RP-HPLC

A simple, fast and precise reverse phase, isocratic HPLC method was developed for the separation and quantification of Sildenafil and Duloxetine in pharmaceutical dosage form. The quantification was carried out using Symmetry C18-ODS 4.6X150mm, 3µm enhanced polar selectivity column and mobile phase comprised of potassium dihydrogen phosphate buffer and acetonitrile and water in proportion of ratio 30:65:5 and degassed under ultra-sonication. The flow rate was 0.6mL/min and the effluent was monitored at 244nm. The retention time of Sildenafil and Duloxetine were 4.3 and 3.4 respectively. The method was validated in terms of linearity, precision, accuracy, and specificity, limit of detection and limit of quantization. Linearity of Sildenafil and Duloxetine were in the range of 100 to 300µg/mL and 30 to 90µg/mL respectively. The percentage recoveries of both the drugs were 98.7% and 99.8% for Sildenafil and Duloxetine respectively from the tablet formulation.The method was found to be precise, accurate and specific during the study. The proposed method enables rapid quantification and simultaneous analysis of both drugs from commercial formulations without any excipients interference. The method can be used for routine analysis of marketed products of Sildenafil and Duloxetine in combined tablet formulation

A simple, rapid, and precise RP-HPLC method for simultaneous analysis of Losartan potassium and Atorvastatin calcium in bulk and its pharmaceutical formulations has been developed and validated. Atorvastatin was separated from losartan by using Grace Smart Altima C-18 column (25 cm × 4.6 mm, 5-μm) with a mobile phase consisting of acetonitrile: 10mM phosphate buffer (55:45 %v/v, pH 3.0) a flow rate of 1mL/min and detection wavelength at 240 nm. Aceclofenac was used as an internal standard in this method. Losartan, atorvastatin and aceclofenac were eluted with retention times of 4.85 min, 8.31min and 9.51 min respectively. The method was validated for accuracy, precision, linearity and sensitivity in accordance with ICH (Q2B) guidelines and the results of all the validation parameters were found to be within the acceptable limits. The calibration plots were linear over the concentration ranges from 200-30000ng/mL (r2 = 0.999) for both the drugs. Accuracy and precision were determined by QC sample covering low, medium, and high concentration levels. Intra and inert-day accuracy were found to be 97.16-102.57% for losartan and 97.01-103.05% for atorvastatin. The limit of quantification was found to be166ng/mL and 179ng/mL for losartan and atorvastain respectively. The method was successfully applied for the assay of the dosage form, recovery of the individual drugs from the combined tablet dosage was found to be >97% for both the drugs. From the results it is suggested that the proposed method is simple, reproducible, accurate and precise.

A New simple, precise, accurate and rapid RP-HPLC method was proposed for determination of Dorzolamide hydrochloride from pure and its dosage form. A Symmetry Hypersil C18   (250 × 4.6mm, 5µ) column in isocratic mode with mobile phase phosphate buffer (pH 6.2): Acetonitrile (60:40) at a flow rate of 1ml/min. The effluent was monitored at 254nm. The retention time was 3.337min for Dorzolamide hydrochloride. The linearity range was found to be 20 – 120 µg/ml. The developed method was validated for parameters like specificity, accuracy, ruggedness and robustness and ascertained values were found to be within limits. The method has significant advantages in terms of shorter analysis time, selectivity and accuracy then previously reported method and indicates that the method can be considered suitable for carrying out quality control &routine determination of Dorzolamide hydrochloride in bulk and pharmaceutical dosage form.

The objective of this work was to develop and validate simple, rapid and accurate chromatographic methods (A and B) for determination of Desvenlafaxine succinate in solid dosage form. In method A - RP-HPLC method was based on Reversed Phase High Performance Liquid Chromatography, on ODS C18 RP column (150 mm × 4.6 mm i.d., 5 µ), using Methanol : 50 mM Phosphate buffer (pH 8.0): Acetonitrile (50:40:10 % v/v) as the mobile phase, at a flow rate of 1 mL/min at ambient temperature. Quantification was achieved by UV detection at 225 nm over a concentration range of 5-25 µg/mL for Desvenlafaxine succinate. The mean retention time for Desvenlafaxine succinate was found to be 4.80 min. The amount of Desvenlafaxine succinate estimated as percentage label claim was found to be 99.83 ± 1.1093. In method B - HPTLC method was based on TLC separation of the drug using silica gel 60 F 254 aluminium sheets and Chloroform : Methanol : Water (60:30:10 v/v/v) as mobile phase. Detection was carried out at 226 nm over the concentration of 1 - 3.5 µg/mL Desvenlafaxine Succinate. The mean Rf value of Desvenlafaxine succinate was found to be 0.63. The amount of Desvenlafaxine succinate was estimated as percentage label claim found to be 101.57 ± 0.92668. Both of these methods were found to be simple, precise, accurate, selective and could be successfully applied for determination of pure laboratory prepared mixture and tablet. Key words: RP-HPLC, HPTLC, Desvenlafaxine succinate, marketed formulation.

A simple, precise, accurate and stability-indicating reverse phase high performance liquid chromatography (RP-HPLC) method is developed for estimation of Isosorbide 5-Mononitrate in bulk drug and tablet dosage form. The method employed, with reverse phase phenomenex® Luna 5µ C18 (2) 100A (250 × 4.60 mm) column in an isocratic mode, with mobile phase of methanol: water: acetonitrile in the ratio 55:28:17 (%v/v/v). The flow rate was 1.0 ml/min and effluent was monitored at 217 nm. Retention time was found to be 4.391±0.015 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 1- 9 µg/ml respectively. The LOD and LOQ values for were found to be 2.5 and 10 ng/ml respectively. No chromatographic interference from tablet excipients and degradants were found. The proposed method was successfully used for estimation of Isosorbide 5-Mononitrate in tablet dosage form.

