Thin-layer Chromatography Densitometric Method Research Articles

Development and validation of a thin-layer chromatographic-densitometric method for the analysis of ciprofloxacin hydrochloride tablets

Ciprofloxacin [1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-(piperazin-1-yl)quinoline-3-carboxylic acid hydrochloride] (Figure 1) is freely soluble in water (3.5 g dL–1) and less soluble in organic solvents such as methanol (0.016 g dL–1) [1, 2]. It is a second-generation fluoroquinolone chemotherapeutic agent used worldwide as antibacterial and has strong effect on Gramnegative bacteria and weak effect on Gram-positive bacteria. It is used in the treatment of infections of the urinary tract, airways, alimentary tract, skin and soft tissues, bones, arthritis, infections caused by Pseudomonas bacteria, and serious general infections [3, 4].

Simultaneous Quantification of Diarylheptanoids in Alnus nepalensis Using a Validated HPTLC Method

Platyphylloside, oregonin and hirsutanonol 5-O-β-D-glucopyranoside are known bioactive metabolites in Alnus nepalensis. In this article, the aforementioned three markers were isolated and simultaneously quantified by the thin-layer chromatography densitometric method. A sensitive, selective and robust qualitative and quantitative densitometric high-performance thin-layer chromatographic method was developed and validated for the determination of above markers in the leaves of A. nepalensis. The separation was performed on silica gel 60F254 high-performance thin-layer chromatography plates using chloroform:methanol:formic acid (75:25:2, v/v) as mobile phase. The quantitation of diarylheptanoids was carried out using the densitometric reflection/absorption mode at 610 nm after post-chromatographic derivatization with vanillin-sulfuric acid reagent. A precise and accurate quantification can be performed in the linear working concentration range of 333-3330 ng/spot with good correlation (r(2) = 0.999). The method was validated for peak purities, precision, robustness, limit of detection and quantitation, etc., as per ICH guidelines. Specificity of quantitation was confirmed using retention factor and spectra correlation of markers in standard and sample tracks.

Separation and determination of lamivudine, tenofovir disoproxil fumarate and efavirenz in tablet dosage form by thin-layer chromatographic-densitometric method

Summary This paper describes the development and validation of high-performance thin-layer chromatography (HPTLC) method for the simultaneous determination of lamivudine (LAM), tenofovir disoproxil fumarate (TDF) and efavirenz (EFV). The separation was carried out on Merck HPTLC aluminum plates precoated with silica gel G60 F 254 (20 × 10 cm) with 250-μm thickness using chloroform–methanol–toluene (9:1.2:0.3, v/v) as mobile phase. HPTLC separation of the three drugs followed by densitometric measurement was carried out in the absorbance mode at 260 nm. The drugs were resolved satisfactorily with R F values of 0.20 ± 0.02, 0.61 ± 0.01 and 0.73 ± 0.02 for LAM, TDF and EFV, respectively. The linear regression analysis data for the calibration plots showed good linear relationship with r 2 = 0.9997, 0.9991 and 0.9990 for LAM, TDF and EFV, respectively, in the concentration range of 400–800 ng spot – 1 for LAM and TDF and 800–1600 ng spot –1 for EFV. The limit of detection and quantitation were 180 and 300 ng spot – 1 , respectively for LAM, 150 and 210 ng spot – 1 , respectively for TDF and 300 and 400 ng spot –1, respectively, for EFV. The method was validated for precision, robustness, limit of detection (LOD), limit of quantitation (LOQ), specificity and accuracy. The method can be used for routine analysis of these drugs in various formulations in quality control laboratories.

An HPTLC-densitometry method for the quantification of pharmacologically active alkaloids inSceletium tortuosumraw material and products

Mesembrine-type alkaloids are reported to be responsible for the psychoactive properties of Sceletium tortuosum. The aim of the study was to develop a simple, rapid, and precise high-performance thin-layer chromatography densitometric method for the simultaneous determination of mesembrenol, mesembranol, mesembrine, and mesebrenone in S. tortuosum raw materials and commercial products. Silica gel 60 F254 pre-coated glass plates were used with a mobile phase of dichloromethane-MeOH-10% NH3 (90:10:0.1, v/v). Densitometric quantification was performed in absorbance mode at 280 nm, and good separation was obtained using a single mobile phase. Regression analyses of the calibration data showed good linear relationships with regression coefficient (r2) values ranging from 0.994 to 0.999. The method was validated for linearity, accuracy, limit of detection, limit of quantification, and precision (repeatability). The results showed good correlation with data obtained from gas chromatography/mass spectrometry anal...

