Bio-Analytical Method Development and Validation of Gamma-Oryzanol in RAT Plasma and Brain Homogenate Using Reversed Phase-High Performance Liquid Chromatography

Abstract

The study was aimed to develop and validate an accurate, precise and reproducible bioanalytical method for qualitative and quantitative estimation of gamma oryzanol in different body fluids i.e. plasma and brain homogenate. Gamma Oryzanol (Gammaoryzanol) is component of rice bran oil used for many medicinal purposes for e.g Anticancer, antihyperlipidemic, antidiabetic, cardiotonic, and anti-stress. Analytical method was developed using a HPLC system with a PDA detector for Gamma-oryzanol standard in which drug elution was performed using c-18 column (4.6 mm x 150 cm, 5 microns) with 1% acetic acid in methanol: acetonitrile (65:35, V/V) mobile phase at 1.2 ml/min flow rate and detected at 326 nm wavelength. Then Oryzanol was spiked in rat plasma and brain homogenate and extracted out with liquid liquid extraction. The bioanalytical method was validated according to the guidelines provided by USFDA. Extraction method was optimized by comparing different extraction procedures i.e. liquid-liquid extraction (LLE), protein precipitation (PP) and solid phase extraction (SPE). Highest recovery of oryzanol was found in LLE (i.e.95% in plasma, 85% in brain homogenate) as compared to other methods. Various Extraction solvents for each body fluid were analysed, out of which highest recovery for brain homogenate was observed in Iso-propyl alcohol (IPA) and for plasma, in acetone:IPA (1:1, v/v). Recovery of oryzanol from plasma was 95% and from brain was 85%. The linear response was observed from 500 ng/ml to 5000 ng/ml. Here, LLOQ was 500ng/ml. The interday and intraday precision (i.e. %RSD) was less than 10% and accuracy was ± 5%. Selectivity and matrix effect was checked and found within acceptance criteria (complete absence of interference was observed). Bio analytical samples were found to be stable over the analysis period, HQC samples were stable up to third cycle in freeze and thaw stability while LQC samples were stable over fourth cycle. The developed bio-analytical method is applicable in pharmacokinetic study. This method can also be used in estimation of oryzanol in novel drug delivery systems and different dosage forms. The method proved to be simple, useful and appropriate, for preclinical and experimental research.