Determination of Sumatriptan and Zolmitriptan in Presence of Their Corresponding Degradation Products by HPTLC Methods

Accurate, sensitive, and precise high performance thin layer chromatographic (HPTLC) methods were developed and validated for the determination of sumatriptan and zolmitriptan in presence of their degradation products. Sumatriptan was separated from its degradation products and analyzed on TLC silica gel 60 F₂₅₄ plates using chloroform–ethyl acetate–methanol–ammonia (4:3:3:0.1, v/v) as a developing system followed by densitometric measurement of the bands at 228 nm. Zolmitriptan was determined using chloroform–ethyl acetate–methanol–ammonia (3:3:3:1, v/v) as a developing system followed by densitometric measurement at 222 nm. The methods were validated over a range of 0.5–4 μg/spot for sumatriptan and 0.5–3 μg/spot for zolmitriptan. The proposed methods were successfully applied for the determination of the studied drugs in bulk powder and in their pharmaceutical formulations.     

Simultaneous HPTLC and RP-HPLC methods for determination of bumadizone in the presence of its alkaline-induced degradation product

Accurate, selective, sensitive and precise HPTLC‐densitometric and RP‐HPLC methods were developed and validated for determination of bumadizone calcium semi‐hydrate in the presence of its alkaline‐induced degradation product and in pharmaceutical formulation. Method A uses HPTLC‐densitometry, depending on separation and quantitation of bumadizone and its alkaline‐induced degradation product on TLC silica gel 60 F₂₅₄ plates, using hexane–ethyl acetate–glacial acetic acid (8:2:0.2, v/v/v) as a mobile phase followed by densitometric measurement of the bands at 240 nm. Method B comprises RP‐HPLC separation of bumadizone and its alkaline‐induced degradation product using a mobile phase consisting of methanol–water–acetonitrile (20:30:50, v/v/v) on a Phenomenex C₁₈ column at a flow‐rate of 2 mL/min and UV detection at 235 nm. The proposed methods were successfully applied to the analysis of bumadizone either in bulk powder or in pharmaceutical formulation without interference from other dosage form additives, and the results were statistically compared with the established method. Copyright © 2011 John Wiley & Sons, Ltd.     

Development and validation of a normal-phase high-performance thin layer chromatographic method for the analysis of sulfamethoxazole and trimethoprim in co-trimoxazole tablets

Pneumocystis carinii pneumonia (PCP) is often the ultimate mortal cause for immunocompromised individuals, such as HIV/AIDS patients. Currently, the most effective medicine for treatment and prophylaxis is co-trimoxazole, a synergistic combination of sulfamethoxazole (SMX) and trimethoprim (TMP). In order to ensure a continued availability of high quality co-trimoxazole tablets within resource-limited countries, Medicines Regulatory Authorities must perform quality control of these products. However, most pharmacopoeial methods are based on high-performance liquid chromatographic (HPLC) methods. Because of the lack of equipment, the Tanzania Food and Drugs Authority (TFDA) laboratory decided to develop and validate an alternative method of analysis based on the TLC technique with densitometric detection, for the routine quality control of co-trimoxazole tablets. SMX and TMP were separated on glass-backed silica gel 60 F₂₅₄ plates in a high-performance thin layer chromatograph (HPTLC). The mobile phase was comprised of toluene, ethylacetate and methanol (50:28.5:21.5, v:v:v). Detection wavelength was 254 nm. The R_f values were 0.30 and 0.61 for TMP and SMX, respectively. This method was validated for linearity, precision, trueness, specificity and robustness. Cochran's criterion test indicated homoscedasticity of variances for the calibration data. The F-tests for lack-of-fit indicated that straight lines were adequate to describe the relationship between spot areas and concentrations for each compound. The percentage relative standard deviations for repeatability and time-different precisions were 0.98 and 1.32, and 0.83 and 1.64 for SMX and TMP, respectively. Percentage recovery values were 99.00% ± 1.83 and 99.66% ± 1.21 for SMX and TMP, respectively. The method was found to be robust and was then successfully applied to analyze co-trimoxazole tablet samples.     

