RP-HPLC

A simple, precise, accurate and reproducible RP-HPLC method for simultaneous estimation of clarithromycin, pantoprazole and Metronidazole in bulk and pharmaceutical formulations. Separation of clarithromycin, pantoprazole and Metronidazole was successfully achieved on a YMC Pack Pro C18 (250 mm x 4.6mm x 5µ) in an isocratic mode utilizing sodium dihydrogen phosphate buffer and methanol (65:35 v/v) at a flow rate of 1.0 mL/min. The method was validated according to ICH guidelines for linearity, sensitivity, accuracy, precision, specificity and robustness. The response was found to be linear in the drug concentration range of 31-93.75 mg/mL for clarithromycin and 5-15 mg/mL for pantoprazole and 50-150 mg/mL for Metronidazole. The correlation coefficient was found to be 0.999 for both the drugs. The limit of detection (LOD) was 0.228, 0.0309 and 0.743 for clarithromycin, pantoprazole and Metronidazole respectively. The limit of quantification (LOQ) was 0.758, 0.1030 and 2.475 for clarithromycin, pantoprazole and Metronidazole respectively. The relative standard deviation (RSD) of six replicates is less than 2%. This HPLC method is applied successfully to the simultaneous quantitative analysis of clarithromycin, pantoprazole and Metronidazole in commercial tablets.

Purpose of this study was to develop a stability-indicating RP-HPLC method for routine analysis of Paracetamol (PARA), Caffeine (CAF) and Ibuprofen (IBU) in their combined solid dosage forms. The new RP-HPLC method was validated as per ICH, FDA and USP guidelines with respect to accuracy, precision, specificity, linearity, solution stability, robustness, sensitivity and system suitability. The method was developed by using a binary gradient mode of phosphate buffer (pH 7.2) and acetonitrile at a flow rate of 1.3mL/min for 15 minutes over C-18 (ODS, 150 x 4.6 mm, 5µm) column at ambient temperature. Injection volume was 20µL for both standard and sample solutions and the eluents were monitored with UV detection at 230nm.Accuracy was determined by the recovery tests of the drugs and found to be within a range of 99.89% to 100.33%. Intraday and inter-day precisions were demonstrated by a relative standard deviation being far less than maximum allowable limit (2.0%, according to FDA). The method showed linear response with a correlation coefficient (r2) value of 0.999 for all three drugs. Forced degradation studies in acidic, basic, oxidation and reduction media were carried out to establish the stability indicating tolerance of this method. Specificity was shown by the separation of drugs with high degree of resolution between them and absence of any interference from the excipients or degradation products. This method was successfully applied to assay the drugs in tablets and capsules. Hence this newly developed method can be considered suitable and reliable for the routine analysis of PARA, CAF and IBU in their solid dosage forms.

A simple, selective and rapid reverse phase high performance liquid chromatographic (RP-HPLC) method for the analysis of Gliclazide in bulk and in tablet dosage form has been developed and validated. Sample was analysed on a Enable C18 (250mm X 4.6 mm i.d, particle size 5μm) column. The mobile phase consist of Methanol: Water (pH 3.5) in the ratio of 85:15v/v which was sonicated to degased and delivered at a flow rate of 1ml/min at ambient temperature. The retention time of Gliclazidewas 3.7+0.02 minutes. Studies were performed using an HPLC system equipped with a UV detector; the response was monitored at 230 nm.The calibration curve was linear over the concentration range of 20-70 μg/ml (r2=0.999). The limit of detection for Gliclazide was found to be 0.2438 μg/ml and the limit of quantification limit was about 0.7388 μg/ml. The accuracy of the method was established based on the recovery studies. The proposed method can be applied to the routine analysis of Gliclazide in bulk and in tablet dosage form.

A simple, sensitive and reproducible stability indicating RP-HPLC method for the simultaneous determination of Phenylephrine and Ebastinein bulk and Pharmaceutical dosage formhas been developed and validated. Chromatographic separation was carried out on kromasil C18 (250×4.6mm, 5mparticle size) column using a mobile phase composed of Phosphate buffer (adjusted to pH 5.0 with dilute OPA): acetonitrile: methanol in the ratio of 30:45:25 %v/v/vat a flow rate of 0.8 ml/min. The analyte was monitored using PDA detector wavelength at 211 nm. The retention time was found to be 2.295 min and 4.225 min for Phenylephrine and Ebastine respectively. The proposed method was found to be having linearity in the concentration range of 25-150µg/ml for both Phenylephrine(r20.99996)and Ebastine(r20.99987)respectively. The developed method has been statistically validated according to ICH guidelines. Stress testing which covered acid, alkali, peroxide, photolytic and thermal degradation was performed on under test to prove the specificity of the method and the degradation was achieved. The proposed method can be successfully applied for the stability indicating RP-HPLC simultaneous determination of Phenylephrine and Ebastine in bulk and combined tablet dosage form and in routine quality control analysis.

