, febrifugal, tonic and anti-acetylcholinesterase properties. Dementia is an age related neurodegenerative syndrome with Alzheimer’s disease, the most prominent cause, which has limited therapeutic options available. Objective: The present study aimed to explore the potential benefits of H. antidysenterica in the management of dementia. Method: Aqueous and methanol extracts of powdered bark of H. antidysenterica were prepared, concentrated and conessine concentration was estimated using High Performance Thin Layer Chromatography (HPTLC) method. Methanol extract of H. antidysenterica (MEHA) was administered at doses 100, 150 and 200 mg/kg (i.p.) to mice (20-30 g) for 14 consecutive days. Scopolamine (Scop; 1 mg/kg) and Lipopolysaccharide (LPS; 250 μg/kg) were given (i.p.) before behavioural trials to induce memory impairment. Learning and memory functions in mice were evaluated. Brain acetylcholinesterase (AChE) activity, glutathione (GSH) and Thiobarbituric Acid Reactive substances (TBARS) levels were estimated. Results: MEHA markedly increased learning and memory of mice. Scop or LPS caused a significant decline of spatial memory in mice, which was attenuated by MEHA (100 and 200 mg/kg). Furthermore, LPS conspicuously increased the lipid peroxidation and compromised antioxidant levels in mice brains. MEHA pre-treatment significantly increased GSH content and decreased TBARS level in the brain of LPS administered mice. AChE activity was significantly decreased by MEHA in the brain of mice. Conclusion: The methanol extract of H. antidysenterica may prove to be a useful remedy in the management of dementia.

Comparison of UV- and Derivative-Spectrophotometric and HPTLC UVDensitometric Methods for the Determination of Amrinone and Milrinone in Bulk Drugs

Background: Spectrophotometry and thin layer chromatography have been commonly applied in pharmaceutical analysis for many years due to low cost, simplicity and short time of execution. Moreover, the latest modifications including automation of those methods have made them very effective and easy to perform, therefore, the new UV- and derivative spectrophotometry as well as high performance thin layer chromatography UV-densitometric (HPTLC) methods for the routine estimation of amrinone and milrinone in pharmaceutical formulation have been developed and compared in this work since European Pharmacopoeia 9.0 has yet incorporated in an analytical monograph a method for quantification of those compounds. Methods: For the first method the best conditions for quantification were achieved by measuring the lengths between two extrema (peak-to-peak amplitudes) 252 and 277 nm in UV spectra of standard solutions of amrinone and a signal at 288 nm of the first derivative spectra of standard solutions of milrinone. The linearity between D252-277 signal and concentration of amironone and 1D288 signal of milrinone in the same range of 5.0-25.0 μg ml/ml in DMSO:methanol (1:3 v/v) solutions presents the square correlation coefficient (r2) of 0,9997 and 0.9991, respectively. The second method was founded on HPTLC on silica plates, 1,4-dioxane:hexane (100:1.5) as a mobile phase and densitometric scanning at 252 nm for amrinone and at 271 nm for milrinone. Results: The assays were linear over the concentration range of 0,25-5.0 μg per spot (r2=0,9959) and 0,25-10.0 μg per spot (r2=0,9970) for amrinone and milrinone, respectively. The mean recoveries percentage were 99.81 and 100,34 for amrinone as well as 99,58 and 99.46 for milrinone, obtained with spectrophotometry and HPTLC, respectively. Conclusion: The comparison between two elaborated methods leads to the conclusion that UV and derivative spectrophotometry is more precise and gives better recovery, and that is why it should be applied for routine estimation of amrinone and milrinone in bulk drug, pharmaceutical forms and for therapeutic monitoring of the drug.

