Validation

A new RP – HPLC method was developed for the simultaneous determination of  Montelukast and Rupatadine fumerate in combined dosage form. An Inertsil C18 column(100 x 4.6, 5µm) was used with mobile phase of composition Methanol : Buffer(0.1% triethyl amine in water with pH adjusted to 3.0 (70:30v/v at pH 4.6) at a flow rate of 1.0 mL/min and injection volume of 20µL with UV detection at 266 nm for separating Montelukast and Rupatadine fumerate. The retention time of Rupatadine fumerate and Montelukast were 5.76 min and 2.86 min respectively. The runtime of the analysis was 6 minutes. The specificity, linearity, precision, accuracy, limit of detection (LOD), limit of quantification (LOQ), ruggedness and robustness of the developed method were studied to validate as per ICH guidelines. The Linearity range for Montelukast and Rupatadine fumerate were 5.0 – 30.0 µg/ml and 5.0 – 30.0 µg/ml, respectively. The percentage recoveries were in the range for Montelukast and Rupatadine fumerate98.80-100.11 % and 99.06-99.44 %, respectively. The developed  method could be used for routine analysis of Montelukast and Rupatadine fumeratein their combined dosage forms.

A new validated RP – HPLC method was developed for the simultaneous determination of Thiocolchicoside and Ketoprofen in combined dosage form. The method developed produced high sensitivity, precision and accuracy. An isocratic C18 (Inertsil ODS, 250 x 4.6 mm, 5µ) column was used with mobile phase of composition Acetonitrile: Phosphate buffer (70: 30 at pH 4.6) at a flow rate of 1.0 mL/min with UV detection at 258.2 nm for separating Thiocolchicoside and Ketoprofen. The retention time of Thiocolchicoside and Ketoprofen were 2.4 min and 3.5 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 Thiocolchicoside and Ketoprofen were found in the range of 2.0 – 12.0 µg/ml and 6.2-38.75 µg/ml, respectively. The percentage recoveries for Thiocolchicoside and Ketoprofen ranged from 99.35- 100.21% and 98.66-99.29 %, respectively. The proposed method could be used for routine analysis of Thiocolchicoside and Ketoprofen in their combined dosage forms. All the proposed methods for Thiocolchicoside and ketoprofenare simple, selective, reproducible and specific with good precision and accuracy. The method was proved to be superior to most of the reported methods. These proposed methods for estimation of selected drugs were successfully applied either in tablet dosage form. More over the low solvent consumption along with short retention time of 2.4 and 3.5 for both Thiocolchicoside and Ketoprofen to be cost effective when compared to other developed method shown in literature reviews. The proposed method can be used as alternative methods to the reported ones for the routine determination of selected drugs under the study in tablet dosage form

The concept of "Quality by Design" (QbD) is getting popularized in pharmaceutical manufacturing industry to understand the product and process to identify the risks involved during manufacturing.  One of the perpetual quality attribute is to have robust analytical method that can provide consistent results though out the life cycle of the product. General considerations during analytical method validation is to perform robustness studies by deliberate changes made in pH of the buffer in mobile phase, change in organic ratio, change in column oven temperature, change in buffer strength and using different column lot numbers etc. However to improve the analytical quality standard, a novel method concept under QbD was introduced which uses single mobile phase for three drug components and estimates using different column chemistries used in pharmaceutical industry viz., C18, C8, phenyl and Cyano column. The validated RP-LC method was successfully applied to the quantitative determination of Chlorpheniramine, Ibuprofen and Pseudoephedrine in tablet dosage form, helping to improve quality control and to assure therapeutic efficacy using all column chemistries.

A new gradient reverse phase Ultra Performance Liquid Chromatography (UPLC) method was developed for the analysis of Dissolution profile samples of Paricalcitol in Paricalcitol Soft Gelatin capsules. The aim of the new method was to achieve proper accuracy and precision for the highly potent low dose drug product formulations. The normal injection loop allows upto 10µL of the sample in normal condition in UPLC systems.  For the current method the loop was modified to handle 50µL of injection volume in order to achieve quantifiable area counts. Efficient separation is achieved on Acquity UPLC HSS T3 (100 mm length × 2.1 mm ID with, 1.8 m particle size. Validation parameters such as specificity, linearity, precision, accuracy, and robustness were evaluated as per ICH guidelines. The validated RP-UPLC method was successfully applied to the Dissolution of Paricalcitol Soft Gelatin Capsules dosage forms.

A simple, accurate, precise and sensitive RP- HPLC method has been developed for the determination of Gatifloxacin and Flurbiprofen Sodium in their pharmaceutical formulation. Chromatographic separation  was carried out on InertsilODS – 3 column (250 mm ×4.6 mm, 5µm ) as stationary phase by using mobile phase consisting of 0.02 M Phosphate buffer (pH 3.5 adjusted with orthophosphoric acid ) : Methanol (80 : 20 v/v). The flow rate was 1.5 ml/min with UV-detection at 245 nm. The retention time was found to be 2.59 min for Gatifloxacin and 5.41min for Flurbiprofen Sodium. The method was validated for various parameters according to ICH guideline. The linear regression analysis data for the calibration plots showed good linear relationship in the concentration range of 30 – 90 µg/ml and 3 – 9 µg/ml and correlation coefficient was found to be 0.9988 and 0.9992 for Gatifloxacin and Flurbiprofen Sodium respectively. The Limit of Detection for Gatifloxacin and Flurbiprofen Sodium were 1.45 and 0.028 respectively. The Limit of Quantification for Gatifloxacin and Flurbiprofen Sodium were 4.39 and 0.28 respectively.

