Validation

A simple, accurate and sensitive RP-HPLC method was developed and validated for the determination of candesartan cilexetil and hydrochlorothiazide in tablet dosage form. The separation of the two drugs was achieved on a Reprospher 100 –C l 8 column (250 x 4.6 mm, 5 µm particle size) with UV detection at 260 nm. The mobile phase consists of two solution, solution (1) orthophosphoric acid (pH 2.2) and the solution (2) which is acetonitrile and purified water in the ratio 55: 45 v/v, respectively. The method was validated in terms of linearity, accuracy, precision, specificity, limit of detection, limit of quantitation and robustness. Linearity was observed in the concentration range 10-70µg/ml for hydrochlorothiazide and 12.8 -89.6µg/ml for candesartan cilexetil. The limit of quantitation was found to be 8.58 and 4.6 µg/ml for hydrochlorothiazide and candesartan cilexetil, respectively whereas the limit of detection, was found to be 2.8 and 1.5 µg /ml for hydrochlorothiazide and candesartan cilexetil, respectively. The % recovery range of candesartan cilexetil was 97.84-101.62% and 100.9-101.53% for hydrochlorothiazide. Variation in HPLC conditions (flow rate, column temperature, mobile phase composition, and wavelength) were used to evaluate the robustness of the method. The method proved to be robust and still produces good results.

The objective of the method was to develop a simple, rapid, efficient, cost effective and reproducible, stability indicating reverse phase high performance liquid chromatography method (RP-HPLC) for the quantification of cinacalcet in bulk and pharmaceutical dosage form. As very few analytical methods have been reported for the analysis of cinacalcet by chromatography, we aimed to develop a method which would be simple with optimum retention time by the use of simple mobile phase, which results in the economic method that can be used for routine analysis of the drug. The RP-HPLC analysis was carried out on Inertsil ODS C18 with a mobile phase of methanol, acetonitrile and water in the ratio of 70:15:15 v/v/v. Detection was carried out at 280 nm using a PDA detector. The method was validated for linearity, accuracy, precision, LOD, LOQ and robustness as per ICH guidelines. The method was found to be linear in the range of 10-50 µg/ml. Limit of detection and limit of quantitation was found to be 0.22 and 0.74 µg/ml respectively. Recovery was found to be in the range 99.7-100.02% and precision less than 1%. The developed method was successfully applied for the estimation of cinacalcet in marketed tablet formulation (PTH 30) and percentage assay was found to be 100.8 %. The developed RP-HPLC method was simple, rapid, accurate, precise and stability indicating for the quantification of cinacalcet in bulk and tablet dosage form.

An accurate, rapid, specific and stability indicating ultra performance liquid chromatographic method was developed and validated for the simultaneous estimation of Verapamil and Trandolapril in a combined pharmaceutical dosage form. The chromatographic separation was attained on Phenonemex Luna C18 (4.0 x 100 mm, 2.6mm) column by isocratic mode with the mobile phase components as 0.03M monobasic potassium phosphate buffer (pH6.5) and acetonitrile (70: 30v/v) at a flow rate of 1.0 mL/min and quantified at 210 nm. The average retention times for Trandolapril and Verapamil were 0.60 and 1.14 min, respectively. The UPLC method proposesoutstanding separation of two drugs with a good resolution of greater than 2.0 and tailing factor less than 2.0 with a run time of 3 minutes. The method shows linearity over the concentration range of 9-45 µg/mL for Verapamil and 0.1-0.5 µg/mL for Trandolapril with a correlation ≥ 0.999. The method is accurate with recoveries in the range of 98.0 -101.0% and precise with %RSD value lesser than2.0% for both the drugs. This method is very fast, cost saving, accurate and specific for the assay of commercially available tablets.

A simple isocratic RP-HPLC method was developed for the estimation of Lurasidone in bulk and pharmaceutical dosage forms by using Waters (Alliance) HPLC (2695 series) System operated with Empower software-2. The optimized chromatographic conditions were found to be buffer of 0.05% Trifluroacetic acid in 0.01 M Potassium dihydrogen orthophosphate solution, mobile phase of buffer and acetonitrile in the ratio 60: 40, Inertsil ODS, C18, 150mm x 4.6mm, 5µ particle size HPLC column at temperature of 30°C, 20 µL of injection volume, 1.0ml/min flow rate and UV detector with detection wavelength 231nm. Retention time and peak area of standard or sample were found to be 5.428min or 5.431 min. and 4128545 or 4126122 respectively. System precision and method precision were found to be less than 2.0%. The method was found to be linear within the limits 2.5-15.0µg/ml with good correlation coefficient 0.9999. The percent of recovery (accuracy) was found from 99.34 to 99.97%. Ruggedness and robustness studies were carried out and the results were found to be satisfactory. Stability of Lurasidone was studied under the different stressed conditions and found to be stable (96.37-93.55%). The % of assay of different dosage of Latuda tablets was found between 99.68 -100.18%. The proposed method was found to be sensitive, precise, accurate, linear, rugged and robust, and applied for the analysis of pharmaceutical formulations and percent of assay was evaluated, hence the proposed method can adopt for the routine analysis in any quality control laboratory.