Haematocarpus validus(Miers) Bakh.f.ex Forman (Khoon phal): An ethnomedicinal fruit crop rich in choline

Haematocarpus validus, a lesser known rare ethnic fruit crop of high nutraceutical and therapeutic value, is used as a hepatoprotective and anti-inflammatory agent in traditional medicine. The study aimed to estimate choline content of methanolic fruit and leaf extract of H. validus for the first timeusing High Performance-Thin Layer Chromatography spectrodensitometric analysis. Choline is an essential nutrient for liver health and it reduces fatty liver build up. For separation and quantitation of choline from methanolic fruit and leaf extract, methanol:water (8:2, v/v) was found suitable as mobile phase. Choline bitartrate was used as reference compound for the preparation of standard curve (r= 0.97874; Y = 8.624 0.080X; SDV = 9.35 %). For fruit extract the amount of choline present in the sample was estimated as 197.81 mg/100g d.wt from the peak area of spectrodensitogram. Choline content was found to be very high in leaf extract (in both defatted and non-defatted leaf extract) and it was >500 mg/100g d.wt. The high content of choline in leaf samples of H. validus substantiates the ethnomedicinal use of its leaf and shoot extract as a hepatoprotective agent.

Highly-porous diatom biosilica stationary phase for thin-layer chromatography

This study showed that a nanostructured, highly-porous stationary phase composed of randomly-deposited biosilica frustules isolated from living cells of diatom Pinnularia sp. significantly improved the conventional thin-layer chromatography (TLC) based separation of the triphenylmethane dyes malachite green and fast green relative to silica gel by two mobile phases (9:1:1 v/v 1-butanol:ethanol:water, 5:1:2 v/v 1-butanol:acetic acid:water). Although both stationary phases were composed of amorphous silica rich in silanol groups with particle size of 10–12 μm, diatom biosilica frustules were highly porous, hollow shells with surface structure dominated by 200 nm pore arrays. Diatom biosilica significantly improved the mobility of both malachite green and fast green, enabling the resolution of these analytes. The diatom biosilica layer had a high void fraction of 96% but reduced the flow velocity and permeability constant by a factor of two relative to silica gel. TLC performance was enhanced, as evidenced by ten-fold reduction in theoretical plate height for both analytes using the 1-butanol:acetic acid:water mobile phase, and an increased difference in retention time between malachite green and fast green (ΔRf = 0.26) using the 1-butanol:ethanol:water mobile phase. Analysis of plate height vs. solvent front position by the modified van Deemter equation suggested that dispersive mass transfer was reduced, leading to improved analyte resolution, and that surface of the frustule decreased boundary layer resistance, leading to increased analyte flux. Overall, the basis for improved chromatographic performance is believed to be the unique nano- and microstructure of the diatom biosilica frustule.

Study of Antioxidant Activity and Composition of Cyanobacteria Metabolites by TLC, HPTLC, and HPLC for the Search of Environmentally Safe Cleaning Agents

The composition of metabolites of cyanobacteria cultures of the Astrakhan region has been studied by thin-layer chromatography, high-performance thin layer chromatography, and high-performance liquid chromatography. Thin layer chromatography analysis of the cyanobacteria metabolites has shown the best separation and quality of chromatographic zones in seven systems: chloroform, ethanol: acetic acid (1: 1), butanol, ethanol: water (1: 9), butanol: acetic acid: water (4: 1: 5), ethanol: water (9: 1), and butanol: acetic acid: water (4: 1: 5). The flavonoid quercetin has been detected in an aqueous–alcoholic extract by high -performance thin-layer chromatography. The high-performance liquid chromatography has allowed identification of aspartic, isomonic, lactic, and fumaric acids in the cyanobacteria metabolites. Antioxidant activity of aqueous and aqueous-alcoholic extracts of the Phormidium ramosum B.-Peters IPPAS B-2022 culture has been detected.

Identification of an isoflavone glycoside hydrolyzing b-glucosidase in the seeds of the desert legume Prosopis cineraria

Legumes, in general, are rich in glycosidic isoflavone conjugates, whereby specific b-glucosidases act to release the free aglycones that could serve in plant-microbe interactions and defense. Prosopis cineraria is an important legume tree, bearing medicinally useful and edible pods, generally growing in extreme arid/semi-arid zones of Rajasthan. However, specific phytochemical-cum-metabolic profiles are lacking for the same. Therefore, the present investigation undertook phytochemical and metabolic screening of the pods/seeds of P. cineraria for the presence of putative isoflavonoids viz. genistein and daidzein, their glycosides and b-glucosidase(s) capable of catalyzing the glycoside hydrolysis. Extraction and identification of these two aglycone forms of the above isoflavonoids were performed with solvent partition chromatography and Fluorescent/High Performance Thin Layer Chromatography, respectively. Furthermore, optimization of the isoflavone conjugate-specific b-glucosidase activity with respect to pH optima, temperature and time were carried out. The partially purified enzyme showed a temperature optima of 50°C and pH optima of 4.5. The enzyme also demonstrated activity towards natural substrates daidzin and genistin which are glycosides of isoflavonoids daidzein and genistein respectively. The methanolic extracts of the seeds of P. cineraria indicated the presence of related isoflavonoids which needs to be further validated.

