TLC-densitometric Method Research Articles

Evaluation of the Stability of the Chromatographic Bands in the TLC-Densitometric Analysis of Selected Pharmaceutical Important Phytosterols and α-tocopherols

In this study the stability of the chromatographic bands of selected phytosterols (stigmasterol, β-sitosterol) and also some vitamins (α-tocopherol and α-tocopherol acetate) obtained by means of thin-layer chromatography combined with densitometry was investigated. The influence of different visualizing reagents such as methylene violet, methyl green, malachite green and the time of storage of the chromatograms since visualization process until their densitometric recording on the properties of obtained chromatographic bands of studied compounds were accurately described. The results revealed that in order to obtain reliable results of TLC-chromatographic analysis of examined drug substances, there is a need to keep strictly the same procedures during conducted TLC-densitometric analysis especially the time of storage of the TLC chromatograms since visualization until their densitometric scanning. The presented TLC-densitometric method can have the advantage in term of stability-indicating method for the determination of phytosterols and also α-tocopherols as drug substances as well as pharmaceutical formulation components.

Chromatographic-Densitometric Analysis of Chosen Fluoroquinolones on TLC Plates Using Mobile Phases with Different Viscosity

TLC-densitometric method was developed for the identification and quantitative determination of selected fluoroquinolones. Silica gel TLC 60 F254 plates were used as the stationary phase. For the separation of examined compounds the suitability of the following four mobile phases with different dynamic viscosity was checked: (I) methanol:ammonia:ethyl acetate:acetonitrile 1:1:2:1 v/v/v/v (1.70 mPa · s), (II) dichloromethane:methanol:isopropanol:ammonia 4:4:5:2 v/v/v/v (2.08 mPa · s), (III) dichloromethane:ethanol:toluene:ammonia:n-butanol:water 6:6:2:1.8:3:0.3 v/v/v/v/v/v (2.27 mPa · s), (IV) 1,4- dioxane:ammonia:tetrahydrofuran 6:3:2 v/v/v (3.24 mPa · s). The calculated values of retardation factors RF, capacity factors k, selectivity factors α, resolution factors Rs, and also asymmetry factors As for the evaluation of separation conditions were used. The obtained results confirmed that dynamic viscosity of the mobile phases is essential and influences the retention parameters of the studied fluoroquinolone chemotherapeutics. The validation results for the mobile phase IV indicate its suitability for the identification and also quantitative analysis of selected drugs in pharmaceutical analysis.

Screening of antidepressant activity and estimation of quercetin from Coccinia indica using TLC densitometry

Context: Coccinia indica Naud (Cucurbitaceae) has been traditionally used for the treatment of depression but these claims have not been validated.Objectives: The objective of this study is to investigate antidepressant activity of various extracts and fractions of C. indica aerial parts, and to estimate content of quercetin in the plant using TLC densitometry.Materials and methods: Coccinia indica aerial parts were successively extracted using solvents in increasing order of polarity, namely n-hexane, chloroform, methanol, and water. Various extracts were evaluated for antidepressant activity at doses of 200 or 400 mg/kg, p.o., upon acute administration in mice using the forced swim test (FST). The bioactive extract was partitioned successively using solvents in increasing order of polarity, namely n-hexane, ethyl acetate, and n-butanol. All fractions were also screened for antidepressant activity at doses of 25 or 50 mg/kg, p.o., upon acute administration in mice.Results: The methanol extract significantly reduced the duration of immobility in FST at dose of 400 mg/kg without affecting locomotor activity in open field test, thus, confirmed its antidepressant activity, which was statistically equivalent to the standard drug (imipramine, 15 mg/kg, i.p.). Ethyl acetate fraction (EAF) exhibited antidepressant activity at 50 mg/kg. Comparative TLC fingerprint studies confirmed the presence of quercetin in methanol extract and EAF. Quercetin was used as a chemical marker to standardize C. indica aerial parts using the validated TLC densitometric method, and the content of quercetin was found to be 0.00172% w/w.Conclusions: The present studies scientifically validated traditional claims of C. indica for antidepressant activity.