Determination of Oxazepam in Pharmaceutical Formulation by HPTLC UV-Densitometric and UV-Derivative Spectrophotometry Methods

Abstract

Methods for determination of oxazepam in pharmaceutical formulation by derivative ultraviolet (UV) spectrophotometry as well as high-performance thin-layer chromatography (HPTLC) UV densitometry were described. For UV-derivative spectrophotometry, some derivatives and wavelengths may be recommended for routine quality control of the drug of interest. On the other hand, HPTLC provided good results, but only when the calibration curve was estimated using nonlinear regression analysis. The HPTLC method was developed with silica F₂₅₄ plates, a mobile phase of benzene/ethanol (5:1, v/v), and densitometric detection at 204 nm receiving R _f  = 0.47. Developed methods were validated and found to be sufficiently precise and reproducible for established conditions.     

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.     

A Rapid and Inexpensive Thin Layer Chromatographic Method for Quantitative Analysis of Bleomycin Complex

Abstract

A densitometric and a spectrophotometric method for rapid but accurate determination of different components of bleomycin injections has been described. The bleomycin components were separated by reversed phase thin layer chromatography on silanized silicagel plates using a mixture of aqueous ammonium nitrate (2.5%) : methanol :: 7 : 3 (v/v) as mobile phase. Assay was done at the absorption maxima of the components (291 nm) by in situ densitometry or by spectroscopy after extracting the drugs from the adsorbent with the mobile phase. Results obtained by both the methods agreed well with each other and with those obtained by an official HPLC method. The densitometric method described was highly suitable for routine quality control of bleomycines as a large number of samples could be analysed within a short time (68 samples/analyst/day).     

Densitometry and indirect normal‐phase HPTLC–ESI–MS for separation and quantitation of drugs and their glucuronide metabolites from plasma

The present work describes novel methods using densitometry and indirect or off‐line high performance thin‐layer chromatography–mass spectrometry (HPTLC–MS) for the simultaneous detection and quantification of asenapine, propranolol and telmisartan and their phase II glucuronide metabolites. After chromatographic separation of the drugs and their metabolites the analytes were scraped, extracted in methanol and concentrated prior to mass spectrometric analysis. Different combinations of toluene and methanol–ethanol–n‐butanol–iso‐propanol were tested for analyte separation and the best results were obtained using toluene–methanol–ammonia (6.9:3.0:0.1, v/v/v) as the elution solvent. All of the drug–metabolite pairs were separated with a homologous retardation factor difference of ≥22. The conventional densitometric approach was also studied and the method performances were compared. Both of the approaches were validated following the International Conference on Harmonization guidelines, and applied to spiked human plasma samples. The major advantage of the TLC–MS approach is that it can provide much lower limits of detection (1.98–5.83 pg/band) and limit of quantitation (5.97–17.63 pg/band) with good precision (?3.0% coefficient of variation) compared with TLC–densitometry. The proposed indirect HPTLC–MS method is simple yet effective and has tremendous potential in the separation and quantitation of drugs and their metabolites from biological samples, especially for clinical studies.     

Different chromatographic methods for simultaneous determination of diloxanide furoate,metronidazole and its toxic impurity

TLC densitometric and RP-HPLC methods are innovative chromatographic methods used for determination of diloxanide furoate, metronidazole and its impurity, 4-nitroimidazole. In the developed TLC densitometric method, appropriate separation was achieved using silica gel 60 F₂₅₄ TLC plates and ethyl acetate/acetone/hexane/ammonia solution (9.5:0.5:0.3:0.3, by volume), as a developing system and the separated bands were UV-scanned at 276 nm. While the developed RP-HPLC method depended on separation of components on C8 column using deionized water containing 0.05 % TEA: methanol (40:60, v/v) as a mobile phase at constant flow rate of 1 mL/min with UV detection at 276 nm. Variables affecting performance of the developed methods were studied and optimized. Regression analysis showed acceptable correlation coefficients in the selected ranges with excellent percentage recoveries. The methods showed no significant interferences from dosage form excipients, and the validity of the proposed methods was further assessed by applying standard addition technique. In addition, results obtained by applying the proposed methods were statistically compared to those obtained by applying the reported method and no significant difference was found between them. The suggested methods were successfully applied for the determination of the cited drugs in bulk powder, laboratory prepared mixtures and commercial tablets.     