Stability-Indicating TLC–Densitometric Method for Simultaneous Determination of Paracetamol and Chlorzoxazone and their Toxic Impurities

A highly sensitive, selective and accurate thin-layer chromatographic (TLC)-densitometric method has been developed and validated for the simultaneous determination of paracetamol (PAR) and chlorzoxazone (CZ) and their toxic impurities, 4-amino phenol (4AP) and 2-amino-4-chlorophenol (2ACP), respectively, which are also considered to be the hydrolytic degradation products and related substances of the studied drugs. A developing system consisting of chloroform-methanol-glacial acetic acid (9.5:0.5:0.25, by volume) was found to be sufficient for chromatographic separation among the four studied components using pre-activated silica gel 60 F254 TLC plates with ultraviolet detection at 225 nm. Calibration curves were constructed in the ranges of 0.3-3, 1-10, 0.06-3 and 0.04-3 µg/band for PAR, CZ, 4AP and 2ACP, respectively, using polynomial equations. The developed method was validated according to International Conference on Harmonization guidelines and demonstrated good accuracy and precision. Moreover, the method was successfully applied for the determination of PAR and CZ in different marketed samples and the results were statistically compared to those obtained by the reported reversed-phase high-performance liquid chromatography method using F-test and Student's-t test. The low detection and quantitation limits of the developed method make it suitable for quality control and stability studies of PAR and CZ in different pharmaceutical formulations.

Development and validation of a TLC-densitometry method for quantitative analysis of nefopam hydrochloride beside its degradation products

A quantitative densitometric thin-layer chromatographic method for determination of nefopam hydrochloride in pharmaceutical preparations has been established and validated. Nefopam from the formulations was separated and identified on silica gel 60 F254 TLC plates with chloroform-methanol-glacial acetic acid (9: 2: 0.1, v/v/v) as mobile phase. Densitometric quantification was performed at absorbance maximum 266 nm. The method was validated for linearity, sensitivity, precision and recovery in accordance with ICH guidelines. The presented method is selective and specific with potential application in pharmaceutical analysis. Nefopam hydrochloride was subjected to acidic and alkaline hydrolysis at different temperatures. As the method could effectively separate the drug from its degradation products, it can be employed as a stability indicating one.

Development and Validation of a Stability-Indicating Method for Determination of Free Sterols in the Asian Medicinal Leech Hirudo manillensis

A rapid, simple, sensitive, selective, precise and robust thin-layer chromatography densitometric method for the determination of free sterols in leech was developed and validated on silica gel layer using carbon tetrachloride-methanol-formic acid (9.5:1.5:0.55, v/v/v). Spectrodensitometric scanning was carried using a Camag TLC scanner III at 366 nm after spraying 2% methanolic sulphuric acid, which gave compact spots for cholesterol (R(F) = 0.35 ± 0.02). The regression analysis data for calibration plot implied a good linear relationship (r(2) = 0.99958) between response and concentration over the range 100-600 ng per spot with respect to peak area. The limits of detection and quantification were found to be 13.8 ± 0.51 and 45.01 ± 1.29 ng per spot, respectively. Validation was in accordance to the International Conference on Harmonization guidelines. Cholesterol was subjected to forced stress conditions of oxidation, hydrolysis and heat. Degradation products resulting from the forced stress did not interfere with detection because the degradant peaks were well separated from the cholesterol peak. The densitometric method can be regarded as stability-indicating and can be used for quality control assay of cholesterol in leech extract.

