An optimized method for the analysis of corticosterone in rat plasma by UV–HPLC

Abstract

Animal models are useful in the study of stress disorders in that they offer the possibility of stimulating a human condition under controlled conditions in a simpler, more readily understandable system. Stress-related activation of the hypothalamic-pituitary-adrenal (HPA) axis characterized by an increase in plasma corticosterone (CT) levels in the rat is an important manifestation of the physiological stress response. Current available methods for the determination of peripheral corticosterone concentrations from trunk blood, is via a commercially available radio immunoassay (RIA) kit. The aim of this study was to optimize and validate a sensitive, specific and cost-effective high performance liquid chromatography (HPLC) method for the determination of CT levels in plasma of rats. A 500 µl plasma sample was extracted with 5 ml dichloromethane and analyzed by HPLC coupled to a diode array detector at 245 nm. The standard curve was linear over a concentration range of 10 - 500 ng/ml (r 2 =0.996). The percentage recovery was above 85%, the relative standard deviation was less than 7% and the limit of quantification was 10 ng/ml. Results from this method were compared with values obtained from a RIA method and the values were in close proximity of each other. We conclude that the current HPLC method that was optimized and validated is suitable for use in subsequent studies in rats.