RP-HPLC method

The present work describes Stability indicating RP-HPLC and Simultaneous equation UV spectrophotometric method for the quantitative determination of Repaglinide and Sitagliptin phosphate. The parameters Specificity, linearity, accuracy, precision, detection limit, quantitation limit, Robustness and system suitability tests were studied and their results were complied to ICH guideline Q2 (R1).  Chromatography was carried out by reverse phase technique on an RP-18 with mobile phase composed of Acetonitrile: Phosphate buffer (65:35; % v/v) adjusted to pH 3.5 with 10% orthophosphoric acid) with flow rate 1 ml/min. Both drugs were eluted, isocratically using detection wavelength 228 nm. Simultaneous equation UV spectrophotometric method was performed and two wavelengths 240 nm (λmax of Repaglinide) and 267 nm (λmax of Sitagliptin phosphate) were selected for the formation of simultaneous equation. The A (1%, 1cm) was determined at both the wavelengths selected for each drug. A set of two simultaneous equations were formed as Cx and Cy. Methanol used as Solvent (diluent) for UV method. For proposed methods, the linearity for both methods were obtained in the concentration range of 0.5-2.5 μg/ml for Repaglinide and 50-250 μg/ml for Sitagliptin phosphate. Statistical analysis by student’s t-test showed no significance difference between the results obtained by these two methods. The suitability of method for the quantitative determination of Repaglinide and Sitagliptin phosphate was proved by validation. The proposed methods and its results had been successfully applied and validated statistically to the simultaneous estimation of Repaglinide and Sitagliptin phosphate in their combination for quality analysis.

A simple, economic, rapid, high range and accurate stability indicating RP-HPLC method was development for simultaneous estimation of Azithromycin and Ambroxol Hydrochloride in their combined tablet dosage form. This method was carried out by using Isocratic peak HPLC instrument with kromasil C-18 Column (250 mm X 4.6mm,5um) with mobile phase consisting a mixture of Methanol: Acetonitrile: Phosphate buffer in the ratio of 70:20:10 (v/v), at a flow rate of 1.1 ml/min with UV detection at 221nm . The retention time for Azithromycin and Ambroxol Hydrochloride are 9.08 and 5.39min respectively. Suitability, specificity, linearity, accuracy, precision, stability, and sensitivity of this method for the quantitative determination of the drugs were proved by validation in accordance with the requirements laid down by International Conference on Harmonization (ICH) Q2 (R1) guidelines. To establish stability indicating nature of the LC method, forced degradation of drug substances was performed under stress conditions like thermal, oxidation, peroxide, UV light, acid and base hydrolysis) The limit of quantification (L.O.Q) for Azithromycin and Ambroxol Hydrochloride are found to be 0.70ug/ml 1.00ug/ml.Then the limit of detection (L.O.D) for Azithromycin and Ambroxol Hydrochloride are found to be 0.15ug/ml and 0.3ug/ml respectively. The results of the study showed that the proposed method is reliable and robust and can be used as quality control tool for the estimation of these drugs in combined pharmaceutical solid dosage forms.

Artemether-lumefantrine (ART-LUME) off late has become the first-line treatment for uncomplicated malaria in many Sub-Saharan Africa, Asia and America. Vigorous monitoring of the therapeutic efficacy of this treatment is needed. This requires high-quality studies following standard protocols; ideally, such studies should incorporate measurement of drug levels in human plasma in biological matrices. A specific and reliable isocratic mode RP-HPLC method has been developed and validated for simultaneous determination of artemether (ART) in combination with lumefantrine (LUME) in human plasma using diode array detector (DAD) at 238 nm. The analyte was separated on NUCLEOSIC-CN Cyano coloumn (150 mm × 4.6 mm, particle size 5 μm) using a mobile phase consisting of acetonitrile and acidic buffer (adjusted to pH 2.5 with H3PO4 ­– 2 %) in the ratio of 37: 63 v/v and flow rate was 1 ml/min. The method is linear over a range of 100-1600 µg/ml (r2 ≥ 0.999) and 1-16µg/ml (r2 ≥0.998) for the assay of ART and LUME respectively. Itraconazole (ITZ) (10µg/ml) was used as internal standard. The retention times of ART and LUME was found to be 4.3 min and 14.3 respectively. Mean extraction recovery for ART and LUME were 87.3 % and 89.1 %, respectively. Inter and intraday coefficients of variation for ART and LUME were ≤ 10%. The lower limits of quantification for ART and LUME were 0.22 and 0.66 μg/ml, respectively. The results of the study showed that the proposed RP-HPLC validated method described is efficient and has the necessary accuracy and precision for the rapid quantitative simultaneous determination of ART and LUME in human plasma and is thus highly suitable for use in pharmacokinetic /bioavailability/bioequivalence studies in healthy human subjects.

A simple rapid, sensitive, selective and reproducible reversed- phase high-performance liquid chromatographic method has been developed and validated for the simultaneous estimation of embtricitabine and tenofovir in pure and Pharmaceutical dosage form. In present work a simple, sensitive and specific method (RP-HPLC assay, stability indicating RP-HPLC) has been developed for the simultaneous estimation of embtricitabine and tenofovir in pure and Pharmaceutical dosage form. A phenomenex BDS C18, column having 5 µm particle size and 150 mm x 4.6 mm in length and gradient mode, with mobile phase containing  potassium dihydrogen phosphate (pH3.0, adjusted with O-phosphoric acid) and  acetonitrile in the ratio of 96:4. The flow rate was 1.0ml/min and effluents were monitored by UV detector at 254nm. The retention times of emtricitabine and tenofovir is 3.1±0.1 & 6.1±0.1 min was recorded at 254nm. The method is linear and the correlation coefficient was found to be 0.999. The method was validated for linearity, precesion, accuracy, solution stability, ruggedness, and post degradation studies were performed. Recoveries from formulations were between 98.3 and 99.5%. The results of specificity studies indicated no interference from excipients, impurities, and degradation products under various stress conditions and assured that the peak response was due to a single component only. Hence, the present method is cost-effective, faster, and can be used for the routine analysis of these drugs in pure and formulations.