Validated HPTLC method for simultaneous estimation of levofloxacin hemihydrate and ornidazole in pharmaceutical dosage form

A simple, rapid, and accurate high-performance thin-layer chromatography (HPTLC) method is described for the simultaneous determination of levofloxacin hemihydrate and ornidazole in tablet dosage form. The method is based on the HPTLC separation of the two drugs followed by densitometric measurements of their spots at 298 nm. The separation is carried out on Merck TLC aluminium sheets of silica gel 60 F254 using n-butanol-methanol-ammonia (5:1:1.5, v/v/v) as mobile phase. The linearity is found to be in the range of 50-250 and 100-500 ng/spot for levofloxacin hemihydrate and ornidazole, respectively. The method is successively applied to pharmaceutical formulation because no chromatographic interferences from the tablet excipients are found. The suitability of this HPTLC method for the quantitative determination of the compounds is proved by validation in accordance with the requirements laid down by International Conference on Harmonization (ICH) guidelines.     

Comparison of high performance TLC and HPLC for separation and quantification of chlorogenic acid in green coffee bean extracts

Two chromatographic methods, high-performance TLC (HPTLC) and HPLC, were developed and used for separation and quantitative determination of chlorogenic acid in green coffee bean extracts. For HPTLC silica gel Kieselgel 60 F 254 plates with ethyl acetate/dichlormethane/formic acid/acetic acid/water (100:25:10:10:11, v/v/v/v/v) as mobile phase were used. Densitometric determination of chlorogenic acid by HPTLC was performed at 330 nm. A gradient RP HPLC method was carried out at 330 nm. All necessary validation tests for both methods were developed for their comparison. There were no statistically significant differences between HPLC and HPTLC for quantitative determination of chlorogenic acid according to the test of equality of the means.     

Quantitative determination of L-DOPA in tablets by high performance thin layer chromatography

A densitometric high performance thin-layer chromatographic (HPTLC) method was developed and validated for quantitative analysis of L-DOPA in tablets. Chromatographic separation was achieved on precoated silica gel F 254 HPTLC plates using a mixture of acetone-chloroform-n-butanol-acetic acid glacial-water (60:40:40:40:35 v/v/v/v/v) as mobile phase. Quantitative analysis was carried out at a wavelength of 497 nm. The method was linear between 100 and 500 ng/microL, with a correlation coefficient of 0.999. The intra-assay variation was between 0.26 and 0.65% and the interassay was between 0.52 and 2.04%. The detection limit was 1.12 ng/microL, and the quantification limit was 3.29 ng/microL. The accuracy ranged from 100.40 to 101.09%, with a CV not higher than 1.40%. The method was successfully applied to quantify L-DOPA in real pharmaceutical samples, including the comparison with HPLC measurements. The method was fast, specific, with a good precision, and accurate for the quantitative determination of L-DOPA in tablets.     

High-performance thin-layer chromatographic method for quantitative determination of 4alpha-methyl-24beta-ethyl-5alpha-cholesta-14,25-dien-3beta-ol, 24beta-ethylcholesta-5,9(11),22E-trien-3beta-ol, and betulinic acid in Clerodendrum inerme

A sensitive, selective, precise, and robust high-performance thin-layer chromatography method was developed and validated for analysis of two new recently isolated sterols, 4alpha-methyl-24beta-ethyl-5alpha-cholesta-14,25-dien-3beta-ol (1) and 24beta-ethylcholesta-5,9(11),22E-trien-3beta-ol (2), and a triterpene, betulinic acid (3), in Clerodendrum inerme extract. The method employed HPTLC plates precoated with silica gel 60F(254 )as the stationary phase. To achieve good separation, an optimised mobile phase consisting of toluene-acetone (94:06, v/v) was used (R(f )0.48, 0.34, and 0.22 for compounds 1, 2, and 3, respectively). Densitometric determination of the above compounds was carried out in reflection/absorption mode at 620 nm. Optimised chromatographic conditions provide well separated compact spots for the compounds 1, 2, and 3. The calibration curves were linear in the concentration range of 100-2500 ng/spot. The method was validated for precision, robustness, and recovery. The limits of detection and quantitation were 5, 6, and 10 microg/mL and 14, 18, and 29 microg/mL, respectively, for 1, 2, and 3. The method reported here is reproducible and convenient for quantitative analysis of these compounds in the aerial parts of C. inerme.     

