Validation

A simple, accurate, precise and specific RP-HPLC method has been developed for the simultaneous estimation of Aceclofenac and Esomeprazole Sodium in bulk. Chromatographic analysis was carried out on C18 column ( 250mm × 4.6mm, 5µm). Mobile phase used is a homogenous mixture of ACN: Methanol in the ratio of 50:50 v/v. The detection was carried out at 285nm. The retention times were found to be 3.00 and 4.41 min for Aceclofenac and Esomeprazole Na respectively. Both the drugs showed linearity in the range of 30 – 70mcg/ml. The correlation coefficient was found to be 0.99 and 0.992 for Aceclofenac and Esomeprazole Na respectively. The developed method was validated as per ICH guidelines.

Two sensitive and reproducible methods are described for the quantitative determination of Glipizide and Metformin Hydrochloride in presence of its degradation products. The first method was based on high performance liquid chromatographic (LC) separation of the drug from its degradation products on the reversed phase, Cosmosil® column [C18 (5 mm, 4.6 x 150 mm, i.d.)] at ambient temperature using a buffer consisting of 10 mM potassium dihydrogen phosphate (pH adjusted to 2.5 with diluted o- phosphoric acid) and acetonitrile 50: 50 v/v as optimized mobile phase in a gradient program. The flow rate was 0.7 ml min-1 and quantitation was achieved UV determination at 225 nm based on peak area with linear calibration curves at concentration range 10-25 µg ml-1 and 20-50 µg ml-1 respectively. The second method was based on   high performance thin-layer chromatographic (HPTLC) separation followed by densitometric measurement of spots at 216 nm. The separation were carried out on Merck HPTLC aluminium sheets of silica gel 60 F 254 using Toluene: Methanol: Ethyl acetate: Formic acid in the ratio of (3:6:3:0.2, v/v/v/v), as mobile phase. This system was found to give compact spots for Glipizide and Metformin hydrochloride after double development (retention factor, RF 0.08 ± 0.02 and RF 0.74 ± 0.02 respectively). The second order polynomial regression analysis data was used forth regression line in the range of 200-1400 ng spot-1 and 200-1400 ng spot-1 respectively. Both the method has been successively applied to pharmaceutical formulation. No chromatographic interference from the tablet exicipients was found. Both the methods were validated in terms of precision, robustness, recovery, limits of detection and quantitation.

A simple, sensitive and accurate method employing HPTLC has been developed and validated as per ICH guidelines for determination of Bosentan in bulk and Tablet dosage form. The absorption maxima of the drug were found to be 288 nm in methanol. The method was validated as per ICH guidelines. The linearity range was found to be 50-300 ng/ml with a regression coefficient of 0.98.Subsequent validation parameters like precision, repeatability in terms of Interday and Intraday precision and recovery studies were evaluated with satisfactory results, the % RSD for these parameters was found to be 1.73%,1.30-0.48%,1.80 – 0.45 % , 1.54 – 1.48 % respectively with an Rf value of 0.23.

The aim of present work was to develop an accurate, precise, reproducible and economical UV spectrophotometric method for estimation of 1H, 1’-H-2, 2’-Bibenzimidazole Impurity In Telmisartan Bulk and Formulation. This method was based on area under curve of UV spectrum between 235 to 254 nm and validated as per ICH guideline Q2 (R1). The method has followed linearity in the range of 5-30μg/ml. The value of correlation coefficient was 0.998. Satisfactory values of Percent relative standard deviation for the intra-day and inter-day precision indicated that method is precise. Results of the recovery studies (97.63% to 98.66 %) showed accuracy of the method. LOD and LOQ were calculated as 0.3221μg/ml and 0.9761 μg/ml, respectively. The developed method can be used for routine estimation of 1H, 1’-H-2, 2’-Bibenzimidazole Impurity In Telmisartan Bulk and Formulation.

Effective and advantageous Hydrotropic Solubilization technique has been developed for the estimation of two drugs i.e, Flurbiprofen and Glipizide in bulk and its pharmaceutical formulations. Hydrotropic Solubilization technique is one of the aqueous solubility enhancing methods employed for the poorly water soluble drugs and found to be simple, precise and cost effective. Solvents like Piperazine, Urea, Sodium Salicylate, Sodium benzoate etc are the commonly used as hydrotropic solventsin different concentrations. The use of these hydrotropic solvents are of proper choice since the use of organic solvents can be reduced as it is hazardous, costlier and causes environmental pollution. In this context, 1M piperazine has been used as a solubilizing agent to enhance solubility of both the drugs, Flurbiprofen and Glipizide. The absorption maximum of Flurbiprofen and Glipizide was found to be at 246nm and276nm in Zero order derivative spectrum (Method A), calculation of Area Under Curve (AUC)(Method B) was done in the wavelength range of 236-256nm for Flurbiprofen and 266-286nm for Glipizide. The Beer-Lambert’s law has been followed in the concentration range of 2-10µg/ml for Flurbiprofen and 5-35µg/ml for Glipizide for both the methods. The methods were validated as per ICH guidelines and all the validation parameters were found to be within the acceptable range. The developed methods were successfully practiced to estimate the amount of Flurbiprofen and Glipizide in bulk and pharmaceutical dosage forms in routine analysis.