Development and validation of a TLC-densitometric method for the quantitation of Glycyrrhizinic Acid in herbal formulation

Development and validation of a TLC-densitometric method for the quantitation of Glycyrrhizinic Acid in herbal formulation

A simple TLC-densitometric method for the quantification of acrylic acid in aqueous solutions

Because acrylic acid is a volatile organic compound, the precise quantification of acrylic acid by thin-layer chromatography (TLC) analysis is difficult. Herein, we report a simple TLC–densitometric method for the quantification of acrylic acid in aqueous solutions following a labeling procedure using the Mizoroki-Heck coupling reaction. To make acrylic acid nonvolatile and highly sensitive for ultraviolet (UV) detection, acrylic acid in an aqueous solution was reacted with iodobenzene in a dimethyl sulfoxide solution using a base (potassium carbonate) and a catalyst (FibreCat 1001), affording trans-cinnamic acid. High-performance liquid chromatography (HPLC) analysis showed that acrylic acid could be labeled over a range of 0.01–10 mM at 60°C for 30 min and the average reaction yield was 96.0%. To quantify acrylic acid by TLC analysis, the reaction mixture was extracted using ethyl acetate and then spotted on a silica gel 60 F254 TLC plate. The spots corresponding to the labeled acrylic acid over a range...

A Validated TLC-Densitometric Method for the Determination of Mesterolone in Bulk Material and in Tablets.

Mesterolone is a synthetic androgenic steroid indicating a weak anabolic activity. A new, simple in use, and economical TLC-densitometric method in normal phase system (NP-TLC) has been developed and validated for the identification and quantitative determination of mesterolone in bulk drug and in tablet formulation. NP-TLC analysis was performed on aluminium plates precoated with silica gel 60F254 as the stationary phase using chloroform-acetone (40 : 10, v/v) as mobile phase. Densitometric analysis was carried out at λ = 745 nm after staining with phosphomolybdic acid. These conditions were found to give visible (dark blue) spot and sharp peak, respectively, for mesterolone at R F 0.75 ± 0.02 and enabled satisfactory separation of mesterolone from its related substance (potential impurity). The proposed NP-TLC-densitometric method was validated for specificity, linearity, precision, accuracy, robustness, and sensitivity according to ICH guideline and other validation requirements. The limit of detection (LOD) and limit of quantification (LOQ) were 61.0 ng·spot−1 and 184.0 ng·spot−1, respectively. The percent content of mesterolone in marketed tablet formulation was found to be 99.40% of label claim. The developed TLC-densitometric method can be successfully used in quality control of mesterolone in bulk material and also tablet formulation.

Post-derivative TLC densitometric stability indicating assay for mesterolone and quantum chemical calculations

A post-derivative TLC densitometric method was developed for the quantification of mesterolone with aid of quantum chemical calculations.

The HPTLC Validated Method Development for the Quantification of Naringin from the Partially Purified Labisia Pumila Dichloromethane Extract

Partially purified fraction E, dichloromethane extract of leaves from Labisia Pumila has been shown to possess anti-ulcer, anti-inflammatory, anti-asthmatic activities among others. Naringin has been identified by LC-MS and HPLC as the major compound present in fraction E. The present study reports the development and validated TLC densitometric method for the quantification of Naringin present in Fraction E. ICH guidelines were followed to develop this method. CAMAG-HPLTC system comprising of a TLC Visualizer and Linomat 5 sample applicator was used in this study. The separation was performed using TLC aluminum plates pre-coated with silica gel 60 F254. Optimized mobile phase consisted of Methanol: Ethyl Acetate (60:40 v/v). Win-CATS-V 1.2.3 software and Video Scan were used to identify and quantify Naringin at 366nm in fluorescence mode. The peak heights at Rf 0.648 ± 0.01 gave a linear range from 200-1000μg/ml correlation coefficient R2± SD = 0.973 ± 0.024. The LOD and LOQ were found to be Naringin 0.74 ± 0.29 μg and 7.73 ± 0.26 μg, respectively. Repeatability gave CV % < 2.0. We recovered 92.56% of Naringin and 642.4 mg/g (64.24%) was found to be present in Fraction E. The HPTLC method was found to be reproducible, accurate and convenient for rapid screening of bioactive constituents present in Fraction E. This developed method will be used for analysis and quality control of drug formulations containing Labisia Pumila.

