Development and Validation of RP-HPLC Method for Quantification of Total, Free and Entrapped Ritonavir in Lipid Nanocarriers and Drug content of Film Coated Fixed Dose Formulation

Abstract

Abstract: Aim: The current study was aimed to develop and validate a simple isocratic, precise, sensitive, accurate and robust reverse phase HPLC analytical method for the quantification of total, free and entrapped ritonavir in lipid nanocarriers and drug content of the film-coated fixed-dose formulation. Materials and Methods: The method was developed by using Inertsil ODS-3V C18 column as stationary phase, orthophosphoric acid (OPA) in water (pH 3.0) and acetonitrile as mobile phase. Further, the method was validated following ICH Q2(R1) guidelines. Ritonavir loaded lipid nanocarriers were developed using hot-emulsion and probe-sonication method. A solid phase extraction (SPE) method was developed for the separation of free and entrapped ritonavir. Total ritonavir was extracted from lipid nanocarriers (LNCs) and film-coated tablets using methanol and analyzed with validated method. Results: The responses were found to be linear and precise over a range of 0.25 μg/mL to 16 μg/mL. The limit of detection (LOD) and limit of quantification (LOQ) of the RP-HPLC method were found to be 14.06 ng/mL and 42.60 ng/mL, respectively which emphasizes the sensitivity of the method. The recovery of ritonavir from LNCs was found to be within 3% of the total drug present in LNCs. Similar, amount of drug recovery was found from assay of marketed formulation i.e., > 97%. Conclusion: The SPE method is capable of separating free and entrapped ritonavir from LNCs. The developed HPLC method is sensitive, robust, economical and gives high recovery. The developed HPLC method can also be employed for routine quantitative analysis of ritonavir in bulk formulation. Key words: HPLC method, Ritonavir, Validation, SPE, NLC, Bulk formulation.