Validation.

ABSTRACTA novel and validated UV spectrophotometric method using differential derivative techniques was developed for the quantification of Orlistat in pharmaceutical formulations. The method was assessed based on various analytical parameters, including linearity, precision, accuracy, sensitivity, ruggedness, and robustness. The assay results indicated a percentage recovery of 98.74%, confirming compliance with Pharmacopeial standards. Linearity studies showed high correlation coefficients (r² ? 0.999) for zero-order, first-order, and second-order derivative methods, ensuring reliable quantification. Precision and repeatability assessments demonstrated low relative standard deviation (%RSD) values, indicating excellent reproducibility. Recovery studies revealed percentage recoveries between 109.81% and 131.16%, highlighting the method's accuracy. Sensitivity analysis, expressed through the limits of detection (LOD) and quantification (LOQ), confirmed the method’s capability to detect low drug concentrations. Ruggedness and robustness evaluations showed that minor variations in experimental conditions did not significantly impact the method’s performance. The validated UV spectrophotometric approach is simple, precise, and cost-effective, making it suitable for routine quality control of Orlistat formulations. Keywords: Orlistat, UV Spectrophotometry, Derivative Spectroscopy, Optimization, Validation.

ABSTRACTThe objective of the study was to develop and evaluate the reverse phase high performance liquid chromatography (RP-HPLC) method for the quantitative determination of potential impurities of Mirabegron active pharmaceutical ingredient. The method uses Puratis C18 column (250 × 4.6mm, 5µm ) with mobile phase A consisted, 20 mM Ammonium acetate, pH adjusted to 4.5 and mobile phase B consisted methanol  with a gradient programme. The column temperature was maintained at 25 °C and the detection was carried out at 247 nm. Efficient and reproducible chromatographic separation was achieved on C18 stationary phase in gradient elution profile. The newly developed HPLC method was validated according to ICH guidelines considering three impurities to demonstrate precision, linearity, accuracy and robustness of the method. The developed HPLC method was found to be accurate and sensitive. The correlation coefficient values are greater than 0.99 for Mirabegron and its three impurities. Detection limit and quantitation limit was 0.04ppm and 0.14ppm respectively, indicating the high sensitivity of the newly developed method. Accuracy of the method was established based on the recovery obtained between 99.67% and 104.98% for all impurities. The result of robustness study also indicates that the method is robust and is unaffected by small variation in chromatographic conditions. The proposed HPLC method provides reliable, reproducible, accurate and sensitive for the quantification of Mirabegron related substances. Keywords: Mirabegron; Impurities; RP-HPLC; Validation.

An isocratic reversed-phase liquid chromatograpic assay method was developed for the quantitative determination of Atorvastatin calcium (ATOR) and Clopidogrel bisulphate (CLOP) in combined dosage form. A Zodiac C18, 250x4.6mm, 5µm column and OPA Buffer: ACN (70: 30,v/v) as mobile phase. The flow rate was 1mL/min and effluents were monitored at 241 nm. The retention times of Atorvastatin and Clopidogrel were 5.8 min and 3.5 min respectively. The correlation coefficient was found to be 0.99947 (for ATOR) and 0.99944 (for CLOP). The proposed method was validated with respect to linearity, accuracy, precision, and robustness. The method was successfully applied to the estimation of Atorvastatin and Clopidogrel in combined capsule dosage form.

A rapid, specific reversed phase HPLC method has been developed for simultaneous determination of pantoprazole and levosulpridein their formulations. Chromatographic separation of these two pharmaceuticals was carried out on an Aligent,Zorbax column (250mmx4.6mm, particle size 5μm)with a 600:4000 (v/v/v) mixture of Potassium dihydrogen orthophosphate (pH-3.0; 0.01M)  buffer adjusted with Ortho phosphoric acid and methanol as mobile phase. The flow rate 1.0mL.min-¹ and the analytes are monitored at 230nm.The assay results were linear from 240-720μg/mL for pantoprazole(r ² >= 0.9999) and 450-1350μg/mL for levosulpride (r ² >= 0.9999), showed intra- and inter-day precision less than 2.0%, and accuracy of 100%.The LOD was 0.00321 and 0.000549μg.mL-¹ for pantoprazole and levosulpride respectively. Separation was complete in less than 5 min. Validation of the RP-HPLC method showed to be robust, precise, accurate and linear over the range of analysis.