Development and validation of a thin-layer chromatography-densitometric method for the quantitation of alliin from garlic (Allium sativum) and its formulations

A selective, precise, and accurate high-performance thin-layer chromatographic (HPTLC) method has been proposed for the analysis of garlic and its formulations for their alliin content. The method involves densitometric evaluation of alliin after resolving it by HPTLC on silica gel plates with n-butanol-acetic acid-water (6 + 2 + 2, v/v) as the mobile phase. For densitometric evaluation, peak areas were recorded at 540 nm after derivatizing the resolved bands with ninhydrin reagent. The relation between the concentration of alliin and corresponding peak areas was found to be linear within the range of 250 to 1500 ng/spot. The method was validated for precision (interday and intraday), repeatability, and accuracy. Mean recovery was 98.36%. The method was applied for the quantitation of alliin in bulbs of Allium sativum Linn. (garlic) and its formulations. The proposed TLC method was found to be precise, specific, sensitive, and accurate and can be used for routine quality control of garlic and its formulations.     

Simultaneous Separation and Determination of Lamivudine and Zidovudine in Pharmaceutical Formulations Using the HPTLC Method

Abstract

A high-performance thin-layer chromatographic method (HPTLC) for the simultaneous determination of lamivudine and zidovudine in a binary mixture has been developed. The method developed was based on HPTLC separation of the two drugs followed by densitometric measurements of spots at 276 and 271 nm for lamivudine and zidovudine, respectively. Separation was carried out on Merck HPTLC silica-gel 60 F₂₅₄ plates, using toluene/chloroform/methanol (1:6:3 v:v) as the mobile phase. Validation of the method was performed based on The International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) guidelines in terms of linearity, accuracy, precision, limit of detection, limit of quantification, and robustness. Second-order polynomial equations were obtained for the regression line in the ranges of 250–1400 and 250–1700 ng/spot for lamivudine and zidovudine respectively. Correlation coefficient (r) values were 0.9998 for both analytes. The method provides sufficient accuracy as indicated by recovery percentages given for lamivudine and zidovudine. For system precision study, the low coefficient of variation values (<2%) for both lamivudine and zidovudine ensured reproducible performance of the instrument. In the method precision study, coefficients of variation <2% were obtained, which showed that the proposed method provides acceptable intraday and interday variation. The detection and quantification limits and were 3.06 and 9.28 ng/spot for lamivudine and 3.34 and 10.13 ng/spot for zidovudine, respectively. Parameters such as mobile-phase composition, volume of mobile phase, time from spotting to development, and time from development to scanning were employed while testing for robustness of the method, and the standard deviation of peak areas was calculated for each parameter. The low coefficient of variation values indicated the robustness of the method. Statistical manipulation did not show any significant effect of one parameter over the others on the robustness of the method.     

HPTLC analysis of ternary mixture containing ketorolac tromethamine,phenylephrine hydrochloride,and chlorpheniramine maleate

Validated and selective high-performance thin-layer chromatography (HPTLC) method was developed for the determination of ketorolac tromethamine (KTC), phenylephrine hydrochloride (PHE), and chlorpheniramine maleate (CPM) in bulk drug and in combined dosage form. The proposed method depends on using HPTLC for separation of the drugs followed by densitometric measurements of their spots at 261?nm. The separation was carried out on Merck HPTLC aluminum sheets of silica gel 60 F254 using chloroform–methanol–ammonia (7.75:2.25:0.1, v/v) as mobile phase. Linear regression lines were obtained over the concentration ranges 0.12–0.50, 0.075–0.27, and 0.09–0.27?µg band^?1 for KTC, PHE, and CPM, respectively, with correlation coefficients higher than 0.999. The method was successfully applied to the analysis of the three drugs in their synthetic mixtures and in their dosage form. The mean percentage recoveries were in the range of 98–102% with percentage relative standard deviation values less than 2%. The method was validated according to ICH guidelines and showed good performances in terms of linearity, precision, accuracy, sensitivity, and stability.     