HPTLC densitometric analysis of arbutin in bulk drug and methanolic extracts of Arctostaphylos uva-ursi

A high-performance thin layer chromatographic densitometric method for the analysis of arbutin was developed and validated in the present investigation. Arbutin was separated on aluminium-backed silica gel 60 F254 plates with methanol : chloroform (3 : 7)% (v/v) as the mobile phase. This system was found to give a compact spot of arbutin at a retention factor (R f) value of 0.32 ± 0.02. The limit of detection and limit of quantification were found to be 35.42 and 106.26 ng/spot, respectively. The proposed method with a high degree of precision and accuracy was employed for the analysis of arbutin in the bulk drug and methanolic extract of Arctostaphylos uva-ursi.

Separation and determination of four oxicams in pharmaceutical formulations by thin-layer chromatographic-densitometric method

A new, simple and rapid thin-layer chromatography (TLC) method with densitometric detection for simultaneous determination of four oxicams, namely, piroxicam, meloxicam, tenoxicam and isoxicam, has been developed. The chromatographic separation was carried out on TLC F254 plates with a mobile phase consisting of ethyl acetate-ethanol-toluene 6:3:1 (v/v/v) + 2 drops of ammonia 25%. Quantitative determination was done by densitometric scanning at wavelength λ = 360 nm. The method was validated with respect to linearity (in the range from 0.05 to 0.50 mg per band), LOD and LOQ, accuracy (about 99.90%) and precision (RSD from 0.33% to 0.80%) in accordance with the International Conference of Harmonization guidelines. The method has been successfully applied to the analysis of drugs in the pharmaceutical formulations.

ISOLATION, CHARACTERIZATION, AND SIMULTANEOUS QUANTIFICATION OF PICROSIDE I AND KUTKOSIDE USING HPTLC FROM PICRORRHIZA KURROA BENTH

Picrorrhiza kurroa Benth is an important plant of Indian System of Medicine for its well-known pharmacological activities. In the present paper, we report our work on isolation and characterization of two important irridoid glycosides, namely, Picroside I and Kutkoside with a modified method as reported earlier for the plant. Further, these two marker compounds were quantified by thin layer chromatography densitometric methods using high performance thin layer chromatography. To the best of our knowledge, this is the first report of simultaneous quantification of these two compounds using HPTLC from this plant. The thin layer chromatography densitometric methods were found to be precise with RSD for intra-day in the range of 2.69–4.16 and 0.45–2.3 and for inter-day in the range of 1.96–4.25 and 0.75–3.09 for different concentrations of Picroside I and Kutkoside, respectively. Instrumental precision was 3.19 and 3.05 (% RSD) for Picroside I and Kutkoside, respectively. Accuracy of the method was checked by conducting recovery studies at three different levels for both the compounds, and the average percentage recoveries obtained were 99.9 and 99.92%, respectively, for Picroside I and Kutkoside. Picrorrhiza kurroa sample was found to contain 3.66% and 4.44% w/w of Picroside I and Kutkoside, respectively.

A new HPTLC densitometric method for analysis of swertiamarin in Enicostemma littorale and commercial formulations

A high-performance thin layer chromatographic densitometric method for the analysis of swertiamarin in 60% methanolic extract of Enicostemma littorale and commercial formulations has been developed and validated in this study. Swertiamarin was separated on aluminium-backed silica gel 60 F254 plates using ethyl acetate : methanol : water (77 : 15 : 8)% v/v as the mobile phase. This system was found to give a compact spot of swertiamarin at Rf value 0.36 ± 0.01. The limit of detection and limit of quantification were found to be 31.25 and 103.12 ng spot−1, respectively. The proposed method was employed with a high degree of precision and accuracy for the estimation of swertiamarin in methanolic extract of Enicostemma littorale and in commercial formulations.

Development and Validation of a Reversed-Phase High-Performance Thin-Layer ChromatographyDensitometric Method for Determination of Atorvastatin Calcium in Bulk Drug and Tablets