A simple and precise stability indicating RP-HPLC method was developed and validated for the simultaneous determination of Hydrochlorothiazide (HCTZ) and Nebivolol Hydrochloride (NBV) inbulk and Pharmaceutical dosage forms. Chromatography was carried out on Thermo Hypersil BDS C 18 (150 x 4.6 mm, 5mparticle size) column using a mobile phase of phosphate buffer (adjusted to pH 6.5 with dilute orthophosphoric acid): acetonitrile (40:60% v/v) at a flow rate of 0.8 ml/min. The analyte was monitored using PDA detector at 282 nm. The retention time was found to be2.4 min and 4.0min for Hydrochlorothiazide and Nebivolol Hydrochloride respectively. The proposed method was found to be having linearity in the concentration range of 6.25-37.5 µg/ml for Hydrochlorothiazide (r2 0.9999) and 2.5-15 µg/ml for Nebivolol Hydrochloride (r2 0.9999) respectively. The mean % recoveries obtained were found to be 99.93 % for Hydrochlorothiazide and 100.03% for Nebivolol Hydrochloride respectively. Stress testing which covered acid, alkali, peroxide, photolytic and thermal degradation was performed on under test to prove the specificity of the method and the degradation was achieved. The developed method has been statistically validated according to ICH guide lines and found to be simple, precise and accurate with the prescribed values. Thus the proposed method was successfully applied for the stability indicating simultaneous determination of Hydrochlorothiazide and Nebivolol Hydrochloridein bulk and Pharmaceutical formulations and in routine quality control analysis.

An accurate, efficient Stability indicating reversed-phase high-perfomance liquid chromatographic (RP-HPLC) method has been developed and validated for the simultaneous estimation of Olmesartan, Cilnidipine and Chlorthalidone. All the drugs were separated on a KROMASIL 250 x 4.6 mm, column packed with 5 µm particles. The mobile phase, optimized through an experimental design, was a 45:55 (v/v) mixture of acetonitrile and Ortho phosphoric acid buffer(0.1%OPA), pumped at a flow rate of 1 ml/min. UV detection was performed at 230 nm. The retention time of Olmesartan, Cilnidipine and Chlorthalidone was found to be 2.280min, 8.356 min and 2.804min respectively. The method was validated in the sample concentration ranges of 20-120 µg/ml for Olmesartan and 5-30 µg/ml for Cilnidipine, and Chlorthalidone 6.25-37.5µg/ml.  The method demonstrated to be robust, resisting to small deliberate changes in pH and flow rate of the mobile phase. The LOD values were 0.08µg/ml, 0.04µg/ml and 0.05 µg/ml, while the LOQ values were 0.24µg/ml, 0.12µg/ml and 0.16µg/ml for Olmesartan, Cilnidipine and Chlorthalidone respectively.

A specific and accurate HPLC method is developed for the determination of etoposidein bulk drugs and in solid capsule dosage form. Best symmetric peak shape obtained with Inertsil ODS C-18 column (250 X 4.6 mm, 5μ) column in an isocratic mode, with retention time 5 min.The mobile phase used was Water : Acetonitrile  60:40(v/v)with flow rate 1.0 ml/min and effluent was monitored at 263 nm. As per ICH guidelines method has validated. Method has found linear in the range of 5-45 µg/ml. The LOD and LOQ were found to be 0.02 and 0.06 µg/ml respectively. Method was found specific with respective of diluents, excipients and degradants.

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.

A new simple, precise, accurate and reproducible RP-HPLC method for simultaneous estimation of gentamicin and clobetasol in bulk and pharmaceutical formulations. Separation of gentamicin and clobetasol was successfully achieved on a Zorbax C18 (150 mm x 4.6mm x 5µ ) in an isocratic mode utilizing disodium hydrogen phosphate buffer and methanol (60:40 v/v) at a flow rate of 1.0 mL/min. The method was validated according to ICH guidelines for linearity, sensitivity, accuracy, precision, specificity and robustness. The response was found to be linear in the drug concentration range of 0.1-0.30 mg/mL for gentamicin and 0.05–0.15 mg/mL for clobetasol. The correlation coefficient was found to be 0.9997 for both the drugs. The LOD and LOQ for gentamicin were found to be 0.3525 µg/mL and 1.1751 µg/mL respectively. The LOD and LOQ for clobetasol were found to be 0.1938 µg/mL and 0.6460 µg/mL respectively.  The proposed method was found to be good percentage recovery for gentamicin and clobetasol, which indicates that the proposed method is highly accurate. The specificity of the method shows good correlation between retention times of standard solution with the sample solution. Therefore, the proposed method specifically determines the analyte in the sample without interference from excipients of pharmaceutical dosage forms.

A new, simple, precise, accurate and reproducible RP-HPLC method was developed and validated for the simultaneous estimation of levodopa, carbidopa and entacapone in bulk and pharmaceutical formulations. Separation of levodopa, carbidopa and entacapone was successfully achieved on a phenomenex C18 (250mm x 4.6mm x 5µm). The mobile phase consisted of 0.1M Ammonium acetate buffer: methanol (60:40 v/v) at a flow rate of 1.0 ml/min. The response was found to be linear in the concentration range of 50µg/ml to150 µg/ml for levodopa, 12.5 µg/ml to 37.5 µg/ml for carbidopa and 200 µg/ml to 600 µg/ml for entacapone. The value of the correlation coefficient was found to be 0.999, 1.000 and 0.999 for levodopa, carbidopa and entacapone respectively. The LOD and LOQ for levodopa were found to be 0.926 µg/ml and 3.088 µg/ml, respectively. The LOD and LOQ for carbidopa were found to be 0.1917 µg/ml and 0.6389 µg/ml, respectively and LOD and LOQ for entacapone were found to be 2.068 µg/ml and 6.893µg/ml respectively.  This method was found to be good percentage recovery for levodopa, carbidopa and entacapone were found to be 99.00, 99.00 and 100.00 respectively indicates that the proposed method is sufficiently accurate. The specificity of the method shows good correlation between retention times of standard with the sample so, the method specifically determines the analyte in the sample without interference from excipients that are commonly present in tablet dosage forms. The method was extensively validated according to ICH guidelines for linearity, range, accuracy, precision, specificity and robustness.