Validation

A new, simple, accurate, precise and robust isocratic RP-HPLC method has been developed and subsequently validated for the determination of Regorafenib in pure form and pharmaceutical dosage forms as per ICH guidelines. The separation achieved on a Symmetry C18 Column, 250 mm x 4.6 mm i.d. and 5µm particle size column as a stationary phase and Methanol: Phosphate buffer (pH adjusted to 4.80 with phosphoric acid) in the ratio of 70:30v/v used as mobile phase at a flow rate of 1.0 ml/min. The UV detection was performed at 268nm. The retention time for Regorafenib was found to be 3.544minutes. The detector response was linear in the concentration range of 0-16µg/ml. The respective linear regression equation being Y= 58945.x + 9634 with R2 = 0.999. The percentage of Regorafenib in pharmaceutical dosage form was found to be within the limits. The limit of detection and the limit of quantification were found to be 0.90µg/ml and 2.90µg/ml respectively. The results of the study showed that, the proposed RP-HPLC method was simple, rapid, precise, accurate and stability indicating, which can be used for the routine determination of Regorafenib in pure form and pharmaceutical dosage forms.

To develop and validate simple, rapid, linear, accurate, precise and economical UV Spectroscopic method for estimation of Ranolazine in tablet dosage form. The drug is freely soluble in analytical grade methanol. The drug was identified in terms of solubility studies and on the basis of melting point which was done on melting point apparatus of Equiptronics. Ranolazine showed absorption maxima were determined in analytical grade methanol. The drug obeyed the Beer’s law and showed good correlation of concentration with absorption which reflect in linearity. The UV spectroscopic method was developed for estimation of Ranolazine in tablet dosage form and also validated as per ICH guidelines. The drug is freely soluble in analytical grade methanol, slightly soluble acetonitrile and very slightly soluble in analytical grade water. So, the analytical grade methanol is used as a diluent in method. The melting point of Ranolazine was found to be 120-122ËšC (uncorrected). It showed absorption maxima 235 nm in analytical grade methanol. On the basis of absorption spectrum the working concentration was set on 8 µg/ml (PPM). The linearity was observed between 2-12 μg/ml (PPM). The results of analysis were validated by recovery studies. The recovery was found to be 98.75, 101 and 98.33% for three levels respectively. The % RSD for precision was found to be 0.6353% which was within acceptance criteria as per ICH guidelines. A simple, rapid, linear, accurate, precise and economical UV Spectroscopic method has been developed for estimation of Ranolazine in tablet dosage form. The method could be considered for the determination of Ranolazine in tablet dosage form in quality control laboratories.

A gradient reversed phase high performance liquid chromatography (RP-HPLC) method has been developed and validated for the determination for related substances of Dapagliflozin drug substance. Chromatographic separation of Dapagliflozin from its process and degradation related substances was achieved on YMC Pack Pro C18, 250mm × 4.6mm 5m i.e A stainless steel column 250 mm long, 4.6 mm internal diameter filled with Octadecyl silane chemically bonded to porous silica particles of 5 mm diameter maintained column oven temperature at 25°C. Orthophosphoric acid buffer is mobile phase A and acetonitrile is mobile phase B. Wavelength for UV detection: 225nm, flow rate: 0.8 ml/min and Injection volume: 20µl. The developed method suitability was checked and validated as per ICH guidelines for specificity, linearity, accuracy, precision, limit of quantification, limit of detection robustness and ruggedness experiments. Dapagliflozin drug substance was subjected to stress conditions of thermal, hydrolysis, humidity, peroxide and photolytic to observe the degradation products. Limit of detection of each RS is less than 0.008%w/w indicating that the developed method is highly sensitive. The experiment results are given in detailed in this research article.

A new simple, accurate, precise and selective stability indicating high performance thin layer chromatographic method has been developed and validated for estimation of Orlistat Tablet.T he mobile phase selected was Toluene: Methanol(8:2v/v) with UV detection at 211nm.The retention factor for Orlistat was found to be 0.60±0.02. The method was validated with respect to linearity, accuracy, precision and robustness as per the ICH guidelines. The drug were subjected to stress condition of hydrolysis (acid, base), oxidation, photolysis and thermal degradation. Results found to be linear in concentration range of 6000-36000 ng/band. The thermal method has been successfully applied for the analysis of drug in pharmaceutical formulation. The % assay (Mean ± S.D) was found to be 99.30±1.10.The developed method can be used for checking the stability of  Orlistst in bulk drug and pharmaceutical dosage form.

Edoxaban Tosylate Monohydrate (EXN) is oral anticoagulant drug indicated to reduce the risk of stroke and systemic embolism (SE) in patients with nonvalvular atrial fibrillation (NVAF) and for the treatment of deep vein thrombosis (DVT) and pulmonary embolism (PE). Sensitive and reproducible UV- Visible spectrophotometric method has been developed and validated for the estimation of Edoxaban Tosylate Monohydrate in its synthetic mixture. Methanol was used as a solvent. Developed method has been validated for linearity range, precision, accuracy, limit of detection, and limit of quantification as per ICH Q2(R1) guidelines. The method was found to be linear in the range of 5-25 μg/mL at λmax 289 nm and the regression coefficient value was found to be 0.9999. For Edoxaban LOD and LOQ values were found to be 0.654 μg/mL and 1.982 μg/mL. The method was successfully applied for estimation of Edoxaban Tosylate Monohydrate in its synthetic mixture and results were found to be in good agreement with the amount of Edoxaban Tosylate Monohydrate present in synthetic mixture.