A convenient, simple, accurate, precise and reproducible RP-HPLC method was developed and validated for the estimation of Linezolid in the bulk drug and pharmaceutical dosage form. The objective was achieved under optimized chromatographic conditions on Welchrom C18 isocratic column (250 mm × 4.6 mm, 5 μm) with Shimadzu LC-20AT Prominence Liquid chromatograph. The mobile phase was a mixture of acetonitrile: water 50:50 v/v, with apparent pH of 6.8. The separation was attained using an isocratic elution method with a flow rate of 1.2 mL/min at room temperature. The detection was made at a wavelength of 254nm by using UV- Visible detector.  The retention time for Linezolid molecule was found to be 2.813.The standard calibration plot was linear over a concentration range of 2-10 µg/mL with r2= 1 and the respective linear regression equation being y= 177.1x + 0.030.The limit of detection and limit of quantification were found to be 0.04µg/mL and 0.014µg/mL respectively. The amount of Linezolid presents in the bulk drug 99.81%and in the formulation 99.81% of the stated amount respectively. The method was validated statistically using the %RSD and the values are found to be within the limits. No interference peaks from Excipients and relative retention time indicated the specificity of the Method.  The recovery studies were performed and mean percentage recoveries were found to be greater than 99% with RSD less than 1.0%. So the proposed method was found to be simple, specific, linear, robust and reproducible. Hence this method was conveniently and easily applied for routine analysis of Linezolid in bulk drug and tablet dosage form.

A simple, precise, rapid, selective, and economic high-performance thin layer chromatography (HPTLC) method has been established for simultaneous analysis of Citicoline Sodium and Methylcobalamin. HPTLC method was developed using on precoated silica gel F254 G60 plates as stationary phase, using methanol: acetonitrile: water: triethylamine (8.5:1.5:1:0.5 v/v/v) as mobile phase. The plates were scanned at approximately 254 nm for both Citicoline sodium and Methylcobalamin respectively. In HPTLC method both the drugs were resolved using proposed mobile phase and Rf value was found to be 0.39 for Citicoline sodium and Rf 0.61 for Methylcobalamin. The method was found to linear in the range 1000-6000 ng/band for citicoline sodium and methylcobalamin respectively. This HPTLC procedure is economic, sensitive, and less time consuming than other chromatographic procedures. It is important tool for analysis of combined dosage form. Proposed method can be successfully applied for the quantitative determination of Citicoline Sodium and Methylcobalamin in Bulk drug and Pharmaceutical dosage form.

Two simple, sensitive selective accurate and economical spectrophotometric methods (Method-A&B) for the determination of Nateglinide in bulk drug and pharmaceutical formulations (tablets) have been described in the present work. Method - A is based on the formation of orange red colored ion-association complex between Nateglinide and Tropaeolineooo (TPooo) exhibiting absorption maximum at 470nm and obeying Beer’s law in the concentration range of 2.5-12.5µg/ml. The Method - B is based on the formation of Ion-association complex between Nateglinide and Alizarine Red S (ARS) to yield an yellow colored chromogen exhibiting absorption maximum at 440nm (Method B) and obeying Beer’s law in the concentration range of 2.5-12.5µg/ml. Statistical analysis of the results has been carried out for the proposed methods revealing high accuracy and good precision. The proposed methods developed by the author could be successfully extended to the commercial pharmaceutical formulations (tablets) containing Nateglinide.

U.V. spectrophotometric method for estimation of Eperisone hydrochloride as individual and in tablet formulation using graphical extrapolation method has been developed. Methanol was selected as solvent for estimation at 258 nm. Linearity was followed in the range of 2.5-17.5 mg/ml with correlation coefficient of 0.997. Detection limit and quantitation limit were found to be 0.082 mg/ml and 0.22 mg/ml .Developed method has followed all the criteria for validation as per ICH norms and found to be accurate, precise, reproducible and sensitive with negligible excipients interference. Accuracy of the developed was assessed using spiking technique on available tablet dosage forms. Results obtained greater than 98% meant good accuracy of method for analysis in any dosage form.