HPLC Quantification of Efavirenz in Nanoparticles

The amount of the EFV in the nanoparticles and filtrate was analyzed by using a reverse phase-HPLC method described in our earlier paper.^{26 (link)} The HPLC apparatus consisted of a pump (LC-10ATvp), system controller (SIL-10ADvp), degasser unit (DGU-14A), refrigerated auto-sampler (SIL-10ADvp), a UV-Vis detector (SPD-10ADvp) and a column heater (Shimadzu Corporation, Columbia, MD). Samples were run through a C₁₈ pre-column and a Gemini C₁₈ reverse-phase [150 × 4 5 mm (I.D.)] with 5 μm particle size packing (Phenomenex, Torrance, CA). The mobile phase consisted of acetonitrile and 25 mM KH₂PO₄ solution (55:45). For HPLC analysis, the flow rate of the mobile phase was at 0.9 ml/min, column oven was set at 35°C, injection volume was 20 μl and detector was set at 212 nm. The retention time for EFV was 10.4 min. For standard curve, EFV stock solution (1 mg/ml) was prepared in acetonitrile. The stock solutions were diluted with acetonitrile to obtain solutions of various concentrations. Standard curve was obtained by injecting 0.1–10 μg/ml of EFV. Limit of detection for EFV was 50 ng/ml. All the experiments were performed in triplicate. The inter-day and intra-day variability for the standard curve was always < 10%.