Validation.

A simple, Accurate, precise method was developed for the simultaneous estimation of the Rilpivirine and Cabotegravir in dosage form. Chromatogram was run through AgilentC18 150 x 4.6mm, 5.0mm.1 Mobile phase containing Buffer 0.1% Ammonium Acetate: Acetonitrile taken in the ratio 55:45v/v was pumped through the column at a flow rate of 1.0 ml/min. Temperature was maintained at 30°C. The optimized wavelength selected was 260 nm. The retention time of Rilpivirine and Cabotegravir was found to be 2.238 min and 2.953 min. %RSD of the Rilpivirine and Cabotegravir was found to be 0.5 and 0.5 respectively. %Recovery was obtained as 99.85% and 99.84% for Rilpivirine and Cabotegravir respectively. LOD and LOQ values obtained from regression equations of Rilpivirine and Cabotegravir were 0.11, 0.3,2, and 0.02, 0.06 respectively.2 The regression equation of Rilpivirine is y = 17712x + 2324.3 and y = 17293x + 1410.5 of Cabotegravir Retention times were decreased and that run time was decreased, so the method developed was simple and economical that can be adopted in regular Quality control test in Industries.  

A straightforward and accurate method was devised for simultaneously determining Drospirenone and Estetrol in tablet dosage form. The chromatogram was generated using a Kromosil C18 column (150 x 4.6 mm, 5.0 mm), with a mobile phase consisting of Buffer 0.01N KH2PO4: Methanol in an 80:20 ratio, flowing through the column at a rate of 1.0 ml/min. This method utilized OPA as the buffer, and the temperature was maintained at 30°C. The optimized wavelength for detection was set at 263.0 nm. The retention times for Drospirenone and Estetrol were determined to be 2.597 min and 2.1336 min, respectively, with %RSD values of 0.4 for both compounds. The %Recovery was calculated as 100.25% for Drospirenone and 100.09% for Estetrol. The LOD and LOQ values were obtained from the regression equations of Drospirenone and Estetrol, resulting in 0.01 and 0.02 for LOD, and 0.08 and 0.25 for LOQ, respectively. The regression equations for Drospirenone were found to be y = 361228x + 6065.6, and for Estetrol, y = 341114x + 46689. The method exhibited reduced retention times and overall run time, indicating its simplicity and cost-effectiveness. This makes it suitable for routine quality control testing in various industries.

The purpose of this research is to develop and validate a precise method for UV-Vis spectrophotometric and Reverse-Phase High Performance Liquid Chromatography (RP-HPLC) for determination of Octenidine dihydrochloride in bulk and pharmaceutical preparation. According to the relevant experiment the maximum wavelength was found to be 285nm and it is used for further process of development of method and its validation. The developed methods used for quantitative estimation of Octenidine dihydrochloride in pharmaceutical preparation and bulk drug which shows the satisfactory results as per ICH guidelines, so these developed and validated methods are found very simple, sensitive and rapid according to the ICH guideline and can be successfully applied to estimate the ODCL in bulk and pharmaceutical dosage form.

A simple, sensitive, selective and rugged liquid chromatography coupled with mass spectrometry (LCMS/MS) method for quantification of Diltiazem and its metabolites, N-desmethyl Diltiazem, desacetyl Diltiazem in human plasma was developed and validated. The chromatography was developed using Luna 5 μ, C18, 100×4.60 mm column having a mobile phase of Acetonitrile: 0.1% formic acid (85:15 % v/v). The flow rate was 0.5 ml/min at a column temperature of 50 ± 5º C. Electron spray ionization technique in positive mode was selected to improve the selectivity and sensitivity required for this application. The retention times of Diltiazem, desmethyl Diltiazem, desacetyl Diltiazem were 2.5, 2.0 and 2.5 minutes respectively. The method was validated for linearity, precision, accuracy, specificity, sensitivity, matrix effect, dilution integrity, ruggedness, injection reproducibility and stability. Calibration curves during the course of validation were found to be linear for Diltiazem, desmethyl Diltiazem, desacetyl Diltiazem in the ranges of 0.604-603.902, 0.303-303.274 and 0.299-299.489 ng/mL with correlation coefficient ≥ 0.9969, 0.9958 and 0.9970 respectively and by using a 1/x2 weighted least square regression analysis of standard plots associated with ten point calibration standards. The precision and mean accuracy were within the acceptable limits. Keywords: Diltiazem; desmethyl Diltiazem, desacetyl Diltiazem; LCMS/MS; Validation.

This presented work is based on application of two multivariate calibration methods for simultaneous UV-Visible spectrophotometric determination of active substances in combined pharmaceutical formulation contained of Tinidazole (TINI) and Norfloxacin (NFX). The methods used were Partial Least Square (PLS) and Principal Component Regression (PCR). The spectra of both NFX and TINI were recorded at concentrations within their linear range 2.0-12.0 μg/mL for NFX and 5.0-30.0 μg/mL for TINI. The 29 set of mixtures were used for calibration and 07 set of mixtures were used for validation in the wavelength range of 260 to 320 nm with the wavelength interval λ= 0.2 nm in methanol. The methods were validated as per International Conference on Harmonization Q2 (R1) (ICH) guidelines. These methods were successfully applied for determination of drugs in pharmaceutical formulation (tablet) with no interference of the excipients as indicated by the recovery study results. The proposed methods are simple, rapid and can be easily used as an alternative analysis tool in the quality control as well as in process control of drugs and formulation.