Zafirlukast, Cyclopentyl (3-(2-methoxy-4-((o-tolylsulfonyl) carbamoyl) benzyl)-1-methyl-1H-indol-5-yl) carbamate (I). A high-performance liquid chromatographic (HPLC) reversed-phase rapid resolution method has been developed and validated for estimation of zafirlukast in a pharmaceutical active pharmaceutical ingredient, which was chromatographed on reversed-phase Hypersil Gold C18 , 2.1 x 100 mm , 1.9 µ column using mixtures of acetonitrile/water and the eluents were monitored at different wavelengths. The method was validated statistically for its linearity, accuracy, robustness and precision. The method employed same chromatographic condition as above and validated in terms of linearity, accuracy, precision, limit of detection (LOD), limit of quantification (LOQ), and solution stability. Present work also describes the development and validation of RP- RRLC method for the determination of impurities of zafirlukast.

The present study is carried out using the UPLC as the analytical technique in developing and validating an accurate, precise, linear and robust analytical method for the simultaneous estimation of Pitofenone hydrochloride, Diclofenac potassium and Fenpiverinium bromide in tablets. The method is optimized with a mixture of 0.01M phosphate buffer (PH4.8) and Acetonitrile in the ratio of 40:60 (V/V) as mobile phase and Agilent SB C18 (250 X 4.6) mm, 5µm as stationary phase. The Chromatographic peaks were detected and measured at 215nm. The retention times of Pitofenone hydrochloride, Diclofenac potassium and Fenpiverinium bromide were found to be 1.0, 1.66 and 2.0 respectively. The developed method was demonstrated to access its suitability for meeting its intended purpose by the Validation with a set of validation parameters as per ICH and USP guidelines.  The method is found to be precise with %RSD - 0.48, 0.69, 0.66 for Pitofenone hydrochloride, Diclofenac potassium and Fenpiverinium bromide respectively: accurate with the recoveries of 99.6 to 101.6, 100.51 to 101 and about 100% for Pitofenone hydrochloride, Diclofenac potassium and Fenpiverinium bromide respectively. The method is proved to be linear from the conc.12.5 to 75ppm for Pitofenone, 250 to750 ppm for Diclofenac and 0.5 to 1.5ppm for Fenpiverinium bromide with the correlation coefficients of 0.999, 0.999 and 0.999 respectively. Hence the developed method could be used for the routine analysis purpose in the evaluation of Pitofenone, Diclofenac potassium and Fenpiverinium bromide Tablets.

An HPLC method has been developed for simultaneous estimation of tenofovir disoproxil and emtricitabine in bulk and in their tablet dosage form. Separation and analysis of both drugs was achieved on Supelco C18 (250 x 4.6 mm; 5 µm particle size) analytical column with temperature set at 25˚C. The best chromatographic condition was found as an isocratic mobile phase consisting of 0.2 M ammonium acetate (pH 4.5) and methanol in a ratio of 65: 35 (% v/v) at a flow rate of 1.2 ml/min for 6 minutes. The retention of emtricitabine and tenofovir disoproxil was found to be 3.020 min and 4.264 min, respectively. The method was validated according to the International Conference on Harmonization guidelines and various validation parameters (system suitability, selectivity, linearity, precision, accuracy, limit of detection, limit of quantification and robustness) were determined. The results of validation parameters are satisfactory. Applicability of the developed and validated HPLC method was checked in tablet dosage form.

The objective of this work was to develop a simple, sensitive, accurate, precise and reproducible high performance liquid chromatography (HPLC) method for the determination of salicylic acid in pharmaceutical dosage forms. Shimadzo Prominance model L20 AD HPLC system equipped with SPD 20A UV-Vis detector was used for the analysis. The separation was done on RESTEX allure C18 column (3 μm, 15 cm × 4.6 mm), for an isocratic elution a mixture of water, methanol and glacial acetic acid (65:35:1, v/v) mobile phase at a wavelength of 254 nm. The flow rate was 1.0mL/min. The RP-HPLC method developed for analysis of salicylic acid was validated with respect to specificity, selectivity, linearity, accuracy, precision and robustness as per the ICH guidelines. The retention time of salicylic acid was 7.575 min. The linearity was established over the concentration ranges of 50-350 μg/mL with correlation coefficients ( r2) 0.999.  The percentage accuracy of salicylic acid ranged from 99.76 -101.66%. The relative standard deviation values for intra-day and inter-day precision was lower than 2.0% and the assay result was found to be in the range 99.57-101.32%. Salicylic acid was subjected to stress conditions such as neutral, acidic, alkaline, oxidation and photolysis degradations as per ICH guidelines. The degradation studies revealed that the drug was found to degrade maximum (1.67%) in alkaline degradation conditions and was highly resistant towards neutral, acidic, oxidative and photolytic degradation conditions.

To develop a new, economical, precise and accurate stability indicating HPTLC method was developed and validated for the determination of Adapalene in bulk drug. The present study deals with development and validation of stability indicating HPTLC method for estimation of Adapalene. Chromatographic separation was performed on aluminium plate pre-coated with Silica Gel 60 F254 using Tetrahydrofuran: 2-Propanol: Water (3:3:3 v/v/v) as a mobile phase. The wavelength selected for densitometric scanning was 230 nm. Regression plots revealed linear relationship in the concentration range of 20-120 ng spot-1. The Rf value of Adapalene was found to be 0.76 (±0.02).The LOD and LOQ were found to be 3.15 and 9.57 ng spot-1respectively. The method was validated as per International Conference on Harmonization (ICH) guidelines, demonstrating to be accurate and precise within the corresponding linearity range of titled analytes. Inherent stability of the drug was studied by exposing drug to acid, alkali, oxidative, photolytic and thermal conditions. Relevant degradation was found to take place under these conditions. The proposed method has been validated as per ICH Q2 (R1) guidelines. This method can be used for routine quality control analysis of Adapalene in bulk drug.