A new, simple, rapid, selective, precise and accurate isocratic reverse phase high performance liquid Chromatography assay method has been developed for estimation of Curcumin in tablet formulations. The separation was achieved by using column Hypersil BDS C18, 150x4.6 mm, 5µ (Make: Thermo), in mobile phase consisted of tetrahydrofuran and citric acid buffer in the ratio of (550:450, v/v). The flow rate was 1.0 mL.min-1 and the separated curcumin was detected using UV detector at the wavelength of 425 nm. The retention time of curcumin, was noted to be 8.05 min respectively, indicative of rather shorter analysis time. The method was validated as per ICH guidelines. The proposed method was found to be accurate, reproducible, and consistent.

A rapid, accurate, linear, and sensitive RP-HPLC method has been developed and validated for estimation of febuxostat in the bulk drug and marketed tablet formulation. The chromatographic separation was achieved on Kromasil C18 Column (250 mm × 4.6 mm, 5 μm particle size) using solvent methanol:water(65 : 35 v/v, pH 3.0 adjusted with OPA)as a mobile phase at flow rate of 1.0 ml/min and25°C column temperature was maintained and analysis were carried out at detection wavelength316 nm. The linearity study was studied in the concentration range 10-50 μg/ml for febuxostat and correlation coefficient was found to be 0.999. The percentage purity of febuxostat was found in the range of 98-101%. The limit of detection and limit of quantification were found to be1.22μg/ml and3.58μg/ml. The method was validated for linearity, precision, accuracy, specificity and selectivity. The obtained results indicates that the proposed method allows selective analysis of febuxostat, in the presence of their degradation products formed under a variety of stress conditions. The developed procedure is also applicable for the determination of instability of the drugs in commercial products.

A sensitive static headspace gas chromatographic method using flame ionization detector was developed and validated for the estimation of Allylamine in Sevelamer hydrochloride tablet dosage form. The chromatographic separation was achieved on capillary column Rtx-5 Amine (30m×0.53mm×1.0μm) with 5% diphenyl/95% dimethyl polysiloxane stationary phase. The critical experimental parameters such as, diluents, columns and sample preparations were studied and optimized. The method was validated as per United States Pharmacopoeia (USP) and International Conference on Harmonization (ICH) guidelines in terms of detection limit, quantification limit, linearity, precision, accuracy, specificity, solution stability and robustness. The method was found to be linear in the range between 10 μg/mL and 75 μg/mL with a correlation coefficient (r2) of 0.99984. The detection and quantification limits obtained for Allylamine were 3.3 μg/mL and 10.0 μg/mL w.r.t sample concentration respectively. The recovery obtained for Allylamine was between 100.0% and 110.0%. The developed method was applied successfully for the estimation of Allylamine in Sevelamer hydrochloride tablet dosage form.

A simple and sensitive stability indicating HPTLC method has been developed and validated for estimation of Palonosetron hydrochloride. Separation of the drug was carried on aluminium plates precoated with silica gel 60 F254 using Ethyl acetate: Methanol: Triethylamine (6:3:1 v/v/v) as mobile phase. The retention factor (Rf) for Palonosetron hydrochloride was found to be 0.50 ± 0.04. The detection was carried at 242 nm. Stress testing of Palonosetron hydrochloride was carried out according to the International conference on harmonization (ICH) guideline Q1A (R2). The drug was subjected to acid, base, neutral hydrolysis, oxidation, thermal degradation and photolysis. Palonosetron hydrochloride showed considerable degradation under oxidative condition. The method was successfully validated according to ICH guidelines Q2 (R1). The data of linear regression analysis indicated a good linear relationship over the range of 250–1500ng/band concentrations with correlation coefficient 0.994. The accuracy of the method was established based on the recovery studies. The LOD and LOQ were 11.81ng/band and 35.78ng/band respectively.

Isosorbide Dinitrate (ISD) is a moderate to long acting oral organic nitrate which acts as a vasodilator profoundly used in the treatment of angina pectoris, a condition which occurs when the oxygen supply to the myocardium is insufficient for its needs. It is slightly soluble in water and propanol, sparingly soluble in ethanol and freely soluble in methanol. The most economical solvent Distilled water was choosen as a solvent. The drug has absorption maximum (method A) at 304nm, Area under curve (method B) at 300-310 and first order derivative method (method C) at 287nm. Linearity for all three methods was found in the range of 5-25 μg/ml with Correlation coefficient is 0.999, Standard error is 0.001. The drug sample was analyzed by UV spectroscopy using distilled water as solvent and the average content of drug present in the formulation was found to be 99.7%. The % recovery of accuracy studies was found to be 99.1 % for method A, 101.5 % for method B, 100.6 % for method C. The %RSD of precision was found to be 1.11% for intraday, 1.22 % for inter day. The force degradation studies of Isosorbide Dinitrate was done on Stress degradation by hydrolysis under alkaline condition by using 0.1N NaOH was found to be 10.09% for 24hrs, 13.3% for 3days, 14.2% for 5days .Stress degradation by hydrolysis under acidic condition by using 0.1N HCl and degradation was found to be  2.68% for 24hrs, 9.7% for 3days, 11.9% for 5days.  Oxidative degradation was done by using hydrogen peroxide (3%v/v) and degradation was found to be 3.22% for 24hrs, 5.06% for 3days, and 8.29% for 5days. The proposed methods can be successfully employed for quality control during manufacture and for assessment of the stability of Isosorbide Dinitrate in bulk samples and its tablet dosage forms.