Thin-layer Chromatography Densitometric Method Research Articles

Development and validation of a high-performance thin-layer chromatography‒densitometric method and mass spectroscopy profiling for the determination of bioactive phytosterol from Manilkara zapota L. P. Royen leaves and correlating its antioxidant and antiinflammatory potential

Development and validation of a high-performance thin-layer chromatography‒densitometric method and mass spectroscopy profiling for the determination of bioactive phytosterol from Manilkara zapota L. P. Royen leaves and correlating its antioxidant and antiinflammatory potential

Straightforward thin-layer chromatography–densitometric method for the determination of phyllanthin in Phyllanthus niruri from different phytogeographical zones

Straightforward thin-layer chromatography–densitometric method for the determination of phyllanthin in Phyllanthus niruri from different phytogeographical zones

Isolation, identification, and quantification of stigmasterol in Hygrophila schulli plant by a validated high-performance thin-layer chromatography‒densitometric method

Isolation, identification, and quantification of stigmasterol in Hygrophila schulli plant by a validated high-performance thin-layer chromatography‒densitometric method

Development and validation of a high-performance thin-layer chromatography densitometric method for the simultaneous determination of novel 1-acridinyl-1,2,3-triazole derivatives

High-performance thin-layer chromatographic (HPTLC) method provides a simple, sensitive, and accurate analytical method for the simultaneous determination of certain synthesized 1-acridinyl-1,2,3-triazole derivatives without interference from starting materials and intermediates. Separation was carried out on Merck HPTLC silica gel 60F254 plates, using chloroform‒methanol (9:1, V/V) and hexane‒ethyl acetate (3:2, V/V) as mobile phases. Validation of the method was performed based on the basis of the International Council for Harmonisation (ICH) guidelines in terms of linearity, sensitivity, limit of detection, limit of quantification, precision, selectivity, and specificity. Least-square equations were calculated for the studied compounds in the ranges of 25–500 and 10–500 ng/spot for ultraviolet (UV) and fluorescence measurements, respectively. Correlation coefficients (r) values were found ranging from 0.9913 to 0.9992 for analytes. The method provides selectivity and specificity which ensure that synthesized compounds are in the pure form without the interference of starting materials and intermediates. The detection limits for the studied compounds ranged from 11.02 to 51.09 ng/spot and 3.84 to 31.95 ng/spot and quantification limits were 33.39–154.82 ng/spot and 11.63–73.67 ng/spot for both spectrophotometric and spectrofluorimetric methods, respectively, indicating applicability for good qualitative and quantitative determination of members of this series at the nanogram concentration levels in biological fluids.

Formulation, Evaluation of Physical Properties, and In Vitro Antioxidant Activity Test of Moringa Leaf (Moringa oleifera L.) Ethanolic Extract Capsules

BACKGROUND: Supplements that contain antioxidants may enhance prevention and treatment effects of a wide range of diseases including COVID-19. Quercetin, a flavonoid compound, is a natural antioxidant that can neutralize free radicals. The current study was conducted to formulate Moringa leaf (Moringa oleifera L) ethanol extract capsules and to determine the quercetin antioxidant activity levels of Moringa ethanol extract capsule formulations. 
 MATERIALS AND METHODS: We tested the total flavonoid levels in solutions with concentrations of 20, 50, 60, 70 and 100 ppm using Thin Layer Chromatography Densitometric method. Evaluation of physical properties of 96% Moringa leaf ethanol extract capsules included moisture content test, granule angle of repose test, granule flow property test, capsule weight uniformity test and capsule disintegration time test. Antioxidant activity test using the DPPH method with two samples, namely 96% Moringa leaf ethanol extract capsules with formulas I, II, III, quercetin as a comparison. 
 RESULT: The results of the evaluation of 96% Moringa leaf ethanol extract capsules showed that formula II (PVP 50 mg) had good physical properties. Testing the antioxidant activity of capsules of ethanol extract of Moringa leaves formulas I, II, III, quercetin obtained IC50 values of 44.0 ppm, 40.2 ppm, 46.4 ppm, and 4.80 ppm, respectively. 
 CONCLUSION: The evaluation of the ethanol extract capsules of Moringa leaf formula II, met the parameters of a good capsule evaluation test requirement and had very strong antioxidant activity seen from the acquisition of the IC50 value. The antioxidant properties of Moringa leaf extract capsules may be able to improve the immune system and clinical trials need to be carried out on patients to become candidates for prevention and therapeutic supplement fora range of diseases including COVID-19.

