A Novel Validated Stability Indacating Method for Simultaneous Estimation of Emtricitabine, Tenofovir and Efavirenz in Tablet Dosage Form by RP-HPLC

Kommana Balaram Kumar*, Yohanes Ayele, Birhanu Motbatnor
^aAssistant Professor, Department of Pharmaceutical analysis and Quality Assurance School of Pharmacy, Haramaya University, Ethiopia.
^bDepartment of clinical pharmacy, H.O.D, School of pharmacy, Haramaya University, Ethiopia
^cLecturer, Department of pharmaceutical analysis and quality assurance School of pharmacy, Haramaya University, Ethiopia

A  B  S  T  R A C T
The objective of the current study was to develop and validate a rapid, precise, specific and stability-indicating reverse phase HPLC  method for the quantitative determination the  of three anti retroviral drugs emtricitabine (200mg), tenofovir (300mg), efavirenz (600 mg) used to treat HIV patients in its combined dosage form. The determination is done for the active pharmaceutical ingredient in its pharmaceutical dosage form in the presence of degradation products. The drug was subjected to stress conditions of acid, alkali, thermal, photolytic, humidity and peroxide. As per international conference on harmonization (ICH) prescribed stress conditions to show the stability-indicating power of the method. All the three drug solutions were scanned from 200-400 nm; it was observed that all the drugs show appreciable absorbance at 270nm. Hence detection was set at 270 nm for method development purpose. Attempts were made to get good separation between all the drugs by varying parameters like, flow rate, pH, buffer molarity, buffer components, type of organic modifier, gradient times, and buffer: organic modifier ratio but could not reduce the elution time of all the three  in isocratic mode. To achieve this, experiments were conducted by changing the columns and mobile shares but unsuccessful in getting good peaks with less run time. Then method was optimized to separate all the three main peaks by changing to Gradient mode. The satisfactory chromatographic separation, with good peak shapes were achieved on Symmetry C18 (4.6 x 150mm, 3.5mm, Make: XTerra) or equivalent with mobile phase potassium di hydrogen sulphate : Methanol and linear gradient programming Time (min)/buffer% 0/30, 5/30, 6/70,12/70,13/30,14/30 with a flow rate of 1.0 ml/min. Several gradient conditions were tried before optimizing the final linear gradient programme. All the System Suitability parameters are within the acceptance limits. The calibration curves for emtricitabine, tenofovir and efavirenz were obtained by plotting the respective peak areas against their concentration. The graphs were found to be linear over the range 7.5-45.0µg/ml for emtricitabine,11.25-67.50µg/ml for tenofovir and 22.5-135.0µg/ml for efavirenz with the correlation coefficient 0.999,0.999 and 0.999 respectively for all the drugs which shows that the good correlation exists between peak areas and concentration of the drug. The low % RSD of intraday and inter day study show that the method is precise. The high % recovery values obtained for these drugs show that the method is accurate. The LOD values of emtricitabine, tenofovir and efavirenz were found to be 0.018µg/ml, 0.81µg/ml and 5.05µg/ml respectively. The LOQ was 0.060µg/ml, 0.252µg/ml and 0.162µg/ml for emtricitabine, tenofovir and efavirenz respectively. The low values of LOD and LOQ show that the method is sensitive and can estimate at micro gram level. The absence of additional peaks indicates the method is specific and the drugs were stable in the diluents for 8 hours which is sufficient to complete the work.
Keywords: Atripla (drugs emtricitabine (200mg), tenofovir (300mg), efavirenz (600 mg)), Forced degradation, Assay, Method Validation, RP-HPLC