Applying novel economic simple green sample preparation procedures on natural and industrial specimens for chromatographic determination of insecticidal residues.

Spraying a tertiary blend of the insecticides (hexythiazox, imidacloprid, and thiamethoxam), on tomato fruits, is a routine in agriculture-attentive countries. A simple green sample preparation technique was developed and applied to the field samples. Specific HP-TLC and RP-HPLC methodologies are established to estimate the residual insecticides and applied to the prepared field specimens. In the planner chromatographic methodology, methanol:chloroform:glacial acetic acid:triethyl amine (8.5:1.5:0.2:0.1, v/v) is recommended as a mobile system. The other one is columnar chromatography; acetonitrile: water (20:80, v/v), pH 2.8, is recommended as a mobile system. The validation parameters were examined following the ICH rules. The means percentages and standard deviations of the accuracy of the HP-TLC method for the determined compounds were 99.66 ± 0.974, 99.41 ± 0.950, and 99.89 ± 0.983, correspondingly. The values were 99.24 ± 0.921, 99.69 ± 0.681, and 99.20 ± 0.692, correspondingly, when they were determined by the RP-HPLC method. The relative standard deviation percentages of the methods' repeatability and intermediate precision ranged from 0.389 to 0.920. Both methods were highly specific having resolution factors of ≥ 1.78 and selectivity factors of ≥ 1.71. They were applied to the field samples perfectly.

Different chromatographic methods for determination of alogliptin benzoate, metformin hydrochloride, and metformin impurity in bulk and pharmaceutical dosage form.

Two simple, sensitive, and reproducible methods were developed for the determination of alogliptin and metformin hydrochloride in presence of metformin impurity "melamin" in pure form and in pharmaceutical formulation. Method (A) was a thin layer chromatographic method in which separation was achieved using ethyl acetate-methanol-formic acid (6:3.8:0.2, by volume) as a developing system followed by densitometric scanning at 230 nm. Method (B) was a high-performance liquid chromatography method; separation was achieved on C18 column, the mobile phase consisted of a mixture of sodium lauryl sulfate buffer 0.1% w/v, pH 3: methanol in the ratio 70:30, v/v and measurement was done at 220 nm. System suitability testing parameters were calculated to ascertain the quality performance of the developed chromatographic methods. The proposed methods have been validated regarding accuracy, precision, and selectivity, moreover they have been successfully applied to Westirizide tablets containing both alogliptin and metformin hydrochloride, results indicate that there was no interference from additives. No significance difference was found when these methods were compared to the reported one.

Validated spectrofluorometric determination of hypoglycemic combination, in pure form and pharmaceutical formulation using 9,10-phenanthraquinone reagent.

The aim of this work is to develop and validate a simple, rapid, highly sensitive and selective spectrofluorometric method for determination of Metformin HCl (MFH) and Glibenclamide (GLB) in their binary mixture without prior separation. The proposed method based on measuring the native fluorescence intensity of GLB at λemission = 348 nm, after excitation at λexcitation = 226 nm and measuring the fluorescence intensity of the fluorescent product produced from the derivatization of MFH using 9,10-phenanthraquinone in alkaline media at λemission = 416 nm after excitation at λexcitation = 240 nm. The proposed spectrofluorometric method allowed sensitive determination of the studied drugs with a limit of quantitation of 0.04 and 0.01 µg mL-1 for MFH and GLB, respectively, providing greater sensitivity than the reported one. Validation of the proposed method was carried out according to ICH guidelines with respect to linearity, accuracy, precision, and selectivity. The developed method was successfully applied for the determination of MFH and GLB in laboratory prepared mixtures and pharmaceutical formulation and the results obtained were statistically compared to those of the reported HPLC method with no significant difference between them.

Development and Validation of Stability Indicating High-Performance Liquid Chromatographic Method for Determination of Cyproheptadine Hydrochloride, its Impurity And Degradation Product.