A simple, specific, accurate and precise reverse phase high performance liquid chromatographic method is developed and validated for the estimation of Dapsone in tablet dosage form. The expected separation and peak shapes were obtained on Luna C18, 15 cm x 4.6 mm (5 μm)  column. To have an ideal separation of the drug under isocratic conditions, mixtures of solvents like methanol and water with or without different buffers indifferent combinations were tested as mobile phases on a Luna C18, 15 cm x 4.6 mm (5 μm) column. A mixture of Methanol:Water in the ratio of 40:60 v/v was proved to be the most suitable of all the combinations since the chromatographic peak obtained was better defined and resolved and almost free from tailing.  The flow rate was 1.0ml/min and effluents were monitored at 260 nm. The retention time for Dapsone was ± 2.4 min. The method was validated for accurate, precise, simple, sensitive and rapid and can be applied successfully for the estimation of Dapsone in bulk and in pharmaceutical formulations without interference and with good sensitivity. And recovery of Dapsone from tablet formulation was found to be 93%. The proposed method was successfully applied for the quantitative determination of Dapsone in tablet formulation.

A simple, sensitive and specific HPLC method with UV detection was developed for the estimation of irbesartan in tablet dosage form. Separation was achieved by Lithosphere 100 RP-18e, 5µ column having 250x4.0 mm internal diameter with mobile phase containing phosphate buffer and acetonitrile in 50:50 (v/v) with an ambient temperature. The flow rate was 0.8mL min-1 and eluent was monitored at 220 nm. The proposed method was found to be rectilinear over theconcentration range of 20µg/ml to 60µg/ml. This method was validated for system suitability, Specificity, Linearity, Precision, Accuracy, Range, Stability studies, Ruggedness, Robustness and Filter validation.  The results of all the validation parameters were well within their acceptance values. Therefore the proposed method can be used for routine analysis of estimation of Irbesartan in its tablet formulation

A new rapid, precise and sensitive reverse phase high performance liquid chromatographic (RP-HPLC) method has been developed and validated for the estimation of Atazanavir and Ritonavir simultaneously in combined dosage form. The two components Atazanavir and Ritonavir were well resolved on an isocratic method, C18 column, utilizing a mobile phase composition of acetonitrile: 0.02M ammonium acetate buffer (40:60), v/v, pH 4.0) at a flow rate of 1.0 mL/min with UV detection at 205 nm. The retention time of Atazanavir and Ritonavir were 2.8 min and 5.7 min respectively. The developed method was validated for specificity, linearity, precision, accuracy, limit of detection (LOD), limit of quantification (LOQ) and robustness as per ICH guidelines. Linearity for Atazanavir and Ritonavir were found in the range of 18.0-42.0 µg/ml and 5.0-14.0 µg/ml, respectively. The percentage recoveries for Atazanavir and Ritonavir ranged from 98.9-101.0 % and 98.2-100.1 %, respectively. The proposed method could be used for routine analysis of Atazanavir and Ritonavir in their combined dosage forms.

A simple, precise and accurate UV Spectrophotometric method has been developed and validated for estimation of Sildenafil in bulk and tablet dosage form. In this method Sildenafil shows λmax at 290nm using 0.1N HCl as a solvent and calibration graphs were plotted over the concentrations ranging from 5 to 30μg/ml of Sildenafil with correlation coefficient 0.999. The proposed method was validated as per ICH Q2 (R1) guidelines for precision, linearity, accuracy and recovery. The limit of detection (LOD) and limit of quantification (LOQ) were found to be 0.113μg/ml and 0.343μg/ml respectively by simple UV spectroscopy. The proposed method was validated.

The present work was defined to develop area under curve method for antipsychotic drug by UV spectrophotometric analysis which was economical, precise and an accurate method for estimation of Quetiapine fumarate. This method was based on area under curve of UV spectrum between 287 to 297 nm and validated as per ICH guideline Q2 (R1). The linearity in the range was found to be 4-14 μg/ml. The result of correlation coefficient was 0.999. The results of percent relative standard deviation for the intra-day and inter-day precision indicated that method is precise. The values of the recovery studies (99.65 % to 101.04 %) showed good accuracy of the method. LOD and LOQ were calculated as 0.3806 and 1.153μg/ml, respectively. The developed method can be applied for routine estimation of Quetiapine fumarate in bulk and tablet dosage forms.

An accurate, rapid, selective and specific reverse phase high performance liquid chromatographic method was developed for the simultaneous estimation of Rosuvastatin and Clopidogrel in a combined formulation. Chromatographic separation was achieved on Inertsil ODS 3 C-18 (250mm x 4.6mm,5µm) column by isocratic elution mode with three mobile phase components, 0.05M potassium phosphate buffer (pH4.2): methanol: acetonitrile (60: 30:10 v/v/v) at a flow rate of 1.0 mL/min and quantified at 238 nm. The average retention times for Rosuvastatin and Clopidogrel were 4.57 and 2.96 min, respectively. The method offers excellent separation of two drugs with resolution > 2.0 and tailing < 1.0 and with no interferences from the excipients. The method is linear over the concentration range of 3.1-18.6 µg/mL for Rosuvastatin and 22.68-136.08 µg/mL for Clopidogrel with a correlation not less than 0.999. The method is accurate with recoveries of both the drugs in between of 98.0 -101.0% and precise with %RSD value less than 2.0% for the Assay of both the drugs. This method is simple, rapid, accurate and specific for the assay of commercial capsules.