Ultra-Performance Liquid Chromatographic and Densitometric Methods for Sensitive Determination of Xipamide and Triamterene in Pure and Pharmaceutical Dosage Forms.

Validated ultra-performance liquid chromatography (UPLC) and thin-layer chromatography (TLC) densitometric methods were prescribed for determination of antihypertensive components. To establish and validate rapid and accurate UPLC and TLC densitometric methods for determination of Xipamide and Triamterene in pure and dosage forms. The first method, UPLC, depended on using an Agilent Zorbax Eclipse Plus C8 (50 mm × 2.1 mm, 1.8 μm) column, a mobile phase composed of acetonitrile-water (70 + 30, v/v) adjusted by acetic acid to obtain pH 3, 0.2 mL/min flow rate, and UV detection at 231.4 nm. The second method was a TLC densitometric method. Separation was achieved by using toluene-methanol-ethyl chloride-acetic acid (7 + 2 + 1 + 0.2, v/v/v) as the mobile phase, pre coated silica gel plates as the stationary phase, and UV detection at 300.0 nm. The obtained results were validated and statistically compared with official and reported methods. The obtained results showed high accuracy and reproducible results with excellent mean recoveries for both drugs. The UPLC method showed shorter retention time for both Xipamide (0.88 min) and Triamterene (0.63 min), a lower detection limit of less than 0.055 µg/mL for both drugs with high selectivity, decreased injection volume (1 µL), and a lower flow rate than any HPLC method. Both proposed methods were sensitive, selective, and effectively applied to pure and dosage forms (Epitens®). Unprecedented sensitive, rapid, and reproducible UPLC and TLC methods were developed for selective determination of mixtures of Xipamide and Triamterene, with LOD os less than 0.076 µg/mL for both drugs.

A validated HPTLC method for the simultaneous determination of seasonal alterations of two antihypertensive monoterpenoid indole alkaloids in Rauvolfia species from northern India

The genus Rauvolfia belongs to the family Apocyanaceae, and is native to the tropical countries of the world. Six species of Rauvolfia have been reported from India, however, only two species viz. R. serpentina and R. tetraphylla grow in the upper Gangetic plains. The monoterpenoid indole alkaloids reserpine and ajmalicine are the major phytochemicals reported from the roots of the above plants and are well known for their potential antihypertensive activity. Presently, R. serpentina has become critically endangered due to its overexploitation. Therefore, this study was designed to develop a validated High Performance Thin Layer Chromatographic densitometric method for determination of reserpine and ajmalicine in R. serpentina and R. tetraphylla in different seasons. Plant materials were collected in March, June, September and December to determine the optimal harvesting time. The densitometric method was validated in terms of instrument precision, range, linearity, specificity, limit of detection and limit of quantitation. Reproducibility of the peak of reserpine and ajmalicine was achieved at Rf 0.56, and 0.79 respectively using optimised mobile phase toluene– ethyl acetate-diethyl amine (7:2:0.6 v/v/v). Our study concluded that maximum level of ajmalicine was found in March while that of reserpine was observed in September. Harvesting of R. tetraphylla at the optimal season may reduce the overexploitation of the endangered species R. serpentina.

Validated green chromatographic methods for determination of amlodipine and celecoxib in presence of methylacetophenone.

Aim: Green, accurate and rapid methods, namely LC-MS/MSand thin layer chromatography-densitometric methods, were developed for determination of amlodipine besylate and celecoxibin presence of its process-related impurities, 4-methylacetophenonein pure and formulated tablets. Results: LC-MS/MS was achieved on ZORBAX Eclipse Plus C18 column using methanol:aqueous solution of 5mM formic acid (95:5v/v). High sensitivity with low limit of detectionvalues 0.00028, 0.00027 and 0.0003 for amlodipine, celecoxib and 4-methylacetophenone, respectively were obtained. While, thin layer chromatography-densitometric was established using methanol:water:ammonia (70:25:1.5, by volume). Good linearity was obtained in the range of 0.1-10μg/band, 1-150μg/band and 0.01-2μg/band for amlodipine, celecoxib and 4-methylacetophenone, respectively. Conclusion: The proposed method validation was achieved according to ICH guidelines. Those methods possess advantages of being ecofriendly methods which permit their application in quality control laboratories.