This work presents a sensitive, accurate and selective RP-HPLC method for simultaneous determination of cyproheptadine HCl (CPH), its impurity B (dibenzosuberone) and CPH oxidative degradation product (10,11-dihydroxy-dibenzosuberone) in bulk powder and in pharmaceutical formulation. The RP-HPLC method depends on isocratic elution using C8 column and mobile phase consisting of 0.05 M KH2PO4 buffer:methanol (35:65, v/v, pH = 4.5) at a flow rate of 2 mL/min, and the eluant was monitored at 245 nm. Good resolution was obtained with tR values of 3.05, 7.54 and 6.17 min for CPH, impurity and oxidative degradate, respectively. The proposed method has been validated as per ICH guidelines using pure forms of CPH, its impurity and degradation product in pharmaceutical formulation with an accuracy of 100.48, 100.16 and 100.11, respectively. Additional spiking experiments yielded an accuracy of 100 ± 1.6%. Repeatability and intermediate precision results indicated acceptable low <2% RSD values. Moreover, the developed method's statistical results were favorably compared to the previously reported method results regarding both accuracy and precision. The developed method can be applied for analysis of the three components in quality control laboratories.

Partial least squares and linear support vector regression chemometric models for analysis of Norfloxacin and Tinidazole with Tinidazole impurity.

The aim of the presented work is to compare two popular chemometric methods which are partial least squares regression (PLSR) and support vector regression (SVR). The comparison shows their characteristics via application of the suggested methods to analysis of Norfloxacin (NF) and Tinidazole (TZ) with the presence of a potential impurity of Tinidazole; 2-Methyl-5-nitro-1H-imidazole (MNZ). For appropriate analysis, a 3 factor 4 level experimental design was constructed, which results in a training set composed of 16 mixtures which contains different concentrations of the three components; achieving symmetry, rotatability and orthogonality in mixture space. In order to validate the prediction ability of the suggested models, an independent test set consisting of 8 in-space and 8 out-of-space mixtures was used. The presented results show high specificity and accuracy of the mentioned multivariate calibration models for analysis of in-space samples of NF and TZ in presence of (MNZ) using UV spectral data. Statistical comparisons of predictive abilities of proposed models against classical least squares CLS model and against each other was performed; whether for analysis of test set mixtures or dosage form. CLS model showed lower predictive ability compared to other models. Results obtained by SVR model are as accurate as PLSR model, however, optimization and implementation of PLSR is faster and easier, hence PLSR could be of choice for this given case study. The developed chemometric models were validated as directed by ICH strategies. The validated methods were efficiently used for estimation of NF and TZ in pure powders and pharmaceuticals which indicates their suitability for application in quality control examination of both of the drugs.

Novel spectral manipulations for determinations of Tolnaftate along with related toxic compounds: Drug profiling and a comparative study.

A comparative study using novel quadruple divisor and mean centering of ratio spectra spectrophotometric methods was developed for resolution of five- component mixture of Tolnaftate, ß-naphthol (Tolnaftate alkaline degradation product and its toxic impurity), methyl(m-tolyl)carbamic acid (Tolnaftate alkaline degradation product), N-methyl-m-toluidine (Tolnaftate toxic impurity) and methyl paraben (as a preservative). For the novel quadruple divisor method, each component in the quinary mixture was determined by dividing the quinary mixture spectrum by a sum of standard spectrum of equal concentration of the other four components as a quadruple divisor. First derivative of each ratio spectra was then obtained which allowed selective determination of each component without interference from other components in the mixture. The second method was mean centering of ratio spectra that depended on utilizing the mean centered ratio spectra in four successive steps leading to enhancement of the signal to noise ratio. The absorption spectra of the five studied components were recorded in the wavelength range of 210-350 nm. The mean centered fourth ratio spectra amplitudes for each component were used for its determination. The developed methods were successfully applied for determination of laboratory prepared quinary mixtures to ensure method's specificity, then, were further applied on Tinea Cure® cream where no interference from excipients. For the first time, Tolnaftate was determined along with its toxic impurity; ß-naphthol, that could be absorbed by the skin, causing systemic toxic effects, unlike Tolnaftate that poorly absorbed, indicating the significance of this work. The proposed methods were statistically compared with each other and with the reference method. Furthermore, ICH guidelines were followed for their validation.

Two validated chromatographic determinations of an antifungal drug, its toxic impurities and degradation product: A comparative study.