Atorvastatin calcium is a synthetic HMG-CoA reductase inhibitor that is used as a cholesterol-lowering agent. A simple, sensitive, selective, and precise RP-HPTLC-densitometric determination of atorvastatin calcium both as bulk drug and from pharmaceutical formulation was developed and validated according to International Conference on Harmonization guidelines. The method used aluminum sheets precoated with silica gel 60 RP18F254S as the stationary phase, and the mobile phase consisted of methanol-water (3.5 + 1.5, v/v). The system gave a compact band for atorvastatin calcium with an Rf value of 0.62 +/- 0.02. Densitometric quantification was carried out at 246 nm. The linear regression analysis data for the calibration plots showed a good linear relationship with r = 0.9992 in the working concentration range of 100-800 ng/band. The method was validated for precision, accuracy, ruggedness, robustness, specificity, recovery, LOD, and LOQ. The LOD and LOQ were 6 and 18 ng, respectively. The drug underwent hydrolysis when subjected to acidic conditions and was found to be stable under alkali, oxidation, dry heat, and photodegradation conditions. Statistical analysis proved that the developed RP-HPTLC-densitometry method is reproducible and selective and that it can be applied for identification and quantitative determination of atorvastatin calcium in bulk drug and tablet formulation.

Simultaneous Quantification of Bergenin, (+)-Catechin, Gallicin and Gallic acid; and Quantification of β-Sitosterol using HPTLC from Bergenia ciliata (Haw.) Sternb. Forma ligulata Yeo (Pasanbheda)

In the present paper we report our work on development and validation of TLC densitometric method for simultaneous quanti fi cation of Bergenin, (+)-Catechin, Gallicin and Gallic acid, and quanti fi cation of β -Sitosterol using HPTLC.To the best of our knowledge, this is the fi rst report of simultaneous quanti fi cation of these four bioactive compounds viz. Bergenin, (+)-Catechin, Gallicin and Gallic acid using HPTLC from this plant. Simultaneous quanti fi cation of Bergenin, (+)-Catechin, Gallicin and Gallic acid was carried out from the methanolic and hydrolysed extract (2N HCl) using the Solvent System of Toluene: Ethyl acetate: Formic acid (6: 6: 1, v/v/v). β -Sitosterol was quanti fi ed from petroleum ether extract using the Solvent System of Toluene : Methanol (9: 1, v/v). The methods were validated using ICH guidelines in terms of precision, repeatability and accuracy. The thin layer chromatography densitometric methods were found to be precise with RSD for intra-day in the range of 2.62–4.26, 0.35–2.5, 0.61–1.83, 1.14–1.57 and 0.15–0.52 and for interday in the range of 1.90–4.27, 0.85–3.05, 0.97–1.45, 0.58–1.27 and 0.26–0.61 for different concentrations of Bergenin, (+)-Catechin, Gallicin, Gallic acid and β -Sitosterol respectively. The linearity ranges for Bergenin, (+)-Catechin, Gallicin and Gallic acid were found to be the same (160 –720 ng/spot) with correlation coef fi cients ( r -values) of 0.999, 0.997, 0.998 and 0.996 respectively. The linearity range for β -Sitosterol was 80–480 ng/spot with correlation coef fi cient ( r -values) of 0.995. Instrumental precision was 3.39, 3.36, 3.05, 1.20 and 0.85 (% RSD) for Bergenin, (+)-Catechin, Gallicin, Gallic acid and β -Sitosterol respectively. Accuracy of the method was checked by conducting recovery studies at three different levels for both the compounds and the average percentage recoveries obtained were 100.38, 101.45, 102.38, 99.9 and 99.92% respectively for Bergenin, (+)-Catechin, Gallicin, Gallic acid and -Sitosterol. The amount of Bergenin, (+)-Catechin and Gallic acid was 0.22, 0.063 and 0.25 % w/w respectively whereas Gallicin was not in detectable amount. Developed method permitted simultaneous quanti fi cation of Bergenin, (+)-Catechin, Gallicin and Gallic acid, and showed good resolution and separation from other constituents of extract and was found to be simple, precise, speci fi c, sensitive and accurate. It can be adopted for routine quality control of herbal material and formulations containing Bergenia ciliata .

Densitometric analysis of celecoxib, etoricoxib and valdecoxib in pharmaceutical preparations

A densitometric thin-layer chromatographic method has been established for the analysis of celecoxib, etoricoxib, and valdecoxib in pharmaceutical formulations. TLC was performed on silica gel 60 F 254 plates with chloroform-acetone-toluene 12:5:2 ( v/v ) as mobile phase. UV detection was performed by densitometric scanning at 254 and 290 nm. The method was validated by determination of linearity, precision, limits of detection, and determination (from 0.0017 to 0.0848 μg per band) and accuracy (from 99.16 to 100.48%).

