Thin-layer Chromatography Densitometry Method Research Articles

A Densitometric Method for Determination of Mangiferin, an Antioxidant Compound, with Thin Layer Chromatography in the Leaf Extracts of Coffee (Coffea Arabica L.)

Mangiferin is one of the antioxidants in Coffea arabica L. leaves that has many pharmacological effects. The content of secondary metabolites in the leaves including mangiferin can be affected by age. A Thin Layer Chromatography (TLC) method for the quantitative analysis of mangiferin in Coffea arabica L. leaves extract was developed and validated. The method was developed using a mobile phase prepared with analytical grade solvents: ethyl acetate, methanol, formic acid, and deionized water (8:2:1:1 v/v/v/v). Regression functions were established over the 199.98-600.00 ng/spot range with r=0.999. The limit of detection (LOD) and limit of quantification (LOQ) were 13.87 and 41.61 ng, respectively. The method was selective with a resolution value of more than 1.5 and specific with the spectra correlation value for purity and identity check of more than 0.99. The percentage RSD was found to be 2.43% for repeatability precision and 2.05% for intermediate precision. The method's accuracy was determined through the standard addition method by adding known quantities of standard mangiferin to the pre-analyzed test solution and the mean recovery was 101.69± 1,21%. This TLC Densitometry method was linear, sensitive, selective and specific, precise, accurate, and can be used for routine analysis of mangiferin. On the young Coffea arabica L. leaves, the concentration of mangiferin ± RSD was 0.830 ± 1.71% w/w, and on the old Coffea arabica L. leaves was 1.128± 1.59% w/w.

The improvement of routine drug quality control analysis of some antihypertensive drugs using high-performance thin-layer chromatography densitometry method with greenness profile evaluation.

The common antihypertensive drugs are B-blockers and diuretics. For the determination of beta-blocker medicines (bisoprolol fumarate and carvedilol) and diuretic drug (Furosemide), new and accurate chromatographic method has been developed. The separation was achieved using a developing system that includes chloroform:methanol:ethyl acetate:ammonia (6:2:2:0.2 by volume) as a mobile phase and the bands were detected at 240nm. The concentration ranges were 5-25, 1-7, and 1-3.5μg/band for bisoprolol fumarate, carvedilol, and furosemide, respectively. This chromatographic approach is the first methodology for simultaneously determining bisoprolol fumarate, carvedilol, and furosemide in their pure forms and in their pharmaceutical dosage forms. The advantages of using known analytical procedures are their simplicity, speed, cost effectiveness, lack of laboriousness, and ability to save time as the three tablets are determined in one step and can be used for routine analysis of the investigated combinations in quality control laboratories. According to International Conference of Harmonization guidelines, the established procedures have been validated, and the results were statistically compared to those obtained by the reported reversed-phase-high-performance liquid chromatography methods using Student's t-test and F-test, with no significant difference between them, indicating that the proposed methods can be used for routine drug quality control analysis.

Quantitative Estimation of Quercetin of Some Selected Edible Plants of West Bengal by High-performance Thin-layer Chromatography Densitometry Method

ABSTRACT Introduction: Quercetin is a polyphenol, widely found in most edible fruits and vegetables. This plant pigment provides cardiovascular, anti-inflammatory, antidiabetic, asthma, and many more health benefits. Objective: The aim of this study is to quantify the quercetin by high-performance thin-layer chromatography (HPTLC) method from the methanolic leaf extracts of three edible plants of Bengal, viz., Nyctanthes arbor-tristis L., Oxalis corniculata L., and Polygonum plebeium R.Br. Materials and Methods: HPTLC was carried out on aluminum-backed plate precoated with silica gel 60 F254, using the solvent system of toluene, ethyl acetate, formic acid, and methanol (5.5:3:1:0.5 v/v/v/v). The method validation was done according to the International Conference on Harmonization guidelines in terms of linearity, limit of detection, limit of quantification, accuracy, precision, and specificity. Results: The desired compound was well separated using the solvent system toluene, ethyl acetate, formic acid, and methanol (5.5:3:1:0.5 v/v/v/v) which gave a sharp and well-defined band with Rf 0.60 for quercetin. The calibration curves showed a good linear correlation coefficient (R 2 = 0.9774). The linearity range was found to be 20–100 (μg/spot). Among the selected plants, the highest amount of quercetin was found in the leaf extracts of O. corniculata L. (220.035 ± 0.06 mg/100 g) and the lowest for N. arbor-tristis L. (31.253 ± 0.99 mg/100 g). Conclusion: The proposed HPTLC method for quercetin estimation was found to be simple, cost-effective, precise, and accurate and can be used for the quality control of plant material. O. corniculata L. was found to be a potent source of quercetin as it possesses a decent amount of quercetin.