Development and Standardization of Polyherbal Formulation for Management of Tuberculosis

Tuberculosis is the second most common cause of death from infectious disease. Current antituberculosis treatments consist of long course of combination of antibiotics and toxic side effects and lead to poor patient compliance. So, the present research work has been undertaken for the development and standardization of polyherbal formulation for tuberculosis. In the present study polyherbal formulation was developed using powder of plants of Ocimum sanctum Linn, Tinospora cordifolia (Willd.) Miers, Alstonia scholaris (L.) R.Br and Trikatu churna. The phytochemical screening was performed for presence of various phytoconstituents. Thin Layer Chromatography densitometric methods were developed using High-Performance Thin-Layer Chromatography for the quantifications of gallic acid and piperine in formulation and found to be 0.049 % and 0.051 % respectively in formulation. In vitro antituberculosis activity was performed using Lowenstein Jensen Medium and percentage inhibition at 50 μg/ml, 500 μg/ml and 1000 μg/ml was found to be 11.71 %, 74.15 % and 94.11 % respectively. Results showed dose dependant in vitro antituberculosis activity. These activities may be due to the presence of various phytoconstituents like flavonoids, tannins and phenoilc in formulations. Above results also support the rational use of above plants as antituberculosis.

Smart TLC–densitometric methods for determination of ophthalmic ternary mixture containing chloramphenicol in the presence of its synthetic precursor: Comparative eco-scaling for greenness assessment

Green analytical methods have gained a growing interest in the field of pharmaceutical research to reduce impacts on the environment and enhance analysts’ health safety. Chloramphenicol (CHL), dexamethasone sodium phosphate (DSP) and tetrahydrozoline HCl (THZ) form an ophthalmic ternary mixture that is co-formulated for conjunctivitis treatment. In the present work, for time saving and higher sensitivity, two green thin-layer chromatography (TLC) methods were developed for the determination of this ophthalmic ternary mixture in the absence or presence of p-nitroacetophenone (PNA), a synthetic precursor of chloramphenicol. In both proposed methods, silica gel 60 F254 plates were used as the stationary phase. The mobile phase used for method (A) was ethanol‒water‒ammonia (7.0:2.5:0.5, V/V), while, for method (B), acetonitrile‒water‒ammonia (10.0:3.0:0.5, V/V) was used as the mobile phase. TLC separation was followed by quantitative determination of the aforementioned drugs at wavelengths 242.0 nm and 220.0 nm. Both methods were validated in compliance with the International Conference on Harmonisation (ICH) guidelines, where both methods were found to be reliable, reproducible, and selective. Statistical comparison of the developed methods was done with a reported high-performance liquid chromatography (HPLC) method where no significant difference was found. Analytical eco-scaling depends on penalty point which was calculated to be 92, 88 and 87 for methods A, B and the reported HPLC, respectively, suggesting that the proposed methods are eco-friendlier with penalty point scoring very high on the scale than the reported one.

Allium cepa fraction attenuates STZ-induced dementia via cholinesterase inhibition and amelioration of oxidative stress in mice.

