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

Quantification of β-Sitosterol by Validated High Performance Thin Layer Chromatography (HPTLC) Densitometric Method in the Flowering Buds of <i>Mesua ferrea</i> Linn

The medium-sized to large-sized tree Mesua ferrea Linn, commonly known as Nagakesara in India, can grow to be around 20 to 30 meters high and has incredibly durable wood. The fruiting season runs from January to March and April to October. The buds are globular, 2mm diagonally, and have scales in the axillary buds. It includes a significant amount of β-sitosterol amides, which are important from a biological perspective. In the current study, we used high-performance thin-layer chromatography (HPTLC) in association with densitometry to quantify these marker chemicals from Mesua ferrea Linn flowering buds. The technique was found to be accurate. For intra-day analysis, the relative standard deviation (RSD) values of β-sitosterol ranged from 1.16 to 1.60%, while for inter-day analyses, the values ranged from 0.68 to 1.77%. By performing a recovery study at three different levels for the standard chemicals, the accuracy of the approach was assessed, and average recoveries were determined to be 99.98%. It was observed that this TLC-densitometric approach was accurate, specific, particular, sensitive, and easy. For the simultaneous analysis of β-sitosterol from flowering buds of Mesua ferrea Linn, it can be employed in regular quality control.

Optimizations of the Conditions for Ceftobiprole Determination in a Complex Matrix

A quick and accurate chromatographic–densitometric method for the determination of ceftobiprole in biological material (whole blood and urine) was developed. Preparation of the test sample required extraction of the drug from the matrix and was carried out by testing methanol or acetone as extracting agents, which were successfully used to isolate ceftobiprole from biological material. Under optimization of the procedure, various stationary and mobile phases were tested. Lastly, HPTLC cellulose plates and a mixture containing ethanol, 2-propanol, glacial acetic acid, and water in the ratio 4:4:1:3 (v/v/v/v) were chosen. Densitometric detection was made at a maximum absorbance of 316 nm. The developed method was validated; a linear function of the ceftobiprole concentration was obtained in the range of 2.4–72 µg/mL (r > 0.99) for both methanol and acetone solutions. The average accuracy of the devised method was measured at nearly 100%; nevertheless, the limit of the quantification was at 8.92 for methanol and 9.14 µg/mL for acetone solution. Therefore, the above method can be successfully used to ceftobiprole in biological material.

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.

HPTLC Analysis and Force Degradation Study of Tapentadol Hydrochloride in Bulk and Its Pharmaceutical Formulation

To achieve easy, sensitive reproducible high-performance thin-layer chromatography (HPTLC) densitometric method was developed and validated for the determination of tapentadol HCl and its pharmaceutical formulation. In this method the ethyl acetate: methanol: ammonia (6:4:0.5v/v/v) were used as mobile phase for chromatographic separation of the drug. The method has been performed on precoated silica Tab 60F254 Merck plates and the Rf value was calculated at 0.47 for tapentadol HCL. The calibration curve was plotted in the concentration range of 500-2500ng/ml. The limits of quantitation and limit of detection were found to be 0.07 and 0.23μg/ml respectively. The correlation coefficient (R2) value was obtained at 0.998 for tapentadol HCL. The percent recoveries were obtained between 99.97 to 101.13% for tapentadol HCl. The method has been validated according to ICH guidelines for linearity, precision, accuracy, specificity, and robustness. The degradation behavior was recorded under acidic, basic, neutral, oxidative, photolytic, and thermal stress conditions. The degradation products were well separated from the pure drug under the optimized conditions. As the method could effectively separate the drug from its degradation products, it can be employed as a stability-indicating method for tapentadol HCl.

Development and Validation of Thin Layer Chromatography-Densitometric Method for Quantification of Kaempferol and Chlorogenic Acid in Methanolic Extract of Dragea Volubilis

The present study aims to prepare a methanolic extract of Dragea volubilis and to quantitatively estimate kaempferol and chlorogenic acid using newly developed and validated method of high-performance thin layer chromatography (HPTLC) using TLC densitometry technique. Dried leaves obtained from D. volubilis were defatted with petroleum ether and extracted in a Soxhlet extractor with methanol. The solvent was evaporated using a Rota evaporator. Quantitative analysis of the extract was done using TLC and HPTLC. A mixture of toluene: ethyl acetate: glacial acetic acid (5.5:4:0.5) for kaempferol and ethyl acetate: acetic acid: water (7:1.5:1.5) for chlorogenic acid was used as a mobile phase. The chromatography was performed on a TLC plate precoated with silica gel GF254 and the developed TLC plates were visualized and quantified at 367 nm and 327 nm for kaempferol and chlorogenic acid, respectively. The percentage yield was found to be 0.0397 ± 0.0001 and 0.0755 ± 0.0000 for kaempferol and 0.0755 ± 0.0000 for chlorogenic acid. Rf. values 0.95 and 0.61 were observed for kaempferol and chlorogenic acid, respectively. The method was validated for instrumental precision, repeatability, coefficient of determination (r2), linearity range (ng), LOD (ng), LOQ (ng), and intra-day and inter-day precision for both kaempferol and chlorogenic acid, and all the parameters were found to be within the acceptable range as per the ICH guidelines. The newly developed HPTLC method was found to be prompt, cost-effective, precisely accurate, and reproducible for the qualitative as well as quantitative analysis of kaempferol and chlorogenic acid in methanolic extract of D. volubilis.