Development and Validation of HPLC, TLC and Derivative Spectrophotometric Methods for the Analysis of Ezetimibe in the Presence of Alkaline Induced Degradation Products

AbstractReversed phase‐high performance liquid chromatography (RP‐HPLC), thin layer chromatography (TLC) densitometry and first derivative spectrophotometry (1D) techniques are developed and validated as a stability‐indicating assay of ezetimibe in the presence of alkaline induced degradation products. RP‐HPLC method involves an isocratic elution on a Phenomenex Luna 5μ C18 column using acetonitrile: water: glacial acetic acid (50:50:0.1 v/v/v) as a mobile phase at a flow rate of 1.5 mL/min. and a UV detector at 235 nm. TLC densitometric method is based on the difference in Rf‐values between the intact drug and its degradation products on aluminum‐packed silica gel 60 F254 TLC plates as stationary phase with isopropanol: ammonia 33% (9:1 v/v) as a developing mobile phase. On the fluorescent plates, the spots were located by fluorescence quenching and the densitometric analysis was carried out at 250 nm. Derivative spectrophotometry, the zero‐crossing method, ezetimibe was determined using first derivative at 261 nm in the presence of its degradation products. Calibration graphs of the three suggested methods are linear in the concentration ranges 1–10 mcg/mL, 0.1–1 mg/mL and 1–16 mcg/mL with a mean percentage accuracy of 99.05 ± 0.54%, 99.46 ± 0.63% and 99.24 ± 0.82% of bulk powder, respectively. The three proposed methods were successfully applied for the determination of ezetimibe in raw material and pharmaceutical dosage form; the results were statistically analyzed and compared with those obtained by the reported method. Validation parameters were determined for linearity, accuracy and precision; selectivity and robustness and were assessed by applying the standard addition technique.

Densitometric TLC analysis of histamine in fish and fishery products

A simple and rapid densitometric thin-layer chromatographic method, suitable for monitoring the histamine content of fish and fishery products according to Commission Regulation (EC) No. 2073/2005, has been introduced. The sample was homogenized with methanol, heated in a water bath, and centrifuged. The sample extracts and standard solutions of histamine were applied to silica gel 60 HPTLC plates and developed by ascending chromatography with acetone-ammonia 95:5 ( v/v ) as mobile phase. Histamine was visualized with ninhydrin. The intensity of the colored spots was measured by densitometry at 410 nm. Successful separation of histamine from other biogenic amines and other ninhydrin-positive components in methanolic fish extracts was achieved. The relative standard deviation of repeatability was between 4.7 and 9.8% in the concentration range from 50 to 300 mg kg −1 and 14% for within-laboratory reproducibility at 100 mg histamine kg −1 . Good correlation was achieved by use of an external standard calibr...

Quantitative Analysis of Oxyresveratrol Content in Artocarpus lakoocha and ‘Puag-Haad’

Objective: To develop a thin-layer chromatography (TLC) densitometric method for the determination of oxyresveratrol content in Artocarpuslakoocha heartwood and in the traditional drug ‘Puag-Haad’. Materials and Methods: Sample solution of A. lakoocha heartwood was prepared by Soxhlet extraction of the plant material in ethanol, whereas the Puag-Haad solution was obtained by dissolving the drug in methanol. Analysis of each sample solution was performed on a Silica gel 60 F₂₅₄ TLC plate (20 × 10 cm) with methylene chloride/methanol (85:15) as the mobile phase. After development, the TLC plate was examined with a TLC scanner in the absorbance mode at 254 nm. The newly developed analytical method was validated using an authentic sample of oxyresveratrol previously isolated from A. lakoocha heartwood, and was used to analyze the oxyresveratrol content in samples of A. lakoocha heartwood and the traditional drug Puag-Haad. Results: A sensitive and reliable TLC densitometric method was successfully developed. The method was validated in terms of accuracy (99.11–102.60%) and precision (1.66–4.23% coefficient of variation). The limits of detection and quantitation were 15.6 and 52 ng/spot, respectively. The amounts of oxyresveratrol in 3 samples of A. lakoocha heartwood collected from its natural habitat were 49.0–182.3 mg/g, whereas those in 11 commercial samples were in the range of 23.4–69.6 mg/g. The oxyresveratrol contents in 2 samples of traditional drug Puag-Haad were 780.1 and 837.5 mg/g. Conclusion: The TLC densitometric method developed in this study is a simple, convenient, sensitive and reliable procedure. It was an effective analytical tool for the evaluation of oxyresveratrol content in both A. lakoocha heartwood and the traditional drug Puag-Haad.