Background An earlier study demonstrated significant antioxidant and anticholinesterase activities of hydromethanol extract (HME) of Allium cepa. The aim of the study was to investigate the component responsible for these activities followed by an in vivo study. Methods In vitro antioxidant and anticholinesterase activities of standardized ethylacetate fraction (EAF) of HME were assessed. Bioactivity-guided fractionation showed that, as compared with its subfractions, EAF had most significant activity in 2,2-diphenyl-1-picrylhydrazyl and Ellman assays. Thus, EAF was further examined using a streptozotocin (STZ)-induced model of Alzheimer's disease in mice. STZ was injected intracerebroventricularly on days 1 and 3 (3 mg/kg) in mice. EAF was thereafter administered (42, 84, and 168 mg/kg b.w./day p.o.) from days 9 to 22. The Morris water maze test was used to evaluate learning and memory in mice. Acetylcholinesterase (AChE) activity and oxidative stress markers were assessed in the brain homogenates of mice. Additionally, histopathological studies were performed to observe effects in the brain at the cellular level. EAF was standardized based on quercetin and quercetin 4'-O-glucoside content using a validated thin layer chromatography densitometric method. Results STZ produced significant (p < 0.05) memory impairment along with oxidative stress and a cholinergic deficit in mice. EAF treatment ameliorated STZ-induced behavioral deficits and biochemical alterations in mice in a significant and dose-dependent manner. Conclusions Our results show that EAF is efficacious in improving memory and learning via AChE inhibition and antioxidant activity in the mice brain. Thus, AC could be explored further to find out a lead candidate for Alzheimer's disease.

Spectrophotometric and spectrodensitometric quantification of a new antiviral combination

Accurate, simple, and selective spectrophotometric and spectrodensitometric methods were developed and adopted to quantify velpatasvir (VPS) and sofosbuvir (SFV) concurrently in their pure forms and tablets. The spectrophotometric technique was based on the first derivative of ratio spectra (1DD) technique and developed to determine VPS and SFV simultaneously in table formulation. However, the spectrodensitometric technique was based on thin-layer chromatography (TLC) and densitometry and developed to determine VPS and SFV simultaneously in tablet dosage form. Chromatographic separation was performed using chloroform:methanol 9.5:0.5 (%, v/v) as the mobile phase on glass-coated TLC plates. Detection was achieved using a 265-nm deuterium lamp in absorbance mode. Both analytical methods were validated according to the International Conference on Harmonization (ICH)-Q2B guidelines. The linearity in the range of concentration ranges of 1–50 μg/mL and 5–80 μg/mL were obtained for VPS and SFV, respectively, using 1DD spectrometric method. However, the linearity in the range of 5–50 and 10–70 μg/band for VPS and SFV, respectively, were recorded using TLC–densitometric method. Accuracy was recorded ˃100% for VPS and SFV using both methods. This is the first TLC–densitometry method that can separate and quantify the studied mixture of the drugs. The proposed analytical methods were found to be accurate, precise, selective, robust and sensitive for simultaneous analysis of VPS and SFV in tablet dosage forms.

Quantitative determination of albendazole forced degradation percentages by densitometric thin layer chromatographic method

Simple densitometric thin layer chromatographic method has been applied in this study to proceed forced degradation test of albendazole by subjecting drug to acidic, alkaline, oxidative, thermal, and photolytic conditions, and determine the degradation percentages of albendazole in these conditions after 1, 2, 4, and 6 hrs. Two different mobile phases have been used in this study to separate degradation products. chloroform: diethyl ether: glacial acetic acid (75: 12.5: 12.5, v/v) as mobile phase 1, and dichloromethane: methanol (90: 10, v/v) as mobile phase 2. Silica gel GF-254 TLC-plates were used, and densitometric Area under curve (AUC) and AUC percentages were measured. Densitometric measurements showed that albendazole is unstable in all stress conditions but in different percentages, with the least susceptibility have been reported with acid condition (just 6.13% of drug lost after 6 hrs. of incubation). Acidic medium caused reversible degradation with homogeneity in the structures of degradation products, Unlike Alkaline medium which showed heterogeneity in structures detected through the change in the number and Rf-values of degradation spots against time. Both mobile phases were suitable for detecting albendazole degradation percentages under different conditions, as quantitative measurements were closely similar for the two phases, but mobile phase 1 was better to differentiate base-induced degradation products and mobile phase 2 was better for photolytic-induced degradation products.