High-performance thin layer chromatography method for quantitative determination of four major anthraquinone derivatives in Rheum emodi

A high-performance thin layer chromatographic (HPTLC) method for the rapid and simple quantification of the four major anthraquinone derivatives i.e. physcion, chrysophanol, emodin and chrysophanol glycoside in Rheum emodi is described. HPTLC of anthraquinone derivatives was performed on pre-coated RP-18 F254S HPTLC plates. For achieving good separation, the mobile phase of methanol-water-formic acid (80:19:1, v/v/v) was used. The densitometric determination of anthraquinone derivatives was carried out at 445 nm in reflection/absorption mode. The calibration curves were linear in the range of 20-100 ng for physcion, 80-400 ng for chrysophanol and emodin, and 200-1000 ng for chrysophanol glycoside. The method was found to be reproducible and convenient for quantitative analysis of anthraquinone derivatives in the methanolic extract of rhizomes of R. emodi collected from three different locations of Western Himalaya, India.     

Estimation of L-dopa from Mucuna pruriens LINN and formulations containing M. pruriens by HPTLC method

A selective, precise, and accurate high-performance thin-layer chromatographic (HPTLC) method has been developed for the analysis of L-dopa in Mucuna pruriens seed extract and its formulations. The method involves densitometric evaluation of L-dopa after resolving it by HPTLC on silica gel plates with n-butanol-acetic acid-water (4.0+1.0+1.0, v/v) as the mobile phase. Densitometric analysis of L-dopa was carried out in the absorbance mode at 280 nm. The relationship between the concentration of L-dopa and corresponding peak areas was found to be linear in the range of 100 to 1200 ng/spot. The method was validated for precision (inter and intraday), repeatability, and accuracy. Mean recovery was 100.30%. The relative standard deviation (RSD) values of the precision were found to be in the range 0.64-1.52%. In conclusion, the proposed TLC method was found to be precise, specific and accurate and can be used for identification and quantitative determination of L-dopa in herbal extract and its formulations.     

HPTLC analysis of organotin compounds in preservative solutions and preservative-treated wood

A high-performance thin-layer chromatographic (HPTLC) method is described for the determination of tributyltin compounds (bis(tri-n-butyltin) oxide, TBTO, and tri-n-butyltin naphthenate, TBTN) and their degradation products (dibutyltin and monobutyltin compounds). The organotin compounds are extracted from wood with ethanol containing 0.5% (v/v) of hydrochloric acid and the separation of the defferent kinds of organotin compounds is achieved by thin-layer chromatography. The sample spots are measured using a scanning densitometer after decomposing the organotin compounds to inorganic tin by ultraviolet irradiation and visualization of the spots with pyrocatechol violet. Applications of the method to detection and quantification of organotin compounds in preservative solutions, in recently impregnated wood, and in wood samples from five-year-old window frames are described.     

Micro Scale Procedure for Analysis of Andrographolide in Andrographis paniculata Leaves

JPC – Journal of Planar Chromatography – Modern TLC - A high-performance thin-layer chromatographic (HPTLC) method has been established for quantitative analysis of andrographolide, the...     

Isolation of gentamicin C compounds from culture filtrates of Micromonospora purpurea

A liquid chromatographic method was developed for the isolation of gentamicin C compounds from commercial fermentation products in order to monitor health hazards (oto- and nephrotoxicity). Chromatography was carried out on silica gel 60 (15-40 microns) with a medium-pressure chromatographic system, employing methanol-25% ammonia solution (85:15, v/v) and methanol-chloroform-25% ammonia solution (20:10:5, v/v) as mobile phases. The eluted fractions were neutralized with 1.0 M hydrochloric acid, concentrated in vacuo and desalted by gel filtration. It was possible to demonstrate by 1H NMR spectroscopy and high-performance liquid and thin-layer chromatography that the separated fractions contained components C1, C1a and C2 in purities of more than 95%.     

