Development and Validation of Stability-indicating assay UHPLC Method for Simultaneous analysis of Dolutegravir, Lamivudine and Tenofovir disoproxil fumarate in Bulk and Pharmaceutical Formulation

Abstract

A stability indicating UHPLC method has been developed to analysis of Dolutegravir (DLT), Lamivudine (LVD), and Tenofovir Disoproxil Fumarate (TDF). The separation was achieved by using UPLC BEH C18 (150mm 2.1mm) with 1.7 µm particle size column at ambient temperature using solvent system in a proportion of (40:60% v/v) acetonitrile:water; pH 6.5 was adjusted with 0.1% OPA. The solvent system was filtered prior to the start of the chromatographic analysis through a 0.2μm membrane (Ultipor N66 Nylon 6, 6) and sonication of it for 20min. A 10μL of fixed volume (working solution) was injected and the chromatogram was studied at a detection wavelength of 262 nm. The proposed method was validated in terms of Linearity, Accuracy, Precision, Ruggedness, Robustness and stability studies. The chromatographic analysis time was approximately less than 3 minutes with complete resolutions of DLT (Rt = 1.35min), LVD (Rt = 0.69min) and TDF (Rt = 2.36min). The method exhibited good linearity range, 3-18 μg/mL, 10-60 μg/mL and 10-60 μg/mL of DLT, LVD and TDF respectively. The % amount of DLT, LVD and TDF in marketed formulation were recorded to be 99.59 ± 0.44, 99.81 ± 0.48 and 99.47 ± 0.59 respectively. The force degradation studies were performed as per ICH guidelines under the acidic, alkali oxidative and neutral conditions for different times. Therefore the developed UHPLC method can be applied for routine qualitative and quantitative analysis of DLT, LVD and TDF in bulk and pharmaceutical formulation and validated as per theICH guidelines and could be employed for the stability studies on pharmaceutical preparation within pharmaceutical industry.