Tolnaftate, a thionoester anti-fungal drug, was subjected to alkaline hydrolysis to produce methyl(m-tolyl)carbamic acid and ß-naphthol (tolnaftate impurity A). N-Methyl-m-toluidine, tolnaftate impurity D, was synthesized and structurally elucidated along with tolnaftate alkaline degradation products using IR, H1 NMR and MS. Two stability-indicating HPTLC and RP-HPLC methods were developed and validated, for the first time, for determination of tolnaftate, its alkaline degradation products and toxic impurities in the presence of methyl paraben, as a preservative in Tinea Cure® cream. The proposed HPTLC method depended on separation of the studied components on TLC silica gel F254 plates using hexane-glacial acetic acid (8:2, v/v) as a developing system and scanning wavelength of 230 nm. The proposed RP-HPLC method was based on separation of the five components on an Eclipse plus C18 column. The mobile phase used was acetonitrile-water containing 1% ammonium formate (40:60, v/v), with a flow rate of 1 mL/min and detection wavelength of 230 nm. The proposed methods allowed the assay of tolnaftate toxic impurities, ß-naphthol and N-methyl-m-toluidine, down to 2%, allowing tracing of ß-naphthol that could be absorbed by the skin causing systemic toxic effects, unlike tolnaftate, indicating the high significance of such determination. International Conference on Harmonization guidelines were followed for validation.

Simultaneous Determination of Thalidomide and Dexamethasone in Rat Plasma by Validated HPLC and HPTLC With Pharmacokinetic Study.

Two validated chromatographic methods have been developed for the simultaneous determination of thalidomide (THD) and dexamethasone (DEX) in rat plasma using paracetamol (PAR) as an internal standard (IS). Chromatographic analysis was achieved firstly by HPLC method on C18 column (150 × 4.6 mm2 i.d., 5 µm) and a mobile phase composed of ethanol:water (containing 0.1% acetic acid) (70:30, v/v) at the flow rate of 0.6 mL min-1. The second method was HPTLC method which depended on using a developing system of methylene chloride:acetone:ethyl acetate (7:4:1, by volume). In both methods, PAR was used as an IS. The developed methods have been validated as per FDA guidelines. All parameters were tested using quality control samples (LQC, MQC and HQC). All the obtained parameters were within the acceptance criteria. In the same way, the two methods were successfully used to study the pharmacokinetic parameters of both THD and DEX after their intra-peritoneal administration. Moreover, results obtained after administration of each drug alone were compared to those obtained after their administration together. The drugs showed drug-drug interactions when administered in combination, meaning that monitoring of such combination is very important.

HPTLC method for Simultaneous Determination of Norfloxacin and Tinidazole in presence of Tinidazole Impurity.

Sensitive, selective and accurate high-performance thin layer chromatographic HPTLC method for quantitative determination of Norfloxacin (NF), Tinidazole (TZ) and 2-Methyl-5-nitroimidazole (MNZ) as potential impurity of Tinidazole is developed and validated in the presented work. Calibration curves were linear over the concentration ranges of 0.4-2.4, 0.4-1.6, 0.2-1.2 µg/band for NF, TZ and MNZ, respectively. The method depends on separation and quantitation of NF, TZ and MNZ on aluminium plates pre-coated with silica gel HPTLC 60F254 as stationary-phase using chloroform: methanol: formic acid (7.5:1: 0.3, by volume) as developing system followed by densitometric measurement of bands at 298 nm. The developed method was validated and proved to meet the requirements delineated by ICH guidelines with respect to linearity, accuracy, precision, specificity and robustness. The validated method was successfully applied for determination of studied drugs in bulk powders and in their pharmaceutical formulation indicating the ability of proposed method to be used for routine quality control analysis of these drugs.

Development and validation of HPTLC and green HPLC methods for determination of furosemide, spironolactone and canrenone, in pure forms, tablets and spiked human plasma.