A simple, specific, accurate, precise and sensitive RP- HPLC method has been developed for the rapid estimation of Alogliptin in bulk and its formulations. The chromatographic separation was carried on Phenomenex Gemini-NX-5 µm C18(2) 110A, LC Column 250 x 4.6 mm, using Acetonitrile:1-octasulphonoic acid (0.005mM) at pH-5 [60:40] (v/v) as mobile phase, at a flow rate of 1.0 ml/min.  The detection was carried out at 220 nm and drug eluted with a retention time of 3.48 min. Beer’s law was obeyed in the concentration range of 2-10?g/ml with correlation coefficient 0.9995. The method has been validated according to ICH guidelines for specificity, linearity, accuracy, precision, robustness, ruggedness, LOD and LOQ. The method was found to be specific, accurate, and precise, robust, rugged and sensitive. The developed method was good linearity, novel, rapid for the estimation of Alogliptin in bulk and tablets dosage form. Thus it can be employed for the routine analysis.

A new simple and sensitive RP-HPLC method was developed and validated as per the ICH guidelines for the estimation of fenoverine in bulk and pharmaceutical dosage form. The chromatographic separation was achieved  on Enable 18H C18  column (250 x 4.6mm, 5µm)  with a mobile phase containing acetonitrile and phosphate buffer pH 7 (55:45) at flow rate of 0.8ml/min using in isocratic elution mode. Detection was carried out at 262nm with the retention time of 4.7mins. Linearity in the calibration plot was achieved over the concentration range of 5-25ng/ml with an r2 value of 0.997. The method was validated for accuracy, precision, specificity and selectivity, robustness, detection and quantification limits and system suitability parameters according to ICH guidelines Q2 R1. The detection limit and quantitation limit were found to be 1.3ng/ml and 4ng/ml respectively. Further the validated method was successfully applied for the analysis of fenoverine in bulk and pharmaceutical dosage forms.

A new simple, accurate, rapid and precise isocratic RP-HPLC was developed and validated for the determination of Rosuvastatin and Ezetimibe in Pharmaceutical tablet dosage form by droping method. The Method employs Shimadzu LC system on Hypersil ODS column (4.6 x 250 mm, 5 µm) and flow rate of 1.5ml/min with an injection volume 20µl.  Buffer, Acetonitrile and Methanol was used as mobile phase in the composition of 40:30:30v/v. The Detection was carried out at 230nm. Linearity ranges for Rosuvastatin and Ezetimibe were 11-33µg/ml, 10-30µg/ml respectively for HPLC. Retention Time of Rosuvastatin and Ezetimibe were found to be 3.7 and 5.7 min respectively. Percent Recovery study values of Rosuvastatin and Ezetimibe were found 99.6-101.1% and 99.9-100.7% respectively. This newly developed method i.e. droping method was successfully utilized for the Quantitative estimation of Rosuvastatin and Ezetimibe in tablet dosage form. This method was validated for selectivity, accuracy, precision, and linearity, Ruggedness, Robustness and Stability Studies as per ICH guidelines.

A simple, sensitive, selective and rugged liquid chromatography coupled with mass spectrometry (LC/MS/MS) method for quantification of Diltiazem and its metabolites, N-desmethyl Diltiazem, desacetyl Diltiazem in human plasma was developed and validated. The chromatography was developed using Luna 5 μ, C18, 100×4.60 mm column having a mobile phase of Acetonitrile: 0.1 % formic acid (85:15 % v/v). The flow rate was 0.5 ml/min at a column temperature of 50 ± 5º C. Electron spray ionization technique in positive mode was selected to improve the selectivity and sensitivity required for this application. The retention times of Diltiazem, desmethyl Diltiazem, desacetyl Diltiazem were 2.5, 2.0 and 2.5 minutes respectively. The method was validated for linearity, precision, accuracy, specificity, sensitivity, matrix effect, dilution integrity, ruggedness, injection reproducibility and stability. Calibration curves during the course of validation were found to be linear for Diltiazem, desmethyl Diltiazem, desacetyl Diltiazem in the ranges of 0.604-603.902, 0.303-303.274 and 0.299-299.489 ng/mL with correlation coefficient ≥ 0.9969, 0.9958 and 0.9970 respectively and by using a 1/x2 weighted least square regression analysis of standard plots associated with ten point calibration standards. The precision and mean accuracy were within the acceptable limits.  

The objective of the present research work was to simultaneously separate the anti-hypertensive agents, Amlodipine and Valsartan and develop a validated analytical method for simultaneous quantitative determination of amlodipine and valsartan in tablet dosage form. A simple, rapid, precise and selective chromatographic method was developed and validated for separation and determination amlodipine and valsartan in tablet preparations. The anti-hypertensive agents were analyzed by Symmetry C18, (150 × 3.4 mm, 5 µ), Shimadzu LC-2010CHT Prominence Liquid Chromatograph and a mobile phase constituted of 10 mM Buffer (pH 3.0): methanol (50:50, v/v). The flow rate was 1.0 mL/min and the analysis were performed using UV- Vis detector at 237nm. The anti-hypertensive agents, Amlodipine and Valsartan were separated within 10 min. Amlodipine and Valsartan showed retention time of 5.06 and 8.28 min respectively. The drugs were found to obey Beer’s law in the concentration range of 100 ppm of amlodipine and 128 ppm of valsartan. The developed assay method is selective, precise and accurate. The method has been successfully applied for determination of Amlodipine and Valsartan in pharmaceutical combination tablet dosage form. This developed method is sensitive, fast and simple with excellent peak symmetry and high resolution.