Development of a new, sensitive, and robust analytical and bio-analytical RP-HPLC method for in-vitro and in-vivo quantification of naringenin in polymeric nanocarriers

Abstract

Background: Naringenin is a flavanone having strong antioxidant potential. It is an anti-hyperlipidemic, anti-depressant, anti-cancer, and neuroprotective agent. However, its major limitation is its low oral bioavailability. Objective: In order to overcome this limitation and to explore its antioxidant potential in autism spectrum disorders, we developed brain targeting PLGA nanocarriers of naringenin. Current study involves development of a sensitive and robust RP-HPLC method for detection and quantification (in vitro and in vivo) of naringenin in nanoparticles. Methods: An isocratic RP-HPLC method was developed using C18 reversed-phase column (250 × 4.6 mm internal diameter and 5 μm particle size). Flow rate of mobile phase was 1 ml/min and temperature of column was 30 °C. Methanol and 0.5% ortho-phosphoric acid in MilliQ water (pH 2) (70:30) was used as mobile phase. The ultraviolet detection wavelength for quantification was at 289 nm. Results: Calibration curve showed linearity within the concentration range from 0.5 to 40 μg/ml (R2 = 1) for the analytical method and for plasma (6.3–200 ng/ml (R2 = 0.9975)) and brain tissue samples (31.25–12,500 ng/ml (R2 = 1)). Limit of detection (LOD) and limit of quantification (LOQ) were 0.15 μg/ml and 0.44 μg/ml for the analytical method. For bio-analytical method, LOD and LOQ were 9.71 ng/ml and 29.44 ng/ml for plasma and 9.06 ng/ml and 27.44 ng/ml for brain sample. Both the method was precise, accurate, and robust. Bio-analytical method showed good recovery from plasma and brain samples (> 95%). Conclusion: This analytical and bio-analytical method was applied to detect entrapment efficiency, in-vitro release, and pharmacokinetic parameters of naringenin nanoparticles.