Two selective and accurate chromatographic methods are presented for simultaneous quantitation of spironolactone (SP) and furosemide (FR) and canrenone (CN), the main degradation product and the main active metabolite of SP. Method A was HPTLC, where separation was completed on silica gel HPTLC F254 plates using ethyl acetate-triethylamine-acetic acid (9:0.7:0.5, by volume) as a developing system and UV detection at 254 nm. Method B was a green isocratic RP-HPLC utilizing a C18 (4.6 × 100 mm) column, the mobile phase consisting of ethanol-deionized water (45: 55, v/v) and UV estimation at 254 nm. Adjustment of flow rate at 1 mL/min and pH at 3.5 with glacial acetic acid was done. Regarding the greenness profile, the proposed RP-HPLC method is greener than the reported one. ICH guidelines were followed to validate the developed methods. Successful applications of the developed methods were revealed by simultaneous determination of FR, SP and CN in pure forms and plasma samples in the ranges of 0.2-2, 0.05-2.6 and 0.05-2 µg/band for method A and 5-60, 2-60 and 2-60 µg/mL for method B for FR, SP and CN, respectively.

Successive ratio subtraction as a novel manipulation of ratio spectra for quantitative determination of a mixture of furosemide, spironolactone and canrenone.

Furosemide and spironolactone are commonly prescribed antihypertensive drugs. Canrenone is the main degradation product and main metabolite of spironolactone. Ratio subtraction and extended ratio subtraction spectrophotometric methods were previously applied for quantitation of only binary mixtures. An extension of the above mentioned methods; successive ratio subtraction, is introduced in the presented work for quantitative determination of ternary mixtures exemplified by furosemide, spironolactone and canrenone. Manipulating the ratio spectra of the ternary mixture allowed their determination at 273.6nm, 285nm and 240nm and in the concentration ranges of (2-16µgmL-1), (4-32µgmL-1) and (1-18µgmL-1) for furosemide, spironolactone and canrenone, respectively. Method specificity was ensured by the application to laboratory prepared mixtures. The introduced method was ensured to be accurate and precise. Validation of the developed method was done with respect to ICH guidelines and its validity was further ensured by the application to the pharmaceutical formulation. Statistical comparison between the obtained results and those obtained from the reported HPLC method was achieved concerning student's t-test and F ratio test where no significant difference was observed.

Quantitative Determination of Synthesized Genotoxic Impurities in Nifuroxazide Capsules by Validated Chromatographic Methods.

Two accurate, selective, and precise chromatographic methods, namely TLC-densitometric and reversed-phase (RP)-HPLC, were developed and validated for the simultaneous determination of nifuroxazide (NIF) and its four synthesized impurities, which are also reported to be its related substances in the range of 10-100 µg/band and 10-100 µg/mL for NIF in the TLC and RP-HPLC methods, respectively. The developed TLC-densitometric method depended on the separation and quantitation of the studied components on silica gel 60 F254 TLC plates. Ethyl acetate-acetone-methanol-ammonia (85 + 25 + 5 + 0.5, v/v/v/v) was used as the developing system, and the separated bands were UV-scanned at 230 nm. On the other hand, the developed RP-HPLC method depended on chromatographic separation using a C8 column at 25°C and an aqueous solution of 0.1% sodium lauryl sulfate-acetonitrile as the mobile phase delivered according to the gradient elution program. Factors affecting the developed methods were studied and optimized. Also, method validation was carried out according to International Conference on Harmonization guidelines. The proposed methods were successfully applied for the determination of the studied drug in its bulk powder and in its pharmaceutical formulation. The developed methods showed no significant difference when compared with the reported RP-HPLC one. Their advantage is being the first stability-indicating methods for NIF and its genotoxic impurities.

Different Chromatographic Methods for Simultaneous Determination of Mefenamic Acid and Two of Its Toxic Impurities.

