An AQbD driven stability indicating HPLC method for simultaneous estimation of lamivudine, tenofovir disoproxil fumarate and efavirenz in plasma†

Abstract

The increasing prevalence of HIV has made us rely on a fixed dose combination (FDC) of anti-retroviral agents including lamivudine (LVD), tenofovir disoproxil fumarate (TDF) and efavirenz (EFZ) to ensure better therapeutic outcomes. In this context, the present study aims to develop and optimize Analytical Quality by Design (AQbD) employing D-optimal and Box–Behnken designs to simultaneously estimate LVD, TDF, and EFZ in a FDC. The optimized mobile phase (pH 4.5) comprised 25% acetonitrile (ACN), 46% methanol, 25% buffer and 4% KOH (30% w/v). The method parameters optimized included a 0.62 ml min⁻¹ flow rate, 10.96 μl injection volume and 36.95 °C column temperature. Under these conditions, retention times (RTs) for LVD, TDF, and EFZ were 4.1 ± 0.2, 5.1 ± 0.3, and 8.6 ± 0.4 min, respectively with an acceptable tailing factor (TF) of 1.55 ± 0.02, 1.43 ± 0.02, and 1.69 ± 0.02 respectively. Forced degradation studies conducted employing the optimized method showed ≤10% peak degradation. The method demonstrated ≤2.00% RSD for the TF, theoretical plates (TP), peak area, robustness and intra/inter-day precision when validated as per ICH Q2 (R1), ICH-Q12 and ICH-Q14 guidelines. The accuracy was observed to be in the range of 99.35 ± 0.43 to 101.22 ± 0.24%, 99.72 ± 0.39 to 101.37 ± 0.23% and 99.64 ± 0.38 to 101.83 ± 0.41% for LVD, TDF, and EFZ respectively. In rabbit plasma, at the lowest spiked concentration (0.25 μg ml⁻¹) the recovery rates for LVD, TDF, and EFZ were 97.81%, 95.92% and 96.38%, confirming the sensitivity of the AQbD driven optimized HPLC method. The study successfully demonstrated the robustness and suitability of the AQbD based analytical method for possible clinical applications.