Guesstimate of thymoquinone diversity in Nigella sativa L. genotypes and elite varieties collected from Indian states using HPTLC technique.

Thymoquinone is a valuable metabolite derived from the Nigella sativa L. seeds and has a variety of therapeutic properties. Thymoquinone was estimated using n-hexane:ethyl acetate (8:2, v/v) green solvent system and computed at a wavelength of 254 nm using the high-performance thin-layer chromatography densitometry method in distinct varieties and genotypes congregated from different geographical regions. Genotype Ajmer Nigella-13 has the paramount thymoquinone content (247.60 µg/100 mg seed) followed by Ajmer Nigella 19 (244.5 µg/100 mg seed), while the lowest amount of thymoquinone was recorded in the genotype Ajmer Nigella-6 (42.88 µg/100 mg seed). The hierarchical cluster analysis found that the collected genotypes and elite varieties were classified into four broad clusters, and the identified chemotypes with elevated thymoquinone proportion were positioned in cluster D. Significant genotypic variation in thymoquinone content is available, that can be used in exploiting pharmaceutical applications of N. sativa L. as well as a breeding programme for specific metabolite improvement perspective.

Quantification of piperine in different varieties of Piper nigrum by a validated high-performance thin-layer chromatography‒densitometry method

Quantification of piperine in different varieties of Piper nigrum by a validated high-performance thin-layer chromatography‒densitometry method

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.

Simultaneous Determination of Paracetamol, Propyphenazone and Caffeine in Presence of Paracetamol Impurities Using Dual-Mode Gradient HPLC and TLC Densitometry Methods.

Two chromatographic methods were validated for the determination of the widely prescribed analgesic and antipyretic drug combination of paracetamol (PC) (recently integrated into the supportive treatment of COVID-19), propyphenazone (PZ) and caffeine (CF) in the presence of two PC impurities, namely 4-aminophenol and 4-nitrophenol. A “dual-mode” gradient high-performance liquid chromatography method was developed, where the separation was achieved via “dual-mode” gradient by changing both the ternary mobile phase composition (acetonitrile: methanol: water) and the flow rate. This enables a good resolution within a relatively shorter analysis time. The analysis was realized using Zorbax Eclipse XDB column C18, 5 μm (250 × 4.6 mm) and the UV detector was set at 220 nm. The other method is a thin-layer chromatography densitometry method, where the separation was achieved using a mobile phase composed of chloroform: toluene: ethyl acetate: methanol: acetic acid (6: 6: 1: 2: 0.1, by volume). Densitometric detection was performed at 220 nm on silica gel 60 F254 plates. The developed methods were fully validated as per the ICH guidelines and proved to be accurate, robust, specific and suitable for application as purity indicating methods for routine analysis of PC in pure form or in pharmaceuticals with PZ and CF in quality control laboratories.

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.