Application and validation of an eco-friendly TLC-densitometric method for simultaneous determination of co-formulated antihypertensive agents

Application and validation of eco-friendly TLC-densitometric method for simultaneous determination of co-formulated antihypertensive agents.

Stability-indicating chromatographic determination of hydroquinone in combination with tretinoin and fluocinolone acetonide in pharmaceutical formulations with a photodegradation kinetic study

Stability indicating HPLC and TLC-densitometric methods for the determination of hydroquinone, tretinoin, fluocinolone acetonide, their degradation products and preservatives.

TLC densitometric method for screening of lycopsamine in comfrey root (Symphytum officinale L.) extracts using retrorsine as a reference compound.

Due to severe toxicity of pyrrolizidine alkaloids, their quantification in medicinal products is very important. The idea of this research was to use retrorsine as a surrogate reference compound instead of lycopsamine reference or lycopsamine isolated from comfrey. A method for the analysis of lycopsamine in extracts of comfrey roots was developed and validated, employing thin layer chromatography, derivatisation with Dann-Mattocks reagent followed by densitometric analysis. The new method showed linearity within 0.70 to 7.0 μg of lycopsamine per application of 10 μL of a solution. It has also been proven to be specific and precise (repeatability RSD 2-4 % within the plate). The method was successfully employed for quantification of lycopsamine in comfrey root and comfrey root medicinal products such as ointments.

Simultaneous identification and quantitative determination of azithromycin, clarithromycin, roxithromycin, spiramycin and troleandomycin by thin-layer chromatography and densitometry

The purpose of this work was to develop a TLC-densitometric method for simultaneous identification and quantitative determination of azithromycin, clarithromycin, roxithromycin, spiramycin, and troleandomycin. The method was developed on TLC aluminium plates precoated with silica gel F254 using solvent system izopropanol:n-hexane:ammonia 25% (8:12:3, v/v/v), which gives compact spots for azithromycin (RF = 0.65), clarithromycin (RF = 0.54), roxithromycin (RF = 0.49), spiramycin (RF = 0.22), and troleandomycin (RF = 0.36). Densitometric analysis was carried out at 478 nm after spraying with (1:4, v/v) sulphuric acid:ethanol and heating at 100°C for 5 min. The linear regression analysis data for the calibration plots showed good linear relationship with correlation coefficient higher than 0.99. The method is distinguished by high sensitivity, with limit of detection (LOD) from 0.34 μg/spot for troleandomycin to 0.67 μg/spot for clarithromycin and limit of quantification (LOQ) from 1.02 μg/spot for troleando...

Two validated liquid chromatographic methods for the simultaneous determination of flumethasone pivalate, its related substance (flumethasone), and clioquinol

Two liquid chromatographic methods were developed and validated. Simple and sensitive thin-layer chromatography (TLC)-densitometric and high-performance liquid chromatographic (HPLC) methods were used for the simultaneous determination of flumethasone pivalate (FP), flumethasone pivalate related substance and impurity, flumethasone (FL), and clioquinol (CL). The proposed TLC-densitometric method has been developed using silica gel plates 60 F254 as a stationary phase with benzene-hexane-acetone-formic acid (5:4:2:0.13, by volume) as a developing system followed by densitometric measurements at 235 nm. The studied components were quantified in the range of 0.3–4, 0.3–3, and 1.5–5 μg band−1, respectively. For HPLC method, chromatographic separation was achieved within 11 min with the required peak symmetry, accuracy, and precision on ODS column using acetonitrile-water (70:30, v/v) as the mobile phase at a flow rate of 1 mL min−1 with ultraviolet (UV) detection at 235 nm. The calibration plots were linear o...