Abstract 4208: Development of 18F-IL2: a PET radiotracer for imaging activated T-cells

Abstract Introduction: Activation of T-cells is accompanied by a strong up-regulation of interleukin-2 (IL2) receptor (CD25). Therefore PET imaging of IL2 receptors might be a suitable imaging biomarker for T-cell activation. 18F-IL2 PET could detect CD25-positive T-cells and the migration of these T-cells to distant sites of inflammation in SCID mice subcutaneously injected with human peripheral blood mononuclear cells1 and NOD mice with insulitis. Also a strong correlation was found between the accumulation of 18F-IL2 and the number of injected activated T-cells in immune-competent rats.2 In tumor bearing mice, 18F-IL2 PET could detect treatment-induced accumulation of activated T-cells in the tumor following local radiotherapy and/or vaccination. Cancer immunotherapy is increasingly obtaining a place in clinical practice. However not all patients benefit. A biomarker for upfront or early response prediction for these immunotherapies might support patient selection before and during therapy. Potentially 18F-IL2 PET might serve this purpose. Therefore we aimed to accommodate the production of 18F-IL2 for use in clinical imaging studies. Material and methods: In order to produce a GMP-compliant tracer the production is being implemented on the Eckert & Ziegler PharmTracer synthesis module. In this synthesis module, disposable cassettes, reactors and vials are used to avoid cross-contamination between productions. First the prosthetic group N-succinimidyl 4-fluorobenzoate (18F-SFB) is produced in 3 steps from cyclotron-produced 18F-fluoride. Subsequently, 18F-SFB is conjugated to human recombinant IL2 (Proleukin®). Various methods for synthesis and purification of 18F-SFB have been evaluated. Also purification of 18F-IL2 has been optimized. Quality control has been performed using ultra performance liquid chromatography (UPLC) and Thin Layer Chromatography (TLC). Results: 18F-SFB was successfully synthesized with the Eckert & Ziegler PharmTracer synthesis module with decay-corrected radiochemical yields comparable to literature (range 28-64%). Major challenges have been encountered, most importantly regarding the purification of the 18F-SFB and 18F-IL2, stability of the IL2 and specific activity. The activated ester 18F-SFB was purified by high performance liquid chromatography (HPLC) to remove any impurities that could interfere with the conjugation. 18F-IL2 has been purified using PD-10 columns with PBS containing 0.05% SDS as mobile phase. Conclusions: Several challenges for the GMP-compliant production of 18F-IL2 have been overcome. In the near future this tracer will be used in preclinical and clinical studies to non-invasively image activated T-cells before and during cancer immunotherapy. This can provide insight in the effects of cancer immunotherapy on the immune response.

Influence of Two Cyclodextrin HP-derivatives on Retention and Migration Distance of Some Isomers in High- Performance Thin-Layer Chromatography and Pressurized Planar Electrochromatography Systems with Non-polar Adsorbents

Separation of several isomer groups, i.e. diastereomers such as quinine and quinidine, cinchonine and cinchonidine, R,S-1-(1-naphthyl)-ethanol, R,S-1-(2-naphthyl)-ethanol, and R,S-1,1’- binaphthyl-2,2’-diamine racemates by means of high-performance thin layer chromatography (HPTLC) as well as pressurized planar electrochromatography (PPEC) techniques and carried out with the use of HPTLC RP-2, HPTLC RP-8 and HPTLC RP-18W plates as well as the acetonitrile - buffer mobile phase with hydroxypropyl-β-cyclodextrin (HP- β-CD) or hydroxypropyl-γ-cyclodextrin (HP-γ- CD) as additives in the mobile phase is presented. The influence of such variables as HP-β-CD concentration in the mobile phase, type of the used adsorbent and dimension of cyclodextrin interior (HP-β-CD and HP-γ-CD) on the migration distance of the solute zones is investigated. It has been observed that an increase in the HP-β-CD concentration brings about an opposite effect in the compared techniques and retention of the solutes diminishes in HPTLC, whereas the migration distances of the majority of isomers decrease in PPEC. The type of applied HP-cyclodextrin has a stronger effect on the separation selectivity of compounds in the PPEC system in comparison with the HPTLC system. Since the isomer differentiation mechanism in the PPEC technique is possible due to the two effects (partition and electrophoresis), whereas the HPTLC system employs only partition, changes in the separation selectivity of solutes in both investigated systems are expected and the former system turns out to be better at distinguishing between enantiomers in the R,S-1-(1-naphthyl)-ethanol and R,S-1-(2-naphthyl)-ethanol mixtures in comparison with the HPTLC-system.