Validation of TLC densitometry method for the quantitative determination of alkaloid in fermented endophytic fungi extract Phyllantus niruri Linn

Endophytic fungi can produce compound that similar with the host, so it can be used as mass production of compound. The optimal and valid assay method is needed to obtain the proper culture condition, which one using densitometric Thin Layer Chromatography (TLC). This research used 11 samples of endophytic fungi culture extract from Phyllantus niruri within various culture condition. Optimization of mobile phase was done using 3 kinds of mobile phase mixture, chloroform: ethyl acetate: methanol (8:8:4); chloroform: ethyl acetate: methanol: NH 4 OH (8:8:4:0.005); chloroform:ethyl acetate:NH 4 OH (8:8:0.005). Validation method measured using some parameters such as linearity, accuracy, and precision. The result of optimization and validation showed that TLC densitometry method can be used for measuring alkaloid level within the endophytic fungi extract of P. niruri using chloroform: ethyl acetate: NH 4 OH (8:8:0.005), with R value is 0.977, percentage of recovery is between 90–110, and RSD ≤7% on each concentration series. While the proper condition of endophytic fungi culture is using sucrose as carbon source and at pH 6 condition . The content of alkaoid reached 25.9 ± 1.4 mg 100 g -1 fresh weight.

Effect of Leaves Infuse (Foeniculum vulgare Mill.) Intake on Reproductive Organs Morphometry of Female Rats (Rattus Sp.) After Parturition

Fennel (Foeniculum vulgare Mill.) traditionally used by people as antifertility agent for female. Fennel leaves (Foeniculum vulgare Mill.) contain phytoestrogen that has role as antiestrogen or synthesized become antiestrogen. Coumarin is the kind phytoestrogen that can be antiestrogen to female reproductive organs such as ovary and utery. The aim of this research is to find out the influence of fennel leaves infuse (Foeniculum vulgare Mill.) on morphometric of ovary and utery in female rats (Rattus sp.) administered after parturition, to find out the effective dose affecting the morphometric of ovary and utery and to find out the total coumarin consisted in fennel leaves (Foeniculum vulgare Mill.). This research was carried out by using CRD (Completely Randomized Design) consisted of 2 groups, those are control group (aquades) and treatment groups (doses of fennel leaves infuse 20 g/300 ml, 40 g/300 ml and 60 g/300 ml). The treatment was administered in the morning and afternoon for 15 days. After 15 days of treatment, the rats were sacrificed and dissected to measure the morphometry of reproductive organs (ovary and utery). The data obtained was analyzed by Analysis of Varian (ANOVA) and continued by LSD (Least Significance Different) test with significance of 5%. The measurement of coumarin level was performed by TLC (Thin Layer Chromatography) densitometry method. The result of this research showed that the administration of infused fennel leaves (Foeniculum vulgare Mill.) at dose of 20 g/300 ml, 40 g/300 ml and 60 g/300 ml significantly decreased ovarian weigh in female rats (Rattus sp.) after parturition (p<0,05), but not decreased the length, width, thick of ovary, length and diameter of utery (p>0,05). No difference was observed between doses 20 g/300 ml, 40 g/300 ml and 60 g/300 ml, and it showed that the dose 20 g/300 ml of fennel leaves infuse was able to be used to decrease the weigth of ovary. Coumarin level observed in leaves is 0,09%, and it is higher than the coumarin content in fruit/seed.

Development and validation of a high-performance thin-layer chromatography—densitometry method for the quantitative estimation of morphine in the classical Ayurvedic formulation Kamini Vidrawan Ras

Kamini Vidrawan Ras is a classical Ayurvedic medicine, referred to in Bhaisjya Ratnavali, a book recognized by the Drugs and Cosmetics Act of India. Its usefulness has been stated in erectile dysfunction, impotence, and premature ejaculation. Opium is the major ingredient of formulation which is highly addictive as it contains narcotic alkaloids (morphine, codeine, and thebaine). Opium is a natural product; hence, the morphine content varies from 4–21%. The aim of this study was to develop a simple, precise, rapid, and reliable high-performance thin-layer chromatography— densitometry method for the quantitative estimation of morphine in the tablets of the said Ayurvedic medicines. Aluminum-backed silica gel F254 (20 cm × 10 cm) was used as the stationary phase and ethyl acetate, methanol, and ammonia (85:10:5, v/v) as the mobile phase. The RF value for morphine was 0.36, and the quantitative evaluation of the bands over plates was performed in the reflectance—absorbance mode at 280 nm. The regression anal...