The ICH guidance in practice: stress degradation studies on stavudine and development of a validated specific stability-indicating HPTLC assay method

A sensitive, selective, precise, and stability-indicating high-performance thin-layer chromatographic (HPTLC) method for the analysis of stavudine both as a bulk drug and in formulations is developed and validated. The solvent system consisted of toluene-methanol-chloroform-acetone (7.0:3.0:1.0:1.0, v/v/v/v). Densitometric analysis of stavudine is carried out in the absorbance mode at 270 nm. This system is found to give compact spots for stavudine (retention factor value of 0.45 +/- 0.05) following development of chromatoplates with the mobile phase. Stavudine is subjected to acid and alkali hydrolysis, oxidation, dry-heat and wet-heat treatment, and photo and UV degradation. The drug undergoes degradation under acidic and basic conditions, oxidation, and wet-heat degradation. Linearity is found to be in the range of 30-1000 ng/spot with a significantly high value of correlation coefficient. The linear regression analysis data for the calibration plots show a good linear relationship with r2 = 0.9997 +/- 0.05 in the working concentration range of 300 to 1000 ng/spot. The mean value of slope and intercept are 0.10 +/- 0.06 and 22.12 +/- 1.08, respectively. The method is validated for precision, robustness, and recovery. The limits of detection and quantitation are 10 and 30 ng/spot, respectively. The proposed HPTLC method is utilized to investigate the kinetics of the acid degradation process. Arrhenius plot is constructed and activation energy is calculated.     

Determination of Ranolazine in Tablet Formulations by High-Performance Thin-Layer Chromatography—Mass Spectrometry Using Reflectance Scanning Densitometry

JPC – Journal of Planar Chromatography – Modern TLC - A new quantitative densitometric high-performance thin-layer chromatographic (HPTLC) method was developed and validated for the...     

Estimation of trans-resveratrol in herbal extracts and dosage forms by high-performance thin-layer chromatography

A simple, sensitive and precise high-performance thin-layer chromatographic (HPTLC) method of analysis of trans-resveratrol in Polygonum cuspidatum root extracts and in dosage forms was developed and validated. The separation was carried out on a TLC aluminium plates precoated with silica gel 60F-254 as the stationary phase, eluted with chloroform-ethylacetate-formic acid (2.5 : 1 : 0.1) as mobile phase. Densitometric analysis of trans-resveratrol was carried out in the absorbance mode at 313 nm. This system was found to give compact spot for trans-resveratrol (Rf value of 0.40+/-0.03). A good linear regression relationship between peak areas and the concentrations was obtained over the range of 0.5-3.0 microg/spot with correlation coefficient 0.9989. The limit of detection and quantification was found to be 9 and 27 ng/spot. The method was validated for precision and recovery. The spike recoveries were within 99.85 to 100.70%. The RSD values of the precision in the range 0.37-1.84%. The proposed developed HPTLC method can be applied for identification and quantitative determination of trans-resveratrol in herbal extracts and dosage forms.     

Quantitative determination of haloperidol in tablets by high performance thin-layer chromatography

A densitometric high performance thin-layer chromatography (HPTLC) method was developed and validated for the quantitative analysis of haloperidol in tablets. Chromatographic separation was achieved on precoated silica gel F 254 HPTLC plates using a mixture of acetone/chloroform/n-butanol/acetic acid glacial/water (5:10:10:2.5:2.5 v/v/v/v/v) as the mobile phase. Quantitative analysis was carried out at a wavelength of 254 nm. The method was linear in the 10-100 ng/microL range, with a determination coefficient of 0.999. The coefficients of variation for precision were not higher than 2.35%. The detection limit was 0.89 ng/microL, and the quantification limit was 2.71 ng/microL. The accuracy ranged from 97.76 to 100.33%, with a CV not higher than 4.50%. This method was successfully applied to quantify haloperidol in real pharmaceutical samples, including the comparison with HPLC measurements. The method was fast, specific, with a good precision and accuracy for the quantitative determination of haloperidol in tablets.     