Two sensitive, accurate and precise chromatographic methods mentioned as TLC-densitometric method and RP-HPLC-DAD method, were developed and validated for the simultaneous determination of mefenamic acid (MEF) and its two toxic impurities, benzoic acid (BA) and 2,3-dimethylaniline (DMA). In the proposed TLC-densitometric method a developing system consisting of chloroform:acetone:acetic acid:ammonia solution(70:30:2:2, v/v/v/v) was used, TLC aluminum plates 60 F254 was used as a stationary phase and the separated bands were UV-scanned at 225 nm. While the proposed RP-HPLC-DAD method depended on chromatographic separation on C18 column using 0.05 M KH2PO4 buffer: acetonitrile (40:60, v/v) as a mobile phase at constant flow rate of 1 mL/min with UV detection at 225 nm. Linear relationships were obtained in the ranges of 0.3-2, 0.3-2 and 0.3-1.8 µg/band (for TLC-densitometric method) and in the ranges of 7-50, 10-50 and 7-50 µg/mL (for HPLC-DAD method) for MEF, BA and DMA, respectively. Factors affecting the developed methods have been studied and optimized. Moreover ,the proposed methods were successfully applied for determination of the studied drug in its pharmaceutical dosage form. The methods showed no significance difference when compared with the reported method using F-test and Student's-t test. The low of detection and quantization limits of the proposed methods get them suitable for quality control and stability studies of MEF in pharmaceutical formulation. The developed methods have advantages of being more selective and sensitive than the published methods.

Validated RP-HPLC and TLC-Densitometric Methods for Analysis of Ternary Mixture of Cetylpyridinium Chloride, Chlorocresol and Lidocaine in Oral Antiseptic Formulation.

This work was concerned with development, optimization, application and validation of reversed phase high performance liquid chromatography (RP-HPLC) and thin layer chromatography (TLC)-densitometric methods for analysis of cetylpyridinium chloride, chlorocresol and lidocaine in Canyon(®) gel. The first developed RP-HPLC method depended on chromatographic separation on a ZORBAX Eclipse Plus C8 column, with elution with a mobile phase consisting of 0.05% phosphoric acid solution : acetonitrile : methanol (15 : 24 : 61, by volume), pumping the mobile phase at a flow rate of 1.00 mL min(-1), with ultraviolet detection at 220 nm. While in the subsequently developed method, the TLC-densitometric method, complete separation of the studied mixture was achieved using methanol : acetone : acetic acid (7 : 3 : 0.2, by volume) as a mobile phase, aluminum plates precoated with silica gel 60 F254 as a stationary phase and 215 nm as the scanning wavelength. Factors affecting the developed methods were studied and optimized; moreover, methods had been validated as per the International Conference of Harmonization guideline and the results indicated that the suggested methods were reproducible, reliable and applicable for rapid routine analysis. Statistical comparison of the two developed methods with the reported HPLC ones using F- and Student's t tests showed no significant difference.

A comparative study of ICH validated novel spectrophotometric techniques for resolving completely overlapping spectra of quaternary mixtures.

A pharmaceutically marketed mixture of Yohimbine, Alpha-tocopheryl acetate, Niacin, and Caffeine co-formulated as a promising therapy for erectile dysfunction. Simultaneous determination of the aforementioned pharmaceutical formulation without prior separation steps was applied using mean centering of ratio spectra and triple divisor spectrophotometric methods. Mean centering of ratio spectra method depended on using the mean centered ratio spectra in three successive steps which eliminated the derivative steps and so the signal to noise ratio was improved. The absorption spectra of the prepared solutions were measured in the wavelength range of 215-300 nm in the concentration ranges of 1-15, 3-15, 1-20, and 3-15 µg mL(-1) for Yohimbine, Alpha-tocopheryl acetate, Niacin, and Caffeine, respectively. The amplitudes of the mean centered third ratio spectra were measured at 250 nm and 268 nm for Yohimbine and Alpha-tocopheryl acetate, respectively and at peak to peak 272-273 and 262-263 nm for Niacin and Caffeine, respectively. In triple divisor method each drug in the quaternary mixture was determined by dividing the spectrum of the quaternary mixture by a standard spectrum of a mixture containing equal concentrations of the other three drugs. First derivative of these ratio spectra was obtained where determination could be achieved without any interference from the other three drugs. Amplitudes of 1-15, 3-15, 1-15, and 3-15 µg mL(-1) were used for selective determination of Yohimbine, Alpha-tocopheryl acetate, Niacin, and Caffeine, respectively. Laboratory prepared mixtures were analyzed by the developed novel methods to investigate their selectivity also, Super Act® capsules were successfully analyzed to ensure absence of interference from additives. The developed methods were validated according to the ICH guidelines. The proposed methods were statistically compared with each other and with the reported methods; using student t-test, F-test, and one way ANOVA, where no significant difference was found with respect to accuracy and precision.