Estimation of Isolated Triterpenoid – Ursolic Acid in Verbena officinalis L. Aerial Parts Using TLC Densitometry

Verbena officinalis L. (Vervain; family-Verbenaceae),a traditionally used and medicinally promising plant, has been reported to contain triterpenoids as major class of phytoconstituents. But no work has ever been carried out on V. officinalis to isolate major chemical constituent(s), and to standardize plant material on the basis of isolated constituent(s) using TLC densitometry.The chloroform extract of V. officinalisaerial parts was prepared by extracting properly identified plant in a Soxhlet apparatus.Preliminary phytochemical screening of chloroform extract showed presence of triterpenoids. Column chromatography of chloroform extract using solvent systems viz., hexane:chloroform, chloroform and chloroform:methanol in different concentrationsyielded five fractions (F1- F5). Preliminary phytochemical screening of various fractions of chloroform extract revealed presence of triterpenoids in F2 and F3 fractions. Therefore, these fractions were further subjected to column chromatography. White colored crystals were obtained in SF1 sub-fraction separated from F2and was designated as VOC-1. Structure of VOC-1 was elucidated by IR and NMRspectral studies and was characterized as ursolic acid. Further, ursolic acid was quantified in V. officinalisaerial parts by validated TLC densitometric method. The content of ursolic acid was found to be 0.1580% in V. officinalis aerial parts.The linearity range, correlation coefficient, intra-day precision, inter-day precision, LOD, LOQ and accuracy were found to be 300-1800 ng, 0.997, 1.2% CV, 1.6% CV, 40 ng/spot, 130 ng/spot and 98.27% respectively.

QUANTIFICATION OF TWO MARKER COMPOUNDS IN GMELINA ARBOREA ROXB. STEM BARK USING VALIDATED THIN LAYER CHROMATOGRAPHIC-DENSITOMETRIC METHOD

Gmelina arborea Roxb. (Verbenaceae) is known as Gambhari in Ayurvedic System of medicine and being used as immuno-modulator since ancient time. Plant sterols such as β-sitosterol and Lupeol are reported to have such activity. A simple, accurate, precise, rapid and reproducible TLC densitometric method was developed for simultaneous estimation of β-sitosterol and Lupeol in plant stem bark. Compounds were separated on silica gel 60 F254 HPTLC plate using Toluene: Ethyl acetate: Formic acid (8.8:1.3:0.1, v/v/v) using Anisaldehyde-Sulphuric acid as the derivatizing reagent. TLC Analysis was performed at 520 nm. Rf value of β-sitosterol and Lupeol were found to be 0.34±0.02 and 0.46±0.01 respectively. Linearity range was 150-350 ng/spot and 300-700 ng/spot with correlation coefficient 0.998 and 0.999 for β -sitosterol and Lupeol, respectively. Method was validated as per ICH guideline. The accuracy was found to be 100.55-102.86% and 99.40-101.56% for β-sitosterol and Lupeol, respectively by recovery studies. The amount of β-sitosterol and Lupeol was found to be 4.4 mg % and 3.6 mg %. This method can be employed for routine quality control analysis of β-sitosterol and Lupeol in stem bark of Gmelina arborea Roxb.

Validated TLC densitometric method for stress degradation study of Asiaticoside from Centella asiatica

Validated TLC densitometric method for stress degradation study of Asiaticoside from Centella asiatica

Validated chromatographic methods for simultaneous determination of tolfenamic acid and its major impurities.

Two accurate, selective and precise chromatographic methods, namely thin-layer chromatography (TLC)-densitometric method and reversed phase high-performance liquid chromatography (RP-HPLC) method, were developed and validated for the simultaneous determination of tolfenamic acid (TOL) and its two major impurities, 2-chlorobenzoic acid (CBA) and 3-chloro-2-methylaniline (CMA), which are also reported to be its related substances. The developed TLC-densitometric method depended on separation and quantitation of the studied drugs on silica gel 60F254 TLC plates. Hexane:chloroform:acetone:acetic acid (75:25:20:0.1, v/v/v/v) was used as a developing system and the separated bands were UV-scanned at 240 nm. Linear relationships were obtained in the range of 10-100 µg band(-1) for the drug and in the range of 0.1-1 µg band(-1) for the studied impurities. The developed RP-HPLC depended on chromatographic separation of the studied drugs on a C18 column using 0.05 M KH2PO4 buffer (pH 3):acetonitrile (45:55, v/v) as a mobile phase delivered at constant flow rate of 1 mL min(-1) with UV detection at 230 nm. Calibration curves for TOL and the two impurities were constructed over the concentration ranges of 10-100 µg mL(-1) for TOL and 0.01-0.1 µg mL(-1) for both CBA and CMA. Factors affecting the developed methods have been studied and optimized. Further, methods validation has been carried out according to International Conference on Harmonization guidelines. The proposed methods were successfully applied for determination of the studied drug in its bulk powder and in pharmaceutical formulation. The methods showed no significant difference when compared with the reported RP-HPLC one. The developed methods have advantages of being more sensitive and specific than the published methods.