Determination of Rutin in Amaranthus spinosus Linn. Whole Plant Powder by HPTLC

A simple, precise and accurate high-performance thin-layer chromatographic method has been established for the determination of rutin in the whole plant powder of Amaranthus spinosus Linn. Rutin has been reported to have anti-diabetic, anti-thrombotic, anti-inflammatory and anti-carcinogenic activity. A methanol extract of the whole plant powder was used for the experimental work. The concentration of rutin in the whole plant powder was found to be 0.15%. Separation was performed on silica gel 60 F₂₅₄ HPTLC plates with ethyl acetate:formic acid:methanol:distilled water in the proportion 10:0.9:1.1:1.7 (v/v), as mobile phase. The determination was carried out using the densitometric absorbance mode at 363 nm. Rutin response was linear over the range 10–60 μg mL^?1. The HPTLC method was evaluated in terms of sensitivity, accuracy, precision and reproducibility.     

High-Performance Thin-Layer Chromatographic (HPTLC) Assessment of the Photodegradation of Bensulfuron-Methyl on Silica Gel

JPC – Journal of Planar Chromatography – Modern TLC - A quantitative instrumental high-performance thin-layer chromatographic (HPTLC) method has been developed for assessment of the...     

High-Performance Thin-Layer Chromatographic Determination of Itopride Hydrochloride in its Pharmaceutical Preparation and in the Bulk Drug

JPC – Journal of Planar Chromatography – Modern TLC - A normal-phase high-performance thin-layer chromatographic (HPTLC) method has been established for quantitative estimation of...     

Development and Validation of an HPTLC Method for the Analysis of Oleanolic Acid from the Roots of Helicteres isora Linn.

JPC – Journal of Planar Chromatography – Modern TLC - A sensitive and reliable high-performance thin-layer chromatographic (HPTLC) method has been developed for the quantitative...     

Simultaneous quantification of withanolides in Withania somnifera by a validated high-performance thin-layer chromatographic method

This paper describes a sensitive, selective, specific, robust, and validated densitometric high-performance thin-layer chromatographic (HPTLC) method for the simultaneous determination of 3 key withanolides, namely, withaferin-A, 12-deoxywithastramonolide, and withanolide-A, in Ashwagandha (Withania somnifera) plant samples. The separation was performed on aluminum-backed silica gel 60F254 HPTLC plates using dichloromethane-methanol-acetone-diethyl ether (15 + 1 + 1 + 1, v/v/v/v) as the mobile phase. The withanolides were quantified by densitometry in the reflection/absorption mode at 230 nm. Precise and accurate quantification could be performed in the linear working concentration range of 66-330 ng/band with good correlation (r2 = 0.997, 0.999, and 0.996, respectively). The method was validated for recovery, precision, accuracy, robustness, limit of detection, limit of quantitation, and specificity according to International Conference on Harmonization guidelines. Specificity of quantification was confirmed using retention factor (Rf) values, UV-Vis spectral correlation, and electrospray ionization mass spectra of marker compounds in sample tracks.     

Quantification of Lupeol in Polyherbal Formulation by High-Performance Thin-Layer Chromatography Method Using Design of Experiments as a Tool to Assess Optimization of Method Development and Extraction Efficiency

JPC – Journal of Planar Chromatography – Modern TLC - A simple, specific, and quantitative high-performance thin-layer chromatographic (HPTLC) method has been developed for the...     

Validated High-Performance Thin-Layer Chromatographic Method for the Simultaneous Determination of Quercetin,Rutin, and Gallic Acid in Amaranthus tricolor L.

JPC – Journal of Planar Chromatography – Modern TLC - A simple, rapid, quantitative, and validated high-performance thin-layer chromatographic (HPTLC) method has been developed for the...