Micelle enhanced fluorimetric and thin layer chromatography densitometric methods for the determination of (+/-) citalopram and its S-enantiomer escitalopram.

Two sensitive and validated methods were developed for determination of a racemic mixture citalopram and its enantiomer S-(+) escitalopram. The first method was based on direct measurement of the intrinsic fluorescence of escitalopram using sodium dodecyl sulfate as micelle enhancer. This was further applied to determine escitalopram in spiked human plasma, as well as in the presence of common and co-administrated drugs. The second method was TLC densitometric based on various chiral selectors was investigated. The optimum TLC conditions were found to be sensitive and selective for identification and quantitative determination of enantiomeric purity of escitalopram in drug substance and drug products. The method can be useful to investigate adulteration of pure isomer with the cheap racemic form.

High-Performance Thin-Layer Chromatography Densitometric Method for the Quantification of Harmine, Harmaline, Vasicine, and Vasicinone in Peganum harmala

Peganum harmala Linn. seed is a reputed drug of the Indian systems of medicine. We report a simple high-performance thin-layer chromatography (HPTLC) densitometric method for the quantification of 4 alkaloids, viz., harmine, harmaline, vasicine, and vasicinone from P. harmala seed. The 4 compounds were resolved in ethyl acetate-methanol-ammonia (7 + 1 + 0.3, v/v/v) mobile phase. The method was validated for precision, repeatability, and accuracy. Instrumental precision was 0.27, 1.53, 0.39, and 1.15% [relative standard deviation (RSD)] and repeatability of the method was 1.01, 0.79, 0.13, and 1.62% RSD for harmine, harmaline, vasicine, and vasicinone, respectively. Accuracy of the method was checked by a recovery study conducted at 3 different levels, and the average recovery was 97.88% for harmine, 97.69% for harmaline, 98.38% for vasicine, and 98.28% for vasicinone. The 4 compounds were quantified in P. harmala seed using the method, and it was found to contain 0.44, 0.096, 0.25, and 0.0007% (w/w) of each, respectively. The proposed HPTLC densitometric method for the quantification of the 4 compounds was found to be simple, precise, specific, sensitive, and accurate. It can be used for routine quality control of P. harmala seed and has the ability to quantify the 4 marker compounds in the formulations containing P. harmala. It also can be used to quantify any of these marker compounds in other herbal drugs.

Effect of hydrolysis on the yield of hederagenin and High-performance thin-layer chromatography densitometric quantification of hederagenin in fruit pericarp of Sapindus spp

Fruit pericarp of Sapindus species are reported to contain glycosides with hederagenin as an aglycone. To free the aglycone from the glycosides, they need to be hydrolyzed, and the commonly used method is hydrolysis with either hydrochloric or sulfuric acid. In the present work, we studied the effect of hydrolysis on the yield of hederagenin from the fruit pericarp of 3 species of Sapindus, viz., S. mukorossi, S. laurifolius, and S. emarginatus. A high-performance thin-layer chromatography densitometric method for the quantification of hederagenin was developed and validated. It involved automated application of samples as bands onto silica gel 60F254 plates, development with toluene-ethyl acetate-formic acid (7 + 3 + 5, v/v/v) mobile phase, detection with anisaldehyde-sulfuric acid reagent, and scanning at 595 nm. The yield of hederagenin ranged from 0.035 to 1.29% (w/w) with different methods of hydrolysis. Hydrolysis with 3.5 M aqueous sulfuric acid under reflux for 6 h gave the maximum yield of hederagenin in all 3 species, with the highest amount in S. emarginatus (1.29%, w/w).