DETERMINATION OF DANOFLOXACIN AND ITS PHOTODEGRADATION PRODUCTS BY THIN-LAYER CHROMATOGRAPHY: KINETIC EVALUATION OF DEGRADATION PROCESS

TLC-densitometric method was developed for determination of danofloxacin (DAN) in presence of its photodegradation products. Silica gel HPTLC Lichrospher 60 F254 plates were used as the stationary phase and methanol:acetone:1 M citric acid:triethylamine (2.8:2:0.2:0.5, v/v/v/v) as the mobile phase. The elaborated method meets the acceptance criteria for specificity, linearity, sensitivity, accuracy, and precision. The linear regression analysis for the calibration curve showed a good linear correlation over the concentration range 1.25– 3.75 µg per band, with determination coefficient, exceeding 0.9806. Accuracy of the method expressed as % recovery at three concentration levels was from 95.56% to 97.30%. Good precision and intermediate precision with % RSD less than 0.92% was also observed. The limits of detection and quantification were respectively 1.10 and 3.40 ng per band. Satisfactory results of validation of the method were also confirmed by determination of DAN in veterinary commercial preparation. The photodegradation process of DAN followed kinetics of the first order reaction for the substrate.

Simultaneous estimation of glycosidic isoflavones in fermented and unfermented soybeans by TLC-densitometric method.

A simple, accurate and rapid high-performance thin-layer chromatographic (HPTLC) method for the simultaneous quantification of three glycosidic isoflavones (daidzin, genistin and glycitin) in soybean (Glycine max L.) has been established and validated. Chromatography was performed on aluminum foil-backed silica gel 60 F254 HPTLC plates and found compact spots for daidzin, genistin and glycitin (Rf value of 0.39, 0.51 and 0.32, respectively) with mobile phase toluene : ethyl acetate : formic acid : acetic acid in the ratio of 1 : 8 : 1 : 0.5, v/v/v/v. Ultraviolet detection was performed densitometrically at the maximum absorbance wavelength, 260 nm. The method was validated for precision, recovery, robustness, specificity, limit of detection (LOD) and limit of quantification (LOQ), in accordance with the ICH guidelines. The LOD (2.9, 19.3 and 3.5 µg mL(-1)), LOQ (9.03, 58.6 and 10.7 µg mL(-1)), recovery (95.9-106.66, 86.97-106.56 and 98.54-105.65%) and precision (≤2.12, ≤0.722 and ≤0.066) were satisfactory for glycosidic form of isoflavones daidzin, genistin and glycitin, respectively. Soybean variety Kh-09 bragg was found to have relatively higher amount of glycosidic isoflavones, namely daidzin, genistin and glycitin 278, 597.5 and 109.4 µg g(-1), respectively, and after fermentation the glycosidic isoflavones concentration in soybean fermented with Bacillus subtilis strain were decreased significantly after 24 h of incubation; conversely, aglycone isoflavones were increased significantly. The method for quantification of isoflavones in unfermented and fermented soybeans, with good resolution has been developed.

Chromatographic determination of clopidogrel bisulfate; detection and quantification of counterfeit Plavix® tablets

Counterfeiting of pharmaceutical products is a global problem. Upon using quality-ensuring methods of international pharmacopoeias, additional impurities and the low content of the active pharmaceutical ingredients were detected. In this paper, two chromatographic HPLC and TLC-densitometric methods were proposed as fast and reliable methods for detection and quantitation of counterfeit Plavix® tablets. These tablets were chemically counterfeited by aspirin, its degradate salicylic acid and metronidazole which were separated, besides the active ingredient clopidogrel bisulfate, by the proposed methods. Also, counterfeit was in packaging, strips and tablets’ color and these could be seen by visual inspection compared to original drug. The proposed methods can easily differentiate genuine from counterfeited tablets without need of prior separation. The proposed methods were validated according to ICH guidelines and applied to eleven batches, also were compared statistically with the reported one.