Separation and quantification of terpenoids of Boswellia serrata Roxb. extract by planar chromatography techniques (TLC and AMD)

An high-performance TLC (HPTLC) method for the separation of boswellic acids, the active constituents in Boswellia serrata extract, has been developed and TLC of these compounds on silica by automated multiple development (AMD) using solvent gradients was performed. Enhancement of the separation of boswellic acids on HPTLC plates was carried out by AMD chromatography. Densitometric analysis of the developed plate was carried out to quantify the four boswellic acids. 11-Keto-beta-boswellic acid (KBA) and acetyl-11-keto-beta-boswellic acid (AKBA) were quantified by densitometric scanning of the developed plate at 254 nm. beta-Boswellic acid (BA) and acetyl-beta-boswellic acid (ABA) were quantified after derivatization with anisaldehyde sulfuric acid reagent at 560 nm. The AMD system provided a clean separation according to polarity for each of the four groups studied and good results were obtained. The proposed HPTLC method for the simultaneous quantification of the major boswellic acids BA, ABA, KBA, and AKBA was found to be simple, precise, specific, sensitive, and accurate and can be used for routine quality control and for the quantification of these compounds in plant materials. The study of market products revealed significant variations in the content of these pharmacologically active compounds in commercial samples.     

Separation and quantification of lignans in Phyllanthus species by a simple chiral densitometric method

A sensitive, selective, and robust high-performance TLC (HPTLC) method using chiral TLC plates for qualitative and quantitative analysis of phyllanthin (A), hypophyllanthin (B), niranthin (C), and nirtetralin (D), the active lignans of Phyllanthus species, was developed and validated. The effectiveness and role of various stationary phases viz TLC silica gel 60F(254), HPTLC silica gel 60F(254), and chiral TLC plates in the quantitation were evaluated. A precoated chiral TLC plate was found suitable for the simultaneous analysis of four pharmacologically active lignans. For achieving good separation, the optimized mobile phase of n-hexane/acetone/1,4-dioxane (9:1:0.5 by volume) was used (R(f) = 0.30, 0.36, 0.41, and 0.48 for compounds A, B, C, and D, respectively). A densitometric determination of the above compounds was carried out in reflection/absorption mode at 620 nm. Optimized chromatographic conditions provide well-separated compact bands for the tested lignans. The calibration curves were found linear in the concentration range of 100-500 ng/band. Recoveries of A-D were 99.98, 100.51, 99.22, and 98.74%, respectively. The method was validated according to ICH guidelines. The method reported here is reproducible and applied for the quantitative analysis of the above lignans in the leaves of four Phyllanthus species, i. e., P. amarus, P. maderaspatensis, P. urinaria, and P. virgatus.     

Simultaneous densitometric determination of shikonin,acetylshikonin, and β‐acetoxyisovaleryl‐shikonin in ultrasonic‐assisted extracts of four Arnebia species using reversed‐phase thin layer chromatography

