Abstract
BackgroundNaringenin 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.ObjectiveIn 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.MethodsAn 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.ResultsCalibration 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%).ConclusionThis analytical and bio-analytical method was applied to detect entrapment efficiency, in-vitro release, and pharmacokinetic parameters of naringenin nanoparticles.
Highlights
Naringenin is a flavanone having strong antioxidant potential
The method which we have developed here has been extrapolated for estimation of the drug in plasma and brain respectively for in-vivo pharmacokinetic studies as well as to quantify the brain uptake of naringenin by calculating the brain:plasma ratio of naringenin
The addition of 0.5% ortho-phosphoric acid improved the separation of naringenin by suppressing its ionization and reduced peak tailing resulting in a sharp symmetric peak with a retention time (Rt) of 7.2 min in the standard solution of 40 μg/ml
Summary
An isocratic RP-HPLC method was developed using C18 reversed-phase column (250 × 4.6 mm internal diameter and 5 μm particle size). Methanol and 0.5% ortho-phosphoric acid in MilliQ water (pH 2) (70:30) was used as mobile phase. 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. Methanol and acetonitrile (HPLC grade) were obtained from Merck (Darmstadt, Germany). Ortho-phosphoric acid (analytical grade) was obtained from Fisher Scientific (Leicestershire, UK). Ultrapure water used for HPLC analysis was obtained from Milli-Q water purification system (Millipore, MA, USA). 0.45 μm solvent filter and 0.22 μm nylon syringe filter (Millipore, MA, USA) respectively were used
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