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.

Novel Rapid Validated TLC- Densitometry for the Simultaneous Determination of Three Co-formulated Drugs used for Deep Tooth Inflammation Treatment

A new use of a tertiary antibiotic combination of ciprofloxacin, clindamycin and metronidazole is currently being studied as an intracranial (intracranial) drug in an effort to disinfect the root canal system to revitalize the tooth with dead pulp. It is necessary to developing analytical method for the new drug combination.
 Herein we developed a rapid validated thin-layer chromatography (TLC)‒densitometric method has been developed for the simultaneous determination of 3 co-formulated drugs used for deep tooth inflammation Treatment. The studied drugs are Ciprofloxacin.HCL (CIP), Clindamycin phosphate (CLI) and Metronidazole(MET). The separation was achieved by using silica gel 60 F254 plates (20*20 cm) as stationary Phase and the developing system of Dichloromethane:n-Hexane:methanol:Ethylamine:Triethylamine (40:20:32:3:5)v/v.
 Densitometry scanning was performed at 220 nm. The Rf values of CIP, CLI and MET were found to be 0.588, 0.776 and 0.898 respectively.
 The method was validated as per the International Conference on Harmonization (ICH) guidelines and was successfully applied for the analysis of ternary lab made mixture, containing the cited ternary mixture without interference from excipients. There is no previously published TLC–densitometry method for the determination of the previously mentioned ternary mixture. The suggested method is novel, rapid, accurate, reproducible and of low cost, so; thus, it can be used for quality control analysis of these formulations.

Gymnemic Acid-Rich Fraction from Gymnema Sylvestre Leaves Ameliorates Post Prandial Hyperglycaemia in In-vitro and In-vivo Studies

Gymnemic acids are triterpenoid glycosides with gymnemagenin as common aglycone found in Gymnema sylvestre (Gurmar). G. sylvestre is often used in herbal formulations because of its beneficial actions in diabetes, hyperlipidemia, and gastric problems. The purpose of the present study is to evaluate gymnemic acid-rich fraction of G. sylvestre in in-vitro and in-vivo model to control postprandial hyperglycemia. The gymnemic acid-rich fraction of G. sylvestre (GARF) was prepared by ultrasonic-assisted extraction using methanol-water (81:19 v/v) at a temperature of 49°C for 50 min using a solid-to-solvent ratio of 1:12.5 g/ml and gymnemic acid content was determined by validated High-performance thin-layer chromatography (HPTLC) densitometric method. The GARF effect was evaluated in the in-vitro model on α-amylase and α-glucosidase enzymes, while the antihyperglycemic effect was studied in carbohydrate-challenged and diabetic rats induced by Streptozotocin (STZ). In vitro studies show that GARF produces dose-dependent enzyme inhibition with IC50 values of 4.34 ± 0.81 mg/ml and 8.78 ± 1.22 mg/ml, respectively. In-vivo studies of normal and diabetic rats showed that GARF treatment (200 mg/kg b.w., p.o.), causes a significant (p < 0.01) blood glucose reduction effect in both models. The current study provides a scientific basis for the use of G. sylvestre in the management of diabetes mellitus.

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.

Study of metabolite variability in Plumbago zeylanica Linn. collected from different localities of the Gangetic plains of India

The metabolite contents of the plants may vary with season and geographic variations, and this induces the biological potential of the plant species. The present study aimed to identify the elite chemotype(s) of Plumbago zeylanica, collected from the natural habitat of Gangetic plains on the basis of plumbagin content. The effect of intra-specific variability in the plumbagin content among the population was estimated by using high-performance thin-layer chromatography (HPTLC) densitometric method. Roots of thirteen naturally occurring populations of P. zeylanica from the entire range were collected, covering the wide topography from foothills up to the highest peak. The method developed for quantification of plumbagin was validated and found to be linear, specific and accurate with precision and accuracy. LOD and LOQ were 1.04 and 3.16 ng/spot. Precision studies (both inter-day and intra-day) were within the standard limit of API, with RSD (%) less than 3%. Quantification of plumbagin within population reveals that it varies from 0.0048 ± 0.0005 μg (NBP-22) to 0.1696 ± 0.003 μg (NBC-25) percent on per dry weight basis. Analysis of variance (ANOVA) suggests that there is a significant difference in plumbagin content among the population. UPGMA cluster analyses based on plumbagin content bifurcate the populations into two major clusters, representing the variation in metabolite content. The first major cluster having NBP-25 and NBP-18 accessions, NBP-25 having maximum plumbagin content was collected from Bankura, West Bengal. The study suggests the presence of chemotaxonomic variation among the population which may be due to the change in environmental/phytogeographical factors.

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.

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.

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...