Validated RP-HPLC-PDA method for simultaneous determination of Zidovudine, Lamivudine, and Nevirapine in pharmaceutical formulation

Abstract

Aim and Objectives: A simple, rapid, and sensitive high performance liquid chromatographic method with ultraviolet detection has been developed and validated according to the International Conference on Harmonization (ICH) guidelines for the quantitation and qualification of zidovudine (ZID), lamivudine (LAM), and nevirapine (NEV) in pharmaceutical dosage forms. Materials and Methods: The proposed method was based on the separation of the drugs in reversed phase mode using Water′s C18 250 cm × 4.6 mm, 5 μ particle size column maintained at an ambient temperature. The optimum mobile phase consisted of Water: Methanol (70:30 v/v), pH adjusted to four with orthophosphoric acid (OPA). The flow rate of mobile phase was set 1.0 mL min -1 and photodiode array detection was performed at 275 nm with a total run time of 8 min which is very short for accurate analysis of simultaneous estimation of three analytes. The method was validated according to ICH guidelines. Results: The method was linear over the concentration range of 25-75 μg mL -1 with limit of quantifications (LOQ) of 13, 0.49, and 0.40 ng mL -1 for ZID, LAM and NEV respectively and limit of detection (LOD) of 4, 0.14, and 0.12 ng mL -1 for ZID, LAM and NEV respectively. Accuracy (% recovery studies) and precision values of both inter and intraday obtained from six different replicates for all the analytes ranged from 99.00% to 100.00% and % relative standard deviation of precision (assay) was between 0.64 and 1.28, respectively. All the three analytes and their combination drug product were exposed to thermal, photolytic, hydrolytic, reductive, oxidative and peroxide stress conditions and the stressed samples were analyzed by the proposed method. There were no interfering peaks from excipients, impurities or degradation products due to variable stress conditions and the proposed method is specific for the simultaneous estimation of ZID, LAM and NEV in the presence of their degradation products. Conclusion: The proposed method can be successfully applied in the quality control and stability samples of pharmaceutical dosage forms.