HPTLC and RP-HPLC methods for simultaneous determination of Paracetamol and Pamabrom in presence of their potential impurities.

Two chromatgraphic methods were developed for determination of Paracetamol (PCM) and Pamabrom (PAM) in presence of P-aminophenol (PAP) and Theophylline (THEO) as potential impurities of both drugs respectively. First method is HPTLC which depends on separation and quantitation of the studied drugs on aluminum plates pre-coated with silica gel 60 F254 as a stationary phase using chloroform:methanol:ethyl acetate:glacial acetic acid (8:0.8:0.6:0.2, v/v/v/v) as mobile phase followed by densitometric measurement of the bands at 254 nm. Second method is RP-HPLC which comprises separation of the studied drugs on a Phenomenex C8 column by gradient elution using mobile phase consisting of sodium dihydrogen phosphate buffer (0.05 M): methanol:acetonitrile (85:10:5, v/v/v) at a flow rate of 1 mL/min for first 7.5 min and (70:20:10, v/v/v) at a flow rate of 1.5 mL/min for the next 5 min. The proposed methods were successfully applied for determination of the potential impurities of PCM and PAM after resolving them from the pure drugs. The developed methods have been validated and proved to meet the requirements delineated by ICH guidelines with respect to linearity, accuracy, precision, specificity and robustness. The validated methods were successfully applied for determination of the studied drugs in their pharmaceutical formulation. The results were statistically compared to those obtained by the reported RP-HPLC method where no significant difference was found; indicating the ability of proposed methods to be used for routine quality control analysis of these drugs.

Validated chromatographic methods for simultaneous determination of tolfenamic acid and its major impurities.

Two accurate, selective and precise chromatographic methods, namely thin-layer chromatography (TLC)-densitometric method and reversed phase high-performance liquid chromatography (RP-HPLC) method, were developed and validated for the simultaneous determination of tolfenamic acid (TOL) and its two major impurities, 2-chlorobenzoic acid (CBA) and 3-chloro-2-methylaniline (CMA), which are also reported to be its related substances. The developed TLC-densitometric method depended on separation and quantitation of the studied drugs on silica gel 60F254 TLC plates. Hexane:chloroform:acetone:acetic acid (75:25:20:0.1, v/v/v/v) was used as a developing system and the separated bands were UV-scanned at 240 nm. Linear relationships were obtained in the range of 10-100 µg band(-1) for the drug and in the range of 0.1-1 µg band(-1) for the studied impurities. The developed RP-HPLC depended on chromatographic separation of the studied drugs on a C18 column using 0.05 M KH2PO4 buffer (pH 3):acetonitrile (45:55, v/v) as a mobile phase delivered at constant flow rate of 1 mL min(-1) with UV detection at 230 nm. Calibration curves for TOL and the two impurities were constructed over the concentration ranges of 10-100 µg mL(-1) for TOL and 0.01-0.1 µg mL(-1) for both CBA and CMA. Factors affecting the developed methods have been studied and optimized. Further, methods validation has been carried out according to International Conference on Harmonization guidelines. The proposed methods were successfully applied for determination of the studied drug in its bulk powder and in pharmaceutical formulation. The methods showed no significant difference when compared with the reported RP-HPLC one. The developed methods have advantages of being more sensitive and specific than the published methods.

Kinetic study and mechanism of Niclosamide degradation.

A spectrophotometric kinetic study of Niclosamide alkaline degradation as a function of drug concentration, alkaline concentration and temperature has been established utilizing double divisor-ratio spectra spectrophotometric method. The developed method allowed determination of Niclosamide in presence of its alkaline degradation products; namely; 2-chloro-4-nitro aniline (DEG I) and 5-chloro salicylic acid (DEG II) with characterization of its degradation mechanism. It was found that degradation kinetic of Niclosamide followed pseudo-first order under the established experimental conditions with a degradation rate constant (k) of 0.0829 mol/h and half life (t1/2) of 8.35 h. The overall degradation rate constant as a function of the temperature under the given conditions obeyed Arrhenius equation where the activation energy was calculated to be 3.41 kcal/mol.

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.