DEVELOPMENT AND VALIDATION OF HPTLC METHOD FOR ESTIMATION OF OLEANOLIC ACID IN ARISTAKA

A Simple, monetary, reproducible, precise High performa nce thin layer chromatography (HPTLC ) method was us ed to determine the content of Oleanolic acid in methanolic extract of Sapindus trifoliatus Linn. using Toluene: Ethyl acetate: Formic aci d (7:2:1 ) as mobile phase. Quantificatio n was performed by ƛ= 529 nm. The validated method showed linear response over conc. range of 400 - 1200 ng. The limit of detection was noticed to be of 10 ng and was stastically tested for repeatability by interday and intraday precision test as per ICH gui delines. Method was found Precise, accurate, specific and recoverable.

Detection and quantification of quinolone signalling molecule: A third quorum sensing molecule of Pseudomonas aeruginosa by high performance-thin layer chromatography

Sophisticated network of quorum sensing involves the production of chemical signals which regulate the combined expression of virulence genes and biofilm formation in Pseudomonas aeruginosa. Two well-characterized acyl homoserine lactone based las and rhl systems together with alkyl quinolone based Pseudomonas quinolone signalling (PQS) are fundamental components of this network. Third signalling molecule, 2-heptyl-3-hydroxy-4-quinolone (PQS) is of paramount importance because of its interconnecting role in quorum sensing hierarchy in P. aeruginosa. Accurate detection of PQS molecule is very important to understand the involvement of this system in infection process of P. aeruginosa. In this study, high performance-thin layer chromatography (HP-TLC) method was developed for detection as well as quantification of PQS signal molecules in P. aeruginosa, which combines conventional method like TLC with sophisticated instrumentation. This method was validated using parameters like linearity, accuracy, precision, reproducibility and sensitivity. Intra- and inter-day accuracy and precision values were determined which were found to be within acceptable level and hence showed reproducibility. Measurement of PQS in the range of 0.01nmol indicated excellent sensitivity of this approach for quantifying PQS molecule. Automated sampling, rapid and simultaneous analysis of large number of samples and minimal errors make this method more suitable for analysis of PQS signalling molecules. Production of PQS was found to be strain dependent since variation in amount of PQS was observed among different P. aeruginosa isolates. Further, PQS production was also dependent on growth phase of P. aeruginosa with maximum production in late stationary phase.

Free radical scavenging activity, kinetic behaviour and phytochemical constituents of Aristolochia clematitis L. roots

Nowadays there is an increasing demand of natural antioxidants. Imbalanced production and consumption of reactive oxygen species, lead to many diseases such as cancer, arteriosclerosis and ageing processes. The protective effects of plants have long been attributed to their antioxidant compounds, like polyphenols, flavonoids, carotenoids, and vitamins C and E. Therefore, the present study was designed to evaluate the antioxidant activity of tannins and crude methanolic extracts of Aristolochia clematitis L. by two complementary test systems (DPPH free radical scavenging and bioautography HPTLC). Besides, phytochemical screening was carried out on roots of A. clematitis and showed the presence of tannins, alkaloids and essential oils.High performance thin layer chromatography (HPTLC) screening provided qualitatively the antioxidant effect of extracts under study. Furthermore, it was found that the tannins and methanolic crude extracts from A. clematitis had a potent DPPH scavenging potency with IC50 values of 0.196 and 0.142mg/mL, respectively. Besides, the kinetic behaviour of DPPH radical scavenging activity of extracts under study allowed us to determine the half life t1/2, time reaction (t) and the remaining DPPH percent.