Cetrizine hydrochloride and Phenylephrine hydrochloride are used in combination in the treatment of allergy. The aim of this project work is to develop selective, accurate, specific and economic RP–HPLC method for simultaneous estimation of Cetrizine hydrochloride and Phenylephrine hydrochloride in Bulk and Tablet dosage form. The reverse phase C18 (250 mm × 4.6 mm i.d) column with 5 µm particle size was used. Acetonitrile: Water was taken as mobile phase was with a flow rate of 1 mL/min and detection was carried out at 222 nm. The retention time of Cetrizine hydrochloride and Phenylephrine hydrochloride was 1.956 min and 4.561 min respectively. The calibration curves were linear (>0.998) in the range of 5-25 μg/ml for Cetrizine hydrochloride and Phenylephrine hydrochloride. The Limit of Detection for Cetrizine hydrochloride and Phenylephrine hydrochloride was found to be 0.26 μg/mL and 0.51μg/mL. The Limit of Quantification was found to be 0.81μg/mL and 1.54 μg/mL respectively for Cetrizine hydrochloride and Phenylephrine hydrochloride. The developed method was simple, selective and precise and can be used for routine analysis of Cetrizine hydrochloride and Phenylephrine hydrochloride in tablet dosage form.

A simple, accurate, precise and rapid reversed-phase high performance liquid chromatographic (RP-HPLC) method has been developed and subsequently validated for the estimation of Liraglutide in bulk and Parenteral Dosage form. The proposed method is based on the separation of drugs in reversed-phase mode using Waters HPLC 22695 model, Inertsil ODS column (250 x 4.6 mm, 5µm particle size).The optimum mobile phase consisted of methanol: phosphate buffer in the ratio of 85:15 v/v(Phosphate buffer pH 5.5) was selected as a mobile phase, flow rate of 1.0 ml/min and UV detection was set at 246 nm. The retention time was 3.25.The method was validated according to ICH guidelines. It was found to be accurate and reproducible. Linearity was obtained in the concentration range of 20-80 μg/ml . The percentage RSD for precision and accuracy of the method was found to be less than 2%. The proposed method can be successfully applied in the quality control of bulk and pharmaceutical dosage forms.

A new simple, accurate, precise and reproducible Reverse Phase-High Performance Liquid Chromatography  method has been developed for the simultaneous estimation of Sitagliptin and Metformin in bulk and pharmaceutical dosage form using Symmetry C18 column (4.6 x 150mm, 3.5mm, Make: XTerra) in isocratic mode. The mobile phase has been prepared by using Potassium Dihydrogen Phosphate and Acetonitrile in different ratio at different pH. Several trials have been performed and it was found that ratio 0f 65:35 of Potassium Dihydrogen Phosphate and Acetonitrile respectively was shown a good peak at pH 5.8 which has been adjusted by using Sodium Hydroxide. The detection was carried out at 254 nm. The method was linear over the concentration range for Sitagliptin 10-30ppl and for Metformin 100-300ppm. The % recoveries of Sitagliptin and Metformin were found to be 99.1 to 100.6% and 98.8 to 100.7% respectively. The validation of method was carried out utilizing International Conference on Harmonization (ICH) guidelines. The described High Performance Liquid Chromatography method was successfully employed for the analysis of pharmaceutical formulations containing combined dosage form.

The present manuscript describes simple, sensitive, rapid, accurate, precise and economical Q-absorbance ratio method for the simultaneous determination of Sparfloxacin and Dexamethasone. Absorbance ratio method uses the ratio of absorbances at two selected wavelengths, one which is an isoabsorptive point and other being the λ-max of one of the two components. Sparfloxacin and Dexamethasone show an isoabsorptive point at 271.2 nm in methanol. The second wavelength used is 241.6 nm, which is the λ-max of Dexamethasone  in methanol. The linearity was obtained in the concentration range of 3-18 μg/ml for Sparfloxacin and 1-6 μg/ml  for Dexamethasone. The concentrations of the drugs were determined by using ratio of absorbances at isoabsorptive point and at the λ-max of Dexamethasone. The method was successfully applied to pharmaceutical dosage form because no interference from the preservative was found. The suitability of this method for the quantitative determination of Sparfloxacin and Dexamethasone was proved by validation. The proposed method was found to be simple and sensitive for the routine quality control application of Sparfloxacin and Dexamethasone in Eye/Ear drops. The results of analysis have been validated statistically and by recovery studies.

A simple, precise, accurate and stability-indicating reverse phase high performance liquid chromatography (RP-HPLC) method is developed for estimation of Levosalbutamol sulphate and Ipratropium Bromide in bulk and nebulizer dosage form. The method employed, with reverse phase Inertsil® 5μ C18 (250 × 4.0 mm) column in an isocratic mode, with mobile phase of acetonitrile: buffer in the ratio 77:23 (%v/v). The flow rate was 1.3 ml/min and effluent was monitored at 210 nm. Retention time was found to be 3.05 min., and 10.59 min. The method was validated in terms of linearity, accuracy, precision, limit of detection (LOD), limit of quantification (LOQ) etc. in accordance with ICH guidelines. Linear regression analysis data for the calibration plot showed that there was good linear relationship between response and concentration in the range of 25 – 150% of the working concentration (r2 > 0.999) respectively. The LOD and LOQ values for were found to be0.72,0.43, 1.24and 0.97 and μg/ml respectively. No chromatographic interference from placebo and degradants were found. The proposed method was successfully used for estimation of Levosalbutamol sulphate and Ipratropium bromide in bulk and nebulizer dosage forms.