A simple, reliable, rapid, precise, sensitive and validated RP-HPLC method has been developed to determine Acetaminophen (AI) and Prasugrel (PRA) in synthetic mixture form. Chromatographic separation achieved isocratically on Luna C18 column (5 µm, 150mm x 4.60mm) and acetonitrile: 0.05M ammonium acetate buffer (pH 4.5) in the ratio of 75:25 (v/v) as the mobile phase, at a flow rate of 0.6 mL/min. Detection was carried out at 245 nm. Parameters such as linearity, precision, accuracy, recovery, specificity and ruggedness are studied as reported in the ICH guidelines. The retention times for AI and PRA was found to be 2.25 ± 0.5 and 8.72 ± 0.5 min respectively. Linearity for AI and PRA was in the range of 75-375 μg/mL and 10-50 μg/mL respectively.  The mean recoveries obtained for AI and PRA were 99.58 and 99.48% respectively and RSD was less than 2. The correlation coefficients for all components are close to 1. Developed method was found to be accurate, precise, selective and rapid for simultaneous estimation of AI and PRA.

The objective of the present research work is to develop a isocratic reversed-phase liquid chromatographic (RP-HPLC) method for the determination of Aspirin in pharmaceutical pharmaceutical dosage forms for its related impurities in presence of esomeprazole. The chromatographic separation was achieved on a RP 18 column (100mm×4.6mm, 5 µm). The isocratic LC method employs mixture of buffer methanol and isopropyl alcohol in the ratio of (84:13:3 v/v) solutions as mobile phase. The buffer solution contains 6.8g of Potassium dihydrogen orthophosphate adjusted to pH 2.5 with orthophosphoric acid .The flow rate was 1.5 ml/min and the detection wavelength was 275 nm. In the developed HPLC method, the resolution between Aspirin and its potential impurity salicylic acid was found to be greater than 4.0. The drug was subjected to stress conditions of hydrolysis, oxidation, photolysis and thermal degradation in presence of esomeprazole. Considerable degradation was found to occur in basic medium and mild degradation observed in acid hydrolysis stress conditions. Degradation product formed during acidic hydrolysis was salicylic acid. The stress samples were assayed against a qualified reference standard and the mass balance was found close to 99.5%. The developed RP-HPLC method was validated with respect to linearity, accuracy, precision and robustness.

A new reverse phase high performance liquid chromatography (RP-HPLC) method for the quantitative determination of Nimorazole was developed and validated as per ICH guidelines. The analyte was injected into an HIBER C18 column (150 mm × 4.6 mm, 5μm), maintained at ambient temperature and effluent was monitored at 297 nm. The mobile phase consisting of acetonitrile: methanol: buffer (2:3:5 v/v/v). The pH of the mobile phase was adjusted to 4.0 by using O-phosphoric acid. The flow rate was maintained at 1.0 mL/min. and retention time was observed 1.76 min. The developed method shows high specificity for Nimorazole. Calibration curve was plotted with a range from 1-5µg/ml (r2>0.999). The lower limit of quantification (LLOQ) was found to be 0.5μg/ml. The method was validated for parameters like accuracy, precision, recovery, linearity, robustness. This RP-HPLC method is suitable for determining the concentration of Nimorazole and it was applied to routine analysis for determination of the Nimorazole from its formulation during pharmacokinetic study.

A simple, accurate and precise HPLC method for the estimation of memantine in bulk and pharmaceutical dosage form has been reported. Chromatography was performed with Shimadzu HPLC equipment comprising an LC-10A VP quaternary pump, a variable-wavelength programmable UV–visible detector, an SPD-10AVP column oven, and an SCL 10AVP system controller. A Rheodyne injector fitted with a 20μL loop was also used and data were recorded and evaluated using Class-VP 5.032 software. The Compound was separated, at ambient temperature (25 ± 2°C), on a  BDS C18, ( 4.6 mm i.d x 250 mm, 5μm reversed phase column with 100% methanol as mobile phase at a flow rate of 1.0mL.min−1. Before use, the mobile phase was filtered through a 0.22-μm Nylon filter. UV detection was performed at 274nm. A linear response was observed in the concentration ranges of 5-25μg/ml with a regression coefficient of 0.9999. The method was then validated for different parameters as per the ICH guidelines. This method can be used for the determination of memantine in quality control of formulation without interference of the excipients.

Quantitative estimation of temozolamide and its pharmaceutical dosage form by UV spectroscopy, RP-HPLC, HPTLC methods was developed. In the UV method (geometric method), temozolamide was quantified at 309nm, 325nm, 340nm in serum and water. The corrected absorbance was calculated. The Recovery studies was found to be 95.5-96.9%. In RP-HPLC method, the drug was resolved using a mobile phase methanol: buffer (5.0ml glacial acetic acid in 1000ml water) (70:30%v/v) on C18 column in isocratic mode. The retention time of temozolamide was found to be 7.30 min. Recovery studies was found to be 99.55-100.98%.  In HPTLC method, the chromatograms were developed by using a mobile phase Chloroform: glacial acetic acid: methanol (2:3:5% v/v) on precoated plate of silica gel 60F254 and quantified by densiometric absorbance mode at 254nm. The Rf value of Temozolamide was 0.47. Recovery studies of 98.99-100.6%, percentage relative standard deviation (%RSD less than 2%) and correlation coefficient (linearity range) that developed methods were accurate and precise. These methods can be employed for the routine analysis of capsules containing temozolamide. Key words: Temozolamide, RP-HPLC, HPTLC, UV spectrophotometry, validation.