Rapid validated thin-layer chromatography–densitometry for the simultaneous determination of three co-formulated drugs used for common cold treatment

A rapid validated thin-layer chromatography (TLC)—densitometric method has been developed for the simultaneous determination of 3 co-formulated drugs used for common cold and cough treatment. The studied drugs are paracetamol, pseudoephedrine, and chlorpheniramine maleate. The separation was achieved using silica gel 60 F254 plates and the developing system of methanol — toluene-acetic acid (44:16:1, v/v). Densitometry scanning was performed at 254 nm. The method was validated as per the International Conference on Harmonization (ICH) guidelines and was successfully applied for the analysis of pharmaceutical preparation containing the cited ternary mixture without interference from excipients. There is no previously published TLC—densitometric method for the determination of the previously mentioned ternary mixture. The suggested method is rapid and of low cost, so it can be used for quality control analysis.

Validated high-performance thin-layer chromatographic–densitometric method for the isolation and standardization of ayapanin in Ayapana triplinervis

A simple, rapid, quantitative, and validated high-performance thin-layer chromatographic (HPTLC) method has been developed for the determination of ayapanin in the extract of Ayapana triplinervis leaves. Densitometric analysis of ayapanin was carried out in the absorbance mode at 254 nm. The method gave spot at RF = 0.56, corresponding to ayapanin. The limit of detection (54.32 ng) and limit of quantification (168.85 ng) per spot were confirmed with the mobile phase petroleum ether and ethyl acetate in a ratio of 60:40 v/v. Linear regression analysis data for the calibration plot for ayapanin showed good linear relationship with a correlation coefficient (r) of 0.997 in the concentration range of 200 ng to 1000 ng per spot. The method was validated for sensitivity, linearity, accuracy, precision, and specificity as per the International Conference on Harmonization (ICH) guidelines. After development and validation, the method was applied for the isolation of ayapanin from the extract. The isolated ayapanin was characterized by Fourier-transform infrared, nuclear magnetic resonance, and mass spectroscopy techniques.

Development and validation of chromatographic methods for the quantification of dabigatran etexilate mesylate in the presence of its risky degradation products

New stability-indicating thin-layer chromatography (TLC)—densitometry and reversed-phase liquid chromatography (RP-LC) methods for the determination of dabigatran etexilate mesylate (DEM) in the presence of its risky degradation products were developed and validated. The first method was a simple economic TLC—densitometric method, where separation was achieved on aluminum plates pre-coated with silica gel gel 60 F254 using methanol—ethyl acetate—benzene (15:20:20, v/v) as the mobile phase. The method gave a compact and well-resolved spot for DEM. Quantification was performed at 225 nm over a concentration range of 0.3–3 µg per spot with a mean percentage recovery of 98.93 ± 0.80. The second method was an RP-LC method. Short analysis time and good efficacy were achieved on a BetaSil C18 (4.6 mm × 250 mm, 5 µm) column using gradient elution. The flow rate was 1.5 mL min−1, and ultraviolet (UV) detection was carried out at 310 and 225 nm using time programming. The responses were linear over a concentration range of 5–100 µg mL−1 with a mean percentage recovery of 99.25 ± 0.74. The limit of quantification and limit of detection were 0.10 and 0.03 µg per spot for the TLC and 0.287 and 0.086 µg mL−1 for the LC method. The proposed methods were used for the identification and quantitative determination of DEM in bulk powder, pharmaceutical dosage forms, and in the presence of degradation products. The International Conference on Harmonization (ICH) guidelines were applied for validation of the methods.

Simultaneous quantification of chlorpheniramine, pseudoephedrine, and ibuprofen in antitussive preparation by high-performance liquid chromatography and thin-layer chromatography–densitometric methods

Simple, accurate, precise, sensitive, and validated high-performance liquid chromatography (HPLC) and thin-layer chromatography (TLC)‒densitometric methods were developed for the simultaneous determination of chlorpheniramine maleate (CPM), pseudoephedrine HCl (PSE), and ibuprofen (IBF) in tablet dosage form. In method A, reversed-phase (RP)-HPLC analysis was performed on Zorbax C8 column (150 mm × 4.6 mm, 5 μm particle size i.d.), using a mobile phase consisting of methanol–acetonitrile–distilled water (pH 4) using orthophosphoric acid in the ratio (80:10:10, v/v) and flow rate of 0.7 mL min−1. Quantification was achieved with ultraviolet (UV) detection at 220 nm. In method B, TLC analysis was carried out on an aluminum-backed sheet of silica gel 60 F254 layer using ethyl acetate‒methanol‒ammonia (8:2:0.8, v/v) as the mobile phase. Quantification was carried out with UV detection at 262 nm. The validation of the proposed methods was applied according to the International Conference on Harmonization (ICH)...