A simple, precise, and rapid high‐performance thin‐layer chromatographic (HPTLC) method for the simultaneous quantification of pharmacologically important naphthoquinone shikonin ( 1 ) together with its derivatives acetylshikonin ( 2 ), and β‐acetoxyisovalerylshikonin ( 3 ) in four species of genus Arnebia (A. euchroma, A. guttata, A. benthamii, and A. hispidissima) from the Indian subcontinent has been developed. In addition, the effect of solvents with varying polarity (hexane, chloroform, ethyl acetate, and methanol) for the extraction of these compounds was studied. HPTLC was performed on precoated RP‐18 F_254S TLC plates. For achieving good separation, mobile phase consisting of ACN/methanol/5% formic acid in water (40:02:08 v/v/v) was used. The densitometric determination of shikonin derivatives was carried out at 520 nm in reflection/absorption mode. The method was validated in terms of linearity, accuracy, precision, robustness, and specificity. The calibration curves were linear in the range of 100–600 ng for shikonin and acetylshikonin, and 100–1800 ng for β‐acetoxyisovalerylshikonin. Lower LOD obtained for compounds 1 – 3 were 18, 15, and 12 ng, respectively, while the LOQ obtained were 60, 45, and 40 ng, respectively.     

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.     

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

Reversed 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.     

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.     

Determination of Sulfanilamide and Sulfisoxazole in Drug Preparations by Quantitative High Performance TLC

Abstract

A densitometric TLC method was developed for quantification of sulfanilamide and sulfisoxazole in creams, supppositories, and tablets. The sulfas were extracted into acidic ethanol, diluted to an appropriate volume, and separated by silica gel HPTLC. The fluorescence quenching of the sulfa zones in samples and standards was compared by in situ scanning. Recoveries of the drugs from authentic samples ranged from 96–105%. Recoveries from products with old expiration dates were low. Identity of the sulfa drugs in products was confirmed by application of fluorescamine and Bratton-Marshall detection reagents.     

Stability-indicating high-performance thin-layer chromatographic determination of levonorgestrel and ethinyloestradiol in bulk drug and in low-dosage oral contraceptives

A stability-indicating high-performance thin-layer chromatography (HPTLC) method was developed and validated for simultaneous determination of steroidal hormones levonorgestrel and ethinyloestradiol both in bulk drug and in low-dosage oral contraceptives. Optimization of conditions for the spectrodensitometric procedure was reached by eluting HPTLC silica gel plates in a 10 cm × 10 cm horizontal chamber. The solvent system consisted of hexane-chloroform-methanol (1.0:3.0:0.25, v/v/v). This system was found to give compact, dense and typical peaks for both levonorgestrel (R_f = 0.65 ± 0.03) and ethinyloestradiol (R_f = 0.43 ± 0.02). Densitometric analysis of the drugs was carried out in the reflectance mode at 225 nm by using a computer controlled densitometric scanner. The calibration curves of levonorgestrel and ethinyloestradiol were linear in the range of 200-800 and 40-160 ng per spot, respectively. The method was validated for precision, robustness and recovery. As the proposed method can effectively separate the drugs from their degradation products, it can be employed as a stability-indicating method.     

Characterization and classification of medicinal plants according to their antioxidant profile estimated by thin layer chromatography assisted by chemometric expertise

This study focuses on the characterization and classification of 42 medicinal plants extracts according to their antioxidant activity. Principal component analysis (PCA), cluster analysis (CA) and the combination of PCA with linear discriminant analysis (PCA-LDA) were used as multivariate exploratory techniques for chromatographic data analysis. For the separation of the compounds a mobile phase containing ethyl acetate: toluene: formic acid: water (30:1.5:4:3 v/v/v/v) and different HPTLC plates (Silica gel 60 and HPTLC Silica gel 60?F₂₅₄) were used. The chromatographic plates were evaluated using the images obtained after spraying the plates with 2-aminoethyldiphenylborate solution (NTS, 0.2% in ethanol) and also after their reaction with 2, 2-diphenyl-1-picrylhydrazyl solution (DPPH^?) (0.2% in ethanol). The score projection on the plane defined by first two components (PC1 and PC2) revealed two large groups of the investigated samples depicted according to their antioxidant capacity. A better classification of samples according to their antioxidant capacity was obtained using the CA and PCA-LDA methodology in all cases. The excellent results obtained in this study concerning the classification of medicinal plants according to their antioxidant capacity using the PCA-LDA methodology applied to TLC chromatograms might lead to a new paradigm in the field of medicinal plant holistic evaluation.