Optimization of RP-HPLC Method for Simultaneous Estimation of Lamivudine and Raltegravir in Binary Mixture by Using Design of Experiment

Veena D. Singh, Sanjay J. Daharwal

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Response surface methodology, box behnken design, RP-HPLC, lamivudine, raltegravir


A simple, sensitive, cost effective and robust RP-HPLC method for the simultaneous estimation of the Lamivudine (LAM) and Raltegravir (RAL) in laboratory prepared binary mixture was developed, optimized and validated. Separation was achieved on phenomenex C18 column (150 X 4.6 mm id, 5μ particle size) and mobile phase was composed of 75% methanol: 15% Acetonitril: 10 % (0.05mM) phosphate buffer (at pH 3.0), with flow rate 1.2 ml/min at 254nm. Developed method was optimized by using Box Behnken Design (BBD) in response surface methodology (RSM). The independent variables such as the concentration of methanol, pH in mobile phase and flow rate were selected for the optimization and Retention time (Rt) were used as responses for both drugs. Derringer’s desirability function was used to concurrently optimize the selected responses. The LOD and LOQ were found to be 1.04 and 3.18 μg/ mL for LAM and 0.36 and 1.08μg/mL of RAL. The percentage recoveries were found to be less than 2% for LAM and RAL. Retention time of LAM and RAL was 3.13±0.07 and 7.27±0.01 minutes respectively. Conclusion: The developed and optimized method was fully validated. The validated method further can be potentially used for estimation of these drugs in combined dosage form.