Determination of biogenic amines in aerobically stored beef using high-performance thin-layer chromatography densitometry

ABSTRACTThe aim of this study was to develop a high-performance thin-layer chromatography densitometry method to simultaneously evaluate levels of different biogenic amines (BAs) in beef stored aerobically at 4°C. Steaks from M. longissimus thoracis et lumborum muscles were overwrapped with polyvinyl chloride film and stored for 0, 4, 7, and 10 days. For this purpose, the solvent system and pH for the derivatization of BAs with dansyl chloride were optimized. Moreover, this method was also validated for linearity, limits of detection and quantification, precision, and recovery. As found using this method in beef samples, spermine content decreased significantly after 7 days of storage, and tyramine and cadaverine were not detected until day 10.

Validated UPLC and TLC-Densitometry Stability Indicating Methods for the Determination of Rafoxanide in the Presence of Its Degradation Products.

Two sensitive and accurate stability-indicating chromatographic methods were developed and validated for the determination of rafoxanide (RFX). Degradation products were identified by mass spectrometry and IR spectroscopy. The first is ultra-performance liquid chromatography method where separation was performed using acetonitrile:0.005 M potassium dihydrogen orthophosphate (pH 3.5) in a ratio of 80:20 by volume as a mobile phase using a Hypersil GOLD™ C8 column 1.9 mm (50 × 2.1 mm), UV detection was adjusted at 220 nm and the flow rate was 0.6 mL min-1. The other is a thin-layer chromatography-densitometry method where separation was achieved using a mobile phase composed of chloroform:ethyl acetate:toluene:ammonia (5:4:3:0.1 by volume) on silica gel 60 F254 plates, and densitometric detection was done at 280 nm. Validation was achieved as per the ICH guidelines. The proposed methods proved to be accurate, robust, specific and suitable for application as stability-indicating methods for routine analysis of RFX in quality control laboratories.

IDENTIFIKASI DAN PENETAPAN KADAR SILDENAFIL SITRAT PADA JAMU KUAT LELAKI YANG BEREDAR DI KOTA MAKASSAR

A studies have been conducted identifying and determining the levels of sildenafil citrate in men vitality herbs that marketed in twon of makassar. This study aims to determine the presence of drugs chemicals substances (BKO) in men vitality herbs that is sildenafil citrate with TLC–Densitometry method. Herbal samples extracted using methanol solvent. Identification test by thin layer chromatography (TLC) using a mobiloe phase of chloroform – ethanol (9.5 : 0,5) indicates that the herbal sample were compared with comparator compounds contains sildenafil citrate. Assay done using a TLC-Densitometry at a maximum wavelength of 292 nm. The results showed levels of sildenafil citrate in men vitality herbs was 0,00827 g/100 g or 0,00827 % per capsule. Key Words : Identifying, Determining, Sildenafil Citrate, Men Vitality Herbs

IDENTIFIKASI DAN PENETAPAN KADAR SILDENAFIL SITRAT PADA JAMU KUAT LELAKI YANG BEREDAR DI KOTA MAKASSAR

A studies have been conducted identifying and determining the levels of sildenafil citrate in men vitality herbs that marketed in twon of makassar. This study aims to determine the presence of drugs chemicals substances (BKO) in men vitality herbs that is sildenafil citrate with TLC–Densitometry method. Herbal samples extracted using methanol solvent. Identification test by thin layer chromatography (TLC) using a mobiloe phase of chloroform – ethanol (9.5 : 0,5) indicates that the herbal sample were compared with comparator compounds contains sildenafil citrate. Assay done using a TLC-Densitometry at a maximum wavelength of 292 nm. The results showed levels of sildenafil citrate in men vitality herbs was 0,00827 g/100 g or 0,00827 % per capsule. Key Words : Identifying, Determining, Sildenafil Citrate, Men Vitality Herbs