Development and validation of simplified high-performance thin-layer chromatography densitometric methods for the determination of sulfalene, sulfadoxine, and pyrimethamine

For quality evaluation purposes, a simple, cost-effective, precise, accurate, and rapid planar chromatographic method was developed and validated for the separation of sulfadoxine, sulfalene, and pyrimethamine using standard thin-layer chromatographic plates and a mobile phase consisting of a mixture of toluene, ethyl acetate, and methanol in the ratio 50:28.5:21.5 (% v/v). Densitometric evaluation was carried out by scanning the plates at a wavelength of λ = 254 nm. The method was validated with respect to specificity, linearity, precision, and accuracy. Linearity was proven in a concentration range of 1.00–3.00 μg per spot for sulfalene/sulfadoxine and 0.050–0.150 μg per spot for pyrimethamine with linear regression coefficients (R2) of 0.980–0.990 for all substances. The values for limit of detection (LOD) were 0.107 ng per spot for sulfadoxine, 0.044 ng per spot for sulfalene, and 1.623 ng per spot for pyrimethamine, whereas those for limit of quantification (LOQ) were found to be 0.324 ng per spot fo...

A Comparative Study of Smart Spectrophotometric Methods and a Densitometric Method for Simultaneous Determination of Binary Mixture of Canagliflozin and Metformin Hydrochloride

Simple and sensitive Spectrophotometric methods and a densitometric thin layer chromatographic method (TLC) are developed and validated for simultaneous determination of binary mixture of metformin hydrochloride (MET) and canagliflozin (CFZ) in combined dosage form. Three spectrophotometric methods manipulating ratio spectra namely ratio subtraction, ratio difference and derivative ratio are used for the determination of MET in the presence of CFZ without preliminary separation, while CFZ is directly determined by measuring its absorbance at λmax 290 nm. The calibration curves follow Beer's law in the concentration range of 1 - 20 and 1 - 30 μg/mL for MET and CFZ respectively. A simple TLC method is developed where separation is performed on TLC silica gel plates 60F254 using toluene: Methylene chloride: methanol: water: glacial acetic acid in the volume ratio (20:15:15:1:0.25) as a mobile phase. Rf values are 0.55 ± 0.05 for CFZ and 0.21 ± 0.03 for MET, the linearity ranges are 0.3-4.5 and 0.5-5.5 μg/band for CFZ and MET respectively. The developed methods are successfully applied for the simultaneous determination of MET and CFZ in bulk and pharmaceutical formulation.

Validated Chromatographic Methods for the Simultaneous Determination of Sulfacetamide Sodium and Prednisolone Acetate in their Ophthalmic Suspension.

Two specific, sensitive, rapid and accurate chromatographic methods have been developed, optimized and validated for the simultaneous determination of sulfacetamide sodium and prednisolone acetate in pure forms and in their binary mixture. The first method is an isocratic Reversed phase-high performance liquid chromatography method where a rapid separation was achieved on a Zorbax ODS column using a green mobile phase of methanol: water (80:20, v/v) and pH adjusted to 5.0 ± 0.2 with orthophosphoric acid. The retention times (tR) were 2.21 and 3.64 min for sulfacetamide sodium and prednisolone acetate, respectively. The separated peaks were detected at 254 nm. The second method is a thin layer chromatography-densitometric method where the two drugs were separated on silica gel plates using a simple mobile phase of chloroform: methanol (90:10, v/v) and scanning of the separated bands was at 254 nm. The retardation factors (Rf) values were 0.37 and 0.64 for sulfacetamide sodium and prednisolone acetate, respectively. The suggested methods were validated in compliance with the ICH guidelines and were successfully applied to determine both drugs in their pure forms, laboratory prepared mixtures and dosage form. The obtained results were statistically compared to the official method where no significant difference was obtained with respect to both accuracy and precision.