The development and validation of a Thin Layer Chromatography densitometry method for the analysis of diclofenac sodium tablets

A Thin Layer Chromatography (TLC) method for the qualitative and quantitative analysis of diclofenac sodium tablets was developed and validated according to ICH and USP guidelines. The method was developed using a mobile phase prepared with environment-friendly solvents: toluene, acetone and glacial acetic acid (10:15:0.2 v/v/v), on pre-coated TLC silica gel 60 F254 glass plates with a saturation time of 25 min and a densitometer detection wavelength of 284 nm in the reflectance absorbance mode. The Rf of diclofenac sodium was at 0.60 and the method was repeatable, robust with good selectivity and specificity. Regression functions were established over the range of 250-600 ng/spot with r2 of 0.993 and 0.999 for the linear and polynomial regressions respectively. The accuracy at nominal concentration was found to be 100.2%, and the results of the assay of three brands for diclofenac sodium tablets were found to meet the USP 95 to 105% assay limits thus demonstrating the usefulness of the method for the assay of these products. The developed assay method for diclofenac sodium in tablets is simple, accurate, and inexpensive, with good precision and should be especially useful in resource constrained countries.

The Development and Validation of a Thin Layer Chromatography Densitometry Method for the Analysis of Diclofenac Sodium Tablets

The Development and Validation of a Thin Layer Chromatography Densitometry Method for the Analysis of Diclofenac Sodium Tablets

Validated Stability Indicating TLC-Densitometric Method for the Determination of Diacerein

This work presents an accurate, sensitive and selective thin-layer chromatography-densitometry method for the simultaneous determination of diacerein in the presence of rhein, the active metabolite and hydrolytic degradation product of diacerein, and emodin, the diacerein impurity, in bulk powder and different pharmaceutical formulations. Chromatographic separation was performed on aluminum plates precoated with 60 F254 silica gel using hexane-ethyl acetate-acetic acid (60:40:0.8, by volume) as a developing system and with detection at 230 nm. The retention factor values of diacerein, rhein and emodin were 0.12, 0.44 and 0.6, respectively. The method was successfully applied for the determination of these compounds with high sensitivity; the linearity ranges were found to be 0.5-10 µg/band (for diacerein and rhein) and 0.5-7 µg/band (for emodin). The developed method was validated according to International Conference on Harmonization guidelines and was applied for the determination of diacerein in different pharmaceutical formulations. Moreover, a statistical comparison between the results of the developed method and those of the reported reversed-phase high-performance liquid chromatography method showed no significant differences. This method can be used for the routine analysis of diacerein, rhein and emodin in quality control laboratories.

A sensitive and specific high-performance thin-layer chromatography-densitometry method for determination of Rebamipide in the bulk material and in pharmaceutical formulation

Rebamipide (RBP), α((4-chlorobenzoyl)-amino)-1,2-dihydro-2-oxo-4-quinoline propanoic acid is antiulcer (gastric cytoprotectant). Literature survey revealed many reverse-phase high-performance liquid chromatography (RP-HPLC) methods for determination of RBP in human plasma, rat plasma, and coionic tissue. Stability-indicating liquid chromatographic assay method and first-order derivative spectrophotometric methods have been established for the determination of RBP in bulk and tablet dosage form. Nowadays, high-performance thin-layer chromatography (HPTLC) is becoming a routine analytical technique due to its low operating cost, high sample throughput, and need for minimum sample clean up. The major advantage of HPTLC is that several samples can be run simultaneously using a small quantity of mobile phase unlike HPLC, thus lowering analysis time and cost per analysis. The intention of the present work is to establish simple, precise, and accurate stability-indicating densitometric thin-layer chromatography (TLC)-densitometry method for analysis of RBP in the bulk material and in tablets. Further the developed method has to be validated as per the International Conference on Harmonisation (ICH) guidelines.